Systems and methods of nitric oxide therapy and treatment monitoring

Abstract

Systems and methods for delivering and monitoring nitric oxide (NO) and / or nitrogen dioxide (NO2) to a patient in medical applications are provided. In some embodiments, systems and methods are provided that are capable of quantifying absorption of NO and / or NO2 by a patient on a breath-by-breath basis. In other embodiments, systems and methods are provided that, based on the measurement of the content of methemoglobin (MetHb) in the blood and, using a MetHb absorption kinetic model, are capable of quantifying absorption of NO and / or NO2 by a patient to whom was delivered a NO and / or NO2 containing gas.

Description

125141.04962 Patent Application - MGH 2025-004-02SYSTEMS AND METHODS OF NITRIC OXIDE THERAPY AND TREATMENT MONITORINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 753,698, filed on February 4, 2025, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] Not applicable.BACKGROUND

[0003] The present disclosure relates generally to systems and methods for delivering and tracking nitric oxide (NO) therapy. In one example, the present disclosure relates to systems and methods for monitoring inhaled NO absorption.

[0004] Inhaled NO has long been used in clinical settings, primarily as a selective pulmonary vasodilator in certain neonatal and adult respiratory conditions. Over time, research has explored additional potential roles for inhaled NO, including effects on cardiac, renal, neurologic, and immune systems, as well as possible antimicrobial activity at higher concentrations. These expanding areas of investigation have prompted interest in more precisely understanding how NO behaves once delivered to the respiratory tract of a patient.

[0005] Despite these potential applications, observed clinical responses to inhaled NO have varied considerably across studies. One challenge in interpreting these findings is the inherent variability in how much NO is ultimately absorbed and becomes available for physiological interaction. Different patients — and even the same patient under different conditions — may absorb NO at markedly different rates due to factors such as delivery technique, lung mechanics, metabolic processes, and underlying disease states.

[0006] Following inhalation, NO rapidly reacts with components of the blood, particularly hemoglobin, forming methemoglobin (MetHb, which is unable to bind and deliver oxygen), as well as nitrate, with a smaller fraction of other nitrogen derivatives that could contribute to downstream biological effects. Because these processes occur quickly and dynamically, it can be difficult to assess, in real time, how much NO a patient actually absorbs during therapy.-1- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0007] Clinically, inhaled NO is typically prescribed based on concentration (e.g., parts per million). However, the administered concentration alone does not necessarily reflect the amount of NO that reaches the bloodstream or tissues. Variables such as minute ventilation, tidal volume, lung volume, hemoglobin concentration, and circulating blood volume can all influence the amount of NO absorbed. Even normal physiologic changes, such as those associated with rest versus exercise, may meaningfully alter NO absorption in a patient.

[0008] A clearer understanding of a patient’s actual absorbed NO dose could potentially help contextualize treatment responses, support efforts to align delivered therapy with intended dosing strategies, and provide insight into how individual physiological differences affect NO availability. Additional knowledge about absorbed dose may also inform efforts to avoid delivering more NO than necessary, which could reduce excess gas usage and limit unnecessary environmental release of nitrogen dioxide (NO2).

[0009] Thus, there is a need for systems and methods to deliver therapies, such as inhaled therapies including NO, across broad and diverse patient populations in a manner that is efficient and effective for clinical practice.SUMMARY

[0010] The present disclosure overcomes the aforementioned drawbacks by providing systems and methods for monitoring delivery of nitric oxide (NO). In one non-limiting example, the systems and methods may monitor or even quantify absorption of inhaled NO. In some nonlimiting examples, the quantification may be implemented on a breath-by-breath basis to address the longstanding challenges associated with inconsistent NO bioavailability, unpredictable dosing, and the absence of standardized techniques for measuring NO uptake. As described herein, variability in pulmonary and extrapulmonary absorption of inhaled NO can lead to inconsistent therapeutic effects, difficulty in dose optimization, and uncertainty in patient response. In some non-limiting examples, the present disclosure provides systems and methods that measure real-time concentrations of NO and / or NO2 together with airway flow characteristics, enabling the calculation of the amount of NO and / or NO2 absorbed by a patient for each individual breath. By integrating sensor data with computational analysis, the disclosed systems can determine actual delivered and absorbed doses, thereby offering a quantitative framework for guiding therapy. In this way, the systems and methods described herein can be used to enable accurate assessment of-2- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02NO absorption, improve dosing precision, enhance patient safety, and reduce waste, ultimately addressing the shortcomings of conventional concentration-based NO delivery approaches.

[0011] In one aspect, the present disclosure provides a system for monitoring nitric oxide (NO) and / or nitrogen dioxide (NO2) containing gas delivered to a patient. The system includes a breathing system configured to provide a first flow of NO and / or NO2 containing gas to the patient. The system further includes a sensor system configured to monitor the first flow of NO and / or NO2 containing gas to the patient and to monitor a second flow of gas exhaled by the patient. The system further includes a controller configured to receive feedback from the sensor system, determine an amount of NO and / or NO2 gas inhaled by the patient and an amount of NO and / or NO2 gas exhaled by the patient to determine an amount of NO and / or NO2 gas absorbed by the patient using the feedback from the sensor system, and generate a report indicating the amount of NO and / or NO2 absorbed by the patient. The system further includes a display configured to communicate the report indicating the amount of NO and / or NO2 absorbed by the patient.

[0012] In some embodiments, the controller is further configured to control the breathing system to adjust the first flow of NO and / or NO2 containing gas and the amount of NO and / or NO2 absorbed by the patient.

[0013] In some embodiments, the breathing system includes a first line through which the first flow of gas flows to the patient and a second line through which the second flow of gas flows.

[0014] In some embodiments, the first line and the second line are in fluid communication with a port. The port is in fluid communication with a patient interface that is configured to deliver NO and / or NO2 containing gas to the patient.

[0015] In some embodiments, the breathing system includes a breathing apparatus configured to provide a flow of gas to the first line, and the system further comprises an NO and / or NO2 gas source configured to inject a gas containing NO and / or NO2 into the first line so that the first flow of gas provided to the patient contains NO and / or NO2.

[0016] In some embodiments, the breathing apparatus is an invasive or non-invasive lung ventilator.

[0017] In some embodiments, the sensor system includes a rapid response NO and / or NO2 sensor configured to sense a first NO and / or NO2 concentration of the first flow of gas and a second NO and / or NO2 concentration of the second flow of gas. Further, the sensor system includes a flow-3- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02rate sensor configured to sense a first flow rate of the first flow of gas and a second flow rate of the second flow of gas.

[0018] In some embodiments, the NO and / or NO2 gas sensor is positioned along a sampling line that is in fluid communication with the port.

[0019] In some embodiments, the breathing system adjusts the first flow of NO and / or NO2 containing gas to adjust the amount of NO and / or NO2 absorbed by the patient by at least one of: (i) adjusting an amount of gas provided to the first line by the breathing apparatus, and (ii) adjusting an amount of gas injected into the first line by the NO and / or NO2 gas source.

[0020] In some embodiments, the system further comprises a user interface configured to receive an input indicating a desired amount of NO and / or NO2 absorbed by the patient. The controller is further configured to adjust the first flow of NO and / or NO2 containing gas to match the amount of NO and / or NO2 absorbed by the patient to the input indicating a desired amount of NO and / or NO2 absorbed by the patient.

[0021] In another aspect, the present disclosure provides a system for monitoring nitric oxide (NO) and / or nitrogen dioxide (NO2) containing gas delivered to a patient. The system comprises a breathing system including a breathing apparatus, a first line, and a second line. The breathing apparatus is configured to provide a first flow of NO and / or NO2 containing gas to the patient via the first line and remove a second flow of gas from the patient via the second line. The system further comprises a sensor system operably coupled to the breathing system. The sensor system comprises a first sensor configured to measure a real-time NO and / or NO2 concentration of the first flow of gas and the second flow of gas and a second sensor configured to measure a real-time flow rate of the first flow of gas and the second flow of gas. The system further comprises a computing device electronically coupled to the sensor system. The computing device is configured to: receive, from the sensor system, a first output from the first sensor and a second output from the second sensor, analyze the first output and the second output to determine an amount of NO and / or NO2 absorbed by the patient, and generate a report indicating the amount of NO and / or NO2 absorbed by the patient.

[0022] In some embodiments, the computing device is further configured to generate a first value indicating the amount of NO and / or NO2 absorbed by the patient.

[0023] In some embodiments, the computing device is further configured to at least one of: (i) compare the first value to a second predetermined value and adjust, based on the comparison-4- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02between the first value and the second predetermined value, the first flow of NO and / or NO2 containing gas to the patient; (ii) display the first value; and (iii) compare the first value to a third predetermined value and generate, based on the comparison between the first value and the third predetermined value, a user notification indicative of the first value and the third predetermined value.

[0024] In some embodiments, the computing device is further configured to determine the first value by: receiving, from a third sensor configured to measure a temperature of the first line and the second line, a third output from the third sensor; estimating, based on the first output, the second output, the third output, and the Ideal Gas Law (PV=nRT), a first amount of NO and / or NO2 in the first flow of gas and a second amount of NO and / or NO2 in the second flow of gas; and determining, based on a difference between the first amount and the second amount, a molar quantity of NO and / or NO2 absorbed by the patient on a breath-by-breath basis.

[0025] In some embodiments, the computing device is further configured to determine the first value by: receiving, from a fourth sensor configured to measure an atmospheric pressure, a fourth output from the fourth sensor; estimating, based on the first output, the second output, the third output, the fourth output, and the Ideal Gas Law (PV=nRT), a first amount of NO and / or NO2 in the first flow of gas and a second amount of NO and / or NO2 in the second flow of gas; and determining, based on a difference between the first amount and the second amount, a molar quantity of NO and / or NO2 absorbed by the patient on a breath-by-breath basis.

[0026] In some embodiments, the system further comprises a display configured to display the report indicating the amount of NO and / or NO2 absorbed by the patient.

[0027] In some embodiments, the system further comprises a user interface configured to receive an input indicating a desired amount of NO and / or NO2 absorbed by the patient. The controller is further configured to adjust the first flow of NO and / or NO2 containing gas to match the first value indicating the amount of NO and / or NO2 absorbed by the patient to the input indicating a desired amount of NO and / or NO2 absorbed by the patient

[0028] In some embodiments, the system further comprises an NO and / or NO2 gas source fluidly coupled to the breathing system, the NO and / or NO2 gas source configured to inject a gas containing NO and / or NO2 into the first line.

[0029] In another aspect, the present disclosure provides a method for delivering a nitric oxide (NO) and / or nitrogen dioxide (NO2) containing gas to a patient and monitoring the NO and / or NO2-5- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02dosage delivered to the patient. The method comprises: (a) delivering a first flow of NO and / or NO2 containing gas to the patient for inhalation via a first line; (b) providing a second line configured to receive a second flow of gas from the patient after exhalation; (c) measuring, with a first sensor, a first flow parameter indicating a real-time NO and / or NO2 concentration of the first flow of gas and the second flow of gas; (d) measuring, with a second sensor, a second flow parameter indicating a flow rate of the first flow of gas and the second flow of gas; (e) analyzing, with a computing device, the first flow parameter and the second flow parameter; and (f) at least one selected from a group of: (i) adjusting, based on the analysis of the first flow parameter and the second flow parameter, the first flow of NO and / or NO2 containing gas to the patient, (ii) displaying the first flow parameter and the second flow parameter, and (iii) generating, based on the analysis of the first flow parameter and the second flow parameter, a user notification indicative of the first flow parameter and the second flow parameter failing to meet a predetermined threshold.

[0030] In some embodiments, step (e) of analyzing the first flow parameter and the second flow parameter comprises: measuring, with a third sensor, a third flow parameter indicating a temperature of the first flow of gas and the second flow of gas; determining, based on the first flow parameter, the second flow parameter, the third flow parameter, and the Ideal Gas Law (PV=nRT), a first amount of NO and / or NO2 in the first flow of gas and a second amount of NO and / or NO2 in the second flow of gas, and determining, based on a difference between the first amount and the second amount, a molar quantity of NO and / or NO2 absorbed by the patient on a breath-by-breath basis.

[0031] In some embodiments, the method further comprises: (g) identifying, based on at least one of the first flow parameter and the second flow parameter, an end of a first respiratory cycle and a start of a second respiratory cycle; and (f) repeating step (a) through step (g) for each respiratory cycle.

[0032] In another aspect, the present disclosure provides a system for monitoring an amount of nitric oxide (NO) absorbed by a patient to whom NO containing gas was delivered. The system comprises a monitoring system configured to monitor an amount of methemoglobin (MetHb) in the patient and generate an output indicating the amount of MetHb in the patient. The monitoring system is configured to quantify the amount of MetHb by at least one of: measuring an absorption of light transmitted through the patient or through the patient’s blood, quantifying hemoglobin-6- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02levels of the patient based on the measurement, analyzing the absorption of light, and determining, based on that analysis, the amount of MetHb in the patient; and / or collecting a sample from the patient and utilizing the principle of blood gas analysis to determine the amount of MetHb in the patient. The system further comprises a computing device configured to: receive the output from the monitoring system; determine the amount of NO absorbed by the patient based on the amount of MetHb in the patient and a MetHb kinetic model; and generate a report indicating the determined amount of NO absorbed by the patient. The system further comprises a display configured to communicate the report indicating the determined amount of NO absorbed by the patient.

[0033] In some embodiments, the NO containing gas was delivered to the patient via extrapulmonary blood gas exchange.

[0034] In some embodiments, the controller is further configured to automatically adjust the NO and / or NO2 delivery based on the determined amount of NO and / or NO2 absorbed by the patient.

[0035] In another aspect, the present disclosure provides a method for delivering a nitric oxide (NO) and / or nitrogen dioxide (NO2) containing gas to a patient and monitoring the NO and / or NO2 dosage delivered to the patient. The method comprises: (a) delivering a flow of NO and / or NO2 containing gas to the patient for inhalation; (b) measuring an amount of MetHb in the patient; (c) determining, based on the amount of MetHb in the patient and a MetHb kinetic model, an amount of NO absorbed by the patient; (d) generating a report indicating the amount of NO absorbed by the patient; and (e) displaying the report.

[0036] In some embodiments, the method further comprises: (f) controlling, based on the amount of NO and / or NO2 absorbed by the patient, the NO and / or NO2 dosage delivered to the patient.

[0037] The foregoing and other aspects and advantages of the present disclosure will appear from the following description. In the description, reference is made to the accompanying drawings that form a part hereof, and in which there is shown by way of illustration one or more embodiment. These embodiments do not necessarily represent the full scope of the invention, however, and reference is therefore made to the claims and herein for interpreting the scope of the invention. Like reference numerals will be used to refer to like parts from Figure to Figure in the following description.-7- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Various objects, features, and advantages of the disclosed subject matter can be more fully appreciated with reference to the following detailed description of the disclosed subject matter when considered in connection with the following drawings, in which like reference numerals identify like elements.

[0039] FIG. 1 shows a schematic illustration of a breathing system according to one embodiment of the present invention.

[0040] FIG. 2 shows a schematic illustration of a breathing system according to one embodiment of the present invention.

[0041] FIG. 3 shows a schematic illustration of a breathing system according to one embodiment of the present invention.

[0042] FIG. 4 shows a schematic illustration of an NO delivery system according to one embodiment of the present invention.

[0043] FIG. 5 is a flowchart illustrating some non-limiting example steps for operating a breathing system according to one embodiment of the present disclosure.

[0044] FIG. 6 is a flowchart illustrating some non-limiting example steps for analyzing the NO concentration and the flow rate according to one embodiment of the present disclosure.

[0045] FIG. 7 is a flowchart illustrating some non-limiting example steps for operating an NO delivery system according to one embodiment of the present disclosure.

[0046] FIG. 8A is a graph showing breath-by-breath method NO absorption against bag method NO absorption in simulations.

[0047] FIG. 8B is a graph showing percentage difference from bag method against bag method NO absorption in simulations.

[0048] FIG. 8C is a graph showing breath-by-breath method NO absorption against bag method NO absorption in swine.

[0049] FIG. 8D is a graph showing percentage difference from bag method against bag method NO absorption in swine.

[0050] FIG. 9A is a graph showing MetHb against time in swine.

[0051] FIG. 9B is a graph showing MetHb against time in swine.

[0052] FIG. 9C is a graph showing MetHb against time in human studies of healthy volunteers.

[0053] FIG. 9D is a graph showing MetHb against time in human studies of healthy volunteers.-8- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0054] FIG 9E is a graph showing MetHb against time in human studies of healthy volunteers.

[0055] FIG. 9F is a graph showing MetHb against time in human studies of healthy volunteers.

[0056] FIG. 10A is a graph showing estimated NO absorption rate against measured NO absorption rate in swine.

[0057] FIG. 10B is a graph showing percentage difference against measured NO absorption rate in swine.

[0058] FIG. 10C is a graph showing estimated NO absorption rate against measured NO absorption rate in human studies.

[0059] FIG. 10D is a graph showing percentage difference against measured NO absorption rate in human studies.

[0060] FIG. 11 A is a graph showing NO absorption by breath against tidal volume.

[0061] FIG. 1 IB is a graph showing NO absorption by breath against inspiratory pause.

[0062] FIG. 11C is a graph showing NO absorption by breath against inspiratory time.

[0063] FIG. 1 ID is a graph showing NO absorption by breath against flow pattern.

[0064] FIG. 1 IE is a graph showing NO absorption by breath against cardiac output.DESCRIPTION

[0065] As described, no standardized methods currently exist to quantify inhaled NO or NO2 absorption. Existing gas-exchange techniques often require alterations to the breathing circuit or collection of gases in external reservoirs, which can disrupt airway pressures, add dead space, or otherwise interfere with respiratory support. Such limitations make the current approaches unsuitable for mechanically ventilated or critically ill patients. There is therefore interest in approaches that can quantify NO and / or NO2 uptake without disturbing standard ventilation systems. Further, a system capable of measuring NO and / or NO2 absorption on a breath-by-breath basis can allow real-time characterization of the amount of NO and / or NO2 that a patient absorbs throughout each respiratory cycle. This provides breath-to-breath insight into physiologic variability and allows continuous monitoring during clinical use.

[0066] FIG. 1 shows a schematic illustration of a nitric oxide (“NO”) and or nitrogen dioxide (NO2) delivery and monitoring system 10 for administering NO and / or NO2 to a patient 11 according to one non-limiting example of the present disclosure. The NO and / or NO2 delivery system 10 can include a breathing system 12, an NO and / or NO2 gas source 14, a sensor system-9- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-0216, and a computing device 18. The breathing system 12 (e.g., a ventilator system, a continuous positive airway pressure (CPAP) system, a High Frequency Oscillatory Ventilator (HFOV), a non-invasive mask with gas flow, etc.) is configured to allow the passage of gas to and from the patient 11. In some examples, the breathing system 12 can provide mechanical ventilation to the patient 11 (i.e., positive pressure to inflate the lungs of the patient 11). In other examples, the patient 11 may be breathing on their own and the breathing system 12 provides a flow path to the airway of the patient 11. The illustrated breathing system 12 includes a breathing apparatus 20, an inspiratory line 22, an expiratory line 24, a port 26, and a patient interface 28. The breathing apparatus 20 is configured to provide a first flow of gas (e.g., air, nitrogen / oxygen, or other gas mixture) via the inspiratory line 22 to the port 26, which provides the first flow of gas to the patient interface 28 and the patient 11. Subsequently, the breathing apparatus 20 is configured to remove a second flow of gas (e.g., exhaled gas). The second flow of gas is provided to the patient interface 28 from the patient 11. The patient interface 28 provides the second flow of gas to the port 26, which provides the second flow of gas to the expiratory line 24. The expiratory line 24 provides the second flow of gas to the breathing apparatus 20 for discarding or re-use in the breathing system 12. In this way, the breathing apparatus 20 can, optionally, be configured to simulate the breathing process for the patient 11. In some examples, the breathing apparatus 20 can be a commercially available mechanical ventilator used in biomedical application (e.g., inhalation therapy). The patient interface 28 is configured to deliver NO and / or NO2 to the patient 11 from the breathing apparatus 20 while maintaining a closed breathing circuit in the NO and / or NO2 delivery system 10. In some examples, the patient interface 28 is an endotracheal tube.

[0067] In other examples, the patient interface 28 is a sealed mask and the breathing apparatus 20 can be a simple gas source that delivers gas via the inspiratory line 22. In this case, the expiratory line 24 may simply be formed as a return to atmosphere, and not be received or processed by the breathing apparatus 20. Alternatively, the expiratory line 24 may be omitted and the patient interface configured to return gas exhaled by the patient to, for example, the atmosphere. In this case, as will be described, the sensor system 16 may be configured to monitor the patient interface as an alternative to or in addition to the port.

[0068] The NO and / or NO2 gas source 14 is fluidly coupled to the breathing system 12 along the inspiratory line 22 at a position located upstream of the port 26 and the patient interface 28. The NO and / or NO2 gas source 14 is configured to inject a gas containing NO and / or NO2 into the-10- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02inspiratory line 22. In some examples, the NO and / or NO2 gas source 14 is coupled at an injection site that is positioned downstream of the breathing apparatus 20. In other examples, the breathing apparatus 20 and the NO and / or NO2 gas source 14 are in-line. In such an example, the breathing apparatus 20 and the NO and / or NO2 gas source 14 can be in fluid communication with a reservoir (not shown) that is configured to receive gas from the breathing apparatus 20 and gas containing NO and / or NO2 from the NO and / or NO2 gas source 14. In this way, the reservoir can fill with NO and / or NO2 gas that is diluted with the gas received from the breathing apparatus 20. In some examples, the NO and / or NO2 gas source 14 can be a tank filled with NO and / or NO2 gas (and other gases such as N2). For example, the NO and / or NO2 gas source 14 can include a pressurized cylinder of NO and / or NO2 gas that, when connected to the NO and / or NO2 delivery system 10 (e.g., the inspiratory line 22 of the breathing system 12), delivers NO and / or NO2 gas to the inspiratory line 22. In other examples, the NO and / or NO2 gas source 14 can include a container filled with NO and / or NO2 gas and a pump in communication with the container to drive NO and / or NO2 gas out of the container and into the inspiratory line 22 (or other portions of the NO and / or NO2 delivery system 10).

[0069] The sensor system 16 is fluidly coupled to the breathing system 12 between the NO and / or NO2 gas source 14 and the patient interface 28. The sensor system 16 can include multiple sensors that can sense parameters of the NO and / or NO2 delivery system 10. For example, the sensor system 16 includes a first sensor (e.g., an NO and / or NO2 sensor 30) and a second sensor (e.g., a flow rate sensor 32). The NO and / or NO2 sensor 30 and the flow rate sensor 32 can each be in fluid communication with a conduit that delivers the gas containing NO and / or NO2 to the patient 11 for inhalation. Relatedly, the NO and / or NO2 sensor 30 and the flow rate sensor 32 can each be in fluid communication with a conduit that returns the gas to the breathing apparatus 20 from the patient 11 for exhalation. In some examples, the conduit for delivering the gas containing NO and / or NO2 and the conduit for returning the gas is the same conduit (e.g., the port 26). In the illustrated example, the flow rate sensor 32 is positioned along the port 26. The NO and / or NO2 sensor 30 is positioned along a sampling line 34 in fluid communication with the port 26. During inhalation, the sampling line 34 is configured to direct a portion of the first flow of gas down the sampling line 34 to be sensed by the NO and / or NO2 sensor 30. Subsequently, the sampling line 34 is configured to discard the portion of the first flow of gas. Likewise, during exhalation, the sampling line 34 is configured to direct a portion of the second flow of gas to the sampling line 34-11- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02to be sensed by the NO and / or NO2 sensor 30 and discard the portion of the second flow of gas. In some examples, the sampling line is heated above a dew point to prevent condensation of water vapor in the NO and / or NO2 sensor 30. In other examples, such as the illustrated example in FIG.2, both of the NO and / or NO2 sensor 30 and the flow rate sensor 32 can be positioned along the port 26. In some examples, the NO and / or NO2 sensor 30 is a dual gas analyzer, using chemiluminescence to quantify NO and laser induced fluorescence to quantify NO2, respectively. In some examples, the flow rate sensor 32 is a pneumotachograph.

[0070] The NO and / or NO2 sensor 30 and the flow rate sensor 32 are configured to sense parameters that can control aspects of the NO and / or NO2 delivery system 10. For example, the NO and / or NO2 sensor 30 can sense a first parameter (i.e., the NO and / or NO2 concentration) of the gas flowing through the port 26 (e.g., gas being delivered to the patient 11 and gas returning to the breathing apparatus 20). Similarly, the flow rate sensor 32 can sense a second parameter (i.e., the flow rate) of the gas flowing through the port 26. Together, the first parameter and second parameter can be used (e.g., by the computing device 18, as will be described in greater detail below), to adjust a flow of gas to the patient 11. While the sensor system 16 includes two sensors, it is also contemplated that a sensor system can include more than, or less than, two sensors for sensing more than, or less than, two parameters that can control aspects of an NO and / or NO2 delivery system. In some examples, the sensor system 16 includes a third sensor (e.g., the temperature sensor 36) that can sense a third parameter (i.e., the temperature of the first flow of gas and the second flow of gas through the port 26). In some examples, the sensor system 16 includes a fourth sensor (e.g., a pressure sensor 37) that can sense a fourth parameter (i.e., the atmospheric pressure).

[0071] The computing device 18 can be in communication with some (or all) of the components within the NO delivery system 10 (e.g., other controllers or systems in the NO delivery system 10). For example, the computing device 18 is configured to receive outputs from components of the NO delivery system 10, analyze the outputs received from the components, and send instructions to components of the NO delivery system 10 (e.g., to cause a component to implement a task). The computing device 18 can be implemented into the NO delivery system 10 in different ways. For example, the computing device 18 can include typical components used such as a processor 38, a controller 40, a memory 42, a display 44, and a user interface 45. In some examples, the computing device 18 can additionally include inputs (e.g., a keyboard, a mouse, a-12- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02graphical user interface, a touch-screen display, etc.), communication devices, etc. In some examples, the computing device 18 can simply be implemented as a controller having a processor and memory.

[0072] In some examples, to receive outputs from components of the NO and / or NO2 delivery system 10, the computing device 18 can be in communication (e.g., wired or wireless) with the sensor system 16. The computing device 18 (e.g., the processor 38 of the computing device 18) is configured to receive an output (or outputs) from the sensor system 16. For example, the computing device 18 can receive a first output from the NO and / or NO2 sensor 30 indicating the NO and / or NO2 concentration of the gas flowing through the port 26. Further, the computing device 18 can receive a second output from the flow rate sensor 32 indicating the flow rate of the gas flowing through the port 26. In some examples, the outputs received by the computing device 18 from the NO and / or NO2 sensor 30 and the flow rate sensor 32 are analog outputs. In such examples, the computing device 18 can include an analog-to-digital converter to convert the outputs to digital outputs. In such examples, the analog-to-digital converter is sampled at between about 20 Hertz to about 160 Hertz, about 60 Hertz to about 250 Hertz, or about 80 Hertz to about 1000 Hertz.

[0073] To analyze the outputs received from the sensor system 16, the computing device 18 can include the processor 38. In that regard, the computing device 18 (eg., the processor 38 of the computing device 18) can be configured to estimate values based on the outputs received from the sensor system 16. For example, the computing device 18 is configured to estimate a first value based on outputs received from the sensor system 16. More specifically, the processor 38 of the computing device 18 is configured to estimate an amount of NO and / or NO2 absorbed by the patient 11 based on the sensed NO and / or NO2 concentration of the first flow of gas and the second flow of gas by the NO and / or NO2 sensor 30 and the sensed flow rate of the first flow of gas and the second flow of gas by the flow rate sensor 32. In some examples, the estimation takes into account additional parameters, such as the ambient and gas flow temperature, the humidity, the atmospheric pressure, and / or content of carbon dioxide. The computing device 18 can use these additional parameters, along with information stored on the memory 40, such as the Ideal Gas Law (PV=nRT), to estimate a first amount of NO and / or NO2 in the first flow of gas and a second amount of NO and / or NO2 in the second flow of gas. In some examples, the computing device 18 can use the sensed flow rate of the first flow of gas and the sensed flow rate of the second flow of-13- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02gas to determine inhalation and exhalation patterns. For example, the computing device 18 can use the inhalation and exhalation patterns, along with the estimation of the first amount of NO and / or NO2 in the first flow of gas and the second amount of NO and / or NO2 in the second gas, to determine a molar quantity of NO and / or NO2 absorbed by the patient 11 on a breath-by-breath basis.

[0074] In some examples, the computing device 18 can be in communication (e.g., wired or wireless) with the breathing system 12 (e.g., the breathing apparatus 20), the NO and / or NO2 gas source 14, and / or other components of the NO and / or NO2 delivery system 10. In some examples, the computing device 18 is configured to compare estimated values to predetermined values (e.g., that are stored in the memory 40) and, based on that comparison, cause a component of the NO and / or NO2 delivery system 10 to implement a task. For example, the computing device 18 is configured to compare an estimated amount of NO and / or NO2 absorbed by the patient 11 to a predetermined value on a breath-by-breath basis. That is, for each breath estimated by the computing device 18, the computing device 18 estimates the amount of NO and / or NO2 absorbed by the patient 11 and compares the estimated value to a predetermined value stored in the memory of the computing device 18. Subsequently, the computing device 18 (e.g., the controller 40 of the computing device 18) is configured to cause a component to implement a task based on the comparison. In a non-limiting example, the computing device 18 may be configured to adjust an amount of NO and / or NO2 delivered to the patient 11 (e g., by causing the breathing apparatus 20 to adjust a flow rate of the first flow of gas delivered to the patient 11, by causing the NO and / or NO2 gas source 14 to adjust an amount of NO and / or NO2 gas delivered to the breathing system 12, etc.) based on the comparison. That is, the computing device 18 can be configured to increase or decrease an amount of NO and / or NO2 delivered to the patient 11 by sending a signal to the breathing system 12 (e.g., the breathing apparatus 20 of the breathing system 12) and / or by sending a signal to the NO and / or NO2 gas source 14. In another non-limiting example, the computing device 18 may be configured to generate a user notification based on the comparison. In some examples, the user notification is an alert (e.g., a repeating sound, a flashing light, etc.) to notify a clinician or user to adjust an amount of NO and / or NO2 gas delivered to the patient 11 when the estimated amount deviates from a predetermined value. In some examples, the user notification can instruct a clinician to manually confirm a recommended adjustment to the NO and / or NO2 gas delivery. In another non-limiting example, the computing device 18 includes a display 42 and the-14- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02computing device 18 may be configured to display the parameters sensed by the sensor system 16 and / or display the values estimated by the computing device 18 on the display 42.

[0075] In some examples, an NO and / or NO2 delivery system can include a Methemoglobin (MetHb) monitor configured to monitor an amount of MetHb in a patient. For example, FIG. 3 illustrates a NO delivery system 110 that is configured to deliver NO and / or NO2 to a patient having a Methemoglobin (MetHb) monitor 150. Unless indicated otherwise, the components, functionality, and advantages of the NO and / or NO2 delivery system 10 illustrated in FIGS. 1-2 apply similarly to the illustrated example of the NO and / or NO2 delivery system 110 in FIG. 3. Correspondingly, similar components and features of the “10” series of reference numerals are denoted in the “110” series of reference numerals, unless otherwise provided.

[0076] Referring to FIG. 3, the NO and / or NO2 delivery system 110 includes a monitoring system configured as the MetHb monitor 150. The MetHb monitor 150 is configured to sense and monitor an amount of MetHb in a patient 111. Further, the MetHb monitor 150 is configured to quantify the amount of MetHb in the patient 111. In some examples, the MetHb monitor 150 is configured to use the principle of optical absorption to quantify the amount of MetHb in the patient 111. For example, the MetHb monitor 150 may be configured to measure an absorption of light (e.g., by blood constituents) transmitted through the patient 111 or the patient’s blood. This measured absorption of light may be used to quantify a hemoglobin level of the patient. The MetHb monitor 116 may further analyze specific absorption characteristics (e g., wave-length absorption peaks, absorbance ratio, etc.) and, based on this analysis, determine the amount of MetHb in the patient 111 (e.g., a proportion of MetHb present in the patient 111). In other examples, the MetHb monitor 150 is configured to collect a sample (e.g., a blood sample) from the patient 111 and use the principle of blood gas analysis to quantify the amount of MetHb in the patient 111. In some examples, the MetHb monitor 150 may be configured to generate an output indicating the amount of MetHb in the patient 11 l.The MetHb monitor 150 can be in communication with a computing device 118. The computing device 118 is configured to receive the output from the MetHb monitor 150. In some examples, the computing device 118 is configured to store a MetHb kinetic model (e.g., on a memory 142 of the computing device 118). The computing device 118 is configured to determine an amount of NO absorbed by the patient 111 based on the amount of MetHb in the patient 111 (e.g., received by the output of the MetHb monitor 150) and the MetHb kinetic model (e g., stored on the memory 142). The computing device 118 can further include a controller 140-15- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02configured to generate a report indicating the determined amount of NO absorbed by the patient 111. In some examples, the computing device 118 is configured to communicate the report indicating the determined amount of NO absorbed by the patient 111 via a display 144. In some examples, the computing device 118 (e.g., the controller 140 of the computing device 118) may be configured to automatically adjust the NO delivery to the patient 111 based on the determined amount of NO absorbed by the patient 111.

[0077] In some examples, an NO and / or NO2 delivery system can inject NO and / or NO2 containing gas into the patient’s blood. For example, FIG.4 illustrates a NO and / or NO2 delivery system 210 that is configured to deliver NO and / or NO2 to a patient 211 via extrapulmonary blood gas exchange (e.g., ECMO). Unless indicated otherwise, the components, functionality, and advantages of the NO and / or NO2 delivery system 10 illustrated in FIGS. 1-2 apply similarly to the illustrated example of the NO and / or NO2 delivery system 210 in FIG. 4. Correspondingly, similar components and features of the “10” series of reference numerals are denoted in the “210” series of reference numerals, unless otherwise provided.

[0078] A NO delivery system can deliver NO containing gas to a patient via extrapulmonary blood gas exchange. That is, in some examples, an NO delivery system includes a pump to collect blood from a patient. The blood can then be injected with NO containing gas before returning to the patient. For example, the NO delivery system 210 includes a pump 260 that is fluidly connected to the patient 211. The pump 260 is configured to pump blood from the patient 211 through a first line 262. An NO gas source 214 is positioned downstream of the pump 260. The NO gas source 214 is configured to inject NO containing gas into the blood flowing through the first line 262 at an injection site 263. The NO containing blood flows through a second line 264 that is fluidly connected to the first line 262 at the injection site 263. The NO containing blood returns to the patient 211 via the second line 264.

[0079] The NO delivery system 210 includes a sensor system 216. The sensor system 216 is configured to sense parameters of the blood that flows through the first line 262 and the second line 264. More specifically, the sensor system 216 includes an NO sensor 230 that is configured to sense an NO concentration of the blood as the blood flows through the first line 262 and the second line 264. Further, the sensor system 216 includes a flow rate sensor 232 that is configured to sense a flow rate of the blood as the blood flows through the first line 262 and the second line 264. While the sensor system 216 is illustrated as having two sensors, it is also contemplated that-16- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02other sensors may also be included in the sensor system 216 (e.g., temperature sensors, pressure sensors, etc.).

[0080] FIG. 5 shows a flowchart of a process or method 300 for delivering NO and / or NO2 gas to a patient and monitoring the NO and / or NO2 dosage delivered to the patient. The method 300 can be implemented using any of the previously described NO and / or NO2 delivery systems or any of the systems described in the examples section herein. In addition, some or all of the blocks of the method 300 can be implemented using one or more computing devices, as appropriate. In some examples, the method 300 can be implemented without the aid of a positive pressure generating apparatus including a CPAP device, a ventilator, etc. In other words, the method 300 can be implemented so that the patient drives inspiration and expiration of the NO and / or NO2 gas.

[0081] At 310, the method 300 can include delivering a first gas containing NO and / or NO2 to a patient for inhalation. In some examples, this can include providing a first line (e.g., an inspiratory line) that runs from a breathing apparatus of a breathing system to a port. The port may include a patient interface that a patient may interact with. A first flow of gas flows from the breathing apparatus and a first gas source that contains NO and / or NO2 can be configured to inject NO and / or NO2 containing gas into the inspiratory line. The first flow of gas flows to the port via the inspiratory line. The first flow of gas flows to the patient interface so that the patient may inhale the first flow of gas via the patient interface.

[0082] At 320, the method 300 can include receiving a second gas exhaled by a patient. In some examples, this can include providing a second line (e.g., an expiratory line) that runs from a port having a patient interface to a breathing apparatus. A second flow of gas flows from the patient down the port to the expiratory line. The second flow of gas flows down the expiratory line to the breathing apparatus.

[0083] At 330, the method 300 can include measuring a NO and / or NO2 concentration and a flow rate of the first gas and the second gas. In some examples, this can include providing a sensor system configured to sense the NO and / or NO2 concentration and the flow rate of the first gas and the second gas. The sensor system may include a first sensor (e.g., a NO and / or NO2 sensor) configured to sense an NO and / or NO2 concentration of the first gas and the second gas and a second sensor (e.g., a flow rate sensor) configured to sense a flow rate of the first gas and the second gas.-17- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0084] At 340, the method 300 can include analyzing the NO and / or NO2 concentration and the flow rate. In some examples, this can include providing a computing device having a processor that is configured to receive the NO and / or NO2 concentration measured by a first sensor and the flow rate measured by a second sensor.

[0085] At 350, the method 300 can include generating a report based on the analysis from step 340. In some examples, this can include providing a computing device with a controller that is in electronic communication with one or more components of the NO and / or NO2 delivery system. For example, the controller can be in electronic communication with a sensor system, a breathing system, and / or an NO and / or NO2 gas source. The controller can be configured to generate a report, which may be communicated, including displayed or transmitted, or may be formed into an alert or flag for clinical action. In a non-limiting example, the controller may generate a user notification based on the analysis, such as alerting a clinician when the estimated NO and / or NO2 absorption deviates from a predetermined value (e.g., when the estimated NO and / or NO2 absorption is outside a therapeutic range), when manual confirmation of a recommended adjustment is advisable. In some examples, the computing device may also include a display to display the sensed parameters and the estimated so that real-time feedback is provided to a user.

[0086] Additionally or alternatively, the report can be used by the system to adjust an amount of NO and / or NO2 delivered to the patient based on the analysis of the NO and / or NO2 concentration and the flow rate. For example, the controller may cause the breathing system to modify a flow rate of the first flow of gas or may cause the NO and / or NO2 gas source to modify an amount of NO and / or NO2 injected into the breathing system. Thus, the system may be configured to operate in a closed-loop configuration.

[0087] Referring now to FIG. 6, one non-limiting example is provided for implementing the analysis. In this non-limiting example, the step 340 can be implemented using a step 342 of measuring a temperature of the first gas and the second gas. In some examples, this can include providing a third sensor (e.g., a temperature sensor) to sense the temperature of the first flow of gas and the second flow of gas. At step 344, the step 340 can include estimating an amount of NO and / or NO2 delivered to the patient. In some examples, this can include receiving the measurements from the sensor system during inhalation and providing the processor to estimate an amount of NO and / or NO2 delivered to the patient based on the measurements. At step 346, the step 340 can include estimating an amount of NO and / or NO2 exhaled by the patient. In some-18- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02examples, this can include receiving the measurements from the sensor system during exhalation and providing the processor to estimate an amount of NO and / or NO2 exhaled by the patient based on the measurements. At step 348, the step 340 can include estimating an amount of NO and / or NO2 absorbed by the patient on a breath-by-breath basis. At step 349, the step 340 can include comparing the estimated NO and / or NO2 absorbed by the patient to a predetermined value. In some examples, this can include providing a predetermined value stored on a memory of the computing device. The predetermined value can be, for example, a predetermined absorption value that a clinician or user inputs into the computing device. This can be a predetermined range of NO absorption values for a specific type of therapy or a predetermined range of NO absorption values for a specific type of patient based on the patient’s weight, age, activity level, etc.

[0088] Referring now to FIG. 7, a flowchart is shown of a process or method 400 for delivering NO and / or NO2 gas to a patient and monitoring the NO and / or NO2 dosage delivered to the patient. The method 400 can be implemented using any of the previously described NO and / or NO2 delivery systems or any of the systems described in the examples section herein. In addition, some or all of the blocks of the method 400 can be implemented using one or more computing devices, as appropriate. In some examples, the method 400 can be implemented without the aid of a positive pressure generating apparatus including a CPAP device, a ventilator, etc. In other words, the method 400 can be implemented so that the patient drives inspiration and expiration of the NO and / or NO2 gas.

[0089] At 410, the method 400 can include delivering NO and / or NO2 to a patient. In some examples, this can include providing a first line (e.g., an inspiratory line) that runs from a breathing apparatus of a breathing system to a port. The port may include a patient interface that a patient may interact with. A first flow of gas flows from the breathing apparatus and a first gas source that contains NO and / or NO2 can be configured to inject NO and / or NO2 containing gas into the inspiratory line. The first flow of gas flows to the port via the inspiratory line. The first flow of gas flows to the patient interface so that the patient may inhale the first flow of gas via the patient interface. In other examples, step 410 can include delivering NO and / or NO2 to a patient via extrapulmonary blood gas exchange (e.g., ECMO). That is, the step 410 can include providing a pump fluidly connected to a patient along a first line. Further, step 410 can include collecting blood from the patient (e.g., via the pump along the first line). Step 410 can further include injecting NO and / or NO2 into the blood flowing through the first line with an NO and / or NO2 gas source. The-19- QBM25141.049621100755084.1125141.04962 Patent Application - MGH 2025-004-02NO and / or NO2 containing blood flows through a second line that is fluidly connected to the first line. Step 410 can include returning the NO and / or NO2 containing blood to the patient via the second line.

[0090] At 420, the method 400 can include measuring an amount of MetHb in a patient. In some examples, this can include using the principle of optical absorption to measure the amount of MetHb in the patient. That is, step 420 can include providing a methemoglobin monitor coupled to the patient. Step 420 can include transmitting light through the patient or through the patient’s blood with the methemoglobin monitor and measuring how the light is absorbed by blood constituents within the patient. Step 420 can further include quantifying overall hemoglobin levels of the patient based on such measurement. Step 420 can further include analyzing specific absorption characteristics (e.g., wave-length absorption peaks, absorbance ratio, etc.) and determining the proportion of MetHb present in the patient (e.g., the blood of the patient) based on those absorption characteristics. In other examples, step 420 can include collecting a sample (e.g., a blood sample) from the patient. With the blood sample, step 420 can include using the principle of blood gas analysis to determine the amount of MetHb in the patient.

[0091] At 430, the method 400 can include determining an amount of NO absorbed by the patient. In some examples, this can include providing a computing device. Step 430 can include receiving, on the computing device, an output from a methemoglobin monitor indicating an amount of MetHb in the patient (e.g., blood of the patient). Step 430 can further include determining the amount of NO absorbed by the patient based on the measured amount of MetHb in the patient from step 420 and a MetHb kinetic model. In some examples, the MetHb kinetic model is stored on the computing device (e.g., a memory of the computing device).

[0092] At 440, the method 400 can include generating a report. In some examples, this can include providing a computing device with a controller that is in electronic communication with one or more components of the NO and / or NO2 delivery system. For example, the controller can be in electronic communication with the methemoglobin monitor, a breathing system, a pump, and / or an NO and / or NO2 gas source. The controller can be configured to generate a report, which may be communicated, including displayed or transmitted, or may be formed into an alert or flag for clinical action. In a non limiting example, the controller may generate a user notification based on the analysis, such as alerting a clinician when the estimated NO absorption deviates from a predetermined value (e.g., when the estimated NO absorption is outside a therapeutic range), when-20- QB1125141.049621100755084.1125141.04962 Patent Application - MGH 2025-004-02manual confirmation of a recommended adjustment is advisable. In some examples, the computing device may also include a display to display the sensed parameters and the estimated parameters so that real time feedback is provided to a user.

[0093] In some examples, the method 400 can include a step 450. At 450, the method 400 can include controlling the NO and / or NO2 dosage delivered to the patient. In some examples, this can include controlling the NO and / or NO2 dosage based on the report generated from step 440 (e.g., based on the determined amount of MetHb and a MetHb kinetic model). For example, the controller may cause a breathing system to modify a flow rate of the first flow of gas, the controller may cause a pump to modify a pump rate, and / or the controller may cause the NO and / or NO2 gas source to modify an amount of NO and / or NO2 injected into the breathing system or blood flowing through the first line. Thus, the system may be configured to operate in a closed-loop configuration.EXAMPLE

[0094] The following example sets forth, in detail, ways in which the breathing system 10 and the method 200 may be used or implemented, and will enable one of skill in the art to more readily understand the principle thereof. The following examples are presented by way of illustration and are not meant to be limiting in any way.

[0095] Overview

[0096] Recent studies uncovered extrapul monary effects of inhaled nitric oxide (NO) therapy. However, no method exists to quantify NO absorption. The present disclosure discloses a breath-by-breath system and method for inhaled NO absorption measurement, which consists of a rapid chemiluminescent NO analyzer and a pneumotachograph. The accuracy of the system was validated in vitro and in an animal model, achieving excellent agreement with the reference gascollection method (linear regression R20.98 and 0.97; mean bias 1.5% ± 15.4 and -3.6% ± 14.4, respectively). A methemoglobin kinetic model is established that followed single-compartment first-order elimination kinetics, and included NO absorption rate, methemoglobin conversion factor, elimination constant, time, hemoglobin, and blood volume. The model was trained and validated in 11 pigs receiving 81 treatments at 20 - 300 parts per million (ppm) and 14 human subjects, including 10 healthy subjects and 4 patients who received 64 treatments at 20 - 300 ppm. The approach yielded stable parameters of elimination constant 0.01 min'1(coefficient of variation [CV] 3.0%) and conversion factor 0.015 g / pmol (CV 2.7%). When comparing methemoglobin--21- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02estimated NO absorption and measured values, linear regression R2reached 0.89, and the mean bias was 1.2% ± 27% in human subjects. Over 80% of estimates fell within ±30% of measured values, indicating strong estimation capability of the model. It is demonstrated that methemoglobin can be a bedside marker for NO absorption and can provide a framework for optimizing NO dosing strategies and advancing dose-response studies in inhaled NO therapy.

[0097] Background

[0098] As a selective pulmonary vasodilator, inhaled nitric oxide (NO) was approved by the U.S. FDA in 1999 for improving oxygenation and reducing the need for extracorporeal membrane oxygenation in term and near-term (> 34-week gestation) neonates with hypoxic respiratory failure associated with clinical or echocardiographic evidence of pulmonary hypertension. Meanwhile, mounting evidence has revealed extrapul monary organ protective effects of the inhaled gas on heart, kidney, brain, and hemostasis. Recent preclinical and clinical studies have also demonstrated that high concentrations of inhaled NO (> 160 ppm) act as a broad antimicrobial agent. The mechanism, which is still under extensive investigation, may involve both direct effects on microorganisms and modulatory effects on the host immune system.

[0099] However, clinical trials evaluating the extrapul monary effects of inhaled NO have shown conflicting results. One of the possible reasons is the variability of its bioavailability attributed to different dosing regimens, gas delivery modalities, absorption and metabolism rates. Inhaled NO therapy demonstrated dose-dependent deposition of NO and its metabolites in extrapulmonary organs. In a murine model of cardiac and hepatic ischemia-reperfusion injury, a U-shaped dose-responsive profile was observed during the administration of nitrite, which is a biological storage reserve of NO. This highlights the need for pharmacokinetic and pharmacodynamic research to guide the dosing, monitoring, and targeting of the therapy, especially when extrapulmonary effects are sought.

[0100] After diffusion in the blood compartment, more than 80% of inspired NO is rapidly inactivated by reaction with oxyhemoglobin to form methemoglobin (MetHb) and nitrate. Most of the MetHb is reduced to ferrous hemoglobin by NADH-cytochrome b5 reductase in erythrocytes. A lesser proportion of the absorbed gas converts to nitrite or combines with plasma proteins, which might preserve NO bioactivity. MetHb is more easily monitored than nitrogen oxides and nitrosothiols and follows kinetics that could be modelled. While conventionally monitored for-22- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02safety concerns, MetHb stands as a potential point-of-care marker of the amount of NO absorption and dosing target.

[0101] Inhaled NO is currently administered as a volumetric concentration measured in parts per million (ppm), with the FDA approving concentrations of up to 20 ppm for clinical use. However, the absorption of inhaled NO depends not only on the concentration delivered but also on individual factors such as minute ventilation, dead space, lung volume, hemoglobin, and blood volume. In a recently published study, healthy volunteers breathing 300 ppm of NO exhibited a doubling of MetHb levels during exercise compared with rest, suggesting that the change in cardiopulmonary conditions significantly influences NO absorption.

[0102] To date, no standardized methods currently exist to quantify inhaled NO absorption. In the present disclosure, we disclose a non-limiting example of a system to quantify NO absorption on a breath-by-breath basis. The aims of the present disclosure include at least: (1) To build and validate a system to quantify breath-by-breath NO absorption; (2) To develop and validate the model of NO absorption and MetHb kinetics allowing bedside estimation of NO absorption; (3) To investigate the contributing factors to the amount of inhaled NO absorption.

[0103] Methods

[0104] System Setup

[0105] To build a system to quantify breath-by-breath NO absorption, a closed breathing circuit was constructed that delivers NO through an endotracheal tube or a sealed mask at constant concentrations. A rapid and sensitive chemiluminescence NO and laser induced fluorescence nitrogen dioxide (NO2) analyzer (CLD 50, Cambustion, Cambridge, UK) and a pneumotachograph (NM3, Philips-Respironics, Murrysville, PA, USA) were connected to a y-piece, allowing continuous monitoring of the NO and NO2 concentrations, airway flow, airway pressure and end tidal carbon dioxide (ETCO2) throughout the respiratory cycle. The NO / NO2 analyzer was connected closer to the patient instead of the y-piece to prevent interference of the NO in the bias flow. The analog output channels of the monitoring devices were connected to an analog-to-digital converter (Powerlab 8 / 35, ADInstruments, Dunedin, New Zealand) and sampled at 100Hz. Digitized signals were recorded as graphs and text files by a software (LabChart v8.1.30, ADInstruments, Dunedin, New Zealand).

[0106] Theory and algorithms-23- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0107] To measure the absorption of inhaled NO in the lung, the difference between the measured amounts of NO entering and leaving the lung was calculated. Since it is infeasible to measure changes in NO content within the lung, absorption was inferred from the inhaled and exhaled amounts of NO measured at the y-piece.

[0108] Accordingly, NO / NO2 absorption was calculated as the integral of the product of airway flow measured by the pneumotachograph and NO / NO2 concentration measured by the NO / NO2 analyzer. The airway flow signal is positive when there is inflow to the subject and negative when there is outflow from the subject. The cumulative amount of NO / NO2 absorption was calculated as Equation 1.rt2 •VNO / NO2-tot=JtlV(t)cNO / NO2(t)dt (1)Where: tl and t2 = start and end of measurement; VNO / NO2-tot=cumulative volume of NO / NO2 absorption; V = airway gas flow; CNO / NO2=concentration of NO / NO2.

[0109] One non-limiting example of breath cycle identification defines the start of a breath cycle identified by airway flow turning from negative to positive and remaining positive for the next 0.3 second. The end of the breath cycle was identified by the start of the next breath cycle. Application of Equation 1 to the time range of the breath cycle generated the breath-by-breath absorption of NO / NO2.tb2VNO / NCh-b_TtblV(t)cNO / NO2(t)dt (2)Where: tbl and tb2 = the start and end of each breath cycle; VNO / No2-b=volume of NO / NO2 absorption in each breath cycle.

[0110] Adjustments - Temperature

[0111] Since airway gas flow calibration was performed at ambient temperature (Tambient.25°C), the measured flow was multiplied by the following factor to obtain the flow at body temperature. For example, at Tbody = 37°C, the correction factor equals:

[0112] Adjustments - Water vapor and carbon dioxide

[0113] The NO / NO2 analyzer used heated sampling line above the dew point to prevent condensation of water vapor. NO signal quench factors for the chemiluminescence measurement were counted per manufacturer instructions, i.e. -1.8% per %H2O and -0.6% per %CO2. The correction of NO absorption depended on the humidity and content of CO2.-24- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0114] If the inspired gas was dry at ambient temperature,>

[0115] If the inspired gas was humidified at body temperature,&>

[0116] Expired gas was humified by the respiratory system,<Where: VNO.ins= volume of NO inhaled; VNO.ex= volume of NO exhaled; PH2O=partial pressure of water vapor; PCO2= partial pressure of CO2; Patm= atmospheric pressure (1 atm or 760 mmHg).

[0117] Final algorithm

[0118] A non-limiting example of a final NO absorption algorithm was defined as: If the inspired gas was humified at body temperature,Vxo-tot

[0119] If the inspired gas was dry at ambient temperature,VNO-tot = VNO-ins >+ 1.04 <

[0120] The molecular amount of NO absorption can be obtained using the ideal gas law:where nNO= molecular amount of NO absorption (mol); VNO= volume of NO absorption (ml); R = ideal gas content [82.1 ml • atm / (mol»K)]; T = temperature [Tbody (°C) + 273.15 (K)] or [Tambient (°C) + 273.15 (K)]. The positive airway pressure superimposed to atmospheric pressure is small enough to be neglected.

[0121] Adjustments-25- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0122] A few areas were identified for optional adjustments of the algorithm, but not included for testing purposes. For example, the present disclosure recognized that there can be variations in intrapulmonaiy gas storage between patients. However, gas-exchange studies indicated an ambiguous impact on the accuracy of pulmonary gas uptake measurements. Thus, adjustments to this end were not included in the testing described below.

[0123] As another example, in one non-limiting implementation, the NO / NO2 analyzer continuously samples gas at a constant flow, which can create a theoretical discrepancy between the measured and the actual amount of NO absorption. However, in the non-limiting example implementation used in the testing described hereafter, adjustments were not made for the analyzer's sampling flow for two reasons. First, only brief measurements were performed to minimize the loss of delivered NO due to analyzer sampling. Second, the accuracy of the pneumotachograph was ± 0.5L / min or ± 3%, whichever is greater, while the NO / NO2 analyzer sampling flow ranged from 0.4 to 0.9 L / min. This sampling flow was therefore close to the measurement error of the pneumotachograph, rendering any adjustment effectively indistinguishable from measurement variability.

[0124] Calibration

[0125] A signaling delay of the NO / NO2 analyzer was caused by the gas transport and mixing within the sampling line of the NO / NO2 analyzer and the response time of the device. Since it is a considerable source of error in pulmonary gas exchange studies, previous studies adopted various methods to calibrate the delay. The calibration was conducted by connecting the tube delivering constant concentration of NO and gas flow to the pneumotachograph and NO / NO2 analyzer before measuring each subject. During each calibration, at least 30 waveforms were recorded for calculation.

[0126] The recorded delay between the NO concentration and flow during the calibration process is composed of three parts. First, the time for NO-containing gas to transport through the volume of the respiratory tube to the sampling line of the NO / NO2 analyzer (TAV) was calculated using the tubing volume (AV) and flow-time integration.AV = ^V(t)dt (12)T V = tl - tO (13)-26- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0127] Second, the time for NO containing gas to transport through the sampling line of NO / NO2 analyzer (Tt) was calculated by subtracting T V from the time difference measured from the start of the flow signal rising to the start of the NO signal rising.

[0128] Third, the rising phase of NO concentration represents the response time of the analyzer and the end of gas transport through the sampling line. Since the rising curve of the NO concentration curve in the system is more sigmoidal shaped than exponential, third-order regression showed the best fitness (R2> 0.88). The rise time (r) was defined as the time reaching 63.2% of the rising curve of NO concentration.

[0129] The signaling delay caused by the NO / NO2 analyzer’s sampling line and response time (Ta) was calculated as the sum of Tt and T:Td = Tt + r (14)

[0130] The mean value of the signaling delay measurements in each calibration process was used for shifting the NO concentration signal, and the standard deviation (SD) was used for error sensitivity analysis.

[0131] The pneumotachograph was calibrated at room temperature using a constant flow generated by a calibrated flow controller with an accuracy of ± 0.4% (MCP-100SLPM-D-DB9M / 5M, Alicat Scientific, AZ, USA).

[0132] Testing the system - Validation

[0133] The breath-by-breath measurement of NO absorption was validated using the gascollection bag method. A simulation lung (ASL 5000, IngMar Medical, PA, USA) was set in spontaneous mode with modifiable tidal volume, respiratory rate, and inspiratory time. An activated charcoal filter was connected as part of the simulation lung to absorb NO. One Ambu valve and a one-way valve were used to separate inhaled and exhaled gas. A 6-liter bag was prefilled with a mixture of air and NO and connected to the inspiratory limb. The other bag was connected to the expiratory limb to collect expired gas. The expired volume of gas was measured by a 1 -liter syringe, and the volume of inspired gas was assumed to be the same as the expired. NO concentration was measured in both bags after mixing, and absorbed NO was calculated as the inspired amount subtracted by the expired amount. Breath-by-breath measurement of NO absorption using our system was conducted simultaneously and compared with the gas-collection method. The same validation test was performed in spontaneously breathing swine.

[0134] Testing the system - Error sensitivity analysis-27- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0135] The accuracy and robustness of the breath-by-breath NO absorption measuring system were examined through error sensitivity analysis. Error ranges of basic measurements, including airway flow, NO concentration, and NO / NO2 analyzer signaling delay, were determined using manufacturer’s instructions or SD of repeated measurements. For each error source, the induced error of the NO absorption measurement was calculated as (breath-by-breath measurement - bag method measurement) / bag method measurement x 100% in the same treatment. Linear regression was fitted for the induced system error against the error of basic measurements.

[0136] In Vivo Application - Animal Study

[0137] An animal study was conducted to develop the model linking measured NO absorption rate to MetHb kinetics, and to identify the ventilatory and physiological determinants of NO absorption. The study was approved by the Massachusetts General Hospital (MGH, Boston, MA, USA) Institutional Animal Care and Use Committee (IACUC). Yorkshire pigs (CBSET Inc., Lexington, MA, USA) were sedated, intubated, and mechanically ventilated (Maquet Servo-I, Getinge, Merrimack, NH, USA) with a tidal volume (VT) of 6 - 10 ml / kg body weight, a respiratory rate (RR) to maintain an ETCO2 between 35 and 45 mmHg and blood pH of 7.3 - 7.5, a positive end expiratory pressure (PEEP) of 5 - 15 cmFLO, and an inspired fraction of oxygen (FiO2) to maintain a partial pressure of oxygen (PaCh) > 60 mmHg. Body temperature was maintained with a combination of a warm fluid mattress and a forced air warming system (Bair Hugger, 3M Medical, St. Paul, MN, USA). Under general anesthesia, the femoral artery and internal or external jugular vein were cannulated, and Swan-Ganz catheter was placed. Bladder catheterization was obtained by transurethral catheterization in female pigs and by minimally invasive surgical cystotomy in male pigs. Inhaled NO therapy was administered at doses of 20 to 300 ppm for 30 to 135 minutes using a custom tank-based system (INOMAX EVOLVE DS, Mallinckrodt Manufacturing LLC, WI, USA), which delivered NO proportional to the airway flow and resulted in constant NO concentrations in the inspiratory limb. Intervals of 30 minutes to 4 hours between treatments allowed the recovery of MetHb to baseline (< 2.0%). NO was delivered through a single-lumen endotracheal tube or a double-lumen tube, with the latter only delivering NO to one lung.

[0138] Parameters recorded during the treatment included NO and NO2 concentrations, airway flow, airway pressure, ETCO2, heart rate, arterial blood pressure, central venous pressure, pulmonary artery pressure, pulmonary artery wedge pressure, and cardiac output. A noninvasive-28- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02pulse co-oximeter (Masimo Rad7, Irvine, CA, US) was used to continuously monitor MetHb and oxyhemoglobin levels. Arterial blood gas samples were obtained before and after each treatment, with arterial blood MetHb concentration tested using co-oximetry (ABL 800 flex, Radiometer Medical, Copenhagen, Denmark).

[0139] NO absorption rate and breath-by-breath absorption was calculated using the system. Meanwhile, breath-by-breath NO absorption was measured and compared before and after manipulating cardiac output and altering ventilatory settings, i.e., tidal volume, inspiratory time, inspiratory pause, flow pattern, and PEEP, individually.

[0140] In Vivo Application Healthy subject study

[0141] To develop and validate the MetHb kinetic model for estimating NO absorption in humans, a healthy subject study was conducted in which NO absorption was measured and MetHb kinetics were continuously monitored. Healthy volunteers were enrolled in a phase I trial (NCT05612074) approved by the MGH institutional review board (IRB) on December 5th, 2022 (2022P002884) to evaluate the safety and kinetics of MetHb under intermittent high-dose inhaled NO administration. The protocol was described in detail by a previous study. Briefly, participants were allocated to two groups with FiO221% or 80% and received inhaled NO at 300 ppm for 30 minutes, three times daily for 5 consecutive days. Subjects underwent interventions at rest on day 1 to 4 and during stationary cycling exercise on day 5. Physiological and laboratory parameters were collected before, during, and after each treatment. MetHb was monitored noninvasively through a pulse co-oximeter (Masimo Rad7, Irvine, CA, US). Subjects breathed through a sealed face mask during treatments. Measurements were identified as valid based on the following criteria: (1) Simultaneous recording of both airway flow and NO concentration at y-piece was conducted; (2) To exclude measurements with gas passing external to the face mask, the percentage of breaths with over 30% difference between expiratory and inspiratory tidal volume should not exceed 30%; (3) The difference of minute ventilation between measurement and treatment period remained within 30% to ascertain the representativeness of the measurement. In addition, healthy volunteers were enrolled and received continuous inhaled NO at 80 ppm for 60-120 minutes with the aforementioned data collected.

[0142] In Vivo Application - Clinical study

[0143] To develop and validate the MetHb kinetic model in clinical settings, we conducted a prospective, observational study (NCT05612074) in which both NO absorption and MetHb-29- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02kinetics were measured in patients receiving inhaled NO therapy. After the approval by the MGH institutional review board on December 26th, 2024 (2024P001999), adult patients who were planned to receive inhaled NO therapy were enrolled after oral consent due to the observational nature of the study. To avoid physiological complexity, we excluded: (1) pregnant or actively lactating patients; (2) patients on renal replacement therapy; (3) patients actively participating in other studies that might interfere with the inhaled NO therapy or data collection; (4) patients who had received inhaled NO therapy within one week.

[0144] Before starting the treatment, the NO absorption monitoring apparatus and a non-invasive co-oximetry were connected to the subject. Continuous recording of non-invasive MetHb, NO concentration, NO2 concentration, airway flow, airway pressure, ETCO2, heart rate, arterial blood pressure, and / or pulmonary artery catheter parameters was conducted during the first hour of the treatment. Arterial blood gas was tested per clinical practice.

[0145] Methemoglobin kinetic model

[0146] The relationship between MetHb kinetics and NO absorption rate, i.e. the amount of NO absorption in unit of time, was investigated in both animal and human studies. Data from inhaled NO treatments were collected and used to develop a MetHb kinetic model assuming a single-compartment, first-order elimination framework. Several assumptions were made: MetHb formation occurs at a constant rate when NO is delivered at a constant concentration over a short period, MetHb remains in the blood compartment, and the elimination rate depends on MetHb concentration.

[0147] During accumulation phase, when NO was delivered, MetHb formation is related to both time and NO absorption rate:<>Where: F (g / pmol) = conversion factor of NO to form MetHb; a (pmol / min) = absorption rate of NO; k (min1) = elimination constant; Va (L) = distribution volume, i.e. blood volume which was estimated according to a previous study in swine and Nadler equation for human; Hb = hemoglobin (g / L); B (%) = baseline of MetHb.

[0148] Elimination phase (after stopping NO delivery):MetHb (%) = (Peak value of MetHb) • e^1' (16)

[0149] Plateau state (steady state during continuous delivery):-30- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0150] Continuous pulse co-oximetry MetHb was used for modelling in animal and human studies, and was adjusted for arterial MetHb by linear regression in the animal study. The model of MetHb kinetics during inhaled NO treatments and post-treatment elimination phase was fitted using a nonlinear least squares model optimized by Limited-memory Broyden-Fletcher-Goldfarb-Shanno with Bound constraints (L-BFGS-B) algorithm. Model performance was evaluated using leave-one-out cross-validation (LOOCV), where each subject’s data was iteratively held out as a test set while training on the remaining subjects. For each fold, F and k were optimized using the training data, and predictions on the held-out subject’s complete time series were evaluated. The following parameters were used to assess model performance: (1) coefficient of variance (CV) of F and k; (2) coefficient of determination (R2) for both training and validation data sets; (3) root mean square error (RMSE). For each LOOCV fold, the NO absorption rate was estimated for the held-out subject using parameters derived from the training set and compared to the measured value.

[0151] Statistical analysis

[0152] According to data distribution and sample size, continuous variables were presented as mean (standard deviation), median (interquartile range) or median (range). Categorical variables were presented as numbers (percentages). Breath-by-breath NO absorption before and after changing ventilatory settings or cardiac output was analyzed using linear mixed-effects model (LMM) with the altered setting as fixed effect, individual animals and treatment pairs as random intercepts. Clinical significance was defined according to potential system error in the error sensitivity analysis. Bonferroni correction was used for multiple comparisons.

[0153] For the comparison of NO absorption before and after ventilatory adjustments, the sample size was determined using linear mixed-effects model power analysis to detect a clinically meaningful difference of 20% in NO absorption between ventilation settings. With over 50 breaths analyzed per setting per subject, five paired comparisons would achieve 80% power at a = 0.05 significance level, accounting for multiple comparisons using Bonferroni correction.

[0154] Sample size determination for the MetHb kinetic model, which is a nonlinear regression model, requires a minimum of 25 animals or subjects considering the 20% variance of time constant in previous literature. However, the design of multiple treatments per subject with washout intervals increases the effective sample size beyond the number of enrolled subjects. For -31- QBM25141.049621100755084.1125141.04962 Patent Application - MGH 2025-004-02the cross-validation, 5- to 10-fold cross-validation should be preferred over LOOCV. Accordingly, at least 10 subjects receiving 2 to 3 treatments each were planned to be enrolled.

[0155] Inhaled NO absorption calculation and statistical analyses were performed using R version 4.2.3 (R CoreTeam, 2024).

[0156] Results

[0157] Validation

[0158] To determine the accuracy of the breath-by-breath NO absorption measurement system, validation tests were performed using spontaneous breaths generated by a simulation lung and by swine (See Table 1 and FIGS. 8A-8D). In particular, the graphs of FIGS. 8A-8D provide a comparison of breath-by-breath and bag method measurement of nitric oxide absorption in spontaneous breath of simulation lung (FIG. 8A and FIG. 8B) and swine (FIGS. 8C and 8D). FIG.8A and FIG. 8C illustrate mean nitric oxide absorption per breath in each treatment measured by the breath-by-breath system compared vs bag method, with error bars indicating standard deviation of within-treatment, between-breath variation. The line represents the linear regression line of the two methods of measurement. In particular, FIG. 8B and FIG. 8D are Bland-Altman plots showing percentage difference (system measurement- bag method measurement) / bag method measurement x 100% of mean nitric oxide absorption per breath, vs bag method. The solid line represents mean bias, and dashed lines indicate 95% limits of agreement.

[0159] The mean NO absorption per breath measured by the breath-by-breath system was compared with that obtained from the reference bag-collection method. Linear regression demonstrated excellent agreement over a variety of NO dose, tidal volume and respiratory rate, with R2= 0.98 for the simulation lung and 0.97 for swine. Bland-Altman analysis showed mean biases of 1.5% (SD 15.4%; 95% limits of agreement, -28.6% to 31.6%) and -3.6% (14.4%; -31.8% to 27.4%), respectively. These results confirm that the breath-by-breath system reliably quantifies NO absorption across a broad spectrum of breathing conditions. Table 1 shows characteristics of treatments tested by the validation system, and values are shown as median (range) unless otherwise specified._ Table 1_ Simulation lungTreatments, n 33 17Breaths per treatment, n 12 (6 - 23) 21 (12 - 43)-32- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02Nitric oxide dose, ppm 70 (16 - 144) 60 (15 - 143)Tidal volume, ml 278 (172-492) 215 (90 -330)rate, min'122 (10 - 39) 34 (19 -47)

[0160] Error sensitivity analysis

[0161] To evaluate the sensitivity of the measurement system to errors in basic measurements, we performed linear regressions between the error percentage of each basic measurement and the resulting system error (See Table 2). The system was sensitive to errors in airway flow measurement (regression slope 4.7, R20.24) and the determination of NO / NO2 analyzer sampling delay (regression slope 2.6, R20.21). This indicates that the ±3% error in airway flow measurement or ±5% error in sampling delay determination would result in approximately 14.1% and 13% system error, respectively. These results suggest the threshold for distinguishing true differences between measured NO absorption and highlight the importance of precise airway flow and NO / NO2 signaling delay calibration. Table 2 shows expected accuracy of component parameters and regression analysis of errors in nitric oxide absorption. In Table 2, slope and error intercept are presented as mean (SD). NO, nitric oxide; NO2, nitrogen dioxide. Percentage of induced error in nitric oxide absorption = slope x (percentage error of basic measurement) + error intercept, f Significantly different R2from zero.Table 2Basic measurements Accuracy / Slope Error R2coefficient of variation intercept (%) Airway flow Greater of ±3% or 4.7 (0.3) 1.5 (0.6) 0.241'0.5L / minNitric oxide concentration ±2% 1.0 (0.5) 1.5 (0.6) 0.01 NO / NO2 analyzer sampling ±5% 2.6 (0.3) 0.9 (0.8) 0.21^ delay

[0162] Methemoglobin kinetics model

[0163] To investigate the relationship between MetHb kinetics and NO absorption, animal and human studies were conducted in which MetHb was continuously monitored before, during and after inhaled NO treatments, and NO absorption was measured using the validated breath-by-breath measurement system.-33- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0164] The animal study included 11 pigs (3-9 months old, 32-84 kg, both males and females) which received 81 inhaled NO treatments at 20-300 ppm for 30-135 min. Six of the animals were healthy, three given intrapulmonary inoculation of Pseudomonas aeruginosa to build a model of ventilator-associated pneumonia and one given hydrochloric acid to build an acute respiratory distress syndrome model. Pulse co-oximetiy monitoring during and after inhaled NO treatments showed that MetHb levels varied based on both NO concentration and whether the gas was delivered to both lungs or single lung with corresponding difference in NO absorption rates (See FIGS. 9 A and 9B).

[0165] The human study included 57 valid NO administrations (80-300 ppm) from 10 healthy subjects and 7 treatments (20-80 ppm) in 4 hospitalized patients. Characteristics of human subjects and treatments are shown in Table 3. As expected, patients with lung conditions exhibited lower NO absorption, smaller changes in MetHb, and reduced tidal volumes compared to healthy volunteers. A lower percentage of inhaled NO was absorbed when delivered via face mask compared to endotracheal tube, indicating a dead space effect. MetHb levels and measured NO absorption exhibited significant difference between healthy volunteers receiving treatments at 250-300 ppm during exercise (median NO absorption rate 115 pmol / min, IQR 128 - 154), at rest (67, 51 - 74) and patients (See FIG. 9C). Table 3 shows characteristics of inhaled nitric oxide therapy in human subjects. In Table 3, values are median (IQR) unless specified. Percentage absorbed (%) = Absorbed NO / inhaled NO x 100%. Nitric oxide is abbreviated as NO. Body mass index is abbreviated as BMI. Mean arterial pressure is abbreviated as MAP. Invasive mechanical ventilation is abbreviated as IMV. Methemoglobin is abbreviated as MetHb.-34- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02Table 3Healthy volunteers Patients(N = 10) (N = 4) Treatments per subject, n 2 (1 - 15) 2 (1 - 2)Inhaled NO dose, n20 - 80 ppm 5 3250 - 300 ppm 5 1Treatment duration, n30 min 5 230 - 120 min 4 3> 120 min 1 2Male sex, n 6 2Age, y 36 (27 - 40) 27 (23 - 67) Height, m 1.73 (1.65 - 1.78) 1.68 (1.65 - 1.70) Weight, kg 60.5 (55.2 - 82.0) 79.4 (54.1 - 92.2) Hemoglobin, g / dl 13.5 (13.1 - 16.0) 10.8 (9.8 - 12.9) Diagnosis, nAcute hypoxemic respiratory failure 0 2Pulmonary hypertension 0 1Lower respiratory tract infection 0 3Vital signsHeart rate, bpm 80 (75 - 89) 78 (68 - 85) MAP, mmHg 83 (76 - 91) 91 (83 - 99) Respiratory parametersIMV, n 0 3Face mask, n 10 1Tidal volume, ml 840 (569 - 968) 448 (377 - 483) Respiratory rate, breaths per minute 12 (9 - 16) 17 (16 - 20) Measured NO absorption20 - 80 ppmRate, pmol / min 13.7 (10.8 - 14.4) 5.8 (3.2 - 14.2) Breath-by-breath, pmol / breath 0.8 (0.6 - 1.0) 0.3 (0.2 - 0.6) Change of MetHb, % 1.6 (1.6 - 1.6) 0.7 (0.5 - 0 9) 250 - 300 ppmRate, pmol / min 70.4 (58.6 - 84.7) 15.0 (13.6 - 16.4) Breath-by-breath, pmol / breath 6.9 (4.1 - 8.7) 0.9 (0.8 - 0.9) Change of MetHb, % 4.3 (3.3 - 5.7) 2.3 (2.2 - 2.4) Percentage absorbed, %IMV 59 (33 - 62)Face mask 60 (46 - 68) 20 (19 - 21)

[0166] MetHb levels followed the single-compartment first-order elimination kinetics (See Table 4 and FIG. 9). In particular, the graphs of FIGS. 9A-9F provide methemoglobin kinetics during and / or post inhaled nitric oxide therapy and measured NO absorption rate. FIG. 9A and-35- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02FIG. 9B illustrate a swine study and FIGS. 9C-9F illustrate healthy volunteers in a human study. Methemoglobin was measured by noninvasive pulse co-oximetry. Nitric oxide was delivered to a single lung in pigs with a double lumen endotracheal tube (DLT). Lines show the mean values of methemoglobin within each group, shades represent the standard deviation, and dashed lines indicate the cessation of treatment. Median values and interquartile range of NO absorption rate are labeled for each group. SLT stands for single lumen endotracheal tube. HV stands for healthy volunteers.

[0167] LOOCV was performed to train and validate the model linking MetHb kinetics, hemoglobin and blood volume to NO absorption rate in both animal and human studies. Two animals were retained for training due to breath-by-breath measurement throughout the treatment and treatments beyond 30 min, leaving 9 animals (68 treatments) for cross validation, while the human study included 14 folds in cross-validation. The fitted MetHb kinetic model yielded stable elimination constant 0.021 min'1in swine (CV 3.2%) and 0.010 in human (CV 3.0%), as well as a conversion factor of 0.015 g / pmol (CV 0.9%) and 0.014 g / pmol (CV 2.7%), respectively. Both training and validation R2exceeded 0.8 with low RMSE values of 0.3% and 0.4% in animal and human studies, respectively. Table 4 shows parameters and performance of methemoglobin kinetics model in swine and human. In Table 4, values are shown as mean (SD) unless specified. Time to reach 95% steady state = t1 / 2x 3. F is a nitric oxide conversion factor, k is an elimination constant. Root mean square error is abbreviated as RMSE.Table 4Swine Human(N= ll) (N = 14)Cross validation folds, n 9 14F, g / pmol 0.0110 (0.0001) 0.0146 (0.0004) k, min10.0210 (0.0004) 0.0101 (0.0003) Training R2, accumulation phase 0.83 (0.02) 0.87 (0.01)Training R2, elimination phase 0.93 (0.01) 0.93 (0.01) Validation R2, accumulation phase 0.81 (0.17) 0.84 (0.23) Validation R2, elimination phase 0.83 (0.14) 0.89 (0.06), N = 11 RMSE, % 0.3 (0.1) 0.4 (0.2)t1 / 2, min 33.0 (0.6) 68.5 (2.1)Time to reach 95% steady state, min 99.0 (1.8) 205.5 (6.3)-36- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0168] To further evaluate the predictability of the model, the estimated NO absorption rates based on the model and directly measured values were compared. In the animal study, linear regression between estimated and measured NO absorption rates yielded a slope of 0.82 (95%CI, 0.72 - 0.92) and R2of 0.80 (See FIG. 10A). Bland-Altman analysis showed a mean bias of -4.7% with 95% limits of agreement from -56.2% to 46.8% (See FIG. 10B), with 70% of estimates within ±30% of measured values. In the human study, linear regression between estimated and measured absorption rates yielded a slope of 1.17 (95%CI, 1.06 - 1.27) andR2of 0.89 (See FIG. 10C). Bland-Altman analysis showed a mean bias of 1.2% with 95% limits of agreement from -51.7% to 54.1%, and 84% of estimates fell within ±30% of measured values (See FIG. 10D). These results validated the MetHb kinetic model as a reliable method for estimating NO absorption rates in both preclinical and clinical settings. In particular, the graphs of FIGS. 10A-10D provide a comparison of inhaled nitric oxide absorption estimated by methemoglobin kinetic model and measured values. FIG. 10A and FIG. 10B illustrate a swine study (9 animals, 68 treatments), and FIG. 10C and FIG.10D illustrate a human study (14 subjects, 64 treatments). Nitric oxide absorption rate was estimated for the held-out subject using parameters derived from the training set and compared to the measured value. In particular, FIG. 10A and FIG. 10C are linear regressions conducted between estimated and measured values with the solid line and dashed lines representing regression line and 95% confidence interval, respectively. In particular, FIG. 10B and FIG. 10D are Bland-Altman plots showing percentage difference [(estimated - measured) / measured] x 100% against measured nitric oxide absorption rate. The solid line represents the mean bias, and the dashed lines indicate 95% limits of agreement. Each data point represents a single treatment, with unique colors and symbols identifying individual subjects. NO stands for nitric oxide.

[0169]

[0170] Contributing factors to NO absorption

[0171] To investigate the contributing factors to NO absorption, animal studies were conducted in 6 pigs receiving 40 inhaled NO treatments at 80 ppm under systematically varied ventilatory settings and / or CO, yielding 9 to 441 breath-by-breath NO absorption measurements per treatment. Linear mixed-effects regression analysis revealed statistically significant factors affecting breath-by-breath NO absorption (See Table 5 and FIG. 11). In particular, FIG. 11 provides a comparison of breath-by-breath nitric oxide absorption between various ventilatory settings and cardiac output. FIG. HA plots tidal volume (ml / kg) vs. NO absorption by breath-37- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02( / / mol / breath). FIG. 1 IB plots inspiratory pause (s) vs. NO absorption by breath ( / / mol / breath). FIG. 11C plots inspiratory time (s) vs. NO absorption by breath ( / / mol / breath). FIG. HD plots flow pattern vs. NO absorption by breath ( / / mol / breath). FIG. 1 IE plots cardiac output (L / min) vs. NO absorption by breath ( / / mol / breath). Each data point represents mean absorption by breath in each treatment, and the whisker represents the standard deviation. Observations prior to and post a single change of ventilatory setting in the same animal were connected by lines. Each animal is assigned a unique symbol. In FIG. HE, grey symbols represent treatments at high positive end expiratory pressure (PEEP, 13-15 cmH20) and black ones at low PEEP (3-5 cmH20). NO stands for nitric oxide. CO stands for cardiac output.

[0172] Tidal volume, inspiratory pause and inspiratory time showed positive effects (17.1% per ml / kg [95%CI, 16.6 - 17.6], 14.6% per second [13.0 - 16.2] and 14.1% per second [11.5 -16.8], respectively) with adjusted p values < 0.001. Conversely, decelerating flow reduced absorption by 7.6% (5.9 - 9.3, adjusted p < 0.001). At the weighted mean cardiac output of 4.6 L / min, higher PEEP decreased NO absorption by 9.5% (8.6 - 10.4, adjusted p < 0.001). A 7.8% (7.3 - 8.3, adjusted p < 0.001) decrease in NO absorption was noted on each IL / min increase of cardiac output at low PEEP (3 - 5 cmEEO). Accounting for a potential system error of 14%, tidal volume, inspiratory pause and inspiratory time demonstrated clinically significant effects on NO absorption. This finding suggests that the inspired volume (and thus the amount of NO delivered into the lungs) and the time available for trachea-alveolus-capillary transfer are two metrics indicating systemic NO absorption. Table 5 shows linear mixed-effect model of contributing factors to NO absorption by breath. In Table 5, N represents the number of treatments. P values were adjusted using Bonferroni correction. Positive end expiratory pressure is abbreviated as PEEP. Cardiac output is abbreviated as CO. signifies at weighted mean cardiac output of 4.6 L / min. signifies at low PEEP.-38- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02Table 5.Factor Coefficient Intercept Adjusted p EffectTidal volume 0.30 0.44 < 0.001 17.1% per ml / kg (N= 12, 1761 breaths) (0.29 to 0.31) (0.12 to 0.76) (16.6 to 17.6) Inspiratory pause 0.09 0.62 < 0.001 14.6% / s(N= 12, 1142 breaths) (0.08 to 0.10) (0.22to l.02) (13.0to l6.2) Inspiratory time 0.08 0.55 < 0.001 14.1% / s(N= 12, 861 breaths) (0.06 to 0.09) (0.24 to 0.89) (11.5 to 16.8) Decelerating flow -0.05 0.60 < 0.001 -7.6%(N = 10, 934 breaths) (-0.06 to -0.04) (0.31 to 0.90) (-9.3 to -5.9) PEEP and CO 0.96(N = 11, 2255 breaths) (0.68 to 1.23)PEEP 0.79 <0.001 -9.5%(0.72 to 0.87) (-10.4 to - CO -0.08 <0.001 -7.8% per (-0.08 to -0.07) (-8.3 to -7Interaction -0.19 <0.001(-0.21 to -0.18)

[0173] Discussion

[0174] In this example, a system to quantify nitric oxide (NO) absorption breath-by-breath during inhaled NO therapy was developed and validated. Using this system, a model linking MetHb kinetics to NO absorption rate was established, providing the first evidence supporting the use of MetHb as a point-of-care marker for estimating NO absorption in clinical settings.

[0175] The breath-by-breath system achieved excellent correlation with the reference bag gascollection method and acceptable bias levels, indicating reliable accuracy across diverse breathing conditions. The mean differences from the bag method (1.5% ± 15.4% for in vitro and -3.6% ± 14.4% for in vivo tests) were comparable to systems measuring the uptake of other gas components using similar principles. Error sensitivity analysis revealed that the system accuracy was mostly affected by airway flow measurement and NO / NO2 analyzer sampling delay determination, which induced errors of ±14% and ±13%, respectively. To minimize these errors, airway gas flow measurement was calibrated using a precision flow controller. The NO / NO2 analyzer sampling delay is one of the main challenges in gas exchange measurement systems. As reported by Noguchi et al and Arieli et al, the delay was decomposed into gas transport lag time and analyzer response time which fitted a third-order correction. The predicted error is also comparable to previously reported gas exchange studies.-39- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0176] A previous study using gas-collection measurement reported NO absorption rate of 0.5 ml / min (20 pmol / min) at 100 ppm among healthy volunteers, which is comparable to the results of this example at 80 ppm. The work was advanced by setting up a system without interfering with the respiratory support, which allows clinical application of the system, especially in critically ill patients. In addition, the breath-by-breath resolution of the system enables real-time measurement approaches and investigation into the interaction between gas delivery and absorption.

[0177] MetHb has been used as a safety parameter of inhaled NO therapy since it reduces the oxygen carrying capacity of hemoglobin. However, it also represents a quantitative marker of NO absorption due to its unique oxidation and reduction kinetics. The reaction between NO and hemoglobin is extremely fast, such that the formation of MetHb is independent of its own concentration, and MetHb remains confined to the vascular space. Under constant rate of NO absorption, the production of MetHb should be constant. The elimination of MetHb follows the first-order kinetics even at levels approaching 20%.

[0178] A previous study modelled MetHb kinetics at different doses of inhaled NO by incorporating time and elimination constant. However, the model should also depend on the NO absorption rate, blood volume, and hemoglobin concentration. The data from this example demonstrated the complexity that the same dose could generate different levels of absorption and MetHb (See Table 3 and FIG. 9), with patients exhibiting lower rates of NO absorption and smaller MetHb changes than healthy subjects. Thus, the model was advanced by including NO absorption rate, blood volume, hemoglobin, and MetHb conversion factor. The model yielded stable parameters, including an elimination constant of 0.01 min1and a MetHb conversion factor of 0.015 g / pmol in human subjects, which are consistent with previous studies. The approach in this example demonstrated strong predictive capability for NO absorption, particularly with human subjects (both healthy volunteers and critically ill patients), achieving R20.89 and a mean bias of 1.2% with 84% of estimated within ±30% of measured values. The wider limits of agreement probably reflected individual variability and larger relative errors for lower levels of absorption. As a result, this model can guide NO dosing strategies and inform future dose-response studies.

[0179] This example identified significant positive effects of tidal volume, inspiratory pause and inspiratory time on NO absorption. While these factors might partially explain the variation in absorption at identical doses observed in both animals (See FIG. 9A) and humans (See FIG. 9C), lung volume could also contribute additional variance which warrants further investigation. These-40- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02findings highlight that systemic NO absorption is primarily governed by ventilatory mechanics: the inspired volume determines the amount of NO delivered to the lungs, while the duration of inspiration and inspiratory pause regulate the time available for gas transfer across the alveolar-capillary membrane.

[0180] Another observation is that healthy lungs absorbed substantially more NO than diseased lungs. Thus, the present disclosure recognizes that NO absorption and MetHb kinetics can be modeled and used as a diagnostic and prognostic marker of lung disease, disease progression and severity. Thus, the systems and methods provided herein can utilize breath-by-breath NO absorption as a noninvasive tool to characterize lung function and generate and communicate reports regarding the same, or related clinical indicators for further action, including adjustments to the prescribed therapy.

[0181] The above-described systems and methods can use breath-by-monitoring for accurate measurement of NO NO2 absorption during inhaled therapy and / or estimate the absorption of NO derived from the MetHb kinetics using a simplified instrumentation. The systems and methods can include models that can be used for monitoring and / or control, such as model between MetHb kinetics and NO absorption rate. Through testing, it was demonstrated that bedside MetHb monitoring can serve as a surrogate marker for NO absorption. Thus, the systems and methods provide a quantitative framework to individualize NO therapy, integrate pharmacokinetics with physiology, and expand the frontier of gas-based therapeutics.

[0182] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The present disclosure is readily extended to other aspects and implementations and may be practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms “mounted,” “connected,” “supported,” and “coupled” and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, “connected” and “coupled” are not restricted to physical or mechanical connections or couplings.-41- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02

[0183] As used herein in the context of computer implementation, unless otherwise specified or limited, the terms “component,” “system,” “module,” “controller,” “framework,” and the like are intended to encompass part or all of computer-related systems that include hardware, software, a combination of hardware and software, or software in execution. For example, a component may be, but is not limited to being, a controller device, a process being executed (or executable) by a controller device, an object, an executable, a thread of execution, a computer program, or a computer. By way of illustration, both an application running on a computer and the computer can be a component. One or more components (or system, module, and so on) may reside within a process or thread of execution, may be localized on one computer, may be distributed between two or more computers or other controller devices, or may be included within another component (or system, module, and so on).

[0184] In the methods described herein, the steps can be carried out in any order without departing from the principles of the disclosure, except when a temporal or operational sequence is explicitly recited. Recitation in a claim to the effect that first a step is performed, and then several other steps are subsequently performed, shall be taken to mean that the first step is performed before any of the other steps, but the other steps can be performed in any suitable sequence, unless a sequence is further recited within the other steps. For example, claim elements that recite “Step A, Step B, Step C, Step D, and Step E” shall be construed to mean step A is carried out first, step E is carried out last, and steps B, C, and D can be carried out in any sequence between steps A and E, and that the sequence still falls within the literal scope of the claimed process. A given step or sub-set of steps can also be repeated.

[0185] Furthermore, specified steps can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed step of doing X and a claimed step of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0186] The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, at least about 99.99%, or at least about 99.999% or more.

[0187] The discussion is presented to enable a person skilled in the art to make and use aspects of the disclosure. Various modifications to the illustrated configurations or processes will be-42- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-02readily apparent to those skilled in the art, and the generic principles herein can be applied to other aspects and applications within the scope of the present disclosure and the understanding of one of skill based thereon. Thus, the present disclosure is not intended to be limited to particular embodiments or aspects shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein. The following detailed description is to be read with reference to the figures, in which like components or elements in different figures have like reference numerals. The figures, which are not necessarily to scale, depict selected aspects and configurations or processes and are not intended to limit the scope of the disclosure. Skilled artisans will recognize the examples provided herein have many useful alternatives and fall within the scope of the disclosure.

[0188] Method examples described herein can be machine or computer-implemented at least in part. Some examples can include a computer-readable medium or machine-readable medium encoded with instructions operable to configure an electronic device to perform methods as described in the above examples. An implementation of such methods can include code, such as microcode, assembly language code, a higher-level language code, or the like. Such code can include computer readable instructions for performing various methods. The code may form portions of computer program products. Further, in an example, the code can be tangibly stored on one or more volatile, non-transitory, or non-volatile tangible computer-readable media, such as during execution or at other times. Examples of these tangible computer-readable media can include, but are not limited to, hard disks, removable magnetic disks, removable optical disks, e.g., compact disks and digital video disks, magnetic cassettes, memory cards or sticks, random access memories (RAMs), read only memories (ROMs), and the like.

[0189] As used in the claims, the phrase "at least one of A, B, and C" means at least one of A, at least one of B, and / or at least one of C, or any one of A, B, or C or combination of A, B, or C. A, B, and C are elements of a list, and A, B, and C may be anything contained in the Specification.

[0190] The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.-43- QB\125141.04962\100755084.1

Claims

125141.04962 Patent Application - MGH 2025-004-02CLAIMS:

1. A system for monitoring nitric oxide (NO) and / or nitrogen dioxide (NO2) containing gas delivered to a patient, the system comprising:a breathing system configured to provide a first flow of NO and / or NO2 containing gas to the patient;a sensor system configured to monitor the first flow of NO and / or NO2 containing gas to the patient and to monitor a second flow of gas exhaled by the patient;a controller configured to receive feedback from the sensor system, determine an amount of NO and / or NO2 gas inhaled by the patient and an amount of NO and / or NO2 gas exhaled by the patient to determine an amount of NO and / or NO2 gas absorbed by the patient using the feedback from the sensor system, and generate a report indicating the amount of NO and / or NO2 absorbed by the patient; anda display configured to communicate the report indicating the amount of NO absorbed by the patient.

2. The system of claim 1, wherein the controller is further configured to control the breathing system to adjust the first flow of NO and / or NO2 containing gas to adjust the amount of NO and / or NO2 absorbed by the patient.

3. The system of claim 2, wherein the breathing system includes a first line through which the first flow of gas flows to the patient and a second line through which the second flow of gas flows.

4. The system of claim 3, wherein the first line and the second line are in fluid communication with a port, the port being in fluid communication with a patient interface that is configured to deliver NO and / or NO2 containing gas to the patient.-44- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-025. The system of claim 4, wherein the breathing system includes a breathing apparatus configured to provide a flow of gas to the first line, andwherein the system further comprises an NO and / or NO2 gas source configured to inject a gas containing NO and / or NO2 into the first line so that the first flow of gas provided to the patient contains NO and / or NO2.

6. The system of claim 5, wherein the breathing apparatus is an invasive or non-invasive lung ventilator.

7. The system of claim 4, wherein the sensor system includes a rapid response NO and / or NO2 sensor configured to sense a first NO and / or NO2 concentration of the first flow of gas and a second NO and / or NO2 concentration of the second flow of gas and a flow rate sensor configured to sense a first flow rate of the first flow of gas and a second flow rate of the second flow of gas.

8. The system of claim 7, wherein the NO and / or NO2 gas sensor is positioned along a sampling line that is in fluid communication with the port.

9. The system of claim 5, wherein the breathing system adjusts the first flow of NO and / or NO2 containing gas to adjust the amount of NO and / or NO2 absorbed by the patient by at least one of(i) adjusting an amount of gas provided to the first line by the breathing apparatus, and (ii) adjusting an amount of gas injected into the first line by the NO and / or NO2 gas source.

10. The system of claim 2, further comprising a user interface configured to receive an input indicating a desired amount of NO and / or NO2 absorbed by the patient and wherein the controller is further configured to adjust the first flow of NO and / or NO2 containing gas to match the amount of NO and / or NO2 absorbed by the patient to the input indicating a desired amount of NO and / or NO2 absorbed by the patient.-45- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-0211. A system for monitoring nitric oxide (NO) and / or nitrogen dioxide (NO2) containing gas delivered to a patient, the system comprising:a breathing system including a breathing apparatus, a first line, and a second line, the breathing apparatus configured to provide a first flow of NO and / or NO2 containing gas to the patient via the first line and remove a second flow of gas from the patient via the second line; a sensor system operably coupled to the breathing system, the sensor system comprising a first sensor configured to measure a real-time NO and / or NO2 concentration of the first flow of gas and the second flow of gas and a second sensor configured to measure a real-time flow rate of the first flow of gas and the second flow of gas; anda computing device electronically coupled to the sensor system, the computing device being configured to:receive, from the sensor system, a first output from the first sensor and a second output from the second sensor,analyze the first output and the second output to determine an amount of NO and / or NO2 absorbed by the patient, andgenerate a report indicating the amount of NO and / or NO2 absorbed by the patient.

12. The system of claim 11, wherein the computing device is further configured to generate a first value indicating the amount of NO and / or NO2 absorbed by the patient.

13. The system of claim 11, wherein the computing device is further configured to at least one of:(i) compare the first value to a second predetermined value and adjust, based on the comparison between the first value and the second predetermined value, the first flow of NO and / or NO2 containing gas to the patient,(ii) display the first value, and(iii) compare the first value to a third predetermined value and generate, based on the comparison between the first value and the third predetermined value, a user notification indicative of the first value and the third predetermined value.-46- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-0214. The system of claim 12, wherein the computing device is further configured to determine the first value by:receiving, from a third sensor configured to measure a temperature of the first line and the second line, a third output from the third sensor,estimating, based on the first output, the second output, the third output, and the Ideal Gas Law (PV=nRT), a first amount of NO and / or NO2 in the first flow of gas and a second amount of NO and / or NO2 in the second flow of gas, anddetermining, based on a difference between the first amount and the second amount, a molar quantity of NO and / or NO2 absorbed by the patient on a breath-by-breath basis.

15. The system of claim 14, wherein the computing device is further configured to determine the first value by:receiving, from a fourth sensor configured to measure an atmospheric pressure, a fourth output from the fourth sensor,estimating, based on the first output, the second output, the third output, the fourth output, and the Ideal Gas Law (PV=nRT), a first amount of NO and / or NO2 in the first flow of gas and a second amount of NO and / or NO2 in the second flow of gas, anddetermining, based on a difference between the first amount and the second amount, a molar quantity of NO and / or NO2 absorbed by the patient on a breath-by-breath basis.

16. The system of claim 11, wherein the system further comprises a display configured to display the report indicating the amount of NO and / or NO2 absorbed by the patient.

17. The system of claim 12, wherein the system further comprises a user interface configured to receive an input indicating a desired amount of NO and / or NO2 absorbed by the patient, andwherein the controller is further configured to adjust the first flow of NO and / or NO2 containing gas to match the first value indicating the amount of NO and / or NO2 absorbed by the patient to the input indicating a desired amount of NO and / or NO2 absorbed by the patient.-47- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-0218. The system of claim 11, wherein the system further comprises an NO and / or NO2 gas source fluidly coupled to the breathing system, the NO and / or NO2 gas source configured to inject a gas containing NO and / or NO2 into the first line.

19. A method for delivering a nitric oxide (NO) and / or nitrogen dioxide (NO2) containing gas to a patient and monitoring the NO and / or NO2 dosage delivered to the patient, the method comprising:(a) delivering a first flow of NO and / or NO2 containing gas to the patient for inhalation via a first line;(b) providing a second line configured to receive a second flow of gas from the patient after exhalation;(c) measuring, with a first sensor, a first flow parameter indicating a real-time NO and / or NO2 concentration of the first flow of gas and the second flow of gas;(d) measuring, with a second sensor, a second flow parameter indicating a flow rate of the first flow of gas and the second flow of gas;(e) analyzing, with a computing device, the first flow parameter and the second flow parameter; and(f) at least one selected from a group of: (i) adjusting, based on the analysis of the first flow parameter and the second flow parameter, the first flow of NO and / or NO2 containing gas to the patient, (ii) displaying the first flow parameter and the second flow parameter, and (iii) generating, based on the analysis of the first flow parameter and the second flow parameter, a user notification indicative of the first flow parameter and the second flow parameter failing to meet a predetermined threshold.-48- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-0220. The method of claim 19, wherein step (e) of analyzing the first flow parameter and the second flow parameter comprises:measuring, with a third sensor, a third flow parameter indicating a temperature of the first flow of gas and the second flow of gas;determining, based on the first flow parameter, the second flow parameter, the third flow parameter, and the Ideal Gas Law (PV=nRT), a first amount of NO and / or NO2 in the first flow of gas and a second amount of NO and / or NO2 in the second flow of gas, anddetermining, based on a difference between the first amount and the second amount, a quantity of NO and / or NO2 absorbed by the patient on a breath-by-breath basis.

21. The method of claim 20, wherein the method further comprises:(g) identifying, based on at least one of the first flow parameter and the second flow parameter, an end of a first respiratory cycle and a start of a second respiratory cycle; and (h) repeating step (a) through step (g) for each respiratory cycle.-49- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-0222. A system for monitoring an amount of nitric oxide (NO) absorbed by a patient to whom NO containing gas was delivered, the system comprising:a monitoring system configured to monitor an amount of methemoglobin (MetHb) in the patient and generate an output indicating the amount of MetHb in the patient, wherein the monitoring system is configured to quantify the amount of MetHb by at least one of:measuring an absorption of light transmitted through the patient, quantifying hemoglobin levels of the patient based on the measurement, analyzing the absorption of light, and determining, based on that analysis, the amount of MetHb in the patient; and / orcollecting a sample from the patient and utilizing the principle of blood gas analysis to determine the amount of MetHb in the patient;a computing device configured to:receive the output from the monitoring system,determine the amount of NO absorbed by the patient based on the amount of MetHb in the patient and a MetHb kinetic model, andgenerate a report indicating the determined amount of NO absorbed by the patient; and a display configured to communicate the report indicating the determined amount of NO absorbed by the patient.

23. The system of claim 22, wherein the NO containing gas is delivered to the patient via extrapulmonary blood gas exchange.-SO- QB\125141.04962\100755084.1125141.04962 Patent Application - MGH 2025-004-0224. The system of claim 22, wherein the NO containing gas is delivered to the patient via at least one of:a) inhalation therapy;b) invasive ventilation;c) non-invasive ventilation.

25. The system of claim 23 or 24, wherein the controller is further configured to automatically adjust the NO delivery based on the determined amount of NO absorbed by the patient.

26. A method for delivering a nitric oxide (NO) and / or nitrogen dioxide (NO2) containing gas to a patient and monitoring the NO and / or NO2 dosage delivered to the patient, the method comprising:(a) delivering NO and / or NO2 to the patient;(b) measuring an amount of MetHb in the patient;(c) determining, based on the amount of MetHb in the patient and a MetHb kinetic model, an amount of NO absorbed by the patient;(d) generating a report indicating the amount of NO absorbed by the patient; and(e) displaying the report.

27. The method of claim 26, wherein the method further comprises:(f) controlling, based on the amount of NO absorbed by the patient, the NO and / or NO2 dosage delivered to the patient.-51- QB\125141.04962\100755084.1

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