WO2022187751A1 - Bradykinin 1 receptor antagonists and uses thereof for prevention and treatment of respiratory complications - Google Patents

Abstract

Methods provide for treatment using bradykinin 1 receptor antagonists, such as R-954 peptide, for prevention and treatment of respiratory complications.

Description

BRADYKININ 1 RECEPTOR ANTAGONISTS AND USES THEREOF FOR PREVENTION AND TREATMENT OF RESPIRATORY COMPLICATIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[OOOI] The present application claims the benefit of priority of U.S. Provisional Patent Application No. 63/157,621 filed March 5, 2021, entitled “Therapeutic Compounds and Methods for Inhibiting Covid-19 Respiratory Complications,” which is incorporated herein by reference in the present application.

[0002] The present application claims the benefit of priority of U.S. Provisional Patent Application No. 63/162,083 filed March 17, 2021, entitled “Therapeutic Compounds and Methods for Inhibiting Covid-19 Respiratory Complications,” which is incorporated herein by reference in the present application.

FIELD OF THE INVENTION

[0003] The present disclosure describes therapeutic compounds and peptides (e.g., R-954), and related treatments and methods for inhibiting the severity of proinflammatory responses and in particular, bradykinin storms.

BACKGROUND

[0004] Recent studies (see, e.g., Roche, et al.) provide strong evidence that COVID-19 complications are due to a bradykinin (BK) storm. Once the virus enters the host cell via the transmembrane protein angiotensin- converting-enzyme-2 (ACE2), the BK-regulating activity of ACE2 is disrupted, causing sudden increase in extracellular BK, and its metabolite des-Arg9-bradykinin (DABK), for the infected and neighboring cells. Heightened DABK traffics through the Bradykinin-i-receptors (BiR), causing cell inflammation and respiratory distress.

[0005] The COVID-19 pandemic has drastically affected the world, infecting over 245 million people and killing almost 5 million as of October 2021 (Elflein). The outbreak began in January 2020 and rapidly escalated, leading to the World Health Organization declaring COVID-19 a pandemic on March 11th, 2020. The vims that caused this outbreak is known as Severe Acute Respiratory Syndrome Coronavirus 2 (Sars-CoV-2).

[0006] COVID’s effect on individuals can vary drastically from mild symptoms to hospitalization and death. COVID tends to have more serious consequences for the elderly and those with cardiovascular disease, diabetes, respiratory problems, and cancer (WHO). COVID-19 was initially thought to induce a cytokine storm: an immune response in which a large number of cytokines are secreted by immune cells and cause symptoms such as fever, inflammation, fatigue, and nausea (Sino biological). However, Joseph A Roche developed a testable hypothesis that a bradykinin storm, not a cytokine storm, is causing severe immune and respiratory responses in COVID-19 patients (Roche). Dysregulated bradykinin signaling is the likely cause of respiratory complications because COVID-19 enters host cells via the transmembrane protein angiotensin converting enzyme 2 (ACE2) and lowers its numbers, which therefore increases des-Arg9-bradykinin (DABK) that stimulates Bi receptors.

[0007] There are currently no blockers for Bi receptors, to which DABK binds strongly, approved by the United States Food and Drug Administration (FDA); there are only FDA-approved blockers for B2 receptors, to which DABK binds weakly.

[0008] The peptide R-954 has been used in studies to determine its effectiveness in reducing the pro-inflammatory response in septic mice (Ruiz). In this study by Stephanie Ruiz, mice injected subcutaneously with R-954 were observed to have increased permeability of vital organs, with the lungs benefitting the most. These mice also had fewer pulmonary lesions, which are known predictors of death and ICU admission in COVID-19 patients (Ruch). The mice that received R-954 also had a higher survival rate.

[0009] Prior research has pointed to the trafficking of des-Arg9- Bradykinin (DABK) through the cell membrane’s bradykinin 1 receptors (BiR) as the main causative mechanism for leaky vessels, inflammation, and respiratory distress caused by the COVID-19 infection.

[0010] Icatibant is a known B2 receptor antagonist approved only for use in attacks of Hereditary Angioedema (a hereditary condition where people have acute episodes of swelling and inflammation) and administered subcutaneously (30 mg dose is typical).

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] An understanding of embodiments described in this disclosure and many of the related advantages may be readily obtained by reference to the following detailed description when considered with the accompanying drawings, of which:

FIG. 1 depicts metabolic conversion of conversion of Bradykinin (BK1-9) to des-Arg9-Bradykinin via kininase I (carboxypeptidase N);

FIG. 2 depicts a DABK ELISA calibration curve in accordance with an experimental approach;

FIGs. 3A, 3B, 3C, 3D, and 3E depict example representations of a host cell and a bystander cell in accordance with some embodiments of the present invention;

FIG. 4 depicts an example representation of R-954 BiR antagonist blocking the trafficking of DABK via BiR receptors in accordance with some embodiments of the present invention;

FIG. 5 is a flowchart of an example process by which bradykinin increases in an extracellular environment in accordance with some embodiments of the present invention;

FIGs. 6A, 6B, and 6C depict example representations of a cell subjected to bradykinin activity and R-954 in accordance with some embodiments of the present invention;

FIGs. 7A, 7B, 7C, 7D, and 7E depict example representations of a cell subjected to R-954 and then a bradykinin storm in accordance with some embodiments of the present invention;

FIG. 8 depicts a chart of measured extracellular DABK in plasma in accordance with some embodiments of the present invention; FIG. 9 depicts a chart representing increase in extracellular DABK versus an original storm concentration in accordance with some embodiments of the present invention;

FIGs. 10A-10C depict fluorescent images (with color inverted) of respective red blood cell layers resulting from an experimental process in accordance with some embodiments of the present invention;

FIGs. 11A, 11B, and 11C depict charts showing the relative green intensity of specimens in respective experimental conditions in accordance with some embodiments of the present invention; and

FIG. 12 is a chart depicting the relative intercellular Ca+2 response of red blood cell layers in respective experimental conditions in accordance with some embodiments of the present invention.

DETAILED DESCRIPTION

[0012] The inventor for this application has recognized that, in accordance with some embodiments described in this disclosure, some types of proinflammatory responses, such as bradykinin storms, may be inhibited through the use of the peptide identified as R-954. The inventor recognized that a Bi blocker would be significantly more effective in stopping a bradykinin storm, therefore significantly reducing inflammation and life-threatening respiratory complications in patients with COVID-19. Treating COVID-19 patients with R-954 could drastically reduce the hyperinflammatory response, prevent Acute Respiratory Distress Syndrome (ARDS) making ventilators unnecessary, and improve survival rates of those infected with coronavirus.

[0013] In inventor’s research, an R-954 peptide (AcOrn[Oic(2),(aMe)Phe(5),dpNal(7),Ile(8)]desArg(9)-bradykinin) was investigated for its ability to inhibit the passage of extracellular DABK through the BiR receptors, to inhibit the onset of respiratory inflammation, which is the most harmful aspect of the disease. If successful, it will provide compelling evidence for the use of R-954 as a treatment or prevention of severe symptoms (such as ARDS) for COVID- 19, and other diseases that cause respiratory distress.

[0014] It will be understood that bradykinin antagonist R-954 has des- Arg9-bradykinin (DABK) at the end.

[0015] Bradykinin storms affect various conditions and diseases that bradykinin antagonist R-954 may be useful in treating, including but not limited to: acute respiratory distress syndrome (ARDS) (including but not limited to inhalational injury from smoke/chemicals, trauma, pancreatitis, drowning, adverse drug reactions, and infections including Covid-19), sepsis, viral pneumonia, bacterial pneumonia, fungal pneumonias, acute pancreatitis, burns, trauma, and/ or toxic inhalation (e.g., from water, smoke, and/or gastric content).

[0016] In accordance with some embodiments of the present invention, methods provide for inhibiting the severity of symptoms of bradykinin storms in patients. For example, disclosed methods provide for inhibiting the severity of symptoms of bradykinin storms caused by one or more of: ARDS, sepsis, Alzheimer’s, and/or cancer. In one or more such embodiments, a treatment method comprises administering the peptide R-954 to a patient.

[0017] In accordance with some embodiments of the present invention, methods provide for inhibiting the severity of respiratory complications in patients. In one or more such embodiments, a treatment method comprises administering the peptide R-954 to a patient.

[0018] In accordance with some embodiments of the present invention, methods provide for inhibiting the severity of respiratory complications caused by Covid-19. In one or more such embodiments, a treatment method comprises administering the peptide R-954 to a patient. [0019] According to some embodiments, a method is provided for treating, preventing, or ameliorating acute respiratory distress syndrome (ARDS) comprising administering to a human or animal subject a therapeutically effective amount of R-954.

[0020] According to some embodiments, a method further comprises administering to the human or animal subject the therapeutically effective amount of R-954 in combination with a therapeutically effective amount of icatibant.

[0021] According to some embodiments, a therapeutically effective amount of R-954 administered to a human patient may be an amount substantially in the range of 0.001 mg to 50 mg. In one or more embodiments, a therapeutically effective amount of R-954 administered to a patient maybe in the range of about 0.007 ^mg to about 1.8 mg. In one or more embodiments, a therapeutically effective amount of R-954 administered to a patient maybe in the range of about 20 mg to about 40 mg. According to some embodiments, a therapeutically effective amount of icatibant administered for a human patient (e.g., in combination with administered R-954) maybe similarly in the range of about 20 mg to about 40 mg.

[0022] According to some embodiments, a method is provided for treating, preventing, or ameliorating a disease or condition associated with bradykinin storm activity comprising administering to a human or animal subject a therapeutically effective amount of R-954 wherein the disease or condition is selected from: sepsis, pneumonia (e.g., viral, bacterial, and/or fungal pneumonia), acute pancreatitis, burns, trauma, and toxic inhalation (e.g., caused by water, smoke, and/ or gastric content inhalation).

[0023] According to some embodiments, the above method may further comprise administering R-954 in combination with a therapeutically effective amount of icatibant.

[0024] According to some embodiments, a method is provided for treating airway and lung inflammation comprising administering R-954 to a patient in need thereof. [0025] According to some embodiments, a method is provided for treating airway and lung inflammation comprising administering R-954 in combination with icatibant to a patient in need thereof.

[0026] According to some embodiments, a method is provided for treating respiratory inflammations caused by Covid-19 comprising administering R-954 to a patient in need thereof.

[0027] According to some embodiments, a method is provided for treating respiratory inflammations caused by Covid-19 comprising administering R-954 in combination with icatibant to a patient in need thereof.

[0028] According to some embodiments, a method is provided for preventing trafficking of DABK via BiR receptors in infected cells of a patient comprising administering R-954 to the patient.

[0029] Some embodiments provide for R-954 for use in a method of treating airway and lung inflammations, the method comprising administering R-954 to a patient suffering respiratory inflammation. [0030] Some embodiments provide for R-954 for use in a method of treating airway and lung inflammations, the method comprising: administering R-954 to a patient suffering ; and administering icatibant to the patient.

[0031] Any processes described in this disclosure do not necessarily imply a fixed order to any depicted actions, steps, and/ or procedures, and embodiments may generally be performed in any order that is practicable unless otherwise and specifically noted.

[0032] Administering a peptide (e.g., R-954) to a patient, in accordance with one or more embodiments described in this disclosure, may comprise inhalational delivery, nasal delivery, intravenous delivery, and/ or oral delivery.

[0033] As noted above, heightened DABK traffics through the BiR, causing cell inflammation and respiratory distress. Inventor discovered that a BiR antagonist to block this DABK passage could prevent acute respiratory distress syndrome of COVID-19, decreasing the disease’s threat. [0034] As discussed further herein, the R-954 BiR antagonist was investigated in-vitro for such use. A bradykinin storm was simulated as 2.5X the normal level of plasma (~3.5ng/ml), with 35.ong/ml R-954 added in three configurations; DABK/no R-954, simultaneous R-954 with DABK, and R-954 prior to DABK. Untreated DABK storm results depict 0.9X/ o.8x the original DABK storm content after 15/3 omin respectively, providing direct evidence for DABK-BiR migration. For simultaneous R-954/DABK, competition of DABK and R-954 for BiR-sites produced a 2.4X increase in extracellular- DABK in 15mm, and 3.2X in 3 omin, highlighting R-954 ’s ability to block DABK-BiR trafficking. Pre-addition of R-954 promoted its binding to BiR-sites before the addition of DABK, and was most successful, producing increased extracellular- DABK of 3.4X/4.5X at 15mm/ 3omin respectively. DABK-induced cell inflammation was measured via increased intracellular-Ca+2 for simulated COVID-19 DABK-storms, with/without pre-addition of R-954. While untreated DABK-storm RBCs exhibited 4.3X intracellular-Ca+2 (vs. normal), those pretreated with R-954 exhibited near-normal intracellular-Ca+2, further demonstrating R-954’s efficacy in blocking DABK-BiR migration, to prevent COVID-19-induced cell inflammation.

A. Background for experimental process 1. DABK and the bradykinin storm

[0035] Bradykinin (BK) and des-Arg9-Bradykin (DABK) are part of the kinin system, which is involved in blood pressure regulation and inflammatory reactions. The kinin system increases vascular permeability and causes vasodilation. Substrates called kininogens bind to kallikreins, which produces the peptide kallidin. Kallidin is then transformed into the peptide Bradykinin. Bradykinin’s biological effects are mediated by Bi and B2 receptors, two g-protein coupled receptors.

[0036] FIG. 1 is an illustration of the metabolic conversion of bradykinin (BK1-9) to des-Arg9-Bradykinin via Kininase I (Carboxypeptidase N). DABK is the active metabolite of bradykinin, and it binds to Bi receptors on the cells in order to produce a proinflammatory response, which is the bradykinin storm believed to cause severe side effects in COVID-19.

2. ELISA detection of extracellular DABK

[0037] In experiments, a human Des-Arg9-Bradykinin ELISA kit ( MyBioSource Catalog #MBSiog4sg ) was used to quantify extracellular DABK concentrations as a function of added R-954 BiR antagonist. The inventor’s own blood was used throughout the research to simulate a bradykinin storm. For each assay, blood was separated into plasma and blood cell layers. To begin each assay grouping, the student’s plasma layer was separated, and the “normal” or control extracellular DABK concentration was measured, according to the DABK ELISA protocol. [0038] For ELISA detection of human DABK, serial dilutions of long/ml DABK were created (i.e., 10, 5, 2.5, 1.25, and 0.625 ng/ml), and their 45onm absorbances read in triplicate using a Molecular Devices plate reader. The calibration curve was created to convert A450 to DABK ng/ml in subsequent R-954 assays, as depicted in FIG. 2. Specifically, absorbance at 450nm that were measured for each of 0.625-iong/ml DABK standards were plotted against concentration, to create the DABK ELISA calibration plot shown in FIG. 2. During the experiment, increased DABK concentration were visible as increasing yellow well color.

[0039] The control or normal DABK level was found to be 1.4 ng/ml. Accordingly, a bradykinin storm was simulated by the addition of DABK to human blood, prior to separation, to a cumulative concentration of 2.5X the normal (Singh, et al,), or 3.5ng/ml. The artificially-created bradykinin storm was created for each R-954-DABK assay configuration, prior to the measure of extracellular DABK via ELISA.

3. COVID-19 induced dysregulation of bradykinin & des-Arg9- bradykinin signaling

[0040] As noted above, recent research by Roche, et al. highlights the role of a Bradykinin storm in the onset of inflammation and respiratory distress, associated with COVID-19 infection and disease progression. Briefly, entry of SARS-CoV at ACE2 upregulates extracellular DABK for the infected and neighboring cells, which are then trafficked through the BiR, causing leaky vessels, inflammation, and respiratory distress.

[0041] FIGs. 3A-3E depict an example representation of COVID-19 induced dysregulation of Bradykinin (BK) 316a, and des-Arg9-bradykinin (DABK, or DABK9). FIG. 3A depicts a view 302 of a cell with host cell plasma membrane 304 prior to infection by SARS-CoV 306. FIG. 3A further shows example ACE2 308a and 308b, which regulate the production of DABK in the extracellular environment. SARS-CoV enters the host cell plasma membrane 304 via ACE2 and thereby downregulates (depletes) ACE2 at the plasma membrane of infected cells. BK 316a and 316b represent the bradykinin peptide that promotes inflation; DABK 314a represents a bioactive metabolite of BK and is associated with lung inflammation. FIG. 3A further shows example BK-Bi-receptor (BiR) 310a and BK-B2-receptors (B2R) 312a and 312b.

[0042] ACE2 depletion by COVID binding to ACE2 cause upregulation (or increase) in extracellular DABK product for both infected and bystander or neighboring cells. FIG. 3B depicts the example view 402 of the host cell of FIG. 3A is infected and after an increase in the production of extracellular DABK for the infected cell. Specifically, FIG. 3B depicts the SARS-CoV 306 in the now-infected host cell plasma membrane 304, and an increase in BK and DABK in the extracellular set 303. As also depicted in FIG. 3B, DABK 314a, 314b, and 314c and BK 316b of set 305 are engaged, respectively, with BiR 310a, 310b, and 310c, and with B2R 312a and 312b.

[0043] FIG. 3C depicts an example view 330 of a bystander cell with cell plasma membrane 332 and plasma membrane elements 337 (including example ACE2, BiR, and B2R). Members of a set of elements 335 are depicted as engaged with one or more of the plasma membrane elements 337. Example view 330 also depicts an example representation of an increased number of elements of BK and DABK in an extracellular set 333 following an increase in extracellular DABK production for the bystander cell after infection of the example cell of FIG. 3B. [0044] As discussed in this disclosure, following infection, DABK is trafficked through the BiR and (minimally) the B2R, into the cell plasma membrane for both host and bystander cells, resulting in leaky blood vessels, inflammation, and respiratory distress. FIGs. 3D and 3E represent this trafficking of DABK 314a and 314c (FIG. 3D) and DABK 340a and 340b (FIG. 3E) through the example BiR and B2R cell membrane receptors for the host and bystander cells, respectively.

4. R-954 peptide for blocking DABK-BiR binding, to inhibit inflammatory response due to BK and DABK dysregulation

[0045] Inventor discovered the potential of the R-954 peptide for blocking DABK-BiR binding, to inhibit inflammatory response due to BK and DABK dysregulation (such as those resulting from COVID-19 respiratory infections).

[0046] As mentioned in this disclosure, R-954 acts as an antagonist for BiR, and may therefore inhibit the passage of bradykinin-storm extracellular DABK through the BiR, which otherwise causes respiratory inflammation often associated with COVID-related deaths.

[0047] Inventor’s research was focused on establishing evidence of successful R-954/B1R binding during an artificially-created bradykinin storm and the resultant inhibiting of DABK passage through the cell membrane, so that there is no measured loss of extracellular DABK. The example representation 400 of FIG. 4 depicts the theorized role of R-954 420a, 420b, 420c and 42od in blocking the trafficking of DABK of extracellular set 403 via the BiR membrane receptors of the set of membrane elements 407 in example cell membrane 404. Without R- 954/B1R binding and halt of DABK membrane passage, it was expected that extracellular DABK would decrease. These two scenarios, along with a control, with no DABK or R-954 added, were the basis for inventor’s research investigation.

[0048] As discussed above, according to Roche, et al., elevated extracellular DABK causes increased BiR stimulation, followed by upregulation of DABK trafficking through the BiR, and ensuing inflammation. This, in turn, increases extracellular BK, and its metabolite, DABK, in a cyclic process. An example of the cyclic production of extracellular DABK from post-inflammation increase in extracellular BK is depicted in example steps 502, 504, 506, 508, 510, and 512 of example method 500 of FIG. 5. Accordingly, inventor modified the hypothesis: Where R-954 is introduced, successful inhibition of DABK-BiR passage should result in significant increase in extracellular DABK; without R-954, extracellular DABK should decrease to a lesser extent.

[0049] Two approaches were used to confirm R-954 inhibition of DABK trafficking through the BiR. In the first scenario described below with respect to FIGs. 6A-6C, R-954 and DABK were added simultaneously to whole blood, so that the respective concentrations were 35ng/ml and 3.5ng/ml (i.e. IOX R-954 and 2.5X DABK storm). In this scenario, R-954 and DABK should compete for cell membrane BiR receptors. Should R- 954 successfully block DABK trafficking, the extracellular DABK should increase measurably with time.

[0050] As depicted in the example view 602 of FIG. 6A, at time t=o, 2.5X DABK (3.5 ng/ml) and 10X R-954 BiR antagonist (35 ng/ml) were added. The extracellular set of elements 603 is shown as including representative DABK 6i4a-d, BK 616a, and R-95462oa-e. Cell membrane 604 is depicted with representative ACE2 6o8a-b, BiR 6ioa-d, and B2R 612a.

[0051] As depicted in the example view 602 of FIG. 6B representing time t=i5 minutes, some of the elements have engaged with receptors. Specifically, R-954620c and 62oe have engaged with BiR 610a and 610c, and DABK 614a and 6i4d have engaged with BiR 610b and 6iod, respectively. Extracellular BK 6i6a-c are also represented. Further, DABK 614b and 614c are depicted as having trafficked through BiR receptors through the cell membrane 604.

[0052] Finally, as depicted in the example view 602 of FIG. 6B representing time t=30 minutes, the increased DABK production has increased the number of DABK elements in the set of extracellular elements 630. [0053] For a second approach, depicted in FIGs. 7A-7E, IOX (35.ong/ml) R-954 was first added to whole blood. After 30 minutes, simulation of a bradykinin storm was created with the addition of 2.5X, 3-5ng/ml DABK. In this scenario, R-954 should first bind to BiR receptors, in the absence of excess DABK. Once the bradykinin storm is artificially created with 2.5X DABK, 30 minutes later, R-954 blocking of the BiR receptors should cause a significant increase in extracellular DABK, where added DABK creates BK, followed by cyclic production of more DABK. DABK should therefore increase dramatically with time, and more so than simultaneous DABK- R-954 addition.

[0054] As depicted in the example view 702 of FIG. 7A, at time t=o,

10X R-954 BiR antagonist (35 ng/ml) was added. The extracellular set of elements 703 is shown as including representative R-95472oa-d. Cell membrane 704 is depicted with representative membrane elements 707 including ACE2, BiR, and B2R.

[0055] As depicted in the example view 702 of FIG. 7B, at a time before 30 minutes had passed since the addition of the R-954, the representative added R-95472oa-d acts as a BiR antagonist, blocking BiR receptors. In the view 702 of FIG. 7C, at approximately time t=30 minutes, 2.5X DABK (3-5 ng/ml), represented as DABK 705, is added to stimulate a bradykinin storm. In the views 702 of FIGs. 7D and 7E, at approximately time t=45 minutes and 60 minutes, respectively, the amount of DABK in the extracellular set of elements 703 has increased.

5. Extracellular DABK results for simulated DABK storm, with R- 954 BiR antagonist

[0056] Most recent research has pointed to a bradykinin storm as the most significant mechanism for COVID-19-induced respiratory inflammation. In this model, excess extracellular DABK binds to, and passes through the BiR, subsequently causing inflammation. In this research, such passage was first confirmed experimentally, where a 2.5X Bradykinin Storm (3.5ng/ml from a normal, pre-measured i.43ng/ml) was created in whole blood. After 15 minutes and 30 minutes, respectively, measured extracellular DABK decreased from the original storm concentration of 3.5ng/ml to 3.mg/ml and 2.96ng/ml, or 0.9X and o.8x the original DABK storm concentration, respectively (see chart 800 of FIG. 8). This decrease in extracellular DABK provides direct evidence for the passage of the bradykinin through the BiR, into the cells, where it would cause inflammation.

[0057] Subsequently, R-954 was investigated for its ability to block trafficking of DABK via the BiR; such trafficking leads to leaky vessels, inflammation, and respiratory distress. Two unique approaches were carried out to confirm R-954 inhibition of BiR passage of DABK. In the first, storm DABK concentration of 3-5ng/ml is simultaneously added to whole blood along with IOX R-954 (35.ong/ml). In this scenario, DABK and R-954 competition for BiR sites should lead to partial BiR blocking, which when combined with extracellular DABK->BK->DABK cyclic production, should lead to measurable increase in extracellular DABK, which should also increase with time. Results confirm this hypothesis, with 8.35ng/ml extracellular DABK measured at 15 minutes, and n.i8ng/ml DABK measured at 30 minutes. This corresponds to a 2.4X and 3.2X increase in extracellular DABK, relative to the original DABK storm concentration of 3.5 ng/ml (see chart 800 of FIG. 8 and chart 900 of FIG. 9)·

[0058] In the second approach, R-954 and DABK were added prophylactically; i.e. first, iox R-954 (35.ong/ml) was added to whole blood, followed by simulation of a 2.5X Bradykinin Storm (2.5X DABK added to 3.5ng/ml) 30 minutes later. In this approach, R-954 should first bind to BiR, which will inhibit the passage of DABK via the BiR, which would begin as excess DABK is added, 30 minutes later. Combined with extracellular DABK- >BK-> DABK cyclic production, this should bring about a dramatic increase in extracellular DABK concentration, relative to the first approach, and overall. Results confirm this hypothesis, and the ability of R-954 to block DABK passage via the BiR. After 15 minutes, extracellular DABK was found to be n.77ng/ml (3.4X the original DABK storm concentration). After 30 minutes, extracellular DABK increased to I5.7ing/ml, or 4.5X of the original storm DABK concentration (see chart 800 of FIG. 8 and chart 900 of FIG. 9).

6. Measure of COVID-19 DABK-BiR-induced cell inflammation via intracellular Ca⁺²

[0059] Previous experiments, and the corresponding results, highlight R-954 ’s success as a BiR antagonist, to block passage of DABK through the receptor, preventing cell inflammation, and associated respiratory distress often associated with COVID-19. In phase 2 of the research, R-954’s ability to concurrently reduce/inhibit cell inflammation, due to passage of DABK through the BiR, during a COVID-19 -induced DABK storm, was investigated. Previous research by Wang, et al. highlights the use of intracellular Ca+2 as an indicator for cell inflammation, detected through staining of red blood cells (RBCs) with Fluo-4 AM staining reagent. [0060] Using this approach, the student’s own blood was freshly drawn, and similarly pre-treated with 35ng/ml R-954, followed by the simulation of an extracellular DABK storm (3.5ng/ml), 30 minutes later. This R-954 - DABK “pre-treatment” sequence was used, as it provided the best DABK-BiR blocking results, and potentially the best inhibition of associated cell inflammation, in earlier portions of the research. Two additional controls were included for intracellular Ca+2 measurement: a non-treated, normal student blood sample, and a student blood sample with a simulated 3-5ng/ml DABK-Storm, no R-954 treatment.

[0061] Intracellular Ca+2 for each RBC condition was measured (in triplicate) via fluorescence microscopy, using the following procedure, adapted from Wang, et al., with minor modification. Briefly, for each treatment condition:

• Normal RBCs

• RBCs from blood treated with 3.5ng/ml DABK Storm, with no treatment

• RBCs from blood pre-treated with 35ng/ ml R-954, and 3-5ng/ ml DABK Storm 30mm later) [0062] RBCs were isolated via centrifugation at io,ooog for 3 min. The buffy coat and plasma were discarded, and the remaining RBCs were washed three times with Tyrode’s solution containing the following (in mM): 135 NaCl, 54 KC1, 10 glucose, 1 MgCl₂, 1.8 CaCl₂ and 10 HEPES. The pH was adjusted to 7.35 using 0.02M NaOH.

[0063] The RBCs were then loaded with 5mM Fluo-4 AM and incubated for 1 hour at 370C. Then, the cells were washed three times with Tyrode at room temperature (~20oC), and the fluorescent images taken within the 24-well culture plate using an excitation wavelength of 490nm, and an emission wavelength of 535nm; the microscopic images for each condition are highlighted in FIG. 10A (fluorescence images of normal RBCs), FIG. 10B (fluorescence images of RBCs treated with a simulated, 2.5X (3.5ng/ml) DABK Storm, no treatment), and FIG. 10C (fluorescence images of RBCs pre-treated with 35ng/ml R-954, then 2.5X (3.5ng/ml) DABK 30 mins later).

[0064] The relative fluorescence of Fluo-AM in the RBCs was used to evaluate cellular inflammation via intracellular Ca⁺² content. Specifically, ImageJ was used to translate each of the above images to RGB indices, per x-distance. See chart 1102 of FIG. 11A, chart 1104 of FIG. 11B, and chart 1106 of FIG. 11C. For each treatment condition, the G-value was extrapolated, and plotted against x-distance. Then, the summative G-value per image was calculated, and corrected for consistent cell count (at the fixed microscope magnification used throughout the experiment) to produce a relative intracellular-Ca⁺² comparison for each of the RBC treatment conditions. See chart 1200 of FIG. 12.

[0065] Intracellular Ca+2 results for the three RBC conditions first highlight the anticipated cellular inflammation that is caused by a COVID- 19 induced DABK storm, with a 552 relative luminescence/intracellular Ca+2 response, which is 4.3 times the response of normal, control RBCs, at 127 (Fig. 13). Pre-treatment of RBCs with 35ng/ml R-954, followed by simulation of the 3-5ng/ml DABK storm produced near-normal luminescence/intracellular Ca+2 response (172, or 1.4X of normal), providing compelling evidence that R-954 can successfully prevent cell inflammation within RBCs, due to its ability to inhibit DABK passage through the BiR during a COVID-19 bradykinin storm. More specifically, phase 1 results that highlight R-954’s ability to block DABK-BiR migration during a COVID-19 DABK storm, combined with phase 2 findings that demonstrate R-954’s ability to reduce/eliminate intracellular cell inflammation that would otherwise ensue in its absence (during a COVID- 19 induced DABK storm), provide convincing evidence for R-954 as a successful BiR antagonist to reduce/eliminate cellular inflammation and associated respiratory distress associated with COVID-19.

[0066] Collectively, the findings of this research (i) support the proposed mechanism of COVID-19-induced inflammation via the trafficking of storm/elevated levels of extracellular DABK through the cell membrane through the BiR, (ii) provide compelling evidence for R-954’s ability to block passage of bradykinin storm extracellular concentrations of DABK via the BiR, and (iii) provide convincing evidence that R-954 can significantly reduce or eliminate cell inflammation that follows a COVID- 19 -induced DABK storm, by blocking the DABK-BiR migration that would otherwise occur, absent of the R-954 BiR antagonist. This newly discovered (BiR) antagonistic property, specific to DABK, could subsequently prevent the most harmful consequences of Covid-19 infection, including fatigue, inflammation, and acute respiratory distress syndrome (ARDS), which often leads to hospitalization, ventilation, and possible death. As such, these results highlight R-954 as a new, effective Covid-19 treatment, to inhibit the disease’s most lethal progression, via blockage of the DABK-BiR migration mechanism.

INTERPRETATION

[0067] Numerous embodiments are described in this patent application and are presented for illustrative purposes only. The described embodiments are not, and are not intended to be, limiting in any sense. The presently disclosed invention(s) are widely applicable to numerous embodiments, as is readily apparent from the disclosure. One of ordinary skill in the art will recognize that the disclosed invention may be practiced with various modifications and alterations, such as structural, logical, software, and/or electrical modifications. Although particular features of the disclosed invention(s) may be described with reference to one or more particular embodiments and/ or drawings, it should be understood that such features are not limited to usage in the one or more particular embodiments or drawings with reference to which they are described, unless expressly specified otherwise.

[0068] The present disclosure is neither a literal description of all embodiments nor a listing of features that must be present in all embodiments.

[0069] Neither the Title (as may be set forth at the beginning of the first page of this disclosure) nor the Abstract (as may be set forth at the end of this disclosure) is to be taken as limiting in any way the scope of the disclosed invention(s).

[0070] Throughout the description and unless otherwise specified, the following terms may include and/ or encompass the example meanings provided below. These terms and illustrative example meanings are provided to clarify the language selected to describe embodiments both in the specification and in the appended claims, and accordingly, are not intended to be limiting.

[0071] The terms “an embodiment”, “embodiment”, “embodiments”, “the embodiment”, “the embodiments”, “one or more embodiments”, “some embodiments”, “one embodiment” and the like mean “one or more (but not all) disclosed embodiments”, unless expressly specified otherwise. [0072] The terms “the invention” and “the present invention” and the like mean “one or more embodiments of the present invention.”

[0073] A reference to “another embodiment” in describing an embodiment does not imply that the referenced embodiment is mutually exclusive with another embodiment (e.g., an embodiment described before the referenced embodiment), unless expressly specified otherwise.

[0074] The terms “including”, “comprising” and variations thereof mean “including but not limited to”, unless expressly specified otherwise. [0075] The terms “a”, “an” and “the” mean “one or more”, unless expressly specified otherwise.

[0076] The term “plurality” means “two or more”, unless expressly specified otherwise.

[0077] The term “herein” means “in the present disclosure, including anything which maybe incorporated by reference”, unless expressly specified otherwise.

[0078] The phrase “at least one of’, when such phrase modifies a plurality of things (such as an enumerated list of things) means any combination of one or more of those things, unless expressly specified otherwise. For example, the phrase at least one of a widget, a car and a wheel means either (i) a widget, (ii) a car, (iii) a wheel, (iv) a widget and a car, (v) a widget and a wheel, (vi) a car and a wheel, or (vii) a widget, a car, and a wheel.

[0079] The phrase “based on” does not mean “based only on”, unless expressly specified otherwise. In other words, the phrase “based on” describes both “based only on” and “based at least on”.

[0080] Where a limitation of a first claim would cover one of a feature as well as more than one of a feature (e.g., a limitation such as “at least one widget” covers one widget as well as more than one widget), and where in a second claim that depends on the first claim, the second claim uses a definite article “the” to refer to the limitation (e.g., “the widget”), this does not imply that the first claim covers only one of the feature, and this does not imply that the second claim covers only one of the feature (e.g., “the widget” can cover both one widget and more than one widget).

[0081] Each process (whether called a method, algorithm or otherwise) inherently includes one or more steps, and therefore all references to a “step” or “steps” of a process have an inherent antecedent basis in the mere recitation of the term “process” or a like term. Accordingly, any reference in a claim to a “step” or “steps” of a process has sufficient antecedent basis.

[0082] When an ordinal number (such as “first”, “second”, “third” and so on) is used as an adjective before a term, that ordinal number is used (unless expressly specified otherwise) merely to indicate a particular feature, such as to distinguish that particular feature from another feature that is described by the same term or by a similar term. For example, a “first widget” may be so named merely to distinguish it from, e.g., a “second widget”. Thus, the mere usage of the ordinal numbers “first” and “second” before the term “widget” does not indicate any other relationship between the two widgets, and likewise does not indicate any other characteristics of either or both widgets. For example, the mere usage of the ordinal numbers “first” and “second” before the term “widget” (1) does not indicate that either widget comes before or after any other in order or location; (2) does not indicate that either widget occurs or acts before or after any other in time; and (3) does not indicate that either widget ranks above or below any other, as in importance or quality. In addition, the mere usage of ordinal numbers does not define a numerical limit to the features identified with the ordinal numbers. For example, the mere usage of the ordinal numbers “first” and “second” before the term “widget” does not indicate that there must be no more than two widgets.

[0083] As used in this disclosure, a “user” may generally refer to any individual and/ or entity that operates a user device.

[0084] A description of an embodiment with several components or features does not imply that any particular one of such components and/or features is required. On the contrary, a variety of optional components are described to illustrate the wide variety of possible embodiments of the present invention(s). Unless otherwise specified explicitly, no component and/ or feature is essential or required.

[0085] Further, although process steps, algorithms or the like may be described or depicted in a sequential order, such processes may be configured to work in one or more different orders. In other words, any sequence or order of steps that may be explicitly described or depicted does not necessarily indicate a requirement that the steps be performed in that order. The steps of processes described in this disclosure may be performed in any order practical. Further, some steps may be performed simultaneously despite being described or implied as occurring non- simultaneously (e.g., because one step is described after the other step). Moreover, the illustration of a process by its depiction in a drawing does not imply that the illustrated process is exclusive of other variations and modifications, does not imply that the illustrated process or any of its steps is necessary to the invention, and does not imply that the illustrated process is preferred.

[0086] While the present invention has been illustrated by a description of various embodiments and while these embodiments have been described in considerable detail, it is not the intention of the applicant to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details, representative apparatus and method, and illustrative examples shown and described. Accordingly, departures maybe made from such details without departing from the spirit or scope of the general inventive concept.

[0087] The present disclosure provides, to one of ordinary skill in the art, an enabling description of several embodiments and/or inventions. Some of these embodiments and/ or inventions may not be claimed in the present application but may nevertheless be claimed in one or more continuing applications that claim the benefit of priority of the present application. Applicant reserves the right to file additional applications to pursue patents for subject matter that has been disclosed and enabled but not claimed in the present application.

REFERENCES

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[0090] “Cytokine Signaling.” Sino Biological, www.sinobiological.com/resource/cytokines/cytokine-signaling. [0091] “Cytokine Storm.” Sino Biological, www.sinobiological.com/resource/cytokines/cytokine-storm.

[0092] “Definition of Cytokine Storm.” National Cancer Institute, www.hopkinsmedicine.org/health/conditions-and- diseases/coronavims/what-coronavims-does-to-the-lungs.

[0093] Elflein, J. (2021, October 29). Coronavirus deaths worldwide by country. Statista. Retrieved October 31, 2021, from https://www.statista.com/statistics/1093256/novel-coronavirus- 2 o i9nco v- deaths- worldwide-by-country/ .

[0094] Hertz L, Huisjes R, Llaudet-Planas E, Petkova-Kirova P, Makhro A, Danielczok JG, Egee S, del Mar Mafm-Pereira M, van Wijk R, Vives Corrons J-L, Bogdanova A and Kaestner L (2017), Is Increased Intracellular Calcium in Red Blood Cells a Common Component in the Molecular Mechanism Causing Anemia? Front. Physiol. 8:673. doi: io.3389/fphys.20i7.oo673

[0095] Hlavinka, Elizabeth. “COVID-19 Storms: Bradykinin In, Cytokine Out?” Medical News and Free CME Online, MedpageToday, 11 Sept. 2020, www.medpagetoday.com/infectiousdisease/Covid19/88560. [0096] Lowe, Derek. Bradykinin and the Coronavirus. 8 Sept. 2020, blogs.sciencemag.org/pipeline/ archives/ 2020/ 09/08/bradykinin-and- the-coronavirus.

[0097] Marceau, Frangois, et al. “Bifunctional Ligands of the Bradykinin B2 and Bi Receptors: An Exercise in Peptide Hormone Plasticity.” ScienceDirect, Elsevier, 23 May 2018, www.sciencedirect.c0m/science/article/abs/pii/S0196978118301050. [0098] Pirahanchi Y, Sharma S. Physiology, Bradykinin. [Updated 2020 Aug 31]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK537187/

[0099] Roche, Joseph A, and Renuka Roche. “A hypothesized role for dysregulated bradykinin signaling in COVID-19 respiratory complications.” FASEB journal : official publication of the Federation of American Societies for Experimental Biology vol. 34,6 (2020): 7265- 7269. doi:io.1096/fj.202000967

[0100] Ruch, Yvon, et al. “CT Lung Lesions as Predictors of Early Death or ICU Admission in COVID-19 Patients.” Clinical Microbiology and Infection, vol. 26, no. 10, 2020, doi:io.ioi6/j.cmi.2020.07.030. [0101] Ruiz, Stephanie, et al. “Kinin Bi Receptor: a Potential Therapeutic Target in Sepsis-Induced Vascular Hyperpermeability.” Journal of Translational Medicine, vol. 18, no. 1, 2020, doi:io.n86/si2967-020-02342-8.

[0102] Singh, Pradeep K., et al. “Increased Plasma Bradykinin Level Is Associated with Cognitive Impairment in Alzheimer's Patients.” Neurobiology of Disease, vol. 139, 2020, p. 104833., doi:io.ioi6/j.nbd.2020.104833.

[0103] Terashima, R., Kimura, M., Higashikawa, A., Kojima, Y., Ichinohe, T., Tazaki, M., Shibukawa, Y., Intracellular Ca²⁺ mobilization pathway via bradykinin Bi receptor activation in rat trigeminal ganglion neurons. The Journal of Physiological Sciences (2019) 69:199-209 [0104] Tisoncik, Jennifer. “Into the Eye of the Cytokine Storm.” Microbiology and Molecular Biology Reviews, vol. 76, no. 1, 2012, www.ncbi.nlm.nih.gov/pmc/articles/PMC3294426/.

[0105] Vara, Vasanthi. “Coronavirus Outbreak: The Countries Affected so Far.” Pharmaceutical Technology, Global Data, 30 Oct. 2020, www.pharmaceutical-technology.com/features/coronavirus-outbreak-the- countries-affected/.

[0106] Wang, J., Wagner-Britz, L., Bogdanova, A., Ruppenthal, S., Wiesen, K., Kaiser, E., Tian, Q., Krause, E., Bernhardt, L, Lipp, P., Philipp, S. E., & Kaestner, L. (2013, June 28). Morphologically homogeneous red blood cells present a heterogeneous response to hormonal stimulation. PloS one. Retrieved October 30, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695909/.

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Claims

WHAT IS CLAIMED IS:

1. A method of treating, preventing, or ameliorating acute respiratory distress syndrome (ARDS) comprising administering to a human or animal subject a therapeutically effective amount of R-954.

2. The method of claim 1, further comprising administering to the human or animal subject the therapeutically effective amount of R-954 in combination with a therapeutically effective amount of icatibant.

3. The method of claim 1, wherein the therapeutically effective amount of R-954 is substantially in a range of 0.001 mg to 50 mg.

4. The method of claim 1, wherein the therapeutically effective amount of R-954 is substantially in a range of 0.005 ^mg to 2.0 mg.

5. The method of claim 1, wherein the therapeutically effective amount of R-954 is substantially in a range of 20 mg to 40 mg.

6. A method of treating, preventing, or ameliorating a disease or condition associated with bradykinin storm activity comprising administering to a human or animal subject a therapeutically effective amount of R-954 wherein the disease or condition is selected from: sepsis, pneumonia, acute pancreatitis, burns, trauma, and toxic inhalation.

7. The method of claim 6, wherein the pneumonia comprises viral pneumonia.

8. The method of claim 6, wherein the pneumonia comprises bacterial pneumonia.

9. The method of claim 6, wherein the pneumonia comprises fungal pneumonia.

10. The method of claim 6, wherein the toxic inhalation is caused by water inhalation.

11. The method of claim 6, wherein the toxic inhalation is caused by gastric contents inhalation.

12. The method of claim 6, wherein the toxic inhalation is caused by smoke inhalation.

13. The method of claim 6, further comprising administering to the human or animal subject the therapeutically effective amount of R-954 in combination with a therapeutically effective amount of icatibant.

14. A method for treating airway and lung inflammation comprising administering R-954 to a patient in need thereof.

15. A method for treating airway and lung inflammation comprising administering R-954 in combination with icatibant to a patient in need thereof.

16. A method for treating respiratory inflammations caused by Covid-19 comprising administering R-954 to a patient in need thereof.

17. A method for treating respiratory inflammations caused by Covid-19 comprising administering R-954 in combination with icatibant to a patient in need thereof.

18. A method for preventing trafficking of DABK via BiR receptors in infected cells of a patient comprising administering R-954 to the patient.

19. R-954 for use in a method of treating airway and lung inflammations, the method comprising: administering R-954 to a patient suffering respiratory inflammation.

20. R-954 for use in a method of treating airway and lung inflammations, the method comprising: administering R-954 to a patient suffering ; and administering icatibant to the patient.