Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses

Definitions

• the present disclosure provides a method for treating pulmonary inflammatory disease comprising administering an effective amount of resiniferatoxin (RTX) by an epidural, peri-ganglionic or an intra-ganglionic administration.
• RTX resiniferatoxin
• RTX acts as an ultrapotent analog of capsaicin, the pungent principal ingredient of the red pepper.
• RTX is a tricyclic diterpene isolated from certain species of Euphorbia.
• a homovanillyl group is an important structural feature of capsaicin and is the most prominent feature distinguishing resiniferatoxin from typical phorbol-related compounds.
• Native RTX has the following structure: [005] RTX and analog compounds such as tinyatoxin and other compounds (20- homovanillyl esters of diterpenes such as 12-deoxyphorbol 13 -phenyl acetate 20-homovanillate and mezerein 20-homovanillate) are described in U.S. Patent Nos.
• RTX is known as a TRPVl agonist.
• TRPVl the transient receptor potential cation channel subfamily V member 1 (also known as Vanilloid receptor-1 (VR1)) is a multimeric cation channel prominently expressed in nociceptive primary afferent neurons (Caterina et al. (1997 ) Nature 389:816-824; Tominaga et al. (1998 ) Neuron 21:531-543).
• Activation of TRPVl typically occurs at the nerve endings via application of painful heat and is up regulated during certain types of inflammatory stimuli.
• TRPVl Activation of TRPVl in peripheral tissues by a chemical agonist results in the opening of calcium channels and the transduction of a pain sensation (Szalllasi et al. (1999 )Mol. Pharmacol. 56:581-587).
• direct application of certain TRPVl agonists to the cell body of a neuron (ganglion) expressing TRPVl opens calcium channels and triggers a cascade of events leading to programmed cell death (“apoptosis”) (Karai et al. (2004) J. of Clin. Invest. 113:1344-1352).
• ARDS acute respiratory distress syndrome
• ICU intensive care unit
• Coronaviruses are a group of viruses that causes diseases in birds, mammals and humans.
• the diseases include respiratory infections and enteric infections which can be mild or lethal.
• Coronaviruses are viruses in the subfamily Orthocoronavirinae, in the family Coronaviridae , in the order Nidovirales.
• the genus Coronavirus includes avian infectious bronchitis virus, bovine coronavirus, canine coronavirus, human coronavirus 299E, human coronavirus OC43, murine hepatitis virus, rat coronavirus, and porcine hemagglutinating encephalomyelitis virus.
• the genus Torovirus includes Berne virus and Breda virus.
• Coronaviruses are enveloped viruses having a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry.
• the genomic size of coronaviruses ranges 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 China pneumonia outbreak in Wuhan was traced to a novel coronavirus, labeled 2019-nCoV by the World Health Organization (WHO), and also known as SARS-CoV-2, which causes Coronavirus disease 2019, or COVID-19.
• WHO World Health Organization
• ARDS was first described in 1967 (Ashbaugh et al. (1967) Lancet 2:319-323) and is characterized by diffuse pulmonary microvascular injury resulting in increased permeability and hypoxemiea caused by intrapulmonary shunts.
• the first two stages of ARDS progression i.e., 12-72 hours after onset
• An early diagnosis may also be facilitated if the initiating stimulus is known as in determination of sepsis, aspiration of gastric contents, multiple transfusions, severe fractures, burns, pancreatitis or severe trauma.
• the present disclosure provides a method for treating pulmonary inflammatory diseases comprising administering an effective amount of resiniferatoxin (RTX) by an epidural, peri -ganglionic or an intra-ganglionic administration.
• RTX resiniferatoxin
• the dose of RTX for an adult human is from about 0.1 pg to about 100 pg.
• Embodiment 1 is a method for treating pulmonary inflammatory disease comprising administering to a subject in need of treatment for pulmonary inflammatory disease an effective amount of resiniferatoxin (RTX) epidurally, peri-ganglionically or intra- ganglionically.
• RTX resiniferatoxin
• Embodiment 2 is a composition comprising resiniferatoxin (RTX) for use in a method of treating a subject in need of treatment for pulmonary inflammatory disease.
• RTX resiniferatoxin
• Embodiment 3 is the composition for use of embodiment 2, wherein the method comprises administering the composition to the subject epidurally, peri-ganglionically or intra- ganglionically.
• 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 in one or more cytokines comprising IL-6, IL-1 b and/or IFNy
• Embodiment 5 is the method or composition for use of any one of the preceding embodiments, wherein the effective amount of RTX results in improved pulmonary function.
• Embodiment 6 is the method or composition for use of any one of the preceding embodiments, wherein the effective amount of RTX results in reduced lung edema.
• Embodiment 7 is the method or composition for use of any one of the preceding embodiments, wherein the subject is an adult human.
• Embodiment 8 is the method or composition for use of any one of the preceding embodiments, wherein the RTX is administered in a dose of from about 0.1 pg to about 100 pg.
• Embodiment 9 is the method or composition for use of embodiment 8, wherein the dose is from about 0.1 pg to about 1 pg, about 1 pg to about 5 pg, about 5 pg to about 10 pg, about 10 pg, to about 20 pg, about 20 pg to about 50 pg, or about 50 to about 100 pg.
• Embodiment 10 is the method or composition for use of any one of the preceding embodiments, wherein the method comprises epidural administration.
• Embodiment 11 is the method or composition for use of any one of embodiments
• the method comprises a peri-ganglionic nerve block.
• Embodiment 12 is the method or composition for use of any one of embodiments
• Embodiment 13 is the method or composition for use of any one of the preceding embodiments, wherein the RTX is administered in a pharmaceutical formulation comprising the RTX and a pharmaceutically acceptable carrier.
• Embodiment 14 is the method or composition for use of embodiment 13 wherein the pharmaceutically acceptable carrier comprises water.
• Embodiment 15 is the method or composition for use of embodiment 13, wherein the pharmaceutically acceptable carrier comprises saline.
• Embodiment 16 is the method or composition for use of any one of embodiments
• RTX is present in the pharmaceutical formulation at a concentration ranging from 1 pg/ml to 100 pg/ml.
• Embodiment 17 is the method or composition for use of embodiment 16, wherein the RTX is present in the pharmaceutical formulation at a concentration ranging from 1 pg/ml to 5 pg/ml, 5 pg/ml to 10 pg/ml, 10 pg/ml to 20 pg/ml, 20 pg/ml to 50 pg/ml, or 50 pg/ml to 100 pg/ml.
• Embodiment 18 is the method or composition of 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 lung rejection and/or dysfunction, bronchitis, sinusitis, asthma, cystic fibrosis, bacterial infection, fungal infection, parasite infection, viral infection, bronchiolitis obliterans syndrome (BOS), primary ciliary dyskinesia (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 disorder or disease, dust-related air
• Embodiment 20 is the method or composition of any one of the preceding embodiments, wherein the pulmonary inflammatory disease is chronic obstructive pulmonary disease (COPD).
• COPD chronic obstructive pulmonary disease
• Embodiment 21 is the method or composition of any one of the preceding embodiments, wherein the pulmonary inflammatory disease is pulmonary arterial hypertension (PAH).
• PAH pulmonary arterial hypertension
• Embodiment 22 is the 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.
• Embodiment 23 is the method or composition of any one of the preceding embodiments, wherein the pulmonary inflammatory disease is associated with COVID-19.
• FIG. 1 A-B show a schematic diagram of the study design (FIG. 1 A) and the treatment plan with a timeline (FIG. IB).
• the arrow indicates that bleomycin (Bleo) (2.5 mg/kg, -0.15 mL) was administered intra-tracheally to the lungs.
• the square shows the location where lung tissue was collected for cytokine measurement.
• FIG. IB - at day 0, Bleo or saline was given intra-tracheally; at day 3, resiniferatoxin (RTX) or vehicle (Veh) was given into epidural space or into stellate ganglia; at day 7, the rats were sacrificed.
• RTX resiniferatoxin
• Veh vehicle
• FIG. 2A-B show the procedure for stellate isolation and administration of Veh or
• FIG. 2A shows step 1 of the procedure - stellate ganglia was exposed. The arrow shows that stellate ganglion was located medially to the origins of internal thoracic and costocervical arteries.
• FIG. 2B shows step 2 of the procedure - RTX (5 pL, 50 mg/mL) was injected into the left and right stellate ganglions. The arrow shows the tip of a 5-pL syringe inside the stellate ganglion.
• FIG. 3 A-C show plasma extravasation was reduced following epidural RTX treatment at the 7-day time point after Bleo administration.
• FIG. 3 A-B shows representative images of the lungs from the Bleo group (FIG. 3A) and the Bleo+RTX group (FIG. 3B).
• FIG. 3C shows Evans blue concentration from Control, Bleo, and Bleo+RTX groups. **P ⁇ 0.01 vs. Control. ## P ⁇ 0.01 vs. Bleo.
• FIG. 4A-C show day 7 lung tissue cytokine levels following day 3 Veh or epidural RTX administration.
• FIG. 4A shows interleukin 6 (IL-6).
• FIG. 4B shows interleukin 1b (IL-Ib).
• FIG. 4C shows interferon g (IFNy). * ⁇ 0.05 and ** ⁇ 0.01 vs. Control. # P ⁇ 0.05 and ## P ⁇ 0.01 vs. Bleo.
• FIG. 5A-C show day 7 plasma cytokine levels following day 3 Veh or epidural
• FIG. 6A-D show Evans blue extravasation was reduced following stellate RTX injection at the 7-day time point after Bleo administration.
• FIG. 6A-C show representative images of the lungs from Sham (FIG. 6A), Bleo+Veh group (FIG. 6B), and Bleo+RTX group (FIG. 6C). Arrows point to areas of Evans blue extravasation.
• FIG. 6D shows mean Evans blue concentration from each group. ** ⁇ 0.01 vs. Sham. # P ⁇ 0.05 vs. Bleo+Veh. $ E ⁇ 0.05 vs. Sham.
• FIG. 7A-H show day 7 arterial blood gases in Sham, Bleo + Veh and Bleo + RTX rats following intra-stellate administration at day 3 post-injury.
• FIG. 7A shows pH.
• FIG. 6B shows partial pressure of carbon dioxide (PCO2).
• FIG. 7C shows partial pressure of oxygen (PO2).
• FIG. 7D shows base excess (BE).
• FIG. 7E shows bicarbonate (HCCb).
• FIG. 7F shows total CO2 (TCO2).
• FIG. 7G shows oxygen saturation (sCk).
• FIG. 7H shows lactate (Lac).
• FIG. 8A-B show day 7 lung tissue cytokine levels following day 3 Veh or stellate
• FIG. 8A shows IL-6.
• FIG. 8B shows IL-Ib.
• FIG. 9A-H show body weight (BW) and individual organ weight among groups.
• FIG. 9A shows body weight.
• FIG. 9B shows heart.
• FIG. 9C shows lung.
• FIG. 9D shows spleen.
• FIG. 9E shows liver.
• FIG. 9F shows kidney.
• FIG. 9G shows heart/BW.
• FIG. 9H shows lung/BW.
• the term “and/or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other.
• the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
• the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass 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); B (alone); and C (alone).
• the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system.
• “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art.
• “about” or “approximately” can mean a range of up to 10% (i.e., ⁇ 10%) or more depending on the limitations of the measurement system.
• about 5 mg can include any number between 4.5 mg and 5.5 mg.
• the terms can mean up to an order of magnitude or up to 5-fold of a value.
• 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” encompasses variation within 10%, 5%, 2%, 1%, or 0.5% of a stated value.
• Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, all ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions such as “not including the endpoints”; thus, for example, “ranging from 1 to 10” includes the values 1 and 10 and all integer and (where appropriate) non-integer values greater than 1 and less than 10.
• pulmonary inflammatory disease is used collectively to refer to those acute and chronic pathological conditions associated with inflammatory processes.
• Non-limiting examples of chronic pathological conditions of the lung include chronic obstructive pulmonary disease (COPD), pulmonary arterial hypertension (PAH), cystic fibrosis, silicosis, asbestosis, asthma, atherosclerosis, chronic bronchitis, chronic inflammation due to 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 worker’s pneumoconiosis, bronchopulmonary dysplasia, inflammatory pseudotumor.
• COPD chronic obstructive pulmonary disease
• PAH pulmonary arterial hypertension
• cystic fibrosis silicosis
• asbestosis asbestosis
• asthma atherosclerosis
• chronic bronchitis chronic inflammation due to chronic bacterial or viral infections
• coronary artery disease
• epidural administration refers to delivery of a drug or pharmaceutical formulation into the epidural space (also known as “extradural space” or “peridural space”) which is the outermost part of the spinal canal. It is the space within the canal (formed by the surrounding vertebrae) lying outside the dura mater (which encloses the arachnoid mater, subarachnoid space, the cerebrospinal fluid, and the spinal cord).
• epidural delivery may include delivery to the epidural space without direct injection into nerves or may include epidural delivery into nerve tissue.
• peri-ganglionic administration refers to delivery of a drug or pharmaceutical formulation into the space surrounding a ganglion.
• Intra-ganglionic administration means administration to a ganglion. Intra- ganglionic administration can be achieved by direct injection into the ganglion and also includes selective nerve root injections, in which the compound passes up the connective tissue sleeve around the nerve and enters the ganglion from the nerve root just outside the vertebral column.
• an effective amount may be used interchangeably and refer to an amount of the therapeutic agent that when administered to a subject, is sufficient to affect a measurable improvement or prevention of a pulmonary inflammatory disease.
• administering an effective dose may improve pulmonary function expressed as partial pressure of CO2 (pCCh), partial pressure of O2 (p0 2 ), and oxygen saturation (sCE) when measured in arterial blood.
• sCE oxygen saturation
• an effective dose may reduce lung edema.
• Therapeutically effective amounts of the therapeutic agents provided herein, when used alone or in combination with an antiviral agent, will vary depending upon the relative activity of the therapeutic agent, and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition in the subject, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. In one embodiment, a therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated and may be ascertained by one skilled in the art using known techniques. In addition, as is known in the art, adjustments for age as well as the body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the disease may be necessary.
• subject and patient refer to human and non-human animals, including vertebrates, mammals and non-mammals.
• the subject can be human, non-human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.
• administering refers to the physical introduction of a therapeutic agent to 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, for example by injection or infusion.
• parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
• the formulation is administered via a non-parenteral route, e.g., orally.
• non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
• Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
• Treating is to be understood broadly and encompasses any beneficial effect, including, e.g., delaying, slowing, or arresting the worsening of symptoms associated with pulmonary inflammatory disease or remedying such symptoms, at least in part. Treating also encompasses bringing about any form of improved patient function, as discussed in detail below. In some embodiments, treatment also means prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those who already have the disease or disorder, as well as those who tend to have the disease or disorder or who should prevent the disease or disorder.
• a “pharmaceutically acceptable vehicle” for therapeutic purposes is a physical embodiment that can be administered to a subject.
• Pharmaceutically acceptable vehicles include pills, capsules, caplets, tablets, oral fluids, injection fluids, sprays, aerosols, troches, dietary supplements, creams, lotions, oils, solutions, pastes, powders, steam, Or it may be a liquid, but is not limited to these.
• An example of a pharmaceutically acceptable vehicle is a buffered isotonic solution such as phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• ICU patients have higher plasma levels of IL-2, IL-7, IL-10, GSCF, IP10, MCP1, MIP1 A, and TNFa as compared to non-ICU patients, indicating that the presence of high circulating cytokine levels is associated with the severity of the disease. It is therefore necessary to interfere with the inflammatory cascade at a higher level (i.e., eliminating the pro- inflammatory efferent pathway) to appropriately control the multimodal aspect of this inflammatory process.
• TRPV1 expressing neuronal system afferent/ efferent neurons
• TRPV1 positive pathways are responsible for pain transmission, inflammation and immunomodulation throughout the entire pulmonary system.
• TRPV1 expressing C-fibers are small diameter unmyelinated fibers in the vagal nerve and responsible for several processes in the airways and lungs.
• Afferent fibers innervating pulmonary structure are also carried by sympathetic fibers with cell bodies located in the dorsal root ganglia of the thoracic segment between T1 and T6. The activation of this thoracic segment has been related to severe pneumonitis.
• RTX is an ultra-potent agonist of the TRPV1 receptor, and it works by inducing neurolysis of TRP VI -expressing neurons in dorsal root ganglia (DRG), dorsal horns (DH) of the spinal cord, or peripheral nerve ending when applied locally as a nerve block.
• DRG dorsal root ganglia
• DH dorsal horns
• the strong binding of RTX to TRP VI receptors forces the opening of the channel gates leading to a slow and sustained increase in intracellular Ca2+, which in turn disrupts the intra-cellular mitochondrial metabolism and results in neural cell or nerve fiber deletion within minutes.
• the inventors have discovered a therapeutic use of RTX, an ultra-potent TRPVl agonist, as an ablating agent of TRPVl positive pulmonary pathways in patients with acute pulmonary inflammatory disease. Such a therapeutic approach targeting TRPVl expressing neurons in the lungs modulates the inflammatory and immune signal activity, leading to reduced mortality and better overall outcomes.
• RTX is delivered epidurally, peri-ganglionically via nerve block or intra-ganglionically.
• the route of administration for an ablative agent such as RTX includes thoracic epidural injections, peri-ganglionic nerve block or intra-ganglionic injections for “chemical” targeted lung denervation.
• RTX is administered by accessing the vagal nerve with a local ablative agent through the neck, going low and away from the carotid bulb. The nerve location could then be confirmed using ultrasound guidance.
• RTX is delivered peri-ganglionically to the stellate ganglion.
• RTX is delivered intra-ganglionically to the stellate ganglion.
• an epidural or peri -ganglionic injection of RTX in subjects with advanced COVID-19 disease supports palliative ventilation therapy by ablating afferent nerves at the thoracic DRG level to increase survival.
• the effective amount of RTX results in a reduction in one or more cytokines comprising IL-6, IL-1 b and/or IFNy In some embodiments, the effective amount of RTX results in improved pulmonary function, such as higher pCk or sCk, or lower pC0 2. In some embodiments, the effective amount of RTX results in reduced lung edema. Such reductions or improvements may occur relative to the condition of the subject prior to the administration of RTX.
• the methods described herein are for use with any subject in whom RTX is effective, e.g., able to bind and activate TRPVl or a homolog thereof, and who is in need of treatment for PD.
• the RTX is administered at a dose of 0.1-100 pg.
• the dose of RTX ranges from 0.1-0.5 pg, 0.5-1 pg, 1-2 pg, 2-5 pg, 5-10 pg, 10-20 pg, 20-30 pg, 30-40 pg, 40-50 pg, 50-60 pg, 60-70 pg, 70-80 pg, 80-90 pg, or 90-100 pg.
• a 2-, 3-, or 4-point peri-ganglionic nerve block technique is used, with a total dosage in any of the ranges listed above, such as a total dosage of 0.5-1 pg, 1-2 pg, 2-5 pg, 5-10 pg, 10-15 pg, 15-20 pg, or 20-25 pg.
• the dosage can be adjusted depending on the proximity of the site of administration to the nerve fiber. For example, where ultrasound or a nerve stimulator is used to ensure that the site of administration is very close to the nerve, a lower dose and/or volume can be used. Alternatively, a nerve block can be accomplished using a larger volume to ensure contact with the desired nerves.
• RTX is specific for the TRPVl receptor and therefore does not affect non-target nerves such as motor neurons that do not have enough TRPV 1 receptors to be sensitive to RTX.
• RTX which may be at the dosages discussed above, is administered with a pharmaceutically acceptable carrier.
• the pharmaceutically acceptable carrier comprises water.
• the pharmaceutically acceptable carrier comprises polysorbate 80.
• the pharmaceutically acceptable carrier comprises polyethylene glycol.
• the pharmaceutically acceptable carrier comprises sugar or sugar alcohol.
• 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 such as ethanol or DMSO, e.g., as a minority or residual component used as an aid in dissolving RTX before dilution in a primarily aqueous composition.
• the concentration of RTX in the formulation may be any suitable value for delivery of the intended dose.
• the concentration of RTX in the pharmaceutical formulation is in the range of 0.1 to 300 pg/ml. In some embodiments, the concentration of RTX in the pharmaceutical formulation is in the range of 0.1-1 pg/ml, 1-5 pg/ml, 5-10 pg/ml, 10-20 pg/ml, 10-30 pg/ml, 20-30 pg/ml, 20-50 pg/ml, 50-100 pg/ml, 100- 150 pg/ml, 150-200 pg/ml, 200-250 pg/ml, or 250-300 pg/ml. In some embodiments, the concentration of RTX in the pharmaceutical formulation is in the range of 5-50 pg/ml, or 8-25 pg/ml.
• a formulation of RTX for delivery into a subject may be prepared by dilution in an appropriate diluent, such as saline.
• the formulation may have any pH suitable for intra-articular administration.
• the pharmaceutical formulation comprising RTX and a pharmaceutically acceptable carrier has a pH in the range of 6 to 7.6.
• 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.
• the pharmaceutical formulation comprising RTX and a pharmaceutically acceptable carrier has a pH of 6.5 or 7.2.
• the formulation comprises polysorbate 80 and dextrose.
• the concentration of polysorbate 80 is 0.03-7% w/v.
• the concentration of polysorbate 80 is 2-4% w/v, and/or the concentration of dextrose is 4-6% w/v.
• the concentration of polysorbate 80 is 3% w/v, and/or the concentration of dextrose is 5% w/v.
• the formulation may further comprise a buffer, such as phosphate buffer (e.g., sodium phosphate buffer).
• the concentration of phosphate buffer is 10-50 mM.
• the concentration of phosphate buffer is 10-30 mM.
• the concentration of phosphate buffer is lOmM. In some embodiments, the concentration of phosphate buffer is 30 mM.
• the formulation may have a pH in the range of 7-7.5, such as about 7.2. In some embodiments, in any of the foregoing formulations, the concentration of RTX may be 10-30 mcg/ml, such as 10 mcg/ml or 25 mcg/ml.
• the formulation further comprises phosphate buffer, e.g., at a concentration and pH shown for phosphate buffer in Table 1.
• the formulation further comprises NaCl, e.g., at a concentration shown for NaCl in Table 1. When both are present, the phosphate buffer and NaCl may be (but are not necessarily) present at a combination of concentrations and phosphate buffer pH shown for an individual formulation.
• formulations in Table 1 include dextrose.
• the concentration of dextrose is 0.05-5% w/v.
• the concentration of dextrose is 0.8-5% w/v.
• the concentration of dextrose is 0.05% w/v.
• the concentration of dextrose is 0.8% w/v.
• the concentration of dextrose is 3.0% w/v.
• the concentration of dextrose is 5.0% w/v.
• formulations in Table 1 include mannitol.
• 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.
• the dextrose or mannitol is omitted from a formulation shown in Table 1.
• the concentration of RTX in a formulation shown in Table 1 is adjusted to any of the RTX concentrations or concentration ranges disclosed herein.
• the concentration of RTX in a formulation shown in Table 1 is adjusted to 0.3-200 mcg/ml.
• the concentration of RTX in a formulation shown in Table 1 is 200 mcg/ml.
• the concentration of RTX in a formulation shown in Table 1 is 0.3-100 mcg/ml.
• the concentration of RTX in a formulation shown in Table 1 is 100 mcg/ml.
• the concentration of RTX in a formulation shown in Table 1 is adjusted to 0.3-50 mcg/ml.
• the concentration of RTX in a formulation shown in Table 1 is 25 mcg/ml. As another example, in some embodiments, the concentration of RTX in a formulation shown in Table 1 is adjusted to 0.3-15 mcg/ml. As another example, in some embodiments, the concentration of RTX in a formulation shown in Table 1 is adjusted to 0.5-10 mcg/ml. As another example, in some embodiments, the concentration of RTX in a formulation shown in Table 1 is adjusted to 0.6-1.5 mcg/ml. The dextrose or mannitol is omitted from any such formulation having an adjusted RTX concentration.
• formulations in Table 1 may be prepared according to the following exemplary methods, which are provided for formulations 3 and 5 but may be adapted to the other formulations by one skilled in the art.
• Formulation 3 may be made by adding 46 mg sodium phosphate monobasic monohydrate, 94.7 mg sodium phosphate dibasic 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 addition of 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 pH is adjusted with hydrochloric acid/sodium hydroxide to 7.2.
• WFI water for injection
• RTX will sometimes precipitate at the interface of aqueous solution and PEG initially, but will go back into solution upon sonication.
• the full mixture in the flask is diluted to volume (100.00 ml) with water (WFI) and this is mixed by an inversion process.
• WFI water
• the full formulation is filtered through a 0.2 pm polytetrafluoroethylene (PTFE) filter.
• Formulation 5 may be made by adding 138 mg sodium phosphate monobasic monohydrate, 284.1 mg sodium phosphate dibasic anhydrous, and 540 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 addition of 3.0 g of polysorbate 80, and 800 mg of dextrose to form the aqueous component. 20 mg of RTX is added the aqueous component in the volumetric flask, and pH is adjusted with hydrochloric acid/sodium hydroxide to 7.2. The solution is then sonicated to dissolve all the solids.
• WFI water for injection
• the RTX may be initially dissolved in a small volume of ethanol or DMSO, and this solution may then be added to the aqueous component.
• the full mixture in the flask is diluted to volume (100.00 ml) with water (WFI) and this is mixed by an inversion process.
• the full formulation is filtered through a 0.2 pm PTFE filter.
• a formulation according to Formulation 11 is prepared using 200 meg RTX, 300 meg Polysorbate 80 (using commercially-available polysorbate 80); 5.4 mg of sodium chloride, 500 meg of dextrose, 1.38 mg sodium phosphate monobasic monohydrate, 2.84 mg sodium phosphate dibasic anhydrous, and water (WFI) to 1 mL, then pH is adjusted with hydrochloric acid/sodium hydroxide to 7.2. As noted above, the dextrose may be omitted.
• a formulation according to Formulation 13 is prepared using 25 meg RTX, 30 mg
• Polysorbate 80 (using commercially-available polysorbate 80); 5.4 mg of sodium chloride, 50 mg of dextrose, 1.38 mg sodium phosphate monobasic monohydrate, 2.84 mg sodium phosphate dibasic anhydrous, water (WFI) to 1 mL, then pH is adjusted with hydrochloric acid/sodium hydroxide to 7.2. As noted above, the dextrose may be omitted.
• the pharmaceutical formulation is in a unit dosage form.
• the preparation is subdivided into unit doses containing appropriate quantities of the active component.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of formulation, such as in vials, ampoules, or pre-loaded syringes.
• the unit dosage form can be, e.g., a solution or a lyophilized composition for reconstitution.
• RTX may be administered as a one-time single dose. In some embodiments, RTX is periodically administered. In some embodiments, RTX is periodically administered to a subject in need of treatment for pulmonary inflammatory disease as needed to reduce the severity of the disease.
• composition and methods for treating pulmonary inflammatory disease comprising administering RTX to a subject via epidural, peri -ganglionic or intra-ganglionic injection.
• One embodiment provides a method of treating a mammalian subject suffering from ARDS.
• RTX can be administered to reduce the patient’s symptoms, or it can be administered to counter the mechanism of the disease itself. It will be appreciated by those skilled in the art that these therapeutic objectives are often related and the treatment can be adjusted for individual patients based on various factors. These factors include the patient’s age, gender, or health status, progression of pulmonary inflammatory disease, degree of dyspnea, amount of tissue damage to the patient’s respiratory tract, patient smoking history, and various environmental factors (e.g., temperature, humidity and air pollution), which may contribute to the patient’s condition.
• the patient’s therapy can be adjusted depending on the dosage, timing, route of administration, and by administering other therapeutic agents simultaneously or sequentially.
• RTX Resiniferatoxin Ameliorates Acute Respiratory Distress Syndrome (ARDS) in Rodent Model of Lung Injury
• ALI/ARDS may be associated with acute cytokine release, pulmonary edema, and in the long term, fibrosis. The mechanisms underlying these pathological changes are not fully understood.
• Example 1 a novel neural component through cardiopulmonary spinal afferents that mediates lung pathology during ALEARDS was studied.
• Afferents are composed of elements that respond to a variety of sensory modalities such as mechanical deformation, heat, cold, pH, and inflammatory mediators. The reflex effects following stimulation of these afferents depend on the type of stimulus and the neural pathway involved. Activation of vagal afferent pathways tends to be sympatho-inhibitory and anti-inflammatory (Komeage et al. (2016) Brain, Behavior, and Immunity 73:441-449;
• Rat Model of Lung Injury Rats were randomized into three groups and evaluated at 1-week post-instillation as follows: sham rats, bleomycin (Bleo)-exposed rats with saline (epidural or intra-stellate injection), and Bleo-exposed rats with RTX (epidural or intra-stellate injection). Bleo (2.5 mg/kg, -0.15 mL) was instilled intra-tracheally to the lungs under 3% isoflurane anesthesia. Sham control rats underwent intra-tracheal instillation of saline.
• RTX Epidural Application of RTX.
• the upper thoracic spinal afferents were ablated by epidural application of RTX.
• the procedure for epidural administration was essentially as described by Shanks et al (2016) Physiological Reports 6:el3742. Briefly, rats were anesthetized using 2%-3% isoflurane: oxygen mixture. Rats were placed in the prone position and a small midline incision was made in the region of the T13-L1 thoracic vertebrae. Following dissection of the superficial muscles, two small holes (approximately 2 mm x 2 mm) were made in the left and right sides of T13 vertebrae. A polyethylene catheter (PE- 10) was inserted into the subarachnoid space via one hole and gently advanced about 4cm approximating the T1 level.
• PE- 10 polyethylene catheter
• RTX resiniferatoxin
• TRPV 1 receptor an ultra-potent agonist of the TRPV 1 receptor into the subarachnoid space via the 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 pg/ml, lOul) was made at a very slow speed ( ⁇ 1 minute) to minimize the diffusion of the drug.
• the catheter was then pulled back to T2, T3 and T4, respectively to perform serial injections (10 pl/each) at each segment.
• the left or right precava vein were separated with a hooked glass or steel rod laterally away from the brachiocephalic artery to expose the internal thoracic artery and the costocervical artery, which are descending branches of the right subclavian artery.
• Stellate ganglia and ansa subclavia are located medially to the origins of the internal thoracic and costocervical arteries.
• RTX 5pl, 50 mg/ml
• the artery on the ventral aspect of the rat tail was used for the collection of small amounts of blood ( ⁇ 0.1 mL) for analyzing arterial blood gas at day 7 post Bleo treatment.
• the animal was restrained with a commercial restrainer so that its tail was accessible.
• the tail was prepared aseptically by alternating alcohol prep pads and iodine prep pads three times and the artery was punctured using a 24 G needle.
• a small volume of blood ( ⁇ 0.1 mL) was gently aspirated into the syringe for blood gas analysis (iSTAT, Abbott, Chicago, IL, USA). After sample collection, the needle was removed, and a gauze swab was pressed firmly on the puncture site to stop bleeding.
• the lung tissues were then centrifuged (1 min, 14,000 rpm) and the Evans Blue content of the lungs in the supernatant was determined in a 96-well microplate reader (infinite M200, TEC AN, Mannedorf, CH, Switzerland) at 620 nm (100 m ⁇ sample per well). Extravasation of Evans Blue was expressed as mg Evans Blue per g of lung tissue, by comparing the experimental values with a known standard.
• Plasma extravasation (Evans Blue) was used to assess vascular permeability after vans Blue.
• FIG. 3A-C Bleo-treated lungs exhibited a wide distribution of Evans Blue areas in both sides. The highest intensity of Evans Blue was shown at the medial aspect of each lung. The Evans Blue areas were reduced following epidural RTX treatment at the 7-day time point after Bleo administration.
• FIG. 4A-C IL-6 (FIG. 4A), IL-Ib (FIG. 4B), and IFNy (FIG. 4C) were elevated following Bleo treatment. These cytokine levels were reduced in RTX treated rats. [00103] Cytokine levels in response to Bleo were also reduced after epidural application of RTX (FIG. 5A-C).
• RTX As shown in FIG. 6A-D, there was a marked reduction in Evans Blue dye in the lung following stellate injection of RTX.
• FIG. 7A-H Arterial blood gas data were evaluated in rats treated with Veh vs RTX intra- stellate. The results show that pCCk was elevated (FIG. 7B) and pCk was reduced (FIG. 7C) as was sCk in Bleo and Veh treated rats (FIG. 7G). Stellate administration of RTX reversed these changes suggesting improved pulmonary function and gas exchange.
• FIG. 8A-B show that IL-6 (FIG. 8A) and IL-Ib (FIG. 8B) levels in lung tissue were significantly reduced after stellate RTX administration.
• FIG. 9A-H show body weight (BW) and individual organ weight among groups.
• vagal and spinal afferents The lung is innervated by a dual sensory system including vagal and spinal afferents. Both vagal and spinal afferent fibers are composed of group A fiber (high conduction velocity) and group C fiber (low conduction velocity) axons. These fibers and their sensory endings express a variety of membrane receptors that mediate ion channel function including traditional Na + , K + and Ca 2+ channels (both voltage and ligand gated).
• a strategy has been developed to modulate the pathological effects of TRPVl afferent neurons.
• the ultrapotent neurotoxin, RTX binds avidly to the TRPVl receptor. Upon activation, TRPVl channels are highly permeable to calcium (Hsu et al.
• RTX-induced TRPV 1 sensory afferent deletion can block the afferent- contained neuropeptide release and reduce inflammatory pain (Karai et al. (2004) The Jounrla of Clinical Investigation 113 : 1344-1352).
• Cardiopulmonary spinal afferents can also be targeted with RTX by either application into the epidural space at thoracic levels T1-T4 11 (with some spread to higher and lower segments) or by injection into the stellate ganglia. While DRGs are considered exclusively sensory in nature, the stellates contain soma for sympathetic efferent fibers and fibers of passage for thoracic afferents as they course through DRGs and enter the spinal cord. It should be noted that in humans the stellate ganglia can be easily identified, and that this type of transcutaneous procedure can be performed with fluoroscopic or ultrasound guidance (intra-ganglionic or nerve ‘block’ approach). Furthermore, intra-stellate injection requires a small volume (10 pi for bilateral injection), which reduces the risk of systemic absorption of RTX and allows a higher dose of RTX to be used for local injection.

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