Patent Application
20230248706
Acute respiratory distress syndrome (ARDS) is a form of acute-onset hypoxemic respiratory failure with bilateral pulmonary infiltrates, which is caused by acute inflammatory edema of the lungs not attributable to left ventricular heart failure. The most common underlying causes of ARDS include sepsis, severe pneumonia, inhalation of harmful substance, burn, as well as major trauma with shock. Endothelial injury characterized by persistently increased lung microvascular permeability resulting in protein-rich lung edema is a hallmark of ARDS. Despite recent advances in the understanding of the pathogenesis, there are currently no effective pharmacological, cell, or gene-based treatment of the disease, and the mortality rate is as high as 40%. Compared to young adult patients, the incidence of acute lung injury (ALI)/ARDS resulting from sepsis, pneumonia, and flu in elderly patients (≥60 yr) is as much as 20-fold greater and the mortality rate is 10-20-fold greater. However, the underlying causes are poorly understood. In addition, crucially little is known about how aging influences mechanisms of endothelial injury, regeneration and vascular repair as well as resolution of inflammatory injury.
COVID-19 caused by SARS-CoV2 infection is considered as a systemic disease that primarily injures the vascular endothelium although the portal for the virus is inhalational. Clinically, soon after onset of respiratory distress from COVID-19, patients develop severe hypoxiemia, and interstitial rather than alveolar edema. Pathological examinations reveal that the lungs have extensive hemorrhages and are expanded with exudatives with high incidence of thrombi in small vessels, pointing to excessive vascular endothelium injury. In addition to respiratory distress, cardiovascular complication with widespread macro- and micro-thromboses is another feature of severe COVID-19. The morbidity and mortality of COVID-19 patients in elderly patients are much higher than that in young adult patients. In New York city, the death rates of COVID-19 patients are 168, 1,540, 5,020, and 12,630 per million people in age group of 18-44, 45-64, 65-74, and ≥75 years old, respectively. In Italy, the mortality rate is less than 0.3%, for 20-39 years old COVID-19 patients, 10.1% for 60-69 years old while more than 25% for ≥70 year old COVID-19 patients. It is unknown why the severity and mortality are so much higher in elderly patients, and there is no effective treatment. The current therapy is largely supportive. Besides virus eradication, novel therapeutics to inhibit injury and promote repair and recovery is also very important.
Disclosed herein are methods and compositions for treatment of COVID-19, and COVID-19-related conditions such as COVID-19-related sepsis, COVID-19-related respiratory distress, and multi-organ failure. In addition, methods and compositions are disclosed herein to treat sepsis, acute respiratory distress syndrome (ARDS), acute inflammatory injury, infection-induced organ failure characterized by vascular injury and also to treat critical limb ischemia, and restenosis, and vascular diseases associated with impaired endothelial regeneration, vascular repair, and vascular regeneration in a subject in need thereof.
In some embodiments, the methods include administering to the subject an effective amount of one or more compounds that inhibit endothelial injury and inflammation. Exemplary compounds include, but are not limited to N-acetyl cysteine (NAC), NOX2 inhibitors (Thienopyridine, NOX2ds-tat), pan-NOX inhibitors (Apocynin, Ebselen, APX-115), Reseveratrol (trans-E-resveratrol, “RV”) nanoparticles and analogues thereof (e.g., RV-loaded nanoparticles comprising poly(D,L-lactic-co-glycolic acid) (PLGA)-b-long linker poly(ethylene glycol) (PEG, e.g. 5,000 Da) copolymer, and RV-loaded nanoparticles comprising poly(D,L-lactic acid) (PLA)-b-PEG copolymer), and NOX2 inhibiting nucleic acid.
In some embodiments, the methods include administering to the subject an effective amount of one or more compounds that promote endothelial regeneration and vascular repair. Exemplary compounds include, but are not limited to Decitabine (e.g. Dacogen, INQOVI) and its analogues (e.g., Vidaza, ONUREG), prolyl hydroxylase (PHD) inhibitors (e.g., roxadustat (FG-4592), molidustat, vadadustat, and desidustat, and dimethyoxalylglycine (DMOG) analogs), Sirtuinl (SIRT1) inhibitors (e.g., Selisistat, AG1031) and its analogues, rabeprazol (e.g., Aciphex) and its analogues, phenazopyridine (e.g., Pyridium) and its analogues; SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid.
In some embodiments, the methods include administering to the subject a combination therapy comprising (a) an effective amount of one or more compounds that inhibits endothelial injury and inflammation, and (b) an effective amount of one or more compounds that promote endothelial regeneration and vascular repair.
By way of example, but not by way of limitation, in some embodiments the combination therapy includes but is not limited to (a) one or more of the inhibitors of inflammatory injury including Dexamethasone, NAC, Apocynin, Ebselen, APX-115, Thienopyridine, or NOX2ds-tat, RV nanoparticles, NOX2 inhibiting nucleic acid, and (b) one or more of the vascular reparative drugs including Decitabine (e.g., Dacogen, INQOVI, Vidaza, ONUREG), Selisistat, AG-1031, rabeprazol, phenazopyridine or DMOG analogues roxadustat, molidustat, vadadustat, and desidustat, SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid.
The disclosed methods may include administering to a subject in need thereof an effective amount of one or more nucleic acid-based therapeutic agents for treating one or more of ARDS, sepsis, COVID-19, and COVID-19 respiratory distress and multi-organ failure. The inhibiting nucleic acid-based therapeutic may be, but is not limited to an antisense oligonucleotide, a small interfering RNA (siRNA), shRNA, and a guide RNA-based genome editing system.
The disclosed methods may be performed on any suitable subject. In some embodiments, the subject is a human and the subject is elderly, e.g., 60 years old or older.
Also disclosed herein are methods and compositions for treating one or more of: cardiovascular diseases including restenosis (to prevent or treat restenosis after percutaneous coronary intervention), and peripheral vascular disease, e.g., critical limb ischemia (to promote angiogenesis) in a subject in need thereof, the method comprising administering to the subject an effective amount of one or more of (a) a HIF1A expressing nucleic acid, (b) a FOXM1 expressing nucleic acid, (c) a SIRT1 inhibiting nucleic acid, (d) an EGLN1 inhibiting nucleic acid, (e) rabeprazol or analogues thereof, (f) Phenazopyridine or analogues thereof, (g) dimethyoxalylglycine (DMOG) analogues thereof (e.g., roxadustat, molidustat, vadadustat, and desidustat), (h) Selisistat or AG-1031 or analogues thereof.
Described herein are methods and compositions useful for inhibition of vascular injury and inflammation, and promotion of endothelial cell regeneration, vascular repair, and resolution of inflammatory injury as well as inhibiting anemia and promoting angiogenesis. In some embodiments, the methods and compositions disclosed herein are particularly useful in the aged subjects.
It has been shown that the incidence of acute respiratory distress syndrome (ARDS) resulting from sepsis is as much as 20-fold greater in elderly patients (e.g., someone who is 60 years of age or older) than in young adult patients, and the mortality rate of elderly COVID-19 patients is also 10-80-fold greater. Persistent endothelial injury leading to tissue edema and severe hypoxiemia is a hallmark of these conditions. The underlying causes are poorly understood and current therapy is merely supportive. In contrast, the methods and compositions disclosed herein show that treatment with N-acetyl cysteine (NAC), or NOX2 inhibiting nucleic acid markedly inhibit sepsis-induced lung inflammation and vascular injury and promote survival in aged mice. Additionally or alternatively, in some embodiments, nanoparticle-based gene therapy with FoxM1, or treatment with rabeprazole, phenazopyridine, Decitabine, DMOG or its analogue FG-4592 (roxadustat), or EX-527 (Selisistat) alone, or genetic deletion of SIRT1 or EGLN1 alone could promote vascular repair and recovery and rejuvenate the aged vasculature for regeneration and repair and, thus, promote recovery and survival of aged mice. In some embodiment, FOXM1 expression was not induced in lungs of elderly COVID-19 patients which was in contrast to FOXM1 induction in lungs of mid-age adult COVID-19 patients. These data for the first time provide unequivocal evidence that these drugs or their combination can be used for effective treatment of elderly patients with COVID-19 and COVID-19 associated respiratory distress and multi-organ failure, sepsis, ARDS, and multi-organ failure as well as critical limb ischemia and restenosis.
The presently disclosed subject matter is described herein using several definitions, as set forth below and throughout the application.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the invention pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a component” should be interpreted to mean “one or more components.”
As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of these terms which are not clear to persons of ordinary skill in the art given the context in which they are used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term “consisting of,” while encompassed by the term “comprising,” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term. The term “consisting essentially of” while encompassed by the term “comprising,” should be interpreted as a “partially closed” transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim.
Ranges recited herein include the defined boundary numerical values as well as sub-ranges encompassing any non-recited numerical values within the recited range. For example, a range from about 0.01 mM to about 10.0 mM includes both 0.01 mM and 10.0 mM. Non-recited numerical values within this exemplary recited range also contemplated include, for example, 0.05 mM, 0.10 mM, 0.20 mM, 0.51 mM, 1.0 mM, 1.75 mM, 2.5 mM 5.0 mM, 6.0 mM, 7.5 mM, 8.0 mM, 9.0 mM, and 9.9 mM, among others. Exemplary sub-ranges within this exemplary range include from about 0.01 mM to about 5.0 mM; from about 0.1 mM to about 2.5 mM; and from about 2.0 mM to about 6.0 mM, among others.
The terms “subject” and “patient” are used interchangeably herein. The subject treated by the presently disclosed methods, uses, and compositions is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, infant, juvenile, adult, and elderly (about 50, about 55, about 60, about 65, about 70 or about 75 years old or older). In some embodiments, an elderly human subject is about 60 years old or older. Further, a “subject” can include a patient diagnosed with or suspected of having a condition or disease.
As used herein, the term “treatment” or “treat” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By “therapeutic regimen” is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
In general, the “effective amount” or “therapeutically effective amount” of an active agent or drug delivery device refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of any encapsulating matrix, the target tissue, the subject's overall condition, and the like.
As used herein the term “analogue” or “functional analogue” refer to compounds having similar physical, chemical, biochemical, or pharmacological properties. Functional analogues are not necessarily structural analogues with a similar chemical structure. An example of pharmacological functional analogues are morphine, heroine, and fentanyl, which have the same mechanism of action, but fentanyl is structurally quite different from the other two. Exemplary analogues of DMOG include, but are not limited to roxadustat, molidustat, vadadustat, and desidustat. Exemplary analogues of Thienopyridine include, but limited to Apocynin, Ebselen, and NOX2ds-tat. These are functional analogues as they all have NOX2 inhibiting activity.
The term “combination therapy” is used in its broadest sense and means that a subject is administered at least two agents. More particularly, the term “in combination” with respect to therapy administration refers to the concomitant administration of two (or more) active agents for the treatment of a disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms.
Further, the presently disclosed compositions can be administered alone or in combination with adjuvants that enhance stability of the agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase activity, provide adjuvant therapy, and the like, including other active ingredients. In some embodiments, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.
When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times.
In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of an agent and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.
As used herein, “genetic therapy” or “gene therapy” involves the transfer of heterologous DNA to the certain cells, target cells, of a mammal, particularly a human, with a disorder or conditions for which therapy or diagnosis is sought. The DNA is introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby is produced. In some embodiments, the heterologous DNA, directly or indirectly, mediates expression of DNA that encodes the therapeutic product. In some embodiments, the heterologous DNA encodes a product, such as a peptide or RNA that mediates, directly or indirectly, expression of a therapeutic product. In some embodiments, genetic therapy is used to deliver a nucleic acid encoding a gene product to replace a defective gene or supplement a gene product produced by the mammal or the cell in which it is introduced. In some embodiments, the introduced nucleic acid encodes a therapeutic compound, such as a growth factor or inhibitor thereof, or a signaling molecule, a transcription factor, etc. that is not generally produced in the mammalian host, or the host cell, or that is not produced in therapeutically effective amounts or at a therapeutically useful time. In some embodiments, the introduced nucleic acid encodes a therapeutic compound, such as an antisense oligo, a small interfering RNA, a guide RNA oligo. In some embodiments, the heterologous DNA encoding the therapeutic product is modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.
As used herein, “heterologous nucleic acid sequence” is generally DNA that encodes RNA and proteins that are not normally produced in vivo by the cell in which it is expressed For example, the heterologous nucleic acid may include encode a gene product that is not typically expressed in the host organism, or in the host cell, or may encode a gene product that is not expressed by the host organism or the host cell at particular time, at a particular stage of development, or under particular conditions the host or the host cell is currently experiencing. In some embodiments, a heterologous nucleic acid sequence mediates or encodes mediators that alter the expression of endogenous genes by affecting transcription, translation, or other regulatable biochemical processes. Any DNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed is herein encompassed by heterologous DNA. Examples of heterologous DNA include, but are not limited to, native or non-native DNA that encodes traceable marker proteins, such as a protein that confers drug resistance, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes, transcription factors, signaling molecules, receptors, and hormones, and DNA that encodes other types of proteins, such as antibodies.
Disclosed herein are compositions and methods useful for the treatment of COVID-19 and COVID-19-associated respiratory distress and multi-organ failure, acute respiratory distress syndrome (ARDS), sepsis, critical limb ischemia, and restenosis in a subject in need thereof. The methods disclosed herein include administering one or more therapeutic compositions to a subject in need thereof.
In some embodiments, the compositions of the present disclosure include one or more compounds that inhibits endothelial injury and inflammation. In some embodiments, the compositions include one or more compounds that promote endothelial regeneration and vascular repair. In some embodiments, the compounds include a combination of (a) one or more compounds that inhibit endothelial injury and inflammation, and (b) one or more compounds that promote endothelial regeneration and vascular repair.
By way of example, but not by way of limitation, compounds that inhibits endothelial injury and inflammation include, but are not limited to N-acetyl cysteine (NAC), NOX2 inhibitors (Thienopyridine, NOX2ds-tat), pan-NOX inhibitors (Apocynin, Ebselen, APX-115), Reseveratrol (trans-E-resveratrol, “RV”) nanoparticles and analogues thereof (e.g., RV-loaded poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles coated with long linker poly(ethylene glycol) (PEG), and RV-loaded poly(D,L-lactic acid) (PLA) nanoparticles coated with long linker PEG), and NOX2 inhibiting nucleic acid.
By way of example, but not by way of limitation, compounds that promote endothelial regeneration and vascular repair include, but are not limited to Decitabine (e.g. Dacogen, INQOVI) and its analogues (e.g., Vidaza, ONUREG), dimethyoxalylglycine (DMOG, a prolyl hydroxylase (PHD) inhibitor) and analogs thereof (e.g., roxadustat (FG-4592), molidustat, vadadustat, and desidustat), Sirtuinl inhibitors (e.g., Selisistat, AG1031) and SIRT1 inhibiting nucleic acid, rabeprazol (e.g., Aciphex) and its analogues, phenazopyridine and its analogues; EGLN1 inhibiting nucleic acid, SIRT1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid.
Thus the compounds (drugs) exemplified above and their analogs can be repurposed or used for treatment of COVID-19 and COVID-19 associated respiratory distress and multi-organ failure, sepsis, ARDS, and multiple organ failure in aging patients or adult patients by either monotherapy or combination therapy.
As used herein the term Resveratrol refers to a compound having the formula C14H12O3, and is represented by the chemical structure:
Resveratrol has been reported to have anti-inflammatory, anti-oxidant and anti-cancer properties. However, its use is widely hindered by its poor solubility. The present invention identifies specific formulation with nanoparticles for treatment of COVID-19 respiratory distress and multi-organ failure, sepsis, and ARDS in patients. The nanoparticles include, but not limited to poly(D,L-lactic-co-glycolic acid) (PLGA)-b-poly(ethylene glycol) (PEG) copolymer, and poly(D,L-lactic acid) (PLA)-b-PEG copolymer. The molecular weight of PLGA is 5,000 to 100,000 Da, e.g., 55,000 Da; the molecular weight of PLA is 5,000-50,000 Da, e.g. 10,000 Da. The molecular weight of PEG is 1,000-10,000. The present invention found PEG2,000 Da (PEGl) is particularly useful. The nanoparticles include but limited to PLGA-b-PEG co-polymer, e.g., PLGA25,000-b-PEG2,000 and PLA-PEG copolymer, e.g., PLA10,000-b-PEG2,000. The estimated dose range is 0.05-50 mg/kg, e.g., 0.4 mg/kg in patients.
As used herein the term N-Acetylcysteine (NAC) also known as Acetylcysteine refers to a compound having the formula C5H9NO3S, and is represented by the chemical structure:
NAC is a drug for treatment of paracetamol overdose and thick mucus in patients with cystic fibrosis or chronic obstructive pulmonary disease. The present invention identifies new indication in treating COVID-19 respiratory distress and multi-organ failure, ARDS, and sepsis as a monotherapy or combination therapy.
As used herein the term Apocynin refers to a compound having the formula C9H10O3, and is represented by the chemical structure:
As used herein the term Ebselen refers to a compound having the formula C13H9NOSe, and is represented by the chemical structure:
As used herein the term Thienopyridine refers to a compound having the formula C7H5NS, and is represented by the chemical structure:
Apocynin also known as acetovanillone is a natural organic compound structurally related to vanillin. It functions as a NOX inhibitor and anti-oxidant. Ebselen is an organoselenium compound. It is a NOX inhibitor and anti-oxidant. Thienopyridine is an NOX2 inhibitor and also inhibits ADP receptor/P2Y12 and thus is used for its anti-platelet activity. The present invention employs their NOX 2 inhibiting activity and/or anti-oxidant activity for treatment of COVID-19 respiratory distress and multi-organ failure, ARDS and sepsis in elderly patients.
As used herein the term decitabine refers to a compound having the formula C8H12N4O4, and is represented by the chemical structure:
Decitabine is a cytidine antimetabolite analogue with potential antineoplastic activity. Decitabine has been shown to incorporate into DNA and inhibit DNA methyltransferase, resulting in hypomethylation of DNA and intra-S-phase arrest of DNA replication. Decitabine is also known as 5-Aza-2′-deoxycytidine, Dacogen, and 5-Azadeoxycytidine. The present invention identifies new indication in treating COVID-19 respiratory distress and multi-organ failure, ARDS, and sepsis in aged subjects, such as age ≥60 years old. The estimated dosage range is 0.01-1 mg/kg, e.g., 0.02 mg/kg in patients. In some embodiments, Decitabine may be more effective on elderly subjects as compared to younger subjects.
As used herein, rabeprazol refers to a compound having the formula C18H21N3O3S and represented by the structure:
Rabeprazole is a proton pump inhibitor that decreases the amount of acid produced in the stomach. Rabeprazole is used short-term to treat symptoms of gastroesophageal reflux disease (GERD) in adults and children who are at least 1 year old. Rabeprazole is used only in adults to treat conditions involving excessive stomach acid, such as Zollinger-Ellison syndrome. Rabeprazole is also used in adults to promote healing of duodenal ulcers or erosive esophagitis (damage to esophagus caused by stomach acid). Raberazole is also known as Aciphex, Habeprazole and Pariets. The present invention identifies new indication in treating COVID-19, ARDS, and sepsis as well as restenosis following PCI, and critical limb ischemia in subjects. The estimated dosage range is 0.5-10 mg/kg, e.g., 1.6 mg/kg in patients.
As used herein, the term phenazopyridine refers to a compound having the formula C12H12ClN5 and represented by the structure:
Phenazopyridine is often used to relieve the symptoms of urinary tract infections. Phenazopyridine is also known as phenazopyridine hydrochloride, phenazopyridine HCl, pyridium, and urodine. The present invention identifies new indication for Phenazopyridine and its analogues in treating COVID-19 respiratory distress and multi-organ failure, ARDS, and sepsis as well as anemia, restenosis following PCI, and critical limb ischemia in aged subjects, such as age ≥60 years old. The estimated dosage range is 1-20 mg/kg, twice a day, e.g., 4 mg/kg twice a day in patients. It is particularly useful in patients at age ≥60 years old.
As used herein dimethyoxalylglycine (DMOG) refers to a compound having the formula C6H9NO5 and represented by the structure:
Exemplary analogues of DMOG include, but are not limited to roxadustat (FG-4592), molidustat, vadadustat, and desidustat. These drugs are current under clinical trials for treatment of anemia associated with kidney failure patients. The present invention identifies new indication in treating COVID-19 respiratory distress and multi-organ failure, ARDS, and sepsis as well as restenosis following PCI, and critical limb ischemia in subjects, it is particularly useful in patients at age ≥60 years old. The estimated roxadustat dosage range is 0.2-20 mg/kg, e.g., 2 mg/kg in patients.
As used herein Selisistat (EX-527) refers to a compound having the formula C13H13ClN2O and represented by the structure:
Selisistat is a Sirtuin 1 (SIRT1)-selective inhibitor. does not inhibit histone deacetylase (HDAC) or other sirtuin deacetylase family members (IC50 values are 98, 19600, 48700, >100000 and >100000 nM for SIRT1, SIRT2, SIRT3, HDAC and NADase respectively). Enhances p53 acetylation in response to DNA damaging agents. The present invention identifies new indication of Selisistat and its analogues in treating COVID-19 respiratory distress and multi-organ failure, ARDS, and sepsis as well as restenosis following PCI, and critical limb ischemia in subjects, it is particularly useful in patients at age ≥60 years old. The estimated dosage range is 0.1-6 mg/kg, e.g., 0.6 mg/kg in patients.
The compounds (drugs) disclosed herein can be used as monotherapy or combination therapy. For example, two or three drugs can be combined in the same dosage or different dosages, respectively. The compounds can be administered to a subject with the same schedule or different schedules via the same route of administration or different route of administration. Exemplary combination therapy includes for example, at least one compound that promotes endothelial regeneration and vascular repair, and at least one compound that inhibits endothelial injury and inflammation.
Exemplary combinations include, but are not limited to e.g., 1) Dexamethasone, with one or more of Decitabine (e.g., Dacogen, INQOVI, Vidaza, ONUREG), NAC, Apocynin, Selisistat, AG-1031, rabeprazol, phenazopyridine, roxadustat, molidustat, vadadustat, desidustat, SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid; 2) NAC, with one or more of Decitabine (e.g., Dacogen, INQOVI, Vidaza, ONUREG), Selisistat, AG-1031, rabeprazol, phenazopyridine, roxadustat, molidustat, vadadustat, desidustat, SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid; 3) Apocynin with one or more of Decitabine (e.g., Dacogen, INQOVI, Vidaza, ONUREG), Selisistat, AG-1031, rabeprazol, phenazopyridine, roxadustat, molidustat, vadadustat, desidustat, SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid; 4) Thienopyridine with one or more of Decitabine (e.g., Dacogen, INQOVI, Vidaza, ONUREG), Selisistat, AG-1031, rabeprazol, phenazopyridine, roxadustat, molidustat, vadadustat, desidustat, SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid; 5) Ebselen with one or more of Decitabine (e.g., Dacogen, INQOVI, Vidaza, ONUREG), Selisistat, AG-1031, rabeprazol, phenazopyridine, roxadustat, molidustat, vadadustat, desidustat, SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid; 6) APX-115 with one or more of Decitabine (e.g., Dacogen, INQOVI, Vidaza, ONUREG), Selisistat, AG-1031, rabeprazol, roxadustat, molidustat, vadadustat, desidustat; 7) NOX2 inhibiting nucleic acid with one or more of Decitabine (e.g., Dacogen, INQOVI, Vidaza, ONUREG), Selisistat, AG-1031, rabeprazol, phenazopyridine, roxadustat, molidustat, vadadustat, desidustat, SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid; 8) NOX2ds-tat with one or more of Decitabine (e.g., Dacogen, INQOVI, Vidaza, ONUREG), Selisistat, AG-1031, rabeprazol, phenazopyridine, roxadustat, molidustat, vadadustat, desidustat, SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid; 9) RV nanoparticles with one or more of Decitabine, AG-1031, rabeprazole, phenazopyridine, roxadustat, molidustat, vadadustat, desidustat, SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid.
Additionally or alternatively, in some embodiments, viral or non-viral (e.g., nanoparticle, liposome) delivery of FOXM1, or HIF1A alone or combination with one or more of Dexamethasone, NAC or NOX inhibitor including Thienopyridine/Apocynin/Ebselen/APX-115/NOX2ds-tat, or NOX2 inhibiting nuclei acid is also useful for treatment. Viral or non-viral (e.g. Nanoparticle) delivery of SIRT1 inhibiting nucleic acid, or EGLN1 inhibiting nucleic acid alone or combination with either Dexamethasone or NAC or NOX inhibitor including Thienopyridine/Apocynin/Ebselen/APX-115/NOX2ds-tat, or RV is useful for treatment. Viral or non-viral (e.g. Nanoparticle) delivery of NOX2 inhibiting nucleic acid alone or combination with either Selisistat, and/or AG-1031, and/or rabeprazol and/or Decitabine (e.g., Dacogen, INQOVI, Vidaza, ONUREG), phenazopyridine and/or roxadustat/molidustat/vadadustat/desidustat is also useful for treatment.
Also disclosed herein are therapeutic compositions comprising a combination of one or more of decitabine and/or analogues thereof, rabeprazol and/or analogues thereof, phenazopyridine and/or analogues thereof, roxadustat and/or analogues thereof (e.g., molidustat/vadadustat/desidustat), Selisistat and/or Sirtuinl inhibitors (e.g. AG1031), NAC, Dexamethasone, Thienopyridine, NOX2ds-tat, Apocynin, Ebselen, APX-115, NOX2 inhibitors, and RV nanoparticles, for the treatment of COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, and ARDS by inhibiting vascular injury and/or promoting vascular repair and rejuvenation in aging patients.
In therapeutic and/or diagnostic applications, the compounds of the disclosure can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams and Wilkins (2000).
Use of pharmaceutically acceptable inert carriers to formulate the compounds herein disclosed for the practice of the disclosure into dosages suitable for administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, capsules, dragees, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion. For nasal or inhalation delivery, the compositions of the disclosure also may be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, sprays, inhalers, vapors; solubilizing, diluting, or dispersing substances, such as saline, preservatives, such as benzyl alcohol; absorption promoters; and fluorocarbons may be included.
Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, the compounds according to the disclosure are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.1 to 1000 mg, from 0.5 to 200 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. A non-limiting dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.
In some embodiments, the pharmaceutical composition comprises a therapeutic nucleic acid. By way of example, but not by way of limitation, in some embodiments, nucleic acid compositions are administered to a subject via delivery methods including viral vectors, liposomes, nanoparticles, or naked nucleic acids, such as naked DNA.
In some embodiments, therapeutic nucleic acids are provided as inhibitory RNA oligonucleotides and include, but are not limited to modified or unmodified antisense oligonucleotides, small interfering RNAs (siRNA), guide RNA oligonucleotides, or a combination thereof, antisense, siRNA or guide RNA expressing plasmid DNA. By way of example, but not by way of limitation, exemplary therapeutic, inhibitory or inhibiting nucleic acids include NOX2 siRNA, Sirtuin 1 (SIRT1) siRNA, and EGLN1 siRNA.
In some embodiments, therapeutic nucleic acids are engineered and formulated to express a therapeutic protein after administration. By way of example, but not by way of limitation, exemplary therapeutic nucleic acids engineered and formulated to express a therapeutic protein include a FOXM1 expressing nucleic acid, and a HIF1A expressing nucleic acid
Thus, in some embodiments, the presently disclosed subject matter provides a pharmaceutical composition comprising one or more of rabeprazol, phenazopyridine, DMOG analogs (e.g., roxadustat, molidustat, vadadustat, desidustat), Selisistat, AG1031, decitabine (e.g., Dacogen, INQOVI, Vidaza, ONUREG), Dexamethasone, NAC, Apocynin, APX-115, Thienopyridine, NOX2ds-tat, Ebselen, and analogues thereof, and optionally additional agents, and a pharmaceutically acceptable carrier. Additionally or alternatively, in some embodiments, the presently disclosed subject matter provides a pharmaceutical composition comprising one or more of a NOX2 inhibiting nucleic acid, a FOXM1 expressing nucleic acid, a HIF-1α expressing nucleic acid, a Sirtuinl inhibiting nucleic acid, or an EGLN1 inhibiting nucleic acid.
Methods:
Embodiments of the technology include treatment methods whereby pharmaceutical compositions disclosed herein (e.g., a composition including one or more of (a) a compound that inhibits endothelial injury and inflammation, and (b) a compound that promotes endothelial regeneration and vascular repair) are administered to a subject in need thereof.
In some embodiments, a subject in need thereof is a subject who has been diagnosed with or is at risk of having COVID-19. In some embodiments, a subject in need thereof includes a subject suffering from one or more COVID-19 related symptoms, including but not limited to: COVID-19-related sepsis, and COVID-19-related respiratory distress and organ failure.
In some embodiments, a subject in need thereof includes a subject suffering from sepsis, acute respiratory distress syndrome (ARDS), acute inflammatory injury, and infection-induced organ failure characterized by increased lung microvascular permeability and inflammation.
In some embodiments, a subject in need thereof includes a subject suffering from cardiovascular diseases including restenosis, and peripheral vascular disease, e.g., critical limb ischemia.
As noted above, the compositions of the present disclosure may be formulated for a desired mode of administration, including but not limited to parenterally, orally, and via inhalation.
In some embodiments of the methods, a composition may be administered a single time, or may be administered multiple times, over the course of one or more days or weeks.
In some embodiments, a subject in need thereof is elderly, e.g., 60 years old or older, or 70 years old or older, or, 80 years old or older, 90 years old or older In some embodiments, the subject is a human.
In some embodiments, the subject is a non-human mammal.
In some embodiments, the methods include administering one or more of the pharmaceutical compositions described herein to a subject of any age. In some embodiments, the methods include administering one or more of the pharmaceutical compositions described herein to an elderly subject. Useful, maybe more effective, or may have a greater therapeutic effect when administered to by way of example only, but not by way of limitation, in some embodiments, compositions comprising Dexamethasone, Resveratrol, NAC, rabeprazole, phenazopyridine, roxadustat, molidustat, vadadustat, and desidustat, EGLN1 inhibiting nucleic acid, HIF1A expressing nucleic acid, FOXM1 expressing nucleic acid may be administered to a subject of any age, with an expectation of a positive therapeutic effect. In some embodiments, by way of example only and not by way of limitation, compositions comprising Decitabine, Apocynin, Ebselen, APX-115, NOX2 inhibiting peptide (NOX2ds-tat), Thienopyridine, Selisistat, and AG-1031, NOX2 inhibiting nucleic acid, SIRT1 inhibiting nucleic acid may be particularly useful in elderly subjects, e.g., subjects at least about 60 years old or older. That is, compositions comprising these exemplary compounds have a greater therapeutic effect on an elderly subject in need thereof as compared to a non-elderly subject (e.g., a teen or someone under about 60 years old, for example).
Applications
Exemplary application of the methods and compositions disclosed herein include but are not limited to the following: (1) treatment of COVID-19 and COVID-related conditions including, but not limited to (2) COVID-related respiratory distress and multi-organ failure in aging patients and also adult patients, treatment of COVID-related sepsis, and septic shock in aging patients and also adult patients; (3) treatment of acute respiratory distress syndrome in aging patients and also adult patients; (4) treatment of sepsis and multiple organ failure associated with sepsis in aging patients and also adult patients; (5) treatment of acute inflammation in aging patients and also adult patients; (6) treatment of restenosis in aging patients and also adult patients; (7) treatment of peripheral ischemic vascular disease (e.g., critical limb ischemia) in aging patients and also adult patients. In some embodiments, one or more of decitabine (e.g. Dacogen, INQOVI) and its analogues (e.g., Vidaza, ONUREG), N-acetyl cysteine (NAC), NOX2 inhibitors (Thienopyridine, NOX2ds-tat,), pan-NOX inhibitors (Apocynin, Ebselen, APX-115), Reseveratrol (trans-E-resveratrol, “RV”) nanoparticles and analogues thereof (e.g., RV-loaded nanoparticles comprising of poly(D,L-lactic-co-glycolic acid) (PLGA)-b-long linker poly(ethylene glycol) (PEG) copolymer, and RV-loaded nanoparticles comprising of poly(D,L-lactic acid) (PLA)-b-long linker PEG copolymer), and NOX2 inhibiting nucleic acid, and one or more of a prolyl hydroxylase (PHD) inhibitor) and DMOG analogs (e.g., roxadustat, molidustat, vadadustat, and desidustat), Sirtuinl inhibitors (e.g., Selisistat and its analogues, AG1031), rabeprazol (e.g., Aciphex) and its analogues, phenazopyridine (e.g., Pyridium) and its analogues; and SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF-1α expressing nucleic acid, FOXM1 expressing nucleic acid is administered to a subject suffering from one or more of the aforementioned diseases. In some embodiments, the subject is an adult human, and in some embodiments the subject is an elderly human, e.g., age 60 years old or older.
Acute respiratory distress syndrome (ARDS) is a form of acute-onset hypoxemic respiratory failure with bilateral pulmonary infiltrates, which is caused by acute inflammatory edema of the lungs not attributable to left heart failure. The most common underlying causes of ARDS include sepsis, severe pneumonia, inhalation of harmful substance, burn, major trauma with shock, as well as viral infection. Endothelial injury characterized by persistently increased lung microvascular permeability resulting in protein-rich lung edema is a hallmark of ARDS. Despite recent advances on the understanding of the pathogenesis, there are currently no effective pharmacological or cell-based treatment of the disease with a mortality rate as high as 40%. Compared to young adult patients, the incidence of ARDS resulting from sepsis, pneumonia, flu in elderly patients (≥60 yr) is as much as 19-fold greater and the mortality rate is 10-20-fold greater (1, 8-12). However, the underlying causes are poorly understood. Also crucially little is known how aging influences mechanisms of endothelial regeneration and resolution of inflammatory lung injury.
COVID-19 caused by SARS-CoV2 infection is considered as a systemic disease that primarily injures the vascular endothelium although the portal for the virus is inhalational. Clinically, soon after onset of respiratory distress from COVID-19, patients develop severe hypoxiemia, and interstitial rather than alveolar edema. Pathological examinations reveal that the lungs have extensive hemorrhages and are expanded with exudatives with high incidence of thrombi in small vessels, pointing to excessive vascular endothelium injury. In addition to respiratory distress, cardiovascular complication with widespread macro and micro-thromboses is another feature of severe COVID-19. The morbidity and mortality of COVID-19 patients in elderly patients are much greater than that in adult patients. In New York city, the death rates of COVID-19 patients are 168, 1540, 5020, and 12630 per million people in age group of 18-44, 45-64, 65-74, and ≥75 years old, respectively. In Italy, the mortality rate of COVID-19 patients at age of 20-39 years is less than 0.3%, 10.1% for 60-69 years old COVID-19 patients while more than 25% for ≥70 years old COVID-19 patients.
In some embodiments, one or more of the aforementioned conditions or diseases is caused by infection, or is exacerbated by infection, which may be bacterial or viral in origin.
Exemplary, non-limiting examples of viral infections and viral agents include influenza, pneumonia, the common cold (e.g., mainly caused by rhinovirus, coronavirus, and adenovirus) encephalitis and meningitis, (e.g., caused by enterovirus and herpes virus), Zika virus, HIV, hepatitis C, polio, Dengue fever, H1N1 swine flu, Ebola, MERS-CoV, SARS virus, SARS-CoV2 (causing COVID-19), and other coronavirus, mumps, human papillomavirus, herpes virus, rotavirus and chicken pox.
Exemplary, non-limiting examples of bacterial infections and bacterial agents include pneumonia, tuberculosis, typhoid, typhus, meningitis, upper respiratory tract infections, eye infections, sinusitis, urinary tract infections, skin infections, and nosocomial infections. These are caused by either gram negative or positive bacterial infections.
In some embodiments, the subject is treated according to the methods of the present disclosure when an infection has been identified or is suspected, but prior to the onset of sepsis, septic shock, ARDS, COVID-19 respiratory distress, respiratory failure or multiple organ failure due to sepsis or infection, etc. Accordingly, the compositions and methods of the present disclosure may be employed prophylactically as well as therapeutically.
Advantages
Current therapies for COVID-19 respiratory distress and multi-organ failure, sepsis and ARDS are merely supportive; there are no effective therapies for these conditions in adult patients, and particularly in aging patients who have much greater morbidity and mortality. Persistent endothelial injury is a prominent feature of these conditions, in particular, COVID-19 is now considered as a systemic disease that primarily injures the vascular endothelium. In contrast the methods and compositions of the present disclosure provide therapeutic relief by, for example, inhibiting injury, especially vascular injury, and cytokine storm, promoting vascular survival, repair, and recovery, and also inhibiting injury and promoting repair and recovery by combination therapy, and therefore fill a much-needed gap in the treatment of these diseases and conditions. Moreover, while the therapies disclosed herein are effective and safe for patients of all age groups, they are surprisingly and unexpectedly potentially more effective in elderly patients than younger patient.
The following Examples are illustrative and are not intended to limit the scope of the claimed subject matter.
Example 1. Therapeutic activation of endothelial regeneration, vascular repair and resolution of inflammation in elderly patients with COVID-19 and COVID-19 respiratory distress and multi-organ failure, sepsis, ARDS, and multi-organ failure, as well as cardiovascular diseases including but not limited to restenosis and critical limb ischemia.
Rationale: Aging is a risk factor of high incidence and great morbidity and mortality of COVID-19 respiratory distress and multi-organ failure, sepsis and ARDS. However, it is unknown how aging influences mechanisms of endothelial regeneration and resolution of inflammatory lung injury.
Objectives: We aimed to investigate the underlying mechanisms and explore therapeutic approach to reactivate vascular repair and resolve inflammatory injury in aged lungs.
Methods. Genetic lineage tracing was used to study endothelial regeneration. Sepsis was induced by either cecal ligation and puncture (CLP) or lipopolysaccharide (LPS). Vascular permeability and inflammation was measured. In vivo BrdU labeling was used to quantify endothelial proliferation. Foxm1 transgenic mice and gene transduction of FoxM1 in lungs of aged mice were used. FDA-approved drug library was screened to identify drugs which could rejuvenate the aged endothelium for regeneration and repair. Autopsy lung samples from COVID-19 patients were employed to validate clinical relevance of our findings in animals.
Measurements and Main Results. Endothelial regeneration was mediated by lung resident endothelial proliferation, which was impaired in aged mice. Aged mice exhibited persistent inflammatory lung injury and great mortality following sepsis challenge. Expression of FoxM1, an important mediator of lung endothelial regeneration in young adult mice, was not induced in aged lungs. Transgenic expression of FoxM1 normalized vascular repair in aged mice and promoted survival following sepsis challenge. In vivo gene transduction of FOXM1 targeting vascular endothelium or repurposing treatment with FDA-approved drug Decitabine was sufficient to reactivate FoxM1-dependent endothelial regeneration in aged mice, reverse aging-impaired resolution of inflammatory injury, and promote survival. In COVID-19 lung autopsy samples, FOXM1 expression was not induced in vascular endothelial cells of elderly patients in contrast to mid-age patients, validating the clinical relevance of the findings in aged mice.
Conclusion. These results show that aging impairs intrinsic endothelial regeneration and vascular repair leading to persistent inflammatory lung injury following sepsis challenge, and therapeutic restoration of FoxM1 expression can reactivate vascular repair and resolution of inflammatory injury in aged mice. Thus, activation of FoxM1-mediated endothelial regeneration and vascular repair represents a potential effective approach for treatment of COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and organ failure, sepsis, ARDS, and multi-organ failure in elderly patients with pneumonia, flu, SARS-CoV2, and other pathological conditions.
Introduction
Acute respiratory distress syndrome (ARDS) is a form of acute-onset hypoxemic respiratory failure with bilateral pulmonary infiltrates, which is caused by acute inflammatory edema of the lungs not attributable to left heart failure (1-3). The most common underlying causes of ARDS include sepsis, severe pneumonia, inhalation of harmful substance, burn, and major trauma with shock. Severe COVID-19 results in severe sepsis, respiratory distress and multi-organ failure. Endothelial injury characterized by persistently increased lung microvascular permeability resulting in protein-rich lung edema is a hallmark of severe COVID-19 including COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, severe sepsis and ARDS (4-7). Despite recent advances on the understanding of the pathogenesis, there are currently no effective pharmacological or cell or gene-based treatment of COVID-19, sepsis and ARDS with a mortality rate as high as 40% (1-3). Compared to young adult patients, the incidence of COVID-19 respiratory distress and multi-organ failure and ARDS resulting from sepsis, pneumonia, flu, and COVID-19 in elderly patients (≥60 yr) is as much as 19-fold greater and the mortality rate is 10-100 fold greater (1, 8-12). However, the underlying causes are poorly understood. Also crucially little is known how aging influences mechanisms of endothelial regeneration and resolution of inflammatory lung injury.
The forkhead box (Fox) transcriptional factors share homology in the winged helix or forkhead DNA-binding domains (13, 14). Among the Fox family, FoxM1 is the first one identified as a proliferation-specific transcriptional factor. FoxM1 is expressed during cellular proliferation and mediates cell cycle progression by transcriptional control of many of the cell cycle genes (15-19). During embryogenesis, FoxM1 is expressed in many types of cells, such as cardiomyocytes, endothelial cells (ECs), hepatocytes, lung epithelium cells, and smooth muscle cells (20-23). In adult mice, FoxM1 is restrictively expressed in intestinal crypts, thymus and testes (15, 16). Although FoxM1 is silenced in terminally differentiated cells (15-17), it can be induced after organ injury. We have reported that FoxM1 is induced in lung ECs in the repair phase but not in the injury phase following sepsis challenge (24). In EC-restricted Foxm1 null mice, pulmonary vascular EC proliferation and endothelial barrier recovery are defective following inflammatory lung injury (24). FoxM1 also mediates re-annealing of the endothelial adherens junctional complex to restore the endothelial barrier function following vascular injury (25). Additionally, we also showed that EC-expressed FoxM1 is the endogenous mediator of exogenous stem/progenitor cells-elicited paracrine effects on vascular repair and resolution of inflammatory lung injury (26). These results demonstrate the critical role of FoxM1 in vascular repair. Other studies also demonstrate the important role of FoxM1 in mediating lung epithelial repair (27) and hepatocyte regeneration (28) after injury in adult mice. Thus, FoxM1 is an important reparative transcription factor. However, it is unknown if FoxM1 can be induced in aged lungs and whether forced expression of FoxM1 in pulmonary vascular ECs is sufficient to reactivate vascular repair to resolve inflammatory lung injury in aged mice following sepsis challenge.
Here we sought to define the cell source of origin mediating endothelial regeneration and determine how aging affects this process as well as vascular repair and resolution of inflammatory lung injury. We further delineated the underlying molecular mechanisms. Our studies demonstrate that aging impairs the intrinsic endothelial regeneration program and thus vascular repair and inflammation resolution. Restored FoxM1 expression in lung ECs in aged mice is necessary and sufficient to re-activate lung endothelial regeneration and vascular repair and thereby resolve inflammatory lung injury and promote survival following sepsis challenge. Thus, therapeutic activation of FoxM1 expression in aged lungs by either repurposed FDA-approved drugs or nanoparticle delivery of FoxM1 gene represent a novel and effective treatment of COVID-19, and COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, septic shock, ARDS, and multi-organ failure in elderly patients to reduce morbidity and mortality.
Methods
Mice. EndoSCL-CreERT2/mTmG lineage tracing mice were generated by breeding the mice carrying a double-fluorescent reporter expressing membrane-targeted tandem dimer Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) after excision (mTmG mice, #007676, the Jackson Laboratory) with EndoSCL-CreERT2 transgenic mice (29-31) (C57BL/6 background) containing tamoxifen-inducible Cre-ERT2 driven by the 5′ endothelial enhancer of the stem cell leukemia locus. Foxm1 transgenic
Foxm1Tg mice were described previously (32, 33). Both male and female mice were used in the experiments. Mice at various ages (3-5 mo. old referred as young or adult; 19-21 mo. old referred as aged, 25 mo. old referred as elderly) were used. The experiments were conducted according to NIH guidelines on the use of laboratory animals. The animal care and study protocols were approved by the Institutional Animal Care and Use Committees of Northwestern University and The University of Illinois at Chicago.
Induction of lung injury. Polymicrobial sepsis was induced by CLP using a 23-gauge needle (34). Briefly, mice were anesthetized with inhaled isofluorane (2.5% mixed with oxygen). When the mice failed to respond to paw pinch, buprenex (0.1 mg/kg) was administered subcutaneously prior to sterilization of the skin with povidone iodine, then a midline abdominal incision was made. The cecum was exposed and ligated with a 4-0 silk tie placed 0.6 cm from the cecum tip, and the cecal wall was perforated with a 23-gauge needle. Control mice underwent anesthesia, laparotomy, and wound closure, but no cecal ligation or puncture. Following the procedure, 500 μl of prewarmed normal saline was administered subcutaneously. Within 5 min following surgery, the mice woke from anesthesia. The recovered mice subcutaneously received a second dose of buprenex at 8h post-surgery.
To induce endotoxemia, mice received a single dose of LPS (0.25-2.5 mg/kg BW, Escherichia coli 055:B5, Santa Cruz, St. Dallas, Tex.) by i.p. injection. The LPS dose was dependent on the aging of the mice (3-9 mo old, 2.5 mg/kg; 19-21 mo. old, 1.0 mg/kg; 25 mo. old, 0.25 mg/kg). All mice were anesthetized with ketamine/xylazine (100/5 mg/kg BW, i.p.) prior to tissue collection. For the survival study, mice were treated with a single dose of LPS (1.5 mg/kg, i.p.) and monitored for 7 days.
Vascular permeability assessment. The Evans blue dye-conjugated albumin (EBA) extravasation assay was performed as previously described (26, 34). Briefly, mice were retro-orbitally injected with EBA at a dose of 20 mg/kg BW at 30 minutes prior to tissue collection. Lungs were perfused free of blood with PBS, blotted dry and weighed. Next, lung tissues were homogenized in 1 ml PBS and incubated with 2 volumes of formamide at 60° C. for 18 hours. The homogenates were then centrifuged at 10,000×g for 30 minutes. The optical density of the supernatant was determined at 620 nm and 740 nm. The extravasated EBA in lung homogenate was presented as μg of Evans blue dye per g lung tissue.
Myeloperoxidase assay. Following perfusion free of blood, lung tissues were collected and homogenized in 50 mmol/L phosphate buffer. Homogenates were centrifuged at 15,000×g for 20 minutes at 4° C. The pellets were resuspended in phosphate buffer containing 0.5% hexadecyl trimethylammonium bromide and subjected to a cycle of freezing and thawing. Subsequently, the pellets were homogenized and the homogenates were centrifuged again. Absorbance was measured at 460 nm every 15 secs for 3 minutes and data expressed as ΔOD460/min/g lung tissue (26, 34).
Cell proliferation. At 8 h prior to tissue collection, BrdU (Sigma-Aldrich, St Louis, Mo.) was injected i.p. into mice at 50 mg/kg BW. Mouse lung cryosections were stained overnight with anti-BrdU (1:3, BD Biosciences, San Jose, Calif.) and incubated with Alexa Fluo 488-conjugated secondary antibody (1:200, Life Technologies, Grand Island, N.Y.). Lung vascular ECs were immunostained with anti-vWF (1:300, Sigma-Aldrich, St Louis, Mo.) and anti-CD31 (1:100, BD Biosciences, San Jose, Calif.) antibodies at 4° C. then the sections were incubated with Alexa Fluor 594-conjugated secondary antibodies (1:200, Life Technologies, Grand Island, N.Y.). The nuclei were counterstained with DAPI (Life Technologies, Grand Island, N.Y.). Three consecutive cryosections from each mouse lung were examined, the average number of BrdU+ nuclei was used (24, 34).
FACS analysis. After perfusion free of blood with PBS, lung tissues were cut into small pieces, and then incubated with 1 mg/ml collagenase A (Roche Applied Science) for 1 h at 37° C. in a shaking water bath (200 rpm). After digestion, the tissue was dispersed to a single cell preparation using the gentle MACS™ Dissociator (Miltenyi Biotec) with lung program 2. The cells were then filtered using a 40 μm Nylon cell strainer and blocked with 20% FBS for 30 min. After incubation with Fc blocker (1 μg/106 cells, BD Biosciences), the cells were immunostained with anti-CD45-PB (1:800, BioLegend) and/or anti-CD31-APC (1:600, BD Biosciences) for 45 min at room temperature. Cells were then analyzed by flow cytometry (Fortessa, BD Biosciences) and sorted by flow-assisted cell sorting (Moflo Asrtios machine, Beckman Coulter). mGFP- or tdTomato-labelled cells were directly analyzed with 488 nm or 561 nm laser wavelengths, respectively.
Molecular analysis. Total RNA was isolated using an RNeasy Mini kit including DNase I digestion (Qiagen, Valencia, Calif.). Following reverse transcription, quantitative RT-PCR analysis was performed using a sequence detection system (ABI ViiA 7 system; Life Technologies, Grand Island, N.Y.). The following primers sets were used for analysis: mouse FoxM1 primers, 5′-CACTTGGATTGAGGACCACTT-3′ (SEQ ID NO: 1) and 5′-GTCGTTTCTGCTGTGATTCC-3′ (SEQ ID NO: 2); mouse cyclophilin primers, 5′-CTTGTCCATGGCAAATGCTG-3′ (SEQ ID NO: 3) and 5′-TGATCTTCTTGCTGGTCTTGC-3′ (SEQ ID NO: 4). Primers for mouse Cdc25c, Ccna2, Ccnb1, Tnf, Il6, Nos2, and Icam1 were purchased from Qiagen. The mouse gene expression was normalized to cyclophilin.
Western blot analysis was performed using an anti-FoxM1 antibody (1:800, sc-376471, Santa Cruz Biotechnology, Santa Cruz) and the same blot was incubated with anti-β-actin antibody (1:3000, BD Biosciences, San Jose, Calif.) as a loading control.
Imaging. Following immunostaining, lung sections were imaged with a confocal microscope system (LSM510; Carl Zeiss, Inc) equipped with a 63×1.2 NA objective lens (Carl Zeiss, Inc.). For lineage tracing studies, the cryosections were directly mounted with Prolong Gold mounting media containing DAPI.
Histology. Lung tissues were fixed by 5 min instillation of 10% PBS-buffered formalin through tracheal catheterization at a trans-pulmonary pressure of 15 cm H2O, and then agitated overnight at room temperature. After paraffin processing, the tissues were sectioned (5 μm) and stained with hematoxylin and eosin.
Transduction of plasmid DNA into lung vascular endothelial cells in mice. To make liposomes, a mixture comprised of dimethyldioctadecylammonium bromide and cholesterol (1:1 molar ratio) was dried using a Rotavaporator (Brinkmann), and dissolved in 5% glucose followed by 20 min sonication as described previously (25, 34). The complex consisting of plasmid DNA expressing human FOXM1 under the control of human CDH5 promoter or empty vector and liposomes was combined at a ratio of 1 μg of DNA to 8 nmol of liposomes. The DNA/liposome complex (50 μg of DNA/mouse) was injected into the retro-orbital venous plexus at 12h post-LPS challenge.
In a separate study, mixture of nanoparticle:plasmid DNA at a ratio of 1 μg DNA to 0.25 mg nanoparticles (15 μg DNA/mouse) was administered to mice of 25 mo. old at 24h post-LPS.
RNAscope in situ hybridization assay and immunostaining: To determine FOXM1 mRNA expression in ECs of COVID-19 patient lungs and control normal donor lungs, a single-plex RNAscope in situ hybridization assay (ACD, Bio-techne, Newark, Calif.) combined with immunofluorescent staining for CD31 as a EC marker was carried out. Briefly, the tissue sections were baked for 1 h at 60° C., deparaffinized, and treated with H2O2 for 10 min at room temperature. Target retrieval was performed for 15 min at 100° C., followed by protease treatment for 15 min at 40° C. The sections were then hybridized with human FOXM1 probe (Cat #446941, target region 308-1244 in NM_001243088.1, ACD, Bio-techne) for 2 h at 40° C. followed by signal amplification for 30 min using RNAscope® Multiplex Fluorescent v2 Assay (Cat #333110, ACD, Bio-techne) as per manufacturer's instructions. The signal was developed by incubating the slides with TSA plus Cyanine 5 system (PerkinElmer, Waltham, Mass.) for 30 min. After RNAscope assay, the slides were incubated in blocking buffer (3% BSA, 1% FBS and 0.1% normal donkey serum) for 1 h followed by incubation with a primary antibody against CD31 (Cat #Ab28364, Abcam, Cambridge, Mass.) at 4° C. overnight. The sections were washed and incubated with appropriate anti-rabbit secondary antibody labeled with Alexa Fluor 488 for 1 h. The slides were then counterstained with DAPI and mounted in Prolong Gold Antifade mounting medium (ThermoFisher Scientific).
To quantify FOXM1 expression, a score system of 0-5 was used. 5 represented highest while 1 represented lowest expression in vascular ECs of each vessel. Fifteen 63×fields each section were randomly selected and examined.
Statistical analysis. Statistical significance was determined by one-way ANOVA with a Dunnett post hoc analysis that calculates P values corrected for multiple comparisons using Prism 7 (Graphpad Software, Inc.). Two-group comparisons were analyzed by the unpaired 2-tailed Student's t test for equal variance. Statistical analysis of the survival study was performed with the log-rank (Mantel-Cox) test. P<0.05 denoted the presence of a statistically significant difference. All bars in dot plot figures represent means.
Results
Cells for lung endothelial regeneration originate from resident ECs following polymicrobial sepsis-induced injury.
The major pathogenic feature of ALI/ARDS leading to deterioration of vascular barrier function is the precipitous loss of ECs (24, 35-37). To trace the changes of pulmonary ECs following sepsis challenge, we employed a murine tamoxifen-inducible lineage tracing model, mTmG/EndoSCL-CreERT2 mice (
To further determine whether bone marrow-derived cells contribute to post-sepsis endothelial regeneration, we transplanted bone marrow cells from mTmG/EndoSCL-CreERT2 mice to lethally irradiated WT mice to generate chimeric mice. We observed a small population (<0.1%) of CD45−GFP+ cells in the chimeric mouse lungs (Sham group). At 144h post-CLP, the percentage of this cell population was unaltered. The CD45+GFP+ population was also remained steady (
Impaired Endothelial Regeneration Leading to Persistent Inflammatory Lung Injury in Aged Mice Following Polymicrobial Sepsis
FACS analysis revealed that the lung GFP+ EC population was markedly decreased at 48h post-CLP in aged (19-21 mo) mice as observed in adult mice. However, the GFP+ EC population in aged mice failed to recover and remained low at 144h post-CLP (
Aging Impairs Lung Vascular Repair and Resolution of Inflammation Following Endotoxemia
To determine if aged mice also exhibit impaired vascular repair following endotoxemia challenge, aged (19-21 mo) and young (3-5 mo) mice were challenged with LPS. Given that aged mice exhibited greater lung injury indicated by greater EBA flux and MPO activity at 24h post-LPS compared to young adult mice (data not shown), we challenged the aged mice with a lower dose of LPS (e.g., 1.0 mg/kg) to induce similar degree of injury during the injury phase (e.g., 24h) as seen in young adult mice with 2.5 mg/kg of LPS (
MPO activity was also similarly increased at 24h post-LPS in these young adult and aged mice (
To further determine how aging affect vascular repair and inflammation resolution, we challenged the mice at various ages (from 3 to 21 mo old) with LPS and EBA flux and MPO activity were assessed at 72h post-LPS. As shown in
Defective Endothelial Proliferation and Inhibited FoxM1 Induction in Aged Lungs Following LPS Challenge
To gain insights into the molecular and cellular mechanisms of impaired vascular repair and inflammation resolution in aged lungs, we first determined lung endothelial proliferation by in vivo BrdU labeling. There was a marked increase of endothelial proliferation in the lungs of young adult mice at 72h post-LPS whereas endothelial proliferation in lungs of aged mice (19-21 mo old) was largely inhibited (
Normalized Vascular Repair and Inflammation Resolution in Aged FOXM1Tg Mice.
To determine if failure of FoxM1 induction is responsible for the impaired vascular repair and inflammation resolution seen in aged mice, we employed the FOXM1Tg mice expressing human FOXM1 under the control of the −800-base pair Rosa26 promoter (32, 33). EBA flux was similar under basal condition, similarly increased at 24h post-LPS challenge in aged FOXM1Tg mice (19-21 mo old) compared to aged WT mice, demonstrating similar degree of lung vascular injury (
To determine the survival effect, the mice were challenged with a higher dose of LPS (e.g., 1.5 mg/kg). Aged WT mice exhibited 100% mortality within 3-4 days whereas all young adult mice survived (
Therapeutic Expression of FoxM1 Restores Endothelial Regeneration and Resolution of Inflammatory Lung Injury in Aged WT Mice Following LPS Challenge
Next, we employed a gene therapy approach to determine if forced FoxM1 expression in lung vascular ECs of aged WT mice can reactivate endothelial proliferation and thus reverse the defective resolution of inflammatory lung injury. A mixture of liposome:plasmid DNA (25, 34) expressing human FOXM1 under the control of human CDH5 promoter (EC-specific) was administered retro-orbitally to 19-20 mo old WT mice at 12h post-LPS challenge (established lung injury). Empty vector DNA was administered to a separate cohort of aged and gender-matched WT mice. As shown in
We also assessed whether the restored vascular repair and inflammation resolution is attributable to reactivated endothelial proliferation (i.e. regeneration) in aged lungs. BrdU labeling study revealed a marked increase of EC proliferation in lungs of FOXM1 plasmid DNA-transduced mice in sharp contrast to vector DNA-transduced mice (
To further determine if forced expression of FoxM1 in mice at very old age (e.g., 25 mo old) can still reactivate the vascular repair program to promote resolution of inflammatory lung injury, we employed our newly developed nanoparticles (which has the potential as a delivery vehicle for gene therapy) to deliver the FOXM1 plasmid DNA to lungs of 25 mo old WT mice. The mixture of nanoparticle:plasmid DNA was administrated retro-orbitally to mice at 24h post-LPS challenge (to ensure the injury response was not affected, i.e. similar degree injury between FOXM1 plasmid DNA- and vector DNA-transduced mice). At 96h post-LPS, lungs were collected for EBA and MPO assessment. As shown in
Failure of FoxM1 Induction in Pulmonary Vascular ECs of Elderly COVID-19 Patients.
To validate the potential clinical relevance of our findings in aged mice, we collected lung autopsy samples from COVID-19 patients (Table S1) and carried out RNAscope in situ hybridization assay to determine FoxM1 expression. FoxM1 expression in pulmonary vascular ECs was markedly induced in middle-aged COVID-19 patients but not in elderly patients (
Conclusion
The present study has demonstrated that lung resident EC mediates endothelial regeneration responsible for vascular repair and resulting resolution of inflammation following vascular injury induced by polymicrobial sepsis and aging impairs these processes leading to persistent inflammatory lung injury and high mortality in aged mice. Aging inhibits FoxM1 induction and resulting endothelial proliferation in aged lungs following sepsis challenge. Transgenic expression of FoxM1 normalizes vascular repair and inflammation resolution and promotes survival in aged mice. Therapeutic gene transduction of FoxM1 in lung ECs of aged mice reactivates FoxM1-dependent endothelial regeneration and vascular repair in aged mice. These therapeutic effects were also evident in mice even at age of 25 mos. old. We also observed marked induction of FOXM1 expression in pulmonary vascular ECs of mid-age COVID19 patients but not in elderly patients.
Thus, therapeutic activation of FoxM1 expression by delivery of FOXM1 expressing nucleic acid or pharmacological drugs may represent an effective approach to restore the endothelial barrier integrity and reverse lung edema in the prevention and treatment of COVID-19 and COVID-19 respiratory distress and multi-organ failure, sepsis and ARDS as well as vascular diseases with diminished FOXM1 expression including but not limited to restenosis and critical limb ischemia in elderly patients and adult patients.
Example 2. Repurposing rabeprazole or phenazopyridine as monotherapy or combination therapy with NAC, or Dexamethasone or NOX2 inhibitors or other drug(s) for the treatment of COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis and ARDS as well as vascular diseases with impaired HIF-1a signaling and/or diminished FOXM1 expression including but not limited to restenosis and critical limb ischemia in elderly patients and adult patients.
Hypoxia-inducible factors (HIFs) comprised of an O2-sensitive α-subunit (mainly HIF-1α and HIF-2α) and a constitutively expressed β-subunit are key transcription factors mediating adaptive responses to hypoxia and ischemia (1,2). Our study shows that hypoxia-inducible factor (HIF)-1α is required for endothelial regeneration and vascular repair and thus resolution of inflammatory lung injury in (young) adult mice (3). FoxM1 expression was not induced in lung vascular ECs in Hif1α EC-specific knockout mice and restoration of FoxM1 in ECs normalized vascular repair and resolution of inflammation in Hif1α EC-specific knockout mice. To identify FDA-approved drug(s) that could activate HIF-1α/FoxM1 signaling and thereby provide novel therapeutic agents for treatment of COVID-19 respiratory distress and multi-organ failure, sepsis and ARDS and vascular diseases including but not limited to restenosis and critical limb ischemia in elderly patients and adult patients, we carried out high-throughput screening of the Prestwick Chemical Library of FDA-approved drugs (1280 compounds) employing stable cell line containing the HIF-response element. Rabeprazole and Phenazopyridine were the 2 activators identified by the inventor, which can induce FoxM1 expression and promote vascular repair and resolution of inflammation in aged mice and also young adult mice following sepsis challenge.
Rabeprazole is a HIF1α activator which can reactivate FoxM1 expression and vascular repair in aged lungs.
To test if Rabeprazole can activate vascular repair and resolution of inflammation in aged mice, we challenged aged mice (22 mo. old) with LPS to induce endotoxemia and inflammatory injury, and then treated with Rabeprazole at 6h and 24h post-LPS. Lungs were collected for Evan blue-conjugated albumin (EBA) assay (a measurement of vascular permeability) and myeloperoxidase (MPO) activity assay at 72h post-LPS. As shown in
Rabeprazole can also facilitate vascular repair in young adult mice.
To test if Rabeprazole can facilitate vascular repair in young adult mice, we challenged 3-5 mos. old mice with LPS to induce endotoxemia and inflammatory injury, and then treated with Rabeprazole (18 mg/kg, oral) at 6h and 24h post-LPS. Lungs were collected for EBA) assay at various times post-LPS. As shown in
Rabeprazole promote HIF-1a/FoxM1-dependent vascular repair.
To determine if Rabeprazole-induced vascular repair is through activation of HIF-1a, WT and Hif1a EC-specific knockout mice (4 mos. old) were challenged with LPS and then treated with Rabeprazole at 6 h and 24h post-LPS. At 52h post-LPS, lungs were collected EBA assay. As shown in
we also determine if Rabeprazole-induced vascular repair is mediated by endothelial FoxM1. WT and Foxm1 EC-specific knockout mice (3-5 mos. old) were challenged with LPS and then treated with Rabeprazole at 6 h and 24h post-LPS. At 52h post-LPS, lungs were collected EBA assay. Rabeprazole-induced vascular repair seen in WT mice was also inhibited in Foxm1 EC KO mice (
Together, these data demonstrate that Rabeprazole can efficiently activate FoxM1-dependent endothelial regeneration, vascular repair and resolution of inflammation in aged mice as well as young adult mice. Thus, Rabeprazole and its analogues can be repurposed for treatment of elderly patients and also adult patients with COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, ARDS, and multi-organ failure to reduce morbidity and mortality as a monotherapy or combination therapy with either Dexamethasone, NAC, NOX2 inhibitors (Apocynin, Ebselen, APX-115, Thienopyridine, NOX2ds-tat, NOX2 inhibiting nucleic acids), Phenazopyridine or its analogues.
Additionally, as rabeprazole can activate endothelial regeneration and vascular repair, it can also be repurposed for treatment of vascular diseases with impaired HIF-1a signaling or diminished FOXM1 expression including restenosis, and critical limb ischemia (to promote angiogenesis).
Besides rabeprazole, we also found another drug, phenazopyridine, which could also promote vascular repair in aged mice (
Combination of rabeprazole or its analogue with Phenazopyridine or its analogue can be repurposed for treatment of vascular diseases associated with impaired HIF-1a signaling and/or diminished FOXM1 expression including but not limited to restenosis, and critical limb ischemia.
Example 3: EGLN1 deficiency normalizes vascular repair and reactivates FoxM1 expression in lungs of aged mice. EGLN1 inhibitors (e.g., roxadustat, molidustat, vadadustat, and desidustat) and Egln1 inhibiting nucleic acid as a monotherapy or combination therapy with one or more of Dexamethasone, RV, NAC, NOX2 inhibitors (Apocynin, Ebselen, APX-115, Thienopyridine, NOX2ds-tat, NOX2 inhibiting nucleic acid) for treatment of COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, ARDS, and multi-organ failure. And also for treatment of vascular diseases associated with impaired HIF-1a signaling and/or diminished FOXM1 expression but not limited to including restenosis and critical limb ischemia.
As O2 sensors, HIF prolyl-4 hydroxylases [prolyl hydroxylase domain-containing enzymes (PHDs), also known as EGLN1-3] use molecular O2 as a substrate to hydroxylate specific proline residues of HIF-α. Hydroxylation promotes HIF-α binding to the von Hippel-Lindau (VHL) ubiquitin E3 ligase resulting in ubiquitination and subsequent degradation by proteasome (1-4). EGLN1 (i.e. PHD2) is responsible for the majority of HIF-α hydroxylation while EGLN2 and EGLN3 play compensatory roles under certain conditions (5-8). To determine the role of EGLN1 in regulating FoxM1 expression and vascular repair in aged mice, WT and Egln1ΔEC mice with EC-restricted disruption of Egln1 at age of 21 months were challenged with LPS. We observed similar degree of vascular injury and lung MPO activity in WT and Egln1ΔEC mice at 15h post-LPS challenge (
Example 4: Dimethyloxalylglycine (DMOG) analogues including roxadustat, molidustat, vadadustat, and desidustat as a monotherapy or combination therapy with one or more of Dexamethasone, RV, NAC, NOX2 inhibitors (Apocynin, Ebselen, APX-115, Thienopyridine, NOX2ds-tat, NOX2 inhibiting nucleic acid) to treat COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, ARDS, and multi-organ failure in elderly patients and adult patients, and also for treatment of vascular diseases associated with impaired HIF-1a signaling and/or diminished FOXM1 expression but not limited to including restenosis and critical limb ischemia.
DMOG is a cell permeable EGLN/PHD inhibitor which stabilizes HIF-α. We next determined whether DMOG can activate the vascular repair program in aged mice. Aged mice (21 mo. old) were challenged with LPS and then treated with DMOG at 12 or 24h post-LPS and lung tissues were collected at 72h post-LPS. As shown in
FG-4592 (i.e., roxadustat) is a DMOG analogue with more specific inhibition of prolyl hydroxylase 2. Roxadustat was recently tested for treatment of anemia in patients with chronic kidney disease (1). We also tested whether FG-4592 treatment could also activate vascular repair in aged mice. Aged mice (21 mo. old) were challenged with LPS and then treated with FG-4592 at 24h post-LPS and lung tissues were collected at 72h post-LPS. As shown in
Example 5: SIRT1 inhibitors (e.g., Selisistat, AG-1031, SIRT1 inhibiting nucleic acid) as a monotherapy or combination therapy with one or more of Dexamethasone, NAC, NOX2 inhibitors (Apocynin, Ebselen, APX-115, Thienopyridine, NOX2ds-tat, NOX2 inhibiting nucleic acid) to treat COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, ARDS, and multi-organ failure in elderly patients as well as for treatment of vascular diseases with hyperactivated SIRT1 signaling and/or diminished FOXM1 expression including but not limited to restenosis and critical limb ischemia.
SIRT1 belongs to NAD+-dependent histone deacetylases (also called sirtuins, SIRT1-7) (1). Via deacetylation of both epigenetic and non-epigenetic targets, SIRT1 regulates the cell cycle, apoptosis, and oxidative stress response, thereby influences cell viability and aging (2). Published study shows that SIRT1 deficiency enhances lung inflammation following sepsis challenge in adult mice (3). To study the role of SIRT1 in regulating FoxM1 expression and vascular repair in aged mice, we first generated a mouse model with EC-restricted disruption of Sirt1 (Sirt1ΔEC) (
Our findings are novel and fundamentally important for drug development. We for the first time show that SIRT1 deficiency promote vascular repair and resolution of lung inflammation in aged mice which is in contrast with the literature that SIRT1 deficiency in young adult mice enhances inflammatory lung injury. Thus, SIRT1 has different (maybe opposite) functions at different ages in response to sepsis challenge. SIRT1 is an important regulator of the inflammatory response in young adult mice while it is a key inhibitor of vascular repair in aged mice. Thus, targeting SIRT1 may be a novel and important strategy to activate the dormant vascular repair process in aged subject to promote vascular repair and resolution of inflammation and thus promote survival.
Next we addressed the possibility of pharmacological inhibition of SIRT1 to activate the intrinsic vascular repair program in aged mice. WT mice at age of 20 months were challenged with LPS and received treatment of EX-527 (i.e., Selisistat) or vehicle. At 72h post-LPS, lung tissues were collected for analysis. Vascular permeability in aged WT mice treated with EX-527 was at basal level in contrast to control WT mice (
Example 6. Aging exaggerates inflammatory lung injury.
It has been shown that the incidence of acute lung injury (ALI)/ARDS resulting from sepsis is as much as 20-fold greater in elderly patients (≥60 yr) than in young adult patients, and the mortality rate of elderly ALI/ARDS patients is also up to 20-fold greater (1-6). The severity and mortality of COVID-19 in elderly patients are 10-100 fold greater (7, 8). Per CDC report, the overall cumulative hospitalization rate of COVID-19 patients in US between Mar. 1, 2020 and May 8, 2020 is 503 per million, with the highest rates in people 65 years and older (1622 per million) and 50-64 years (790 per million). 8 out of 10 deaths from SARS-CoV2 infection reported in the U.S have been in adults 65 years old and older. In Italy, the death rates of COVID-19 patients by May 6, 2020 are 0.1%-0.9%, 2.5%, 10.1%, and 25% or more in age group of 20-49, 50-59, 60-69, and ≥70 years old, respectively. However, the underlying causes of aging effects are poorly understood and current therapy is supportive. Here, our present invention provides a treatment that could markedly inhibit lung injury and inflammation and promote survival. Furthermore, combination therapy with the injury inhibitors and reparative activators is likely an effective therapeutic approach for COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, and ARDS in elderly patients. Coupled with anti-viral therapy, this novel cocktail therapy may hold great promise for effective treatment of COVID-19 and promote survival.
To determine the injury response of young adult and aged mice, we challenged adult (3 mo. old) and aged (19 mo. old) mice with the same dose of lipopolysaccharide (LPS) (5 mg/kg, i.p., LPS from Sigma Aldrich). At 24h post-LPS, lung tissues were collected for EBA and MPO activity assays. As shown in
Example 7. NOX2 is markedly increased in aged lung ECs and inhibition of NOX2 markedly inhibits inflammatory lung injury in aged mice. Thus, NOX2 inhibitors including but limited to Thienopyridine, NOX2 inhibiting peptide or nucleic acids, or pan-NOX inhibitors Apocynin, Ebselen, or APX-115 as a monotherapy or combination therapy with either Selisistat, AG-1031, and/or rabeprazol, and/or phenazopyridine, and/or DMOG analogues roxadustat or molidustat, or vadadustat, or desidustat, and/or decitabine as well as SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, HIF-1a expressing nucleic acid, or FOXM1 expressing nucleic acid are useful to treat COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, ARDS, and multi-organ failure in elderly patients as well as vascular diseases associated with NOX2 hyperactivity.
NADPH oxidase (NOX) family of enzymes including NOX1-5 and dual oxidase DUOX1 and 2 catalyze the reduction of 02 to reactive oxygen species (ROS), and excessive ROS have been associated with tissue damage (1, 2). NOX2 also known as gp91phox was first discovered in phagocytes, and serves as an important inflammatory mediator against invading bacteria (3). NOX 4 which most generates H2O2 is highly expressed in fibroblasts and vascular smooth muscle cells and play an important role in vascular remodeling and pulmonary fibrosis (4). Inhibition of NOX4 is under clinical trial for human idiopathic pulmonary fibrosis. We have studied the expression changes of NOX2 and NOX in lungs of aged mice at basal and following sepsis challenge. As shown in
Together, these data for the first time demonstrate that aging-dependent increase of NOX2 expression in lung ECs are responsible for the augmented inflammatory lung injury in aged mice in response to LPS challenge whereas NOX4 is protective. Marked increase of NOX2 induced by NOX4 deficiency accounts for the great mortality in NOX4-deficient mice. Thus, inhibition of NOX2 in aged subjects by NOX2 inhibitors including but not limited Thienopyridine, NOX2 inhibiting peptide (e.g., NOX2ds-tat) and NOX2 inhibiting nucleic acid including antisense, siRNA, shRNA and guide RNA, and pan-NOX inhibitor Apocynin, Ebselen, APX-115 as a monotherapy to inhibit inflammatory lung injury and as a combination therapy with one or more of Selisistat, AG-1031, and/or rabeprazol, and/or phenazopyridine, and/or DMOG analogues roxadustat or molidustat, or vadadustat, or desidustat, and/or decitabine or SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, FOXM1 expressing nucleic acid, or HIF-1a expressing nucleic acid to promote vascular repair and thus effectively treat COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, ARDS, and multi-organ failure in elderly patients. Selective inhibition of NOX4 will worsen the disease and increase mortality.
Example 8. N-Acetylcysteine (NAC) as a monotherapy or combination therapy with one or more of Selisistat, AG-1031 and their analogues, and/or rabeprazol, and/or phenazopyridine, and/or DMOG analogues roxadustat or molidustat, or vadadustat, or desidustat, and/or decitabine (e.g. Dacogen, INDOVI) or azacytidine (e.g., Vidiaz, ONUREG), or SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, FOXM1 expressing nucleic acid, or HIF-1a expressing nucleic acid to treat COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, ARDS, and multi-organ failure in elderly patients.
To further understand the pathogenic role of aging-induced NOX2 in promoting inflammatory lung injury in aged mice, we cultured human lung microvascular ECs (HLMVECs) and passaged many times. We found that HLMVECs at passage 16 became senescent evident by prominent β-galactosidase staining (
NAC treatment normalizes inflammatory lung injury in aged mice to the levels similar to young adult mice.
To determine whether NAC treatment in aged mice can attenuate lung injury in aged mice, aged mice (21.5 mos. old) and young adult WT mice (3 mos. old) were challenged with LPS (2 mg/kg, i.p.) and then treated with NAC (120 mg/kg, oral) or PBA at 2 h post-LPS. Lung tissues were collected at 24h post-LPS for analyses. As shown in
Thus, NAC can be used as a monotherapy or more importantly, combination therapy with one or more of Selisistat, AG-1031 and their analogues, and/or rabeprazol, and/or phenazopyridine, and/or DMOG analogues roxadustat or molidustat, or vadadustat, or desidustat, and/or decitabine (e.g., Dacogen, INQOVI) or azacitidine (e.g., Vidiaz, ONUREG), or SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, FOXM1 expressing nucleic acid, or HIF-1a expressing nucleic acid to treat COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, ARDS, and multi-organ failure in elderly patients.
Example 9. Resveratrol as a monotherapy or combination therapy with one or more of rabeprazole, or phenazopyridine, or roxadustat or molidustat, or vadadustat, or desidustat, or decitabine, or EGLN1 inhibiting nucleic acid, FOXM1 expressing nucleic acid, or HIF-1a expressing nucleic acid to treat COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, ARDS, and multi-organ failure in patients.
Resveratrol has been reported to have anti-inflammatory, anti-oxidant and anti-cancer properties. However, its use is widely hindered by its poor solubility. We formulated 3 RV-loaded nanoparticles comprised of PLGA (MW=25,000 Da), and PLGA-PEG600 (PEGs), or PLGA-PEG2000 (PEGl) (
Example 10. Decitabine and its analogues (e.g., Dacogen, INQOVI, Vidaza, NUREG) as a monotherapy or combination therapy with one or more of Resveratrol, NAC, NOX2 inhibitor (e.g. Thienopyridine, NOX2 inhibiting peptide (e.g., NOX2ds-tat) and pan-NOX inhibitor Apocynin, Ebselen, APX-115), Selisistat, AG-1031, rabeprazole, phenazopyridine, or DMOG analogues roxadustat or molidustat, or vadadustat, or desidustat, or NOX2 inhibiting nucleic acid, SIRT1 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, or HIF-1a expressing nucleic acid for the treatment of COVID-19, COVID-19 sepsis, COVID-19 respiratory distress and multi-organ failure, sepsis, ARDS, and multiple organ failure in elderly patients as well as vascular diseases associated with diminished FOXM1 expression including but not limited to restenosis and critical limb ischemia.
Decitabine has no effect on sepsis-induced lung injury in young adult mice. Decitabine is used for treatment of patients with myelodysplastic syndrome (MDS). Previously study has shown that 5′-Aza-2′-deoxycytidine (Aza, Decitabine) (1 mg/kg, i.p.) has no effects on sepsis-induced inflammatory lung injury except reduced lung injury by combined use of both Aza and Trichostatin A (TSA, a histone deacetylase inhibitor) in young adult mice (8-10 weeks old) (1). Our study also shows that decitabine at various doses (0.25, 0.5, and 5 mg/kg, i.p.) has no effect on sepsis-induced inflammatory lung injury in young adult mice (3-5 mo. old) evident by lung MPO activity (
Decitabine reactivation of FoxM1-dependent endothelial regeneration and vascular repair in lungs of aged mice but not of young adult mice.
We next explored the possibility of Decitabine reactivation of FoxM1-dependent endothelial regeneration in aged lungs which will have great translational potential for treatment of ARDS and severe COVID-19 in elderly patients. At 24 h and 48h post-LPS challenge, the aged (21-22 mos. old) mice were treated with Decitabine (0.2 mg/kg, i.p.) or vehicle (PBS) and lung tissues were collected at 96h post-LPS for analyses. EBA assay demonstrated normalized vascular repair in Decitabine-treated aged mice in contrast to vehicle-treated aged mice (
BrdU immunostaining revealed that pulmonary vascular EC proliferation was drastically increased in Decitabine-treated aged mice, indicating reactivation of endothelial regeneration in aged lungs (
Quantitative RT-PCR analysis shows FoxM1 expression was markedly induced in lungs of Decitabine-treated mice at 72h post-LPS compared to vehicle-treated mice (
Together, these data suggest that decitabine and its analogues can be repurposed to reactivate endothelial regeneration and vascular repair and promote recovery and thereby reduce morbidity and mortality of elderly patients with either COVID-19 and COVID-19 respiratory distress, sepsis, and/or multi-organ failure, sepsis, ARDS, or multiple organ failure as a monotherapy or combination therapy with one or more of Resveratrol, NAC, NOX2 inhibitors (e.g. Thienopyridine, NOX2 inhibiting peptide (e.g., NOX2ds-tat), pan-NOX inhibitors (e.g., Apocynin, Ebselen, APX-115), Selisistat, AG-1031, rabeprazol, phenazopyridine, or DMOG analogues roxadustat or molidustat, or vadadustat, or desidustat, NOX2 inhibiting nucleic acid, EGLN1 inhibiting nucleic acid, SIRT1 inhibiting nucleic acid, HIF-1a expressing nucleic acid, or FOXM1 expressing nucleic acid.
In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.
This application claims the benefit of U.S. Provisional Application No. 63/044,356, filed Jun. 26, 2020, the entire content of which is incorporated herein by reference in its entirety.
This invention was made with government support under HL123957, HL125350, HL133951, HL140409, and HL077806, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/070767 | 6/24/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63044356 | Jun 2020 | US |
