Treatment of COVID-19-associated pulmonary inflammatory disease by administering resiniferatoxin

Abstract

Providing treatment for pulmonary inflammatory disease associated with COVID-19 by administering resiniferatoxin. [Solution] Disclosed is a method for treating pulmonary inflammatory diseases, comprising administering an effective amount of resiniferatoxin (RTX) via epidural, periganglionic, or intraganglionic administration. In some embodiments, the dose of RTX for an adult is about 0.1 μg to about 100 μg. The method for treating pulmonary inflammatory diseases comprises administering an effective amount of resiniferatoxin (RTX) via epidural, periganglionic, or intraganglionic administration to a subject in need of treatment for the pulmonary inflammatory disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 002,165, filed March 30, 2020, and U.S. Provisional Application No. 63 / 122,858, filed December 8, 2020, the disclosures of each of which are incorporated by reference herein in their entirety.

[0002] Throughout this application, various publications, patents, and / or patent applications are referenced. The disclosures of such publications, patents, and / or patent applications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0003] Technical Field The present disclosure provides methods for treating pulmonary inflammatory diseases comprising administering an effective amount of resiniferatoxin (RTX) by epidural, periganglionic, or intraganglionic administration. [Background technology]

[0004] background RTX acts as an ultra-potent analog of capsaicin, the main component of chili peppers that irritates the tongue and nose. RTX is a tricyclic diterpene isolated from certain species of Euphorbia. The homovanillyl group is a key structural feature of capsaicin and is the most prominent feature that distinguishes resiniferatoxin from typical phorbol-related compounds. Naturally occurring RTX has the following structure: [ka] It has.

[0005] RTX and analog compounds (e.g., tiniatoxin and other compounds (20-homovanillyl esters of diterpenes such as 12-deoxyphorbol 13-phenylacetate 20-homovanillate and mezerein 20-homovanillate) are described in U.S. Pat. Nos. 4,939,194; 5,021,450; and 5,232,684. Other resiniferatoxin-type phorboid vanilloids have also been identified (Szallasi et al. (1999) Brit. J. Pharmacol. 128:428-434).

[0006] RTX is known as a TRPV1 agonist. TRPV1 is a transient receptor potential cation channel subfamily V member 1 (also known as vanilloid receptor-1 (VR1)), a multimeric cation channel that is prominently expressed in nociceptive primary afferent neurons (Caterina et al. (1997) Nature 389:816-824; Tominaga et al. (1998) Neuron 21:531-543). TRPV1 activation typically occurs in nerve endings via the application of painful heat and is upregulated during certain types of inflammatory stimuli. Activation of TRPV1 in peripheral tissues by chemical agonists results in the opening of calcium channels and the transmission of pain perception (Szalllasi et al. (1999) Mol. Pharmacol. 56:581-587). However, direct application of certain TRPV1 agonists to the cell bodies (ganglia) of TRPV1-expressing neurons opens calcium channels and triggers a cascade of events that leads to programmed cell death ("apoptosis") (Karai et al. (2004) J. of Clin. Invest. 113:1344-1352).

[0007] Respiratory failure due to acute respiratory distress syndrome (ARDS) is one of the major causes of mortality (53%) associated with infection with the novel coronavirus SARS-CoV-2 (COVID-19 disease) (Ruan et al. (2020) Intensive Care Med Mar 3:10-3), and can also result from other diseases and disorders, including other viral diseases or lung injuries. Approximately 10% of patients require intensive care unit (ICU) care with respiratory support, with a reported ICU mortality rate of 79% (Huang et al. (2020) Lancet Vol. 394, Issue 1). 10233, P497-506).

[0008] Coronaviruses are a group of viruses that cause disease in birds, mammals, and humans. These diseases include respiratory and enteric infections that can be mild or fatal. Coronaviruses are viruses in the order Nidovirales, family Coronaviridae, and subfamily Orthocoronavirinae. The genus Coronavirus includes avian infectious bronchitis virus, bovine coronavirus, canine coronavirus, human coronavirus 299E, human coronavirus OC43, mouse hepatitis virus, rat coronavirus, and porcine hemagglutinating encephalomyelitis virus. The genus Torovirus includes Berne virus and Breda virus. Coronaviruses are enveloped viruses with a single-stranded, positive-sense RNA genome and a nucleocapsid with helical symmetry. Coronavirus genome sizes range from approximately 26 to 32 kilobases, which is believed to be the largest for an RNA virus. It is interesting to note that the 2019-2020 pneumonia outbreak in Wuhan, China, was traced by the World Health Organization (WHO) to a novel coronavirus, termed 2019-nCoV, also known as SARS-CoV-2, that causes coronavirus disease 2019, or COVID-19.

[0009] ARDS, first described in 1967 (Ashbaugh et al. (1967) Lancet 2:319-323), is characterized by diffuse pulmonary microvascular injury resulting in hypoxemia caused by increased permeability and intrapulmonary shunting. The first two stages of ARDS progression (i.e., 12 to 72 hours after onset) represent the most critical time window for intervention, as the syndrome can be reversed if its initiating factors and inflammatory mediators can be controlled. Early diagnosis can also be facilitated if the initiating stimulus is known, such as in diagnosed sepsis, aspiration of gastric contents, multiple transfusions, severe fractures, burns, pancreatitis, or severe trauma. Progression to the third stage of ARDS results in increasing pulmonary hypertension, an increased heart rate to compensate for hypoxemia, and mechanical ventilation support therapy is generally required. Pathologically, the cellular infiltrate becomes denser with continued neutrophil infiltration and an increase in mononuclear cell, lymphocyte and fibroblast infiltrates. The severity of the disease increases with age, with 80% of deaths observed in patients over 60-65 years of age (CDC COVID-19 Response Team (2020) MMWR Morb Mortal Wkly Rep 69:343-346), whereas younger infected individuals appear to be less susceptible and exhibit moderate to mild symptoms (Wu et al. (2020) JAMA Published online February 24, 2020). Once the lower respiratory tract is affected, respiratory distress progresses very rapidly, with reported time to death being as rapid as 14 days from initial symptoms, despite the availability of palliative support through mechanical ventilation. Severity and mortality in susceptible populations infected with COVID-19 have been reported to be associated with a cytokine storm, in which exacerbated production of pro-inflammatory substances is released into the lung microenvironment over a short period of time (Mehta et al. (2020) Lancet Vol. 395, Issue 1). 10229, P1033-1034). Novel life-saving strategies are desperately needed to reduce the high mortality rates associated with acute respiratory distress, including such distress associated with later-stage viral infections. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 4,939,194 [Patent Document 2] U.S. Patent No. 5,021,450 [Patent Document 3] U.S. Patent No. 5,232,684 [Non-patent literature]

[0011] [Non-Patent Document 1] Szallasi et al. (1999) Brit. J. Pharmacol. 128:428-434 [Non-patent document 2] Caterina et al. (1997) Nature 389:816-824 [Non-patent document 3] Tominaga et al. (1998) Neuron 21:531-543 [Non-patent document 4] Szallasi et al. (1999) Mol. Pharmacol. 56:581-587 [Non-Patent Document 5] Karai et al. (2004) J. of Clin. Invest. 113:1344-1352 [Non-patent document 6] Ruan et al. (2020) Intensive Care Med Mar 3:10-3 [Non-Patent Document 7] Huang et al. (2020) Lancet Vol. 394, Issue 10233, P497-506 [Non-patent document 8] Ashbaugh et al. (1967) Lancet 2:319-323 [Non-Patent Document 9] CDC COVID-19 Response Team (2020) MMWR Morb Mortal Wkly Rep 69:343-346 [Non-Patent Document 10] Wu et al. (2020) JAMA Published online February 24, 2020 [Non-Patent Document 11] Mehta et al. (2020) Lancet Vol. 395, Issue 10229, P1033-1034 Summary of the Invention [Means for solving the problem]

[0012] Abstract The present disclosure provides methods for treating pulmonary inflammatory diseases, comprising administering an effective amount of resiniferatoxin (RTX) via epidural, periganglionic, or intraganglionic administration. In some embodiments, the dose of RTX for an adult is about 0.1 μg to about 100 μg.

[0013] Embodiment 1 is a method for treating a pulmonary inflammatory disease, the method comprising administering an effective amount of resiniferatoxin (RTX) epidurally, periganglionally, or intraganglionally to a subject in need of treatment for the pulmonary inflammatory disease.

[0014] Embodiment 2 is a composition comprising resiniferatoxin (RTX) for use in a method of treating a subject in need of treatment for a pulmonary inflammatory disease.

[0015] Embodiment 3 is a composition for use according to embodiment 2, wherein the method comprises administering the composition epidurally, periganglionally, or intraganglionally to the subject.

[0016] Embodiment 4 is the method of embodiment 1 or the composition for use of embodiment 2 or 3, wherein the effective amount of RTX results in a reduction of one or more cytokines, including IL-6, IL-1β, and / or IFNγ.

[0017] Embodiment 5 is a composition for a method or use according to any one of the preceding embodiments, wherein the effective amount of RTX results in improved lung function.

[0018] Embodiment 6 is a composition for a method or use according to any one of the preceding embodiments, wherein the effective amount of RTX results in reduced pulmonary edema.

[0019] Embodiment 7 is a composition for a method or use according to any one of the preceding embodiments, wherein the subject is an adult.

[0020] Embodiment 8 is a composition for the method or use according to any one of the preceding embodiments, wherein the RTX is administered at a dose of about 0.1 μg to about 100 μg.

[0021] Embodiment 9 is a composition for the method or use according to embodiment 8, wherein the dose is about 0.1 μg to about 1 μg, about 1 μg to about 5 μg, about 5 μg to about 10 μg, about 10 μg to about 20 μg, about 20 μg to about 50 μg, or about 50 to about 100 μg.

[0022] Embodiment 10 is a method or composition for use according to any one of the preceding embodiments, wherein the method comprises epidural administration.

[0023] Embodiment 11 is a method or composition for use according to any one of embodiments 1 to 9, wherein the method comprises a periganglionic nerve block.

[0024] Embodiment 12 is a method or composition for use according to any one of embodiments 1 to 9, wherein the method comprises intraganglionic administration.

[0025] Embodiment 13 is a composition for the method or use according to any one of the preceding embodiments, wherein the RTX is administered in a pharmaceutical formulation comprising the RTX and a pharmaceutically acceptable carrier.

[0026] Embodiment 14 is a composition for the method or use of embodiment 13, wherein the pharmaceutically acceptable carrier comprises water.

[0027] Embodiment 15 is a composition for the method or use of embodiment 13, wherein the pharmaceutically acceptable carrier comprises saline.

[0028] Embodiment 16 is a composition for the method or use according to any one of embodiments 13 to 15, wherein the RTX is present in the pharmaceutical formulation at a concentration ranging from 1 μg / ml to 100 μg / ml.

[0029] Embodiment 17 is a composition for the method or use according to embodiment 16, wherein the RTX is present in the pharmaceutical formulation at a concentration ranging from 1 μg / ml to 5 μg / ml, 5 μg / ml to 10 μg / ml, 10 μg / ml to 20 μg / ml, 20 μg / ml to 50 μg / ml, or 50 μg / ml to 100 μg / ml.

[0030] Embodiment 18 is a method or composition according to any one of the preceding embodiments, wherein the pulmonary inflammatory disease is selected from the group consisting of acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), pulmonary arterial hypertension (PAH), chronic inflammatory lung disease, pulmonary fibrosis, pulmonary vasculitis, pulmonary sarcoidosis, inflammation and / or infection associated with lung transplantation, acute or pulmonary rejection and / or dysfunction, bronchitis, sinusitis, asthma, cystic fibroma, bacterial infection, fungal infection, parasitic infection, viral infection, bronchiolitis obliterans syndrome (BOS), primary ciliary dyskinesia (PCD), pulmonary alveolar proteinosis, idiopathic pulmonary fibrosis (IPF), eosinophilic pneumonia, eosinophilic bronchitis, inflammation and / or infection associated with mechanical ventilation, ventilator-associated pneumonia, asbestos-related airway disorder or disease, dust-related airway disorder or disease, silicosis, and radiation- or chemical-related airway disease or disorder, and any combination thereof.

[0031] Embodiment 19 is a method or composition according to any one of the preceding embodiments, wherein the pulmonary inflammatory disease is acute respiratory distress syndrome (ARDS).

[0032] Embodiment 20 is a method or composition according to any one of the preceding embodiments, wherein the pulmonary inflammatory disease is chronic obstructive pulmonary disease (COPD).

[0033] Embodiment 21 is a method or composition according to any one of the preceding embodiments, wherein the pulmonary inflammatory disease is pulmonary arterial hypertension (PAH).

[0034] Embodiment 22 is a method or composition of any one of the preceding embodiments, wherein the pulmonary inflammatory disease is inflammation and / or infection associated with mechanical ventilation and / or ventilator-associated pneumonia.

[0035] Embodiment 23 is a method or composition of any one of the preceding embodiments, wherein the pulmonary inflammatory disease is associated with COVID-19. [Brief explanation of the drawings]

[0036] [Figure 1A-1B] Figures 1A-B show a schematic diagram of the study design (Figure 1A) and the treatment plan and timeline (Figure 1B). In Figure 1A, the arrow indicates intratracheal administration of bleomycin (Bleo) (2.5 mg / kg, approximately 0.15 mL) into the lungs. The box indicates the location where lung tissue was collected for cytokine measurement. As shown in Figure 1B, on day 0, Bleomycin or saline was administered intratracheally; on day 3, resiniferatoxin (RTX) or vehicle (Veh) was administered into the epidural space or stellate ganglion; and on day 7, the rats were sacrificed.

[0037] [Figure 2A-2B] Figures 2A-B show the stellate ganglion isolation procedure and the administration of Veh or RTX. Figure 2A shows step 1 of the procedure—the stellate ganglion was exposed. The arrow indicates that the stellate ganglion was positioned medially toward the origin of the internal thoracic and costocervical arteries. Figure 2B shows step 2 of the procedure—RTX (5 μL, 50 mg / mL) was injected into the left and right stellate ganglia. The arrow indicates the tip of the 5 μL syringe inside the stellate ganglion.

[0038] [Figure 3A-3C] Figures 3A-C show that plasma extravasation was reduced after epidural RTX treatment 7 days after Bleo administration. Figures 3A-B show representative images of lungs from the Bleo group (Figure 3A) and the Bleo + RTX group (Figure 3B). Figure 3C shows the concentrations of Evans blue from the control, Bleo, and Bleo + RTX groups. **P<0.01 vs. control. ##P<0.01 vs. Bleo.

[0039] [Figures 4A-4C]Figures 4A-C show lung tissue cytokine levels on day 7 after Veh or epidural RTX administration on day 3. Figure 4A shows interleukin 6 (IL-6). Figure 4B shows interleukin 1β (IL-1β). Figure 4C shows interferon gamma (IFNγ). *P<0.05 and **P<0.01 compared to control. #P<0.05 and ##P<0.01 compared to Bleo.

[0040] [Figures 5A-5C] Figure 5A-C shows plasma cytokine levels on day 7 after Veh or epidural RTX administration on day 3.

[0041] [Figures 6A-6D] Figures 6A-D show that Evans blue extravasation was reduced after stellate ganglion RTX injection at 7 days after Bleo administration. Figures 6A-C show representative images of lungs from the sham (Figure 6A), Bleo+Veh (Figure 6B), and Bleo+RTX (Figure 6C) groups. Arrows point to areas of Evans blue extravasation. Figure 6D shows the mean Evans blue concentration from each group. **P<0.01 vs. sham. #P<0.05 vs. Bleo+Veh. $P<0.05 vs. sham.

[0042] [Figures 7A-7D] Figures 7A-H show arterial blood gases on day 7 in sham, Bleo+Veh, and Bleo+RTX rats after intrastellate ganglion administration on day 3 post-injury. Figure 7A shows pH. Figure 7B shows carbon dioxide partial pressure (PCO2). Figure 7C shows oxygen partial pressure (PO2). Figure 7D shows base excess (BE). Figure 7E shows bicarbonate (HCO3). Figure 7F shows total CO2 (TCO2). Figure 7G shows oxygen saturation (sO2). Figure 7H shows lactate (Lac). *P<0.05 vs. sham. †P<0.05 vs. Bleo+Veh. [Figures 7E-7H]Figures 7A-H show arterial blood gases on day 7 in sham, Bleo+Veh, and Bleo+RTX rats after intrastellate ganglion administration on day 3 post-injury. Figure 7A shows pH. Figure 7B shows carbon dioxide partial pressure (PCO2). Figure 7C shows oxygen partial pressure (PO2). Figure 7D shows base excess (BE). Figure 7E shows bicarbonate (HCO3). Figure 7F shows total CO2 (TCO2). Figure 7G shows oxygen saturation (sO2). Figure 7H shows lactate (Lac). *P<0.05 vs. sham. †P<0.05 vs. Bleo+Veh.

[0043] [Figure 8A-8B] Figures 8A-B show lung tissue cytokine levels on day 7 after Veh or stellate ganglion RTX administration on day 3. Figure 8A shows IL-6. Figure 8B shows IL-1β.

[0044] [Figures 9A-9D] Figures 9A-H show body weight (BW) and individual organ weights between groups. Figure 9A shows body weight. Figure 9B shows heart. Figure 9C shows lung. Figure 9D shows spleen. Figure 9E shows liver. Figure 9F shows kidney. Figure 9G shows heart / BW. Figure 9H shows lung / BW. Compared with sham rats, lung wet weight (WLW) and the ratio of WLW to BW were significantly higher in Bleo+Veh rats, which was significantly reduced by intrastellate ganglion injection of RTX. These data suggest that intrastellate ganglion injection of RTX reduces pulmonary edema after Bleo. [Figures 9E-9H] Figures 9A-H show body weight (BW) and individual organ weights between groups. Figure 9A shows body weight. Figure 9B shows heart. Figure 9C shows lung. Figure 9D shows spleen. Figure 9E shows liver. Figure 9F shows kidney. Figure 9G shows heart / BW. Figure 9H shows lung / BW. Compared with sham rats, lung wet weight (WLW) and the ratio of WLW to BW were significantly higher in Bleo+Veh rats, which was significantly reduced by intrastellate ganglion injection of RTX. These data suggest that intrastellate ganglion injection of RTX reduces pulmonary edema after Bleo. DETAILED DESCRIPTION OF THE INVENTION

[0045] Detailed Description Reference will now be made in detail to certain specific embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the invention as defined by the appended claims.

[0046] Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to particular compositions or process steps, as such may vary. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a conjugate" includes a plurality of conjugates, reference to "a cell" includes a plurality of cells, etc. The use of alternatives (e.g., "or") herein is understood to mean either one or both of the alternatives or any combination thereof.

[0047] The term "and / or" as used herein should be taken to mean the specific disclosure of each of the specified features or components, whether or not the others are present. For example, the term "and / or" when used herein in a phrase such as "A and / or B" means "A and B." and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or," when used in a phrase such as "A, B, and / or C," refers to each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); It is intended to encompass B (alone); as well as C (alone).

[0048] As used herein, the terms "comprising," "including," "having," and "containing," and grammatical variations thereof, when used herein, are intended to be open-ended, such that one item or multiple items in a list do not exclude other items, and such other items may be substituted for or added to the listed items. Wherever aspects are described herein with the wording "comprising," it is understood that otherwise similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0049] As used herein, the term "about" refers to a particular value or composition that is within an acceptable error range for that value or composition as determined by one of ordinary skill in the art, where the error range depends in part on how the value or composition is measured or determined (i.e., the limitations of the measurement system). For example, "about" or "approximately" can mean within one or more standard deviations per practice in the art. Alternatively, "about" or "approximately" can mean up to 10% (i.e., ±10%) or a greater range, depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to five times the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for that particular value or composition. In some embodiments, "about" includes variations within 10%, 5%, 2%, 1%, or 0.5% of the stated value.

[0050] Numerical ranges are inclusive of the numbers defining the range. Measured and measurable values ​​are understood to be approximations, taking into account significant digits and error associated with measurement. Also, all ranges should be interpreted as including the endpoints unless an exclusionary expression such as "not including the endpoints" is present; thus, for example, "ranging from 1 to 10" includes the values ​​1 and 10, and includes all numbers and (where appropriate) non-integer values ​​greater than 1 and less than 10.

[0051] The use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," "includes," and "including" is not intended to be limiting. It is to be understood that the foregoing general and detailed descriptions are exemplary and explanatory only and are not limiting of the teachings. Unless specifically noted above in the specification, embodiments herein that describe various components as "comprising" are also contemplated as "consisting of" or "consisting essentially of" the described components; embodiments herein that describe various components as "consisting of" are also contemplated as "including" or "consisting essentially of" the described components; and embodiments herein that describe various components as "consisting essentially of" are also contemplated as "consisting of" or "including" the described components (this interchangeability does not apply to the use of these terms in the claims).

[0052] The section headings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any way. In the event that any document incorporated by reference conflicts with any term defined herein, the present specification shall control. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. To the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0053] definition As used herein, "pulmonary inflammatory disease" refers collectively to those acute and chronic pathological conditions associated with inflammatory processes. Non-limiting examples of pulmonary inflammatory diseases include acute respiratory distress syndrome (ARDS), pneumonia, pneumonitis, bronchitis, lung infection, neonatal atelectasis, conditions associated with inflammatory lung injury (e.g., chemotherapy-induced (e.g., bleomycin) lung injury, pancreatitis-induced lung injury, hyperoxia-induced lung injury, amiodarone-induced pneumonitis, radiation pneumonitis, chlorine gas or smoke inhalation injury, bronchiolitis obliterans / obstructive pneumonia (BOOP), viral and mycoplasmal pneumonia (e.g., Legionella and CMV pneumonia), pneumoconiosis, pulmonary vasculitis, pulmonary sarcoidosis, respiratory tract bacterial infection, respiratory tract fungal infection, respiratory tract parasitic infection, respiratory tract viral infection, ventilator-associated inflammation and / or infection, ventilator-associated pneumonia). Non-limiting examples of chronic pathological conditions of the lung include chronic obstructive pulmonary disease (COPD), pulmonary arterial hypertension (PAH), cystic fibroma, silicosis, asbestosis, asthma, atherosclerosis, chronic bronchitis, chronic inflammation due to chronic bacterial or viral infection, coronary artery disease, idiopathic pulmonary fibrosis (IPF), familial pulmonary fibrosis (FPF), desquamative interstitial pneumonitis (DIP), hypersensitivity pneumonitis, interstitial pneumonitis, collagen vascular disease, sarcoidosis, coal workers' pneumoconiosis, bronchopulmonary dysplasia, and inflammatory pseudotumor.

[0054] As used herein, "epidural administration" refers to the delivery of a drug or pharmaceutical formulation into the epidural space (also known as the "extradural space" or "peridural space"), the outermost portion of the spinal canal. It is the space within the spinal canal (formed by the surrounding vertebrae) that is outside the dura mater (which encases the arachnoid mater, subarachnoid space, cerebrospinal fluid, and spinal cord). For example, epidural delivery can include delivery to the epidural space without direct injection into a nerve, or it can include epidural delivery to neural tissue.

[0055] As used herein, "peri-ganglionic administration" refers to the delivery of a drug or pharmaceutical formulation to the space surrounding a nerve ganglion.

[0056] "Intra-ganglionic administration" means administration into a ganglion. Intra-ganglionic administration can be achieved by direct injection into the ganglion, and also includes selective nerve root injection, in which the compound passes through the connective tissue sleeve around the nerve and enters the ganglion at the nerve root just outside the vertebra.

[0057] The terms "effective amount," "therapeutically effective amount," or "effective dose," or related terms, may be used interchangeably and refer to an amount of a therapeutic agent sufficient to affect a measurable improvement or prevention of pulmonary inflammatory disease when administered to a subject. For example, administration of an effective dose may improve pulmonary function, expressed as partial pressure of CO (pCO), partial pressure of O (pO), and oxygen saturation (sO), as measured in arterial blood. In another example, an effective dose may reduce pulmonary edema. The therapeutically effective amount of a therapeutic agent provided herein, when used alone or in combination with an antiviral agent, will vary depending on the relative activity of the therapeutic agent and the subject and disease state being treated, the subject's weight, age, and sex, the severity of the disease state in the subject, the mode of administration, and the like, which can be readily determined by one of ordinary skill in the art. In one embodiment, the therapeutically effective amount depends on certain aspects of the subject to be treated and the disorder to be treated, and can be ascertained by one of ordinary skill in the art using known techniques. Additionally, as is known in the art, adjustments for age, weight, general health, sex, diet, time of administration, drug interactions, and severity of disease may be necessary.

[0058] The terms "subject" and "patient," as used herein, refer to humans and non-human animals, including vertebrates, mammals and non-mammals. In one embodiment, the subject may be a human, non-human primate, simian, ape, murine (e.g., mouse and rat), cow, pig, horse, dog, cat, goat, wolf, ranine, or fish.

[0059] The terms "administering," "administered," and grammatical variations refer to the physical introduction of a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration (e.g., by injection or infusion). The phrase "parenteral administration," as used herein, refers to a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In one embodiment, the formulation is administered via a non-parenteral route (e.g., orally). Other non-parenteral routes include topical, epidermal, or mucosal routes of administration (e.g., intranasal, vaginal, rectal, sublingual, or topical). Administration can also be, for example, single, multiple, and / or over one or more extended periods of time.

[0060] "Treating" should be understood broadly and encompasses any beneficial effect, including, for example, at least partially delaying, slowing the worsening, or halting symptoms associated with pulmonary inflammatory disease, or remedying such symptoms. Treating also encompasses resulting in some form of improved patient function, as discussed in detail below. In some embodiments, treating also means prolonging survival compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or disorder, as well as those prone to have the disease or disorder, or those in whom the disease or disorder is to be prevented.

[0061] "Pharmaceutically acceptable vehicle" for therapeutic purposes A pharmaceutically acceptable vehicle is a physical embodiment that can be administered to a subject. Pharmaceutically acceptable vehicles include, but are not limited to, pills, capsules, caplets, tablets, oral fluids, injectable solutions, sprays, aerosols, lozenges, dietary supplements, creams, lotions, oils, liquids, pastes, powders, and steam. Alternatively, it can be a liquid. An example of a pharmaceutically acceptable vehicle is a buffered isotonic solution (e.g., phosphate-buffered saline (PBS)).

[0062] Clinical signs of COVID-19 have been observed to be consistent with those observed in viral pneumonia. These pulmonary changes are likely responsible for both systemic and local immune responses that result in a hyperinflammatory state. Patient mortality is suspected to be related to a virus-driven cytokine storm similar to that seen in SARS-CoV-2 infection. The cytokine storm is the result of a severe immune response in the lungs, as measured by high levels of inflammatory markers (c-reactive protein, serum ferritin) and cytokine levels (IL-6, IL-2, IL-7, IL-10, GSCF, IP10, MCP1, MIP1A, IL-1β, IFNγ, and TNFα) in plasma. ICU patients have higher plasma levels of IL-2, IL-7, IL-10, GSCF, IP10, MCP1, MIP1A, and TNFα compared with non-ICU patients. This indicates that the presence of high circulating cytokine levels is associated with disease severity. Therefore, it is necessary to intervene in the inflammatory cascade at a higher level (ie, eliminate pro-inflammatory efferent pathways) to adequately control multiple aspects of this inflammatory process.

[0063] The underlying physiological events associated with morbidity, disease severity, and mortality may be explained by the involvement of TRPV1-expressing neural systems (afferent / efferent neurons). TRPV1-positive pathways are responsible for pain transmission, inflammation, and immune regulation throughout the pulmonary system.

[0064] Afferent innervation of the pulmonary system is primarily provided by the vagus nerve and its branches. TRPV1-expressing C fibers are small-diameter, unmyelinated fibers in the vagus nerve that carry several processes in the airways and lungs. Afferent fibers that innervate pulmonary structures are also transmitted by sympathetic fibers with cell bodies located in the dorsal root ganglia of the thoracic spinal cord segments between T1 and T6. Activation of this thoracic spinal cord segment is associated with severe pneumonitis.

[0065] RTX is an ultrapotent agonist of the TRPV1 receptor, which functions by inducing neurolysis of TRPV1-expressing neurons in the dorsal root ganglion (DRG), dorsal horn (DH) of the spinal cord, or peripheral nerve terminals when applied locally as a nerve block. Strong binding of RTX to the TRPV1 receptor opens the channel gate, resulting in a slow, sustained increase in intracellular Ca2+, which in turn disrupts intracellular mitochondrial metabolism and causes neuronal or nerve fiber ablation within minutes. The inventors have discovered the therapeutic use of RTX (an ultrapotent TRPV1 agonist) as an ablating agent of TRPV1-positive pulmonary pathways in patients with acute pulmonary inflammatory disease. This therapeutic approach, targeting TRPV1-expressing neurons in the lung, modulates inflammatory and immune signaling activity, resulting in reduced mortality and better overall outcomes.

[0066] Exemplary Compositions for Use and Related Methods Provided herein are methods and compositions for use in treating pulmonary inflammatory diseases in which RTX is delivered epidurally, periganglionally via a nerve block, or intraganglionally. In various embodiments, an ablative agent such as RTX is used. Routes of administration of the RTX (agent) include thoracic epidural injection, periganglionic nerve block, or intraganglionic injection for "chemical" targeted pulmonary denervation. In one embodiment, RTX is administered via the neck with a local ablative agent, accessing the vagus nerve low and away from the carotid sinus. Nerve location can then be confirmed using ultrasound guidance. In some embodiments, RTX is delivered periganglionally to the stellate ganglion. In some embodiments, RTX is delivered intraganglionally to the stellate ganglion.

[0067] In some embodiments, epidural or periganglionic injection of RTX in subjects with advanced COVID-19 disease supports palliative mechanical ventilation by ablating afferent nerves at the level of the DRG in the thoracic spinal cord, increasing survival.

[0068] In some embodiments, an effective amount of RTX results in a reduction of one or more cytokines, including IL-6, IL-1β, and / or IFNγ. In some embodiments, an effective amount of RTX results in improved pulmonary function (e.g., higher pO2 or sO2, or lower pCO2). In some embodiments, an effective amount of RTX results in reduced pulmonary edema. Such reduction or improvement may occur in a subject's condition prior to administration of RTX.

[0069] The methods described herein are for use in any subject in need of PD treatment in which RTX is effective, e.g., capable of binding to and activating TRPV1 or its homologs. In some embodiments, the RTX is administered at a dose of 0.1 to 100 μg. In some embodiments, the dose of RTX ranges from 0.1 to 0.5 μg, 0.5 to 1 μg, 1 to 2 μg, 2 to 5 μg, 5 to 10 μg, 10 to 20 μg, 20 to 30 μg, 30 to 40 μg, 40 to 50 μg, 50 to 60 μg, 60 to 70 μg, 70 to 80 μg, 80 to 90 μg, or 90 to 100 μg. In some embodiments, a two-point, three-point, or four-point periganglionic nerve block technique is used, with a total dose in any of the ranges listed above (e.g., a total dose of 0.5-1 μg, 1-2 μg, 2-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, or 20-25 μg).

[0070] The dosage can be adjusted depending on the proximity of the administration site to the nerve fiber.For example, when ultrasound or nerve stimulators are used to ensure that the administration site is very close to the nerve, a lower dosage and / or volume can be used.Alternatively, nerve block can be achieved using a larger volume to ensure contact with the desired nerve.Notably, RTX is specific to TRPV1 receptors, and therefore does not affect non-target nerves, such as motor neurons, that do not have sufficient TRPV1 receptors to be sensitive to RTX.

[0071] Numerous examples of formulations of RTX are available in the literature. For example, see Ueda et al. (2008) J. of Cardiovasc. Pharmacol. 51:513-520, and US 2015 / 0190509 A1. Any suitable formulation of RTX for parenteral administration (e.g., injection) can be used.

[0072] In some embodiments, RTX (which may be in the dosages discussed above) is administered with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises water. In some embodiments, the pharmaceutically acceptable carrier comprises polysorbate 80. In some embodiments, the pharmaceutically acceptable carrier comprises polyethylene glycol. In some embodiments, the pharmaceutically acceptable carrier comprises a sugar or sugar alcohol. In some embodiments, the pharmaceutically acceptable carrier comprises mannitol. In some embodiments, the pharmaceutically acceptable carrier comprises dextrose. In some embodiments, the pharmaceutically acceptable carrier comprises a pharmaceutically acceptable buffer. In some embodiments, the pharmaceutically acceptable carrier comprises a phosphate buffer. In some embodiments, the pharmaceutically acceptable carrier comprises a pharmaceutically acceptable salt. In some embodiments, the pharmaceutically acceptable carrier comprises NaCl. In some embodiments, the pharmaceutically acceptable carrier comprises an organic solvent (e.g., ethanol or DMSO) as a minor or remaining component used to aid in dissolving the RTX, e.g., prior to dilution in a primarily aqueous composition.

[0073] The concentration of RTX in the formulation can be any appropriate value for delivery of the intended dose. In some embodiments, the concentration of RTX in the pharmaceutical formulation is in the range of 0.1 to 300 μg / ml. In some embodiments, the concentration of RTX in the pharmaceutical formulation is in the range of 0.1 to 1 μg / ml, 1 to 5 μg / ml, 5 to 10 μg / ml, 10 to 20 μg / ml, 10 to 30 μg / ml, 20 to 30 μg / ml, 20 to 50 μg / ml, 50 to 100 μg / ml, 100 to 150 μg / ml, 150 to 200 μg / ml, 200 to 250 μg / ml, or 250 to 300 μg / ml. In some embodiments, the concentration of RTX in the pharmaceutical formulation is in the range of 5 to 50 μg / ml or 8 to 25 μg / ml.

[0074] Starting from a concentrated stock solution, a formulation of RTX for delivery to a subject can be prepared by dilution in an appropriate diluent (eg, saline).

[0075] The formulation may have any pH suitable for intra-articular administration. In some embodiments, the pharmaceutical formulation comprising RTX and a pharmaceutically acceptable carrier has a pH in the range of 6 to 7.6. In some embodiments, the pharmaceutical formulation comprising RTX and a pharmaceutically acceptable carrier has a pH in the range of 6 to 6.4, 6.3 to 6.7, 6.4 to 6.8, 6.8 to 7.2, 7 to 7.4, or 7.2 to 7.6. In some embodiments, the pharmaceutical formulation comprising RTX and a pharmaceutically acceptable carrier has a pH of 6.5 or 7.2.

[0076] In some embodiments, the formulation comprises polysorbate 80 and dextrose. In some embodiments, the polysorbate 80 concentration is 0.03 to 7% w / v. In some embodiments, the polysorbate 80 concentration is 2 to 4% w / v and / or the dextrose concentration is 4 to 6% w / v. In some embodiments, the polysorbate 80 concentration is 3% w / v and / or the dextrose concentration is 5% w / v. The formulation may further comprise a buffer (e.g., a phosphate buffer (e.g., a sodium phosphate buffer)). In some embodiments, the phosphate buffer concentration is 10 to 50 mM. In some embodiments, the phosphate buffer concentration is 10 to 30 mM. In some embodiments, the phosphate buffer concentration is 10 mM. In some embodiments, the phosphate buffer concentration is 30 mM. The formulation may have a pH in the range of 7 to 7.5 (e.g., about 7.2). In some embodiments, the concentration of RTX in any of the aforementioned formulations is 10 to 30 mcg / ml (e.g., 10 mcg / ml or 25 mcg / ml). In some embodiments, the formulation further comprises a phosphate buffer (e.g., at the concentration and pH shown for the phosphate buffer in Table 1). In some embodiments, the formulation further comprises NaCl (e.g., at the concentration shown for NaCl in Table 1). When both are present, the phosphate buffer and NaCl may (but are not necessarily) present at the combination of concentration and phosphate buffer pH shown for the particular formulation.

[0077] Exemplary formulations of RTX are shown in the table below.

[0078] [Table 1-1] [Table 1-2] [Table 1-3]

[0079] In some embodiments, the formulations in Table 1 contain dextrose. In some embodiments, the concentration of dextrose is 0.05-5% w / v. In some embodiments, the concentration of dextrose is 0.8-5% w / v. In some embodiments, the concentration of dextrose is 0.05% w / v. In some embodiments, the concentration of dextrose is 0.8% w / v. In some embodiments, the concentration of dextrose is 3.0% w / v. In some embodiments, the concentration of dextrose is 5.0% w / v.

[0080] In some embodiments, the formulations in Table 1 include mannitol. In some embodiments, the concentration of mannitol is 0.8-3.0% w / v. In some embodiments, the concentration of mannitol is 0.8% w / v. In some embodiments, the concentration of mannitol is 3.0% w / v.

[0081] In some embodiments, the dextrose or mannitol is omitted from the formulations shown in Table 1.

[0082] In some embodiments, the concentration of RTX in the formulation shown in Table 1 is adjusted to any of the RTX concentrations or concentration ranges disclosed herein. For example, in some embodiments, the concentration of RTX in the formulation shown in Table 1 is adjusted to 0.3 to 200 mcg / ml. In some embodiments, the concentration of RTX in the formulation shown in Table 1 is 200 mcg / ml. In some embodiments, the concentration of RTX in the formulation shown in Table 1 is 0.3 to 100 mcg / ml. In some embodiments, the concentration of RTX in the formulation shown in Table 1 is 100 mcg / ml. In some embodiments, the concentration of RTX in the formulation shown in Table 1 is adjusted to 0.3 to 50 mcg / ml. In some embodiments, the concentration of RTX in the formulation shown in Table 1 is 25 mcg / ml. As another example, in some embodiments, the concentration of RTX in the formulation shown in Table 1 is adjusted to 0.3 to 15 mcg / ml. As another example, in some embodiments, the concentration of RTX in the formulations shown in Table 1 is adjusted to 0.5 to 10 mcg / ml. As another example, in some embodiments, the concentration of RTX in the formulations shown in Table 1 is adjusted to 0.6 to 1.5 mcg / ml. The dextrose or mannitol is omitted from any such formulations having the adjusted RTX concentration.

[0083] The formulations in Table 1 can be prepared according to the following exemplary method. This is provided for formulations 3 and 5, but can be adapted to other formulations by those skilled in the art. Formulation 3 can be made by adding 46 mg sodium dihydrogen phosphate monohydrate, 94.7 mg disodium hydrogen phosphate anhydrous, and 860 mg NaCl to a 100 ml volumetric flask. 50 ml of water for injection (WFI) is added to dissolve the components in the flask, followed by 1.0 g of polysorbate 80 to form the aqueous component. 20 mg of RTX is added to the aqueous component in the volumetric flask, and the pH is adjusted to 7.2 with hydrochloric acid / sodium hydroxide. 30 mL of PEG 300 is then added, and the solution is sonicated to dissolve the solids. It should be noted that RTX sometimes precipitates at the interface of the aqueous solution and PEG initially, but returns to solution upon sonication. The complete mixture in the flask is diluted to the required volume (100.00 ml) with water (WFI) and mixed by an end-over-end mixing process. The complete formulation is filtered through a 0.2 μm polytetrafluoroethylene (PTFE) filter.

[0084] Formulation 5 can be made by adding 138 mg of sodium dihydrogen phosphate monohydrate, 284.1 mg of disodium hydrogen phosphate anhydrous, and 540 mg of NaCl to a 100 ml volumetric flask. 50 ml of water for injection (WFI) is added to dissolve the components in the flask, followed by 3.0 g of polysorbate 80 and 800 mg of dextrose to form the aqueous component. 20 mg of RTX is added to the aqueous component in the volumetric flask, and the pH is adjusted to 7.2 with hydrochloric acid / sodium hydroxide. The solution is then sonicated to dissolve all solids (alternatively, RTX can be first dissolved in a small amount of ethanol or DMSO, and then this solution can be added to the aqueous component). The complete mixture in the flask is diluted to a predetermined volume (100.00 ml) with water (WFI) and mixed by end-over-end mixing. The complete formulation is filtered through a 0.2 μm PTFE filter.

[0085] A formulation according to Formulation 11 is prepared using 200 mcg RTX, 300 mcg polysorbate 80 (commercially available polysorbate 80 is used); 5.4 mg sodium chloride, 500 mcg dextrose, 1.38 mg sodium phosphate monobasic monohydrate, 2.84 mg sodium phosphate dibasic anhydrous, and water (WFI) to 1 mL, then the pH is adjusted to 7.2 with hydrochloric acid / sodium hydroxide. As noted above, the dextrose may be omitted.

[0086] A formulation according to Formulation 13 is prepared using 25 mcg RTX, 30 mg polysorbate 80 (commercially available polysorbate 80 is used); 5.4 mg sodium chloride, 50 mg dextrose, 1.38 mg sodium phosphate monobasic monohydrate, 2.84 mg disodium phosphate dibasic anhydrous, water (WFI) to 1 mL, and then the pH is adjusted to 7.2 with hydrochloric acid / sodium hydroxide. As noted above, the dextrose may be omitted.

[0087] In some embodiments, the pharmaceutical preparation is in unit dosage form.In this form, the preparation is divided into unit doses containing appropriate amounts of active ingredients.The unit dosage form can be a packaged preparation (the package contains discrete amounts of the preparation, such as in a vial, an ampoule, or a pre-filled syringe).The unit dosage form can also be, for example, a liquid formulation or a lyophilized composition for reconstitution.

[0088] Further details on techniques for formulation and administration may be found in Gennaro, A., ed., Remington's Pharmaceutical Sciences, 18th ed. (1990) (Mack Publishing Co., Easton, Pa.).

[0089] In some embodiments, RTX can be administered as a single dose.In some embodiments, RTX is periodically administered.In some embodiments, RTX is periodically administered to the subject in need of pulmonary inflammatory disease treatment, if necessary, to reduce the severity of the disease.

[0090] The present invention provides a method for treating pulmonary inflammatory diseases, comprising administering RTX to a subject via epidural, periganglionic or intraganglionic injection.One embodiment provides a method for treating a mammalian subject suffering from ARDS.

[0091] In exemplary embodiments, RTX can be administered to reduce the patient's symptoms, or it can be administered to combat the mechanism of the disease itself. Those skilled in the art will understand that these therapeutic goals are often related, and that the treatment can be adjusted for each patient based on various factors. These factors include the patient's age, sex, or health condition, the progression of the pulmonary inflammatory disease, the degree of respiratory distress, the amount of tissue damage to the patient's airway, the patient's smoking history, and various environmental factors (e.g., temperature, humidity, and air pollution), which may contribute to the patient's condition. Patient treatment can be adjusted depending on the dosage, timing, and route of administration, and by administering other therapeutic agents simultaneously or sequentially. [Example]

[0092] 1. Resiniferatoxin (RTX) ameliorates acute respiratory distress syndrome (ARDS) in a rodent model of lung injury Respiratory failure due to ARDS is one of the major causes of mortality associated with acute lung injury (ALI), including COVID-19. ALI / ARDS can be associated with acute cytokine release, pulmonary edema, and, in the long term, fibrosis. The mechanisms underlying these pathological changes are not fully understood. In Example 1, a novel neural component via cardiopulmonary spinal afferent fibers that mediates pulmonary pathology during ALI / ARDS was examined.

[0093] Sensory neurons that innervate the heart and lungs enter the central nervous system by one of two routes: via the vagus nerve to the brainstem (medulla oblongata), where their cell bodies reside in the inferior ganglia, or directly to the spinal cord, where their cell bodies reside in the dorsal root ganglia (DRG). Afferent fibers are composed of elements that respond to various sensory modalities, such as mechanical deformation, heat, cold, pH, and inflammatory mediators. The reflex effects following stimulation of these afferent fibers depend on the type of stimulus and the neural pathway involved. Activation of vagal afferent pathways tends to be sympathoinhibitory and anti-inflammatory (Komeage et al. (2018) Brain, Behavior, and Immunity 73:441-449; Bonaz et al. (2016) The Journal of Physiology 594:5781-5790). On the other hand, activation of spinal afferent fibers tends to be sympathoexcitatory and proinflammatory (Shanks et al. (2019) Hypertension 74:910-920; Shanks et al. (2018) Physiological Reports 6:313-742; Alawi et al. (2010) Pharmacol. Ther. 125:181-195; Lazar et al. (2018) Pancreas 47:110-115; Abdulla et al. (2017) Acta Physiol (Osf) 220:404-416; Wang et al. (2017) The Journal of Physiology 595:2519-2534). We hypothesized that ablation of pulmonary afferent innervation (thoracic spinal cord) by application of RTX, an ultrapotent selective afferent neurotoxin, would modify the pathological process, including pulmonary edema and local pulmonary inflammation, associated with progressive ALI.

[0094] method A rat model of lung injury was performed. Rats were randomized into three groups and evaluated 1 week after instillation as follows: sham rats, bleomycin (Bleo)-exposed rats with saline (epidural or stellate ganglion injection), and Bleo-exposed rats with RTX (epidural or intrastellate ganglion injection). Bleo (2.5 mg / kg, approximately 0.15 mL) was instilled intratracheally into the lungs under 3% isoflurane anesthesia. Sham control rats received intratracheal instillation of saline. Animals were treated with RTX or vehicle (Veh; phosphate-buffered saline) via either the epidural T1-T4 DRG route (6 μg / ml, 10 μl / ganglion) or intrastellate ganglion administration (50 μg / ml, 5 μl / side) 3 days after Bleo delivery (Figure 1A-B).

[0095] Epidural application of RTX. Upper thoracic spinal cord afferent fibers were ablated by epidural application of RTX. The epidural administration procedure was essentially as described by Shanks et al. (2018) Physiological Reports 6:e13742. Briefly, rats were anesthetized using a 2%-3% isoflurane:oxygen mixture. The rat was placed in the prone position, and a small midline incision was made in the region of the T13-L1 thoracic vertebrae. After incision of the superficial muscles, two small holes (approximately 2 mm x 2 mm) were made on the left and right sides of the T13 thoracic vertebra. A polyethylene catheter (PE-10) was inserted into the subarachnoid space through one of the holes and gently advanced approximately 4 cm, approaching the T1 level. The upper thoracic sympathetic afferent ganglion was ablated by injecting resiniferatoxin (RTX; Sigma-Aldrich) (a highly potent agonist of the TRPV1 receptor) into the subarachnoid space via a catheter. RTX (1 mg; Sigma-Aldrich) was dissolved in a 1:1:8 mixture of ethanol, Tween® 80 (Sigma-Aldrich), and isotonic saline. The initial injection of RTX (6 μg / ml, 10 μl) was performed at a very slow rate (approximately 1 minute) to minimize drug diffusion. The catheter was then retracted to T2, T3, and T4, respectively, and a series of injections (10 μl each) were performed at each segment. The catheter was then withdrawn, and the same injection was repeated on the other side. The foramen at T13 thoracic vertebra was sealed using silicone gel. The skin over the muscle was closed with 3-0 polypropylene simple interrupted sutures and betadine was applied to the wound. For post-procedural pain control, buprenorphine (0.05 mg / kg) was injected subcutaneously immediately after surgery and twice daily for 2 days.

[0096] Intrastellate ganglion injection of RTX. Rats were anesthetized with a 2%–3% isoflurane:oxygen mixture. After cannulation of the trachea, artificial respiration was initiated (Model 683, Harvard Apparatus, South Natick, MA). The skin was incised from the rostral end of the sternum to the level of the third rib. Portions of the superficial and deep pectoral muscles and the first intercostal muscle were cut and dissected. To locate the left or right stellate ganglion, the left or right superior vena cava was separated laterally from the brachiocephalic trunk with a hooked glass or steel rod to expose the internal thoracic artery, the descending branch of the right subclavian artery, and the costocervical artery. The stellate ganglion and subclavian snare were positioned medial to the origin of the internal thoracic artery and the costocervical artery. RTX (5 μl, 50 mg / ml) was then injected bilaterally into the ganglia over 30 seconds using a 5 μl Hamilton syringe (Microliter #95, Hamilton, Reno, NV, USA). Images of this procedure are shown in Figure 2A-B. After these maneuvers, the rib cage between the first and second intercostal spaces was closed with continuous 4-0 Dexon II coated braided polyglycolic acid absorbable sutures, and the skin was closed with 3-0 polypropylene sutures and retracted from the chest. Betadine was applied to the wound, and the rat was allowed to recover from anesthesia. For postprocedural pain management, buprenorphine (0.05 mg / kg) was injected subcutaneously immediately after surgery and twice daily for 2 days.

[0097] Blood gas analysis. The artery on the ventral aspect of the rat tail was used to collect a small amount of blood (approximately 0.1 mL) for arterial blood gas analysis 7 days after Bleo treatment. The animals were restrained in a commercially available restrainer to allow access to their tails. The tail was aseptically prepared by alternating alcohol and iodine prep pads three times, and the artery was punctured using a 24G needle. A small amount of blood (approximately 0.1 mL) was gently aspirated into a syringe for blood gas analysis (iSTAT, Abbott, Chicago, After sample collection, the needle was removed and a gauze swab was applied firmly to the puncture site to stop bleeding.

[0098] Cytokine assay. Lung and plasma cytokines were measured with R&D cytokine ELISA assay (Minneapolis, MN, USA) according to the manufacturer's instructions. Organ weights were assessed postmortem.

[0099] Pulmonary plasma extravasation, tissue extraction, and quantification of Evans Blue. Rats were anesthetized with pentobarbitone (40 mg / kg). Evans Blue, 20 mg / kg (10 mg / ml dissolved in saline + 100 IE / ml heparin) was administered intravenously. Ten minutes later, rats were euthanized by transcardial perfusion with PBS (0.01 M, pH 7.4). Lungs were harvested and initially photographed. Lung samples were then immediately weighed, placed in 2 ml of N,N'-dimethylformamide, cut into small pieces, and heated overnight in a 50°C water bath. The lung tissue was then centrifuged (1 min, 14,000 rpm), and the lung Evans Blue content in the supernatant was analyzed using a 96-well microplate reader (infinite M200, TECAN, Mannedorf, CH, Switzerland). and ) at 620 nm (100 μl sample / well). Evans Blue extravasation was expressed as Evans Blue / g lung tissue by comparing experimental values ​​with known standards.

[0100] Statistics. Statistical evaluation was performed using GraphPad Prism (GraphPad Software, San Diego, CA. Version 8). Differences between treatments were determined using a mixed effects model for repeated measures ANOVA. For comparisons between the three groups (sham, Bleo+Veh, and Bleo+RTX experiments), both Tukey's and Bonferroni's corrections for multiple comparisons were used.

[0101] result Plasma extravasation (Evans Blue) was used to assess vascular permeability after ALI. As shown in Figure 3A-C, Bleo-treated lungs showed widespread Evans Blue areas on both sides. The highest intensity of Evans Blue was observed on the medial side of each lung. The Evans Blue areas were reduced after epidural RTX treatment at 7 days after Bleo administration.

[0102] Three pro-inflammatory tissue cytokines prevalent in the lungs after Bleo treatment are shown in Figures 4A-C. IL-6 (Figure 4A), IL-1β (Figure 4B), and IFNγ (Figure 4C) were elevated after Bleo treatment. These cytokine levels were reduced in RTX-treated rats.

[0103] Cytokine levels in response to Bleo were also reduced after epidural application of RTX (Fig. 5A-C).

[0104] Plasma extravasation in response to Bleo was reduced after stellate ganglion injection of RTX. As shown in Figures 6A-D, Evans blue dye was significantly reduced in the lungs after stellate ganglion injection of RTX.

[0105] Arterial blood gas data were assessed in rats treated with Veh versus RTX (Figures 7A-H). The results show increased pCO2 (Figure 7B) and decreased pO2 (Figure 7C) as well as sO2 (Figure 7G) in Bleo- and Veh-treated rats. Stellate ganglion administration of RTX revealed these changes, suggesting improved pulmonary function and gas exchange.

[0106] 8A-B show that IL-6 (FIG. 8A) and IL-1β (FIG. 8B) levels in lung tissue were significantly reduced after stellate ganglion RTX administration.

[0107] Figures 9A-H show body weight (BW) and individual organ weights between groups. Compared with sham rats, lung wet weight (WLW) and the ratio of WLW to BW were significantly higher in Bleo- and Veh-treated rats, which were significantly reduced by intrastellate ganglion injection of RTX. The data suggest that intrastellate ganglion injection of RTX reduces pulmonary edema after Bleo treatment.

[0108] These data demonstrate that ablation of TRPV1 afferent sensory fibers in the presence of ALI using RTX delivered by either of two different routes targeting cardiopulmonary afferent fibers results in a rapid reduction in pulmonary microvascular permeability and a reduction in tissue and plasma inflammatory markers. Although pulmonary function was not directly measured in this series of experiments, arterial blood gas data suggest improved gas exchange. The improved body weight and reduced lung weight in rats with lung injury after stellate ganglion administration of RTX suggest potential clinical benefits from reduced pulmonary edema and protective effects on non-pulmonary organs that are otherwise affected by the induced systemic inflammatory process in the lung.

[0109] The lungs are innervated by a dual sensory system that includes vagus and spinal afferent fibers. Both vagus and spinal afferent fibers consist of axons of group A fibers (high conduction velocity) and group C fibers (slow conduction velocity). These fibers and their sensory nerve endings are connected to the classical Na + , K. + and Ca 2+TRPV1 neurons express various membrane receptors that mediate ion channel function (both voltage-gated and ligand-gated), including ATP channels. A strategy to modulate the pathological effects of TRPV1 afferent neurons has been developed. RTX, an ultrapotent neurotoxin, binds strongly to TRPV1 receptors. Upon activation, TRPV1 channels become highly permeable to calcium (Hsu et al. (1985) Journal of Applied Physiology 118:1533-1543; Brown et al. (2015) Pain 156:1018-1024). After initial stimulation, high intracellular calcium levels mediate inhibition of neuronal function. Site-specific delivery of RTX can be used to intervene in a variety of conditions, alleviating pain, inflammation, fibrosis, and plasma extravasation. RTX-induced depletion of TRPV1 afferents has been shown to block afferent-restricted neuropeptide release and reduce inflammatory pain (Karai et al. (2004) The Journal of Clinical Investigation 113:1344-1352). Cardiopulmonary spinal nerve afferent fibers can also be targeted with RTX by either application to the epidural space at the level of the T1-T4 thoracic vertebrae (with some spreading to higher and lower segments) or injection into the stellate ganglion. While the DRG is considered exclusively sensory in nature, the stellate ganglion contains the cell bodies of sympathetic efferent fibers and the transit fibers of thoracic spinal afferent fibers as they pass through the DRG and enter the spinal cord. It should be noted that in humans, the stellate ganglion can be easily identified, and this type of percutaneous procedure can be performed with fluoroscopic or ultrasound guidance (intraganglionic or nerve "block" approach). Furthermore, intrastellate ganglion injections require only small volumes (10 μl for bilateral injections), which reduces the risk of systemic absorption of RTX and allows higher doses of RTX to be used for local injections.

[0110] The complete disclosures of all publications cited herein are hereby incorporated by reference in their entirety, as if each were individually indicated and incorporated herein by reference.

[0111] Various modifications and alterations to the embodiments disclosed herein will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Illustrative embodiments and examples are provided by way of example only and are not intended to limit the scope of the invention. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for treating a pulmonary inflammatory disease, the method comprising administering an effective amount of resiniferatoxin (RTX) epidurally, periganglionally, or intraganglionally to a subject in need of treatment for the pulmonary inflammatory disease. (Item 2) A composition comprising resiniferatoxin (RTX) for use in a method of treating a subject in need of treatment for a pulmonary inflammatory disease. (Item 3) 3. The composition for use according to item 2, wherein the method comprises administering the composition epidurally, periganglionally, or intraganglionally to the subject. (Item 4) 4. The method according to item 1 or the composition for use according to item 2 or 3, wherein the effective amount of RTX results in a reduction of one or more cytokines including IL-6, IL-1β and / or IFNγ. (Item 5) The method or composition for use according to any one of the preceding items, wherein the effective amount of RTX results in improved lung function. (Item 6) The method or composition for use according to any one of the preceding items, wherein the effective amount of RTX results in reduced pulmonary edema. (Item 7) The method or composition for use according to any one of the preceding items, wherein the subject is an adult. (Item 8) The method or composition for use according to any one of the preceding items, wherein the RTX is administered at a dose of about 0.1 μg to about 100 μg. (Item 9) Item 9. The method or composition for use according to Item 8, wherein the dose is about 0.1 μg to about 1 μg, about 1 μg to about 5 μg, about 5 μg to about 10 μg, about 10 μg to about 20 μg, about 20 μg to about 50 μg, or about 50 to about 100 μg. (Item 10) The method or use of any one of the preceding items, wherein the method comprises epidural administration. Composition for. (Item 11) 10. The method or composition for use according to any one of items 1 to 9, wherein the method comprises a periganglionic nerve block. (Item 12) 10. The method or composition for use according to any one of items 1 to 9, wherein the method comprises intraganglionic administration. (Item 13) The method or composition for use according to any one of the preceding items, wherein the RTX is administered in a pharmaceutical formulation comprising the RTX and a pharmaceutically acceptable carrier. (Item 14) Item 14. The composition for the method or use according to item 13, wherein the pharmaceutically acceptable carrier comprises water. (Item 15) Item 14. The composition for the method or use according to item 13, wherein the pharmaceutically acceptable carrier comprises saline. (Item 16) 16. The method or composition for use according to any one of items 13 to 15, wherein the RTX is present in the pharmaceutical formulation at a concentration ranging from 1 μg / ml to 100 μg / ml. (Item 17) Item 17. The method or composition for use according to item 16, wherein the RTX is present in the pharmaceutical formulation at a concentration ranging from 1 μg / ml to 5 μg / ml, 5 μg / ml to 10 μg / ml, 10 μg / ml to 20 μg / ml, 20 μg / ml to 50 μg / ml, or 50 μg / ml to 100 μg / ml. (Item 18) Item 11. The method or composition of any one of the preceding items, wherein the pulmonary inflammatory disease is selected from the group consisting of acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), pulmonary arterial hypertension (PAH), chronic inflammatory lung disease, pulmonary fibrosis, pulmonary vasculitis, pulmonary sarcoidosis, inflammation and / or infection associated with lung transplantation, acute or lung rejection and / or dysfunction, bronchitis, sinusitis, asthma, cystic fibroma, bacterial infection, fungal infection, parasitic infection, viral infection, bronchiolitis obliterans syndrome (BOS), primary ciliary dyskinesia (PCD), pulmonary alveolar proteinosis, idiopathic pulmonary fibrosis (IPF), eosinophilic pneumonia, eosinophilic bronchitis, inflammation and / or infection associated with mechanical ventilation, ventilator-associated pneumonia, asbestos-related airway disorder or disease, dust-related airway disorder or disease, silicosis, and radiation- or chemical-related airway disease or disorder, and any combination thereof. (Item 19) The method or composition of any one of the preceding items, wherein the pulmonary inflammatory disease is acute respiratory distress syndrome (ARDS). (Item 20) The method or composition of any one of the preceding items, wherein the pulmonary inflammatory disease is chronic obstructive pulmonary disease (COPD). (Item 21) The method or composition of any one of the preceding items, wherein the pulmonary inflammatory disease is pulmonary arterial hypertension (PAH). (Item 22) 23. The method or composition of claim 22, wherein the pulmonary inflammatory disease is inflammation and / or infection associated with mechanical ventilation and / or ventilator-associated pneumonia. The method or composition of any one of the preceding items, wherein the pulmonary inflammatory disease is associated with COVID-19.

Claims

[Claim 1] The invention described in the specification.

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