Treatment of COVID-19-related pneumonia by administering resiniferatoxin

Abstract

Disclosed is a method for treating a pulmonary inflammatory disease, 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 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 based on U.S. Provisional Application No. 63 / 002,165, filed Mar. 30, 2020, and U.S. Provisional Application No. 63 / 122,858, filed Dec. 8, 2020, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Throughout this application, various publications, patents, and / or patent applications are referenced. The disclosures of the above - mentioned publications, patents, and / or patent applications are incorporated herein by reference in their entirety to more fully describe the state of the art to which this disclosure pertains.

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

Background Art

[0004] Background RTX acts as a super - potent analog of capsaicin, the main component of chili peppers that stimulates the tongue and nose. RTX is a tricyclic diterpene isolated from certain species of Euphorbia. The homovanillyl group is an important structural feature of capsaicin and the most prominent feature that distinguishes resiniferatoxin from typical phorbol - related compounds. Natural RTX has the following structure:

Chemical formula

[0005] RTX and its analog compounds (e.g., thinyatoxin and other compounds such as 20-homovannillyl esters of diterpenes, including 12-deoxyphorbol 13-phenylacetate 20-homovannillate and mezelein 20-homovannillate) are described in U.S. Patents No. 4,939,194; No. 5,021,450; and No. 5,232,684. Other phoroid vanilloids of the resiniferatoxin type 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)) and is a multi-mercation 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 at nerve terminals 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 calcium channel opening and transmission of pain sensation (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 triggers a cascade of events that open calcium channels and lead 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 ventilator support, and an ICU mortality rate of 79% has been reported (Huang et al. (2020) Lancet Vol. 394, Issue 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, which can be mild or fatal. Coronaviruses belong to 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 bovine torovirus (Breda virus). Coronaviruses are enveloped viruses with a single-stranded positive-sense RNA genome and a helical nucleocapsid. The genome size of coronaviruses ranges from approximately 26 to 32 kilobases, which is considered the largest for an RNA virus. It is worth noting that the pneumonia outbreak in Wuhan, China in 2019-2020 was traced back to a novel coronavirus that causes coronavirus disease 2019, or COVID-19, which was called 2019-nCoV by the World Health Organization (WHO) and is also known as SARS-CoV-2.

[0009] ARDS, first described in 1967 (Ashbaugh et al. (1967) Lancet 2:319-323), is characterized by diffuse pulmonary microvascular injury resulting in increased permeability and hypoxemia caused by intrapulmonary shunts. The first two stages of ARDS progression (i.e., 12–72 hours after onset) are the most critical timeframe 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 a diagnosis of sepsis, aspiration of gastric contents, multiple transfusions, severe fracture, burns, pancreatitis, or severe trauma. As ARDS progresses to the third stage, pulmonary hypertension increases, heart rate increases to compensate for hypoxemia, and mechanical ventilation is generally required. Pathologically, cellular infiltrations become denser with continued neutrophil infiltration and an increase in mononuclear, lymphocyte, and fibroblast infiltrations. The severity of the above-mentioned 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). On the other hand, 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, the respiratory distress described above progresses very rapidly, and the time to reported death is as short as 14 days from the first symptom, even with the availability of palliative support with mechanical ventilation. Severity and mortality in susceptible populations infected with COVID-19 have been reported to be associated with cytokine storms, in which the aggravated production of pro-inflammatory substances is released into the pulmonary microenvironment over a short period of time (Mehta et al. (2020) Lancet Vol. 395, Issue 10229, P1033-1034). Novel life-saving strategies are urgently needed to mitigate the high mortality rates associated with acute respiratory distress (including such distress linked to 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 Problems]

[0012] Abstract The present disclosure provides a method for treating a pulmonary inflammatory disease, comprising administering an effective amount of resiniferatoxin (RTX) by epidural, periganglionic or intraganglionic administration. In some embodiments, the dose of RTX for an adult is from 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, periganglionically or intraganglionically to a subject having a need for treatment of a pulmonary inflammatory disease.

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

[0015] Embodiment 3 is the composition for use according to Embodiment 2, wherein the method comprises administering the composition epidurally, periganglionically or intraganglionically to the subject.

[0016] Embodiment 4 is a composition for use according to Embodiment 1 or 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 the method or use of any one of the above embodiments, wherein the effective amount of RTX produces improved lung function.

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

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

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

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

[0022] Embodiment 10 is a composition for the method or use of any one of the above embodiments, the method comprising epidural administration.

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

[0024] Embodiment 12 is a composition for the method or use described in any one of Embodiments 1 to 9, wherein the method includes intraganglionic administration.

[0025] Embodiment 13 is a composition for the method or use according to any one of the above 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 described in Embodiment 13, wherein the pharmaceutically acceptable carrier comprises water.

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

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

[0029] Embodiment 17 is a composition for use according to Embodiment 16, wherein the RTX is present in the pharmaceutical preparation at concentrations 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 above embodiments, wherein the inflammatory lung 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 (BOS), primary ciliary dysplasia (PCD), alveolar proteinosis, idiopathic pulmonary fibrosis (IPF), eosinophilic pneumonia, eosinophilic bronchitis, inflammation and / or infection associated with mechanical ventilation, ventilator-associated pneumonia, asbestos-related airway disorders or diseases, dust-related airway disorders or diseases, silicosis, and radiation or chemical-related airway diseases or disorders, and any combination thereof.

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

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

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

[0034] Embodiment 22 is the method or composition according to any one of the above embodiments, wherein the inflammatory pneumonia is an inflammation and / or infection associated with mechanical ventilation, and / or ventilator-associated pneumonia.

[0035] Embodiment 23 is a method or composition according to any one of the above embodiments, wherein the pneumonic inflammatory disease is related to COVID-19. [Brief explanation of the drawing]

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

[0037] [Figure 2A-2B] Figures 2A and 2B show the procedure for stellate ganglion isolation and administration of Veh or RTX. Figure 2A shows step 1 of the procedure described above - the stellate ganglion was exposed. The arrow indicates that the stellate ganglion was positioned medially towards the origins 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 lung images 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 compared to control. ##P<0.01 compared to Bleo.

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

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

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

[0042] [Figures 7A-7D] Figures 7A-H show arterial blood gases at day 7 in rats treated with sham, Bleo+Veh, and Bleo+RTX after intrastellate ganglion administration on day 3 post-injury. Figure 7A shows pH. Figure 6B shows partial carbon dioxide pressure (PCO2). Figure 7C shows partial oxygen 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 for sham. †P<0.05 for Bleo+Veh. [Figure 7E-7H]Figures 7A-H show arterial blood gases at day 7 in rats treated with sham, Bleo+Veh, and Bleo+RTX after intrastellate ganglion administration on day 3 post-injury. Figure 7A shows pH. Figure 6B shows partial carbon dioxide pressure (PCO2). Figure 7C shows partial oxygen 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 for sham. †P<0.05 for Bleo+Veh.

[0043] [Figure 8A-8B] Figures 8A and 8B show the cytokine levels in lung tissue on day 7 after administration of Veh or stellate ganglion RTX 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 the heart. Figure 9C shows the lungs. Figure 9D shows the spleen. Figure 9E shows the liver. Figure 9F shows the kidneys. Figure 9G shows the heart / BW ratio. Figure 9H shows the lung / BW ratio. Compared to sham rats, the wet weight (WLW) of the lungs and the WLW to BW ratio were significantly higher in Bleo+Veh rats, which were significantly reduced by intrastellate ganglion injection of RTX. These data suggest that intrastellate ganglion injection of RTX reduces pulmonary edema after Bleo. [Figure 9E-9H] Figures 9A–H show body weight (BW) and individual organ weights between groups. Figure 9A shows body weight. Figure 9B shows the heart. Figure 9C shows the lungs. Figure 9D shows the spleen. Figure 9E shows the liver. Figure 9F shows the kidneys. Figure 9G shows the heart / BW ratio. Figure 9H shows the lung / BW ratio. Compared to sham rats, the wet weight (WLW) of the lungs and the WLW to BW ratio were significantly higher in Bleo+Veh rats, which were significantly reduced by intrastellate ganglion injection of RTX. These data suggest that intrastellate ganglion injection of RTX reduces pulmonary edema after Bleo. [Modes for carrying out the invention]

[0045] Detailed explanation Herein, specific embodiments of the present invention are referenced in detail. Examples of such embodiments are illustrated in the accompanying drawings. While the present invention is described in conjunction with the illustrated embodiments, it should be understood that they are not intended to limit the invention to those embodiments. In contrast, the present invention is intended to encompass all possible substitutions, modifications, and equivalents that may be included within the invention as defined by the accompanying claims.

[0046] Before describing this instruction in detail, it should be understood that this disclosure is not limited to any particular composition or process, for such things can vary. It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include references to the plural unless the context clearly indicates otherwise. Thus, for example, a reference to “a conjugate” includes multiple conjugates, and a reference to “a cell” includes multiple cells. The use of options herein (for example, “or”) is understood to mean any one or both of those options or any combination thereof.

[0047] As used herein, the terms "and / or" should be understood to mean the specific disclosure of each identified feature or component, with or without the others. For example, when the term "and / or" is used herein in a phrase such as "A and / or B," it is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when the term "and / or" is used in a phrase such as "A, B, and / or C," it is intended to include each of the following situations: 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); B (alone); and C (alone).

[0048] Where used herein, the terms “comprising,” “including,” “having,” and “containing,” as well as their grammatical variations, are intended to be non-limiting, so as to whether one or more items in a list exclude other items, and such other items may be used in place of or in addition to the listed items. Wherever a circumstance is described herein with the word “comprising,” it is understood that other similar circumstances are also provided, which are described with respect to “consisting of” and / or “consisting essentially of.”

[0049] As used herein, the term “about” means a particular value or composition that is within the acceptable margin of error of that value or composition as determined by those skilled in the art, the margin of error depending in part on how the value or composition is measured or determined (i.e., the limits of the measuring system). For example, “about” or “approximately” may mean within 1 or a standard deviation greater than 1 for the practice of the art. Alternatively, “about” or “approximately” may mean a range of up to 10% (i.e., ±10%) or greater, depending on the limits of the measuring system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may mean up to one decimal place or up to five times the value. Where 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 the acceptable margin of error of that particular value or composition. In some embodiments, “about” includes variations of 10%, 5%, 2%, 1%, or 0.5% or less of the stated value.

[0050] A numerical range includes the digits that define that range. It is understood that the measured and measurable values ​​are approximations, taking into account significant figures and errors associated with the measurement. Furthermore, all ranges should be interpreted as including the endpoints unless there is an exclusionary expression such as "not including the endpoints"; therefore, for example, "ranging from 1 to 10" includes the values ​​1 and 10, and all digits 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,” and “including” is not intended to be limiting. It should be understood that the above general and detailed descriptions are both illustrative and descriptive, and not limiting to the teachings. Unless specifically noted in the above specification, embodiments in this specification that describe “comprise” various components are also intended to “consist of” or “essentially consist of” those components; embodiments in this specification that describe “consist of” various components are also intended to “contain” or “essentially consist of” those components; and embodiments in this specification that describe “essentially consist of” various components are also intended to “consist of” or “contain” those components (this interchangeability does not apply to the use of these terms in the claims).

[0052] The section headings used herein are for structural purposes only and should not be construed as limiting the desired subject matter in any way. In the event of any conflict between any reference cited herein and any term defined herein, this specification shall prevail. While this instruction is described with various embodiments, it is not intended to be limited to such embodiments. In contrast, this instruction includes various substitutions, modifications, and equivalents as will be understood by those skilled in the art.

[0053] definition As used herein, “pulmonary inflammatory disease” is used collectively to refer to acute and chronic pathological conditions associated with inflammatory processes. Non-exclusive examples of pulmonary inflammatory disease include acute respiratory distress syndrome (ARDS), pneumonia, pneumonitis, bronchitis, lung infections, neonatal atelactasis, and conditions associated with inflammatory lung injury (e.g., chemotherapy agent (e.g., bleomycin)-induced lung injury, pancreatitis-induced lung injury, oxygen overload-induced lung injury, amiodarone-induced pneumonitis, radiation pneumonitis, injury from chlorine gas or smoke inhalation, bronchiolitis obliterans / obstructive pneumonia (BOOP), viral and mycoplasma pneumonia (e.g., Legionella and CMV lung), pneumoconiosis, pulmonary vasculitis, pulmonary sarcoidosis, respiratory bacterial infections, respiratory fungal infections, respiratory parasitic infections, respiratory viral infections, ventilator-associated inflammation and / or infections, and ventilator-associated pneumonia). Non-limiting examples of chronic pathological conditions of the lungs include chronic obstructive pulmonary disease (COPD), pulmonary arterial hypertension (PAH), cystic fibroma, silicosis, asbestosis, asthma, atherosclerosis, chronic bronchitis, chronic inflammation resulting from chronic bacterial or viral infections, coronary artery disease, idiopathic pulmonary fibrosis (IPF), familial pulmonary fibrosis (FPF), desquamative interstitial pneumonitis (DIP), hypersensitivity pneumonitis, interstitial pneumonitis, collagen vascular disease, sarcoidosis, coal miner's pneumoconiosis, bronchopulmonary dysplasia, and inflammatory pseudotumor.

[0054] As used herein, “epidural administration” means the delivery of a drug or pharmaceutical preparation into the epidural space (also known as the “extradural space” or “peridural space”), which is the outermost part of the spinal canal. It is the space within the spinal canal (formed by the surrounding vertebrae) that lies outside the dura mater (which encloses the arachnoid membrane, subarachnoid space, cerebrospinal fluid, and spinal cord). For example, epidural administration may include delivery to the epidural space without direct injection into nerves, or it may include epidural delivery to nerve tissue.

[0055] As used herein, “peri-ganglionic administration” means the delivery of a drug or pharmaceutical preparation into the space surrounding a ganglion.

[0056] "Intra-ganglionic administration" refers to administration into the ganglia. Intra-ganglionic administration can be achieved by direct injection into the ganglia and also includes selective nerve root injection (where the compound passes through the connective tissue sleeve surrounding the nerve and enters the ganglia from 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 may refer to an amount of therapeutic agent sufficient to have a measurable effect on the improvement or prevention of a pneumonic inflammatory disease when administered to a subject. For example, an effective dose may improve pulmonary function, expressed as partial pressure of CO2 (pCO2), partial pressure of O2 (pO2), and oxygen saturation (sO2) when measured in arterial blood. In another example, an effective dose may reduce pulmonary edema. The therapeutically effective amount of therapeutic agents provided herein, when used alone or in combination with antiviral agents, will vary depending on the relative activity of the therapeutic agent and on the subject being treated and the disease state, the subject’s weight, age, and sex, the severity of the disease state in the subject, the mode of administration, etc., which can be readily determined by those skilled in the art. In one embodiment, the therapeutically effective amount will depend on a particular aspect of the subject being treated and the disorder being treated, which can be determined by those skilled in the art using known techniques. Furthermore, as is well known in the field, adjustments may be necessary regarding age, weight, general health status, sex, diet, administration time, drug interactions, and disease severity.

[0058] The terms “subject” and “patient” as used herein mean human and non-human animals (including vertebrates, mammals and non-mammals). In one embodiment, the subject may be a human, a non-human primate, a monkey (simian), an ape, a rodent (e.g., mouse and rat), a cattle, a pig, a horse, a dog, a cat, a goat, a wolf, a ranine, or a piscine.

[0059] The terms “administering,” “administered,” and their grammatical variations refer to the physical introduction of a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration (e.g., by injection or infusion). As used herein, “parenteral administration” means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In one embodiment, the formulation is administered via a non-parenteral route (e.g., orally). Other non-parenteral routes of administration include topical, cutaneous, or mucosal routes of administration (e.g., intranasal, vaginal, rectal, sublingual, or topical). Administration may also be, for example, one dose, multiple doses, and / or over one or more extended periods.

[0060] "Treatment" should be understood broadly and encompass any beneficial effect, including, for example, delaying, slowing the progression of, or halting symptoms associated with inflammatory pneumonia, or remedying such symptoms, at least in part. Treatment also encompasses bringing about any form of improved patient function, as will be discussed in detail below. In some embodiments, treatment also means prolonging survival compared to the survival expected if treatment is not received. Those in need of treatment include individuals who already have the disease or disorder, as well as those who are prone to developing the disease or disorder, or those who should be prevented from developing the disease or disorder.

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

[0062] The 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 leading to an over-inflammatory state. Patient mortality is suspected to be related to a virus-driven cytokine storm similar to that seen in SARS-CoV-2 infection. A 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 the plasma. ICU patients have higher plasma levels of IL-2, IL-7, IL-10, GSCF, IP10, MCP1, MIP1A, and TNFα compared to non-ICU patients. This suggests that the presence of high circulating cytokine levels is associated with disease severity. Therefore, it is necessary to appropriately control the diverse aspects of this inflammatory process by interfering with the inflammatory cascade at a higher level (i.e., by eliminating pro-inflammatory centrifugal pathways).

[0063] Underlying physiological events related to disease prevalence, disease severity, and mortality may be explained by the involvement of TRPV1-expressing nervous systems (afferent / efferent neurons). TRPV1-positive pathways are responsible for pain transmission, inflammation, and immunomodulation throughout the pulmonary system.

[0064] Afferent innervation of the pulmonary system is primarily carried out by the vagus nerve and its branches. TRPV1-expressing C fibers are small-diameter, unmyelinated fibers in the vagus nerve that are responsible for several processes in the airways and lungs. Afferent fibers innervating the pulmonary structures are also transmitted by sympathetic nerve fibers associated with cell bodies located in the dorsal root ganglia of the thoracic spinal segments between T1 and T6. Activation of these thoracic spinal segments 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 ganglia (DRG), spinal dorsal horn (DH), or peripheral nerve terminals when applied topically as a nerve block. The potent binding of RTX to the TRPV1 receptor opens the channel gate, resulting in a slow, sustained increase in intracellular Ca2+, which then disrupts intracellular mitochondrial metabolism, leading to the removal of nerve cells or nerve fibers within minutes. We have discovered the therapeutic use of RTX (an ultrapotent TRPV1 agonist) as an ablating agent for the TRPV1-positive pulmonary pathway in patients with acute inflammatory lung disease. Such a therapeutic approach targeting TRPV1-expressing neurons in the lung modulates inflammatory and immune signaling activity, resulting in reduced mortality and better overall outcomes.

[0066] Compositions and related methods for illustrative use Methods for treating inflammatory pneumonia and compositions for use in treating such conditions are provided herein, in which RTX is delivered epidurally, perigantinely via nerve block, or intraganglionically. In various embodiments, routes of administration of an ablative agent such as RTX include epidural injection into the thoracic spinal cord for "chemical" targeted denervation, perigantine nerve block, or intraganglionic injection. In one embodiment, RTX is administered by traversing the neck with a local ablative agent, descending and moving away from the carotid sinus to access the vagus nerve. The location of the nerve may then be confirmed using ultrasound guidance. In some embodiments, RTX is delivered perigantinely to the stellate ganglion. In some embodiments, RTX is delivered intraganglionically 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 dissecting afferent nerves at the level of the thoracic spinal cord's DRG, thereby increasing survival.

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

[0069] The methods described herein are for use in any subject for whom RTX is effective, for example, in which it can bind to and activate TRPV1 or its homolog, and which requires treatment for PD. In some embodiments, the RTX is administered in doses 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 for the total dose within any of the ranges listed above (e.g., total doses 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 above dosage may be adjusted depending on the proximity of the injection site to the nerve fiber. For example, lower doses and / or volumes may be used if ultrasound or neurostimulants are used to ensure that the injection site is very close to the nerve. Alternatively, nerve blocks may be achieved using larger volumes to ensure contact with the desired nerve. Notably, RTX is specific to the TRPV1 receptor and therefore does not affect non-target neurons, such as motor neurons, which do not have sufficient TRPV1 receptors to be sensitive to RTX.

[0071] Numerous examples of RTX formulations are available in the literature. See, for example, 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) may be used.

[0072] In some embodiments, RTX (which may be in the dosages considered 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 includes an organic solvent (e.g., ethanol or DMSO) as one of the fewer or remaining components used as an aid in dissolving RTX, for example, before dilution in a predominantly aqueous composition.

[0073] The concentration of RTX in the above formulation may be any appropriate value for the delivery of the intended dose. In some embodiments, the concentration of RTX in the above pharmaceutical formulation is in the range of 0.1 to 300 μg / ml. In some embodiments, the concentration of RTX in the above 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 above 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, formulations of RTX for delivery to subjects can be prepared by dilution in a suitable diluent (e.g., physiological saline).

[0075] The above formulations 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 concentration of polysorbate 80 is 0.03–7% w / v. In some embodiments, the concentration of polysorbate 80 is 2–4% w / v, and / or the concentration of dextrose is 4–6% w / v. In some embodiments, the concentration of polysorbate 80 is 3% w / v, and / or the concentration of dextrose is 5% w / v. The formulation may further contain a buffer (e.g., phosphate buffer (e.g., sodium phosphate buffer)). In some embodiments, the concentration of phosphate buffer is 10–50 mM. In some embodiments, the concentration of phosphate buffer is 10–30 mM. In some embodiments, the concentration of phosphate buffer is 10 mM. In some embodiments, the concentration of phosphate buffer is 30 mM. The formulation may have a pH in the range of 7–7.5 (e.g., about 7.2). In some embodiments, the concentration of RTX in any of the formulations described above is 10–30 mcg / ml (e.g., 10 mcg / ml or 25 mcg / ml). In some embodiments, the formulation further comprises phosphate buffer (e.g., at the concentrations and pH shown for phosphate buffer in Table 1). In some embodiments, the formulation further comprises NaCl (e.g., at the concentrations shown for NaCl in Table 1). If both are present, the phosphate buffer and NaCl may (but not necessarily) be present in the combinations of concentrations and phosphate buffer pH shown for each individual formulation.

[0077] Exemplary RTX formulations 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 to 5% w / v. In some embodiments, the concentration of dextrose is 0.8 to 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 contain mannitol, and in some embodiments, the mannitol concentration is 0.8-3.0% w / v. In some embodiments, the mannitol concentration is 0.8% w / v. In some embodiments, the mannitol concentration 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 formulations 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 formulations shown in Table 1 is adjusted to 0.3 to 200 mcg / ml. In some embodiments, the concentration of RTX in the formulations shown in Table 1 is 200 mcg / ml. In some embodiments, the concentration of RTX in the formulations shown in Table 1 is 0.3 to 100 mcg / ml. In some embodiments, the concentration of RTX in the formulations shown in Table 1 is 100 mcg / ml. In some embodiments, the concentration of RTX in the formulations shown in Table 1 is adjusted to 0.3 to 50 mcg / ml. In some embodiments, the concentration of RTX in the formulations shown in Table 1 is 25 mcg / ml. As another example, in some embodiments, the concentration of RTX in the formulations 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 0.5–10 mcg / ml. As yet another example, in some embodiments, the concentration of RTX in the formulations shown in Table 1 is adjusted to 0.6–1.5 mcg / ml. The dextrose or mannitol mentioned above is omitted from any such formulation having the adjusted RTX concentration.

[0083] The formulations in Table 1 may be prepared according to the following exemplary method, which is provided for formulations 3 and 5, but can be adapted to other formulations by those skilled in the art. Formulation 3 can be prepared 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. Add 50 ml of water for injection (WFI) to dissolve the components in the flask, and then add 1.0 g of polysorbate 80 to form the aqueous components. Add 20 mg of RTX to the aqueous components in the volumetric flask and adjust the pH to 7.2 with hydrochloric acid / sodium hydroxide. Then add 30 mL of PEG 300 and sonicate the solution to dissolve the solid. It should be noted that RTX initially precipitates occasionally at the interface between the aqueous solution and PEG, but returns to the solution upon sonication. The complete mixture in the flask is diluted with water (WFI) to a predetermined volume (100.00 ml), and then mixed by inversion. The complete formulation is filtered through a 0.2 μm polytetrafluoroethylene (PTFE) filter.

[0084] Preparation 5 can be prepared by adding 138 mg of sodium dihydrogen phosphate monohydrate, 284.1 mg of anhydrous disodium hydrogen phosphate, 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, and then 3.0 g of polysorbate 80 and 800 mg of dextrose are added to form the aqueous components. 20 mg of RTX is added to the aqueous components 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 (or RTX may be first dissolved in a small amount of ethanol or DMSO, and then this solution may be added to the aqueous components). The complete mixture in the flask is diluted with water (WFI) to a predetermined volume (100.00 ml) and mixed by inversion. The complete preparation is filtered through a 0.2 μm PTFE filter.

[0085] Prepare the formulation according to Formulation 11 using 200 mcg RTX, 300 mcg polysorbate 80 (use commercially available polysorbate 80); 5.4 mg sodium chloride, 500 mcg dextrose, 1.38 mg sodium dihydrogen phosphate monohydrate, 2.84 mg disodium hydrogen phosphate anhydrous, and 1 mL of water (WFI), then adjust the pH to 7.2 with hydrochloric acid / sodium hydroxide. As noted above, dextrose may be omitted.

[0086] Prepare the formulation according to Formulation 13 by mixing 25 mcg RTX, 30 mg polysorbate 80 (use commercially available polysorbate 80); 5.4 mg sodium chloride, 50 mg dextrose, 1.38 mg sodium dihydrogen phosphate monohydrate, and 2.84 mg disodium hydrogen phosphate anhydrous, using water (WFI) to make 1 mL, and then adjust the pH to 7.2 with hydrochloric acid / sodium hydroxide. As noted above, dextrose may be omitted.

[0087] In some embodiments, the pharmaceutical formulation is in unit dose form. In such a form, the preparation is divided into unit doses containing an appropriate amount of the active ingredient. The unit dose form may be a packaged preparation (the package may include discontinuous volume formulations, such as in vials, ampoules, or pre-loaded syringes). Alternatively, the unit dose form may be, for example, a liquid formulation or a lyophilized composition for reconstitution.

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

[0089] In some embodiments, RTX may be administered as a single dose. In some embodiments, RTX is administered periodically. In some embodiments, RTX is administered periodically to subjects who require treatment for inflammatory pneumonia as needed to reduce the severity of the disease.

[0090] Compositions for treating inflammatory pneumonia and methods comprising the step of administering RTX to a subject via epidural, periganglionic, or intraganglionic injection are provided herein. One embodiment provides a method for treating a mammalian subject suffering from ARDS.

[0091] In exemplary embodiments, RTX may be administered to alleviate a patient's symptoms, or it may be administered to counteract the mechanism of the disease itself. Those skilled in the art will understand that these therapeutic objectives are often related and that the above treatment may be modified for individual patients based on various factors. These factors include the patient's age, sex, or health status, the progression of the inflammatory pneumonia, the degree of dyspnea, the amount of tissue damage to the patient's airways, the patient's smoking history, and various environmental factors (e.g., temperature, humidity, and air pollution), which may contribute to the patient's condition. Treatment of the patient may be modified depending on the dosage, timing, and route of administration, and by administering other therapeutic agents simultaneously or consecutively. [Examples]

[0092] 1. Resiniferatoxin (RTX) improves acute respiratory distress syndrome (ARDS) in rodent models of lung injury. Respiratory failure due to ARDS is one of the leading causes of mortality associated with acute lung injury (ALI), including COVID-19. ALI / ARDS can be associated with acute cytokine release, pulmonary edema, and, over time, fibrosis. The mechanisms underlying these pathological changes are not fully understood. In Example 1, we tested novel neural components mediated by cardiopulmonary spinal afferent fibers during pulmonary pathology during ALI / ARDS.

[0093] Sensory neurons innervating the heart and lungs enter the central nervous system via one of two pathways: via the vagus nerve to the brainstem (medulla oblongata) where the cell bodies reside in the inferior ganglia, or directly to the spinal cord where the cell bodies reside in the dorsal root ganglia (DRG). Afferent fibers consist of elements that respond to various sensory modalities such as mechanical deformation, heat, cold, pH, and inflammatory mediators. The reflex effects of these afferent fibers after stimulation depend on the type of stimulation and the neural pathway involved. Activation of the vagus nerve's afferent pathway tends to be sympathetic inhibitory 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 sympathetic excitatory and pro-inflammatory (Shanks et al. (2019) Hypertension 74:910-920; Shanks et al. (2018) Physiological Reports 6:313742; 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 the application of RTX, an extremely potent selective afferent neurotoxin, to detach the afferent innervation (thoracic spinal cord) of the lungs would alter the pathological processes, including pulmonary edema and focal pneumonia associated with progressive ALI.

[0094] method A rat model of lung injury. Rats were randomized into three groups and evaluated one week after administration 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 administered intratracheally into the lungs under 3% isoflurane anesthesia. Sham control rats received intratracheal administration of saline. Three days after Bleo delivery, 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 / one side) (Figure 1A-B).

[0095] Epidural administration of RTX. Afferent fibers of the upper thoracic spinal cord were dissected by epidural administration of RTX. The procedure for epidural administration was essentially as described by Shanks et al. (2018) Physiological Reports 6:e13742. Briefly, rats were anesthetized with a 2%-3% isoflurane:oxygen mixture. The rats were placed in a prone position, and a small midline incision was made in the T13-L1 thoracic vertebral region. 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 to approach the T1 level. The upper thoracic sympathetic afferent ganglia were dissected 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 first injection of RTX (6 μg / ml, 10 μl) was given very slowly (approximately 1 minute) to minimize drug diffusion. The catheter was then withdrawn to T2, T3, and T4, respectively, and a series of injections (10 μl / each) were given in each segment. The catheter was withdrawn, and the same injections were repeated on the other side. The foramen of the T13 thoracic vertebra was sealed using silicone gel. The skin over the muscle was closed with a simple interrupted suture of 3-0 polypropylene, and betadine was applied to the wound. For postoperative pain management, buprenorphine (0.05 mg / kg) was administered subcutaneously immediately after surgery and twice daily for two days.

[0096] Intrastellate ganglion injection of RTX. Rats were anesthetized with a 2%-3% isoflurane:oxygen mixture. After inserting a cannula into 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. Parts 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 artery using a hook-shaped glass or steel rod to expose the internal thoracic artery and costocervical artery, which are descending branches of the right subclavian artery. The stellate ganglion and subclavian loop are positioned midway between the origins of the internal thoracic artery and costocervical artery. Next, RTX (5 μl, 50 mg / ml) was injected into the ganglia bilaterally over 30 seconds using a 5 μl Hamilton syringe (Microliter #95, Hamilton, Reno, NV, USA). Images of this procedure are shown in Figures 2A-B. After these careful procedures, the thoracic cavity 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 withdrawn from the chest. Betadine was applied to the wound to allow the rats to recover from anesthesia. For postoperative pain management, buprenorphine (0.05 mg / kg) was administered subcutaneously immediately after surgery and twice daily for two days.

[0097] Blood gas analysis. A small amount of blood (approximately 0.1 mL) was collected from the ventral artery of a rat's tail 7 days after Bleo treatment for arterial blood gas analysis. The animal was restrained with a commercially available restraint device to allow access to its tail. The tail was aseptically prepared by alternating three passes of alcohol and iodine prep pads, 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, IL, USA). After sample collection, the needle was removed, and a gauze swab was firmly pressed against the puncture site to stop bleeding.

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

[0099] Evans blue was extracted from lung plasma, tissue, and quantified. Rats were anesthetized with pentobarbiton (40 mg / kg). Evans blue, 20 mg / kg (10 mg / ml (dissolved in physiological saline) + 100 IE / ml heparin) was administered intravenously. Ten minutes later, the rats were euthanized by transcardiac perfusion with PBS (0.01 M, pH 7.4). The lungs were collected and first photographed. The lung samples were then immediately weighed, placed in 2 ml of N,N'-dimethylformamide, cut into pieces, and heated overnight in a 50°C water bath. Next, the lung tissue was centrifuged (1 minute, 14,000 rpm), and the Evans Blue content in the supernatant was determined at 620 nm (100 μl sample / well) using a 96-well microplate reader (infinite M200, TECAN, Mannedorf, CH, Switzerland). The Evans Blue overflow was expressed as Evans Blue / g lung tissue by comparing the experimental value with a known standard.

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

[0101] result Vascular permeability after ALI was evaluated using plasma spillage (Evans Blue). As shown in Figures 3A-C, the Bleo-treated lungs showed a broad distribution of Evans Blue areas bilaterally. The highest intensity of Evans Blue was shown on the medial side of each lung. These Evans Blue areas were reduced after epidural RTX treatment at 7 days post-Bleo administration.

[0102] Three pro-inflammatory tissue cytokines widely found 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 (Figure 5A-C).

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

[0105] Arterial blood gas data were evaluated in Veh-treated rats compared to RTX-treated stellate ganglion (Figures 7A-H). The results showed an increase in pCO2 (Figure 7B) and a decrease in pO2 (Figure 7C), similar to sO2 in Bleo-treated and Veh-treated rats (Figure 7G). RTX administration to the stellate ganglion revealed these changes, suggesting improved lung function and gas exchange.

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

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

[0108] The above data demonstrate that detachment of TRPV1 afferent sensory fibers in the presence of ALI using RTX delivered via one of two different pathways targeting cardiopulmonary afferent fibers results in a rapid reduction in pulmonary microvascular permeability, as well as a reduction in tissue and plasma inflammatory markers. Although lung function was not directly measured in this series of experiments, arterial blood gas data suggest improved gas exchange. 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 otherwise affected by the systemic inflammatory process induced in the lungs.

[0109] The lungs are innervated by a dual sensory system, which includes afferent fibers of the vagus nerve and spinal nerves. Both the vagus nerve and spinal nerve afferent fibers consist of axons of group A fibers (high conduction velocity) and group C fibers (low conduction velocity). These fibers and their sensory nerve endings are traditionally Na + , K + and Ca 2+It expresses various membrane receptors that mediate ion channel function (both voltage- and ligand-opening types), including TRPV1 channels. Strategies have been developed to modulate the pathological effects of TRPV1 afferent neurons. RTX, a highly potent neurotoxin, strongly binds 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). Following initial stimulation, high intracellular calcium levels mediate the inhibition of neuronal function. Site-directed delivery of RTX can be used to intervene under various conditions to alleviate pain, inflammation, fibrosis, and plasma spillage. RTX-induced deprivation of afferent TRPV1 sensation has been shown to block restricted afferent 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 thoracic vertebrae T1-T4 11 (extending somewhat to higher and more slender segments) or injection into the stellate ganglion. While the DRG is considered to be of primarily sensory nature, the stellate ganglion contains cell bodies of sympathetic efferent fibers and passing fibers of thoracic spinal afferent fibers as they run through the DRG into the spinal cord. It should be noted that in humans, the stellate ganglion is readily identifiable, and this type of percutaneous procedure can be performed with fluoroscopy or ultrasound guidance (intra-ganglion or nerve "block" approach). Furthermore, intrastellate ganglion injection requires only a small amount (10 μl for bilateral injection), which reduces the risk of systemic absorption of RTX and allows higher doses of RTX to be used for local injection.

[0110] The complete disclosures of all publications cited herein are incorporated herein by reference in whole, as each of them is shown and invoked herein individually.

[0111] Various modifications and changes to the embodiments disclosed herein will be apparent to those skilled in the art without departing from the scope and spirit of this disclosure. Illustrative embodiments and examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. In certain embodiments, for example, the following are provided: (Item 1) A method for treating an inflammatory pneumonia, the method comprising the step of administering an effective amount of resiniferatoxin (RTX) epidurally, periganidally, or intraganglionally to a subject in need of treatment for an inflammatory pneumonia. (Item 2) A composition comprising resiniferatoxin (RTX) for use in a method of treating subjects requiring treatment for inflammatory pneumonia. (Item 3) The composition for use according to item 2, wherein the method comprises the step of administering the composition to the subject epidurally, periganally, or intraganglionally. (Item 4) The effective amount of RTX is a composition for use according to item 1 or item 2 or 3, which results in a reduction of one or more cytokines including IL-6, IL-1β and / or IFNγ. (Item 5) The effective amount of RTX is a composition for the method or use described in any one of the preceding items, which produces improved lung function. (Item 6) The effective amount of RTX is a composition for the method or use described in any one of the preceding items, which produces reduced pulmonary edema. (Item 7) The subject is an adult, and the composition is for use or according to any one of the preceding items. (Item 8) The RTX is administered in a dose of approximately 0.1 μg to approximately 100 μg, and is a composition for use according to any one of the above items. (Item 9) The aforementioned dose is approximately 0.1 μg to approximately 1 μg, approximately 1 μg to approximately 5 μg, approximately 5 μg to approximately 10 μg, approximately 10 μg to approximately 20 μg, approximately 20 μg to approximately 50 μg, or approximately 50 to approximately 100 μg, the method or composition for use according to item 8. (Item 10) The method described above includes epidural administration, and the composition for use according to any one of the items described above. (Item 11) The method described above includes a periganglionic nerve block, or a composition for use according to any one of items 1 to 9. (Item 12) The method described above includes intraganglionic administration, and the composition for use according to any one of items 1 to 9. (Item 13) The RTX is administered in a pharmaceutical formulation comprising the RTX and a pharmaceutically acceptable carrier, or a composition for use according to any one of the preceding items. (Item 14) The pharmaceutically acceptable carrier is a composition for use according to item 13, comprising water. (Item 15) The pharmaceutically acceptable carrier is a composition for the method or use described in item 13, comprising physiological saline. (Item 16) The RTX is present in the pharmaceutical preparation at a concentration ranging from 1 μg / ml to 100 μg / ml, and the composition for use according to any one of items 13 to 15. (Item 17) The composition for use according to item 16, wherein the RTX is present in the pharmaceutical preparation at concentrations 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) The inflammatory lung 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 (BOS), primary ciliary dysplasia (PCD), alveolar proteinosis, idiopathic pulmonary fibrosis (IPF), eosinophilic pneumonia, eosinophilic bronchitis, inflammation and / or infection associated with mechanical ventilation, ventilator-associated pneumonia, asbestos-related airway disorders or diseases, dust-related airway disorders or diseases, silicosis, and radiation or chemical-related airway diseases or disorders, and any combination thereof, and is the method or composition described in any one of the above items. (Item 19) The method or composition according to any one of the above items, wherein the pneumonic disease is acute respiratory distress syndrome (ARDS). (Item 20) The method or composition according to any one of the above items, wherein the pneumonic inflammatory disease is chronic obstructive pulmonary disease (COPD). (Item 21) The method or composition according to any one of the above items, wherein the inflammatory pneumonia is pulmonary arterial hypertension (PAH). (Item 22) The method or composition according to any one of the preceding items, wherein the inflammatory pneumonia is an inflammation and / or infection associated with mechanical ventilation, and / or ventilator-associated pneumonia. (Item 23) The inflammatory pneumonia is related to COVID-19, and is a method or composition according to any one of the preceding items.

Claims

1. A composition comprising resiniferatoxin (RTX) for use in a method for treating acute respiratory distress syndrome (ARDS), the method comprising the step of administering the composition epidurally, periganidally, or intraganglionally to a subject in need of treatment for ARDS.

2. A composition comprising resiniferatoxin (RTX) for use in a method of treating a subject in need of treatment for ARDS.

3. The composition for use according to claim 2, wherein the method comprises the step of administering the composition to the subject extradurally, periganglarly, or intraganglarly.

4. A composition for use according to any one of claims 1 to 3, wherein an effective amount of RTX results in a reduction of one or more cytokines, including IL-6, IL-1β, and / or IFNγ.

5. A composition for use according to any one of claims 1 to 4, comprising an effective amount of RTX, which produces improved lung function.

6. A composition for use according to any one of claims 1 to 5, wherein an effective amount of RTX produces reduced pulmonary edema.

7. The composition for use according to any one of claims 1 to 6, wherein the subject is an adult.

8. The composition for use according to any one of claims 1 to 7, characterized in that the RTX is administered in a dose of about 0.1 μg to about 100 μg.

9. The composition for use according to claim 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.

10. The composition for use according to any one of claims 1 to 9, comprising the method of epidural administration.

11. The composition for use according to any one of claims 1 to 9, comprising the method described above, including periganglionic administration.

12. The composition for use according to any one of claims 1 to 9, comprising the method of intraganglionic administration.

13. The composition for use according to any one of claims 1 to 12, characterized in that the RTX is administered in a pharmaceutical formulation comprising the RTX and a pharmaceutically acceptable carrier.

14. The composition for use according to claim 13, wherein the pharmaceutically acceptable carrier comprises water.

15. The composition for use according to claim 13, wherein the pharmaceutically acceptable carrier comprises physiological saline.

16. The composition for use according to any one of claims 13 to 15, wherein the RTX is present in the pharmaceutical preparation at a concentration ranging from 1 μg / ml to 100 μg / ml.

17. The composition for use according to claim 16, wherein the RTX is present in the pharmaceutical preparation at concentrations 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.

18. The ARDS is a composition according to any one of claims 1 to 17, relating to COVID-19.

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