WO2021216399A1

WO2021216399A1 - Pulmonary aminosterol compositions and methods of using the same to treat microbial infections

Info

Publication number: WO2021216399A1
Application number: PCT/US2021/027882
Authority: WO
Inventors: Denise Barbut; Machael ZASLOFF
Original assignee: Enterin, Inc.
Priority date: 2020-04-20
Filing date: 2021-04-19
Publication date: 2021-10-28

Abstract

The present application relates generally to method of treating microbial infections, such as coronavirus infection, using lactate or dilactate salts of aminosterols, administered via inhalation or pulmonary administration.

Description

PULMONARY AMINOSTEROL COMPOSITIONS AND METHODS OF USING THE SAME TO TREAT MICROBIAL INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application the benefit of U.S. Provisional Application No. 63/012,854, filed April 20, 2020, and U.S. Provisional Application No. 63/015,470, filed April 24, 2020, the contents of which are incorporated herein by reference in their entirety.

FIELD

[0002] The present application relates generally to a methods of treating microbial infections using inhaled or pulmonary-administered aminosterols and compositions related to the same.

BACKGROUND

[0003] Aminosterols are amino derivatives of a sterol. Exemplary aminosterols include squalamine and aminosterol 1436 (also known as trodusquemine and MSI-1436).

[0004] There is a need in the art for novel methods of treating microbial infections, and in particular pulmonary microbial infections and viral infections, for example, viral infections by a coronavirus. Activity of aminosterols against other viral infections has previously been described. See, for example, USPN 8,729,058; USPN 9,867,835; USPN 10,478,444; and US 16/559,530; the disclosures of which are hereby incorporated by reference.

[0005] There is a need in the art for new methods of treating microbial infections. The present disclosure satisfies this need.

SUMMARY

[0006] In a first aspect, the present disclosure is directed to methods of treating a subject in need, wherein the subject has been diagnosed with a coronavirus infection, is suspected of having a coronavirus infection, and/or is at risk of developing a coronavirus infection, wherein the coronavirus is SARS-CoV-2, which is the vims responsible for COVID-19. The method comprises administering to the subject a therapeutically effective amount of a composition comprising at least one aminosterol via inhalation or pulmonary administration, wherein the aminosterol is in a lactate or dilactate salt form.

[0007] It is expected that administration of the aminosterol lactate or dilactate salt dosage via inhalation or pulmonary administration will increase survival and/or decrease undesirable side effects of COVID-19. In other aspects, administration of the aminosterol lactate or dilactate salt dosage form can improve respiratory symptoms, shorten duration of vims shedding, reduce duration of hospitalization, interfere with viral replication, stimulate tissue regeneration, or any combination thereof.

[0008] Inhalation or pulmonary administration can be via a device or method selected from the group consisting of a nebulizer, pressurized nebulizer, jet nebulizer, ultrasonic nebulizer, vibrating mesh nebulizer, soft mist inhaler, metered dose inhaler (MDI), pressurized metered-dose inhaler, Dry Powder Inhaler, and Intratracheal Nebulizing Catheter.

[0009] In another aspect, the methods described herein can use a single dose Dry Powder Inhaler (DPI) device. The device can deliver the composition in the form of a dry powder contained in a capsule, and additionally the device can be a breath actuated inhaler. The device can deliver the composition in the form of a dry powder dispensed from a capsule containing the composition.

[0010] For all of the methods described herein, the composition can be in a dry powder dosage form or alternatively the composition can be in a liquid dispersion dosage form. Other dosage forms are also described herein.

[0011] In one aspect, the composition can be administered to a mechanically ventilated subject. For example, the composition can be administered to the subject using an inhaler or nebulizer device connected to the ventilator via an actuator device. In another aspect, the device can be connected to the ventilator using a spacer chamber.

[0012] In one embodiment, the composition is administered to the mechanically ventilated subject using a dry powder inhaler, soft mist inhaler, or intratracheal nebulizing catheter adapted for in-line use.

[0013] In another embodiment, the coronavirus infection is correlated with pneumonia and/or a lung infection. Correlation may comprise comorbid COVID-19 and pneumonia as conformed by a commercially available test of Table 1 and X-ray or CT-scan, respectively.

[0014] The method can additionally comprise administering one or more compounds selected from the group consisting of bronchodilators, inhaled corticosteroids, antibiotics, pulmonary surfactant, mucolytics, biologicals, genes, prostanoids, surfactants, heparin, morphine, furosemide, and combinations thereof. For example, (a) the bronchodilator can be albuterol, formoterol, arformoterol, fenoterol, metaproterenol, or ipratropium; (b) the inhaled corticosteroid can be budesonide, beclomethasone, or fluticasone; (c) the antibiotic can be tobramycin, amikaci, amikacin, fosfomycin, colistin, ciprofloxacin, ribavirin, and amphotericin B; (d) the surfactant can be Exosurf, Survanta, Curosurf, Infasurf, and KL4; (e) the mucolytic can be N-acetylcysteine or dornase alfa; (f) the biological can be a monoclonal antibody; (g) the gene can be an siRNA; and (h) the prostanoid can be epoprostenol, iloprost, or treprostinil.

[0015] In another aspect, the subject has a comorbidity selected from the group consisting of diabetes, hypertension, cardiovascular disease, cancer, prior cancer treatment, cerebrovascular disease, chronic obstructive pulmonary disease (COPD), chronic kidney disease, sarcoidosis, obstructive lung disease, idiopathic pulmonary fibrosis (IPF), asthma, chronic bronchitis, emphysema, cystic fibrosis/bronchiectasis, and pneumonia. In another aspect, the subject has a lung disease or respiratory disorder.

[0016] The present disclosure is also directed to methods of treating a subject in need, wherein the subject has a microbial infection. The methods comprise administering to the subject a therapeutically effective amount of a composition comprising at least one aminosterol via inhalation or pulmonary administration, wherein the aminosterol is in a lactate or dilactate salt form.

[0017] The inhalation or pulmonary administration can be via any suitable device, such as a nebulizer (including pressurized nebulizers), a metered dose inhaler (including pressurized MDIs), or a Dry Powder Inhaler, including but not limited to a single dose Dry Powder Inhaler (DPI) device. In one aspect, the device delivers the composition in the form of dry powder contained in a capsule. In another aspect, the device is a hand-held device. In a further aspect, the device can be a breath actuated inhaler. Other devices that can be used to administer the composition include soft mist inhalers and intratracheal nebulizing catheters.

[0018] In one aspect, the composition is in a dry powder dosage form, a liquid dispersion dosage form or any other dosage form described herein.

[0019] The microbial infection can be, for example, a viral infection, fungal infection, or bacterial infection. In addition, the microbial infection can be correlated with pneumonia and/or a lung infection.

[0020] In one aspect, the microbial infection is a viral infection. An exemplary viral infection is a coronavirus infection. For example, a coronavirus can be selected from the group consisting of an Alphacoronavirus; a Colacovirus such as Bat coronavirus CDPHE15; a Decacovirus such as Bat coronavirus HKU10 or Rhinolophus ferrumequinum alphacoronavirus HuB-2013; a Duvinacovirus such as Human coronavirus 229E; a Luchacovirus such as Lucheng Rn rat coronavirus; a Minacovirus such as a Ferret coronavirus or Mink coronavirus 1; a Minunacovirus such as Miniopterus bat coronavirus 1 or Miniopterus bat coronavirus HKU8; a Myotacovirus such as Myotis ricketti alphacoronavirus Sax-2011; a nyctacovirus such as Nyctalus velutinus alphacoronavirus SC- 2013; a Pedacovirus such as Porcine epidemic diarrhea virus or Scotophilus bat coronavirus 512; a Rhinacovirus such as Rhinolophus bat coronavirus HKU2; a Setracovirus such as Human coronavirus NL63 or NL63 -related bat coronavirus strain BtKYNL63-9b; a Tegacovirus such as Alphacoronavirus 1; a Betacoronavirus; a Embecovirus such as Betacoronavirus 1, Human coronavirus OC43, China Rattus coronavirus HKU24, Human coronavirus HKU1 or Murine coronavirus; a Hibecovirus such as Bat Hp-betacoronavirus Zhejiang2013; a Merbecovirus such as Hedgehog coronavirus 1, Middle East respiratory syndrome-related coronavirus (MERS-CoV), Pipistrellus bat coronavirus HKU5 or Tylonycteris bat coronavirus HKU4; a Nobecovirus such as Rousettus bat coronavirus GCCDC1 or Rousettus bat coronavirus HKU9, a Sarbecovirus such as a Severe acute respiratory syndrome-related coronavirus, Severe acute respiratory syndrome coronavirus (SARS-CoV) or Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19); a Deltacoronavirus; an Andecovirus such as Wigeon coronavirus HKU20; a Buldecovirus such as Bulbul coronavirus HKU11, Porcine coronavirus HKU15, Munia coronavirus HKU13 or White-eye coronavirus HKU16; a Herdecovirus such as Night heron coronavirus HKU19; a Moordecovirus such as Common moorhen coronavirus HKU21; a Gammacoronavirus; a Cegacovirus such as Beluga whale coronavirus SW1; and an Igacovirus such as Avian coronavirus.

In a particular aspect, the microbial infection is caused by a coronavirus which is SARS-CoV-2.

[0021] In another aspect, the subject has a comorbidity selected from the group consisting of diabetes, hypertension, cardiovascular disease, cancer, prior cancer treatment, cerebrovascular disease, chronic obstructive pulmonary disease (COPD), chronic kidney disease, sarcoidosis, obstructive lung disease, idiopathic pulmonary fibrosis (IPF), asthma, chronic Bronchitis, emphysema, cystic fibrosis/bronchiectasis, and pneumonia. In another aspect, the subject has a lung disease or respiratory disorder. For all of the methods described herein, the subject can have a lung disease or respiratory disorder.

[0022] Further, for the methods described herein, optionally the method can additionally comprise administering (a) an antimicrobial drug; and/or (b) an antiviral drug if the subject has a viral infection; and/or (c) an antibacterial drug if the subject has a bacterial infection; and/or (d) an antifungal drug if the subject has a fungal infection.

[0023] In another aspect, the lactate or dilactate salt of the aminosterol is a pharmaceutically acceptable grade of the aminosterol.

[0024] The compositions described herein can further comprises one or more of an aqueous carrier, a buffer, a sugar, and/or a polyol compound.

[0025] In one aspect of the methods, the subject is a human. In another aspect, the subject is at risk, or is a member of a patient population at risk, of developing the microbial infection to be treated.

In another aspect of the methods described herein, the lactate or dilactate salt of the aminosterol is administered at a very low dose of about 50 mg or less. In yet another aspect, the lactate or dilactate salt of the aminosterol is administered at a very low dose of about 45 mg or less, about 40 mg or less, about 35 mg or less, about 30 mg or less, about 25 mg or less, about 20 mg or less, about 15 mg or less, about 14 mg or less, about 13 mg or less, about 12 mg or less, about 11 mg or less, about 10 mg or less, about 9 mg or less, about 8 mg or less, about 7 mg or less, about 6 mg or less, about 5 mg or less, about 4 mg or less, about 3 mg or less, about 2 mg or less, about 1 mg or less, about 0.5 mg or less, and greater than 0 mg, and including about 5 mg. In one aspect, the lactate or dilactate salt of the aminosterol is administered by nebulization at a concentration of about 0.02% in about 0.9% saline up to a total single dose of about 5 mg.

[0026] In yet another aspect, the lactate or dilactate salt of the aminosterol is administered at about 9.5 mg or less, about 9 mg or less, about 8.5 mg or less, about 8 mg or less, about 7.5 mg or less, about 7 mg or less, about 6.5 mg or less, about 6 mg or less, about 5.5 mg or less, about 5 mg or less, about 4.5 mg or less, about 4 mg or less, about 3.5 mg or less, about 3 mg or less, about 2.5 mg or less, about 2 mg or less, about 1.5 mg or less, about 1 mg or less, and greater than about 0 mg.

[0027] For all of the methods described herein, the aminosterol can be selected from the group consisting of squalamine lactate, squlamine dilactate, aminosterol 1436 lactate, and aminosterol 1436 dilactate.

[0028] In another embodiment, the aminosterol is selected from the group consisting of: (a) a lactate or dilactate salt of an isomer of squalamine; and/or (b) a lactate or dilactate salt of an isomer of aminosterol 1436; and/or (c) a lactate or dilactate salt of an aminosterol comprising a sterol nucleus and a polyamine attached at any position on the sterol, such that the molecule exhibits a net charge of at least + 1; and/or (d) a lactate or dilactate salt of an aminosterol comprising a bile acid nucleus and a polyamine, attached at any position on the bile acid, such that the molecule exhibits a net charge of at least + 1; and/or (e) a lactate or dilactate salt of an aminosterol derivative modified to include one or more of the following: (i) substitutions of the sulfate by a sulfonate, phosphate, carboxylate, or other anionic moiety chosen to circumvent metabolic removal of the sulfate moiety and oxidation of the cholesterol side chain; (ii) replacement of a hydroxyl group by a non-metabolizable polar substituent, such as a fluorine atom, to prevent its metabolic oxidation or conjugation; and (iii) substitution of one or more ring hydrogen atoms to prevent oxidative or reductive metabolism of the steroid ring system; and/or (f) a lactate or dilactate salt of a derivative of squalamine modified through medical chemistry to improve bio-distribution, ease of administration, metabolic stability, or any combination thereof; and/or (g) a lactate or dilactate salt of a derivative of aminosterol 1436 modified through medical chemistry to improve bio-distribution, ease of administration, metabolic stability, or any combination thereof; and/or (h) a lactate or dilactate salt of a synthetic aminosterol.

[0029] In another aspect, for all of the method described herein, the aminosterol can be a lactate or dilactate salt of any of the following aminosterol compounds:

Compound 3,

[0030] In yet another aspect, for all of the methods described herein the aminosterol can be selected from the group consisting of:

(a) an aminosterol having the formula:

Compound VI (ENT-06), wherein the compound is in a lactate or dilactate salt form;

(b) an aminosterol having the formula:

C25 (R) Compound VI (ENT-06), wherein the compound is in a lactate or dilactate salt form;

(c) an aminosterol having the formula:

Compound IV (D5 ENT-06), wherein the compound is in a lactate or dilactate salt form;

(d) an aminosterol having the formula:

C25 (R) Compound IV (D5 ENT-06), wherein the compound is in a lactate or dilactate salt form; (e) an aminosterol having the formula:

Compound V (D4 ENT-06), wherein the compound is in a lactate or dilactate salt form; (f) an aminosterol having the formula:

C25 ( R ) Compound V (D4 ENT-06), wherein the compound is in a lactate or dilactate salt form;

(g) an aminosterol which is a prodrug comprising a compound of formula:

wherein:

R¹ is H, an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C₆ alkyl, optionally substituted C1-C₆ alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C1-C₆ alkenyl; and R² is H or -C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; provided that at least one of R¹ and R² is not H, and wherein the compound is in a lactate or dilactate salt form;

(h) an aminosterol which is a prodrug comprising a compound of formula:

C25 (R) Compound VI-P, wherein the compound is in a lactate or dilactate salt form;

(i) an aminosterol which is a prodrug comprising a compound of formula:

Compound IV-P, wherein:

R¹ is H, an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C₆ alkyl, optionally substituted C1-C₆ alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C1-C₆ alkenyl; and

R² is H or -C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C₆ alkyl, optionally substituted C1-C₆ alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; provided that at least one of R¹ and R² is not H, and wherein the compound is in a lactate or dilactate salt form;

(j) an aminosterol which is a prodrug comprising a compound of formula:

C25 (R) Compound IV-P, where the compound is in a lactate or dilactate salt form;

(k) an aminosterol compound having the formula:

Compound V-P, wherein: R¹ is H, an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C1-C6 alkenyl; and R² is H or - C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; provided that at least one of R¹ and R² is not H, wherein the compound is in a lactate or dilactate salt form;

(1) an aminosterol compound having the formula:

C25 (R) Compound V-P, wherein the compound is in a lactate or dilactate salt form.

[0031] In yet another aspect, for all of the methods described herein the aminosterol can be selected from the group consisting of:

(a) an aminosterol having the formula:

(Compound III) (ENT-03), wherein the compound is in a lactate or dilactate salt form; (b) an aminosterol having the formula:

(C25 (R) Compound III) (ENT-03), wherein the compound is in a lactate or dilactate salt form; (c) an aminosterol having the formula:

(Compound IV), wherein the compound is in a lactate or dilactate salt form;

(d) an aminosterol having the formula:

(C25 ( R ) Compound IV) (D5 ENT-03), wherein the compound is in a lactate or dilactate salt form;

(e) an aminosterol having the formula:

Compound V (D4 ENT-03), wherein the compound is in a lactate or dilactate salt form;

(f) an aminosterol having the formula:

C25 (R) Compound V (D4 ENT-03), wherein the compound is in a lactate or dilactate salt form;

(g) an aminosterol which is a prodrug comprising a compound having the formula:

(Compound III-P), wherein:

R¹ is H, an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; and

R² is H or -C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; provided that at least one of R¹ and R² is not H, and wherein the compound is in a lactate or dilactate salt form;

(h) an aminosterol which is a prodrug comprising a compound of formula:

(C25 (R) Compound III-P), wherein the compound is in a lactate or dilactate salt form;

(i) an aminosterol which is a prodrug comprising a compound having the formula:

(Compound IV-P), wherein:

R² is H or -C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C₆ alkyl, optionally substituted C1-C₆ alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C₆ alkenyl; provided that at least one of R¹ and R² is not H, wherein the compound is in a lactate or dilactate salt form;

(j) an aminosterol which is a prodrug comprising a compound having the formula:

(C25 (R) Compound IV-P), wherein the compound is in a lactate or dilactate salt form;

(k) an aminosterol compound having the formula:

Compound V-P, wherein: R¹ is H, an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C1-C6 alkenyl; and R² is H or - C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; provided that at least one of R¹ and R² is not H, wherein the compound is in a lactate or dilactate salt form; or (1) an aminosterol compound having the formula:

[0032] In yet another aspect, for all of the methods described herein the aminosterol can be a crystalline form of the dilactate salt of 3P-(N-[3-aminopropyl]-l,4-butanediamine)-7a, 24R- dihydroxy-5a-cholestane-24-sulfate.

[0033] In yet another aspect, for all of the methods described herein the aminosterol can be an amorphous form of the dilactate salt of 3P-(N-[3-aminopropyl]-l,4-butanediamine)-7a, 24R- dihydroxy-5a-cholestane-24-sulfate.

[0034] Also encompassed by the disclosure is a hand-held inhaler device comprising a lactate or dilactate salt of an aminosterol as described herein. In one aspect, the device is a single dose Dry Powder Inhaler (DPI). In another aspect, the device delivers aminosterol in the form of dry powder contained in a capsule. Further, the device can be a breath actuated inhaler.

[0035] Both the foregoing summary and the following description of the drawings and detailed description are exemplary and explanatory. They are intended to provide further details of the disclosure, but are not to be construed as limiting. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 shows the X-ray Structure of steroid 34.

[0037] Figures 2A-2D show that squalamine boosts survival rates and decreases viral titer loads against Yellow Fever (YF), an RNA virus in animal models. Treatment of YF in Syrian hamsters. Survival of Syrian hamsters treated before (Fig. 2A) or after (Fig. 2C) viral inoculation. Dosing regimens are indicated and described in Materials and Methods (Zasloff, 2011). Serum ALT was sampled at day 6 post-infection for the group receiving squalamine before (Fig. 2B) or after (Fig. 2D) viral inoculation [treatment groups (n = 10) and D5W (5% dextrose) placebo (n = 20)]. All animals that had survived 14 d remained alive until day 21 and were designated as cured.

[0038] Figures 3A-C shows cationic amphipathic design of protein SARS CoV-2 Protein 3a (Figure 3A) and structurally related cytolysins NK-lysin (Human) (Figure 3B) and Naegleriapore A1 (Figure 3C).

DETAILED DESCRIPTION

I. Overview

[0039] The present disclosure is directed to the surprising discovery that lactate and dilactate salts of aminosterols are surprisingly effective at treating microbial infections by inhaled or pulmonary administration. In one aspect, the method is used to treat a pulmonary microbial infection.

[0040] "Pulmonary application", "pulmonary administration", and/or “inhalation” or “inhaled administration” refers to any means of applying a composition of the present invention to the pulmonary system of a subject. The present invention is not limited to any particular modes of administration. Indeed, a variety of modes are contemplated to be useful for pulmonary administration including those described herein.

[0041] Thus, in one aspect encompassed is a method of treating a microbial infection comprising administering via inhalation a therapeutically effective amount of at least one lactate or dilactate salt of an aminosterol. The microbial infection can be for example bacterial, fungal, viral, protozoan, or any combination thereof.

[0042] Surprisingly, the compositions can be administered at very low doses. For example, the lactate or dilactate salt of the aminosterol can be administered at a very low dose of about 50 mg or less. In yet another aspect, the lactate or dilactate salt of the aminosterol is administered at a very low dose of about 45 mg or less, about 40 mg or less, about 35 mg or less, about 30 mg or less, about 25 mg or less, about 20 mg or less, about 15 mg or less, about 14 mg or less, about 13 mg or less, about 12 mg or less, about 11 mg or less, about 10 mg or less, about 9 mg or less, about 8 mg or less, about 7 mg or less, about 6 mg or less, about 5 mg or less, about 4 mg or less, about 3 mg or less, about 2 mg or less, about 1 mg or less, about 0.5 mg or less, and greater than 0 mg, and including about 5 mg. In one aspect, the lactate or dilactate salt of the aminosterol is administered by nebulization at a concentration of about 0.02% in about 0.9% saline up to a total single dose of about 5 mg.

[0043] In yet another aspect, the lactate or dilactate salt of the aminosterol is administered at about 9.5 mg or less, about 9 mg or less, about 8.5 mg or less, about 8 mg or less, about 7.5 mg or less, about 7 mg or less, about 6.5 mg or less, about 6 mg or less, about 5.5 mg or less, about 5 mg or less, about 4.5 mg or less, about 4 mg or less, about 3.5 mg or less, about 3 mg or less, about 2.5 mg or less, about 2 mg or less, about 1.5 mg or less, about 1 mg or less, and greater than about 0 mg.

[0044] In another aspect, the very low dose of the lactate or dilactate aminosterol salt, administered via inhalation or via pulmonary administration, does not have a measurable effect when administered orally. [0045] As used herein, the term "microorganism" refers to any species or type of microorganism, including but not limited to, bacteria, viruses, archaea, fungi, protozoans, mycoplasma, prions, and parasitic organisms. The term microorganism encompasses both those organisms that are in and of themselves pathogenic to another organism (e.g., animals, including humans, and plants) and those organisms that produce agents that are pathogenic to another organism, while the organism itself is not directly pathogenic or infective to the other organism.

[0046] The present invention is not limited by the number of doses of aminosterol lactate or dilactate salt administered. In some embodiments, multiple doses are administered on separate days. In some embodiments, the multiple doses are administered on the same day.

[0047] A “subject in need” is a human or animal at risk of a microbial infection, or which has contracted a microbial infection. As used herein, the terms "at risk for disease" and "at risk for infection" refer to a subject that is predisposed to experiencing a particular disease and/or infection. This predisposition may be genetic (e.g., a particular genetic tendency to experience the disease, such as heritable disorders), or due to other factors (e.g., environmental conditions, exposures to detrimental compounds present in the environment, etc.). Thus, it is not intended that the present invention be limited to any particular risk (e.g., a subject may be "at risk for disease" simply by being exposed to and interacting with other people that carry a risk of transmitting a pathogen), nor is it intended that the present invention be limited to any particular disease and/or infection.

[0048] In one aspect, the present invention encompasses a method of treating a subject, wherein the subject is susceptible to or has an infection by one or more microorganisms.

[0049] In one aspect, the present invention encompasses a method of treating a subject, wherein the subject is susceptible to or has an infection by one or more gram-positive or gram negative bacterial species. In another aspect, the present invention encompasses a method of treating a subject, wherein the subject is susceptible to or has an infection by one or more viruses. In yet another aspect, the present invention encompasses a method of treating a subject, wherein the subject is susceptible to or has an infection by one or more fungi. In one aspect, the present invention encompasses a method of treating a subject, wherein the subject is susceptible to or has an infection by one or more protozoan. In all of the methods described herein, the infection can be but is not limited to a pulmonary infection.

[0050] Compositions and methods of the present invention also find use in the treatment and/or prevention of a host of respiratory infections (e.g., respiratory infections of the upper respiratory tract (e.g., nose, ears, sinuses, and throat) and the lower respiratory tract (e.g., trachea, bronchial tubes, and lungs)).

II. Microorganisms

[0051] The microbial infection can be caused for example by a virus, bacteria, fungus, or protozoan. The present invention is not limited by the type of microbe treated.

[0052] The viral infection to be treated or prevented can be caused by any virus, including but not limited to, "African Swine Fever Viruses," Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Bimaviridae, Birnaviridae, Bunyaviridae, Caliciviridae, Caulimoviridae, Circoviridae, Coronaviridae, Cystoviridae, Dengue, EBV, HIV, Deltaviridae, Filviridae, Filoviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Iridoviridae, Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Myoviridae, Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Paramyxoviridae, Prions, Parvoviridae, Phycodnaviridae, Picomaviridae (e.g. Rhinovirus, Poliovirus), Poxviridae (such as Smallpox or Vaccinia), Potyviridae, Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV- II, Lentivirus), Rhabdoviridae, Tectiviridae, Togaviridae (e.g., Rubivirus), or any combination thereof. In another embodiment of the invention, the viral infection is caused by a virus selected from the group consisting of herpes, pox, papilloma, corona, influenza, hepatitis, sendai, sindbis, vaccinia viruses, west nile, hanta, or viruses which cause the common cold. In another embodiment of the invention, the condition to be treated is selected from the group consisting of AIDS, viral meningitis, Dengue, EBV, hepatitis, and any combination thereof.

[0053] In one aspect, the viral infection is caused by a coronavirus. For example, the coronavirus can be selected from the group consisting of an Alphacoronavirus; a Colacovirus such as Bat coronavirus CDPHE15; a Decacovirus such as Bat coronavirus HKU10 or Rhinolophus ferrumequinum alphacoronavirus HuB-2013; a Duvinacovims such as Human coronavirus 229E; a Luchacovirus such as Lucheng Rn rat coronavirus; a Minacovirus such as a Ferret coronavirus or Mink coronavirus 1; a Minunacovirus such as Miniopterus bat coronavirus 1 or Miniopterus bat coronavirus HKU8; a Myotacovirus such as Myotis ricketti alphacoronavirus Sax-2011; a nyctacovirus such as Nyctalus velutinus alphacoronavirus SC- 2013; a Pedacovirus such as Porcine epidemic diarrhea virus or Scotophilus bat coronavirus 512; a Rhinacovirus such as Rhinolophus bat coronavirus HKU2; a Setracovirus such as Human coronavirus NL63 or NL63 -related bat coronavirus strain BtKYNL63-9b; a Tegacovirus such as Alphacoronavirus 1; a Betacoronavirus; a Embecovirus such as Betacoronavirus 1, Human coronavirus OC43, China Rattus coronavirus HKU24, Human coronavirus HKU 1 or Murine coronavirus; a Hibecovirus such as Bat Hp-betacoronavirus Zhejiang2013; a Merbecovirus such as Hedgehog coronavirus 1, Middle East respiratory syndrome-related coronavirus (MERS- CoV), Pipistrellus bat coronavirus HKU5 or Tylonycteris bat coronavirus HKU4; aNobecovirus such as Rousettus bat coronavirus GCCDC1 or Rousettus bat coronavirus HKU9, a Sarbecovirus such as a Severe acute respiratory syndrome-related coronavirus, Severe acute respiratory syndrome coronavirus (SARS-CoV) or Severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2, COVID-19); a Deltacoronavirus; an Andecovirus such as Wigeon coronavirus HKU20; a Buldecovirus such as Bulbul coronavirus HKU 1 1 , Porcine coronavirus HKU 15, Munia coronavirus HKU 13 or White-eye coronavirus HKU 16; a Herdecovirus such as Night heron coronavirus HKU 19; a Moordecovirus such as Common moorhen coronavirus HKU21 ; a Gammacoronavirus; a Cegacovirus such as Beluga whale coronavirus SW1; and an Igacovirus such as Avian coronavirus. In one aspect, the coronavirus is SARS-CoV-2.

[0054] The terms "bacteria" and "bacterium" refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included within this term are prokaryotic organisms that are Gram-negative or Gram-positive. Examples of bacteria include, but are not limited to, bacterial cells of a genus of bacteria selected from the group comprising Salmonella, Shigella, Escherichia, Enter obacter, Serratia, Proteus, Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella, Hafiiia, Ewingella, Kluyvera, Morganella, Planococcus, Stomatococcus, Micrococcus, Staphylococcus, Vibrio, Aeromonas, Plessiomonas, Haemophilus, Actinobacillus, Pasteurella, Mycoplasma, Ureaplasma, Rickettsia, Coxiella, Rochalimaea, Ehrlichia, Streptococcus, Enterococcus, Aerococcus, Gemella, Lactococcus, Leuconostoc, Pedicoccus, Bacillus, Corynebacterium, Arcanobacterium, Actinomyces, Rhodococcus, Listeria, Erysipelothrix, Gardnerella, Neisseria, Campylobacter, Arcobacter, Wolinella, Helicobacter, Achromobacter, Acinetobacter, Agrobacterium, Alcaligenes, Chryseomonas, Comamonas, Eikenella, Flavimonas, Flavobacterium, Moraxella, Oligella, Pseudomonas, Shewanella, Weeksella, Xanthomonas, Bordetella, Franciesella, Brucella, Legionella, Afipia, Bartonella, Calymmatobacterium, Cardiobacterium, Streptobacillus, Spirillum, Peptostreptococcus, Peptococcus, Sarcinia, Coprococcus, Ruminococcus, Propionibacterium, Mobiluncus, Bifidobacterium, Eubacterium, Lactobacillus, Rothia, Clostridium, Bacteroides, Porphyromonas, Prevotella, Fusobacterium, Bilophila, Leptotrichia, Wolinella, Acidaminococcus, Megasphaera, Veilonella, Norcardia, Actinomadura, Norcardiopsis, Streptomyces, Micropolysporas, Thermoactinomycetes, Mycobacterium, Treponema, Borrelia, Leptospira, and Chlamydiae.

[0055] The term "fungi" is used in reference to eukaryotic organisms such as molds and yeasts, including dimorphic fungi.

[0056] Compositions and methods of the present invention can be utilized to treat (e.g., kill and/or inhibit growth of) organisms capable of forming biofilms including, but not limited to, dermatophytes (e.g, Microsporum species such as Microsporum canis, Trichophyton species such as Trichophyton rubrum and Trichophyton mentagrophytes), yeasts (e.g., Candida albicans, Candidaparapsilosis, Candida glabrata, Candida tropicalis, and other Candida species including drug resistant Candida species), Epidermophytonfloccosum, Malasseziafuurfur (Pityropsporon orbiculare, Pityropsporon ovale) Cryptococcus neoformans, Aspergillusfumigatus and other Aspergillus species, Zygomycetes (Rizopus, Mucor), hyalohyphomycosis (Fusarium species), Paracoccidiodes brasiliensis, Blastmyces dermatitides, Histoplasma capsulatum, Coccidiodes immitis, Sporothrix schenckii, and Blastomyces. [0057] The present invention also provides compositions and methods for treating (e.g., killing and/or inhibiting growth of) microorganisms that heretofore display resistance to a broad spectrum of antibiotics (e.g., species of the genus Acinetobacter ).

[0058] Acinetobacter species are generally considered nonpathogenic to healthy individuals. However, several species persist in hospital environments and cause severe, life-threatening infections in compromised patients. The spectrum of antibiotic resistances of these organisms together with their survival capabilities make them a threat to hospitals as documented by recurring outbreaks both in highly developed countries and elsewhere. Infections occur in immunocompromised individuals, and the strain A. baumannii is the second most commonly isolated nonfermenting bacteria in human specimens. Acinetobacter is frequently isolated in nosocomial infections and is especially prevalent in intensive care units, where both sporadic cases as well as epidemic and endemic occurrence is common. A. baumannii is a frequent cause of nosocomial pneumonia, especially of late-onset ventilator associated pneumonia. It can cause various other infections including skin and wound infections, bacteremia, and meningitis. A. hvoffi is also causative of meningitis. A. baumannii can survive on the human skin or dry surfaces for weeks.

[0059] Acinetobacter species are innately resistant to many classes of antibiotics, including penicillin, chloramphenicol, and often aminoglycosides. Thus, in some embodiments, compositions and methods of the present invention are utilized to treat (e.g., kill and/or inhibit growth of) bacteria of th e Acinetobacter species (e.g., individually or in combination with other treatments (e.g., carbapenems, polymyxin B, and/or sulbactam)).

III. Exemplary Respiratory Infections/Diseases

[0060] In one aspect of the disclosure, encompassed are methods of treating pulmonary and/or respiratory infections with an inhaled lactate or dilactate aminosterol composition. "Respiratory infection" and "pulmonary infection" refer to a microbial infection (e.g., bacterial, viral, fungal, etc.) of the respiratory tract. In humans, the respiratory tract comprises the upper respiratory tract (e.g., nose, throat or pharynx, and larynx); the airways (e.g., voice box or larynx, windpipe or trachea, and bronchi); and the lungs (e.g., bronchi, bronchioles, alveolar ducts, alveolar sacs, and alveoli).

[0061] "Respiratory disease", "pulmonary disease," "respiratory disorder", "pulmonary disorder," "respiratory condition", "pulmonary condition," "pulmonary syndrome," and "respiratory syndrome" refer to any one of several ailments that involve inflammation and affect a component of the respiratory system including especially the trachea, bronchi and lungs.

[0062] In another aspect of the disclosure, encompassed are methods of treating such conditions comprising administration of an inhaled lactate or dilactate aminosterol composition. Examples of such ailments include acute alveolar disease, obstructive respiratory disease (e.g., asthma; bronchitis; and chronic obstructive pulmonary disease, referred to as COPD), upper airway disease (e.g., such as otitis media, and rhinitis/sinusitis), cystic fibrosis (CF), insterstitial lung disease, allergy, and respiratory infection (e.g., pneumonia, pneyumocystis carinii, and respiratory syncitial virus (RSV)).

[0063] Specific examples of acute alveolar disease include acute lung injury (ALI), acute respiratory distress syndrome (ARDS), meconium aspiration syndrome (MAS) and respiratory distress syndrome (RDS). ALI is associated with conditions that either directly or indirectly injure the air sacs of the lung, the alveoli. ALI is a syndrome of inflammation and increased permeability of the lungs with an associated breakdown of the lungs' surfactant layer. The most serious manifestation of ALI is ARDS. Among the causes of ALI are complications typically associated with certain major surgeries, mechanical ventilator induced lung injury (often referred to as VILI), smoke inhalation, pneumonia, and sepsis.

[0064] Cystic fibrosis (CF) is a life-threatening disorder that causes severe lung damage due to a defective transmembrane protein called CFTR responsible for the balance of electrolytes.

Thick mucus forms plugging the tubes, ducts and passageways in the lungs. This environment is ideal for opportunistic bacteria to establish biofilm communities, leading to respiratory infections. Systemically-administered antibiotics can decrease the frequency and severity of exacerbations; however, the bacteria are never be completely eradicated from the airways and the lungs. Nebulized antibiotics are used, but resistance emergence and/or colonization of different resistant species is a major concern. Cystic fibrosis (CF) results in the functional impairment of innate respiratory defense mechanisms, providing an environment for colonization of pathogenic bacterial species such as Staphylococcus aureus and Haemophilus influenzae, and a number of opportunistic species such as Pseudomonas aeruginosa, Achromobacter xylosoxidans, Stenotrophomonas maltophilia, Ralstonia spp. , Pandoraea spp., and the Burkholderia cepacia complex (Bcc) species. The Bcc comprises a group of at least 17 phylogenetically related saprophytic gram-negative bacilli, most of which can form biofilm. They are particularly difficult to treat and are associated with increased rates of morbidity and mortality in CF patients. They also are among the most antimicrobial-resistant bacterial species encountered in human infections. Once established, the infection and associated inflammation are rarely eliminated, resulting in progressive lung disease ending in pulmonary failure and death.

A. Coronavirus

[0065] In one aspect, a method of treating or preventing an infection by a coronavirus in a subject is provided, comprising administering a therapeutically effective amount of the aminosterol compound of any embodiment herein or the composition of any embodiment herein to the subj ect.

[0066] In some embodiments, the coronavirus comprises a virus selected from the group consisting of an Alphacoronavirus; a Colacovirus such as Bat coronavirus CDPHE15; a Decacovirus such as Bat coronavirus HKU10 or Rhinolophus ferrumequinum alphacoronavirus HuB-2013; a Duvinacovirus such as Human coronavirus 229E; a Luchacovirus such as Lucheng Rn rat coronavirus; a Minacovirus such as a Ferret coronavirus or Mink coronavirus 1; a Minunacovirus such as Miniopterus bat coronavirus 1 or Miniopterus bat coronavirus HKU8; a Myotacovirus such as Myotis ricketti alphacoronavirus Sax-2011; a nyctacovirus such as Nyctalus velutinus alphacoronavirus SC-2013; a Pedacovirus such as Porcine epidemic diarrhea virus or Scotophilus bat coronavirus 512; a Rhinacovirus such as Rhinolophus bat coronavirus HKU2; a Setracovirus such as Human coronavirus NL63 or NL63 -related bat coronavirus strain BtKYNL63-9b; a Tegacovirus such as Alphacoronavirus 1; a Betacoronavirus; a Embecovirus such as Betacoronavirus 1, Human coronavirus OC43, China Rattus coronavirus HKU24, Human coronavirus HKU 1 or Murine coronavirus; a Hibecovirus such as Bat Hp- betacoronavirus Zhejiang2013; a Merbecovirus such as Hedgehog coronavirus 1, Middle East respiratory syndrome-related coronavirus (MERS-CoV), Pipistrellus bat coronavirus HKU5 or Tylonycteris bat coronavirus HKU4; a Nobecovirus such as Rousettus bat coronavirus GCCDC1 or Rousettus bat coronavirus HKU9, a Sarbecovirus such as a Severe acute respiratory syndrome-related coronavirus, Severe acute respiratory syndrome coronavirus (SARS-CoV) or Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19); a Deltacoronavirus; an Andecovirus such as Wigeon coronavirus HKU20; a Buldecovirus such as Bulbul coronavirus HKU11, Porcine coronavirus HKU 15, Munia coronavirus HKU 13 or White- eye coronavirus HKU 16; a Herdecovirus such as Night heron coronavirus HKU 19; a Moordecovirus such as Common moorhen coronavirus HKU21 ; a Gammacoronavirus; a Cegacovirus such as Beluga whale coronavirus SW1; and an Igacovirus such as Avian coronavirus.

[0067] In some embodiments, the coronavirus is encoded by a polynucleotide comprising the sequence of SARS-CoV-2, or a polynucleotide having at least 80% sequence identity to the polynucleotide comprising the sequence of SARS-CoV-2. In some embodiments, the coronavirus comprises or is characteristic of human coronavirus 229E, human coronavirus OC43, SARS-CoV, HCoVNL63, HKU1, MERS-CoV, or SARS-CoV-2. In some embodiments, the coronavirus comprises or is characteristic of SARS-CoV-2.

[0068] In some embodiments, the subject is deemed at risk for severe illness and/or serious complications from the infection. In some embodiments, the subject is about age 50 or older, about age 55 or older, about age 60 or older, or about age 65 or older. In some embodiments, the subject suffers from one or more pre-existing conditions selected from the group consisting of diabetes, asthma, a respiratory disorder, high blood pressure, and heart disease. In some embodiments, the subject is immunocompromised. In some embodiments, the subject is immunocompromised due to AIDS, cancer, a cancer treatment, hepatitis, an auto-immune disease, steroid receiving, immunosenescence, or any combination thereof. [0069] In some embodiments, administration increases the chance of survival for the subject following exposure to a coronavirus. In some embodiments, the chance of survival is increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, as measured using any clinically recognized technique. In other aspects, administration can improve respiratory symptoms, shorten duration of virus shedding, reduce duration of hospitalization, interfere with viral replication, stimulate tissue regeneration, or any combination thereof, all by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, as measured using any clinically recognized technique..

[0070] In some embodiments, the subject is exposed to or is anticipated to be exposed to an individual who is contagious for a coronavirus. In some embodiments, the individual who is contagious for a coronavirus has one or more symptoms selected from the group consisting of fever, cough, shortness of breath, diarrhea, sneezing, runny nose, and sore throat. In some embodiments, the subject is a healthcare worker, aged 60 years or older, frequent traveler, military personnel, caregiver, or a subject with a preexisting condition that results in increased risk of mortality with infection.

[0071] In some embodiments, the method further comprises administering one or more antiviral drugs. In some embodiments, the one or more antiviral drugs are selected from the group consisting of chloroquine, hydroxychloroquine, darunavir, galidesivir, interferon beta, lopinavir, ritonavir, remdesivir, and triazavirin (or any other antiviral drug described herein).

[0072] In some embodiments, administration reduces the risk of transmission of coronavirus. In some embodiments, the reduction in risk of transmission is by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, as measured using any clinically recognized technique. In some embodiments, the medically recognized technique comprises PCR (polymerase chain reaction), a test of Table 1 below, or immunoassay.

[0073] Cells that are rich in a cell-surface receptor called angiotensin-converting enzyme 2 (ACE2) are more readily invaded by coronaviruses because the virus requires that receptor (ACE2) to enter a cell. In one aspect a method of treating and or preventing infection by a coronavirus (i.e., SARS-CoV-2) in a subject is provided, comprising administering to an ACE2- rich tissue of the subject an aminosterol disclosed herein. ACE-2 rich tissues may include for example lung, alveoli, renal, neural cortex, brain stem, and digestive tract tissue. In some embodiments, the subject overexpresses ACE2.

B. COVID-19

[0074] Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease was first identified in December 2019 in Wuhan, the capital of China's Hubei province, and has since spread globally, resulting in the ongoing 2019-20 coronavirus pandemic. Symptoms include fever, cough, shortness of breath, diarrhea, sneezing, runny nose, blood clotting, low blood O2, and sore throat.

[0075] In one aspect, the subject has been diagnosed with COVID-19, is at risk of contracting COVID-19, or is suspected of suffering from COVID-19. The method comprises administering to the subject a therapeutically effective amount of a composition comprising at least one aminosterol via inhalation or pulmonary administration, wherein the aminosterol is in a lactate or dilactate salt form. It is expected that administration of the aminosterol lactate or dilactate salt dosage via inhalation or pulmonary administration will increase survival and/or decrease undesirable side effects of COVID-19. For example, administration can improve respiratory symptoms, shorten duration of virus shedding, reduce duration of hospitalization, interfere with viral replication, stimulate tissue regeneration, or any combination thereof.

[0076] In some embodiments, of the present methods, the subject has been diagnosed with and/or tested positive for COVID-19. COVID-19 tests include any commercially available test known in the art, for example those in Table 1 below, or a positive test result may be determined using, for example, polymerase chain reaction (PCR) or immunoassay. In one embodiment, the subject has been diagnosed with COVID-19, is at risk of contracting COVID-19, or is suspected of suffering from COVID-19. Table 1: Commercially available tests for SARS-CoV-2 and COVID-19 Company Name Test Name

Table 1: Commercially available tests for SARS-CoV-2 and COVID-19 Company Name Test Name

[0077] In one aspect, the composition can be administered to a mechanically ventilated subject. For example, the composition can be administered to the subject using an inhaler or nebulizer device connected to the ventilator via an actuator device. In another aspect, the device can be connected to the ventilator using a spacer chamber. In one embodiment, the composition is administered to the mechanically ventilated subject using a dry powder inhaler, soft mist inhaler, or intratracheal nebulizing catheter adapted for in-line use.

[0078] In another embodiment, the coronavirus infection is correlated with pneumonia and/or a lung infection.

[0079] The method can additionally comprise administering one or more compounds selected from the group consisting of bronchodilators, inhaled corticosteroids, antibiotics, pulmonary surfactant, mucolytics, biologicals, genes, prostanoids, surfactants, heparin, morphine, furosemide, and combinations thereof. For example, (the bronchodilator can be albuterol, formoterol, arformoterol, fenoterol, metaproterenol, or ipratropium; (b) the inhaled corticosteroid can be budesonide, beclomethasone, or fluticasone; (c) the antibiotic can be tobramycin, amikaci, amikacin, fosfomycin, colistin, ciprofloxacin, ribavirin, and amphotericin B; (d) the surfactant can be Exosurf, Survanta, Curosurf, Infasurf, and KL4; (e) the mucolytic can be N-acetylcysteine or dornase alfa; (f) the biological can be a monoclonal antibody; (g) the gene can be an siRNA; and (h) the prostanoid can be epoprostenol, iloprost, or treprostinil.

[0080] In another aspect, the COVID-19 subject has a comorbidity selected from the group consisting of diabetes, hypertension, cardiovascular disease, cancer, prior cancer treatment, cerebrovascular disease, chronic obstructive pulmonary disease (COPD), chronic kidney disease, sarcoidosis, obstructive lung disease, idiopathic pulmonary fibrosis (IPF), asthma, chronic Bronchitis, emphysema, cystic fibrosis/bronchiectasis, and pneumonia. In another aspect, the subject has a lung disease or respiratory disorder. In another embodiment, the subject has a comorbidity selected from any of the symptoms of COVID-19 discussed below. In some embodiments, symptoms to be treated by the present methods, or evaluated during the practice of the present methods may include any of the symptoms of COVID-19, including those discussed below, for example, increased immune response, cytokine storm, ischemia, encephalitis, stroke, loss of smell or taste, respiratory distress, and kidney failure or injury.

[0081] Subjects with COVID-19 may suffer from deleterious immune response as a result of the virus. Subjects may have a cytokine storm. In some embodiments, the subject has an elevated cytokine selected from the group consisting of interferons (IFN) such as Type I IFNs (IFN-a and IFN-b), type II IFN (IFN-g), Lambda IFNs (lFN-lI, IFN-/,2, and IFN-/,3); interleukins such as IL-la and IL-Ib; chemokines such as CXCL8 (IL-8), CCL2 (monocyte chemoattractant protein 1 [MCP-1]), and CCL11 (eotaxin); colony-stimulating factors (CSFs), such as granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony- stimulating factor (M-CSF), and granulocyte colony-stimulating factor (G-CSF); tumor necrosis factor (TNF); and combinations thereof. The elevated cytokine level may be determined using any medically recognized technique, for example, mass spectrometry, enzyme-linked immunosorbant assay (ELISA), or immunohistochemistry. The elevation may be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, relative to the subject before infection by coronavirus or relative to a healthy subject. In some embodiments, the method reduces the elevated cytokine by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, of its elevation relative to the subject before infection by coronavirus or relative to a healthy subject or a subject without COVID-19.

[0082] In some embodiments, the method further comprises administering one or more immunosuppressant drugs with the aminosterol. The immunosuppressant drug may treat or ameoleorate the immune response to coronavirus and reduce or eliminate the cytokine storm. In some embodiments, the immunosuppressant drug is one or more of tocilizumab; sarilumab; calcineurin inhibitors such as Tacrolimus and Cyclosporine; Antiproliferative agents such as Mycophenolate Mofetil, Mycophenolate Sodium and Azathioprine; mTOR inhibitors such as Sirolimus; and steroids such as Prednisone. [0083] In some embodiments, the subject has reduced blood O2. The reduction may be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, relative to the subject before infection by coronavirus or relative to a healthy subject or a subject without COVID-19. In some embodiments, the method increases the reduced blood C by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, of its reduction relative to the subject before infection by coronavirus or relative to a healthy subject. The increase in blood O2 may be measured using a medically recognized technique, for example, pulse oximeter or blood gas (ABG test).

[0084] In some embodiments the subject is suffering from acute respiratory distress syndrome (ARDS). Acute respiratory distress syndrome (ARDS) is a type of respiratory failure characterized by rapid onset of widespread inflammation in the lungs. Symptoms include shortness of breath, rapid breathing, and bluish skin coloration. For those who survive, a decreased quality of life is common. In some embodiments, the subject with COVID-19 is suffering from ischemia. In some embodiments the ischemia comprises ischemia of the kidney or myocardial ischemia.

[0085] In some embodiments, the method results in improvement in respiratory symptoms in the subject as measured by one or more medically recognized tests, for example, Respiratory Distress Scale. The improvement by comprise in increase or decrease in score by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, relative to the subject before administration of aminosterol.

[0086] “Viral shedding” refers to the expulsion and release of virus progeny following successful reproduction during a host-cell infection. Once replication has been completed and the host cell is exhausted of all resources in making viral progeny, the viruses may begin to leave the cell by several methods. In some embodiments, the method reduces viral shedding in the subject. Viral shedding may be measured by, for example, RT-PCR. The reduction may be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, relative to the subject before administration of aminosterol.

[0087] In some embodiments, the subjected is hospitalized and the method results in a reduction in hospital stay relative to a subject no receiving aminosterol administration. The reduction may be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, relative to a subject that does not receive aminosterol.

[0088] In some embodiments, the subject is suffering from the blood clot. The blood club may comprise lung thrombi, arterial or white thrombi, venous or red thrombi. In some embodiments, the subject has elevated D-dimer, relative to the subject before infection by coronavirus or relative to a healthy subject or a subject without COVID-19. The elevation may be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, relative to the subject before infection by coronavirus or relative to a healthy subject. In some embodiments, the method reduces the elevated D-dimer by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, of its elevation relative to the subject before infection by coronavirus or relative to a healthy subject or a subject without COVID-19.

[0089] In some embodiments, the subject is in renal failure. Kidney failure is common in COVID-19 subjects and 27% of 85 hospitalized patients in Wuhan, China had kidney failure (Diao et al., 2020). Those with acute kidney injury (AKI), were more than five times as likely to die as COVID-19 patients without it. In some embodiments, the subject has AKI. Diagnosis of kidney failure in COVID-19 subjects can be confirmed by blood tests such as BUN, creatinine, and GFR that measure the buildup of waste products in the blood. Urine tests may be ordered to measure the amount of protein, detect the presence of abnormal cells, or measure the concentration of electrolytes. In some embodiments, the subject is diabetic, thus being at increased risk for kidney injury.

[0090] In some embodiments the subject is suffering from encephalitis. Encephalitis is inflammation of the brain. Encephalopathy occurring in COVID-19 patients has been observed (Wadman et ah, 2020). Encephalitis symptoms may be assessed in subjects using a medically recognized technique, for example, Magnetic resonance imaging (MRI), Computed tomography scan (also called a CT or CAT scan), blood tests, urine and stool tests, sputum culture, electroencephalogram (EEG), or spinal tap (also called a lumbar puncture). Antiviral medications are often used to treat encephalitis. In one embodiment, the method further comprises administration of one or more antiviral encephalitis drugs. Antiviral encephalitis drugs may include, without limitation acyclovir, ganciclovir, and foscamet.

[0091] Seizures, “sympathetic storm,” loss of sense of smell have also been observed in patients. Sympathetic storm includes an increase in circulating corticoids and catecholamines or a stress response and symptoms can include alterations in level of consciousness, increased posturing, dystonia, hypertension, hyperthermia, tachycardia, tachypnea, diaphoresis, and agitation.

[0092] Ocular manifestations of COVID-19 have also been identified as symptoms. These include epiphora, conjunctival congestion, or chemosis (Wu et ah, 2020). Viral conjunctivitis is known to present with upper respiratory infections (colds, flus, etc.) and may be a symptom of COVID-19. In some embodiments, the symptom to be treated, prevented, or evaluated is conjunctivitis. Conjunctivitis may be assessed by a medically recognized technique such as slit-lamp, acuity testing, visual analogue scale, McMonnies/Chapman-Davies scale (MC-D), Validated bulbar redness scale (VBR), and Institute for Eye Research scale (IER).

[0093] More than half of COVID-19 patients hospitalized in two Chinese centers had elevated levels of enzymes indicating injury to the liver or bile ducts (Zhang et ah, 2020). Alanine transaminase (ALT), Aspartate transaminase (AST), Alkaline phosphatase (ALP), and Gamma- glutamyl transpeptidase (GGT) are liver enzymes that may be elevated. The elevation may be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, relative to the subject before infection by coronavirus or relative to a healthy subject. In some embodiments, the method reduces the elevated liver enzyme by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, of its elevation relative to the subject before infection by coronavirus or relative to a healthy subject or a subject without COVID-19.

[0094] In some embodiments, the method treats, prevents, or reverses gastro-intestinal tract damage resulting from COVID-19. In some embodiments, the method results in altering gene expression in the gut. As shown in 62/896,950, aminosterols increase the transcriptome in the subject, the entire disclosure of 62/896,950 is hereby incorporated by reference. In some embodiments, the methods result in an increase in gene transcription in the subject. In some embodiments, the methods result in an increase in protein synthesis or translation in the subject.

[0095] For example, in the methods of the invention, expression of at least one gene in the gut can be increased by about 50%, about 75%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 550%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, or about 1000%.

[0096] In some embodiments, the gene or protein is selected from one or more of caspase 14, collagen type XVII alpha 1, corneodesmosin, cornifelin, cy statin E/M, dermokine, desmocollin 1, desmoglein 1 beta, filaggrin, gap junction protein beta 4, gap junction protein beta 6, H19 imprinted maternally expressed transcript, hornerin, kallikrein related-peptidase 7 (chymotryptic stratum, keratin 1, keratin 10, keratinocyte differentiation associated protein, keratinocyte expressed proline- rich, late comified envelope 1A1, late cornified envelope 1A2, late cornified envelope IB, late cornified envelope 1C, late cornified envelope IE, late comified envelope IF, late comified envelope 1G, late cornified envelope 1H, late cornified envelope II, late cornified envelope 1J, late cornified envelope 1L, late cornified envelope 1M, late comified envelope 3C, late comified envelope 3E, late cornified envelope 3F, lectin galactose binding soluble 7, loricrin, sciellin, myoglobin, myosin binding protein C slow-type, myosin heavy polypeptide 1 skeletal muscle, myosin heavy polypeptide 8 skeletal muscle, myosin light chain phosphorylatable fast ske, myosin light polypeptide 3, myozenin 1, myozenin 2, and ti tin-cap.

IV. Combination Therapy

[0097] In some embodiments, the lactate or dilactate aminosterol compositions may be administered alone or in combination with one or more other therapeutic agents, such as an antimicrobial agent.

The additional therapeutic or antimicrobial agent can be administered via any pharmaceutically acceptable method, including but not limited to pulmonary or inhaled administration. In addition, an example of an additional therapeutic agent is one known to treat the condition the aminosterol is being administered to treat.

[0098] Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously. Administration “in combination” further includes the separate administration of one of the compounds or agents administered first, followed by the second. In some embodiments, co-administration can be via the same or different route of administration. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary.

[0099] The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent. In other embodiments, co-administration is preferable to treat and/or prevent infection by more than one type of infectious agent (e.g., bacteria and/or viruses). [0100] In some embodiments, the second agent is an antimicrobial agent selected from the group comprising, but not limited to, antibiotics, antibodies, antibacterial enzymes, peptides, and lanthione- containing molecules. In some embodiments, the antimicrobial interferes with or inhibits cell wall synthesis. In some embodiments, the antimicrobial is selected from the group including, but not limited to, b-lactams, cephalosporins, glycopeptides, aminoglycosides, sulfonomides, macrolides, folates, polypeptides and combinations thereof. In some embodiments, the antimicrobial interferes with protein synthesis (e.g., glycosides, tetracyclines and streptogramins).

[0101] In some embodiments, co-administration with a composition comprising a lactate or dilactate aminosterol permits administering a lower dose of an antimicrobial agent than would be administered without such co-administration.

[0102] Examples of drugs that are typically administered via aerosol to mechanically ventilated subjects are shown in Table 2 below.

Table 2

V. Aminosterol Compounds

[0103] The present disclosure is directed to methods of administering via inhalation at least one aminosterol lactate or dilactate salt. The aminosterol can be any aminosterol described herein, when it is in a lactate or dilactate salt form and formulated for inhalation or pulmonary administration.

[0104] U.S. Patent No. 6,962,909 discloses various aminosterols, and this disclosure is specifically incorporated by reference with respect to its teaching of aminosterol compounds. Any aminosterol known in the art, including those described in U.S. Patent No. 6,962,909, can be used in the disclosed methods, when it is in a lactate or dilactate salt form and formulated for inhalation or pulmonary administration.

[0105] In some embodiments, the aminosterol is squalamine lactate, squalamine dilactate, aminosterol 1436 lactate, aminosterol 1436 dilactate, or a combination thereof, where the aminosterol is formulated for inhalation or pulmonary administration.

[0106] Useful aminosterol compounds comprise a bile acid nucleus and a polyamine, attached at any position on the bile acid, such that the molecule exhibits a net positive charge contributed by the polyamine. Thus, in some embodiments, the disclosed methods comprise administering a therapeutically effective amount of one or more aminosterols having the chemical structure of Formula I, when the compund is in a lactate or dilactate salt form and formulated for inhalation or pulmonary administration:

Formula I wherein,

W is 24 S -OSO3 or 24R-0S0₃;

X is 3p-H₂N-(CH2)4-NH-(CH2) 3-NH- or 3a-H₂N-(CH₂) ₄-NH-(CH₂) 3-NH-; Y is 20R- CH₃; and Z is 7a or 7b -OH.

[0107] In another embodiment of the invention, the aminosterol is selected from the following group, where the compound is in a lactate or dilactate salt form and formulated for pulmonary or inhalation administration:

Compound 3;

Compound 7 (aminosterol 1436); and

Compound 8 (Squalamine).

[0108] Variants or derivatives of known aminosterols, such as squalamine, Aminosterol 1436, or an aminosterol isolated from Squalus acanthias, may be used in the disclosed compositions and methods, when the variant or derivative is in a lactate or dilactate salt form and formulated for inhalation or pulmonary administration.

[0109] In yet another embodiment of the invention, the aminosterol is a derivative of squalamine or aminosterol 1436 modified through medical chemistry to improve biodistribution, ease of administration, metabolic stability, or any combination thereof, and wherein the aminosterol is in a lactate or dilactate salt form and formulated for inhalation or pulmonary administration. In another embodiment, the aminosterol is modified to include one or more of the following: (1) substitutions of the sulfate by a sulfonate, phosphate, carboxylate, or other anionic moiety chosen to circumvent metabolic removal of the sulfate moiety and oxidation of the cholesterol side chain; (2) replacement of a hydroxyl group by a non-metabolizable polar substituent, such as a fluorine atom, to prevent its metabolic oxidation or conjugation; and (3) substitution of various ring hydrogen atoms to prevent oxidative or reductive metabolism of the steroid ring system.

[0110] In yet another embodiment, the aminosterol comprises a sterol nucleus and a polyamine, attached at any position on the sterol, such that the molecule exhibits a net charge of at least + 1, the charge being contributed by the polyamine.

[0111] In yet another embodiment, the aminosterol comprises a bile acid nucleus and a polyamine, attached at any position on the bile acid, such that the molecule exhibits a net positive charge being contributed by the polyamine. [0112] In some embodiments, the aminosterol is selected from the group consisting of the following: (a) squalamine lactate or dilactate salt; (b) a squalamine lactate or dilactate salt isomer; (c) Aminosterol 1436 lactate or dilactate salt; (d) an aminosterol comprising a sterol or bile acid nucleus and a polyamine, attached at any position on the sterol or bile acid, such that the molecule exhibits a net charge of at least + 1, the charge being contributed by the poly amine, in a lactate or dilactate salt form; (e) an aminosterol which is a derivative of squalamine modified through medical chemistry to improve biodistribution, ease of administration, metabolic stability, or any combination thereof, in a lactate or dilactate salt form; (f) an aminosterol modified to include one or more of the following and in a lactate or dilactate salt form: (i) substitutions of the sulfate by a sulfonate, phosphate, carboxylate, or other anionic moiety chosen to circumvent metabolic removal of the sulfate moiety and oxidation of the cholesterol side chain; (ii) replacement of a hydroxyl group by a non- metabolizable polar substituent, such as a fluorine atom, to prevent its metabolic oxidation or conjugation; and (iii) substitution of various ring hydrogen atoms to prevent oxidative or reductive metabolism of the steroid ring system; (g) an aminosterol in a lactate or dilactate salt form and that can inhibit the formation of actin stress fibers in endothelial cells stimulated by a ligand known to induce stress fiber formation, having the chemical structure of Formula I (above); or (h) any combination thereof.

A. Human squalamine aminosterol compounds

[0113] In one aspect, the aminosterol is a human squalamine molecule having the following formula:

Compound VI (ENT-06), where the compound is in a lactate or dilactate salt form.

[0114] In one embodiment the aminosterol has the formula:

C25 (R) Compound VI (ENT-06), where the compound is in a lactate or dilactate salt form.

[0115] In one aspect an aminosterol is provided having the formula:

Compound IV (D5 ENT-06), where the compound is in a lactate or dilactate salt form.

[0116] In one embodiment, the aminosterol has the formula:

C25 (R) Compound IV (D5 ENT-06), where the compound is in a lactate or dilactate salt form.

[0117] In another aspect, the aminosterol compound has the formula:

Compound V (D4 ENT-06), where the compound is in a lactate or dilactate salt form.

[0118] In some embodiments, the aminosterol compound has the formula:

C25 (R) Compound V (D4 ENT-06), where the compound is in a lactate or dilactate salt form.

[0119] In one embodiment, the aminosterol is a prodrug comprising a compound of formula:

wherein:

R¹ is H, an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C1-C6 alkenyl; and

R² is H or -C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; provided that at least one of R¹ and R² is not H, and where the compound is in a lactate or dilactate salt form.

[0120] In one embodiment, the aminosterol is a prodrug comprises a compound of formula:

C25 (R) Compound VI-P, where the compound is in a lactate or dilactate salt form.

[0121] In one aspect, the aminosterol comprises prodrug having the formula:

Compound IV-P, wherein:

R² is H or -C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; provided that at least one of R¹ and R² is not H, and wherein the compound is in a lactate or dilactate salt form.

[0122] In one embodiment, the aminosterol is a prodrug comprising a compound of the following formula:

C25 (R) Compound IV-P, where the compound is in a lactate or dilactate salt form.

[0123] In another aspect, the disclosure encompasses an aminosterol compound having the formula:

Compound V-P, wherein: R¹ is H, an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C1-C6 alkenyl; and R² is H or -C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; provided that at least one of R¹ and R² is not H, where the compound is in a lactate or dilactate salt form. [0124] In one embodiment, the aminosterol compound has the formula:

C25 (R) Compound V-P, where the compound is in a lactate or dilactate salt form.

[0125] The aminosterols may comprise an asymmetric carbon atom. As such, aminosterols of this disclosure can exist as either individual enantiomers, or mixtures of the two enantiomers. Accordingly, an aminosterol of the present disclosure can include both racemic mixtures, and also individual respective stereoisomers or diastereoisomers that are substantially free from another possible stereoisomer. The term “substantially free of other stereoisomers” as used herein means less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of other stereoisomers, or less than “about X”% of other stereoisomers (wherein X is a number between 0 and 100, inclusive) are present.

[0126] In some embodiments, the aminosterol is a derivative of any of the aminosterols disclosed herein, or a derivative of Compound VI (ENT-06), modified through medicinal chemistry to improve biodistribution, ease of administration, metabolic stability, or any combination thereof, where the aminosterol is in a lactate or dilactate salt form. In some embodiments, the aminosterol is modified to include one or more of the following:

(1) substitutions of the carboxylate by a sulfonate, phosphate, or other anionic moiety chosen to circumvent metabolic removal of the sulfate moiety and oxidation of the cholesterol side chain;

(2) replacement of a hydroxyl group by a non-metabolizable polar substituent, such as a fluorine atom, to prevent its metabolic oxidation or conjugation; and/or (3) substitution of various ring hydrogen atoms to prevent oxidative or reductive metabolism of the steroid ring system. B. Human aminosterol 1436 compounds

[0127] The aminosterol which is in a lactate or dilactate form and formulated for inhalation or pulmonary administration can be a human aminosterol 1436 compound. In one aspect such an aminosterol is provided having the formula:

(Compound III) (ENT-03), where the compound is in a lactate or dilactate salt form.

[0128] In one embodiment the aminosterol has the formula:

(C25 (R) Compound III) (ENT-03), where the compound is in a lactate or dilactate salt form.

[0129] In one aspect an aminosterol is provided having the formula:

(Compound IV), where the compound is in a lactate or dilactate salt form. [0130] In one embodiment, the aminosterol has the formula:

(C25 (R) Compound IV) (D5 ENT-03), where the compound is in a lactate or dilactate salt form.

[0131] In another aspect, the aminosterol compound has the formula:

Compound V (D4 ENT-03), where the compound is in a lactate or dilactate salt form.

[0132] In some embodiments, the aminosterol compound has the formula:

C25 (R) Compound V (D4 ENT-03), where the compound is in a lactate or dilactate salt form.

[0133] In one embodiment, the aminosterol is a prodrug comprising a compound of formula:

(Compound III-P), wherein:

[0134] In one embodiment, the aminosterol is a prodrug comprising a compound of formula:

(C25 (R) Compound III-P), where the compound is in a lactate or dilactate salt form.

[0135] In one aspect, the aminosterol is a prodrug comprising a compound having the formula:

(Compound IV-P), wherein:

R² is H or -C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; provided that at least one of R¹ and R² is not H, where the compound is in a lactate or dilactate salt form.

[0136] In one embodiment, the aminosterol is a prodrug comprising a compound of formula:

(C25 (R) Compound IV-P), where the compound is in a lactate or dilactate salt form.

[0137] In another aspect, the aminosterol compound havs the formula:

Compound V-P, wherein: R¹ is H, an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C1-C6 alkenyl; and R² is H or -C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; provided that at least one of R¹ and R² is not H, where the compound is in a lactate or dilactate salt form.

[0138] In one embodiment, the aminosterol compound has the formula:

[0139] The aminosterols of the present disclosure may comprise an asymmetric carbon atom.

As such, aminosterols of this disclosure can exist as either individual enantiomers, or mixtures of the two enantiomers. See for example the stereochemistry at C25 in the natural molecule depicted above. The present disclosure encompasses both the mixture at C25 and the natural R- orientation of the compound. Accordingly, an aminosterol of the present disclosure can include both racemic mixtures, and also individual respective stereoisomers that are substantially free from another possible stereoisomer. The term “substantially free of other stereoisomers” as used herein means less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of other stereoisomers, or less than “about X”% of other stereoisomers (wherein X is a number between 0 and 100, inclusive) are present.

[0140] In some embodiments, the aminosterol is a derivative of any of the aminosterols disclosed herein, modified through medical chemistry to improve biodistribution, ease of administration, metabolic stability, or any combination thereof. In some embodiments, the aminosterol is modified to include one or more of the following: (1) substitutions of the carboxylate by a sulfonate, phosphate, or other anionic moiety chosen to circumvent metabolic removal of the sulfate moiety and oxidation of the cholesterol side chain; (2) replacement of a hydroxyl group by a non-metabolizable polar substituent, such as a fluorine atom, to prevent its metabolic oxidation or conjugation; and/or (3) substitution of various ring hydrogen atoms to prevent oxidative or reductive metabolism of the steroid ring system.

C. Aminosterol Dilactate Compounds

[0141] In one embodiment, the aminosterol is a crystalline or amorphous dilactate compound as described in US Patent Nos. 7,981,876 and 8,716,270, specifically incorporated by reference.

[0142] For example, the aminosterol used in the inhalation or pulmonary methods described herein can be a crystalline form of the dilactate salt of 3P-(N-[3-aminopropyl]-l,4- butanediamine)-7a, 24R-dihydroxy-5a-cholestane-24-sulfate. In one aspect, an X-ray powder diffraction pattern of the salt has major diffraction angles of 12.5°, 16.6°, and 18.8° with relative peak intensities of 890, 829 and 756, respectively. In another aspect, an X-ray powder diffraction pattern of the salt has major diffraction angles of 10.2°, 13.0°, and 16.6° with relative peak intensities of 1826, 2305 and 1817, respectively. In another aspect, an X-ray powder diffraction pattern of the salt has major diffraction angles of 13.1 °, 17.7° and 18.3° with relative peak intensities of 939, 937 and 967, respectively. In another aspect, an X-ray powder diffraction pattern of the salt has major diffraction angles of 12.6°, 15.7° and 18.8° with relative peak intensities of 977, 891 and 1333, respectively. [0143] In another example, the aminosterol used in the inhalation or pulmonary methods described herein can be an amorphous form of the dilactate salt of 3P-(N-[3-aminopropyl]-l,4- butanediamine)-7a, 24R-dihydroxy-5a-cholestane-24-sulfate. In one aspect, an X-ray powder diffraction pattern of the salt has major diffraction angles of 15.5 to 15.6 degrees, 17.3 to 17.5 degrees, and 21.3 to 21.5 degrees, with relative peak intensities of 286, 391, and 107, respectively.

VI. Routes of Administration

[0144] In one aspect, a method of treating a subject in need is provided, wherein the subject has been diagnosed with a coronavirus infection, is suspected of having a coronavirus infection, and/or is at risk of developing a coronavirus infection, wherein the coronavirus is SARS-CoV-2, wherein the method comprises: administering to the subject a therapeutically effective amount of a composition comprising at least one aminosterol via intranasal, inhalation, or pulmonary administration, wherein the aminosterol is in a lactate or dilactate salt form.

[0145] In one embodiment, the composition is administered intranasally (IN) and the dose of the aminosterol for the subject is at a range of from about 0.001 mg up to about 6 mg/day, or any amount in-between these two values. In yet a further embodiment, the aminosterol composition is administered intranasally and the dose of the aminosterol for the subject is a dose which is sub therapeutic when administered orally or by injection.

[0146] In one embodiment, administering comprises nasal administration. Nasal administration may be accomplished via insufflation of solids, liquids or powders, inhalation of a gas, or via inhalation of a mist comprising the at least one aminosterol in a suitable carrier and optionally excipients. Suitable carriers and excipients are known to the skilled artisan and include buffers such as sodium phosphate, sodium citrate, and citric acid; solubilizers such as glycols, small quantities of alcohol, transcutol (diethylene glycol monoethyl ether), medium chain glycerides, labrasol (saturated polyglycolyzed Cx-C io glyceride), surfactants and cyclodextrins; preservatives such as parabens, phenyl ethyl alcohol, benzalkonium chloride, EDTA (ethylene diaminetetraaceticacid), and benzoyl alcohol; antioxidants such as sodium bisulfite, butylated hydroxytoluene, sodium metabisulfite and tocopherol; humectants such as glycerin, sorbitol and mannitol; surfactants such as polysorbet; bioadhesive polymers such as mucoadhesives; and penetration enhancers such as dimethyl sulfoxide (DMSO).

[0147] Nasal administration via inhalation of a mist may employ the use of metered-dose spray pumps. Typical volumes of aminosterol-comprising mist, delivered via a single pump of a metered-dose spray pump may be about 20-100 mΐ, 100-150 mΐ, or 150-200 mΐ. Such pumps offer high reproducibility of the emitted dose and plume geometry. The particle size and plume geometry can vary within certain limits and depend on the properties of the pump, the formulation, the orifice of the actuator, and the force applied.

VII. Compositions

[0148] In another aspect, provided herein are compositions comprising a lactate or dilactate salt of an aminosterol compound disclosed herein, and one or more pharmaceutically acceptable carriers and/or excipients. Administration of an aminosterol disclosed herein may comprise administration of the composition.

A. Pharmaceutical Carriers

[0149] While it is possible for a lactate or dilactate salt of an aminosterol compound to be administered alone, it is typical to administer it as a pharmaceutical formulation, together with one or more pharmaceutically acceptable carriers. The carrier(s) should be "acceptable" in the sense of being compatible with the lactate or dilactate salt of the aminosterol, and not unduly deleterious to the recipients thereof. Thus, in addition to being pharmaceutically acceptable for administration, the carrier(s) also should be compatible with the lactate or dilactate salt of the aminosterol.

[0150] Generally, the formulations are prepared by contacting a lactate or dilactate salt of an aminosterol described herein, with liquid carriers or finely divided solid carriers or both, depending on the mode of administration. Then, if necessary, the product is shaped into the desired formulation.

[0151] The carrier can comprise minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as gelatin, serum albumin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

[0152] Inhalable dosage forms may be administered. Inhalable or pulmonary dosage forms may include aerosols, dry powders, liquids or sprays. In some embodiments, the administration is with a metered dose inhaler. Inhalation refers to the delivery of the aminosterol through a respiratory passage, through the subject’s airways, such as the subject’s nose or mouth.

[0153] In instances where aerosol administration is appropriate, a lactate or dilactate salt of an aminosterol described herein can be formulated for administration as an aerosol using standard procedures. The term "aerosol" includes any gas-borne suspended phase of a compound described herein which is capable of being inhaled into the bronchioles or nasal passages, and includes dry powder aerosols, aqueous aerosols, and pulmonary aerosols. Specifically, “aerosol” includes a gas- born suspension of droplets comprising a compound described herein, as may be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. “Aerosol” also includes a dry powder formulation of a composition comprising a compound described herein suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones, Drug Delivery to the Respiratory Tract (Ellis Horwood, 1987); Gonda, Critical Reviews in therapeutic Drug Carrier Systems, 6:273-313 (1990); and Raeburn et al,. Pharmacol. Toxicol. Methods, 27:143-159 (1992).

[0154] A metered dose inhaler in the present context means a device capable of delivering a metered or bolus dose of drug, such as aminosterol, to the lungs. One example of the inhalation device can be a pressurized metered dose inhaler, a device which produces the aerosol clouds for inhalation from solutions, solids, and/or suspensions of drugs in chlorofluorocarbon (CFC) and/or hydrofluoroalkane (HFA) solutions.

[0155] The inhalation device can be also a dry powder inhaler. In such case, the aminosterol is inhaled in solid composition, usually in the form of a powder with particle size less than 10 micrometers in diameter or less than 5 micrometers in diameter.

[0156] The metered dose inhaler can be a soft mist inhaler (SMI), in which the aerosol cloud containing a respiratory drug can be generated by passing a solution containing the respiratory drug through a nozzle or series of nozzles. The aerosol generation can be achieved in SMI, for example, by mechanical, electromechanical or thermomechanical process. Examples of soft mist inhalers include the Respimat^® Inhaler (Boeringer Ingelheim GmbH), the AERx^® Inhaler (Aradigm Corp.), the Mystic™ Inhaler (Ventaira Pharmaceuticals, Inc.) and the Aira™ Inhaler (Chrysalis Technologies Incorporated). For a review of soft mist inhaler technology, see e.g. M. Hindle, The Drug Delivery Companies Report, Autumn/Winter 2004, pp. 31-34. The aerosol for SMI can be generated from a solution of the aminosterol further containing pharmaceutically acceptable excipients. The solution can be, for example, a solution of aminosterol in water, ethanol or a mixture thereof. Preferably, the diameter of the aminosterol-containing aerosol particles is less than about 10 microns, or less than about 5 microns, or less than about 4 microns.

[0157] The inhalatory formulation administered can include a hydrophobic substance in order to reduce sensitivity to humidity. Such hydrophobic substance is preferably leucine, which makes the particle disaggregation easier.

[0158] In case of production of a solid product in powder form, this can occur using different techniques, well consolidated in the pharmaceutical industry. The preparation of fine particles through spray-drying represents a preferred method according to the invention. In case of industrial production, this technique is undoubtedly preferred to freeze-drying, which at the moment is the most expensive drying process, both for the apparatus used, and for the yield and production times.

[0159] Aminosterol concentration in an aerosolable composition, such as a dry powder, used in a metered dose inhaler can range from about 500 pg/g to about 2500 pg/g. Aminosterol concentration in an aerosolable composition, such as a solution, used in a metered dose inhaler can range from about 500 pg/ml to about 2500 pg/ml, or from about 800 pg/ml to about 2200 pg/ml, or from about 1000 pg/ml to about 2000 pg/ml.

[0160] The dose of aminosterol that can be administered using a metered dose inhaler, for example, a dry powder inhaler, in a single event (a single pump or discharge of the inhaler) can be from about 20 mg or less, about 10 mg or less, about 9.5 mg or less, about 9 mg or less, about 8.5 mg or less, about 8 mg or less, about 7.5 mg or less, about 7 mg or less, about 6.5 mg or less, about 6 mg or less, about 5.5 mg or less, about 5 mg or less, about 4.5 mg or less, about 4 mg or less, about 3.5 mg or less, about 3 mg or less, about 2.5 mg or less, about 2 mg or less, about 1.5 mg or less, about 1 mg or less, and greater than about 0 mg. The pharmaceutically effective amount of aminosterol during pulmonary or inhaled administration may be, for example, any of the aforementioned amounts.

[0161] The aminosterol dose can be administered in 20 breaths or less, or in 10 breaths or less, or than 5 breaths or less. Preferably, aminosterol is administered in 3, 2 or 1 breaths. The total time of a single administering event can be less than 5 minutes, or less than 1 minute, or less than 30 seconds; aminosterol can be administered a single time per day or several times per day.

B. Dosage Forms & Dosing Period

[0162] The aminosterol compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

[0163] Formulations or compositions of the present technology may be packaged together with, or included in a kit with, instructions or a package insert. "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0164] The composition may comprise encapsulated aminosterol. Encapsulation with a coating that allows for time-release of aminosterol upon inhalation or pulmonary administration to the subject.

[0165] Pharmaceutical compositions according to the present technology may also comprise one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art. [0166] Examples of filling agents include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents include various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel^® PHI 01 and Avicel^® PHI 02, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™).

[0167] Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil^® 200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.

[0168] Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame. Examples of flavoring agents are Magnasweet^® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like.

[0169] Examples of preservatives include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0170] Suitable diluents include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and/or mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel^® PHI 01 and Avicel^® PHI 02; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose^® DCL21; dibasic calcium phosphate such as Emcompress^®; mannitol; starch; sorbitol; sucrose; and glucose.

[0171] Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.

[0172] In an exemplary embodiment, the dosage can be provided as a sterile filled syringe containing 1ml of the lactate or dilactate aminosterol at the desired dosage, e.g., about 10 mg/ml. It can be diluted prior to use in normal saline to a concentration of about 0.2 mg/ml (i.e. into 50 mis normal saline). In one aspect, the total dose administered will be based on the volume of liquid delivered by nebulizer. So, for example, for the lowest dose of 0.5mg, only 2.5mls of this would be placed in the nebulizer reservoir and delivered at the rate permissible by the nebulizer.

[0173] In one aspect, the method comprises administering a very low dose of the lactate or dilactate aminosterol salt, as described herein. For example, the very low dose can be about 50 mg or less. In other embodiments, the very low dose of the lactate or dilactate aminosterol salt can be about 10 mg or less, about 9.5 mg or less, about 9 mg or less, about 8.5 mg or less, about 8 mg or less, about 7.5 mg or less, about 7 mg or less, about 6.5 mg or less, about 6 mg or less, about 5.5 mg or less, about 5 mg or less, about 4.5 mg or less, about 4 mg or less, about 3.5 mg or less, about 3 mg or less, about 2.5 mg or less, about 2 mg or less, about 1.5 mg or less, about 1 mg or less, and greater than about 0 mg.

[0174] Aminosterol doses can be de-escalated (reduced) if any given aminosterol dose induces a persistent undesirable side effect, such as diarrhea, vomiting, or nausea. In another embodiment, a dose of an aminosterol can be varied plus or minus a defined amount to enable a modest reduction in a dose to eliminate adverse events, or a modest increase in a dose if clinical results suggest this is desirable - e.g., no or minimal adverse events and potential increased efficacy with a modest increase in dose. For example, in one embodiment an aminosterol dose can be increased or decreased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%.

[0175] The pharmaceutical composition comprising a lactate or dilactate aminosterol salt can be administered for any suitable period of time, including as a maintenance dose for a prolonged period of time. Dosing can be done on an as needed basis using any pharmaceutically acceptable dosing regimen. Aminosterol dosing can be no more than lx per day, once every other day, once every three days, once every four days, once every five days, once every six days, once a week, or divided over multiple time periods during a given day (e.g., twice daily). In an exemplary embodiment, dosing is lx/day.

[0176] In other embodiments, the composition can be administered: (1) as a single dose, or as multiple doses over a period of time; (2) at a maintenance dose for an indefinite period of time; (3) once, twice or multiple times; (4) daily, every other day, every 3 days, weekly, or monthly; (5) for a period of time such as about 1, about 2, about 3, or about 4 weeks, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 months, about 1 year, about 1.5 years, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, about 20, about 20.5, about 21, about 21.5, about 22, about 22.5, about 23, about 23.5, about 24, about 24.5, or about 25 years, or (6) any combination of these parameters, such as daily administration for 6 months, weekly administration for 1 or more years, etc.

[0177] Yet another exemplary dosing regimen includes periodic dosing, where an effective dose can be delivered once every about 1, about 2, about 3, about 4, about 5, about 6 days, or once weekly.

[0178] In an exemplary embodiment, the aminosterol dose is taken in the morning, i.e. on an empty stomach preferably within about two hours of waking up and may be followed by a period without food, such as for example about 60 to about 90 minutes. In other embodiments, the aminosterol dose is taken within about 15 min, about 30 min, about 45 min, about 1 hr, about 1.25 hrs, about 1.5 hrs, about 1.75 hrs, about 2 hrs, about 2.25 hrs, about 2.5 hrs, about 2.75 hrs, about 3 hrs, about 3.25 hrs, about 3.5 hrs, about 3.75 hrs, or about 4 hrs within waking up. In yet further embodiments, the aminosterol dose is followed by about period without food, wherein the period is at least about 30 min, about 45 mins, about 60 mins, about 1.25 hrs, about 1.5 hrs, about 1.75 hrs, or about 2 hrs.

VIII. Patient Populations

[0179] The disclosed aminosterols and compositions comprising the same can be used to treat a range of subjects, including human and non-human animals, including mammals, as well as immature and mature animals, including human children and adults. The human subject to be treated can be an infant, toddler, school-aged child, teenagers, young adult, adult, or elderly patient.

[0180] In embodiments disclosed herein relating to prevention, particular patient populations may be selected based on being “at risk for” the development of any of the conditions disclosed herein.

For example, genetic markers of the condition or family history may be used as signs to identify subjects likely to develop the particular condition. Thus, in some embodiments, prevention may involve first identifying a patient population at risk of developing the condition. Alternatively, certain symptoms are considered early signs of particular disorders. Thus, in some embodiments, a patient population may be selected for being “at risk” for developing the condition based on age and experiencing symptoms associated with the condition. Further genetic or hereditary signs may be used to refine the patient population.

[0181] In some embodiments, the subject is a male or non-pregnant female adult >18 years of age; Laboratory confirmed SARS-CoV-2 infection as determined by PCR (or a test in Table 1), or other commercial or public health assay in any specimen <72 hours prior to randomization; has crackles/rales; has Sp02 < 94% on room air; has acute respiratory failure requiring mechanical ventilation and/or supplemental oxygen; and/or has radiographic infiltrates.

IX. Kits

[0182] Aminosterol formulations or compositions of the disclosure may be packaged together with or included in a kit along with instructions or a package insert. Such instructions or package inserts may address recommended storage conditions, such as time, temperature and light, taking into account the shelf-life of the lactate or dilactate aminosterol salt. Such instructions or package inserts may also address the particular advantages of the the lactate or dilactate aminosterol salt, such as the ease of storage for formulations that may require use in the field, outside of controlled hospital, clinic or office conditions.

[0183] The disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more the lactate or dilactate aminosterol salt pharmaceutical compositions disclosed herein. The kits may include, for instance, containers filled with an appropriate amount of a lactate or dilactate aminosterol salt pharmaceutical composition, either as a powder, a tablet, to be dissolved, or as a sterile solution. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the lactate or dilactate aminosterol salt may be employed in conjunction with other therapeutic compounds. [0184] In other aspects, a kit comprising a pulmonary delivery (liquid or dry powder) device as described herein is disclosed. In one aspect, the kit may comprise one or more devices as disclosed herein, comprising a disclosed low dose lactate or dilactate aminosterol salt composition, wherein the device is sealed within a container sufficient to protect the device from atmospheric influences. The container may be, for example, a foil, or plastic pouch, particularly a foil pouch, or heat-sealed foil pouch. Suitable containers sufficient to adequately protect the device will be readily appreciated by one of skill in the art.

[0185] In one aspect, the kit may comprise one or more devices as disclosed herein, wherein the device may be sealed within a first protective packaging, or a second protective packaging, or a third protective packaging, that protects the physical integrity of the product. One or more of the first, second, or third protective packaging may comprise a foil pouch. The kit may further comprise instructions for use of the device. In one aspect, the kit contains two or more devices.

[0186] In one aspect, the kit may comprise a device as disclosed herein, and may further comprise instructions for use. In one aspect, the instructions may comprise visual aid/pictorial and/or written directions to an administrator of the device.

[0187] In another aspect, the kit encompasses a single dose Dry Powder Inhaler (DPI) device. The device delivers medicine in the form of dry powder contained in a capsule. Such single dose Dry Powder Inhalers (e.g., a “Handihaler”) are easier to use than metered dose inhalers because they do not require hand and breath co-ordination. “Handihaler” is a breath actuated inhaler that means that one draws the medication from the device using force of one’s own breath.

[0188] Examples of other devices include but are not limited to pressurized metered-dose inhalers and nebulizers (jet, ultrasonic, and vibrating mesh).

X. Definitions

[0189] The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

[0190] Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. Any suitable materials and/or methodologies known to those of ordinary skill in the art can be utilized in carrying out the methods described herein.

[0191] The terms “pharmacologically effective amount” or “therapeutically effective amount” of a composition, aminosterol or agent, as provided herein, refer to a nontoxic but sufficient amount of the composition, aminosterol or agent to provide the desired response. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein. For convenience only, exemplary dosages are provided herein. Those skilled in the art can adjust such amounts in accordance with the methods disclosed herein to treat a specific subject suffering from a specified symptom or disorder. The therapeutically effective amount may vary based on the route of administration and dosage form.

[0192] As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consisting essentially of’ when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.

[0193] The term “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used,

“about” will mean up to plus or minus 10% of the particular term. For example, in some embodiments, it will mean plus or minus 5% of the particular term. Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number, which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0194] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0195] “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the disclosure and that causes no significant adverse toxicological effects to the patient.

[0196] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, wetting agents (e.g., sodium lauryl sulfate), isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like. Examples of carriers, stabilizers and adjuvants have been described and are known in the art (See e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa.

(1975), incorporated herein by reference).

[0197] “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0198] As used in the description of the disclosure and the appended claims, the singular forms “a”, “an” and “the” are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless the context clearly indicates otherwise. Also, as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0199] As used herein the term “aminosterol” refers to an amino derivative of a sterol. [0200] The term “administering” as used herein includes prescribing for administration as well as actually administering and includes physically administering by the subject being treated or by another.

[0201] As used herein “subject,” “patient,” or “individual” refers to any subject, patient, or individual, and the terms are used interchangeably herein. In this regard, the terms “subject,” “patient,” and “individual” includes mammals, and, in particular humans. When used in conjunction with “in need,” the term “subject,” “patient,” or “individual” intends any subject, patient, or individual having or at risk for a specified symptom or disorder.

[0202] The terms “treatment,” “treating,” or any variation thereof includes reducing, ameliorating, or eliminating (i) one or more specified symptoms and/or (ii) one or more symptoms or effects of a specified disorder. The terms “prevention,” “preventing,” or any variation thereof includes reducing, ameliorating, or eliminating the risk of developing (i) one or more specified symptoms and/or (ii) one or more symptoms or effects of a specified disorder.

[0203] “Prodrug” a prodrug is a medication or compound that, after administration, is metabolized (i.e., converted within the body) into a pharmacologically active drug. In some embodiments, a prodrug comprises a derivative of Compound VI, wherein the alcohol and/or the carboxylate has been esterified.

[0204] “Optionally substituted” refers to a group selected from that group and a substituted form of that group. A “substituted” group, refers to that group substituted with a chemical substituent, for example be replacement of a C-H bond with a bond between that C and the substituent. In one embodiment, substituents are selected from, for example, CF3, OCF3, halo, haloaryl, C1-C₆ alkoxy, acyl, propionyl, butyrl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, carboxyl ester, carboxyl ester amino, (carboxyl ester)oxy, haloalkyl, aryloxy, haloalkoxy, hydroxyl, thiol, dihydroxy, aminohydroxy, carboxy, amido, sulfoxy, sulfonyl, haloaryloxy, aryl, benzyl, benzyloxy, heteroaryl, nitrile, C1-C₆ alkyl, C1-C₆ alkenyl, C1-C₆ alkynyl, C₃-C₆ cycloalkyl, C1-C₆ haloalkyl, C1-C₆ haloalkenyl, C1-C₆ haloalkynyl, C₃-C₆ halocycloalkyl, C₆-C1₀ aryl, C₃-C8 cycloalkyl, C2-C10 heterocyclyl, C1-C10 heteroaryl, -N3, nitro, -CO2H or a C1-C6 alkyl ester thereof, or combinations thereof.

XI. Examples

Example 1: Synthetic Procedures

[0205] Squalamine lactate 39b was synthesized according to Schemes 1 and 2 below.

Scheme 1: Synthesis of intermediate 34

[0206] SQM-4 is commercially available from Pfizer Center One, who prepares it from a proprietary soy derived steroid that is available in multi-ton quantities. Also, it has been synthesized by Bridge Organics from the inexpensive and abundant steroid chenodeoxycholic acid by a modification of the published route (Zhang 2005). Referring to Scheme 1, SQM-4 was converted to SQM-5 by reaction with ethylene glycol under / oluenesulfonic acid catalysis in dichloromethane at 40 °C (4-8 h) and crystallization from acetonitrile in 85% yield. Reduction of SQM-5 to the diol SQM-6 was achieved by slow addition (160 min) to a suspension of lithium aluminum hydride in tetrahydrofuran at 10 °C, followed by one hour of stirring. SQM-6 was dissolved in dichloromethane, treated with 4-dimethylaminopyridine (4 equiv.), and reacted with benzoyl chloride (3 equiv.) that was added dropwise while maintaining the internal temperature at 25 °C. The resulting dibenzoate SQM-7 was selectively hydrolyzed to SQM-8 by treating with sodium hydroxide (2 equiv.) in a mixture of methanol and tetrahydrofuran over four hours at 20 °C. The product was isolated by crystallization from isopropanol and silica gel chromatography of the mother liquor to afford SQM-8 in 93% overall yield for three steps. The standard TEMPO/bleach procedure was used to provide the aldehyde SQM-9 in 85-97% yield. The aldehyde is isolated by crystallization (two crops), without the need for chromatography.

The final reaction was accomplished by adding a toluene solution of SQM-9 to a solution of diisopropyl zinc (at least 2 equivalents), in toluene or xylene. The stereochemistry is controlled by using a chiral aminoalcohol as the Ligand (Scheme 1). Most of the desired 24 -R Steroid 34 can be isolated by trituration of the crude reaction mixture and subsequent crystallization (which upgrades the isomer ratio, >96% de). The mother liquors can then be chromatographed to get more material, resulting in an overall yield of about 70% for this step. An X-ray diffraction was performed on Steroid 34, which confirmed the 24f?-assignment (Figure 1).

Scheme 2: Synthesis of Squalamine Lactate

[0207] Deprotection of the ketal (34) was achieved with Amberlyst 15 ion exchange resin or equivalent in acetone to afford compound 35, which was not isolated. The reaction mixture was filtered, pyridine was added, and the acetone was removed by distillation and methylene chloride was added. Sulfur trioxide-pyridine complex was added in one portion and the reaction mixture was heated to about 40 °C for about 60 min to afford 36. Ethyl acetate was added to remove pyridine by distillation. Potassium chloride was added to form the potassium salt and this was precipitated with methyl /-butyl ether. Claracel was added as a filter aid in the isolation of 36. Squalamine is produced by coupling Compound 36 with azidospermidine (38, Weis 1999, Chellquist 2014) to generate an imine at ambient temperature in methanol. The reaction mixture is then cooled to about -75 °C and treated with sodium borohydride to produce azido-spermidine squalamine (ratio of 3-polyamine orientation b to a: 95: 5). This intermediate, which is not isolated, is treated potassium hydroxide at 70 °C in methanol and then hydrogenated in the presence of Raney nickel to yield squalamine. The crude squalamine was precipitated from 2- butanol in methyl t-butyl ether, dissolved in ethanol and water, combined with lactic acid, crystallized, and filtered to afford squalamine lactate with removal of most of the a-polyamine isomer.

[0208] Squalamine lactate and phosphate were characterized as described in Table 3.

[0209] Container/Closure System - Squalamine lactate is packaged in a 4-mil thickness polyethylene bag secured with a tie. The polyethylene bag is placed inside a larger Mylar bag (48GaPET/PE/0.00035 Foil/LLDPE, 4.5 mil thickness) with a silica gel packet placed between the two bags. The outer bag is heat sealed and placed in HDPE pails.

[0210] Stability - Accelerated Stability Conclusions: An example of squalamine lactate drug substance lot#61106 stability is shown below in Table 4 under accelerated conditions. This supports stability at 25 °C for up to two years.

NA: Not Applicable per protocol NMT : Not More Than LOQ: Limit of Quantitation ND: Not detected

¹ For Information Only, Testing performed under non-GMP conditions

² Initial results collected under protocol TTP-OIT-MOO 17, Release testing

³ Testing not performed due to testing outside stability pull window per Catalent PR 562647

⁴ AE u equals heat of fusion in units of J/g

Example 2: Preparation of Squalamine Lactate Inhalable Formulation

[0211] The following steps describe a general method of manufacture of Squalamine lactate for inhalation (COV-ENT-10:

• Combine squalamine lactate drug substance, COV-ENT-1, with a sufficient amount of Water for Injection USP (WFI) to prepare a solution equivalent to 10 mg of squalamine free base per ml of solution (corrected for purity)

• Stir at room temperature until dissolved

• 0.2um filtration of the solution in to bioprocess container.

• 0.2um sterile filtration of the solution into a sterilized bioprocess container.

• Filling into the syringes (3ml Becton Dickinson glass syringes with butyl-rubber plunger stopper)

• 100% visual inspection per HBT SOP

• Bulk packaging of the syringes and shipment to J-Star Research Inc in NJ for HPLC Assay and Impurity testing

• Released against the squalamine lactate oral inhalation specification o Sterility (contracted to Nelson Labs, Salt Lake City, UT) o Endotoxin o Particulate Matter

[0212] Lot Analysis and Stability testing were conducted, results are shown in Tables 5 and 6 below. Regarding the stability testing results in Table 6, there is no stability testing available on the current formulation, but there is considerable stability data on an aqueous formulation for use in an ophthalmic indication (IND 066897), and which contains additional excipients (benzalkonium chloride and Disodium Edetate Dihydrate). The data in Table 6 below indicate that the squalamine lactate in solution is stable for at least 30 months.

Table 6: Stability Testing of Inhalable Squalamine Lactate Opthalmic Formulation

Example 3: Toxicology Studies for Squalamine Lactate

[0213] In this Example, the principal objective is to determine whether inhaled COV- ENT-1 causes bronchospasm, and if so, at what total dosing. In addition, what damage, if any occurs at the maximal tolerable dose. Of minimal concern is the systemic exposure resulting from passage of the inhaled squalamine into the systemic circulation, since the maximal dose being considered, 5-10 mg, is several hundred-fold below the maximal tolerated dose administered intravenously.

[0214] The study will precede a full 14 day GLP study that will include the usual histopathology, clinical chemistry and TK conducted at low, medium, and high doses. These studies are being conducted by the inhalation toxicology division of Charles River Labs as outlined below.

[0215] Study 1: A Maximum Tolerated Dose Inhalation Toxicity Study of A Nebulized Aerosol Formulation In The Beagle Dog

[0216] Groups 1 to 3: 1 male and 1 female will receive escalating single doses until the maximum tolerated dose is considered reached (assumes 3 doses). A minimum of 24 hours washout will be allowed between each group’s dose.

[0217] Group 4: 1 male and 1 female will receive the maximum tolerated dose followed by a 14-day observation period. Filter Analysis: 3 occasions pretreatment, 3 occasions during escalating phase, 1 occasion during MTD (7 occasions total). Oronasal administration will be used with atmosphere generation using liquids and nebulizers for 1 hr.

[0218] Inhalation Measurements to be conducted as follows: (1) Pretreatment validation: Aerosol concentration, chamber homogeneity and particle size analysis. (2) Treatment: Aerosol concentration (2 times during exposure) and particle size analysis (once during MTD).

[0219] The total population of text subjects is 3/sex, with 1/sex spare. The animals were 5-7 months old on arrival. The pretreatment period was 3 weeks, and mortality/morbidity is observed twice daily. Detailed observations for Groups 1-3 are taken daily, and detailed observations for Group 4 are recorded weekly. Food consumption for all animals is recorded daily. Weighing for Groups 1-3 is at randomization, during the last week of the acclimation period and on the day before each dose (Groups 1 to 3). Group 4 will be weighed weekly during the observation period.

[0220] Clinical Pathology Parameters: Hematology, Coagulation, Clinical Chemistry: Once pretest for all animals. Prior to and following each dose for Groups 1 to 3. Days 1 and 14 of the observation period for Group 4 (standard parameters).

[0221] Terminal Procedures: Escalating and MTD phases: Animals sacrificed with no processing or retention of tissues.

[0222] Study 2: 5-Day Nose-Only Inhalation Toxicity Range-Finding Study in Sprague Dawley

[0223] Inhalation of squalamine lactate will be tested in rats as follows. (1) Compliance: Non-GLP; (2) Species/Strain: Sprague Dawley; (3) Test Article Preparation: Weekly;

(4) Filter Analysis (optional): If requested, 1 occasion pre-treatment, twice weekly during treatment (3 occasions total); (5) Dosing Regimen: Once daily for 5 days; (6) Route of Administration: Nose only; (7) Atmosphere Generation: Liquids: Nebulizers, up to a 6- hr exposure; (8) Characterization of Exposures: Test article concentrations in the exposure atmospheres will be determined using standard gravimetric methods (adjusted to % active and/or % solids); (9) Particle Size Measurement: Particle size determinations will be conducted at least two times. The aerosol size will be expressed in terms of the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD).

[0224] Total Population: 40 animals on study, 48 animals ordered. The age of the animals at initiation of exposure will be 7-9 weeks, and the pre-treatment period is two weeks. Mortality/morbidity will be observed twice daily, and detailed clinical observations will be recorded weekly, beginning during the pretest, prior to the first exposure, and prior to scheduled necropsy. Cage-side observations will be recorded prior to and after each daily exposure. In addition, the observations will be recorded weekly, beginning during pretest, prior to the 1st exposure, prior to the final exposure and prior to the scheduled necropsy (fasted).

Table 9

Food Consumption: Prior to the 1st exposure, prior to the final exposure Necropsy: Complete macroscopic examination of all animals found dead, euthanized in extremis, or at the scheduled necropsy with collection of select tissues from the CRL standard tissue list.

Standard weight determinations of selected organs (brain, kidneys, liver, lungs, spleen, and thymus) for all animals from all groups at the scheduled necropsy.

[0225] Study 3: A 14-Day Inhalation Toxicity Study Of A Nebulized Aerosol Formulation In The Beagle Dog Followed By A 14-Day Recovery

[0226] Toxicity study in beagle dogs will be carried out as follows: (1) Compliance: GLP; (2) Species/Strain: Beagle Dog; (3) 3 occasions pretreatment and 2 occasions during treatment (5 occasions total); (4) Dose Formulation Analysis: Achieved concentrations; (5) Dosing Regimen: once daily for 14 days followed by a 14-day recovery period; (6) Route of Administration: Oronasal; (7) Atmosphere Generation: Liquids, nebulizer, up to 1 hour; (8) Inhalation Measurements; Pretreatment validation: Aerosol concentration, chamber homogeneity and particle size analysis; and (9) Treatment: Aerosol concentration (2 times during exposure) and particle size analysis (weekly).

[0227] The Total Population of animals is 17/sex, with 1/sex spares. The animals are 5-7 months old on arrival. The Pretreatment Period is 3 weeks, and Mortality/Morbidity is observed twice daily. Detailed clinical observations are recorded weekly, starting 1 week prior to dosing. Body weight is recorded weekly, and food consumption is recorded daily. Pretest and Week 2: At the end of recovery only if treatment related findings observed.

[0228] Electrocardiography (ECG): Pretest, Day 1 and Week 2 at 1 to 4 hours post-dose. At the end of recovery only if treatment related findings observed. (1) Clinical Pathology Parameters: Hematology, Coagulation, Clinical Chemistry: Pretest, Day 14 and at the end of recovery (standard parameters). (2) Urinalysis: Pretest, Day 14 and at the end of recovery (standard parameters). (3) Toxicokinetics Sample, Collection and Analysis: Days 1 and 14: Samples will be taken from all animals at 6 time points (time points to be determined by sponsor). (4) Estimated number of samples collected: 384. [0229] Toxicokinetic Reporting: Non-compartmental analysis of the test item analytes (1) in blood matrix concentrations will be performed using validated computer software Phoenix for each dose level, gender, and occasion (2) following standard operating procedures. Results will be tabulated and graphically displayed using Phoenix and appropriately interpreted by a PK Scientist.

[0230] Terminal Procedures: Day 15/29 Macroscopic examination, organ weights and tissue retention. Histopathology examination - all animals for respiratory tract only. Remaining tissues retained in fixative.

Study 4: A 14-Day Inhalation Toxicity Study Of A Nebulized Aerosol [0231] A 14-Day Inhalation Toxicity Study of A Nebulized Aerosol in rats will be completed as follows. (1) Compliance: GLP; (2) Species/Strain: Sprague Dawley Rat; (3) Test Item Preparation: Weekly; (4) Analysis of Test Item: Method development/validation if required at additional cost. Method development and validation must be completed prior to sample analysis in order to have no impact on data delivery or integrity; (5) Filter Analysis (Optional): If requested, 2 occasions pre-treatment, weekly during treatment (4 occasions total); (6) Dose Formulation Analysis: Achieved concentration: 2 occasions; (7) Dosing Regimen: Once daily for 14 days; (8) Route of Administration: Nose only; (9) Atmosphere Generation: Liquids: Nebulizers, up to a 6-hr exposure; and (10) Inhalation Measurements: Aerosol concentration (2 times during each exposure) and particle size analysis (weekly).

Table 11

Test System:

[0232] The total population of test subjects is 80/sex, with 10/sex spares. The age of the animals is 5-7 weeks upon arrival, and the source is Charles River. In addition, the following parameters are noted: (1) Pretreatment Period: Two weeks; (2) Mortality/Morbidity: Twice daily; (3) Detailed Observations: Weekly, excluding TK animals, starting 1 week prior to dosing; (4) Body Weight: Weekly; (5) Food Consumption: Weekly starting Day 1, excluding TK animals; (6) Clinical Pathology Parameters: Hematology, Coagulation, Clinical Chemistry: At termination (standard parameters), excluding TK animals; and (7) Toxicokinetics Sample Collection: Days 1 and 14 Samples will be taken from 3 rats/sex/TA group at 6 time points and at 2 time points from both control TK animals (time points to be determined by sponsor) for a total of 240 samples. All toxicokinetic animals will be discarded without further evaluation following euthanasia.

Table 12

Toxicokinetic Sample 240 study samples analyzed, plus optional 24 samples if ISR Analysis: required on this study, see below.

Terminal Day 15. Macroscopic examination, organ weights and tissue

Procedures: retention. Histopathology examination - Respiratory tract and gross lesions from the control and high dose (standard tissues). Gross lesions (assumes 2/animal) from the low and intermediate dose groups. All other tissues to be retained.

Example 4: Squalamine boosts survival rates and decreases viral titer loads against Yellow Fever

[0233] In 2011 it was reported that squalamine is unambiguously active in vitro and in vivo against a broad spectrum of human viral pathogens, including both RNA- and DNA-enveloped viruses (Zasloff, 2011). In particular, squalamine could both protect and treat animals from lethal doses of a human strain of Yellow Fever (Figure 2), and partially protect from Eastern equine encephalitis. Both of these viruses are single positive strand RNA viruses, as is SARS-CoV-2. In vitro, antiviral activity was demonstrated against human strains of dengue, hepatitis B, and hepatitis D (Figure 2). Example 5: Squalamine inhibited the viral cytopathic in COVID-19 patients

[0234] Recently, in response to the emerging Covid-19 pandemic, squalamine was assayed against SARS-CoV-2E-29E in the laboratory of a French collaborator. At 1 OmM (7pg/ml) squalamine inhibited the viral cytopathic effect by 10-50% at multiplicities of infection ranging from 0.1-0.001.

Example 6: A Multicenter Randomized Study to Evaluate the Safety and Efficacy of Inhaled SL-1 (squalamine lactate) in Improving Outcome in Patients with COVID-19 Pneumonia

[0235] The purpose of this study is to evaluate the safety, tolerability and efficacy of SL-1 in subjects with Covid-19 infection. This study will be conducted as a randomized safety, tolerability and efficacy study. Study duration will be approximately 4 weeks. SL-1 will be administered daily via nebulizer in escalating doses for up to 15 days. The study will be conducted on an in-patient basis with assessments performed at screening and daily thereafter up to 15 days and at 28 days (study termination).

[0236] Primary Safety and Tolerability Objective: To determine the safety and tolerability of daily inhaled SL-1 for up to 15 days in subjects with Covid-19 infection as determined by Adverse Events and Serious Adverse Events.

[0237] Primary Efficacy Objectives: To determine the efficacy of inhaled SL-1 in improving outcome from Covid-19 infection as determined by a 7-point ordinal disease severity scale disease severity at 15 days.

[0238] Secondary Efficacy Objectives: To determine the effect of inhaled SL-1 on outcome from Covid-19 infection as determined by improvement in the 7-point ordinal disease severity at 28 days. Duration of mechanical ventilation, duration of hospitalization, oxygenation status, ventilator use time from treatment initiation to death and virologic measures will also be assessed out to 28 days.

[0239] Inclusion Criteria for Subjects: (1) Subject (or legally authorized representative) provides written informed consent prior to initiation of any study procedures; (2) Understands and agrees to comply with planned study procedures; (3) Agrees to the collection of nasopharyngeal swabs and venous blood per protocol; (4) Male or non-pregnant female adult >18 years of age at time of enrollment; (5) Laboratory confirmed SARS-CoV-2 infection as determined by PCR, or other commercial or public health assay in any specimen <72 hours prior to randomization; (6) Hospitalized patients with illness of any duration, and at least one of the following: (i) Clinical assessment (presence of crackles/rales) AND Sp02 < 94% on room air OR (ii) Acute respiratory failure requiring mechanical ventilation and/or supplemental oxygen OR (iii) Radiographic infiltrates by imaging (CT, CXR); and (7) Women of childbearing age must agree to use contraception for the duration of the study.

[0240] Exclusion Criteria: (1) Pregnant or breast feeding; (2) Uncontrolled, clinically significant heart diseases such as arrhythmias, angina or uncompensated congestive heart failure; and (3) Non-hospitalized patients.

[0241] Study Product, Dose, Route, Regimen: SL-1 will be administered in inhaled form via nebulizer over 5-50 minutes, given every 8 hours for up to 15 days. The first group of subjects will be dosed 0.5mg SL-1 (in 2.5mls of normal saline). Dosing will be increased every 4 patients in increments of 0.5mg then l.Omg until a maximum dose of 5mg (25mls of normal saline).

[0242] Study Endpoints: (1) Safety and Tolerability Endpoints: Adverse Events and Serious Adverse Events out to 28 days, as evaluated with subject report, clinical assessment and vital signs, chemical chemistry and electrocardiograms (EKG). (2) Primary Efficacy Endpoint: The primary efficacy endpoint is the severity rating on the following 7-point ordinal disease severity scale at 15 days (i) Not hospitalized, no limitations on activities; (ii) Not hospitalized, limitation on activities; (iii) Hospitalized, not requiring supplemental oxygen; (iv) Hospitalized, requiring supplemental oxygen; (v) Hospitalized, on non-invasive ventilation or high flow oxygen devices; (vi) Hospitalized, on invasive mechanical ventilation or ECMO; (vii) Death.

[0243] Secondary Efficacy Endpoints: To determine the effect of inhaled SL-1 on outcome from Covid-19 infection as determined by improvement in the 7-point ordinal disease severity scale at 28 days. Duration of mechanical ventilation, duration of hospitalization, oxygenation status, ventilator use, time from treatment initiation to death and virologic measures will also be assessed out to 28 days. (1) The severity rating on the 7-point ordinal disease severity scale at days 3, 6, 9, 12, and 28; (2) Time to discharge or to a National Early Warning Score (NEWS) of < 2 maintained for 24 hours, whichever occurs first (at 3, 6, 9, 12, 15, and 28 days); (3) Change from baseline in NEWS at days 3, 6, 9, 12, 15, and 28; (4) Number of oxygen free days over 28 days; (5) Incidence and duration of new oxygen use over 28 days; (6) New mechanical ventilation over 28 days; (7) Ventilator free days over 28 days; (8) Duration of hospitalization; (9) SARS-CoV-2 in nasopharyngeal sample at days 3, 6, 9, 12, 15, and 28; (10) Quantitative SARS-CoV-2 virus in blood at days 3, 6, 9, and 12.

[0244] Statistical Considerations: (1) Safety Population: All enrolled subjects who have received at least 1 dose of study drug. (2) Efficacy Evaluable Population: All subjects in the Safety Population who complete the study with no major protocol violations. (3) Analysis Methods: Descriptive summary statistics will be presented for the safety and tolerability endpoints overall. Continuous variables will be summarized using the number of observations, mean, standard deviation, median, minimum, and maximum. Categorical variables will be summarized using frequency counts and percentages. Summary statistics will be provided for Stage 1. Inferential statistics will also be performed in Stage 2. (4) Stage 2 Primary Efficacy Endpoint Analysis: The primary efficacy endpoint analysis is a treatment group comparison of the patients’ day 15 7-point ordinal disease severity scale using a Cochran-Mantel- Haenszel (CMH) row mean score difference test controlling for the baseline score. The primary efficacy analysis will compare the pooled Stage 2 placebo groups to the pooled Stage 2 SL- 1 groups at the two- sided 0.05 Type I error rate. (5) Sample Size Justification: For Stage 1, the sample size of 4 subjects per group (2 on SL-1, 2 on placebo) is designed to provide an initial assessment of safety and tolerability for the initial doses of SL-1. The Stage 2 sample size of 28 patients in an SL-1 treatment group (i.e., the 2 SL-1 treatment groups combined) provides a 95% probability that an AE with an underlying rate of 10% will occur in these patients. Additionally, in Stage 2, a sample size of 28 patients per group (i.e., pooling the Stage 2 placebo groups and the SL-1 groups) will provide approximately 80% power to detect a treatment difference at the 2-sided 0.05 Type I error rate based on a using a CMH row mean score difference test controlling for the baseline score. This assumes the following day 15 distribution for the seven-category disease severity score.

[0245] Rational for Study and Background: The ability of squalamine to alter the electrostatic charge in so drastic of a fashion as to cause displacement of membrane-anchored proteins, led to the hypothesis that squalamine might have, as a consequence, antiviral properties. Many viruses enter cells through engagement of the p-GTPases, such as Racl to influence the actin cytoskeleton (Pelkmans and Helenius 2003, Pelkmans, Fava et al. 2005, Zamudio-Meza, Castillo-Alvarez et al. 2009). Displacement of key proteins anchored through electrostatic forces (of host or viral origin) from the cytoplasmic face of the plasma membrane might interfere with virus entry (Pelkmans and Helenius 2003, Pelkmans, Fava et al. 2005, Mercer and Helenius 2008), viral protein synthesis (Ahola, Lampio et al. 1999), virion assembly (Chukkapalli, Hogue et al. 2008, Chukkapalli, Oh et al. 2010), virion budding (Stansell, Apkarian et al. 2007), or other steps in the viral replication cycle (Agirre, Barco et al. 2002). Certain viruses seem to require the presence of the anionic phospholipid phosphatidylserine in the target cell plasma membrane as part of the fusion process, and charge neutralization of the anionic phospholipid could, in principle, interrupt these events (Ahola, Lampio et al. 1999, Coil and Miller 2004, Coil and Miller 2005, Mercer and Helenius 2008, Soares, King et al. 2008). Because squalamine, after parenteral administration, is cleared from the circulation over the course of hours by the liver, from which it is excreted through the biliary system, an initial extensive survey of its potential antiviral activity on viruses that infect the liver was focused upon.

[0246] As yet there is only a limited understanding of the biology surrounding SARS-CoV-2 infectivity. However, a plausible hypothesis can be proposed to explain how squalamine might inhibit the viral cytopathic effect. Of the several proteins encoded by the virus, protein 3 a appears to be required for the majority of the cytopathic effect caused by a SARS CoV2 infection (Freundt, Yu et al. 2010). Upon synthesis within an infected cell protein 3a binds to the plasma membrane as well to the Golgi system (Yount, Roberts et al. 2005, Oostra, de Haan et al. 2006). Vesiculation and disruption of the cell membrane then follows. Protein 3a has been implicated as the protein most likely to initiate the severe inflammatory reaction associated with pneumonia (or ARDS) associated with SARS CoV2 (Chen, Moriyama et al. 2019). Protein 3a (also called “viroporin 3 a”) appears to provoke inflammation through activation of the NLRP3 inflammasome (Chen, Moriyama et al. 2019).

[0247] From an analysis of the recently published protein sequence of Protein 3a (Swiss Prot database), Protein 3a exhibits the classical characteristics of a cationic, amphipathic lytic protein, such as NK-lysin (Figure 3B), the cytolytic protein secreted by activated NK cells (Andersson, Holmgren et al. 1996), or the lytic peptide Naegleriapore (Figure 3C) produced by the cytopathic intracellular protozoan Naegleria fowled (Andersson, Holmgren et al. 1996, Bruhn, Riekens et al. 2003).

[0248] As can be seen in Figure 3 A, Protein 3a, when displayed as a linear sequence, the N- terminal half of the protein is more heavily represented by positively charged amino acids (RED) while the other half of the protein is more heavily represented by negatively charged amino acids (GREEN). A similar pattern can be seen in the primary sequences of NK-lysin and Naegleriapore, as other examples. This structural feature, namely the spatial segregation of cationic and anionic residues in a protein, conveys upon the protein or peptide the property of binding to membrane surfaces that are composed of anionic phospholipids (Zasloff 2002). In addition, the protein exhibits amphipathic behavior, namely, the property of being soluble in both aqueous and membrane phases. By virtue of electrostatic force, the cationic amphipathic protein is drawn to the membrane surface. Upon binding to the membrane, it distorts the normal architecture of the binding site. As the surface concentration of these proteins increase the normal physical integrity of the membrane is progressively disrupted. The cell eventually lyses. Squalamine, by virtue of its biophysical characteristics, can displace amphipathic proteins and peptides also such as the SARS CoV2 peptide 3a. It is via this mechanism that squalamine displaces alpha-synuclein, the intra neuronal protein that causes damage to the enteric nervous system in Parkinson’s disease, and the basis for the development of oral squalamine for that condition (Pemi, Galvagnion et al. 2017).

[0249] Although it remains to be proven, it is proposed that squalamine reduces the cytopathic effect of SARS-CoV2, in part, by, in effect, blocking the interaction of Protein 3a with target membranes within the infected cell.

[0250] Dosing Rationale: In general, the antiviral activity of squalamine falls within the range seen for antibacterial activity, namely between less than 3 pg/ml to values exceeding 50 pg/ml (Alhanout, Malesinki et al. 2010, Nicol, Mlouka et al. 2019). (In the case of bacteria and fungi, squalamine targets the outer leaflet of the microbial plasma membranes, which contains the bulk of the anionic phospholipid of the membrane). With respect to the strain of coronavirus we have tested (HCoV 229E), as noted above, a concentration (in vitro ) of about 7 pg/ml resulted in a significant decrease in viral cytopathic effect.

[0251] To estimate the target human dosing range for the proposed clinical trial, a published study was used which compares the efficacy of inhaled squalamine and colistin for the treatment of a pulmonary Ps. aeruginosa infection in rats (Hraiech, Bregeon et al. 2012). In this study, the inoculum had an MIC against squalamine of between 2-8 pg/ml.

[0252] The dose administered to the rat was estimated to be 0.15 mg/kg, based on the respiratory parameters of the rat and the rate of drug delivery in the inhalation chamber. The weight of the rat lung is about 1.3g (or about 0.5% of total body weight) and that of the human lung is 1.3 kg (or about 1.7% of total body weight). The approximate total amount of squalamine delivered to the rat lung was 0.05 mg and a tissue concentration of 0.05 mg/1.3 g, or 23 pg/g tissue (assuming instantaneous delivery). If it is scaled accordingly to humans, to reach a comparable tissue concentration (assuming PK parameters in the human and rat lungs are not profoundly different, which is unlikely), we will require about 23 mg per dose in humans.

[0253] It can also be estimated as an effective human dose of squalamine by another approach, using the known effective dosing regimen of inhaled colistin in gram-negative pneumonia. The standard colistin dosing regimens in adults’ range between 75 to 150 mg, twice or three times per day (Kofteridis, Alexopoulou et al. 2010). In the rat experiment the animals were exposed to a solution of 3 mg of squalamine or 160 mg of colistin for 4 hours, every 12 hrs. By this estimate,

3 mg of squalamine was therapeutically equivalent to 160 mg of colistin when administered in an inhalation chamber. From this experiment and the known effective dosing regimens of colistin in man, It can be estimated that 3 mg per treatment in humans would suffice to treat a lung infection based on equivalence with colistin. Since the treatments in the animal experiment were not administered directly into the airway of the animal, but were estimated from respiratory parameters of the rat, and the duration of nebulization in the inhalation chamber, the dose received was likely an overestimate. Based on the above assumptions it is estimated that a reasonable human inhaled dose is between about 5 to about lOmg.

[0254] A second variable is the concentration of inhaled squalamine. Squalamine is a membrane active compound. There is considerable clinical data that establishes a window of safety with respect to potential injury upon administration to mucosal epithelial surfaces. As outlined in detail in IND 066897, 0.2% squalamine lactate has been applied topically as a twice daily eye drop in two Phase 2 clinical trials for retinopathy in over 70 patients for 36 weeks. No significant adverse reactions were observed in these studies, other than a mild stinging sensation that resolved several minutes after application to eye. In addition, Applicant is currently evaluating the oral administration of squalamine (as the phosphate salt, ENT-01, IND 130770) in the treatment of constipation in Parkinson’s patients, and have treated over 100 patients without clinical evidence of injury to the GI tract, which, of course, is lined by a single layer of epithelial cells. ENT-01 is administered once daily at doses up to 250 mg.

[0255] This study proposes to administer squalamine lactate by nebulization at a concentration of 0.02% in 0.9% saline up to a total single dose of 5mg. This dose was well tolerated in both dogs and rats when administered by inhalation at the proposed concentration for a duration that supports the 14-day maximal dosing period in the planned clinical trial.

[0256] The study will be conducted in two stages. Stage 1 will be single-blind (patient will be blinded, but the physicians and sponsor will be unblinded), whereas Stage 2 will be double-blind (patient and physician will be blinded, sponsor will be unblinded). In Stage 1, 24 patients will be treated in groups of 4, randomized 1 : 1 to SL-1 or placebo and treated with escalating doses. The first group (n=4) will be treated with SL-1 or placebo at 0.5mg (in 2.5mls normal saline). The second group (n=4) will be treated with SL-1 or placebo at l.Omg (in 5 ml normal saline). Subsequent groups (each group n=4) will be treated with SL-1 or placebo at 2.0mg (in lOmls normal saline), 3.0mg (in 15mls normal saline), 4.0mg (in 20mls normal saline) or 5.0mg (in 25mls normal saline). In Stage 2, 28 patients will be randomized 1:1 to SL-1 or placebo at the highest dose without any drug-related adverse events, and another 28 patients will be randomized 1 : 1 to SL-1 or placebo at the dose just below the highest dose without any drug-related adverse events.

*SOC: Standard of Care

Note: Based on a review of the safety and tolerability data of each dosing group, the DSMB will have the option of allowing an additional 4 patients to be randomized to a dosing group (see SectionError! Reference source not found.).

a: To balance the distribution of oxygen support between the two groups as an indicator of severe respiratory failure, randomization in Stage 2 will be stratified on the basis of respiratory support methods at the time of enrollment: no oxygen support with nasal duct or mask, or high flow oxygen, noninvasive ventilation, or invasive ventilation including ECMO. [0257] Approximately 80 subjects will be entered at up to 10 sites in the USA. Hospitalized patients with illness of any duration will be assessed for eligibility on the basis of a positive reverse transcriptase polymerase chain reaction (RT-PCR) assay or other commercial or public health assay for SARS-CoV-2 in a respiratory tract sample. Patients who test positive in any sample <72 hours prior to randomization will be eligible. In addition to a positive SARS-CoV-2 test, patients will be required to have crackles/rales and an Sp02 < 94% on room air, or respiratory failure requiring supplemental oxygen/ventilation, or radiographic infiltrates by imaging (CT, CXR). All eligible patients will receive standard of care. Standard of care will include supplemental oxygen, noninvasive and invasive ventilation, antibiotics, vasopressors and ECMO. In addition, patients will receive study medication/placebo via standard nebulizer, administered every 8 hours for up to 15 days. A concentration of 0.2mg/ml will be used. The lowest dose group will receive 0.5mg in 2.5mls of normal saline, delivered by standard nebulizer over 5-10 minutes. The highest dosing group will receive 5mg in 25mls of normal saline, delivered over 40-50 minutes, or however long it takes to nebulize that volume of liquid. Thus, the variable is duration of delivery, with larger amounts of compound requiring longer dosing duration. Medication will be discontinued if the patient improves to the point where they can be discharged.

[0258] Patients will be assessed daily for 15 days or until discharge, and again on days 12, 15, and 28. All data will be entered into an EDC daily. Subjects who prematurely discontinue study drug for any reason will be asked to have an Early Termination (ET) visit the day the last study drug dose is administered.

I/E: Inclusion/Exclusion criteria; Demo: Demographics; PE: Physical Examination; Labs: CBC and chemistry; Pregnancy testing: For women of child-bearing potential, using urine pregnancy kit (UPT); Sp02: peripheral Oxygen Saturation; 7-POS: 7-Point Ordinal Disease Severity Scale; NEWS: National Early Warning Score; AE: Adverse Event; LOC: Level Of Consciousness; CT*: CT scan is optional alternative to CXR; CXR: Chest X-Ray; CoV-2 N/P: Quantitative SARS-CoV-2 PCR from Nasopharyngeal swab sample; CoV-2 Blood: Quantitative SARS-CoV-2 PCR from blood sample. IP: Investigational product or placebo administration.

[0259] 7-Point Ordinal Disease Severity Scale: This is an assessment of the clinical status and it will serve as the primary endpoint. The scale is as follows: (1) Not hospitalized, no limitations on activities; (2) Not hospitalized, limitation on activities; (3) Hospitalized, not requiring supplemental oxygen; (4) Hospitalized, requiring supplemental oxygen; (5) Hospitalized, on non-invasive ventilation or high flow oxygen devices; (6) Hospitalized, on invasive mechanical ventilation or ECMO; (7) Death.

[0260] NEWS: The NEW Score has demonstrated the ability to discriminate patients at risk of poor outcomes and is being used as an efficacy measure. This score is based on 7 clinical parameters: (1) Respiration rate; (2) Oxygen saturation; (3) Any supplemental oxygen; (4) Temperature; (5) Systolic blood pressure; (6) Heart rate; (7) Level of consciousness.

[0261] Quantitative Nasopharyngeal SARS-CoV-2 RNA testing (RT-PCR): Nasopharyngeal or throat swabs will be used. Any FDA approved or commercial testing method will be acceptable.

[0262] Quantitative blood SARS-CoV-2 RNA testing (RT-PCR): Blood samples will be used. Any FDA approved or commercial testing method will be acceptable.

[0263] Study Population: Hospitalized patients with illness of any duration will be assessed for eligibility on the basis of a positive reverse transcriptase polymerase chain reaction (RT- PCR) assay or other commercial or public health assay for SARS-CoV-2 in a respiratory tract sample. Patients who test positive in any sample <72 hours prior to randomization will be eligible. Approximately 80 subjects will be entered at up to 10 sites in the USA.

[0264] Concomitant and Prohibited Medication: (1) Hydroxychloroquine; (2) Non-study drug anti -viral agents such as lopinavir, ritonavir, ribavirin or interferon- 1b; and (3) Any other medication not approved for the treatment of SARS-CoV-2.

[0265] All study medication will be supplied by Enterin, Inc. as a sterile filled syringe containing 1ml of SL-1 at lOmg/ml. It will be diluted at the hospital prior to use in normal saline to a concentration of 0.2mg/ml (i.e. into a volume of 50mls normal saline). The total dose administered will be based on the volume of liquid delivered to the patient by nebulizer. So, for example, for the lowest dose of 0.5mg, only 2.5mls of this would be placed in the nebulizer reservoir and delivered at the maximal rate permissible by the nebulizer.

[0266] Study medication will be supplied in blinded, single-use syringes packaged in kits. Each kit will contain either drug product syringes only or placebo syringes only. Labelling will clearly identify that the syringes contain an investigational drug product but will not include any patient- specific information. The kit box labelling will copy with regulatory requirements for investigational drug labelling and will include a pre-assigned individual medication ID number for randomization assignment and storage conditions. Individual syringes within the kit will not be labelled with the medication ID number and should not be removed from the kit until time of use. Labelling will include a tamper-proof emergency use method for unblinding.

[0267] Storage, Dispensing and Reconciliation of Study Drug and Identity of Investigational Products: All study medication should be stored at room temperature until dispensed. Storage in hospital should be in a locked and secure location accessible only to site staff involved with this study. If a hospital becomes aware that study medication has not been properly handled, the sponsor must be contacted immediately. In such an event, study medication should not be utilized until the sponsor provides further direction. Neither the investigator nor any study personnel will distribute any study medication to any person not participating in this study. The study medication will be administered at the discretion and direction of the investigator in accordance with the conditions specified in this protocol. It is the investigator’s responsibility to ensure that accurate records of study medication issuance and return are maintained. The sponsor is responsible for the tracking and accountability of study medication dispensed to hospitals and will inform hospitals how to return or destroy study medication once it is no longer needed at the site.

*modified to address hospitalized patients with existing respiratory symptoms

[0268] Criteria for Dose Limiting Toxicity (DLT) Endpoints: Any new wheezing during the inhalation or worsening of pre-existing wheezing or difficulty breathing accompanied by a deterioration of oxygenation will be considered a DLT. If wheezing occurs during study drug inhalation, the inhalation will be discontinued. Persistent wheezing will be treated with standard medical therapy such as bronchodilators and/or oxygen. The patient will be terminated from the study and the DSMB notified. All subjects enrolled in the study will have SARS-CoV-2 infection and respiratory symptoms.

[0269] Treatment Emergent Adverse events: A treatment-emergent adverse event (TEAE) is defined as any event not present prior to the initiation of the treatments or any event already present that worsens in either intensity or frequency following exposure to the treatments. An adverse event is typically collected after signing the informed consent form and could be related or unrelated to the study drug. A TEAE is for after the subject actually takes the study drug. Separate summaries for adverse events that occur during treatment (summary of treatment emergent adverse events) will be provided.

[0270] Laboratory Abnormalities: Clinical labs will be performed by each hospital in their own laboratories. Labs to be drawn during the study include serum chemistries, a hematology panel and coagulation panel. A serum pregnancy test must be performed, and the result must be negative prior to the entry of women of child-bearing potential. Clinical laboratory reports must be reviewed by a physician for out-of-range values within 12 hours of receipt. Out-of-range values will be evaluated using the following notations: (1) NS: Not clinically significant; (2) LE: Laboratory Error; (3) PT: Subject abnormal; relates to the subject's usual state of health; (4) CS: Clinically Significant. This value cannot be explained by any of the other flags.

[0271] Adverse Event Assessment and Recording: All adverse events, exacerbations of concomitant illnesses, or events known to be related to underlying disease processes or concomitant medications are to be recorded on the CRF throughout the study. If a pre-existing condition worsens on study, the date on which the exacerbation began should be recorded. Onset dates for study treatment-related adverse events must be on or after the date of initial study treatment use. The need to record an adverse event on the CRF is not dependent on whether the adverse event is associated with the use of the study medication. In order to avoid vague, ambiguous or colloquial expressions, the adverse event should be recorded in standard medical terminology. Adverse event recording will include the date of onset, severity, duration, whether or not the study medication was discontinued because of the event, the treatment given and the outcome. The investigator must also assess whether the event was related to the study medication, concurrent drug therapy, underlying disease, a combination of these factors, or if it is unknown. Subjects with an adverse event should be carefully followed to determine outcome.

[0272] The investigator will use the National Cancer Institute (NCI) definitions to grade the severity of the event. (1) Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not indicated; (2) Grade 2: Moderate; minimal, local, or non- invasive intervention indicated; limiting age-appropriate ADL; (3) Grade 3: Severe or medically significant but not immediately life-threatening; hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care ADL; (4) Grade 4: Life-threatening consequences; urgent intervention indicated; (5) Grade 5: Death.

[0273] The relationship or association of the study medication in causing or contributing to the adverse event will be characterized as remote, possible or probable as defined below: (1) Not related: Evidence indicates no plausible direct relationship to the study medication; (2) Remote: Suggests other conditions are reasonably likely to account for the event including concurrent illness, progression or expression of the disease state, or reaction to concurrent medication; (3) Possible: Suggests that the association of the event with the study medication is unknown; however, the adverse event is not reasonably supported by other conditions; (4) Probable: Suggests that a reasonable temporal sequence of the event with medication administration exists and, based upon the investigator's clinical experience, the association of the event with study medication seems likely; (5) Definite: Suggests that based upon the investigator’s experience, the association of the event with the study medication seems very certain. Procedures such as surgery should not be recorded as adverse events. However, the medical condition for which the procedure was performed should be reported if it meets the definition of adverse event.

[0274] Reporting Requirements: Any adverse event, including both observed or volunteered problems, complaints, or symptoms that begins any time between the start of the first dose and within 30 days after the end of the last dose are to be recorded briefly on the appropriate CRF and in detail in the source documents. A check list of adverse events may not be used during this study. The following are specific definitions that are relevant to meeting your reporting obligations and which are included in the FDA Regulations, 21CFR Part 312.32, and International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Guidelines:

[0275] Adverse Event: Any untoward medical occurrence in a subject administered a pharmaceutical product and which does not necessarily have to have a causal relationship with this treatment. An adverse event can be any unfavorable sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of the investigational drug, whether or not considered related to the investigational drug.

[0276] Serious Adverse Event: An untoward event or reaction that at any dose: (1) results in death; (2) is life-threatening; (3) prolongs existing hospitalization; (4) results in permanent or significant disability or incapacity; (5) requires intervention to prevent permanent impairment/damage.

[0277] Life-threatening: An event which a subject was at risk of death at the time of event. There is a distinction between the severity and the seriousness of an adverse event. Severe is a measurement of intensity, thus a severe reaction is not necessarily a serious adverse event. For example, a headache may be severe in intensity, but would not be serious unless it met one of the criteria for serious adverse events listed previously.

[0278] Serious Adverse Events: Adverse events (AEs) and serious adverse events (SAEs) will be collected from the start of study treatment until 28 days. Medical occurrences that began prior to the start of study treatment, but after obtaining informed consent were recorded on the Medical History/Current Medical Conditions CRF. The investigator or site staff will be responsible for the detection and documentation of events meeting the criteria and definition of an adverse event or serious adverse event, as provided in the study protocol, However, any SAEs assessed as related to study participation (e.g., dosing, protocol mandated procedures, invasive tests, or change in existing therapy) or related to a concomitant medication will be recorded from the time a subject consent to participate in the study up to and including any follow-up contact.

[0279] Stopping Rules: During the 15-day dosing period, it is anticipated that no more than 2 subjects out of 40 (5.0%) will have an adverse event of grade 4 or 5 that is at least possibly related to SL-1. Should there be more than 2 subjects with an adverse event grade 4 or 5 that is at least possibly related to SL-1 in the cohort of subjects, the study will be put on an immediate clinical hold. In addition, individual safety patient study stopping rules will include: (1)

Reaching dose-limiting tolerability (DLT) before clinical improvement; (2) Having a non-DLT respiratory adverse event > grade 3 within 24 hours of taking study medication that is at least possibly attributable to SL-1.

Example 7

[0280] In an exemplary example, squalamine lactate is formulated into a dry powder formulation and administered using a single dose, hand held Dry Powder Inhaler (DPI), breath actuated device. The device delivers the squalamine lactate in the form of dry powder contained in a capsule to a subject in need. Each dose of the formulation is proposed to deliver about 10 mg of squalamine lactate.

[0281] The formulation is proposed to be administered to a subject diagnosed with COVID-19, at risk of developing COVID-19, or suspected to be suffering from COVID-19. It is expected that administration of the aminosterol lactate salt dosage via inhalation or pulmonary administration will increase survival and/or decrease undesirable side effects of COVID-19. For example, administration can administration can improve respiratory symptoms, shorten duration of virus shedding, reduce duration of hospitalization, interfere with viral replication, stimulate tissue regeneration, or any combination thereof.

Example 8

[0282] A mechanically ventilated subject diagnosed with COVID-19 is administered squalamine lactate formulated into a liquid dispersion. The dispersion is delivered to the ventilated subject using an intratracheal nebulizing catheter adapted for in-line use. Each dose of the formulation is proposed to deliver about 20 mg of squalamine lactate.

[0283] It is expected that administration of the aminosterol lactate salt dosage via inhalation or pulmonary administration will increase survival and/or decrease undesirable side effects of COVID-19. For example, administration can administration can improve respiratory symptoms, shorten duration of virus shedding, reduce duration of hospitalization, interfere with viral replication, stimulate tissue regeneration, or any combination thereof.

* * * *

[0284] While certain embodiments have been illustrated and described, it should be understood that changes and modifications can be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the following claims.

[0285] The embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified.

[0286] The present disclosure is not to be limited in terms of the particular embodiments described in this application. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and compositions within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, or compositions, which can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0287] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0288] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof, inclusive of the endpoints. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0289] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0290] Other embodiments are set forth in the following claims.

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Claims

WHAT IS CLAIMED IS:

1. A method of treating a subject in need, wherein the subject has been diagnosed with a coronavirus infection, is suspected of having a coronavirus infection, and/or is at risk of developing a coronavirus infection, wherein the coronavirus is SARS-CoV-2, wherein the method comprises: administering to the subject a therapeutically effective amount of a composition comprising at least one aminosterol via inhalation or pulmonary administration, wherein the aminosterol is in a lactate or dilactate salt form.

2. A method of inhibiting viral replication of SARS-CoV-2 in a subject, wherein the method comprises: administering to the subject a therapeutically effective amount of a composition comprising at least one aminosterol via inhalation or pulmonary administration, wherein the aminosterol is in a lactate or dilactate salt form.

3. The method of claim 1 or 2, wherein following administration, viral load in the subject is reduced by about about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, relative to the subject before administration of aminosterol, wherein the reduction is measured by a medically recognized technique selected from the group consisting of tunable resistive pulse sensing (TRPS), flow cytometry, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and transmission electron microscopy (TEM).

4. The method of any one of claims 1-3, wherein inhalation or pulmonary administration is via a device or method selected from the group consisting of a nebulizer, pressurized nebulizer, a jet nebulizer, constant-output jet nebulizer, inspiratory synchronized jet nebulizer, ultrasonic nebulizer, constant-output ultrasonic nebulizer, vibrating-mesh nebulizer, constant-output vibrating-mesh nebulizer, vibrating mesh nebulizer, soft mist inhaler, metered dose inhaler (MDI), pressurized metered-dose inhaler, Dry Powder Inhaler, and Intratracheal Nebulizing Catheter.

5. The method of any one of claims 1-4, wherein:

(a) the method uses a single dose Dry Powder Inhaler (DPI) device for administration; and/or

(b) the device delivers the composition in the form of a dry powder contained in a capsule; and/or

(c) the device is a breath actuated inhaler.

6. The method of any one of claims 1-5, wherein the composition is in a dry powder dosage form.

7. The method of any one of claims 1-5, wherein the composition is in a liquid dispersion dosage form.

8. The method of any one of claims 1-7, wherein the composition is administered to a mechanically ventilated subject, and optionally wherein:

(a) the composition is administered to the subject using an inhaler or nebulizer device connected to the ventilator via an actuator device; and/or

(b) the device is connected to the ventilator using a spacer chamber; and/or

(c) the ventilation comprises delivery of a tidal volume selected from the group consisting of about 100 mL to about 200 ml, about 200 ml to about 300 ml, about 300 ml to about 400 ml, about 400 ml to about 500 ml, about 500 ml to about 600 mL, about 600 ml to about 700 mL, or greater than about 700 ml; and/or

(d) the ventilation comprises inspiratory flow of about 10 L/min to about 20 L/min, about 20 L/min to about 30 L/min, about 30 L/min to about 40 L/min, about 40 L/min to about 50 L/min, about 50 L/min to about 60 L/min, or greater than about 60 L/min; and/or

(e) the composition is administered to the mechanically ventilated subject using a dry powder inhaler, soft mist inhaler, or intratracheal nebulizing catheter adapted for in-line use.

9. The method of any one of claims 1-8, wherein the composition comprising at least one aminosterol is heated before administering.

10. The method of any one of claims 1-9, wherein the coronavirus infection is correlated with pneumonia and/or a lung infection.

11. The method of any one of claims 1-10, wherein the method additionally comprises administering one or more compounds selected from the group consisting of bronchodilators, inhaled corticosteroids, antibiotics, pulmonary surfactant, mucolytics, biologicals, genes, prostanoids, surfactants, heparin, morphine, furosemide, and combinations thereof.

12. The method of claim 11, wherein:

(a) the bronchodilator is one or more selected from albuterol, formoterol, arformoterol, fenoterol, metaproterenol, and ipratropium; and/or

(b) the inhaled corticosteroid is one or more selected from budesonide, beclomethasone, and fluticasone; and/or

(c) the antibiotic is one or more selected from tobramycin, amikaci, amikacin, fosfomycin, colistin, ciprofloxacin, ribavirin, and amphotericin B; and/or

(d) the surfactant is one or more selected from Exosurf, Survanta, Curosurf, Infasurf, and KL4; and/or

(e) the mucolytic is one or more selected from N-acetylcysteine and dornase alfa; and/or

(f) the biological is a monoclonal antibody; and/or

(g) the gene is an siRNA; and/or

(h) the prostanoid is one or more selected from epoprostenol, iloprost, and treprostinil.

13. The method of any one of claims 1-12, wherein the subject has a comorbidity selected from one or more of diabetes, hypertension, cardiovascular disease, cancer, prior cancer treatment, cerebrovascular disease, chronic obstructive pulmonary disease (COPD), chronic kidney disease, sarcoidosis, obstructive lung disease, idiopathic pulmonary fibrosis (IPF), asthma, chronic Bronchitis, emphysema, cystic fibrosis/bronchiectasis, and pneumonia.

14. The method of any one of claims 1-13, wherein the subject has a lung disease or respiratory disorder.

15. The method of any one of claims 1-14, wherein the lactate or dilactate salt of the aminosterol is a pharmaceutically acceptable grade of the aminosterol.

16. The method of any one of claims 1-15, wherein the composition further comprises one or more of an aqueous carrier, a buffer, a sugar, and/or a polyol compound.

17. The method of any one of claims 1-16, wherein the subject is a human.

18. The method of any one of claims 1-17, wherein the lactate or dilactate salt of the aminosterol is administered at a very low dose of about 50 mg or less.

19. The method of claim 18, wherein:

(a) the lactate or dilactate salt of the aminosterol is administered at a very low dose of about 45 mg or less, about 40 mg or less, about 35 mg or less, about 30 mg or less, about 25 mg or less, about 20 mg or less, about 15 mg or less, about 14 mg or less, about 13 mg or less, about 12 mg or less, about 11 mg or less, or about 10 mg or less; or

(b) the lactate or dilactate salt of the aminosterol is administered at about 9.5 mg or less, about 9 mg or less, about 8.5 mg or less, about 8 mg or less, about 7.5 mg or less, about 7 mg or less, about 6.5 mg or less, about 6 mg or less, about 5.5 mg or less, about 5 mg or less, about 4.5 mg or less, about 4 mg or less, about 3.5 mg less, about 3 mg or less, about 2.5 mg or less, about 2 mg or less, about 1.5 mg or less, about 1 mg or less, and greater than 0 mg.

20. The method of any one of claims 1-19 wherein the aminosterol is selected from the group consisting of squlamine lactate, squlamine dilactate, aminosterol 1436 lactate, and aminosterol 1436 dilactate.

21. The method of any one of claims 1-19 wherein the aminosterol is selected from the group consisting of:

(a) a lactate or dilactate salt of an isomer of squalamine; and/or

(b) a lactate or dilactate salt of an isomer of aminosterol 1436; and/or

(c) a lactate or dilactate salt of an aminosterol comprising a sterol nucleus and a polyamine attached at any position on the sterol, such that the molecule exhibits a net charge of at least + 1; and/or

(d) a lactate or dilactate salt of an aminosterol comprising a bile acid nucleus and a polyamine, attached at any position on the bile acid, such that the molecule exhibits a net charge of at least + 1; and/or

(e) a lactate or dilactate salt of an aminosterol derivative modified to include one or more of the following:

(i) substitutions of the sulfate by a sulfonate, phosphate, carboxylate, or other anionic moiety chosen to circumvent metabolic removal of the sulfate moiety and oxidation of the cholesterol side chain;

(ii) replacement of a hydroxyl group by a non-metabolizable polar substituent, such as a fluorine atom, to prevent its metabolic oxidation or conjugation; and

(iii) substitution of one or more ring hydrogen atoms to prevent oxidative or reductive metabolism of the steroid ring system; and/or

(f) a lactate or dilactate salt of a derivative of squalamine modified through medical chemistry to improve bio-distribution, ease of administration, metabolic stability, or any combination thereof; and/or

(g) a lactate or dilactate salt of a derivative of aminosterol 1436 modified through medical chemistry to improve bio-distribution, ease of administration, metabolic stability, or any combination thereof; and/or

(h) a lactate or dilactate salt of a synthetic aminosterol.

22. The method of any one of claims 1-19 wherein the aminosterol is a lactate or dilactate salt of any of the following aminosterol compounds:

Compound 4,

23. The method of any one of claims 1-19 wherein the aminosterol is selected from the group consisting of:

(a) an aminosterol having the formula:

Compound VI (ENT-06), wherein the compound is in a lactate or dilactate salt form; (b) an aminosterol having the formula:

(c) an aminosterol having the formula:

(d) an aminosterol having the formula:

C25 (R) Compound IV (D5 ENT-06), wherein the compound is in a lactate or dilactate salt form;

(e) an aminosterol having the formula:

C25 (R) Compound V (D4 ENT-06), wherein the compound is in a lactate or dilactate salt form;

(g) an aminosterol which is a prodrug comprising a compound of formula:

wherein:

(h) an aminosterol which is a prodrug comprising a compound of formula:

(i) an aminosterol which is a prodrug comprising a compound of formula:

Compound IV-P, wherein:

(j) an aminosterol which is a prodrug comprising a compound of formula:

C25 (R) Compound I V-P, where the compound is in a lactate or dilactate salt form;

(k) an aminosterol compound having the formula:

Compound V-P, wherein: R¹ is H, an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C₆ alkyl, optionally substituted C1-C₆ alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C1-C₆ alkenyl; and R² is H or -C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C₆ alkyl, optionally substituted C1-C₆ alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C₆ alkenyl; provided that at least one of R¹ and R² is not H, where the compound is in a lactate or dilactate salt form;

24. The method of any one of claims 1-19 wherein the aminosterol is selected from the group consisting of:

(a) an aminosterol having the formula:

(Compound IV), wherein the compound is in a lactate or dilactate salt form;

(d) an aminosterol having the formula:

(e) an aminosterol having the formula:

(f) an aminosterol having the formula:

(g) an aminosterol which is a prodrug comprising a compound having the formula:

(Compound III-P), wherein:

(h) an aminosterol which is a prodrug comprising a compound of formula:

(i) an aminosterol which is a prodrug comprising a compound having the formula:

(Compound IV-P), wherein:

(C25 (R) Compound I V-P), wherein the compound is in a lactate or dilactate salt form;

(k) an aminosterol compound having the formula:

Compound V-P, wherein: R¹ is H, an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, and optionally substituted C1-C6 alkenyl; and R² is H or -C(0)R³, wherein R³ is an optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkynyl, optionally substituted heterocyclyl, optionally substituted C3-C8 cycloalkyl, or optionally substituted C1-C6 alkenyl; provided that at least one of R¹ and R² is not H, wherein the compound is in a lactate or dilactate salt form;

(1) an aminosterol compound having the formula:

C25 ( R ) Compound V-P, wherein the compound is in a lactate or dilactate salt form.

25. The method of any one of claims 1-19 wherein:

(a) the aminosterol is a crystalline form of the dilactate salt of 3b-(N-[3- aminopropyl]-l,4-butanediamine)-7a, 24R-dihydroxy-5a-cholestane-24-sulfate; or

(b) the aminosterol is an amorphous form of the dilactate salt of 3b-(N-[3- aminopropyl]-l,4-butanediamine)-7a, 24R-dihydroxy-5a-cholestane-24-sulfate.

26. A composition comprising an aminosterol according to any one of claims 20-24, wherein the composition is useful in a method according to any one of claims 1-19.

27. A hand-held inhaler device comprising an aminosterol according to any one of claims 20-24.

28. The device of claim 27, wherein:

(a) the device is a single dose Dry Powder Inhaler (DPI); and/or

(b) the device delivers medicine in the form of dry powder contained in a capsule; and/or

(c) the device is a breath actuated inhaler.