Methods for treating acute respiratory inflammatory conditions and cytokine storm syndrome

Abstract

The present invention provides a pharmaceutical composition for use in treating, improving, or preventing acute respiratory diseases or conditions or cytokine storms in subjects requiring such treatment. [Solution] A pharmaceutical composition containing an effective amount of conditioned medium (CM) derived from mesenchymal stem cells (MSCs) that have been exposed to chemotherapy.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 049,663, filed on July 9, 2020, titled "ANTI CANCER THERAPY - ACTIVATED CELL THERAPY FOR TREATING ACUTE RESPIRATORY INFLAMMATORY CONDITIONS AND CYTOKINE STORM SYNDROME", the content of which is hereby incorporated by reference in its entirety.

[0002] Technical Field The present invention is in the field of treating cytokine storm and inflammation.

Background Art

[0003] Inflammatory pneumonia remains a leading cause of admission to the intensive care unit and is associated with high mortality. Respiratory failure is often caused by acute pulmonary distress syndrome (ARDS), defined by non-cardiogenic pulmonary edema, acute hypoxemia, and the need for mechanical ventilation. ARDS primarily develops from severe lung trauma, viral or bacterial pneumonia, sepsis, aspiration of gastrointestinal contents with resulting lung infections, and other conditions including acute pancreatitis. While improvements in intensive care over the past 60 years have improved patient survival rates, mortality rates remain at 30-40%. Furthermore, in many patients, a combination of an active immune system activation caused by viral or bacterial infection and an inadequate anti-inflammatory response leads to cytokine storm syndrome (CSS). Damage to the respiratory epithelium, accompanied by resulting hyperactivation of macrophages and lymphocytes, induces the massive production of pro-inflammatory cytokines such as interferons, tumor necrosis factor, interleukins, and chemokines. However, the initiation of the regenerative process is limited and insufficient. Therefore, this uncontrolled inflammation leads to widespread localized alveolar damage, as well as increased systemic levels of inflammatory cytokines that can induce pulmonary capillary damage and promote multiple organ failure.

[0004] In recent years, the outbreak of coronavirus disease 2019 (COVID-19), first identified in Hubei Province, China in December 2019, spread worldwide and became a pandemic. This disease is induced by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Severe symptoms of this disease include fever, dry cough, and difficulty breathing. Some patients with these symptoms progress to ARDS, CSS, and severe pneumonia leading to multiple organ failure. The average global mortality rate from COVID-19 is 4–7%, but in high-risk patients, including the elderly and those with certain comorbidities (e.g., diabetes, autoimmune diseases, hypertension), the mortality rate can reach 15%. COVID-19 is characterized by a long incubation period, is generally asymptomatic, and the onset of the first symptoms varies from 2 to 14 days. Nevertheless, the majority of patients infected with the virus develop only mild symptoms or remain asymptomatic. Currently, the treatment of COVID-19 is maintained with three main approaches: antiviral therapy to treat infection, including the recently approved remdesivir (Gilead Sciences, Inc.); anti-cytokine therapy to treat CSS, including anti-IL-6 and anti-IL-1 therapies; and finally, intensive supportive care with mechanical ventilation for patients with ARDS. Furthermore, research into the physiological mechanisms and pathology of the disease is needed to select the most appropriate type and timing of treatment, thereby improving patient survival and effectively managing the pandemic.

[0005] Evidence continues to grow suggesting that a subgroup of patients with severe COVID-19 develop life-threatening CSS. In particular, a cytokine profile similar to that of secondary hemophagocytic lymphohistiocytosis (sHLH), an acute inflammatory syndrome characterized by severe and fatal hypercytokinemia accompanied by multiple organ failure, has been demonstrated in COVID-19 patients. sHLH is typically induced by viral infection and occurs in about 4% of sepsis cases. sHLH associated with the severity of COVID-19 disease is characterized by elevated levels of interleukin (IL)-2, IL-7, granulocyte colony-stimulating factor (GCSF), and other pro-inflammatory cytokines. Furthermore, predictors of mortality in COVID-19 patients have been shown to include elevated levels of ferritin and IL-6 in the blood, further suggesting that mortality may be due to hyperinflammation activated by the virus. Therefore, effective COVID-19 treatment should include effective anti-inflammatory agents to suppress the hyperactivation of the immune system.

[0006] Mesenchymal stem cells (MSCs) are pluripotent stem cells found in bone marrow and mesenchymal tissues. MSCs acquire potent anti-inflammatory properties by secreting factors including PGE2, TSG6, TGFβ, and IL-10, thereby inducing broad immunosuppressive activity. Furthermore, MSCs possess excellent regenerative capabilities and have been demonstrated to effectively homing to damaged tissue, reducing inflammation and immune cell influx, improving angiogenesis, and stimulating tissue repair. Moreover, activation of MSCs with chemotherapeutic agents further enhances their regenerative capacity. There is a need for safe and effective medicines or therapies to prevent or reduce COVID-19 infection and ARDS and CCS. [Overview of the project]

[0007] The present invention provides a method for treating, improving or preventing an acute respiratory disease or condition or cytokine storm in a subject requiring such treatment, comprising administering a pharmaceutical product to the subject comprising an effective amount of MSCs in contact with paclitaxel or a conditioned medium derived from MSCs in contact with paclitaxel.

[0008] According to a first aspect, a method is provided for treating, improving or preventing an acute respiratory disease or condition in a subject requiring such treatment, comprising administering to the subject a pharmaceutical composition comprising an effective amount of mesenchymal stem cells (MSCs) contacted with chemotherapy or a conditioned medium (CM) derived from MSCs contacted with chemotherapy, thereby treating, improving or preventing an acute respiratory disease or condition in the subject.

[0009] According to a first aspect, a method is provided for treating, improving or preventing a cytokine storm in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an effective amount of MSCs exposed to chemotherapy or a conditioned medium (CM) derived from MSCs exposed to chemotherapy, thereby treating, improving or preventing a cytokine storm in the subject.

[0010] According to some embodiments, the acute respiratory disease or condition is acute respiratory distress syndrome (ARDS).

[0011] According to some embodiments, the acute respiratory disease or condition is selected from severe pulmonary trauma, sepsis, pulmonary infection, and acute pancreatitis.

[0012] According to some embodiments, the subject is suffering from a lung infection.

[0013] According to some embodiments, lung infections are selected from bacterial infections, viral infections, and infections resulting from aspiration of gastrointestinal contents.

[0014] According to some embodiments, the viral infection is a coronavirus infection.

[0015] According to some embodiments, the coronavirus is selected from human coronavirus (HcoV)-NL63, HCoV-OC43, HCoV-229E, HCoV-HKUI, severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2.

[0016] According to some embodiments, the acute respiratory disease or condition is an acute lung infection, and the method is a method for treating an acute lung infection in a person in need.

[0017] According to some embodiments, the acute respiratory disease or condition includes at least one of cytokine storm, cytokine release syndrome, secondary hemophagocytic lymphohistiocytosis (sHLH), acute respiratory distress syndrome (ARDS), perpetual fever, cytopenia, or hyperferritinemia.

[0018] According to some embodiments, the subjects do not suffer from pulmonary fibrosis.

[0019] According to some embodiments, the subject is suffering from cytokine release syndrome.

[0020] According to some embodiments, the method includes administering the substance to a conditioned medium.

[0021] According to some embodiments, the method further includes administering additional drugs or therapies to a target.

[0022] According to some embodiments, additional drugs or therapies are selected from anti-cytokine therapy, antiviral drugs, analgesics, corticosteroids, and mechanical ventilation.

[0023] According to some embodiments, the antiviral drug is selected from remdesivir, ribavirin, oseltamivir, zanamivir, and interferon alpha 2b.

[0024] According to some embodiments, the anti-cytokine therapy is selected from anti-IL-6, anti-IL6R, and anti-IL1.

[0025] According to some embodiments, treating comprises reducing an inflammatory cytokine selected from IL-6, TNFα, IL-2, IL-3, G-CSF, MIP-1α, and INFγ, improving respiratory parameters, or a combination thereof.

[0026] According to some embodiments, treating comprises reducing at least one inflammatory cytokine selected from IL-6, TNFα, IL-2, IL-3, G-CSF, MIP-1α, and INFγ in a subject.

[0027] According to some embodiments, the reduction is a reduction in the blood of the subject.

[0028] According to some embodiments, the composition is administered by oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal, or rectal administration.

[0029] According to some embodiments, the composition is administered by intratracheal or intravenous administration.

[0030] According to some embodiments, the chemotherapy is selected from paclitaxel, cisplatin, and gemcitabine.

[0031] According to some embodiments, the chemotherapy is selected from paclitaxel and cisplatin.

[0032] According to some embodiments, the chemotherapy is paclitaxel.

[0033] Further embodiments and the full scope of applicability of the present invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific examples illustrate preferred embodiments of the present invention, various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description, and these are given merely as examples.

[0034] In this specification, the present invention will be described merely as an example with reference to the accompanying drawings. While the drawings are referenced in detail and specifically here, the details shown are illustrative and intended solely for illustrative purposes to provide what is considered to be the most useful and readily understandable explanation of preferred embodiments of the present invention. In this regard, there is no intention to provide structural details of the present invention in more detail than is necessary for a basic understanding of the invention, and the description provided in conjunction with the drawings will make it clear to those skilled in the art how several forms of the present invention can be actually embodied. [Brief explanation of the drawing]

[0035] [Figure 1A-1E] Paclitaxel-activated MSC-derived therapy improves acute pneumonia. Figures 1A–1E each demonstrate computed tomography (CT) evaluation of pneumonia in the upper panel and hematoxylin and eosin (H&E) staining of the lung in the lower panel. 10–12-week-old C57Bl mice were intratracheally administered either (Figures 1B–1E) 5 mg / kg of lipopolysaccharide (LPS) or (Figure 1A) vehicle control (4 mice / group). Mice were either untreated or intratracheally (IT) administered conditioned medium (CM) derived from (1C) control mesenchymal stem cells (MSCs), (1D) cisplatin-activated MSCs, or (1E) paclitaxel-activated MSCs. Treatment was administered daily for 5 days. Pneumonia was evaluated by CT prior to the endpoint. Subsequently, the lungs were resected and processed for histopathology. The hematoxylin and eosin (H&E) staining of lung sections is demonstrated. Representative images from each panel are shown. [Figure 2]Paclitaxel-activated MSC-derived therapy restores respiratory rate in mice with acute pneumonia. The bar graph shows respiratory rate. 10-12 week old C57Bl mice were either intratracheally administered (IT) with 5 mg / kg of lipopolysaccharide (LPS) or treated with a vehicle control (4 mice / group). Mice were either untreated or administered IT with conditioned medium (CM) from control MSCs, cisplatin, or paclitaxel-activated MSCs. Treatment was administered daily for 5 days. Prior to the endpoint, pneumonia was assessed by CT scan and respiratory rate was recorded. [Figure 3] Patronized media containing paclitaxel-activated MSCs inhibit the infiltration of immune cells into lungs with acute inflammation. The bar graph shows the number of immune cells. 10-12 week old C57Bl mice were administered intratracheally (IT) with either 5 mg / kg of lipopolysaccharide (LPS) or a vehicle control. Mice were either untreated or received IT with CM from MSCs activated by cisplatin or paclitaxel. Treatment was administered daily for 5 days. At the endpoint, bronchoalveolar lavage fluid (BALF) was collected and immune cells were counted. The number of cells in BALF is shown (n=4 mice / group). [Figure 4A-4B] Chemotherapy-activated MSC-derived therapy reduces pneumonia. (4A) Bar graph showing the percentage of immune cells. Cells in Figure 3 were immunostained for MDSCs. The cells were then analyzed by flow cytometry to determine the percentage of cells in each sample. (4B) Bar graph of immune cells differentiated into macrophages, as detailed in Materials and Methods. Bone marrow-derived macrophages were cultured with a conditioned medium derived from chemotherapy-activated MSCs, and the macrophage state was evaluated by flow cytometry. The graph shows the percentage of pro-regenerative M2 macrophages. [Figures 5A-5F]Paclitaxel-activated MSC-derived therapy resulted in better recovery from acute pneumonia compared to other chemotherapeutic agents. Figures 5A–5F demonstrate computed tomography (CT) evaluation of pneumonia, respectively. 10–12-week-old C57Bl mice were intratracheally administered (5B–5F) 5 mg / kg of lipopolysaccharide (LPS) or (5A) vehicle control (4 mice / group). Mice were either untreated or intratracheally (IT) administered (5C) control mesenchymal stem cells (MSCs), (5D) gemcitabine, or (5E) cisplatin, or (5F) paclitaxel-activated MSC-derived conditioned medium (CM). Treatment was administered daily for 5 days. Pneumonia was evaluated by CT prior to the endpoint. Representative images from each panel are shown. [Figures 6A-6D] Intratracheal and intraperitoneal administration of paclitaxel-activated MSC-derived therapy improves acute pneumonia in mice. 10-12 week old C57Bl mice were intratracheally administered either (6B-D) 5 mg / kg of lipopolysaccharide (LPS) or (6A) vehicle control (4 mice / group). Mice were either untreated or received either (6C) intraperitoneal (IP) (100 μl) or (6D) intratracheal (IT) (70 μl) paclitaxel-activated MSC-derived conditioned medium (CM) daily for 5 days. Pneumonia was assessed by CT prior to endpoint (upper panel). Lungs were then resected and processed for histopathology. Hematoxylin and eosin (H&E) staining of the lungs was demonstrated (lower panel). Representative images from each panel are shown. [Figure 7] Intratracheal and intraperitoneal administration of paclitaxel-activated MSC-derived therapy improves respiratory rate in mice with acute pneumonia. The bar graph shows respiratory rate. 10-12 week old C57Bl mice were intratracheally administered 5 mg / kg of lipopolysaccharide (LPS) or vehicle control (4 mice / group). Mice were either untreated or administered paclitaxel-activated MSC-derived conditioned medium (CM) via intraperitoneal injection (IP, 100 μl) or intratracheal infusion (IT, 70 μl). Treatment was administered daily for 5 days. Respiratory rate was recorded before the endpoint. [Figure 8]A volcano plot shows that paclitaxel-activated MSC-derived therapy reduces plasma levels of pro-inflammatory cytokines and increases anti-inflammatory cytokines in mice with acute pneumonia. 10-12 week old C57Bl mice were intratracheally administered 5 mg / kg of lipopolysaccharide (LPS). Mice were either untreated or administered paclitaxel-activated MSC-derived conditioned medium (CM) by intratracheal infusion (IT, 70 μl) (4 mice / group). Treatment was administered daily for 5 days. Peripheral blood samples were collected and pooled from each group at the endpoint. Expression levels of various inflammatory cytokines in plasma were evaluated using the Proteome Profiler Mouse XL Cytokine Array (R&D Systems). Data are presented as volcano plots. [Figures 9A-9F] Paclitaxel-activated MSC-derived therapy is superior to anti-IL-6R blockade therapy in the treatment of acute pneumonia. 10-12 week old C57Bl mice were intratracheally administered (9B-D) 5 mg / kg of lipopolysaccharide (LPS) or (9A) vehicle control (4 mice / group). Mice were either untreated or (9C-D) treated with anti-mouse IL-6R antibody (10 mg / kg; BioXcell) and received (9C) intraperitoneal (IP) injection or (9D) intratracheal (IT, 70 μl) injection of paclitaxel-activated MSC-derived conditioned medium (CM). Treatment was administered daily for 5 days. Prior to the endpoint, pneumonia was assessed by CT (upper panel). The lungs were then resected and processed for histopathology. Hematoxylin and eosin (H&E) staining of the lungs was demonstrated (lower panel). Representative images for each panel are shown. [Figure 10A-10C]Paclitaxel-activated MSC-derived therapy improves pulmonary physiological function in mice with acute pneumonia more effectively than anti-IL-6R blockade therapy. (10A-C) Bar graphs show (10A) inflammatory tissue and inspiration (10B) and respiratory rate (10C) calculated using CTAn software (Bruker Corporation). 10-12 week old C57Bl mice were intratracheally administered 5 mg / kg of lipopolysaccharide (LPS) or a vehicle control (4 mice / group). Mice were either untreated or treated with anti-mouse IL-6R antibody (10 mg / kg; BioXcell) and received conditioned medium (CM) derived from paclitaxel-activated MSCs via intraperitoneal (IP) injection or intratracheal (IT, 70 μl) infusion. Treatment was administered daily for 5 days. Pulmonary function was assessed by CT prior to endpoints. [Figure 11A-11I] Paclitaxel-activated MSC-derived therapy is safe and non-toxic to healthy mice. Ten 12-week-old C57Bl mice were either untreated or administered paclitaxel-activated MSC-derived conditioned medium (CM) via intraperitoneal injection (IP, 100 μl) or intratracheal infusion (IT, 70 μl) (4 mice / group). Treatment was administered daily for 8 days. At the endpoint, organs were harvested, fixed, and examined for pathological lesions and histopathological changes in the tissues. Representative images of the following organs examined are provided: (11A) brain, (11B) spleen, (11C) stomach, (11D) heart, (11E) kidney, (11F) small intestine, (11G) lung, (11H) liver, (11I) colon. [Modes for carrying out the invention]

[0036] In some embodiments, the present invention provides methods for treating, improving, or preventing acute respiratory diseases or conditions in subjects requiring such treatment. The present invention further relates to methods for treating, preventing, or improving cytokine storms in subjects requiring such treatment.

[0037] Surprisingly, mesenchymal stem cells (MSCs), macrophages, fibroblasts, and T cells, or their conditioned media, activated by anticancer drugs (referred to here as "repair cells"), have been found to be remarkably effective in treating or managing acute diseases or conditions. In particular, and surprisingly, conditioned media (CM) derived from MSCs contacted with paclitaxel were found to be superior to CM derived from untreated MSCs, MSCs contacted with cisplatin, and MSCs contacted with gemcitabine. While it was known that MSCs activated by anticancer drugs could improve their ability to treat chronic injuries such as fibrosis, it was not known that these cells and their secretomes were well-suited for treating acute conditions such as infections and cytokine storms. Furthermore, it was previously unknown that paclitaxel activation was particularly well-suited for treating these types of diseases.

[0038] A first aspect provides a method for treating, improving or preventing an acute disease or condition in a subject requiring such treatment, comprising administering to the subject mesenchymal stem cells (MSCs) or a conditioned medium derived from MSCs that has been exposed to chemotherapy, thereby treating, improving or preventing an acute disease or condition in the subject.

[0039] In another aspect, a method is provided for treating, improving or preventing an acute disease or condition in a subject in need thereof, comprising administering to the subject mesenchymal stem cells (MSCs) contacted with paclitaxel or a conditioned medium derived from MSCs contacted with paclitaxel, thereby treating, improving or preventing an acute disease or condition in the subject.

[0040] In another aspect, a method is provided for treating, improving or preventing systemic inflammatory response syndrome (SIRS) in a subject in need thereof, comprising administering to the subject mesenchymal stem cells (MSCs) or a conditioned medium derived from MSCs that has been exposed to chemotherapy, thereby treating, improving or preventing SIRS in the subject.

[0041] In another aspect, a method is provided for treating, improving or preventing systemic inflammatory response syndrome (SIRS) in a subject in need thereof, comprising administering to the subject mesenchymal stem cells (MSCs) contacted with paclitaxel or MSC-derived conditioned medium contacted with paclitaxel, thereby treating, improving or preventing SIRS in the subject.

[0042] In another aspect, a method is provided for treating, improving or preventing systemic inflammatory response syndrome (SIRS) in a subject in need thereof, comprising administering to the subject mesenchymal stem cells (MSCs) contacted with cisplatin or a conditioned medium derived from MSCs contacted with cisplatin, thereby treating, improving or preventing SIRS in the subject.

[0043] In some embodiments of the present invention, a method is provided for treating, improving or preventing an acute condition or disease in a subject of need, comprising the step of administering to the subject a pharmaceutical composition comprising an effective amount (which may be a therapeutic and / or prophylactic effective amount) of a conditioned medium (CM) derived from a cell culture of repair cells treated with an anticancer drug.

[0044] In some embodiments of the present invention, a method is provided for treating, improving or preventing an acute condition or disease in a subject of need, comprising the step of administering to the subject a pharmaceutical composition containing an effective amount (which may be a therapeutic and / or prophylactic effective amount) of repair cells treated with an anticancer drug.

[0045] In some embodiments of the present invention, a method is provided for treating, improving or preventing an acute respiratory disease or acute respiratory condition in a subject requiring the treatment, comprising the step of administering to the subject a pharmaceutical composition comprising an effective amount (which may be a therapeutic and / or prophylactic effective amount) of a conditioned medium (CM) derived from a cell culture of repair cells treated with an anticancer drug. In some embodiments, the anticancer drug is paclitaxel. In some embodiments, the anticancer drug is cisplatin.

[0046] In some embodiments of the present invention, a method is provided for treating, improving or preventing an acute respiratory disease or acute respiratory condition in a subject requiring the treatment thereof, comprising the step of administering to the subject requiring the treatment a pharmaceutical composition comprising an effective amount (a therapeutic and / or prophylactic effective amount) of repair cells treated with an anticancer drug. In some embodiments, the anticancer drug is paclitaxel. In some embodiments, the anticancer drug is cisplatin.

[0047] In some embodiments, this method is a treatment method. In some embodiments, this method is a method of improvement. In some embodiments, this method is a method of prevention. In some embodiments, this method is a therapeutic method.

[0048] In another embodiment, MSCs contacted with paclitaxel or CMs derived from MSCs contacted with paclitaxel are provided for use in the treatment, improvement, or prevention of acute diseases or conditions.

[0049] In another embodiment, MSCs contacted with paclitaxel or CMs derived from MSCs contacted with paclitaxel are provided for use in the treatment, improvement, or prevention of acute respiratory diseases or conditions.

[0050] In another embodiment, MSCs contacted with paclitaxel or CMs derived from MSCs contacted with paclitaxel are provided for use in the treatment, improvement, or prevention of SIRS.

[0051] As used herein, the terms “chemotherapy,” “chemotherapeutic agent,” and “chemotherapeutic drug” are synonymous and interchangeable. These terms refer to anticancer drugs or compounds, or combinations thereof, that are toxic to actively dividing cells, particularly cancer cells. Chemotherapy can inhibit mitosis, induce DNA damage, or otherwise kill actively dividing cells. Examples of chemotherapy are well known in the art, and any such compound or combination of compounds can be used to activate MSCs. Examples of chemotherapy include, but are not limited to, paclitaxel, cisplatin, gemcitabine, 5-fluorouracil, dacarbazine, doxorubicin, cyclophosphamide, methotrexate, docetaxel, viorelbine, epirubicin, and many others. Examples of chemotherapy for activating MSCs can also be found in international patent application WO2020049552, which is incorporated in its entirety herein by reference. In some embodiments, the chemotherapy is selected from paclitaxel, cisplatin, and gemcitabine. In some embodiments, the chemotherapy is selected from paclitaxel and cisplatin. In some embodiments, the chemotherapy is paclitaxel. In some embodiments, the chemotherapy is cisplatin. In some embodiments, the chemotherapy is gemcitabine.

[0052] In some embodiments, MSCs are administered. In some embodiments, a composition is administered. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises MSCs. In some embodiments, the pharmaceutical composition comprises CMs. In some embodiments, the pharmaceutical composition comprises MSCs and CMs. In some embodiments, the pharmaceutical composition is provided for the use described above. In some embodiments, the pharmaceutical composition is a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0053] As used herein, the terms “carrier,” “excipient,” or “adjuvant” refer to any component of a pharmaceutical composition that is not an activator. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation aid, or simply a sterile aqueous medium such as saline. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt, gelatin, and talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline and Ringer's solution; ethyl alcohol and phosphate buffer, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances that can function as carriers in this specification include sugars, starches, cellulose and their derivatives, tragacanth powder, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifiers, and other non-toxic compatible substances used in other pharmaceutical formulations. Wetting and lubricating agents such as sodium lauryl sulfate, as well as colorants, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier can be used to formulate the compositions intended herein.In this regard, suitable pharmaceutically acceptable carriers, excipients, and diluents are well known to those skilled in the art, for example, in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the "Inactive Ingredient Guide" of the US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, all of which are incorporated herein by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents useful for the composition include distilled water, physiological saline, Ringer's solution, dextrose solution, Hanks' solution, and DMSO. These additional inactive ingredients, as well as effective formulations and administration procedures, are well known in the art and are described in standard texts, such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al., Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005), each of which is incorporated herein by reference in its entirety.The compositions described herein may also include artificially created structures such as liposomes, ISCOMS, sustained-release particles, and other vehicles that extend the half-life of peptides or polypeptides in serum. Examples of liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, and lamellar layers. Liposomes for use with the peptides described herein are formed from standard vesicle-forming lipids, typically containing neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipids is generally determined considering factors such as liposome size and blood stability. Various methods are available for preparing liposomes, as discussed in Coligan, JE et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York; see also U.S. Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0054] The carriers may, in total, constitute about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0055] In some embodiments, the pharmaceutical composition contains an effective amount or number of MSCs. In some embodiments, the pharmaceutical composition contains an effective amount of CMs. In some embodiments, the pharmaceutical composition contains effective amounts of both MSCs and CMs. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is sufficient to treat, prevent, or improve at least one symptom of an acute disease or condition or cytokine storm.

[0056] The term "therapeutic dose" refers to the amount of an active compound that is effective in treating a disease or disorder in a mammal. The term "therapeutic dose" refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome. The exact dosage form and regimen are determined by the physician based on the patient's condition.

[0057] Unless otherwise indicated, the term “effective dose” as used herein refers to the amount of compound or composition sufficient to obtain the desired response. For therapeutic purposes, the effective dose is also the amount in which the therapeutically beneficial effect of the compound or composition outweighs any toxic or adverse effects. The specific effective dose or sufficient dose will vary depending on factors such as the specific condition being treated, the patient’s physical condition (e.g., the patient’s weight, age, or sex), the type of mammal or animal being treated, the duration of treatment, the nature of any concurrent therapies, and the specific formulation used. The effective dose may be expressed, for example, in grams, milligrams, or micrograms, or in milligrams per kilogram of body weight (mg / kg), by the concentration of the active ingredient. Alternatively, the effective dose may be expressed by the concentration of the active component, such as molar concentration, mass concentration, volume concentration, gravimetric concentration, mole fraction, mass fraction, and mixing ratio. Furthermore, those skilled in the art can calculate the human equivalent dose (HED) of a medicinal product (such as the compounds of this disclosure) based on doses determined from animal models. For example, the maximum safe starting dose for human use can be estimated according to industry guidance issued by the U.S. Food and Drug Administration (FDA) titled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers."

[0058] Unless otherwise indicated, the “therapeutic dose” of a compound or composition is the amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or condition, or to delay or minimize one or more symptoms associated with the disease or condition. The therapeutic dose of a compound or composition is the amount of one or more therapeutic agents, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease or condition. The term “therapeutic dose” may include the amount that improves the overall treatment, reduces or avoids the symptoms or causes of the disease or condition, or enhances the therapeutic effect of another therapeutic agent.

[0059] Unless otherwise indicated, the “preventive effective dose” of a compound or composition is the amount sufficient to prevent or prevent the recurrence of a disease or condition, or one or more symptoms associated with the disease or condition. The preventive effective dose of a compound or composition means the amount of the therapeutic agent, alone or in combination with other agents, that provides a preventive effect in preventing the disease. The term “preventive effective dose” may include an amount that improves overall prevention or enhances the preventive effect of another preventive agent.

[0060] In some embodiments, acute respiratory disease or condition is a disease. In some embodiments, acute respiratory disease or condition is a condition. In some embodiments, acute respiratory disease or condition is selected from the group consisting of acute respiratory distress syndrome (ARDS) and acute lung injury (ALI). In some embodiments, acute respiratory disease or condition is ARDS. In some embodiments, acute respiratory disease or condition is not lung injury. In some embodiments, acute respiratory disease or condition is acute lung injury. In some embodiments, acute lung injury is not chronic lung injury. In some embodiments, acute lung injury does not include fibrosis. In some embodiments, acute respiratory disease or condition does not include fibrosis. In some embodiments, fibrosis is pulmonary fibrosis.

[0061] In some embodiments, an acute disease is not a chronic disease. In some embodiments, an acute disease is one that has recently developed. In some embodiments, an acute respiratory disease or condition is short-lived. In some embodiments, short means less than 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week. Each possibility represents a distinct embodiment of the invention. In some embodiments, recent means less than 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week. Each possibility represents a distinct embodiment of the invention. In some embodiments, an acute disease does not include fibrosis.

[0062] In some embodiments, ARDS develops from one or more conditions associated with severe lung trauma, viral or bacterial pneumonia, sepsis, aspiration of gastrointestinal contents with lung infection, coronavirus disease, or acute pancreatitis. In some embodiments, the acute respiratory disease or condition is selected from severe lung trauma, viral or bacterial pneumonia, sepsis, aspiration of gastrointestinal contents with lung infection, coronavirus disease, or acute pancreatitis. In some embodiments, the acute respiratory disease or condition is selected from severe lung trauma, sepsis, lung infection, and acute pancreatitis. In some embodiments, the acute respiratory disease or condition is severe lung trauma. In some embodiments, the acute respiratory disease or condition is sepsis. In some embodiments, the acute respiratory disease or condition is lung infection. In some embodiments, the acute respiratory disease or condition is acute pancreatitis. In some embodiments, the subject has a lung infection. In some embodiments, the acute respiratory disease or condition is systemic inflammatory response syndrome (SIRS).

[0063] In some embodiments, the lung infection is selected from bacterial infections, viral infections, and infections resulting from aspiration of gastrointestinal contents. In some embodiments, the lung infection is a bacterial infection. In some embodiments, the lung infection is a viral infection. In some embodiments, the lung infection is an infection resulting from aspiration of gastrointestinal contents. In some embodiments, the infection is pneumonia. In some embodiments, the lung infection causes SIRS. In some embodiments, the lung infection causes a cytokine storm. In some embodiments, SIRS is a cytokine storm. In some embodiments, SIRS is cytokine release syndrome.

[0064] In some embodiments, ARDS is associated with one or more of the following: non-cardiogenic pulmonary edema, acute hypoxemia, or the need for mechanical ventilation. In some embodiments, ARDS is associated with non-cardiogenic pulmonary edema. In some embodiments, ARDS is associated with acute hypoxemia. In some embodiments, ARDS is associated with the need for mechanical ventilation. In some embodiments, ARDS is associated with SIRS. In some embodiments, ARDS is associated with cytokine storm.

[0065] In some embodiments, the viral infection is a coronavirus infection. In some embodiments, the disease is a coronavirus infection. In some embodiments, the coronavirus disease is COVID-19 disease, HCoV-NL63, HCoV-OC43, HCoV-229E, HCoV-HKUI, SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus), CoVMERS (Middle East Respiratory Syndrome Coronavirus), or SARS-CoV-2. In some embodiments, the coronavirus is selected from human coronavirus (HcoV)-NL63, HCoV-OC43, HCoV-229E, HCoV-HKUI, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-1), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-1. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the coronavirus is MERS-CoV.

[0066] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a subject susceptible to coronavirus infection. In some embodiments, the subject is a bird. In some embodiments, the subject is a feline. In some embodiments, the subject is a primate.

[0067] In some embodiments, the subject is infected with the coronavirus. In some embodiments, the subject has confirmed coronavirus infection. Coronavirus infection can be confirmed by any method known in the art. Typically, the diagnosis is made by PCR testing. Methods for performing PCR testing are known in the art and are described, for example, in the WHO interim guidance of March 19, 2020: Laboratory testing for coronavirus disease (COVID-19) in suspected human cases (apps.who.int / iris / rest / bitstreams / 1271387 / retrieve), which is incorporated herein by reference in its entirety.

[0068] In some embodiments, subjects have a severe COVID-19 illness. Severe coronavirus illness / infection may include subjects having one or more of the following conditions: cytokine storm (also known as cytokine release syndrome, or CRS), secondary hemophagocytic lymphohistiocytosis (sHLH), acute respiratory distress syndrome (ARDS), persistent fever, cytopenia, and hyperferritinemia. In some embodiments, subjects are at risk of developing coronavirus illness and are at risk of developing one or more of the following conditions: cytokine storm (also known as cytokine release syndrome, or CRS), secondary hemophagocytic lymphohistiocytosis (sHLH), acute respiratory distress syndrome (ARDS), persistent fever, cytopenia, and hyperferritinemia. In some embodiments, the treatment improves, cures, or reduces at least one of the symptoms of cytokine storm (CCS), secondary hemophagocytic lymphohistiocytosis (sHLH), acute respiratory distress syndrome (ARDS), persistent fever, cytopenia, and hyperferritinemia.

[0069] In some embodiments, the infection is a severe infection. In some embodiments, the infection is pneumonia. In some embodiments, the subject is in the first phase of coronavirus infection. In some embodiments, the first phase is the first stage. In some embodiments, the first phase is an early infection. In some embodiments, the first phase is pre-symptomatic. In some embodiments, the first phase includes an upper respiratory tract infection. In some embodiments, the first phase includes upper respiratory tract symptoms. In some embodiments, the subject is in the second phase of coronavirus infection. In some embodiments, the second phase is the second stage. In some embodiments, the second phase is the lung phase. In some embodiments, the second stage includes two parts, IIA and IIB. In some embodiments, stage IIA includes pneumonia without hypoxia. In some embodiments, stage IIB includes pneumonia with hypoxia. In some embodiments, the second phase includes a lower respiratory tract infection. In some embodiments, the second stage includes pneumonia. In some embodiments, the subject is in either phase 1 or phase 2.

[0070] In some embodiments, the subject is in Phase 3. In some embodiments, Phase 3 is Stage 3. In some embodiments, Stage 3 is the third stage. In some embodiments, Phase 3 is the hyperinflammatory phase. In some embodiments, Phase 3 includes a cytokine storm. In some embodiments, Phase 3 includes ARDS. In some embodiments, Phase 3 includes admission to the ICU. In some embodiments, Phase 3 includes mechanical ventilation. In some embodiments, Phase 3 is the acute phase of infection. In some embodiments, the subject has entered the acute phase of infection. In some embodiments, Stage 3 is the severe stage of infection.

[0071] In some embodiments, subjects are at risk of coronavirus infection. In some embodiments, subjects at risk are unvaccinated subjects. In some embodiments, subjects at risk are immunocompromised subjects. In some embodiments, subjects at risk are subjects with at least one comorbidity. In some embodiments, subjects at risk are subjects in areas / locations with high infection rates. In some embodiments, high infection rates are infection rates greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. Each possibility represents a distinct embodiment of the invention. In some embodiments, high infection rates are infection rates greater than 10%. In some embodiments, subjects at risk are frontline workers. In some embodiments, subjects at risk are healthcare workers. In some embodiments, subjects at risk are care facility workers. In some embodiments, subjects at risk are subjects who cannot be vaccinated. In some embodiments, the subjects at risk are all people during the pandemic. In some embodiments, the subjects do not have comorbidities.

[0072] In some embodiments, the subject has at least one comorbidity. In some embodiments, the comorbidity is a comorbidity with coronavirus. In some embodiments, the comorbidity is selected from hypertension, diabetes mellitus, coronary heart disease, cerebrovascular disease, obesity, dyslipidemia, asthma, chronic obstructive pulmonary disease (COPD), chronic liver disease, and chronic kidney disease. In some embodiments, the comorbidity is hypertension. In some embodiments, the comorbidity is diabetes mellitus. In some embodiments, the comorbidity is coronary heart disease. In some embodiments, the comorbidity is cerebrovascular disease. In some embodiments, the comorbidity is obesity. In some embodiments, the comorbidity is dyslipidemia. In some embodiments, the comorbidity is asthma. In some embodiments, the comorbidity is COPD. In some embodiments, the comorbidity is chronic liver disease. In some embodiments, the comorbidity is chronic kidney disease.

[0073] In some embodiments, the subject has one or more of the following conditions: cytokine storm (also known as cytokine release syndrome, or CRS), secondary hemophagocytic lymphohistiocytosis (sHLH), acute respiratory distress syndrome (ARDS), persistent fever, cytopenia, and hyperferritinemia. In some embodiments, the subject suffers from a cytokine storm. In some embodiments, the subject suffers from cytokine release syndrome. In some embodiments, the subject suffers from sHLH. In some embodiments, the subject suffers from ARDS. In some embodiments, the subject suffers from persistent fever. In some embodiments, the subject suffers from cytopenia. In some embodiments, the subject suffers from hyperferritinemia.

[0074] As used herein, the term “cytokine release syndrome” (CRS) refers to a systemic inflammatory response triggered in response to a stimulus or a combination of stimuli. Cytokine release syndrome may have the characteristics of a cytokine storm, while a cytokine storm is characterized by the immediate onset of CRS in response to a stimulus. It should be noted that, in most cases, these terms are used interchangeably, as severe CRS may be referred to as a cytokine storm. These conditions occur when a large number of lymphocytes are activated and release pro-inflammatory cytokines throughout the body. This results in a cascade of lymphocyte activation and cytokine production. Cytokine storms often lead to organ failure and are frequently lethal. Methods for identifying cytokine storms are well known in the art and any such method may be used. Generally, blood sampling and identification of the presence of high levels of circulating pro-inflammatory cytokines are performed. In some embodiments, the subject suffers from CRS. In some embodiments, the subject suffers from a cytokine storm.

[0075] As used herein, the terms “treatment” or “to treat” a disease, disorder, or condition encompass alleviation of at least one symptom thereof, reduction of its severity, or inhibition of its progression. Treatment does not necessarily mean a complete cure of the disease, disorder, or condition. For a treatment to be effective, any useful composition or method herein needs to reduce the severity of the disease, disorder, or condition, reduce the severity of its associated symptoms, or result in an improvement in the quality of life of the patient or subject.

[0076] In some embodiments, the treatment improves, cures, reduces, or prevents at least one of the following conditions: cytokine storm (CCS), secondary hemophagocytic lymphohistiocytosis (sHLH), acute respiratory distress syndrome (ARDS), perpetual fever, cytopenia, and hyperferritinemia. In some embodiments, the treatment includes treating at least one symptom. In some embodiments, the treatment includes treating a cytokine storm. In some embodiments, the treatment includes reducing inflammation. In some embodiments, the treatment includes treating an infection. In some embodiments, the treatment includes treating sHLH. In some embodiments, the treatment includes treating ARDS. In some embodiments, the treatment includes treating perpetual fever. In some embodiments, the treatment includes treating cytopenia. In some embodiments, the treatment includes treating hyperferritinemia.

[0077] In some embodiments, subjects are at risk of developing severe COVID-19 disease and one or more of the following conditions: cytokine storm (also known as cytokine release syndrome or CRS), secondary hemophagocytic lymphohistiocytosis (sHLH), acute respiratory distress syndrome (ARDS), persistent fever, cytopenia, and hyperferritinemia.

[0078] In some embodiments, the treatment prevents at least one of the following symptoms: cytokine storm (also known as cytokine release syndrome, or CRS), secondary hemophagocytic lymphohistiocytosis (sHLH), acute respiratory distress syndrome (ARDS), persistent fever, cytopenia, and hyperferritinemia. In some embodiments, prevention includes preventing at least one symptom. In some embodiments, prevention includes preventing a cytokine storm. In some embodiments, prevention includes preventing increased inflammation. In some embodiments, prevention includes preventing infection. In some embodiments, prevention includes preventing symptomatic disease. In some embodiments, prevention includes preventing sHLH. In some embodiments, prevention includes preventing ARDS. In some embodiments, prevention includes preventing persistent fever. In some embodiments, prevention includes preventing cytopenia. In some embodiments, prevention includes preventing hyperferritinemia.

[0079] In some embodiments, the method includes administering MSCs treated with paclitaxel. In some embodiments, the method includes administering CMs derived from MSCs treated with paclitaxel. In some embodiments, the method includes administering MSCs and their CMs. In some embodiments, administration means administering a composition. In some embodiments, the composition does not contain an anticancer drug. In some embodiments, the composition does not contain paclitaxel. In some embodiments, the composition is configured for systemic administration. In some embodiments, the composition is configured for topical administration. In some embodiments, topical is topical to the lungs. In some embodiments, topical is intratracheal. In some embodiments, topical is direct to the lungs. In some embodiments, administration to the lungs includes inhalation. In some embodiments, the composition is configured for administration to the lungs. In some embodiments, the composition is configured as an aerosol.

[0080] As used herein, the terms “administering,” “dosing,” and similar terms refer to any method of delivering a composition containing an active agent to a subject in a manner that produces a therapeutic effect in a sound medical practice. One aspect of this subject provides intratracheal administration of a therapeutically effective dose of the composition of this subject to a patient in need. Other preferred routes of administration may include parenteral, oral, subcutaneous, intravenous, intramuscular, inhalation, or intraperitoneal. In some embodiments, the administration is systemic. In some embodiments, the administration is topical. In some embodiments, the administration is by inhalation.

[0081] The dosage will depend on the recipient's age, health condition, and weight, as well as the type, frequency, and nature of any concurrent treatments, if any.

[0082] Methods for producing MSCs and CMs of the present invention are provided below herein. In some embodiments, contact with paclitaxel is at least 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours. Each possibility represents a distinct embodiment of the present invention. In some embodiments, contact with paclitaxel is at least 24 hours. In some embodiments, contact with paclitaxel is at most 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 144 hours, or 168 hours. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the contact was about 24 hours.

[0083] In some embodiments, contact occurs during culture. In some embodiments, contact occurs ex vivo. In some embodiments, contact occurs in vitro. In some embodiments, contact is transient. In some embodiments, after contact, the medium containing the anticancer drug (i.e., paclitaxel) is removed. In some embodiments, a fresh medium is added, and the fresh medium is then used as a conditioned medium. In some embodiments, the conditioned medium is derived from MSC cultures of at least 12, 16, 18, 24, 48, 60, 72, 84, or 96 hours. Each possibility represents a distinct embodiment of the invention. In some embodiments, the conditioned medium is derived from MSC cultures of at least 24 hours. In some embodiments, the conditioned medium is derived from MSC cultures of up to 72, 84, 96, 108, 120, 132, 144, 156, or 168 hours. Each possibility represents a distinct embodiment of the invention. In some embodiments, the acclimatization medium is derived from a culture of MSCs for approximately 24 hours.

[0084] In some embodiments, the MSCs are human MSCs. In some embodiments, the MSCs are mammalian MSCs. In some embodiments, the MSCs are derived from bone marrow, fat, dental pulp, umbilical cord, or placenta. In some embodiments, the MSCs are derived from bone marrow. Methods for identifying and isolating MSCs are well known in the art and are provided below. Any such method may be used to isolate MSCs to be contacted with an anticancer drug (i.e., paclitaxel).

[0085] In some embodiments, the method further includes administering additional drugs or therapies.

[0086] In some embodiments, additional drugs or therapies are anti-cytokine therapy, anti-IL-6, anti-IL-1, antiviral drugs, interferon-alpha-2b, analgesics, corticosteroids, or mechanical ventilation. In some embodiments, the antiviral drug may be remdesivir, ribavirin, oseltamivir, or zanamivir. In some embodiments, additional drugs or therapies are selected from anti-cytokine therapy, antiviral drugs, analgesics, corticosteroids, and mechanical ventilation. In some embodiments, the antiviral drug is selected from remdesivir, ribavirin, oseltamivir, and zanamivir. In some embodiments, the antiviral drug is selected from remdesivir, ribavirin, oseltamivir, zanamivir, and interferon-alpha-2b. In some embodiments, the additional drug or therapy is interferon-alpha-2b. In some embodiments, the anti-cytokine therapy is selected from anti-IL-6, anti-IL-6R, and anti-IL-1. In some embodiments, the anti-cytokine therapy is anti-IL-6. In some embodiments, the anti-cytokine therapy is anti-IL6R. In some embodiments, the anti-cytokine therapy is anti-IL2.

[0087] In some embodiments, the treatment relates to the reduction of one or more pro-inflammatory cytokines, such as IL-6 (normal: <7 pg / ml; ARDS: >400 pg / ml), TNFα (normal: <5 pg / ml; ARDS: 200-400 pg / ml), IL-2 (normal: <5 pg / ml; ARDS: 200-400 pg / ml), G-CSF (normal: <20 pg / ml; ARDS: 200-2000 pg / ml), MIP-1α (normal: <100 pg / ml; ARDS: 200-2000 pg / ml), or INFγ (normal: <15 pg / ml; ARDS: 50-10000 pg / ml), as well as the improvement of respiratory parameters such as oxygen saturation (SpO2) and respiratory rate.

[0088] In some embodiments, the treatment includes the reduction of pro-inflammatory cytokines. In some embodiments, the treatment includes the reduction of at least one pro-inflammatory cytokine. In some embodiments, the treatment includes the reduction of at least two pro-inflammatory cytokines. In some embodiments, the pro-inflammatory cytokine is selected from IL-6, TNFα, IL-2, IL-3, G-CSF, MIP-1α, and INFγ. In some embodiments, the pro-inflammatory cytokine is IL-6. In some embodiments, the pro-inflammatory cytokine is TNFα. In some embodiments, the pro-inflammatory cytokine is IL-2. In some embodiments, the pro-inflammatory cytokine is IL-3. In some embodiments, the pro-inflammatory cytokine is G-CSF. In some embodiments, the pro-inflammatory cytokine is MIP-1α. In some embodiments, the pro-inflammatory cytokine is INFγ.

[0089] In some embodiments, the treatment includes improving respiratory parameters. In some embodiments, the parameter is respiratory rate. In some embodiments, the parameter is inhalation volume.

[0090] In some embodiments, the treatment includes an increase in at least one anti-inflammatory cytokine. In some embodiments, the treatment includes an increase in at least two anti-inflammatory cytokines. In some embodiments, the anti-inflammatory cytokine is selected from IL-10 and acidic FGF. In some embodiments, the anti-inflammatory cytokine is IL-10. In some embodiments, the anti-inflammatory cytokine is acidic FGF.

[0091] In some embodiments, the treatment involves changes in immune cell infiltration. In some embodiments, the infiltration is directed towards the site of acute inflammation. In some embodiments, the infiltration is directed towards the site of infection. In some embodiments, the infiltration is directed towards the lungs. In some embodiments, the changes include at least one of a decrease in CD4 helper cells, an increase in B cells, an increase in cytotoxic T cells, an increase in bone marrow-derived suppressor cells (MDSCs), and an increase in M2 macrophages. In some embodiments, the changes include at least one of a decrease in CD4 helper cells, an increase in B cells, an increase in cytotoxic T cells, and an increase in bone marrow-derived suppressor cells (MDSCs). In some embodiments, the changes are a decrease in CD4 helper cells. In some embodiments, the changes are an increase in B cells. In some embodiments, the changes are an increase in cytotoxic T cells. In some embodiments, the changes are an increase in MDSCs. In some embodiments, the changes are an increase in M2 macrophages. In some embodiments, M2 are anti-inflammatory macrophages. In some embodiments, CD4 T helper cells are effector cells. In some embodiments, the effector cells are activated effector cells. In some embodiments, the cytotoxic T cells are CD8 T cells. In some embodiments, the cytotoxic T cells are activated T cells.

[0092] In some embodiments, an increase is observed in the subject. In some embodiments, a decrease is observed in the subject. In some embodiments, a change is observed in the subject. In some embodiments, an improvement is observed in the subject. In some embodiments, an increase in the subject is observed in the subject's blood. In some embodiments, increases, decreases, changes, and / or improvements are observed compared to untreated subjects. In some embodiments, increases, decreases, changes, and / or improvements are observed compared to subjects treated with MSCs or CMs that have not been exposed to contact. In some embodiments, increases, decreases, changes, and / or improvements are observed compared to subjects treated with MSCs exposed to an anticancer drug other than paclitaxel or CMs. In some embodiments, the anticancer drug other than paclitaxel is cisplatin. In some embodiments, the anticancer drug other than paclitaxel is gemcitabine. In some embodiments, increases, decreases, changes, and / or improvements are significant. In some embodiments, significant means statistically significant.

[0093] In some embodiments, prior to treatment, the subject has elevated blood ferritin levels (normal: males -12 to 300 ng / ml, females -12 to 150 ng / ml, ARDS: males -270 ng / ml, females -680 ng / ml) and / or IL-6 levels (normal: <7 pg / ml; ARDS: >400 pg / ml). In some embodiments, the treatment reduces IL-6 levels to normal levels, i.e., <7 pg / ml. In some embodiments, the treatment substantially reduces the patient's IL-6 levels by up to four times. In some embodiments, the treatment substantially reduces the patient's IL-6 levels by up to two times. In some embodiments, the treatment substantially reduces the patient's IL-6 levels by more than two times.

[0094] In some embodiments, the subject has elevated or increased levels of one or more of the following pro-inflammatory cytokines: interleukin (IL)-2, IL-3, granulocyte colony-stimulating factor (GCSF), and other pro-inflammatory cytokines. In some embodiments, the treatment reduces the level of at least one pro-inflammatory cytokine to a normal level. In some embodiments, the treatment reduces the level of at least one pro-inflammatory cytokine in the patient by more than twofold. In some embodiments, the treatment reduces the level of at least one pro-inflammatory cytokine in the patient by more than fourfold. In some embodiments, the subject has elevated or increased levels of interleukin (IL)-2, IL-3, granulocyte colony-stimulating factor (GCSF), and other pro-inflammatory cytokines.

[0095] In some embodiments, the anticancer drug is paclitaxel or cisplatin. In some embodiments, the anticancer drug is paclitaxel. In some embodiments, the anticancer drug is cisplatin. In some embodiments, the anticancer drug is not cisplatin. In some embodiments, the anticancer drug is not gemcitabine.

[0096] In some embodiments, the composition is administered orally, sublingually, subcutaneously, intramuscularly, intravenously, topically, locally, intratracheally, intranasally, transdermally, and rectally. In some embodiments, the composition is administered systemically. In some embodiments, systemic administration is intravenous (iv) administration. In some embodiments, the composition is administered topically. In some embodiments, topical administration is intratracheally.

[0097] In some embodiments, the composition is administered by intratracheal or intravenous (IV) administration. In some embodiments, the composition may be administered by continuous infusion. In some embodiments, a minipump may be used.

[0098] Where used herein, “preventing” or “inhibiting” or “inhibiting” or “prophylactic” are interchangeable to mean reducing the likelihood of a subject acquiring a condition that may be fatal (e.g., if the subject has not yet experienced or shown all symptoms of the disease, or if the symptoms are mild, the onset of the disease is reduced or suppressed). The biological and physiological parameters for identifying such patients are known to the physician and depend on the disease or disorder. In the case of severe Covid-19 or ARDS, prophylactic measures are taken if the patient exhibits a cytokine storm (also known as cytokine release syndrome or CRS), secondary hemophagocytic lymphohistiocytosis (sHLH), perpetual fever, cytopenia, hyperferritinemia, or high levels of pro-inflammatory cytokines in plasma (e.g., IL-6, TNFα, IL-2, IL-3, G-CSF, MIP-1α, or INFγ), reduced respiratory parameters, or any combination thereof. In some embodiments, measures to prevent severe ARDS may be initiated when the patient has one or more of the following: more than 30 breaths per minute, blood oxygen saturation less than 93%, and a heart rate greater than 120 beats per minute.

[0099] The terms “treatment” or “to treat” a subject include applying or administering secretory cytoproducts of repair cells or repair cells themselves to a subject for the purpose of stabilizing, treating, curing, reducing, alleviating, altering, restoring, curing, curing, mitigating, relieving, improving, or influencing a disease or condition, symptoms of a disease or condition, or risk (or susceptibility) to a disease or condition. The terms refer to any evidence of success in treating or improving an acute condition or disease, including any objective or subjective parameters, e.g., reduction; remission; reduction in the rate of exacerbation; reduction in the severity of the disease; stabilization, reduction of symptoms, or making the injury, condition or state more tolerable to the subject; or slowing the rate of exacerbation or decline. In some embodiments, the terms “to treat” may include extending the subject’s life expectancy and / or delaying the need for further treatment and / or preventing death.

[0100] "Repair cells," "repair mesenchymal stem cells," "repair macrophage cells," or "repair fibroblast cells," etc., as used herein, refer to cells, mesenchymal stem cells, macrophages, or other cells that have been pre-trained with an anticancer agent using standard media for at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 48 ​​hours, or 72 hours or longer. These repair cells or the conditioned medium therefrom may be used in the treatment methods described herein. Examples of such anticancer agents include gemcitabine, paclitaxel, paclitaxel and anakinra, cisplatin, 5-FU, dacarbazine, temozoloamine-targeted drugs (e.g., bortezomib), radiation, anti-angiogenic agents, and immune checkpoint inhibitor antibodies. The terms “activating composition” or “repairing composition” include cells activated by anticancer therapy or either or both of the conditioned medium or their conditioned medium. The conditioned medium may be diluted, concentrated, dried or lyophilized. Activated cells are any of the activated mesenchymal stem cells and / or activated macrophages and / or T cells and / or fibroblasts and / or nerve tissue cells (such as astrocytes and glial cells) and / or adipose tissue-derived cells and / or other tissue-resident cells or any other activated cells that can be used to treat acute conditions, diseases or disorders such as ARDS or severe coronavirus disease. The number of activated MSCs or any other activated or repair cells of the present invention, or the conditioned medium, dosage form and administration regimen produced therefrom, and, in the case of topical administration, the location thereof, depend on the type of acute disease, condition or disorder, the location of the organ being treated, and the severity of the disease. In most cases, though not exclusively, the conditioned medium should contain at least 10 per ml. 5 ~10 6 It is produced from individual cells. In some embodiments, the treatment involves at least 1 to 10 × 10 per treatment. 6This is achieved by using individual cells. In some embodiments, the reparative composition, which is the pharmaceutical composition of the present invention, comprises reparative cells or their CMs pre-activated with an anticancer agent and a pharmaceutically acceptable carrier, and can be administered once daily, every other day, every three days, or once a week until the patient recovers. In some embodiments, the reparative cells or their CMs can be administered routinely to patients who have recovered from ARDS, AI, CRS, or coronavirus disease to prevent subsequent damage to the lungs or other body organs.

[0101] It should be noted that in some embodiments of the present invention, the conditioned medium may be concentrated, for example, by centrifugation at 1,100,000 g to obtain exosomes and other products. In some cases, the conditioned medium may be freeze-dried, for example, to be stored as a dry product for later reconstitution and use. It should be noted that in the treatment methods of the present invention, the term “conditioned medium” or “CM” also refers to the secretome, the dry product, the supernatant, and the uncontacted or diluted conditioned medium.

[0102] In one embodiment of the present invention, an "activating composition" (also interchangeably referred to herein as a "repairing composition") is provided, as defined below. According to some embodiments of the present invention, the activating composition comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts activated by an anticancer agent such as a chemotherapeutic agent. In other embodiments, the activating composition comprises mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts activated by a specific anticancer agent, e.g., a chemotherapeutic agent, which comprises the supernatant or conditioned medium of a preparation separated using the secretome of such activated cells, for example by centrifugation. The term "secretome" refers to extracellular vesicles such as proteins, metabolites, enzymes, lipids, sugar molecules, and exosomes secreted into the extracellular space by cells. In some embodiments of the present invention, cells may be collected from a patient, treated with an anticancer agent, and returned to the patient. In some embodiments of the present invention, cells may be collected from a patient, treated with an anticancer agent, and their CM administered to the patient.

[0103] Extracellular vesicles, or exosomes, are bleb formations or secretions of small vesicles from cells containing proteins, miRNAs, RNAs, DNA, and other biomolecules that can move between cells. These vesicles have been shown to function under physiological and pathological conditions.

[0104] In some embodiments of the present invention, an activation composition is provided comprising mesenchymal stem cells and / or macrophages activated by a chemotherapeutic agent or other anticancer agent, wherein the mesenchymal stem cells and / or macrophages and / or T cells and / or fibroblasts activated by the chemotherapeutic agent are isolated from a conditioned medium, where the conditioned medium is used as therapy. In some embodiments, the anticancer agent is paclitaxel. In some embodiments, the anticancer agent is cisplatin.

[0105] Routes of administration of the compositions of the present invention include oral (e.g., as tablets, capsules, or ingestible solutions), topical, mucosal (e.g., as nasal sprays or aerosols for inhalation), nasal, parenteral (e.g., in injectable forms), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, vaginal, intraventricular, intracerebral, subcutaneous, ocular (e.g., intravitreal or anterior chamber), percutaneous, rectal, buccal, epidural, and sublingual. The compositions of the present invention can be formulated specifically for any of these routes of administration.

[0106] Different composition / formulation requirements may exist depending on the delivery system. It should be understood that not all compositions need to be administered via the same route. Similarly, if a composition contains multiple active components, those components may be administered via different routes. For example, the pharmaceutical compositions of the present invention can be formulated to be delivered parenterally, for example, as a nasal spray or aerosol for inhalable or ingestible solutions using a minipump or via a mucosal route, or in an injectable form for delivery via intravenous, intramuscular or subcutaneous routes. Alternatively, the formulation may be designed to be delivered via multiple routes.

[0107] In some embodiments, the formulation may be administered as an enteric-coated formulation. As the enteric coating layer polymer, one or more solutions or dispersions of, for example, methacrylic acid copolymer, cellulose acetate phthalate, cellulose acetate butyrate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate trimellitate, carboxymethyl ethylcellulose, shellac, or other suitable enteric coating layer polymers may be used individually or in combination.

[0108] Where appropriate, the pharmaceutical composition may be administered by inhalation, topically in the form of suppositories or pessaries, topically in the form of lotions, solutions, creams, ointments or powders, by use as a skin patch, or orally in the form of tablets containing excipients such as starch or lactose, capsules alone or mixed with excipients, or elixirs containing ovules, fragrances or colorants, solutions or suspensions, or the pharmaceutical composition may be administered parenterally, for example, by intravenous, intramuscular, or subcutaneous injection. For buccal or sublingual administration, the composition may be administered in the form of tablets or lozenges and may be formulated in the conventional manner.

[0109] When the compositions of the present invention are administered parenterally, such administration includes one or more of the following methods: intravenous, intra-arterial, intraperitoneal, subarachnoid, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, or subcutaneous drug administration; and / or by the use of infusion techniques.

[0110] The pharmaceutical compositions of the present invention can be administered parenterally, for example, by infusion or injection. Pharmaceutical compositions suitable for injection or infusion may be in the form of a sterile aqueous solution, dispersion, or sterile powder containing the active ingredient, and may be prepared as needed for the preparation of such sterile solutions or dispersions suitable for infusion or injection. This preparation may optionally be encapsulated in liposomes. In any case, the final preparation must be sterile, liquid, and stable under manufacturing and storage conditions. To improve storage stability, such preparations may contain preservatives that prevent microbial growth. Prevention of microbial action can be achieved by the addition of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, or ascorbic acid. Often, isotonic substances such as sugars, buffers, or sodium chloride are recommended to ensure osmotic pressure similar to that of body fluids, especially blood. Long-term absorption of such injectable mixtures can be achieved by the introduction of absorption retarders such as aluminum monostearate or gelatin.

[0111] The dispersion can be prepared in a liquid carrier or intermediate such as glycerin, liquid polyethylene glycol, triacetin oil, or mixtures thereof. The liquid carrier or intermediate may be a solvent or liquid dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, etc.), vegetable oils, non-toxic glycerin esters, or suitable mixtures thereof. Suitable fluidity can be maintained by liposome formation, administration of a suitable particle size in the case of a dispersion, or addition of a surfactant.

[0112] For parenteral administration, the composition is best used in the form of a sterile aqueous solution, which may contain other substances, such as salts or glucose sufficient to make the solution isotonic with blood. The aqueous solution needs to be properly buffered as needed (preferably to pH 3-9). The preparation of suitable parenteral formulations under sterile conditions is readily achieved by standard pharmaceutical techniques well known to those skilled in the art.

[0113] A sterile injection solution can be prepared by mixing the repair composition with a suitable solvent and one or more of the aforementioned carriers, followed by sterile filtration. In the case of sterile powders suitable for use in the preparation of sterile injection solutions, the preparation method may include vacuum drying and freeze-drying.

[0114] As used herein, the term “approximately” in combination with a value refers to a range of ±10% of the reference value. For example, a length of approximately 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0115] It should be noted that the singular forms “a,” “an,” and “the” as used herein and in the appended claims include multiple references unless the context explicitly indicates otherwise. For example, a reference to “polynucleotide” includes multiple such polynucleotides, and a reference to “polypeptide” includes one or more polypeptides and their equivalents that are known to those skilled in the art. It should be further noted that the claims may be constructed to exclude any optional element. Thus, this statement is intended to serve as a prerequisite for the use of exclusive terms such as “solely” and “only” in relation to the description of elements of the claims or the use of “negative” limitations.

[0116] Where a convention similar to “at least one of A, B, and C” applies, such a configuration is generally intended in the sense that a person skilled in the art will understand the convention (for example, “a system having at least one of A, B, and C” includes, but is not limited to, systems of A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together). A person skilled in the art will further understand that virtually any disjunctive word and / or phrase presenting two or more alternative terms in the specification, claims, or drawings should be understood to constrain the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B”.

[0117] For clarity, it is understood that certain features of the Invention described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the Invention described in the context of a single embodiment may be provided separately or in any preferred subcombination. All combinations of embodiments relating to the Invention are specifically encompassed by the Invention and are disclosed herein as if every combination were individually and explicitly disclosed. Furthermore, all subcombinations of various embodiments and their elements are also specifically encompassed by the Invention and are disclosed herein as if every such subcombination were individually and explicitly disclosed herein.

[0118] Further objectives, advantages, and novel features of the present invention will become apparent to those skilled in the art by considering the following examples, which are not intended to be limiting. In addition, each of the various embodiments and aspects of the present invention described above and claimed in the following claims will be experimentally supported in the following examples.

[0119] Each of the various embodiments and aspects of the present invention described above and claimed in the following claims is experimentally supported in the following examples.

[0120] Examples In general, the nomenclature used herein and the experimental procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are described in detail in the literature. For example, "Molecular Cloning: A Laboratory Manual," Sambrook et al., (1989); "Current Protocols in Molecular Biology," Volumes I-III, Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA," Scientific American Books, New York; Birren et al. (eds.), "Genome Analysis: A Laboratory Manual Series," Volumes 1-4, Cold Spring Harbor Laboratory Press, New York York (1998); the method described below, U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III, Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique", Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology", Volumes I-III, Coligan, JE, ed. (1994); Stites et al.(eds.), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual," CSHL Press (1996); all of these are incorporated by reference. Other general reference materials are provided herein.

[0121] material and method Preparation of chemotherapy-trained MSCs or MSC-derived conditioned media: Mouse MSCs were isolated from bone marrow aspirate and cultured in minimal essential medium alpha (αMEM) culture dishes supplemented with 10% FBS, 1% L-glutamine, 1% sodium pyruvate, and 1% streptomycin. MSC purification was performed based on their adherence to plastic culture dishes. The medium was changed every 3 days until hematopoietic cells were washed away, leaving the adhered homogeneous MSC culture. The resulting mouse MSCs were augmented and passaged, and cells up to 10 passages were used in experiments.

[0122] To generate chemotherapy-activated MSCs, cultured MSCs were exposed to paclitaxel (100 nM), cisplatin (3 μM), gemcitabine (10 nM), or a vehicle control for 24 hours. To generate MSC-derived conditioned medium (CM), chemotherapy-educated MSCs (as described above) were placed in serum-free medium in 1 x 10⁶ units. 6 The cells were reseeded at a concentration of cells / ml. CM was collected after 72 hours.

[0123] Animal Models, Treatments, and Live Imaging: Animal use and experimental protocols were approved by the TAnimal Care and Use Committee of the Technion (Envigo, Israel). A mouse model of acute pneumonia was induced by a single intratracheal infusion of 5 mg / kg of lipopolysaccharide from Escherichia coli (Sigma Aldrich, Israel), as previously reported. Mice were either untreated or treated with anti-IL-6R antibody (10 mg / kg; BioXcell, NH, USA), control inactivated cell composting (CM) or activated cell therapy (CM). Treatments were administered once daily for 5 days. The effectiveness of the delivery route was determined using intratracheal or intraperitoneal administration. Pneumonia was evaluated by computed tomography (CT) screening using a SKY SCAN1276 micro-CT scanner (Bruker Corporation, MA, USA). After 5 days, mice were anesthetized and sacrificed. Bronchoalveolar lavage (BAL) was performed by washing the lungs with Hanks equilibrium salt solution (HBSS) supplemented with 100 μM ethylenediaminetetraacetic acid (EDTA), obtained from Biological Industries Ltd, Israel. Flow cytometry was used for the analysis of bone marrow-derived suppressor cells (using CD11b+ and Gr1+ surface markers). Lungs and additional organs were collected for histopathological evaluation. For safety testing, healthy mice were treated with activated cell products under the same conditions as above for eight consecutive days. Histopathological examination was performed on the mice.

[0124] Isolation and Differentiation of Bone Marrow-Derived Macrophages: Bone marrow-derived macrophages (BMDMs) were isolated as described above. Briefly, bone marrow cells were washed from the bone marrow and then cultured at a concentration of 500,000 cells / ml in the presence of macrophage colony-stimulating factor (MCSF, 10 ng / ml, PeproTech, Israel) to induce macrophage differentiation. After 7 days, the medium was aspirated and replaced with fresh medium. The resulting population represented M0 macrophages. Macrophage skewing was achieved by treating M0 macrophages with control MSCs or MSCs activated with paclitaxel or cisplatin using CM. M1 (CD11b+F4 / 80+, CD11c+, CD206-) and M2 (CD11b+F4 / 80+, CD11c-, CD206+) phenotypes were confirmed using flow cytometry.

[0125] Cytokine array: Levels of inflammatory cytokines in the plasma of mice with LPS-induced pneumonia or mice treated with CM of paclitaxel-activated MSCs were measured using the Proteome Profiler Mouse XL Cytokine Array (ARY028, R&Dsystems, MN) according to the manufacturer's instructions. The signals corresponding to each factor in the array were quantified by densitometry. The ratios between the expression levels of various factors in the plasma of diseased mice and mice treated with CM of paclitaxel-activated MSCs were calculated.

[0126] Histology: Lungs from control or CM-treated mice were harvested at the endpoint and subsequently fixed with paraformaldehyde. Paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin (H&E) solution.

[0127] Statistical analysis: Data were expressed as mean ± standard deviation (SD). Statistical significance was assessed using one-way ANOVA, followed by Tukey ad hoc statistical tests using GraphPad Prism5 software (LaJolla, CA). In some experiments, Student's t-tests were used when comparing only two groups. All group differences were compared to one another, and a p-value of less than 0.05 was considered statistically significant.

[0128] Example 1: Chemotherapy-activated MSC-conditioned medium increases the healing of acute respiratory inflammation. MSC therapy is currently being evaluated for the treatment of several medical conditions, including those requiring regenerative medicine. In recent years, the therapeutic activity of MSC therapy has been tested for its ability to suppress acute inflammation and cytokine storms. For example, recent studies have evaluated the ability of MSCs to inhibit the pathogenesis of COVID-19 associated with acute pneumonia and cytokine storms. However, to date, such therapies have not demonstrated any therapeutic benefits due to partially insufficient activity and high variability in treatment outcomes.

[0129] The following experiments were conducted using cell therapy products from MSCs stimulated by anticancer agents that induce regenerative, anti-inflammatory, and repair activities of MSCs. To investigate the therapeutic effect of activated cell therapy on acute pneumonia, a lipopolysaccharide (LPS)-induced model of acute pneumonia was implemented. Specifically, 10-12 week old C57Bl mice were infused with a single dose of lipopolysaccharide (LPS; 5 mg / kg) derived from E. coli. The mice were randomly divided into groups and either left untreated or received intratracheal treatment once daily with MSC-derived conditioned medium activated by different chemotherapy agents. On day 4 of the experiment, lung injury and intrapulmonary inflammation were assessed by CT scans, and one day after the mice were sacrificed, the BALF was collected and the organs were processed for histological evaluation.

[0130] The effects of paclitaxel and cisplatin-activated MSC products on respiratory inflammatory load were compared. For this purpose, CT scans and histological analyses of lungs from diseased or treated mice demonstrated that intratracheal administration of paclitaxel-activated MSC-derived conditioned medium significantly reduced pneumonia and improved respiratory function compared to the lungs of diseased mice or mice treated with unactivated conditioned medium (control MSCs) (Figure 1A-E). Cisplatin-activated MSC therapy also resulted in a reduction of pneumonia, but to a much smaller degree compared to paclitaxel-activated therapy. Similar results were further supported by additional experiments comparing the effects of paclitaxel-activated therapy with gemcitabine and cisplatin therapy in mice with LPS-induced pneumonia. CT analysis showed that paclitaxel-activated MSC therapy was the most potent treatment compared to gemcitabine and cisplatin (Figure 5A-E). These data were supported by analyses of respiratory rate and function. Specifically, paclitaxel activation therapy nearly completely restored the respiratory rate of treated mice, although this was reduced by severe pneumonia. Cisplatin activation therapy resulted in only a partial improvement in the respiratory rate of treated mice (Figure 2).

[0131] To further investigate the severity of pneumonia, BALF (branch-associated lymphocytes) were collected and analyzed. As predicted, acute pneumonia induced the recruitment and homing of inflammatory cells to the lungs, as assessed by the number of immune cells in the BALF. However, BALF obtained from mice treated with paclitaxel activation therapy showed a dramatic reduction in the number of infiltrating inflammatory cells (Figure 3). Cisplatin-activated cell-derived conditioned medium also resulted in a partial reduction in lung cell infiltration, but it was less effective than paclitaxel activation therapy.

[0132] Next, flow cytometry analysis of cells in BALF further confirmed that treatment with paclitaxel-activated MSC-conditioned medium improved the inflammatory state of the pneumonia (Figures 4A-4B). Specifically, bone marrow-derived suppressor cells increased in the lungs of mice treated with paclitaxel-activated MSC-conditioned medium (Figure 4A). This data was further supported by the observation of a shift in macrophages from a naive state to an anti-inflammatory (M2) state when mouse bone marrow was differentiated into macrophages in the presence of paclitaxel-activated MSC-conditioned medium (Figure 4B). Overall, these results indicate that paclitaxel-activated MSC-conditioned medium can be used as an effective treatment for inducing immunosuppression and treating acute pneumonia caused by infection.

[0133] Next, the administration routes of the treatment were evaluated. A conditioned medium derived from paclitaxel-activated MSCs was administered once daily to mice with LPS-induced acute pneumonia, either systemically via intraperitoneal injection or locally via intratracheal infusion. The severity of pneumonia was assessed by CT and histological evaluation (Figures 6A-6D). The conditioned medium derived from paclitaxel-activated MSCs was found to improve the inflammatory state of the lungs and restore respiratory function compared to untreated affected mice. Furthermore, local intratracheal infusion of the conditioned medium yielded significantly better results than systemic administration, resulting in reduced immune cell infiltration into the lungs and a larger respiratory area. Notably, the IP injection volume was almost 1.5 times greater than the intratracheal infusion volume. Additionally, the healing properties of activated cell therapy were confirmed by measuring the respiratory rate of treated and control mice. Specifically, pneumonia caused a dramatic impairment of respiratory function and reduced the respiratory rate of diseased mice, but paclitaxel-activated cell therapy restored respiratory capacity in treated mice (Figure 7). Intratracheal infusion of a conditioned medium derived from paclitaxel-activated cells resulted in better recovery of respiratory function in affected mice compared to untreated control mice. In summary, these results suggest that paclitaxel-activated cell therapy is effective in treating acute inflammatory lung disease caused by infection.

[0134] Example 2: Chemotherapy-activated MSCs suppress cytokine storm syndrome. Cytokine storm syndrome (CSS) is another characteristic of uncontrolled acute immune activation and subsequent depletion of immune cells. It is a life-threatening condition that leads to multiple organ failure. Because patients with COVID-19 infection can develop CSS, there is an urgent need for treatments to suppress or block the transmission of CSS. To evaluate whether a conditioned medium derived from paclitaxel-activated MSCs can suppress hypercytokinemia and prevent tissue damage, we analyzed circulating levels of over 100 inflammatory cytokines and chemokines in mice with acute pneumonia treated with paclitaxel-activated MSCs or in untreated control mice. The results demonstrated that acute respiratory inflammation induced massive secretion of pro-inflammatory mediators, such as IL-2, IL-3, IL-5, IL-6, IL-22, CCL17, tissue necrosis factor alpha (TNFα), interferon-gamma (IFNγ), macrophage inflammatory protein-1-alpha (MIP-1α), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and growth factors EGF, HGF, and FGF. CM treatment was administered intratracheally daily for 5 days. The therapeutic effect was evaluated in terms of cytokine storm and lung tissue status as endpoints. Treatment with paclitaxel-activated MSC-derived conditioned medium dramatically reduced plasma levels of all the above-mentioned pro-inflammatory mediators overexpressed in diseased mice (Figure 8 and Table 1). Furthermore, this treatment significantly upregulated circulating plasma levels of IL-10, a major immunosuppressive regulator, and thus further inhibited pneumonia. Overall, this treatment proved highly effective in controlling cytokine storms induced by acute pneumonia and preventing lung tissue damage.

[0135] [Table 1]

[0136] Siltuximab (anti-IL-6), sarilumab, and tocilizumab (anti-IL-6R) are monoclonal antibodies with significant immunosuppressive effects and are approved for the treatment of various inflammatory conditions such as autoimmune diseases, rheumatoid arthritis, and lymphoproliferative disorders, as well as various malignancies such as multiple myeloma, renal cancer, and prostate cancer. Furthermore, tocilizumab is approved for the treatment of cytokine release syndrome after CAR-T therapy. Anti-IL-6R therapy is currently under FDA review for the treatment of COVID-19-induced pneumonia and cytokine storms. Recent clinical studies in Italy and China have shown rapid improvement in ARDS and CSS in patients treated with anti-IL-6R therapy. Based on these data, the effect of chemotherapy-activated MSCs on CSS in mice with acute respiratory inflammation was compared with anti-IL-6R therapy. CT and histological staining demonstrated that anti-IL-6R therapy is effective in suppressing acute pneumonia (Figure 9A-C). Intraperitoneal injection of anti-IL-6R reduced inflammation (Figure 10A), increased respiratory function (Figures 10B-C), and inhibited inflammatory cell infiltration in the lungs (data not shown). However, CT scans revealed diffuse peripheral turbidity in the lungs, H&E staining still detected immune cell infiltration in the alveoli, and respiratory volume did not fully recover to healthy levels. In contrast, conditioned medium derived from paclitaxel-activated MSCs resulted in greater lung recovery and prevented extensive lung damage. CT scans showed no peripheral shadows and only small inflammatory areas concentrated near the main bronchi (Figures 9D, 10A). Histological analysis of the lungs detected minimal immune cell infiltration comparable to that of healthy lungs. Respiratory rate also significantly improved in treated mice. Furthermore, treatment with chemotherapy-activated cell products resulted in improved lung function compared to anti-IL-6R therapy. Respiratory volume recovered to levels seen in healthy mice (Figure 10B), and respiratory rate substantially increased compared to untreated diseased mice or diseased mice treated with anti-IL6R antibodies (Figure 10C). In summary, chemotherapy-activated MSC-derived CMs were found to be superior to anti-IL-6R therapy in treating acute pneumonia.While not bound by any single theory, this may be due to the repair and immunosuppressive properties of chemotherapy-activated MSCs, and their ability to suppress IL-6, increase IL-10 levels, and thus inhibit pneumonia.

[0137] Example 3: The chemotherapy-activated MSC product is nontoxic. The safety profile associated with the administration of paclitaxel-activated MSC-derived CM was evaluated. To test this, toxicity studies were conducted. For this purpose, naive C57Bl mice were treated with a conditioned medium derived from paclitaxel-activated MSCs for eight consecutive days (twice as long as a normal experimental course). As described in the Methods section, treatment was either systemic by intraperitoneal injection or local to the lungs by intratracheal infusion. At the endpoint, the mice were euthanized, and organs such as the brain (Figure 11A), heart (Figure 11D), lungs (Figure 11G), liver (Figure 11H), stomach (Figure 11C), spleen (Figure 11B), kidneys (Figure 11E), small intestine (Figure 11F), and colon (Figure 11I) were fixed and external histopathological evaluation was performed. Regardless of the delivery route, paclitaxel activation therapy did not result in any pathological abnormalities, and all organs and tissues examined were found to be in a normal state (Figures 11A-I). Therefore, these results provide further evidence that the conditioned medium derived from paclitaxel-activated MSCs is safe and non-toxic.

[0138] While the present invention has been described in conjunction with its specific embodiments, it is evident that many alternative, modified, and variant forms will be apparent to those skilled in the art. Therefore, it is intended to encompass all such alternative, modified, and variant forms that fall within the spirit and broad scope of the appended claims.

[0139] All publications, patents, and patent applications referenced herein are incorporated herein by reference in the same manner as each individual publication, patent, or patent application is incorporated herein by reference specifically and individually. Furthermore, any citation or specification of any reference in this application should not be construed as an acknowledgment that such reference is available as prior art to the present invention. Section headings should not necessarily be construed as being restrictive to the extent that they are used.

Claims

1. A pharmaceutical composition for use in treating, improving, or preventing a cytokine storm in a subject, comprising an effective amount of a conditioned medium (CM) derived from mesenchymal stem cells (MSCs) that have been contacted with chemotherapy.

2. A pharmaceutical composition for use in treating, improving or preventing an acute respiratory disease or condition selected from acute respiratory distress syndrome (ARDS) and pulmonary infections in a subject, comprising an effective amount of a conditioned medium (CM) derived from mesenchymal stem cells (MSCs) that have been contacted with chemotherapy.

3. The pharmaceutical composition for use according to claim 2, wherein the acute respiratory disease or condition is acute respiratory distress syndrome (ARDS).

4. The pharmaceutical composition for use according to claim 2, wherein the acute respiratory disease or condition is a pulmonary infection.

5. The pharmaceutical composition for use according to claim 1, wherein the subject is suffering from a lung infection.

6. The pharmaceutical composition for use according to claim 4 or 5, wherein the lung infection is selected from bacterial infections, viral infections, and infections resulting from aspiration of gastrointestinal contents.

7. The pharmaceutical composition for use according to claim 6, wherein the viral infection is coronavirus infection.

8. The pharmaceutical composition for use according to claim 7, wherein the coronavirus is selected from human coronavirus (HCoV)-NL63, HCoV-OC43, HCoV-229E, HCoV-HKUI, severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2.

9. A pharmaceutical composition for use according to any one of claims 2 to 8, wherein the acute respiratory disease or condition is an acute lung infection, and the use is a use in a method for treating an acute lung infection in a subject.

10. A pharmaceutical composition for use according to any one of claims 2 to 8, wherein the acute respiratory disease or condition comprises at least one of cytokine storm, cytokine release syndrome, secondary hemophagocytic lymphohistiocytosis (sHLH), acute respiratory distress syndrome (ARDS), perpetual fever, cytopenia, or hyperferritinemia.

11. A pharmaceutical composition for use according to any one of claims 1 to 8, wherein the subject is not suffering from pulmonary fibrosis.

12. A pharmaceutical composition for use according to any one of claims 1 to 8, wherein the subject is suffering from cytokine release syndrome.

13. The pharmaceutical composition for use according to any one of claims 1 to 8, wherein the pharmaceutical composition comprises the conditioned culture medium.

14. A pharmaceutical composition for use according to any one of claims 1 to 8, for use in combination with an additional drug or therapy.

15. The pharmaceutical composition for use according to claim 14, wherein the additional drug or therapy is selected from anti-cytokine therapy, antiviral drugs, analgesics, corticosteroids, and mechanical ventilation.

16. The pharmaceutical composition for use according to claim 15, wherein the antiviral agent is selected from remdesivir, ribavirin, oseltamivir, zanamivir, and interferon alpha 2b.

17. The pharmaceutical composition for use according to claim 16, wherein the anti-cytokine therapy is selected from anti-IL-6, anti-IL6R, and anti-IL1.

18. A pharmaceutical composition for use according to any one of claims 1 to 8, wherein the treatment comprises reducing pro-inflammatory cytokines selected from IL-6, TNFα, IL-2, IL-3, G-CSF, MIP-1α, and INFγ, improving respiratory parameters, or a combination thereof.

19. The pharmaceutical composition for use according to claim 18, wherein the treatment comprises reducing at least one pro-inflammatory cytokine selected from IL-6, TNFα, IL-2, IL-3, G-CSF, MIP-1α, and INFγ in the subject.

20. The pharmaceutical composition for use according to claim 18, wherein the reduction is a reduction in the blood of the subject.

21. The pharmaceutical composition for use according to any one of claims 1 to 8, wherein the pharmaceutical composition is formulated for oral, sublingual, subcutaneous, intramuscular, intravenous, local, topical, intratracheal, intranasal, transdermal, or rectal administration.

22. The pharmaceutical composition for use according to claim 21, wherein the composition is formulated for intratracheal or intravenous administration.

23. A pharmaceutical composition for use according to any one of claims 1 to 8, wherein the chemotherapy is selected from paclitaxel, cisplatin, and gemcitabine.

24. The pharmaceutical composition for use according to claim 23, wherein the chemotherapy is selected from paclitaxel and cisplatin.

25. The pharmaceutical composition for use according to claim 24, wherein the chemotherapy is paclitaxel.

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