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WO2024150064A1 - Thérapie par molécules à protéine d'ancrage lipidique - Google Patents

Thérapie par molécules à protéine d'ancrage lipidique Download PDF

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Publication number
WO2024150064A1
WO2024150064A1 PCT/IB2024/000013 IB2024000013W WO2024150064A1 WO 2024150064 A1 WO2024150064 A1 WO 2024150064A1 IB 2024000013 W IB2024000013 W IB 2024000013W WO 2024150064 A1 WO2024150064 A1 WO 2024150064A1
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subject
apoa
group
binding protein
lipid binding
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PCT/IB2024/000013
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English (en)
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WO2024150064A9 (fr
Inventor
Cyrille TUPIN
Constance PEYROTTES
Ronald Barbaras
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Abionyx Pharma Sa
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Publication of WO2024150064A1 publication Critical patent/WO2024150064A1/fr
Publication of WO2024150064A9 publication Critical patent/WO2024150064A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

  • the condition is a gram-positive bacterial infection, a gram-negative bacterial infection, a viral infection, such as a SARS-CoV-2 (COVID-19) infection or an influenza virus infection, acute myocardial infarction (AMI), Alzheimer's disease, chronic inflammatory bowel disease (IBD), a cardiovascular disease (CVD), stroke, transient ischemic attack, cytokine release syndrome (CRS, cytokine storm), organ transplant, such as heart transplant rejection, ischemia reperfusion-induced tissue injury, post-operative inflammation, psoriasis, sepsis (e g., septic shock), including but not limited to sepsis wherein the subject has an abnormal level of at least two of TNFa, IL-6, IL-8, TREM-1 , and a kynurenine pathway biomarker, such as an abnormal level of TREM-1 and at least one of quinolinic acid
  • the methods of the disclosure comprise treating a subject with a lipid binding protein molecule, for example an apolipoprotein such as ApoA-l, or an apolipoprotein mimetic.
  • a lipid binding protein molecule for example an apolipoprotein such as ApoA-l, or an apolipoprotein mimetic.
  • the lipid binding protein molecule can be administered at a high dose, which is typically an aggregate of two or more individual doses administered over one or more days, particularly where the indication is an acute indication such as sepsis.
  • the high dose is typically higher than a dose that would be used to treat a chronic condition, such as familial hypercholesterolemia.
  • the high dose is typically administered over a relatively short period of time, for example, over a period of one day to two weeks or one day to three weeks, and typically comprise miiitinio aHnqjnjstrations of the lipid binding protein molecule, for example two to 20 individual doses.
  • the individual doses can be separated by less than one day (e.g., twice daily administration), or one day or more (e.g., once daily administration).
  • the lipid binding protein molecule is a component of a lipid binding protein-based complex.
  • Lipid binding protein-based complexes can comprise amphipathic molecules such as lipids, for example a sphingomyelin and/or a negatively charged lipid.
  • An exemplary lipid binding protein-based complex that can be used in the methods of the disclosure is CER-001.
  • CER-001 is a negatively charged lipoprotein complex, and comprises recombinant human ApoA-l, sphingomyelin (SM), and 1 , 2-dihexadecanoyl-sn-glycero-3-phospho-(T-rac- glycerol) (Dipalmitoylphosphatidyl-glycerol; DPPG).
  • SM sphingomyelin
  • DPPG 2-dihexadecanoyl-sn-glycero-3-phospho-(T-rac- glycerol)
  • CER-001 therapy was observed to have broad pleiotropic effects in an LPS-induced acute kidney injury animal model and in a clinical trial with septic human patients at high risk of acute kidney injury (AKI).
  • CER-001 therapy resulted in, inter alia, a reduction in levels of various inflammatory cytokines such as IL-6 and a reduction in levels of markers of the kynurenine pathway.
  • the studied markers are associated with various conditions in addition to sepsis. Without being bound by theory, it is believed that the pleiotropic effects of CER-001 can be extended beyond septic patients to provide a clinical benefit to subjects having other conditions, for example the conditions described herein.
  • the disclosure provides a method of treating a subject with or at risk of a grampositive bacterial infection, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of a gramnegative bacterial infection, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of a viral infection, such as a SARS-CoV-2 (COVID-19) infection or an influenza virus infection, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a viral infection such as a SARS-CoV-2 (COVID-19) infection or an influenza virus infection
  • COVID-19 SARS-CoV-2
  • influenza virus infection e.g., a lipid binding protein molecule
  • the disclosure provides a method of treating a subject with or at risk of acute myocardial infarction (AMI), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • AMI acute myocardial infarction
  • the disclosure provides a method of treating a subject with or at risk of Alzheimer's disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of chronic inflammatory bowel disease (IBD), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • IBD chronic inflammatory bowel disease
  • the disclosure provides a method of treating a subject with or at risk of a cardiovascular disease (CVD), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • CVD cardiovascular disease
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject having or at risk of a stroke, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject having or at risk of a transient ischemic attack, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of cytokine release syndrome (CRS, cytokine storm), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of organ transplant, such as heart transplant rejection, comprising administering to the subject a lipid binding protein molecule (e g., ApoA-l).
  • a lipid binding protein molecule e g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of ischemia reperfusion-induced tissue injury, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of postoperative inflammation, comprising administering to the subject a lipid binding protein molecule (e g., ApoA-l).
  • a lipid binding protein molecule e g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of psoriasis, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of sepsis (e.g., septic shock), including but not limited to sepsis wherein the subject has an abnormal level of at least two (e.g., two, three, four, or five) of TNFa, IL-6, IL-8, TREM-1 , and a kynurenine pathway biomarker, such as an abnormal level of TREM-1 and at least one of quinolinic acid, kynurenic acid, kynurenine, tryptophan, and kynurenine/tryptophan ratio, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of sepsis- induced acute kidney injury (AKI), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with a vitamin deficiency, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with inflammatory bowel disease (IBD), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with kidney disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with infections, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with stress, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with thyroid disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with diabetes, comprising administering to the subject a lipid binding protein molecule (e g., ApoA-l).
  • a lipid binding protein molecule e g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with nephrotic syndrome, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with lupus, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with cirrhosis, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with liver disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with heart failure, comprising administering to the subject a lipid binding protein molecule (e g., ApoA-l).
  • a lipid binding protein molecule e g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with malnutrition, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of attention- deficit/hyperactivity disorder (ADHD), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of a central nervous system (CNS) disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of COVID-19 cognitive decline, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of depression or major depressive disorder, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of epilepsy, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of HIV- associated neurocognitive disorder, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of Huntington's disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of inflammatory bowel disease (IBD), comprising administering to the subject a lipid binding protein molecule (e g., ApoA-l).
  • IBD inflammatory bowel disease
  • the disclosure provides a method of treating a subject with or at risk of long-term cognitive decline (“brain fog’’), such as can occur after sepsis, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of mortality or neurological deficit following cardiac arrest, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of multiple sclerosis (MS), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • MS multiple sclerosis
  • the disclosure provides a method of treating a subject with or at risk of Parkinson's disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of schizophrenia, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of vascular endothelial disorder, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA- I)-
  • the disclosure provides a method of treating a subject with or at risk of a urinary tract infection, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of a blood infection, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of a post- surgical infection, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of gastrointestinal perforation, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of a perforated duodenal ulcer, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of a perforated bowel, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of septic shock (e.g., a subject with sepsis who has not yet progressed to the stage of septic shock), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of septic shock following trauma, for example abdominal trauma.
  • a subject having septic shock has hypotension (e.g., systolic arterial pressure ⁇ 90 mm Hg or mean arterial pressure (MAP) ⁇ 65 mm Hg) requiring the use of vasopressors despite intravenous fluid resuscitation.
  • hypotension e.g., systolic arterial pressure ⁇ 90 mm Hg or mean arterial pressure (MAP) ⁇ 65 mm Hg
  • the disclosure provides a method of treating a subject with or at risk of pneumonia, e.g., hospital acquired pneumonia, comprising administering to the subject a lipid binding protein molecule (e g., ApoA-l).
  • a lipid binding protein molecule e g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of a pancreatitis, e.g., necrotizing pancreatitis, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the present disclosure provides dosing regimens for lipid binding protein molecule-based therapy (e g., ApoA-l therapy) for subjects described herein.
  • lipid binding protein molecule-based therapy e g., ApoA-l therapy
  • the dosing regimens of the disclosure typically entail multiple administrations of ApoA-l to a subject (e.g., administered daily or twice in one day).
  • the ApoA-l therapy can be continued for a predetermined period, e.g., for one week or less (e.g., one day, two days, three days, four days, five days, six days, or seven days) or a period longer than one week (e.g., two weeks or three weeks).
  • administration of ApoA-l to a subject can be continued until one or more symptoms of a condition are reduced or continued until the levels (e.g., serum levels) of one or more relevant biomarkers are reduced, for example reduced to a normal level or reduced relative to a baseline measurement taken prior to the start of ApoA-l therapy.
  • the therapy can in some embodiments be continued until the subject has recovered from the infection.
  • the dosing regimens of the disclosure can entail administering a lipid binding protein molecule (e.g., ApoA-l) to a subject according to an initial “induction” regimen, optionally followed by administering the lipid binding protein molecule to the subject according to a “consolidation” regimen.
  • a lipid binding protein molecule e.g., ApoA-l
  • the induction regimen typically comprises administering multiple doses of the lipid binding protein molecule (e.g., ApoA-l) to the subject, for example six doses over three days, eight doses over four days, 10 doses over five days, 12 doses over six days, or 14 doses over seven days.
  • the lipid binding protein molecule e.g., ApoA-l
  • the consolidation regimen typically comprises administering one or more doses of a lipid binding protein molecule (e.g., ApoA-l) to the subject following the final dose of the induction regimen, for example one or more days after the final dose of the induction regimen.
  • the first dose of the consolidation regimen is administered on the third day after the final dose of the induction regimen.
  • a dosing regimen can comprise administration of a lipid binding protein molecule (e.g., ApoA-l) to a subject according to an induction regimen on days 1 , 2, and 3, and administration of the lipid binding protein molecule to the subject according to a consolidation regimen on day 6.
  • the consolidation regimen comprises two doses of the lipid binding protein molecule.
  • a lipid binding protein molecule e.g., ApoA-l
  • a standard of care therapy for the subject is administered in combination with a standard of care therapy for the subject’s disease or condition.
  • an antihistamine e.g., dexchlorpheniramine, hydroxyzine, diphenhydramine, cetirizine, fexofenadine, or loratadine
  • a lipid binding protein molecule e.g., ApoA-l
  • the antihistamine can reduce the likelihood of allergic reactions.
  • lipid binding protein molecules and lipid binding protein-based complexes that can be used in methods and dosing regimens of the disclosure are described in Section 6.1 and specific embodiments 382 to 407 and 678 to 698, infra.
  • Further features of subjects who can be treated according to the methods and dosing regimens of the disclosure are described in Section 6.2 and specific embodiments 1 to 201 , 332 to 381 , 651 to 672, and 700 to 707, infra.
  • Exemplary combination therapies are described in Section 6.4 and specific embodiments 637 to 650, infra.
  • FIG. 1 A shows quinolinic acid levels for individual endotoxemic pigs (“LPS”) and individual endotoxemic pigs treated with one dose of CER-001 at 20 mg/kg (“20 mg’’), in a study described in Example 1.
  • FIG. 1B shows quinolinic acid levels for individual endotoxemic pigs (“LPS’’) and individual endotoxemic pigs treated with two doses of CER-001 at 20 mg/kg, total 40 mg/kg (“40 mg’’), in a study described in Example 1.
  • FIG. 1C shows quinolinic acid levels for all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg, in a study described in Example 1.
  • FIG. 2A shows kynurenic acid levels for individual endotoxemic pigs (“LPS”) and individual endotoxemic pigs treated with one dose of CER-001 at 20 mg/kg (“20 mg”), in a study described in Example 1.
  • FIG. 2B shows kynurenic acid levels for individual endotoxemic pigs (“LPS”) and individual endotoxemic pigs treated with two doses of CER-001 at 20 mg/kg each (“40 mg”), in a study described in Example 1.
  • FIG. 2C shows kynurenic acid levels for all three groups of endotoxemic pigs: LPS, 20mg, and 40 mg, in a study described in Example 1.
  • FIG. 3A shows tryptophan levels for individual endotoxemic pigs (“LPS”) and individual endotoxemic pigs treated with one dose of CER-001 at 20 mg/kg (“20 mg”), in a study described in Example 1.
  • FIG. 3B shows tryptophan levels for individual endotoxemic pigs (“LPS”) and individual endotoxemic pigs treated with two doses of CER-001 at 20 mg/kg each (“40 mg”), in a study described in Example 1.
  • FIG. 3C shows tryptophan levels for all three groups of endotoxemic pigs: LPS, 20mg, and 40 mg, in a study described in Example 1.
  • FIG. 4A shows kynurenine levels for individual endotoxemic pigs (“LPS”) and individual endotoxemic pigs treated with one dose of CER-001 at 20 mg/kg (“20 mg”), in a study described in Example 1.
  • FIG. 4B shows kynurenine levels for individual endotoxemic pigs (“LPS”) and individual endotoxemic pigs treated with two doses of CER-001 at 20 mg/kg each (“40 mg”), in a study described in Example 1.
  • FIG. 4C shows kynurenine levels for all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg, in a study described in Example 1.
  • FIG. 5A shows kynurenine/tryptophan ratios for first cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg, in a study described in Example 1.
  • FIG. 5B shows kynurenine/tryptophan ratios for second cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg, in a study described in Example 1 .
  • FIG. 6A shows relative fold gene expression of indoleamine 2,3-dioxygenase 1 (IDO1) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg, in a study described in Example 1.
  • IDO1 indoleamine 2,3-dioxygenase 1
  • FIG. 6B shows relative fold gene expression of aromatic-L-amino-acid/L-tryptophan decarboxylase (DDC) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg, in a study described in Example 1 .
  • DDC aromatic-L-amino-acid/L-tryptophan decarboxylase
  • FIG. 6C shows relative fold gene expression of kynurenine formamidase isoform X1 (AFMID) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg, in a study described in Example 1.
  • AFMID kynurenine formamidase isoform X1
  • FIG. 6D shows relative fold gene expression of kynurenine 3-monooxygenase (KMO) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg, in a study described in Example 1.
  • KMO kynurenine 3-monooxygenase
  • FIG. 6E shows relative fold gene expression of kynurenine-oxoglutarate transaminase 3 (KYAT3) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg, in a study described in Example 1.
  • KYAT3 kynurenine-oxoglutarate transaminase 3
  • FIG. 6F shows relative fold gene expression of interleukin-6 (IL-6) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg, in a study described in Example 1.
  • IL-6 interleukin-6
  • FIG. 7 shows a schematic of the clinical study of Example 2.
  • FIG. 8A shows lipopolysaccharide (LPS) changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 8B shows LPS changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 8C shows LPS changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 8D shows LPS changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 8E shows lipopolysaccharide (LPS) changes from baseline for each subject in the standard of care (SOC) group and the three experimental groups (CER-001) in the clinical study of Example 2.
  • SOC standard of care
  • CER-001 three experimental groups
  • FIG. 8F shows lipopolysaccharide (LPS) changes from baseline for each subject in the standard of care (SOC) group and each of the three experimental groups (CER-001) in the clinical study of Example 2.
  • LPS lipopolysaccharide
  • FIG. 9A shows endotoxin activity assay (EAA) changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • EAA endotoxin activity assay
  • FIG. 9B shows EAA changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 9C shows EAA changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 9D shows EAA changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 9E shows endotoxin activity assay (EAA) changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 10A shows TNF-alpha changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 10B shows TNF-alpha changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 10C shows TNF-alpha changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 10D shows TNF-alpha changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 10E shows TNF-alpha changes from baseline for each subject in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 10F shows TNF-alpha changes from baseline for each subject in each of the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 11A shows MCP-1 changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 11B shows MCP-1 changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 11C shows MCP-1 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 11D shows MCP-1 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 11E shows MCP-1 changes from baseline for each subject in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 11F shows MCP-1 changes from baseline for each subject in each of the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 12A shows IL-6 changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 12B shows IL-6 changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 12C shows IL-6 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 12D shows IL-6 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 12E shows IL-6 changes from baseline for each subject in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 12F shows IL-6 changes from baseline for each subject in each of the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 13A shows IL-8 changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 13B shows IL-8 changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 13C shows IL-8 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 13D shows IL-8 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 13E shows IL-8 changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 13F shows IL-8 changes from baseline for the standard of care (SOC) group (Group A) and each of the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 14A shows IL-10 changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 14B shows IL-10 changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 14C shows IL-10 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 14D shows IL-10 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 15A shows TREM-1 changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 15B shows TREM-1 changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 15C shows TREM-1 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 15D shows TREM-1 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 15E shows TREM-1 changes from baseline for each subject in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 15F shows TREM-1 changes from baseline for each subject in each of the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 16A shows VCAM changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 16B shows VCAM changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 16C shows VCAM changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 16D shows VCAM changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 16E shows VCAM changes from baseline for each subject in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 16F shows VCAM changes from baseline for each subject in each of the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 17A shows ICAM changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 17B shows ICAM changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 17C shows ICAM changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 17D shows ICAM changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 17E shows ICAM changes from baseline for each subject in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 17F shows ICAM changes from baseline for each subject in each of the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 18A shows ferritin changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 18B shows ferritin changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 18C shows ferritin changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 18D shows ferritin changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 19A shows white blood cell count changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 19B shows white blood cell count changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 19C shows white blood cell count changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 19D shows white blood cell count changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 20A shows C-reactive protein (CRP) changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 20B shows CRP changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 20C shows CRP changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 20D shows CRP changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 20E shows C-reactive protein (CRP) changes from baseline for each subject in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 20E shows C-reactive protein (CRP) changes from baseline for each subject in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 20F shows C-reactive protein (CRP) changes from baseline for each subject in the standard of care (SOC) group (Group A) and each of the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 21A shows KIM-1 changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 21 B shows KIM-1 changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 21C shows KIM-1 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 21 D shows KIM-1 changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 22A shows serum albumin changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 22B shows serum albumin changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 22C shows serum albumin changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 22D shows serum albumin changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • FIG. 22E shows serum albumin changes from baseline for each subject in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 23A shows serum creatinine changes from baseline for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 23B shows serum creatinine changes from baseline for each of Groups A-D in the clinical study of Example 2.
  • FIG. 23C shows serum creatinine changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 23D shows serum creatinine changes as a percentage of peak for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D), broken out by whether the subject was enrolled from the ICU or the nephrology department of the center, in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 23E shows the AUC for serum creatinine (mean ⁇ SEM) for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 23F shows the AUC for serum creatinine (95% confidence interval) for the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 24A shows estimated glomerular filtration rate (eGFR) changes from baseline for all subjects in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 24B shows eGFR changes from baseline for all subjects in each of Groups A-D in the clinical study of Example 2.
  • FIG. 24C shows eGFR changes from baseline only for subjects in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) who entered the clinical study of Example 2 with AKI.
  • SOC standard of care
  • FIG. 24D shows eGFR changes from baseline for r the same subjects as in FIG. 24C.
  • FIG. 24E shows eGFR changes as a percentage of peak for all subjects in the standard of care (SOC) group (Group A) and aggregated Groups B-D in the clinical study of Example 2.
  • FIG. 24F shows eGFR changes as a percentage of peak only for subjects entering the study with AKI, in the standard of care (SOC) group (Group A) and aggregated Groups B-D in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 25 shows P/F changes from baseline for all subjects in the standard of care (SOC) group (Group A) and the three experimental groups (Groups B-D) in the clinical study of Example 2.
  • FIG. 26 shows survival proportions for all subjects after days in ICU for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • FIG. 27A shows survival proportions over 30 days for all subjects for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • FIG. 27B shows survival proportions over 30 days for all subjects who entered the study from the center's ICU, for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • FIG. 28A shows the evolution of AKI staging for the standard of care group (Group A, “SOC”) in the clinical study of Example 2.
  • FIG. 28B shows the evolution of AKI staging for aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • FIG. 29 shows days on mechanical ventilation for all subjects who entered the study from the center's ICU, for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • SOC standard of care group
  • CER-001 aggregated Groups B-D
  • FIG. 30 shows days on vasopressor therapy for all subjects who entered the study from the center's ICU, for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • SOC standard of care group
  • CER-001 aggregated Groups B-D
  • FIG. 31 A shows days on dialysis for all subjects who entered the study from the center’s ICU, for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • SOC standard of care group
  • CER-001 aggregated Groups B-D
  • FIG. 31 B shows days on dialysis for all subjects who entered the study for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • FIG. 32 shows days alive without organ support for all subjects who entered the study from the center's ICU, for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • FIG. 33A shows changes in daily average mean arterial pressure (MAP) for all subjects who entered the study from the center's ICU, for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • MAP mean arterial pressure
  • FIG. 33B shows changes in daily average mean arterial pressure (MAP) for each subject who entered the study from the center's ICU, for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • MAP mean arterial pressure
  • FIG. 34 shows change in daily average heart rate (HR) for all subjects who entered the study from the center’s ICU, for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • HR daily average heart rate
  • FIG. 35 shows change in daily average P/F ratio for all subjects who entered the study from the center's ICU, for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • SOC standard of care group
  • CER-001 aggregated Groups B-D
  • FIG. 36 shows the survival curve of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 37 shows serum VCAM levels of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 38 shows serum ICAM levels of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 39 shows serum TNF-a levels of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 40 shows serum MCP-1 levels of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 41 shows serum II-6 levels of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 42 shows systemic classical pathway complement activation in pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 43 shows systemic alternative pathway complement activation in pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 44 shows systemic lectin pathway complement activation in pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 45A shows representative hematoxylin and eosin (HE) staining of hepatic tissue of pigs upon challenge with LPS, as described in Example 3.
  • FIG. 45B shows representative HE staining of hepatic tissue of pigs upon challenge with LPS and CER-001 treatment (20 mg/kg), as described in Example 3.
  • FIG. 45C shows representative HE staining of hepatic tissue of pigs upon challenge with LPS and CER-001 treatment (20 mg/kg x 2), as described in Example 3.
  • FIG. 45D shows liver injury quantified from images of stained liver of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 45E shows serum levels of ALT enzyme of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 46A shows representative HE staining of renal tissue of pigs upon challenge with LPS, as described in Example 3.
  • FIG. 46B shows representative HE staining of renal tissue of pigs upon challenge with LPS and CER-001 treatment (20 mg/kg), as described in Example 3.
  • FIG. 46C shows representative HE staining of renal tissue of pigs upon challenge with LPS and CER-001 treatment (20 mg/kg x 2), as described in Example 3.
  • FIG. 46D shows tubular pathological score quantified from images of stained kidney of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 46E shows glomerular pathological score quantified from images of stained kidney of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 47 shows serum levels of creatinine of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 48 shows urinary output of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 49A shows serum Cystatin C levels of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 49B shows urinary Cystatin C levels of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 50A shows serum KIM-1 levels of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 50B shows urinary KIM-1 levels of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 51 shows serum LPS levels of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 52A shows a western blot of LPS and p-actin protein expression in livers of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 52B shows a densitometric analysis of LPS and p-actin protein expression in livers of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 53 shows endotoxin levels in bile of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 54 shows serum levels of human ApoA-l of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 55 shows levels of human ApoA-l in bile of pigs upon challenge with LPS and CER-001 infusions, as described in Example 3.
  • FIG. 56A shows mean ApoA-l levels for the control group and the aggregated study groups in the clinical study of Example 2.
  • FIG. 56B shows ApoA-l levels for each subject broken out by study group in the clinical study of Example 2.
  • FIG. 56C shows changes from baseline of ApoA-l levels for each subject in the clinical study of Example 2.
  • FIG. 56D shows changes from baseline of ApoA-l levels for each subject broken out by study group in the clinical study of Example 2.
  • FIG. 57A shows changes from baseline of aspartate transaminase (AST) levels for each subject in the clinical study of Example 2.
  • FIG. 57B shows changes from baseline of alanine transaminase (ALT) levels for each subject in the clinical study of Example 2.
  • FIG. 58 shows MTT cell viability assay results for cultured endothelial cells upon challenge with LPS and CER-001 infusions, as described in Example 4.
  • FIG. 59 summarizes endothelial nitric oxide synthase (eNOS)-based (eNOS(phosphoS1177)) FACS results for cultured endothelial cells upon challenge with LPS and CER-001 infusions, as described in Example 4.
  • eNOS endothelial nitric oxide synthase
  • FIG. 60 shows eNOS(phosphoS1177)-based FACS results for cultured endothelial cells upon challenge with LPS and CER-001 infusions in one representative of three independent experiments, compared to basal and VEFG (positive control) cells, as described in Example 4.
  • FIG. 61 shows MTT cell viability assay results for PBMCs from healthy donors upon challenge with LPS and CER-001 infusions, as described in Example 4.
  • FIG. 62 shows TNF-a synthesis for PBMCs from healthy donors upon challenge with LPS and CER-001 infusions, as described in Example 4.
  • FIG. 63 shows CD14-based FACS results for PBMCs from healthy donors upon challenge with LPS and CER-001 infusions in one representative of three independent experiments, as described in Example 4.
  • FIG. 64 summarizes CD14-based FACS results for PBMCs from healthy donors upon challenge with LPS and CER-001 infusions, as described in Example 4.
  • FIG. 65 shows days until ICU discharge for all subjects who entered the study from the center’s ICU, for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”) in the clinical study of Example 2.
  • SOC standard of care group
  • CER-001 aggregated Groups B-D
  • FIG. 66 shows changes in serum quinolinic acid (QA) levels from baseline (day 1) for subjects in the standard of care (SOC) group (Group A) and aggregated Groups B-D in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 67 shows changes in serum kynurenine/tryptophan ratios (Kyn/Trp) levels from baseline (day 1 ) for subjects in the standard of care (SOC) group (Group A) and aggregated Groups B-D in the clinical study of Example 2.
  • FIG. 68 shows changes in serum serotonin levels from baseline (day 1 ) for subjects in the standard of care (SOC) group (Group A) and aggregated Groups B-D in the clinical study of Example 2.
  • FIG. 69 shows the overall trial design of Example 5.
  • FIG. 70 shows the study participation for an individual subject of Example 5.
  • the present disclosure provides methods for treating subjects having or at risk of various conditions with lipid binding protein molecules.
  • the condition is a gram-positive bacterial infection, a gram-negative bacterial infection, a viral infection, such as a SARS-CoV-2 (COVID- 19) infection or an influenza virus infection, acute myocardial infarction (AMI), Alzheimer's disease, chronic inflammatory bowel disease (IBD), a cardiovascular disease (CVD), stroke, transient ischemic attack, cytokine release syndrome (CRS, cytokine storm), organ transplant, such as heart transplant rejection, ischemia reperfusion-induced tissue injury, post-operative inflammation, psoriasis, sepsis (e.g., septic shock), including but not limited to sepsis wherein the subject has an abnormal level of at least two of TNFa, IL-6, IL-8, TREM-1 , and a kynurenine pathway biomarker, such as an abnormal level of TREM- 1 and at
  • the methods comprise administering a high dose of a lipid binding protein molecule.
  • the disclosure provides a method of treating a subject with or at risk of a grampositive bacterial infection, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of a gramnegative bacterial infection, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of a viral infection, such as a SARS-CoV-2 (COVID-19) infection or an influenza virus infection, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a viral infection such as a SARS-CoV-2 (COVID-19) infection or an influenza virus infection
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of acute myocardial infarction (AMI), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • AMI acute myocardial infarction
  • the disclosure provides a method of treating a subject with or at risk of Alzheimer's disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of chronic inflammatory bowel disease (IBD), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • IBD chronic inflammatory bowel disease
  • the disclosure provides a method of treating a subject with or at risk of a cardiovascular disease (CVD), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • CVD cardiovascular disease
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject having or at risk of a stroke, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject having or at risk of a transient ischemic attack, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of cytokine release syndrome (CRS, cytokine storm), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • CRS cytokine release syndrome
  • ApoA-l lipid binding protein molecule
  • the disclosure provides a method of treating a subject with or at risk of organ transplant, such as heart transplant rejection, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of ischemia reperfusion-induced tissue injury, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of postoperative inflammation, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of psoriasis, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of sepsis (e.g., septic shock), including but not limited to sepsis wherein the subject has an abnormal level of at least two of TNFa, IL-6, IL-8, TREM-1 , and a kynurenine pathway biomarker, such as an abnormal level of TREM-1 and at least one of quinolinic acid, kynurenic acid, kynurenine, tryptophan, and kynurenine/tryptophan ratio, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of sepsis- induced acute kidney injury (AKI), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with a vitamin deficiency, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with inflammatory bowel disease (IBD), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with kidney disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with infections, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with stress, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with thyroid disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with diabetes, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with nephrotic syndrome, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with lupus, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with cirrhosis, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with liver disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with heart failure, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • the disclosure provides a method of treating a subject with or at risk of hypoalbuminemia associated with malnutrition, comprising administering to the subject a lipid binding protein molecule (e g., ApoA-l).
  • a lipid binding protein molecule e g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of attention- deficit/hyperactivity disorder (ADHD), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of a central nervous system (CNS) disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of COVID-19 cognitive decline, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of depression or major depressive disorder, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of epilepsy, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of HIV- associated neurocognitive disorder, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of Huntington's disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of inflammatory bowel disease (IBD), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • IBD inflammatory bowel disease
  • the disclosure provides a method of treating a subject with or at risk of long-term cognitive decline (“brain fog”), such as can occur after sepsis, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of mortality or neurological deficit following cardiac arrest, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of multiple sclerosis (MS), comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of Parkinson's disease, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of schizophrenia, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA-l).
  • a lipid binding protein molecule e.g., ApoA-l
  • the disclosure provides a method of treating a subject with or at risk of vascular endothelial disorder, comprising administering to the subject a lipid binding protein molecule (e.g., ApoA- I).
  • the condition is associated with abnormal levels of one or more of TREM- 1 , albumin, interleukin 10 (IL-10), a kynurenine pathway biomarker (such as tryptophan, serotonin, formylkynurenine, kynurenine, kynurenic acid, 2-amino-3-carboxymuconate-semialdehyde, 3- hydroxykynurenine, xanthurenic acid, anthralinic acid, 3-hydroxyanthralinic acid, quinolinic acid, picolinic acid, quinaldic acid, or kynurenine/tryptophan ratio), TNF-a, MCP-1 , IL-6, IL-8, IL-10, VCAM-1 , or ICAM- 1.
  • TREM- 1 a kynurenine pathway biomarker
  • IL-10 interleukin 10
  • a kynurenine pathway biomarker such as tryptophan, serotonin, formylkyn
  • the condition is associated with an abnormal level of 2-amino-3- carboxymuconate-semialdehyde.
  • the condition is associated with an abnormal level of 3-hydroxyanthranilic acid.
  • the condition is associated with an abnormal level of 3- hydroxykynurenine.
  • the condition is associated with an abnormal level of albumin.
  • the condition is associated with an abnormal level of anthranilic acid.
  • the condition is associated with an abnormal level of formylkynurenine.
  • the condition is associated with an abnormal level of ICAM-1.
  • the condition is associated with an abnormal level of IL-6.
  • the condition is associated with an abnormal level of IL-8.
  • the condition is associated with an abnormal level of kynurenic acid.
  • the condition is associated with an abnormal level of kynurenine.
  • the condition is associated with an abnormal level of kynurenine/tryptophan ratio.
  • the condition is associated with an abnormal level of MCP-1.
  • the condition is associated with an abnormal level of picolinic acid.
  • the condition is associated with an abnormal level of quinaldic acid.
  • the condition is associated with an abnormal level of quinolinic acid.
  • the condition is associated with an abnormal level of serotonin.
  • the condition is associated with an abnormal level of TNFa.
  • the condition is associated with an abnormal level of TREM-1.
  • the condition is associated with an abnormal level of tryptophan.
  • the condition is associated with an abnormal level of VCAM-1 .
  • the condition is associated with an abnormal level of xanthurenic acid.
  • the lipid binding protein molecule is provided as a component of a lipid binding protein-based complex.
  • the lipid binding protein-based complex is an Apomer, a Cargomer, a HDL based complex, or a HDL mimetic-based complex.
  • the lipid binding protein-based complex is CER-001.
  • methods of the disclosure comprise administering a lipid binding protein molecule (e.g., ApoA-l) to a subject in two phases.
  • a lipid binding protein molecule e.g., ApoA-l
  • the lipid binding protein molecule e.g., ApoA-l
  • the lipid binding protein molecule is administered in an initial, intense “induction” regimen.
  • the induction regimen is followed by a less intense “consolidation” regimen.
  • a lipid binding protein molecule e.g., ApoA-l
  • can be administered to a subject in a single phase for example according to an administration regimen corresponding to the dose and administration frequency of an induction or consolidation regimen described herein.
  • Induction regimens that can be used in the methods of the disclosure are described in Section 6.3.1 and consolidation regimens that can be used in the methods of the disclosure are described in Section 6.3.2.
  • the dosing regimens of the disclosure comprise administering a lipid binding protein molecule (e.g., ApoA-l) as monotherapy or as part of a combination therapy with one or more medications, for example in combination with a standard of care therapy for the subject’s disease or condition.
  • Combination therapies are described in Section 6.4.
  • Lipid binding protein molecules that can be used, either directly or in a lipid binding protein-based complexes described herein, include apolipoproteins such as those described in Section 6.1.1.1 and apolipoprotein mimetic peptides such as those described in Section 6.1.1.2.
  • a mixture of lipid binding protein molecules can be used, optionally as members of a complex.
  • the mixture of lipid binding protein molecules can comprise one or more apolipoproteins.
  • the mixture of lipid binding protein molecules can comprise one or more apolipoprotein mimetic peptides.
  • the mixture of lipid binding protein molecules can comprise one or more apolipoproteins and one or more apolipoprotein mimetic peptides.
  • Suitable apolipoproteins from which the lipid binding protein molecule can be chosen, and that can be included in lipid binding protein-based complexes disclosed herein, include apolipoproteins ApoA- I, ApoA-ll, ApoA-IV, ApoA-V, ApoB, ApoC-l, ApoC-ll, ApoC-lll, ApoD, ApoE, ApoJ, ApoH, and any combination of two or more of the foregoing.
  • apolipoproteins Polymorphic forms, isoforms, variants and mutants as well as truncated forms of the foregoing apolipoproteins, the most common of which are Apolipoprotein A- iMiiano (APOA-IM), Apolipoprotein A-lparis (ApoA-lp), and Apolipoprotein A-lzaragoza (ApoA-lz), can also be used.
  • Apolipoproteins mutants containing cysteine residues are also known, and can also be used (see, e.g., U.S. Publication No. 2003/018132).
  • the apolipoproteins may be in the form of monomers or dimers, which may be homodimers or heterodimers.
  • apolipoproteins can be modified in their primary sequence to render them less susceptible to oxidations, for example, as described in U.S. Publication Nos. 2008/0234192 and 2013/0137628, and
  • the apolipoproteins can include residues corresponding to elements that facilitate their isolation, such as His tags, or other elements designed for other purposes.
  • the apolipoprotein or apolipoprotein containing complex is soluble in a biological fluid (e.g., lymph, cerebrospinal fluid, vitreous humor, aqueous humor, blood, or a blood fraction (e.g., serum or plasma).
  • the lipid binding protein molecule comprises covalently bound lipid- binding protein monomers, e.g., dimeric apolipoprotein A-hiiano, which is a mutated form of ApoA-l containing a cysteine.
  • the cysteine allows the formation of a disulfide bridge which can lead to the formation of homodimers or heterodimers (e.g., ApoA-lMiiano-ApoA-ll).
  • the apolipoprotein molecules comprise ApoA-l, ApoA-ll, ApoA-IV, ApoA-
  • V ApoB, ApoC-l, ApoC-ll, ApoC-lll, ApoD, ApoE, ApoJ, or ApoH molecules or a combination thereof.
  • the apolipoprotein molecules comprise or consist of ApoA-l molecules.
  • said ApoA-l molecules are human ApoA-l molecules.
  • said ApoA-l molecules are recombinant.
  • the ApoA-l molecules are not ApoA-lMiiano.
  • the ApoA-l molecules are Apolipoprotein A-lMiiano (APOA-IM), Apolipoprotein A-lparis (ApoA-lp), or Apolipoprotein A-lzaragoza (ApoA-lz) molecules.
  • Apolipoproteins can be purified from animal sources (and in particular from human sources) or produced recombinantly as is well-known in the art, see, e.g., Chung et al., 1980, J. Lipid Res. 21(3):284- 91 ; Cheung et al., 1987, J. Lipid Res. 28(8):913-29. See also U.S. Patent Nos. 5,059,528, 5,128,318, 6,617,134; U.S. Publication Nos. 2002/0156007, 2004/0067873, 2004/0077541 , and 2004/0266660; and PCT Publications Nos. WO 2008/104890 and WO 2007/023476. Other methods of purification are also possible, for example as described in PCT Publication No. WO 2012/109162, the disclosure of which is incorporated herein by reference in its entirety.
  • the ApoA-l is recombinant ApoA-l produced by a mammalian host cell.
  • the host cell can be from any mammalian cell line.
  • Polynucleotides encoding the ApoA-l can be codon optimized for expression in recombinant host cells.
  • host cells are mammalian host cells, including, but not limited to, Chinese hamster ovary cells (e.g. CHO-K1 ; ATCC No. CCL 61 ; CHO-S (e.g., GIBCO Life Technologies Inc., Rockville, MD, Catalog #11619012)), VERO cells, BHK (ATCC No. CRL 1632), BHK 570 (ATCC No.
  • the mammalian cells such as CHO-S cells, are adapted for growth in serum-free medium. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, Va.
  • the recombinant ApoA-l is produced by a CHO cell, for example a CHO-S cell.
  • a CHO cell for example a CHO-S cell.
  • recombinant polypeptides e.g., recombinant ApoA-l
  • a mammalian host cell such as a CHO cell
  • the resulting recombinant ApoA-l can have one or more structural features (e.g, glycosylation pattern) that are different from ApoA-l purified from human plasma.
  • the apolipoprotein can be in prepro- form, pro- form, or mature form.
  • the apolipoprotein can comprise ApoA-l (e.g., human ApoA-l) in which the ApoA-l is preproApoA-l, proApoA- I, or mature ApoA-l.
  • ApoA-l e.g., human ApoA-l
  • the ApoA-l is preproApoA-l, proApoA- I, or mature ApoA-l.
  • the ApoA-l has at least 90% sequence identity to SEQ ID NO:1 : PPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQE FWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHEL QEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEK AKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO:1)
  • the ApoA-l has at least 95% sequence identity to SEQ ID NO:1 . In other embodiments, the ApoA-l has at least 98% sequence identity to SEQ ID NO:1 . In other embodiments, the ApoA-l has at least 99% sequence identity to SEQ ID NO:1 . In other embodiments, the ApoA-l has 100% sequence identity to SEQ ID NO:1.
  • the ApoA-l has at least 95% sequence identity to amino acids 25 to 267 of SEQ ID NO:2: MKAAVLTLAVLFLTGSQARHFWQQDEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNL KLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEE MELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLE ALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO:2)
  • the ApoA-l has at least 98% sequence identity to amino acids 25 to 267 of SEQ ID NO:2. In other embodiments, the ApoA-l has at least 99% sequence identity to amino acids 25 to 267 of SEQ ID NO:2. In other embodiments, the ApoA-l has 100% sequence identity to amino acids 25 to 267 of SEQ ID NO:2.
  • SEQ ID NO:3 DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVT QEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLH ELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLS EKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO:3).
  • the host cell comprises a nucleotide sequence encoding the amino acid sequence of a mature ApoA-l protein, which can be operably linked to a signal sequence for secretion of the ApoA-l from the host cell and/or to a proprotein sequence.
  • the nucleotide sequence encodes an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 97% or 100%) to SEQ ID NO:2 (human prepro-ApoA-l), which comprises a signal sequence (amino acids 1-18 of SEQ ID NO:2) and a propeptide sequence (amino acids 19-24 of SEQ ID NO:2).
  • SEQ ID NO:2 human prepro-ApoA-l
  • Other signal sequences suitable for directed secretion of ApoA-l can be either heterologous to ApoA-l, e.g., a human albumin signal peptide or a human IL-2 signal peptide, or homologous to ApoA-l.
  • the nucleotide sequence encodes an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2. In some embodiments, the nucleotide sequence encodes an amino acid sequence having at least 98% sequence identity to SEQ ID NO:2. In some embodiments, the nucleotide sequence encodes an amino acid sequence having at least 99% sequence identity to SEQ ID NO:2. In some embodiments, the nucleotide sequence encodes an amino acid sequence having 100% sequence identity to SEQ ID NO:2. [0341] In some embodiments, ApoA-l is produced by a CHO cell (e.g., CHO-S cell) engineered to express the amino acid sequence of SEQ ID NO:2.
  • a CHO cell e.g., CHO-S cell
  • the engineered cell can comprise a nucleotide sequence encoding SEQ ID NO:2 operably linked to a promoter, for example a constitutive promoter.
  • the engineered cell comprises a nucleotide sequence encoding SEQ ID NO:2 operably linked to a simian cytomegalovirus immediate early promoter.
  • recombinant ApoA-l can be produced by culturing any of the mammalian host cells described herein under conditions in which ApoA-l is expressed and secreted.
  • the ApoA-l can be recovered from the supernatant of a cultured mammalian host cell, and optionally purified to yield mature, biologically active ApoA-l.
  • the apolipoprotein molecule(s) can comprise a chimeric apolipoprotein comprising an apolipoprotein and one or more attached functional moieties, such as for example, one or more CRN-001 complex(es), one or more targeting moieties, a moiety having a desired biological activity, an affinity tag to assist with purification, and/or a reporter molecule for characterization or localization studies.
  • An attached moiety with biological activity may have an activity that is capable of augmenting and/or synergizing with the biological activity of a compound incorporated into a complex of the disclosure.
  • a moiety with biological activity may have antimicrobial (for example, antifungal, antibacterial, anti-protozoal, bacteriostatic, fungistatic, or antiviral) activity.
  • an attached functional moiety of a chimeric apolipoprotein is not in contact with hydrophobic surfaces of the complex.
  • an attached functional moiety is in contact with hydrophobic surfaces of the complex.
  • a functional moiety of a chimeric apolipoprotein may be intrinsic to a natural protein.
  • a chimeric apolipoprotein includes a ligand or sequence recognized by or capable of interaction with a cell surface receptor or other cell surface moiety.
  • a chimeric apolipoprotein includes a targeting moiety that is not intrinsic to the native apolipoprotein, such as for example, S. cerevisiae a-mating factor peptide, folic acid, transferrin, or lactoferrin.
  • a chimeric apolipoprotein includes a moiety with a desired biological activity that augments and/or synergizes with the activity of a compound incorporated into a complex of the disclosure.
  • a chimeric apolipoprotein may include a functional moiety intrinsic to an apolipoprotein.
  • an apolipoprotein intrinsic functional moiety is the intrinsic targeting moiety formed approximately by amino acids 130-150 of human ApoE, which comprises the receptor binding region recognized by members of the low density lipoprotein receptor family.
  • Other examples of apolipoprotein intrinsic functional moieties include the region of ApoB-100 that interacts with the low density lipoprotein receptor and the region of ApoA-l that interacts with scavenger receptor type B 1.
  • a functional moiety may be added synthetically or recombinantly to produce a chimeric apolipoprotein.
  • apolipoprotein with the prepro or pro sequence from another preproapolipoprotein (e.g., prepro sequence from preproapoA-ll substituted for the prepro sequence of preproapoA-l).
  • apolipoprotein for which some of the amphipathic sequence segments have been substituted by other amphipathic sequence segments from another apolipoprotein.
  • chimeric refers to two or more molecules that are capable of existing separately and are joined together to form a single molecule having the desired functionality of all of its constituent molecules.
  • the constituent molecules of a chimeric molecule may be joined synthetically by chemical conjugation or, where the constituent molecules are all polypeptides or analogs thereof, polynucleotides encoding the polypeptides may be fused together recombinantly such that a single continuous polypeptide is expressed.
  • a chimeric molecule is termed a fusion protein.
  • a "fusion protein” is a chimeric molecule in which the constituent molecules are all polypeptides and are attached (fused) to each other such that the chimeric molecule forms a continuous single chain.
  • the various constituents can be directly attached to each other or can be coupled through one or more linkers.
  • One or more segments of various constituents can be, for example, inserted in the sequence of an apolipoprotein, or, as another example, can be added N-terminal or C-terminal to the sequence of an apolipoprotein.
  • a fusion protein can comprise an antibody light chain, an antibody fragment, a heavy-chain antibody, or a single-domain antibody.
  • a chimeric apolipoprotein is prepared by chemically conjugating the apolipoprotein and the functional moiety to be attached.
  • Means of chemically conjugating molecules are well known to those of skill in the art. Such means will vary according to the structure of the moiety to be attached, but will be readily ascertainable to those of skill in the art.
  • Polypeptides typically contain a variety of functional groups, e.g., carboxylic acid (--COOH), free amino (-NH2), or sulfhydryl (--SH) groups, that are available for reaction with a suitable functional group on the functional moiety or on a linker to bind the moiety thereto.
  • a functional moiety may be attached at the N-terminus, the C-terminus, or to a functional group on an interior residue (i.e. , a residue at a position intermediate between the Island C-termini) of an apolipoprotein molecule.
  • the apolipoprotein and/or the moiety to be tagged can be derivatized to expose or attach additional reactive functional groups.
  • fusion proteins that include a polypeptide functional moiety are synthesized using recombinant expression systems. Typically, this involves creating a nucleic acid (e.g., DNA) sequence that encodes the apolipoprotein and the functional moiety such that the two polypeptides will be in frame when expressed, placing the DNA under the control of a promoter, expressing the protein in a host cell, and isolating the expressed protein.
  • a nucleic acid e.g., DNA
  • a nucleic acid encoding a chimeric apolipoprotein can be incorporated into a recombinant expression vector in a form suitable for expression in a host cell.
  • an "expression vector” is a nucleic acid which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide.
  • the vector may also include regulatory sequences such as promoters, enhancers, or other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are known to those skilled in the art (see, e.g., Goeddel, 1990, Gene Expression Technology: Meth. Enzymol.
  • an apolipoprotein has been modified such that when the apolipoprotein is incorporated into a complex of the disclosure, the modification will increase stability of the complex, confer targeting ability or increase capacity.
  • the modification includes introduction of cysteine residues into apolipoprotein molecules to permit formation of intramolecular or intermolecular disulfide bonds, e.g., by site-directed mutagenesis.
  • a chemical crosslinking agent is used to form intermolecular links between apolipoprotein molecules to enhance stability of the complex.
  • Intermolecular crosslinking prevents or reduces dissociation of apolipoprotein molecules from the complex and/or prevents displacement by endogenous apolipoprotein molecules within an individual to whom the complexes are administered.
  • an apolipoprotein is modified either by chemical derivatization of one or more amino acid residues or by site directed mutagenesis, to confer targeting ability to or recognition by a cell surface receptor.
  • Lipid binding protein molecules and complexes comprising lipid binding protein molecules can be targeted to a specific cell surface receptor by engineering receptor recognition properties into an apolipoprotein.
  • lipid binding protein molecules or complexes may be targeted to a particular cell type known to harbor a particular type of infectious agent, for example by modifying a apolipoprotein of other lipid binding protein molecule to render it capable of interacting with a receptor on the surface of the cell type being targeted.
  • lipid binding protein molecules or complexes may be targeted to macrophages by altering a apolipoprotein or other lipid binding protein molecule to confer recognition by the macrophage endocytic class A scavenger receptor (SR-A).
  • SR-A macrophage endocytic class A scavenger receptor
  • SR-A binding ability can be conferred to a lipid binding protein molecule or complex by modifying a apolipoprotein or other lipid binding protein molecule by site directed mutagenesis to replace one or more positively charged amino acids with a neutral or negatively charged amino acid.
  • SR-A recognition can also be conferred by preparing a chimeric apolipoprotein that includes an N- or C-terminal extension having a ligand recognized by SR-A or an amino acid sequence with a high concentration of negatively charged residues.
  • Lipid binding protein molecules and complexes comprising lipid binding protein molecules can also interact with apolipoprotein receptors such as, but not limited to, ABCA1 receptors, ABCG1 receptors, Megalin, Cubulin and HDL receptors such as SR-B1.
  • apolipoprotein receptors such as, but not limited to, ABCA1 receptors, ABCG1 receptors, Megalin, Cubulin and HDL receptors such as SR-B1.
  • Peptides, peptide analogs, and agonists that mimic the activity of an apolipoprotein can also be used as the lipid binding protein molecule or in the complexes described herein, either alone, in combination with one or more other lipid binding proteins.
  • apolipoprotein peptide mimetics can also be used as the lipid binding protein molecule or in the complexes described herein, either alone, in combination with one or more other lipid binding proteins.
  • Non-limiting examples of peptides and peptide analogs that correspond to apolipoproteins, as well as agonists that mimic the activity of ApoA-l, ApoA-lw, ApoA-ll, ApoA-IV, and ApoE, that are useful as lipid binding protein molecules and/or are suitable for inclusion in the complexes and compositions described herein are disclosed in U.S. Pat.
  • WO/2010/093918 to Dasseux et al., the disclosures of which are incorporated herein by reference in their entireties.
  • These peptides and peptide analogues can be composed of L-amino acid or D-amino acids or mixture of L- and D-amino acids. They may also include one or more non-peptide or amide linkages, such as one or more well-known peptide/amide isosteres.
  • Such apolipoprotein peptide mimetic can be synthesized or manufactured using any technique for peptide synthesis known in the art, including, e.g., the techniques described in U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166.
  • the lipid binding protein molecules comprise apolipoprotein peptide mimetic molecules and optionally one or more apolipoprotein molecules such as those described above.
  • the apolipoprotein peptide mimetic molecules comprise an ApoA-l peptide mimetic, ApoA-ll peptide mimetic, ApoA-IV peptide mimetic, or ApoE peptide mimetic or a combination thereof.
  • a lipid binding protein is a component of (e.g., formulated as) a lipid binding protein-based complex, for example complexed with one or more amphipathic molecules such as lipids.
  • Lipid binding protein-based complexes that can be used include HDL and HDL mimetic-based complexes.
  • lipid binding protein-based complexes can comprise a lipoprotein complex as described in U.S. Patent No. 8,206,750, PCT publication WO 2012/109162, PCT publication WO 2015/173633 A2 (e.g., CER-001 ) or US 2004/0229794 A1 , the contents of each of which are incorporated herein by reference in their entireties.
  • lipoproteins and “apolipoproteins” are used interchangeably herein, and unless required otherwise by context, the term “lipoprotein” encompasses lipoprotein mimetics.
  • lipid binding protein and “lipid binding polypeptide” are also used interchangeably herein, and unless required otherwise by context, the terms do not connote an amino acid sequence of particular length.
  • Lipoprotein complexes can comprise a protein fraction (e.g., an apolipoprotein fraction) and a lipid fraction (e.g., a phospholipid fraction).
  • the protein fraction includes one or more lipid-binding protein molecules, such as apolipoproteins, peptides, or apolipoprotein peptide analogs or mimetics, for example one or more lipid binding protein molecules described in Section 6.1.1.
  • the lipid fraction typically includes one or more phospholipids which can be neutral, negatively charged, positively charged, or a combination thereof.
  • phospholipids which can be neutral, negatively charged, positively charged, or a combination thereof.
  • Exemplary phospholipids and other amphipathic molecules which can be included in the lipid fraction are described in Section 6.1.3.
  • the lipid fraction contains at least one neutral phospholipid (e.g., a sphingomyelin (SM)) and, optionally, one or more negatively charged phospholipids.
  • the neutral and negatively charged phospholipids can have fatty acid chains with the same or different number of carbons and the same or different degree of saturation.
  • the neutral and negatively charged phospholipids will have the same acyl tail, for example a C16:0, or palmitoyl, acyl chain.
  • the weight ratio of the apolipoprotein fraction: lipid fraction ranges from about 1 :2.7 to about 1 :3 (e.g., 1 :2.7).
  • any phospholipid that bears at least a partial negative charge at physiological pH can be used as the negatively charged phospholipid.
  • Non-limiting examples include negatively charged forms, e.g., salts, of phosphatidylinositol, a phosphatidylserine, a phosphatidylglycerol and a phosphatidic acid.
  • the negatively charged phospholipid is 1 ,2-dipalmitoyl-sn-glycero-3-[phospho-rac- (1-glycerol)], or DPPG, a phosphatidylglycerol.
  • Preferred salts include potassium and sodium salts.
  • a lipoprotein complex used in the methods of the disclosure is a lipoprotein complex as described in U.S. Patent No. 8,206,750 or WO 2012/109162 (and its U.S. counterpart, US 2012/0232005), the contents of each of which are incorporated herein in its entirety by reference.
  • the lipid binding protein molecule component of the lipoprotein complex is as described in Section 6.1 and preferably in Section 6.1.1 of WO 2012/109162 (and US 2012/0232005), the lipid component is as described in Section 6.2 of WO 2012/109162 (and US 2012/0232005), which can optionally be complexed together in the amounts described in Section 6.3 of WO 2012/109162 (and US 2012/0232005).
  • a lipoprotein complex of the disclosure is in a population of complexes that is at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% homogeneous, as described in Section 6.4 of WO 2012/109162 (and US 2012/0232005), the contents of which are incorporated by reference herein.
  • a lipid binding protein-based complex comprises 1 to 8 apolipoprotein molecules (e g., 1 to 6, 1 to 4, 1 to 2, 2 to 8, 2 to 6, 2 to 4, 4 to 8, 4 to 6, or 6 to 8 apolipoprotein molecules).
  • the complex comprises 1 apolipoprotein molecule.
  • the complex comprises 2 apolipoprotein molecules.
  • the complex comprises 3 apolipoprotein molecules.
  • the complex comprises 4 apolipoprotein molecules.
  • the complex comprises 5 apolipoprotein molecules.
  • the complex comprises 6 apolipoprotein molecules.
  • the complex comprises 7 apolipoprotein molecules.
  • the complex comprises 8 apolipoprotein molecules.
  • the complex comprises 1 to 8 ApoA-l equivalents (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 8, 2 to 6, 2 to 4, 4 to 6, or 4 to 8 ApoA-l equivalents).
  • Lipid binding proteins can be expressed in terms of ApoA-l equivalents based upon the number of amphipathic helices they contain. For example, ApoA-h, which typically exists as a disulfide-bridged dimer, can be expressed as 2 ApoA-l equivalents, because each molecule of ApoA-h contains twice as many amphipathic helices as a molecule of ApoA-l.
  • a peptide mimetic that contains a single amphipathic helix can be expressed as a 1/10-1/6 ApoA-l equivalent, because each molecule contains 1/10-1/6 as many amphipathic helices as a molecule of ApoA-l.
  • a lipoprotein complex that can be used in the methods of the disclosure comprises 2-4 ApoA-l equivalents, 2 molecules of charged phospholipid, 60 molecules of lecithin and 40 molecules of SM.
  • a lipoprotein complex that can be used in the methods of the disclosure consists essentially of 2-4 ApoA-l equivalents, 2 molecules of charged phospholipid, 50-80 molecules of lecithin and 20-50 molecules of SM.
  • a lipoprotein complex that can be used in the methods of the disclosure consists essentially of 2-4 ApoA-l equivalents, 2 molecules of charged phospholipid, 50 molecules of lecithin and 50 molecules of SM.
  • a lipoprotein complex that can be used in the methods of the disclosure consists essentially of 2-4 ApoA-l equivalents, 2 molecules of charged phospholipid, 80 molecules of lecithin and 20 molecules of SM.
  • a lipoprotein complex that can be used in the methods of the disclosure consists essentially of 2-4 ApoA-l equivalents, 2 molecules of charged phospholipid, 70 molecules of lecithin and 30 molecules of SM.
  • a lipoprotein complex that can be used in the methods of the disclosure consists essentially of 2-4 ApoA-l equivalents, 2 molecules of charged phospholipid, 60 molecules of lecithin and 40 molecules of SM.
  • a lipoprotein complex that can be used in the methods of the disclosure consists essentially of about 90 to 99.8 wt % lecithin and about 0.2 to 10 wt % negatively charged phospholipid, for example, about 0.2-1 wt %, 0.2-2 wt %, 0.2-3 wt %, 0.2-4 wt %, 0.2- 5 wt %, 0.2-6 wt %, 0.2-7 wt %, 0.2-8 wt %, 0.2-9 wt % or 0.2-10 wt % total negatively charged phospholipid(s).
  • HDL-based or HDL mimetic-based complexes can include a single type of lipid-binding protein, or mixtures of two or more different lipid-binding proteins, which may be derived from the same or different species.
  • the complexes will preferably comprise lipid-binding proteins that are derived from, or correspond in amino acid sequence to, the animal species being treated, in order to avoid inducing an immune response to the therapy.
  • lipid-binding proteins of human origin are preferably used for treatment of human patients.
  • the use of peptide mimetic apolipoproteins may also reduce or avoid an immune response.
  • the lipid component includes two types of phospholipids: a sphingomyelin (SM) and a negatively charged phospholipid.
  • SM sphingomyelin
  • exemplary SMs and negatively charged lipids are described in Section 6.1.3.1.
  • Lipid components including SM can optionally include small quantities of additional lipids.
  • Virtually any type of lipids may be used, including, but not limited to, lysophospholipids, galactocerebroside, gangliosides, cerebrosides, glycerides, triglycerides, and cholesterol and its derivatives.
  • such optional lipids will typically comprise less than about 15 wt% of the lipid fraction, although in some instances more optional lipids could be included. In some embodiments, the optional lipids comprise less than about 10 wt%, less than about 5 wt%, or less than about 2 wt%. In some embodiments, the lipid fraction does not include optional lipids.
  • the phospholipid fraction contains egg SM or palmitoyl SM or phytosphingomyelin and DPPG in a weight ratio (SM: negatively charged phospholipid) ranging from 90:10 to 99:1 , more preferably ranging from 95:5 to 98:2. In one embodiment, the weight ratio is 97:3.
  • the molar ratio of the lipid component to the protein component of complexes of the disclosure can vary, and will depend upon, among other factors, the identity(ies) of the apolipoprotein comprising the protein component, the identities and quantities of the lipids comprising the lipid component, and the desired size of the complex.
  • apolipoproteins such as ApoA-l are thought to be mediated by the amphipathic helices comprising the apolipoprotein, it is convenient to express the apolipoprotein fraction of the lipid :apolipoprotein molar ratio using ApoA-l protein equivalents. It is generally accepted that ApoA-l contains 6-10 amphipathic helices, depending upon the method used to calculate the helices. Other apolipoproteins can be expressed in terms of ApoA-l equivalents based upon the number of amphipathic helices they contain.
  • ApoA-h which typically exists as a disulfide-bridged dimer
  • 2 ApoA-l equivalents can be expressed as 2 ApoA-l equivalents, because each molecule of ApoA-h contains twice as many amphipathic helices as a molecule of ApoA-l.
  • a peptide apolipoprotein that contains a single amphipathic helix can be expressed as a 1/10-1/6 ApoA-l equivalent, because each molecule contains 1/10-1/6 as many amphipathic helices as a molecule of ApoA-l.
  • the lipid:ApoA-l equivalent molar ratio of the lipoprotein complexes (defined herein as “Ri”) will range from about 105:1 to 110:1. In some embodiments, the Ri is about 108:1. Ratios in weight can be obtained using a MW of approximately 650-800 for phospholipids.
  • the molar ratio of lipid : ApoA-l equivalents ranges from about 80:1 to about 110:1 , e.g., about 80:1 to about 100:1.
  • the RSM for complexes can be about 82:1 .
  • lipoprotein complexes used in the methods of the disclosure are negatively charged complexes which comprise a protein fraction which is preferably mature, full-length ApoA-l, and a lipid fraction comprising a neutral phospholipid, sphingomyelin (SM), and negatively charged phospholipid.
  • SM sphingomyelin
  • the lipid component contains SM (e.g., egg SM, palmitoyl SM, phytoSM, or a combination thereof) and negatively charged phospholipid (e.g., DPPG) in a weight ratio (SM : negatively charged phospholipid) ranging from 90:10 to 99:1 , more preferably ranging from 95:5 to 98:2, e.g., 97:3.
  • SM negatively charged phospholipid
  • the ratio of the protein component to lipid component can range from about 1 :2.7 to about 1 :3, with 1 :2.7 being preferred. This corresponds to molar ratios of ApoA-l protein to lipid ranging from approximately 1 :90 to 1 :140. In some embodiments, the molar ratio of protein to lipid in the complex is about 1:90 to about 1 :120, about 1 :100 to about 1 :140, or about 1 :95 to about 1 :125. [0390] In particular embodiments, the complex comprises CER-001 , CSL-111 , CSL-112, CER-522, ETC-216, or ETC-642. In a preferred embodiment, the complex is CER-001.
  • CER-001 as used in the literature and in the Examples below refers to a complex described in Example 4 of WO 2012/109162.
  • WO 2012/109162 refers to CER-001 as a complex having a 1:2.7 lipoprotein weighttotal phospholipid weight ratio with a SM:DPPG weightweight ratio of 97:3.
  • Example 4 of WO 2012/109162 also describes a method of its manufacture.
  • CER- 001 refers to a lipoprotein complex whose individual constituents can vary from CER-001 as described in Example 4 of WO 2012/109162 by up to 20%.
  • the constituents of the lipoprotein complex vary from CER-001 as described in Example 4 of WO 2012/109162 by up to 10%.
  • the constituents of the lipoprotein complex are those described in Example 4 of WO 2012/109162 (plus/minus acceptable manufacturing tolerance variations).
  • the SM in CER-001 can be natural or synthetic.
  • the SM is a natural SM, for example a natural SM described in WO 2012/109162, e.g., chicken egg SM or plant SM.
  • the SM is a synthetic SM, for example a synthetic SM described in WO 2012/109162, e.g., synthetic palmitoylsphingomyelin, for example as described in WO 2012/109162.
  • Methods for synthesizing palmitoylsphingomyelin are known in the art, for example as described in WO 2014/140787 and WO 2024/003612, the contents of which are incorporated herein by reference in their entireties.
  • the lipoprotein in CER-001 apolipoprotein A-l (ApoA- I), preferably has an amino acid sequence corresponding to amino acids 25 to 267 of SEO ID NO:1 of WO 2012/109162.
  • ApoA-l can be purified by animal sources (and in particular from human sources) or produced recombinantly.
  • the ApoA-l in CER-001 is recombinant ApoA-l.
  • CER- 001 used in a dosing regimen of the disclosure is preferably highly homogeneous, for example at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% homogeneous, as reflected by a single peak in gel permeation chromatography. See, e.g., Section 6.4 of WO 2012/109162.
  • SEQ ID NO:1 of WO 2012/109162 is identified herein as SEQ ID NO:2.
  • Amino acids 25 to 267 of SEQ ID NO:1 of WO 2012/109162 are identified herein as SEQ ID NO:3.
  • CSL-111 is a reconstituted human ApoA-l purified from plasma complexed with soybean phosphatidylcholine (SBPC) (Tardif et a/., 2007, JAMA 297:1675-1682).
  • SBPC soybean phosphatidylcholine
  • CSL-112 is a formulation of ApoA-l purified from plasma and reconstituted to form HDL suitable for intravenous infusion (Diditchenko et al., 2013, DOI 10.1161/ATVBAHA.113.301981 ).
  • ETC-216 (also known as MDCO-216) is a lipid-depleted form of HDL containing recombinant ApoA-lMiiano( Nicholls et al., 2011 , Expert Opin Biol Ther. 11(3):387-94. doi: 10.1517/14712598.2011.557061).
  • ETC-642 is complex of a 22-amino acid amphipathic peptide (ESP-2418) complexed with sphingomyelin and 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (Dipalmitoylphosphatidylcholine, DPPC ( Di Bartolo et al., 2011 , Atherosclerosis 217:395-400).
  • ESP-2418 22-amino acid amphipathic peptide
  • DPPC Di Bartolo et al., 2011 , Atherosclerosis 217:395-400
  • CER- 522 is a lipoprotein complex comprising a combination of three phospholipids and a 22 amino acid peptide, CT80522:
  • the phospholipid component of CER-522 consists of egg sphingomyelin, 1 ,2-dipalmitoyl-sn- glycero-3-phosphocholine (Dipalmitoylphosphatidylcholine, DPPC) and 1 ,2-dipalmitoyl-sn-glycero-3- [phospho-rac-(l-glycerol)] (Dipalmitoylphosphatidyl- glycerol, DPPG) in a 48.5:48.5:3 weight ratio.
  • the ratio of peptide to total phospholipids in the CER-522 complex is 1 :2.5 (w/w).
  • the lipoprotein complex is delipidated HDL.
  • Most HDL in plasma is cholesterol-rich.
  • the lipids in HDL can be depleted, for example partially and/or selectively depleted, e.g., to reduce its cholesterol content.
  • the delipidated HDL can resemble small a, preP- 1 , and other prep forms of HDL. A process for selective depletion of HDL is described in Sacks et al., 2009, J Lipid Res. 50(5): 894-907.
  • a lipoprotein complex comprises a bioactive agent delivery particle as described in US 2004/0229794.
  • a bioactive agent delivery particle can comprise a lipid binding polypeptide (e.g., an apolipoprotein as described previously in this Section or in Section 6.1.1 ), a lipid bilayer (e.g., comprising one or more phospholipids as described previously in this Section or in Section 6.1.3.1 ), and a bioactive agent (e.g., an anti-cancer agent), wherein the interior of the lipid bilayer comprises a hydrophobic region, and wherein the bioactive agent is associated with the hydrophobic region of the lipid bilayer.
  • a bioactive agent delivery particle as described in US 2004/0229794.
  • a bioactive agent delivery particle does not comprise a hydrophilic core.
  • a bioactive agent delivery particle is disc shaped (e.g., having a diameter from about 7 to about 29 nm).
  • Bioactive agent delivery particles include bilayer-forming lipids, for example phospholipids (e.g., as described previously in this Section or in Section 6.1.3.1 ).
  • a bioactive agent delivery particle includes both bilayer-forming and non-bilayer-forming lipids.
  • the lipid bilayer of a bioactive agent delivery particle includes phospholipids.
  • the phospholipids incorporated into a delivery particle include dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG).
  • DMPC dimyristoylphosphatidylcholine
  • DMPG dimyristoylphosphatidylglycerol
  • the lipid bilayer includes DMPC and DMPG in a 7:3 molar ratio.
  • the lipid binding polypeptide is an apolipoprotein (e.g., as described previously in this Section or in Section 6.1.1 ).
  • the predominant interaction between lipid binding polypeptides, e.g., apolipoprotein molecules, and the lipid bilayer is generally a hydrophobic interaction between residues on a hydrophobic face of an amphipathic structure, e.g., an a-helix of the lipid binding polypeptide and fatty acyl chains of lipids on an exterior surface at the perimeter of the particle.
  • Bioactive agent delivery particles may include exchangeable and/or non-exchangeable apolipoproteins.
  • the lipid binding polypeptide is ApoA-L
  • bioactive agent delivery particles include lipid binding polypeptide molecules, e.g., apolipoprotein molecules, that have been modified to increase stability of the particle.
  • the modification includes introduction of cysteine residues to form intramolecular and/or intermolecular disulfide bonds.
  • bioactive agent delivery particles include a chimeric lipid binding polypeptide molecule, e.g., a chimeric apolipoprotein molecule, with one or more bound functional moieties, for example one or more targeting moieties and/or one or more moieties having a desired biological activity, e.g., antimicrobial activity, which may augment or work in synergy with the activity of a bioactive agent incorporated into the delivery particle.
  • a chimeric lipid binding polypeptide molecule e.g., a chimeric apolipoprotein molecule
  • one or more bound functional moieties for example one or more targeting moieties and/or one or more moieties having a desired biological activity, e.g., antimicrobial activity, which may augment or work in synergy with the activity of a bioactive agent incorporated into the delivery particle.
  • amphipathic molecule is a molecule that possesses both hydrophobic (apolar) and hydrophilic (polar) elements.
  • Amphipathic molecules that can be used in complexes described herein include lipids (e.g., as described in Section 6.1.3.1), detergents (e.g., as described in Section 6.1.3.2), fatty acids (e.g., as described in Section 6.1.3.3), and apolar molecules and sterols covalently attached to polar molecules such as, but not limited to, sugars or nucleic acids (e.g., as described in Section 6.1.3.4).
  • the complexes can include a single class of amphipathic molecule (e.g., a single species of phospholipids or a mixture of phospholipids) or can contain a combination of classes of amphipathic molecules (e.g., phospholipids and detergents).
  • the complex can contain one species of amphipathic molecules or a combination of amphipathic molecules configured to facilitate solubilization of the lipid binding protein molecule(s).
  • the amphipathic molecules included in comprise a phospholipid, a detergent, a fatty acid, an apolar moiety or sterol covalently attached to a sugar, or a combination thereof (e.g., selected from the types of amphipathic molecules discussed above).
  • the amphipathic molecules comprise or consist of phospholipid molecules.
  • the phospholipid molecules comprise negatively charged phospholipids, neutral phospholipids, positively charged phospholipids or a combination thereof.
  • the phospholipid molecules contribute a net charge of 1-3 per apolipoprotein molecule in the complex.
  • the net charge is a negative net charge.
  • the net charge is a positive net charge.
  • the phospholipid molecules consist of a combination of negatively charged and neutral phospholipids.
  • the molar ratio of negatively charge phospholipid to neutral phospholipid ranges from 1 :1 to 1 :3. In some embodiments, the molar ratio of negatively charged phospholipid to neutral phospholipid is about 1 :1 or about 1 :2.
  • the amphipathic molecules comprise neutral phospholipids and negatively charged phospholipids in a weight ratio of 95:5 to 99:1.
  • Lipid binding protein-based complexes can include one or more lipids.
  • one or more lipids can be saturated and/or unsaturated, natural and/or synthetic, charged or not charged, zwitterionic or not.
  • the lipid molecules e.g., phospholipid molecules
  • the lipid molecules can together contribute a net charge of 1-3 (e.g., 1-3, 1-2, 2-3, 1 , 2, or 3) per lipid binding protein molecule in the complex. In some embodiments, the net charge is negative. In other embodiments, the net charge is positive.
  • the lipid comprises a phospholipid.
  • Phospholipids can have two acyl chains that are the same or different (for example, chains having a different number of carbon atoms, a different degree of saturation between the acyl chains, different branching of the acyl chains, or a combination thereof).
  • the lipid can also be modified to contain a fluorescent probe (e.g., as described at yorkilipids.com/product-category/products/fluorescent-lipids/).
  • the lipid comprises at least one phospholipid.
  • Phospholipids can have unsaturated or saturated acyl chains ranging from about 6 to about 24 carbon atoms (e.g., 6-20, 6-16, 6-12, 12-24, 12-20, 12-16, 16-24, 16-20, or 20-24).
  • a phospholipid used in a complex of the disclosure has one or two acyl chains of 12, 14, 16, 18, 20, 22, or 24 carbons (e.g., two acyl chains of the same length or two acyl chains of different length).
  • Non-limiting examples of acyl chains present in commonly occurring fatty acids that can be included in phospholipids are provided in Table 1 , below:
  • Lipids that can be present in the complexes of the disclosure include, but are not limited to, small alkyl chain phospholipids, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine 1- myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2- stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine dioleophosphatidylethanolamine, dilauroylphosphatidylglycerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinosito
  • Synthetic lipids such as synthetic palmitoylsphingomyelin or N- palmitoyl-4-hydroxysphinganine-1-phosphocholine (a form of phytosphingomyelin) can be used to minimize lipid oxidation.
  • a lipid binding protein-based complex includes two types of phospholipids: a neutral lipid, e.g., lecithin and/or sphingomyelin (abbreviated SM), and a charged phospholipid (e.g., a negatively charged phospholipid).
  • a “neutral” phospholipid has a net charge of about zero at physiological pH.
  • neutral phospholipids are zwitterions, although other types of net neutral phospholipids are known and can be used.
  • the molar ratio of the charged phospholipid (e.g., negatively charged phospholipid) to neutral phospholipid ranges from 1 :1 to 1 :3, for example, about 1 :1 , about 1 :2, or about 1 :3.
  • the neutral phospholipid can comprise, for example, one or both of the lecithin and/or SM, and can optionally include other neutral phospholipids.
  • the neutral phospholipid comprises lecithin, but not SM.
  • the neutral phospholipid comprises SM, but not lecithin.
  • the neutral phospholipid comprises both lecithin and SM. All of these specific exemplary embodiments can include neutral phospholipids in addition to the lecithin and/or SM, but in many embodiments do not include such additional neutral phospholipids.
  • SM includes sphingomyelins derived or obtained from natural sources, as well as analogs and derivatives of naturally occurring SMs that are impervious to hydrolysis by LCAT, as is naturally occurring SM.
  • SM is a phospholipid very similar in structure to lecithin, but, unlike lecithin, it does not have a glycerol backbone, and hence does not have ester linkages attaching the acyl chains. Rather, SM has a ceramide backbone, with amide linkages connecting the acyl chains.
  • SM can be obtained, for example, from milk, egg or brain.
  • SM analogues or derivatives can also be used.
  • Non-limiting examples of useful SM analogues and derivatives include, but are not limited to, palmitoylsphingomyelin, N-palmitoyl-4-hydroxysphinganine-1-phosphocholine (a form of phytosphingomyelin), palmitoylsphingomyelin, stearoylsphingomyelin, D-erythro-N-16:0-sphingomyelin and its dihydro isomer, D-erythro-N-16:0-dihydro-sphingomyelin.
  • Synthetic SM such as synthetic palmitoylsphingomyelin or N-palmitoyl-4-hydroxysphinganine-1 -phosphocholine (phytosphingomyelin) can be used in order to produce more homogeneous complexes and with fewer contaminants and/or oxidation products than sphingolipids of animal origin. Methods for synthesizing SM are described in U.S. Publication No. 2016/0075634.
  • Sphingomyelins isolated from natural sources can be artificially enriched in one particular saturated or unsaturated acyl chain.
  • milk sphingomyelin (Avanti Phospholipid, Alabaster, Ala.) is characterized by long saturated acyl chains (/.e., acyl chains having 20 or more carbon atoms).
  • egg sphingomyelin is characterized by short saturated acyl chains (i.e., acyl chains having fewer than 20 carbon atoms).
  • milk sphingomyelin comprises C16:0 (16 carbon, saturated) acyl chains
  • about 80% of egg sphingomyelin comprises C16:0 acyl chains.
  • the composition of milk sphingomyelin can be enriched to have an acyl chain composition comparable to that of egg sphingomyelin, or vice versa.
  • the SM can be semi-synthetic such that it has particular acyl chains.
  • milk sphingomyelin can be first purified from milk, then one particular acyl chain, e.g., the C16:0 acyl chain, can be cleaved and replaced by another acyl chain.
  • the SM can also be entirely synthesized, by e.g., large-scale synthesis. See, e.g., Dong et al., U.S. Pat. No. 5,220,043, entitled Synthesis of D-erythro- sphingomyelins, issued Jun. 15, 1993; Weis, 1999, Chem. Phys. Lipids 102 (1-2):3-12.
  • SM can be fully synthetic, e.g., as described in U.S. Publication No. 2014/0275590.
  • the lengths and saturation levels of the acyl chains comprising a semi-synthetic or a synthetic SM can be selectively varied.
  • the acyl chains can be saturated or unsaturated, and can contain from about 6 to about 24 carbon atoms. Each chain can contain the same number of carbon atoms or, alternatively each chain can contain different numbers of carbon atoms.
  • the semisynthetic or synthetic SM comprises mixed acyl chains such that one chain is saturated and one chain is unsaturated. In such mixed acyl chain SMs, the chain lengths can be the same or different.
  • the acyl chains of the semi-synthetic or synthetic SM are either both saturated or both unsaturated.
  • both acyl chains comprising the semi-synthetic or synthetic SM are identical.
  • the chains correspond to the acyl chains of a naturally-occurring fatty acid, such as for example oleic, palmitic or stearic acid.
  • SM with saturated or unsaturated functionalized chains is used.
  • both acyl chains are saturated and contain from 6 to 24 carbon atoms.
  • Non-limiting examples of acyl chains present in commonly occurring fatty acids that can be included in semi-synthetic and synthetic SMs are provided in Table 1 , above.
  • the SM is palmitoyl SM, such as synthetic palmitoyl SM, which has C16:0 acyl chains, or is egg SM, which includes as a principal component palmitoyl SM.
  • functionalized SM such as phytosphingomyelin
  • Lecithin can be derived or isolated from natural sources, or it can be obtained synthetically.
  • suitable lecithins isolated from natural sources include, but are not limited to, egg phosphatidylcholine and soybean phosphatidylcholine.
  • lecithins include, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine 1 -myristoyl -2-palmitoylphosphatidylcholine, 1-palmitoy1-2- myristoylphosphatidylcholine, 1 -palmitoyl -2-stearoylphosphatidylcholine, 1 -stearoyl -2- palmitoylphosphatidylcholine, 1 -palmitoyl -2-oleoylphosphatidylcholine, 1 -oleoyl -2- palmitylphosphatidylcholine, dioleoylphosphatidylcholine and the ether derivatives or analogs thereof.
  • Lecithins derived or isolated from natural sources can be enriched to include specified acyl chains.
  • identity(ies) of the acyl chains can be selectively varied, as discussed above in connection with SM.
  • both acyl chains on the lecithin are identical.
  • the acyl chains of the SM and lecithin are all identical.
  • the acyl chains correspond to the acyl chains of myristitic, palmitic, oleic or stearic acid.
  • the complexes of the disclosure can include one or more negatively charged phospholipids (e.g., alone or in combination with one or more neutral phospholipids).
  • negatively charged phospholipids are phospholipids that have a net negative charge at physiological pH.
  • the negatively charged phospholipid can comprise a single type of negatively charged phospholipid, or a mixture of two or more different, negatively charged, phospholipids.
  • the charged phospholipids are negatively charged glycerophospholipids.
  • Suitable negatively charged phospholipids include, but are not limited to, a 1 ,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], a phosphatidylglycerol, a phospatidylinositol, a phosphatidylserine, a phosphatidic acid, and salts thereof (e.g., sodium salts or potassium salts).
  • the negatively charged phospholipid comprises one or more of phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and/or phosphatidic acid.
  • the negatively charged phospholipid comprises or consists of a salt of a phosphatidylglycerol or a salt of a phosphatidylinositol.
  • the negatively charged phospholipid comprises or consists of 1 ,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1- glycerol)], or DPPG, or a salt thereof.
  • the negatively charged phospholipids can be obtained from natural sources or prepared by chemical synthesis. In embodiments employing synthetic negatively charged phospholipids, the identities of the acyl chains can be selectively varied, as discussed above in connection with SM.
  • both acyl chains on the negatively charged phospholipids are identical.
  • the acyl chains all types of phospholipids included in a complex of the disclosure are all identical.
  • the complex comprises negatively charged phospholipid(s), and/or SM all having C16:0 or C16:1 acyl chains.
  • the fatty acid moiety of the SM is predominantly C16:1 palmitoyl.
  • the acyl chains of the charged phospholipid(s), lecithin and/or SM correspond to the acyl chain of palmitic acid.
  • the acyl chains of the charged phospholipid(s), lecithin and/or SM correspond to the acyl chain of oleic acid.
  • Examples of positively charged phospholipids that can be included in the complexes of the disclosure include N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide, 1 ,2-di-O-octadecenyl-3- trimethylammonium propane, 1 ,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl- sn-glycero-3-ethylphosphocholine, 1 ,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1 ,2-distearoyl-sn- glycero-3-ethylphosphocholine, 1 ,2-dipal
  • the lipids used are preferably at least 95% pure, and/or have reduced levels of oxidative agents (such as but not limited to peroxides).
  • Lipids obtained from natural sources preferably have fewer polyunsaturated fatty acid moieties and/or fatty acid moieties that are not susceptible to oxidation.
  • the level of oxidation in a sample can be determined using an iodometric method, which provides a peroxide value, expressed in milli-equivalent number of isolated iodines per kg of sample, abbreviated meq O/kg.
  • the level of oxidation, or peroxide level is low, e.g., less than 5 meq O/kg, less than 4 meq O/kg, less than 3 meq O/kg, or less than 2 meq O/kg.
  • Complexes can in some embodiments include small quantities of additional lipids.
  • Virtually any type of lipids can be used, including, but not limited to, lysophospholipids, galactocerebroside, gangliosides, cerebrosides, glycerides, triglycerides, and sterols and sterol derivatives (e.g., a plant sterol, an animal sterol, such as cholesterol, or a sterol derivative, such as a cholesterol derivative).
  • a complex of the disclosure can contain cholesterol or a cholesterol derivative, e.g., a cholesterol ester.
  • the cholesterol derivative can also be a substituted cholesterol or a substituted cholesterol ester.
  • the complexes of the disclosure can also contain an oxidized sterol such as, but not limited to, oxidized cholesterol or an oxidized sterol derivative (such as, but not limited to, an oxidized cholesterol ester).
  • an oxidized sterol such as, but not limited to, oxidized cholesterol or an oxidized sterol derivative (such as, but not limited to, an oxidized cholesterol ester).
  • the complexes do not include cholesterol and/or its derivatives (such as a cholesterol ester or an oxidized cholesterol ester).
  • the complexes can contain one or more detergents.
  • the detergent can be zwitterionic, nonionic, cationic, anionic, or a combination thereof.
  • Exemplary zwitterionic detergents include 3-[(3- Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3- Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and N,N- dimethyldodecylamine N-oxide (LDAO).
  • nonionic detergents include D-(+)-trehalose 6- monooleate, N-octanoyl-N-methylglucamine, N-nonanoyl-N-methylglucamine, N-decanoyl-N- methylglucamine, 1-(7Z-hexadecenoyl)-rac-glycerol, 1-(8Z-hexadecenoyl)-rac-glycerol, 1-(8Z- heptadecenoyl)-rac-glycerol, 1-(9Z-hexadecenoyl)-rac-glycerol, 1-decanoyl-rac-glycerol.
  • Exemplary cationic detergents include (S)-O-methyl-serine dodecylamide hydrochloride, dodecylammonium chloride, decyltrimethylammonium bromide, and cetyltrimethylammonium sulfate.
  • Exemplary anionic detergents include cholesteryl hemisuccinate, cholate, alkyl sulfates, and alkyl sulfonates.
  • the complexes can contain one or more fatty acids.
  • the one or more fatty acids can include short-chain fatty acids having aliphatic tails of five or fewer carbons (e.g. butyric acid, isobutyric acid, valeric acid, or isovaleric acid), medium-chain fatty acids having aliphatic tails of 6 to 12 carbons (e.g, caproic acid, caprylic acid, capric acid, or lauric acid), long-chain fatty acids having aliphatic tails of 13 to 21 carbons (e g., myristic acid, palmitic acid, stearic acid, or arachidic acid) , very long chain fatty acids having aliphatic tails of 22 or more carbons (e.g., behenic acid, lignoceric acid, or cerotic acid), or a combination thereof.
  • short-chain fatty acids having aliphatic tails of five or fewer carbons e.g. butyric acid, isobutyric acid,
  • the one or more fatty acids can be saturated (e.g., caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, or cerotic acid), unsaturated (e.g., myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, or docosahexaenoic acid) or a combination thereof.
  • Unsaturated fatty acids can be cis or trans fatty acids.
  • unsaturated fatty acids used in the complexes of the disclosure are cis fatty acids.
  • the complexes can contain one or more amphipathic molecules that comprise an apolar molecule or moiety (e.g., a hydrocarbon chain, an acyl or diacyl chain) or a sterol (e.g., cholesterol) attached to a sugar (e.g., a monosaccharide such as glucose or galactose, or a disaccharide such as maltose or trehalose).
  • a sugar e.g., a monosaccharide such as glucose or galactose, or a disaccharide such as maltose or trehalose.
  • the sugar can be a modified sugar or a substituted sugar.
  • Exemplary amphipathic molecules comprising an apolar molecule attached to a sugar include dodecan-2-yloxy-B-D-maltoside, tridecan-3-yloxy-B-D-maltoside, tridecan-2-yloxy-B-D-maltoside, n-dodecyl-B-D-maltoside (DDM), n-octyl- B-D-glucoside, n-nonyl-li-D-glucoside, n-decyl- -D-maltoside, n-dodecyl-P-D-maltopyranoside, 4-n- Dodecyl-a,a-trehalose, 6-n-dodecyl-a,a-trehalose, and 3-n-dodecyl-a,a-trehalose.
  • DDM dodecan-2-yloxy-B-D-maltoside
  • DDM n-dodecyl-B-
  • the apolar moiety is an acyl or a diacyl chain.
  • the sugar is a modified suoar or a substituted sugar. 6.1.4.
  • Lipid binding protein molecules and lipid binding protein-based complexes comprising a lipid binding protein molecule can be formulated for the intended route of administration, for example according to techniques known in the art (e g., as described in Allen et al., eds., 2012, Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press, London, UK).
  • CER-001 intended for administration by infusion can be formulated in a phosphate buffer with sucrose and mannitol excipients, for example as described in WO 2012/109162.
  • Subjects who can be treated according to the methods described herein are preferably mammals, most preferably human.
  • the subject has or is at risk of a condition that is associated with an abnormal level of TREM-1.
  • Such conditions include, but are not limited to, acute myocardial infarction (AMI), Alzheimer's Disease, chronic irritable bowel disease (IBD), cardiovascular diseases (CVDs), stroke, transient ischemic attack, organ transplant rejection (such as heart transplant rejection), ischemia reperfusion-induced tissue injury, post-operative inflammation, psoriasis, sepsis (e.g., septic shock), and sepsis-induced acute kidney injury (AKI).
  • AMI acute myocardial infarction
  • IBD chronic irritable bowel disease
  • CVDs cardiovascular diseases
  • stroke transient ischemic attack
  • organ transplant rejection such as heart transplant rejection
  • ischemia reperfusion-induced tissue injury post-operative inflammation
  • psoriasis sepsis
  • sepsis e.g., septic shock
  • sepsis-induced acute kidney injury AKI
  • the subject has or is at risk of a condition that is associated with an abnormal level of albumin.
  • a condition that is associated with an abnormal level of albumin.
  • Such conditions include, but are not limited to, hypoalbuminemia (which can be caused by, for example, liver disease, heart failure, malnutrition or a vitamin deficiency, inflammatory bowel disease, kidney disease, infections, stress, thyroid disease, diabetes, nephrotic syndrome, lupus, or cirrhosis).
  • the subject has or is at risk of a condition that is associated with an abnormal level of a kynurenine pathway biomarker.
  • a condition that is associated with an abnormal level of a kynurenine pathway biomarker.
  • Such conditions include, but are not limited to, Alzheimer's Disease, attention deficit/hyperactivity disorder (ADHD), CNS diseases, COVID-19 cognitive impairment, depression and major depressive disorder, epilepsy, HIV-associated neurocognitive disorder, Huntington’s Disease, long-term cognitive impairment after sepsis, mortality and neurological outcome following cardiac arrest, multiple sclerosis (MS), Parkinson's Disease, schizophrenia, sepsis, and sepsis- induced AKI.
  • ADHD attention deficit/hyperactivity disorder
  • CNS diseases COVID-19 cognitive impairment
  • depression and major depressive disorder epilepsy
  • HIV-associated neurocognitive disorder Huntington’s Disease
  • MS multiple sclerosis
  • Parkinson's Disease schizophrenia, sepsis, and sepsis- induced AKI.
  • the subject has or is at risk of a condition that is associated with an abnormal level of IL-10.
  • a condition that is associated with an abnormal level of IL-10.
  • Such conditions include, but are not limited to, autoimmune diseases, IBD, rheumatoid arthritis (RA), systemic lupus erythematosus, Type I diabetes, MS, pemphigus vulgaris, ulcerative colitis (UC), Sjogren’s syndrome, Grave’s disease, myasthenia gravis, psoriasis, autoimmune lymphoproliferative syndrome (ALPS), cytokine release syndrome (CRS, cytokine storm), sepsis, and sepsis-induced AKI.
  • autoimmune diseases IBD, rheumatoid arthritis (RA), systemic lupus erythematosus, Type I diabetes, MS, pemphigus vulgaris, ulcerative colitis (UC), Sjogren’s syndrome, Grave’s disease, myasthenia gravis
  • the subject has or is at risk of a condition that is associated with an abnormal level of TNFa.
  • conditions include, but are not limited to, CRS, sepsis, and sepsis-induced AKI.
  • the subject has or is at risk of a condition that is associated with an abnormal level of MCP-1. Such conditions include, but are not limited to, CRS, sepsis, and sepsis-induced AKI. [0448] In some aspects, the subject has or is at risk of a condition that is associated with an abnormal level of IL-6. Such conditions include, but are not limited to, CRS, sepsis, and sepsis-induced AKI. [0449] In some aspects, the subject has or is at risk of a condition that is associated with an abnormal level of IL-8. Such conditions include, but are not limited to, CRS, sepsis, and sepsis-induced AKI.
  • the subject has or is at risk of a condition that is associated with an abnormal level of VCAM-1 and/or ICAM-1.
  • Such conditions include, but are not limited to, vascular endothelial disorder.
  • the subject has or is at risk of a bacterial infection.
  • bacteria that commonly cause infection and sepsis include Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Klebsiella pneumoniae, and Pseudomonas aeruginosa (GBD 2019 Antimicrobial Resistance Collaborators, 2023, Lancet 400(10369):2221-2248).
  • bacteria that can cause infection and sepsis include Acinetobacter baumanni, Bacteroides fragilis, and Proteus mirabilis.
  • the subject has or is at risk of a gram-positive bacterial infection.
  • the subject has or is at risk of a gram-negative bacterial infection.
  • the subject has or is at risk of a viral infection, such as a SARS-CoV-2 (COVID- 19) infection or an influenza virus infection.
  • a viral infection such as a SARS-CoV-2 (COVID- 19) infection or an influenza virus infection.
  • the subject has or is at risk of acute myocardial infarction (AMI).
  • AMI acute myocardial infarction
  • the subject has or is at risk of Alzheimer’s disease.
  • the subject has or is at risk of chronic inflammatory bowel disease (IBD).
  • IBD chronic inflammatory bowel disease
  • the subject has or is at risk of a cardiovascular disease (CVD).
  • CVD cardiovascular disease
  • the subject is having or is at risk of a stroke or transient ischemic attack (TIA).
  • TIA stroke or transient ischemic attack
  • Subjects having or who have had a TIA commonly referred to as a “mini-stroke,” are at risk of a stroke, particularly within the days following the TIA.
  • a subject experiencing or who has experienced a TIA is administered a lipid binding protein according to an administration regimen described herein.
  • the subject has or is at risk of cytokine release syndrome (CRS, cytokine storm).
  • CRS cytokine release syndrome
  • CRS is secondary to an infection, such as a bacterial infection or a viral infection.
  • the subject has or is at risk of organ transplant, such as heart transplant rejection.
  • the subject has or is at risk of ischemia reperfusion-induced tissue injury.
  • the subject has or is at risk of post-operative inflammation.
  • the subject has or is at risk of psoriasis.
  • the subject has or is at risk of sepsis, including but not limited to sepsis wherein the subject has an abnormal level of at least two of TNFa, IL-6, IL-8, TREM-1 , and a kynurenine pathway biomarker, such as an abnormal level of TREM-1 and at least one of quinolinic acid, kynurenic acid, kynurenine, tryptophan, and kynurenine/tryptophan ratio.
  • sepsis is secondary to an infection, such as a bacterial infection or a viral infection.
  • a subject with sepsis has a documented or suspected infection and an acute change in total SOFA score (see, Vincent et al. 1996, Intensive Care Med, 22:707-710) of greater than or equal to 2 points.
  • the subject has septic shock.
  • a subject having septic shock has hypotension (e.g., systolic arterial pressure ⁇ 90 mm Hg or mean arterial pressure (MAP) ⁇ 65 mm Hg) requiring the use of vasopressors for more than one hour despite intravenous fluid resuscitation.
  • a subject having septic shock has hypotension requiring vasopressor treatment to maintain a mean arterial pressure of 65 mm Hg or greater and serum lactate level greater than 2 mmol/L (>18 mg/dL) despite adequate fluid resuscitation.
  • the subject has or is at risk of sepsis-induced acute kidney injury (AKI).
  • AKI sepsis-induced acute kidney injury
  • the subject has or is at risk of hypoalbuminemia.
  • the subject has or is at risk of hypoalbuminemia associated with a vitamin deficiency.
  • the subject has or is at risk of hypoalbuminemia associated with inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the subject has or is at risk of hypoalbuminemia associated with kidney disease.
  • the subject has or is at risk of hypoalbuminemia associated with infections.
  • the subject has or is at risk of hypoalbuminemia associated with stress.
  • the subject has or is at risk of hypoalbuminemia associated with thyroid disease.
  • the subject has or is at risk of hypoalbuminemia associated with diabetes.
  • the subject has or is at risk of hypoalbuminemia associated with nephrotic syndrome.
  • the subject has or is at risk of hypoalbuminemia associated with lupus.
  • the subject has or is at risk of hypoalbuminemia associated with cirrhosis.
  • the subject has or is at risk of hypoalbuminemia associated with liver disease.
  • the subject has or is at risk of hypoalbuminemia associated with heart failure.
  • the subject has or is at risk of hypoalbuminemia associated with malnutrition.
  • the subject has or is at risk of attention-deficit/hyperactivity disorder (ADHD).
  • ADHD attention-deficit/hyperactivity disorder
  • CNS central nervous system
  • the subject has or is at risk of COVID-19 cognitive decline.
  • the subject has or is at risk of depression or major depressive disorder.
  • the subject has or is at risk of epilepsy.
  • the subject has or is at risk of HIV-associated neurocog nitive disorder.
  • the subject has or is at risk of Huntington's disease.
  • the subject has or is at risk of inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the subject has or is at risk of long-term cognitive decline (“brain fog”), such as can occur after sepsis.
  • cognitive fog long-term cognitive decline
  • the subject has or is at risk of mortality or neurological deficit following cardiac arrest.
  • the subject has or is at risk of multiple sclerosis (MS).
  • MS multiple sclerosis
  • the subject has or is at risk of Parkinson's disease.
  • the subject has or is at risk of schizophrenia.
  • the subject has or is at risk of vascular endothelial disorder.
  • the subject has or is at risk of acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • the subject has a SOFA score of 1 to 24 before treatment with a lipid binding protein molecule, e.g., a score of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 (see, Vincent ef al. 1996, Intensive Care Med, 22:707-710).
  • the subject has acute kidney injury (AKI) or is at risk of AKI, for example due to a viral infection or a bacterial infection (e.g., septic subjects).
  • AKI acute kidney injury
  • a viral infection or a bacterial infection e.g., septic subjects.
  • the subject can have CRS or be at risk of CRS, and/or be in need of reduction in serum levels of one or more inflammatory markers such as IL-6.
  • the subject has CRS.
  • the subject has CRS secondary to an infection, for example a viral infection or a bacterial infection.
  • the subject is at risk of CRS, for example due to a viral infection or a bacterial infection.
  • the subject is a subject in need of a reduction in serum levels of one or more inflammatory markers, for example a subject with elevated levels of the one or more inflammatory markers compared to normal levels.
  • exemplary inflammatory cytokines include interleukin 6 (IL-6), C- reactive protein, D-dimer, ferritin, interleukin 8 (IL-8), granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemoattractant protein (MCP) 1 , triggering receptor expressed on myeloid cells-1 (TREM-1 ), and tumor necrosis factor a (TNFa).
  • the one or more cytokines comprise IL-6.
  • the one or more cytokines comprise a combination of the foregoing, for example, 2, 3, 4, 5, 6, 7, 8, or all 9 of interleukin 6 (IL-6), C-reactive protein, D-dimer, ferritin, interleukin 8 (IL-8), granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemoattractant protein (MCP) 1 , TREM-1 , and tumor necrosis factor a (TNFa).
  • IL-6 interleukin 6
  • C-reactive protein D-dimer
  • ferritin interleukin 8
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • MCP monocyte chemoattractant protein
  • TREM-1 tumor necrosis factor a
  • the methods of the disclosure typically entail multiple administrations of a lipid binding protein molecule (e.g., ApoA-l), e.g., two to 20 individual doses, although in some embodiments, a single dose can be used.
  • an administration regimen can include two or more, three or more, or four or more individual doses of a lipid binding protein molecule (e.g., ApoA-l), e.g, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more than twenty individual doses.
  • an administration regimen comprises or consists of a single dose.
  • an administration regimen comprises or consists of two individual doses. In some embodiments, an administration regimen comprises or consists of three individual doses. In some embodiments, an administration regimen comprises or consists of four individual doses. In some embodiments, an administration regimen comprises or consists of five individual doses. In some embodiments, an administration regimen comprises or consists of six individual doses. In some embodiments, an administration regimen comprises or consists of seven individual doses. In some embodiments, an administration regimen comprises or consists of eight individual doses. In some embodiments, an administration regimen comprises or consists of nine individual doses. In some embodiments, an administration regimen comprises or consists of ten individual doses. In some embodiments, an administration regimen comprises or consists of eleven individual doses.
  • an administration regimen comprises or consists of twelve individual doses. In some embodiments, an administration regimen comprises or consists of thirteen individual doses. In some embodiments, an administration regimen comprises or consists of fourteen individual doses. In some embodiments, an administration regimen comprises or consists of fifteen individual doses. In some embodiments, an administration regimen comprises or consists of sixteen individual doses. In some embodiments, an administration regimen comprises or consists of seventeen individual doses. In some embodiments, an administration regimen comprises or consists of eighteen individual doses. In some embodiments, an administration regimen comprises or consists of nineteen individual doses. In some embodiments, an administration regimen comprises or consists of twenty individual doses.
  • the amount of lipid binding protein molecule delivered by each dose can be from 5 mg/kg to 40 mg/kg on a protein weight basis. In some embodiments, the amount of lipid binding protein molecule delivered by each dose can be from 5 mg/kg to 10 mg/kg on a protein weight basis. In some embodiments, the amount of lipid binding protein molecule delivered by each dose can be from 5 mg/kg to 20 mg/kg on a protein weight basis. In some embodiments, the amount of lipid binding protein molecule delivered by each dose can be from 10 mg/kg to 30 mg/kg on a protein weight basis. In some embodiments, the amount of lipid binding protein molecule delivered by each dose can be from 10 mg/kg to 20 mg/kg on a protein weight basis.
  • a dose is 5 mg/kg on a protein weight basis. In some embodiments, a dose is 6 mg/kg on a protein weight basis. In some embodiments, a dose is 7 mg/kg on a protein weight basis. In some embodiments, a dose is 8 mg/kg on a protein weight basis. In some embodiments, a dose is 9 mg/kg on a protein weight basis. In some embodiments, a dose is 10 mg/kg on a protein weight basis. In some embodiments, a dose is 11 mg/kg on a protein weight basis. In some embodiments, a dose is 12 mg/kg on a protein weight basis. In some embodiments, a dose is 13 mg/kg on a protein weight basis.
  • a dose is 14 mg/kg on a protein weight basis. In some embodiments, a dose is 15 mg/kg on a protein weight basis. In some embodiments, a dose is 16 mg/kg on a protein weight basis. In some embodiments, a dose is 17 mg/kg on a protein weight basis. In some embodiments, a dose is 18 mg/kg on a protein weight basis. In some embodiments, a dose is 19 mg/kg on a protein weight basis. In some embodiments, a dose is 20 mg/kg on a protein weight basis. In some embodiments, a dose is 21 mg/kg on a protein weight basis. In some embodiments, a dose is 22 mg/kg on a protein weight basis.
  • a dose is 23 mg/kg on a protein weight basis. In some embodiments, a dose is 24 mg/kg on a protein weight basis. In some embodiments, a dose is 25 mg/kg on a protein weight basis. In some embodiments, a dose is 26 mg/kg on a protein weight basis. In some embodiments, a dose is 27 mg/kg on a protein weight basis. In some embodiments, a dose is 28 mg/kg on a protein weight basis. In some embodiments, a dose is 29 mg/kg on a protein weight basis. In some embodiments, a dose is 30 mg/kg on a protein weight basis. In some embodiments, a dose is 31 mg/kg on a protein weight basis.
  • a dose is 32 mg/kg on a protein weight basis. In some embodiments, a dose is 33 mg/kg on a protein weight basis. In some embodiments, a dose is 34 mg/kg on a protein weight basis. In some embodiments, a dose is 35 mg/kg on a protein weight basis. In some embodiments, a dose is 36 mg/kg on a protein weight basis. In some embodiments, a dose is 37 mg/kg on a protein weight basis. In some embodiments, a dose is 38 mg/kg on a protein weight basis. In some embodiments, a dose is 39 mg/kg on a protein weight basis. In some embodiments, a dose is 40 mg/kg on a protein weight basis.
  • An exemplary dosing regimen for subjects having sepsis comprises twice daily administration of a lipid binding protein molecule (e.g., ApoA-l) for five days.
  • ApoA-l e.g., as CER-001
  • the ApoA-l is administered at a dose of 20 mg/kg on a protein weight basis.
  • the two daily doses can be administered, for example, approximately 12 hours apart. In some embodiments, two daily doses are administered 11-13 hours apart. In some embodiments, two daily doses are administered as a morning dose and an evening dose (which may be more or less than 12 hours apart).
  • the subject has septic shock.
  • the lipid binding protein molecule is administered according to an induction and, optionally, a consolidation regimen as described in Sections 6.3.1 and 6.3.2, respectively.
  • the lipid binding protein molecule can be administered in a single phase, e.g., according to an administration regimen described in this Section.
  • the subject is not treated with the lipid binding protein molecule according to a maintenance regimen, e.g., a regimen comprising long-term (e.g., one month or longer) administration of the lipid binding protein molecule.
  • the subject is treated with the lipid binding protein molecule according to a maintenance regimen, e.g., a regimen comprising long-term (e.g., one month or longer) administration of the lipid binding protein molecule, for example when the subject has a chronic condition such as Alzheimer’s disease.
  • a maintenance regimen e.g., a regimen comprising long-term (e.g., one month or longer) administration of the lipid binding protein molecule, for example when the subject has a chronic condition such as Alzheimer’s disease.
  • the lipid binding protein molecule (e.g., ApoA-l) administration regimens of the disclosure can last up to one week, one week, or more than one week (e.g., two weeks or three weeks).
  • a lipid binding protein molecule (e.g., ApoA-l) administration regimen can comprise administering: five individual doses of ApoA-l over one week; six individual doses of ApoA-l over one week; seven individual doses of ApoA-l over one week; eight individual doses of ApoA-l over one week; nine individual doses of ApoA-l over one week; ten individual doses of ApoA-l over one week; twelve individual doses of ApoA-l over one week; fourteen individual doses of ApoA-l over one week; ten individual doses of ApoA-l over two weeks; twelve individual doses of ApoA-l over two weeks; fourteen individual doses of ApoA-l over two weeks.
  • the methods of the disclosure comprise administering seven individual doses of ApoA-l over one week, e.g., on days 1 , 2, 3, 4, 5, 6, and 7.
  • the methods of the disclosure comprise administering multiple individual doses over a period of four to six days, for example one to two individual doses per day for four to six days. In some embodiments, the methods of the disclosure comprise administering multiple individual doses over four days, for example one to two individual doses per day for four days. In some embodiments, the methods of the disclosure comprise administering multiple individual doses over five days, for example one to two individual doses per day for five days. In some embodiments, the methods of the disclosure comprise administering multiple individual doses over six days, for example one to two individual doses per day for six days. In some embodiments of the methods described in this paragraph, one individual dose is administered per day.
  • two individual doses are administered per day.
  • a plurality of doses of a lipid binding protein molecule are administered no more than one day apart.
  • two or more individual doses are administered approximately 12 hours apart.
  • two individual doses are administered approximately 12 hours apart.
  • three individual doses are administered approximately 12 hours apart.
  • two individual doses are administered approximately 12 hours apart and a third individual dose is administered approximately one day later.
  • three individual doses are administered approximately 12 hours apart and a fourth individual dose is administered approximately one day later.
  • a lipid binding protein molecule (e.g., ApoA-l) is administered to a subject (e.g., over a period of 0.5 to 1 hour) at hours 0 and 12, for example at a dose of 10 mg/kg or 15 mg/kg.
  • a lipid binding protein molecule (e.g., ApoA-l) is administered to a subject (e.g., over a period of 0.5 to 1 hour) at hours 0 and 12, 24, and 48, for example at a dose of 10 mg/kg or 15 mg/kg.
  • a lipid binding protein molecule (e.g., ApoA-l) is administered daily, e.g., daily for at least 5 days, at least 6 days, at least 7 days, or more than 7 days (e.g., daily for up to one week or daily for up to two weeks).
  • a lipid binding protein molecule (e.g., ApoA-l) is administered less frequently, e.g., every other day, two times per week, three times per week, or once a week.
  • an administration window can be provided, for example, to accommodate slight variations to a multi-dosing per week dosing schedule. For example, a window of ⁇ 2 days or ⁇ 1 day around the dosage date can be used.
  • a lipid binding protein molecule (e.g., ApoA-l) can be administered in the methods of the disclosure for a pre-determined period of time, e.g. , for one week.
  • administration of a lipid binding protein molecule can be continued until one or more symptoms of a condition (are reduced or continued until the serum levels of one or more inflammatory markers are reduced, for example reduced to a normal level or reduced relative to a baseline value for the subject, e.g., a baseline value measured prior to the start of lipid binding protein molecule (e.g., ApoA-l) therapy.
  • Reference or “normal” levels of various markers for example, inflammatory markers are known in the art.
  • the Mayo Clinic Laboratories test catalog (www.mayocliniclabs.com/test-catalog) provides the following reference values: IL-6: ⁇ 1.8 pg/ml; C-reactive protein: ⁇ 8.0 mg/ml; D-dimer: ⁇ 500 ng/mL Fibrinogen Equivalent Units (FEU); ferritin: 24-336 mcg/L (males), 11-307 mcg/L (females); IL-8 ⁇ 57.8 pg/mL; TNF- a ⁇ 5.6 pg/mL; albumin: 3.5-5.0 g/dL; tryptophan: 17-80 nmol/mL; serotonin: ⁇ 330 ng/mL.
  • FEU Fibrinogen Equivalent Units
  • the methods of the disclosure typically comprise administering a high dose of a lipid binding protein molecule (e.g., ApoA-l).
  • the high dose can be the aggregate of multiple individual doses (e.g., two, three, four, five, six, seven, eight, nine or 10 individual doses), for example administered over one or multiple days (e.g., a period of one day, a period of two days, a period of three days, four days, five days, six days, seven days, eight days, nine days, 10 days, eleven days, 12 days, 13 days, 14 days, or 15 days).
  • the individual doses of a high dose are in some embodiments administered daily, twice daily, or two to three days apart.
  • the high dose is an amount effective to increase the subject’s HDL and/or ApoA-l blood levels and/or improve the subject’s vascular endothelial function, e g., measured by circulating vascular cell adhesion molecule 1 (VCAM-1) and/or intercellular adhesion molecule 1 (ICAM- 1) levels.
  • VCAM-1 circulating vascular cell adhesion molecule 1
  • IAM- 1 intercellular adhesion molecule 1
  • the high dose or an individual dose is an amount which increases the subject’s HDL and/or ApoA-l levels by at least 25%, at least 30%, or at least 35% 2 to 4 hours after administration.
  • the high dose is an amount effective to reduce serum levels of one or more inflammatory markers, for example, one or more of IL-6, C-reactive protein, D-dimer, ferritin, IL-8, GM-CSF, MCP1 , TREM-1 , or TNF-a.
  • the serum levels of the one or more inflammatory markers are reduced from an elevated range to a normal range, and/or reduced by at least 20%, at least 40%, or at least 60%.
  • the dose of a lipid binding protein molecule (e.g., ApoA-l) administered to a subject (e.g., an individual dose which when aggregated with one or more other individual doses forms a high dose) can in some embodiments range from 4 to 40 mg/kg (e.g., 10 to 40 mg/kg) on a protein weight basis (e.g., 5, 10, 15, 20, 25, 30, 35, or 40 mg/kg or any range bounded by any two of the foregoing values, e.g., 10 to 20 mg/kg, 15 to 25 mg/kg, 20 to 40 mg/kg, 25 to 35 mg/kg, or 30 to 40 mg/kg).
  • a protein weight basis e.g., 5, 10, 15, 20, 25, 30, 35, or 40 mg/kg or any range bounded by any two of the foregoing values, e.g., 10 to 20 mg/kg, 15 to 25 mg/kg, 20 to 40 mg/kg, 25 to 35 mg/kg, or 30 to 40 mg/kg.
  • protein weight basis means that a dose of a lipid binding protein molecule (e.g., ApoA-l) to be administered to a subject is calculated based upon the amount of the lipid binding protein molecule (e.g., ApoA-l) to be administered and the weight of the subject. For example, a subject who weighs 70 kg and is to receive a 20 mg/kg dose of CER-001 would receive an amount of CER-001 that provides 1400 mg of ApoA-l (70 kg x 20 mg/kg).
  • the same amount of lipid binding protein can be administered for each individual dose.
  • the amount of lipid binding protein can vary between individual doses.
  • one or more individual doses can be administered at a first dose amount and, subsequently, one or more individual doses can be administered at a different, second dose amount.
  • a first dose amount can be 5 mg/kg (on a protein weight basis) and the second dose amount can be a higher amount, for example 10 mg/kg (on a protein weight basis).
  • a first dose amount can be 10 mg/kg (on a protein weight basis) and the second dose amount can be a higher amount, for example 15 mg/kg (on a protein weight basis).
  • a first dose amount can be 10 mg/kg (on a protein weight basis) and the second dose amount can be a higher amount, for example 20 mg/kg (on a protein weight basis).
  • a first dose amount can be 15 mg/kg (on a protein weight basis) and the second dose amount can be a higher amount, for example 20 mg/kg (on a protein weight basis).
  • a first dose amount can be 20 mg/kg (on a protein weight basis) and the second dose amount can be a lower amount, for example 10 mg/kg (on a protein weight basis).
  • a first dose amount can be 20 mg/kg (on a protein weight basis) and the second dose amount can be a lower amount, for example 5 mg/kg (on a protein weight basis).
  • a first dose amount can be 10 mg/kg (on a protein weight basis) and the second dose amount can be a lower amount, for example 5 mg/kg (on a protein weight basis).
  • a lipid binding protein e.g., ApoA-l, optionally in the form of CER-001
  • each individual dose amount is the same; alternatively, different individual dose amounts can be used, for example as described in the preceding paragraph.
  • a lipid binding protein e.g., ApoA-l, optionally in the form of CER-001
  • a lipid binding protein is administered at dose of 5 mg/kg to 20 mg/kg (on a protein weight basis) one to two times a day for four days.
  • a lipid binding protein e.g., ApoA-l, optionally in the form of CER-001
  • a lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001 ) is administered at dose of 5 mg/kg to 20 mg/kg (on a protein weight basis) one to two times a day for six days.
  • the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001) is administered at dose of 5 mg/kg (on a protein weight basis) one to two times a day for four to six days.
  • the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001) is administered at dose of 5 mg/kg (on a protein weight basis) one to two times a day for four days.
  • the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001 ) is administered at dose of 5 mg/kg (on a protein weight basis) one to two times a day for five days. In some embodiments, the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001 ) is administered at dose of 5 mg/kg (on a protein weight basis) one to two times a day for six days. In some embodiments, the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001 ) is administered at dose of 10 mg/kg (on a protein weight basis) one to two times a day for four to six days.
  • the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001) is administered at dose of 10 mg/kg (on a protein weight basis) one to two times a day for four days. In some embodiments, the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001 ) is administered at dose of 10 mg/kg (on a protein weight basis) one to two times a day for five days. In some embodiments, the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001) is administered at dose of 10 mg/kg (on a protein weight basis) one to two times a day for six days.
  • the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001 ) is administered at dose of 15 mg/kg (on a protein weight basis) one to two times a day for four to six days. In some embodiments, the lipid binding protein (e.g., ApoA-l, optionally in the form of CER- 001 ) is administered at dose of 15 mg/kg (on a protein weight basis) one to two times a day for four days. In some embodiments, the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001) is administered at dose of 15 mg/kg (on a protein weight basis) one to two times a day for five days.
  • the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001) is administered at dose of 15 mg/kg (on a protein weight basis) one to two times a day for six days. In some embodiments, the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001) is administered at dose of 20 mg/kg (on a protein weight basis) one to two times a day for four to six days. In some embodiments, the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001) is administered at dose of 20 mg/kg (on a protein weight basis) one to two times a day for four days.
  • the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001 ) is administered at dose of 20 mg/kg (on a protein weight basis) one to two times a day for five days. In some embodiments, the lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001 ) is administered at dose of 20 mg/kg (on a protein weight basis) one to two times a day for six days. In some embodiments of the methods described in this paragraph, the lipid binding protein is administered once a day. In other embodiments of the methods described in this paragraph, the lipid binding protein is administered twice a day.
  • a lipid binding protein (e.g., ApoA-l, optionally in the form of CER-001) is administered to a subject according to one of the following regimens: two individual doses per day at a first dose amount (e.g., 5 mg/kg, 10 mg/kg 15 mg/kg, or 20 mg/kg) for at least 3 days, then one individual dose per day at a second dose amount (e.g., 5 mg/kg, 10 mg/kg 15 mg/kg, or 20 mg/kg) that is the same or higher than first dose amount for up to 15 days (e.g., up to 5 days, up to one week, up to 10 days, for one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days); two individual doses per day at a first dose amount (e.g., 5 mg/kg, 10 mg/kg 15 mg/kg, or 20 mg/kg
  • the first dose amount is the same as the second dose amount, e.g., in some embodiments the first and second dose amount is 5 mg/kg, 10 mg/kg, 15 mg/kg or 20 mg/kg. In other embodiments, the second dose amount is higher than the first dose amount. For example, in some embodiments, the first dose amount is 5 mg/kg and the second dose amount is 10 mg/kg, 15 mg/kg, or 20 mg/kg; the first dose amount is 10 mg/kg and the second dose amount is 15 mg/kg or 20 mg/kg; or the first dose amount is 15 mg/kg and the second dose amount is 20 mg/kg.
  • a lipid binding protein molecule e.g., ApoA-l
  • the unit dosage used in the methods of the disclosure can in some embodiments vary from 300 mg to 4000 mg (e.g., 600 mg to 4000 mg) per administration (on a protein weight basis).
  • the dosage of a lipid binding protein molecule is 300 mg to 400 mg, 300 mg to 500 mg, 300 mg to 600 mg, 300 mg to 800 mg, 300 mg to 1000 mg, 300 mg to 1200 mg, 300 mg to 1500 mg, 300 mg to 2000 mg, 300 mg to 2400 mg, 300 mg to 3000 mg, 400 mg to 500 mg, 400 mg to 600 mg, 400 mg to 800 mg, 400 mg to 1000 mg, 400 mg to 1200 mg, 400 mg to 1500 mg, 400 mg to 2000 mg, 400 mg to 2400 mg, 400 mg to 3000 mg, 400 mg to 4000 mg, 500 mg to 600 mg, 500 mg to 800 mg, 500 mg to 1000 mg, 500 mg to 1200 mg, 500 mg to 1500 mg, 500 mg to 2000 mg, 500 mg to 2400 mg, 500 mg to 3000 mg, 500 mg to 4000 mg, 600 mg to 800 mg, 600 mg to 1000 mg, 600 mg to 1200 mg, 500 mg to 1500 mg, 500 mg to 2000 mg, 500 mg to 2400 mg, 500 mg to 3000 mg, 500 mg to 4000 mg, 600 mg to 800 mg
  • a high dose of a lipid binding protein molecule e.g., ApoA-l
  • a high dose is 600 mg to 40 g (on a protein weight basis).
  • a high dose is 3 g to 35 g or 5 g to 30 g (on a protein weight basis).
  • a lipid binding protein molecule (e.g., ApoA-l) is preferably administered as an IV infusion.
  • a stock solution of CER-001 can be diluted in normal saline such as physiological saline (0.9% NaCI) to a total volume between 125 and 250 ml.
  • subjects weighing less than 80 kg will have a total volume of 125 ml whereas subjects weighing at least 80 kg will have a total volume of 250 ml.
  • doses of CER-001 are administered in a total volume of 250 ml.
  • a lipid binding protein molecule (e.g., ApoA-l) can be administered over a period ranging from one-hour to 24- hours.
  • administration can be by slow infusion with a duration of more than one hour (e.g., up to 2 hours or up to 24 hours), by rapid infusion of one hour or less, or by a single bolus injection.
  • a lipid binding protein molecule e.g., ApoA-l
  • a dose of a lipid binding protein molecule is administered as an infusion over a 24-hour period.
  • induction regimens suitable for use in the methods of the disclosure entail administering multiple doses of a lipid binding protein molecule (e.g., ApoA-l) over multiple consecutive days, e.g., three consecutive days, four consecutive days, five consecutive days, or six consecutive days.
  • lipid binding protein molecule e.g., ApoA-l
  • lipid binding protein molecule e.g., ApoA-l
  • lipid binding protein molecule e.g., ApoA-l
  • lipid binding protein molecule e.g., ApoA-l
  • induction regimens suitable for use in the methods of the disclosure entail twice daily administration of a lipid binding protein molecule (e.g., ApoA-l) such as twice daily administration on multiple consecutive days. Twice daily administration can comprise, for example, two doses approximately 12 hours apart or a morning dose and an evening dose (which may be more or less than 12 hours apart).
  • a lipid binding protein molecule e.g., ApoA-l
  • Twice daily administration can comprise, for example, two doses approximately 12 hours apart or a morning dose and an evening dose (which may be more or less than 12 hours apart).
  • the induction regimen comprises two doses of a lipid binding protein molecule (e.g., ApoA-l) per day for three consecutive days. In an embodiment, the induction regimen comprises two doses of a lipid binding protein molecule (e.g., ApoA-l) per day for four consecutive days. In an embodiment, the induction regimen comprises two doses of a lipid binding protein molecule (e.g., ApoA-l) per day for five consecutive days. In an embodiment, the induction regimen comprises two doses of a lipid binding protein molecule (e.g., ApoA-l) per day for six consecutive days.
  • a lipid binding protein molecule e.g., ApoA-l
  • a therapeutic dose of a lipid binding protein molecule (e.g., ApoA-l) administered by infusion in the induction regimen can range from 4 to 40 mg/kg (e.g., 4 to 30 mg/kg) on a protein weight basis (e.g., 4, 5, 6, 7, 8, 9, 10, 12 15, 20, 25, 30 or 40 mg/kg, or any range bounded by any two of the foregoing values, e.g., 5 to 15 mg/kg, 10 to 20 mg/kg, or 15 to 25 mg/kg).
  • the dose of a lipid binding protein molecule (e.g., ApoA-l) used in the induction regimen is 5 mg/kg.
  • the dose of a lipid binding protein molecule (e.g., ApoA-l) used in the induction regimen is 10 mg/kg. In some embodiments, the dose of a lipid binding protein molecule (e.g., ApoA-l) used in the induction regimen is 15 mg/kg. In some embodiments, the dose of a lipid binding protein molecule (e.g., ApoA-l) used in the induction regimen is 20 mg/kg. In some embodiments, the induction regimen comprises six doses of a lipid binding protein molecule (e.g., ApoA-l) administered over three days at a dose of 5 mg/kg, 10 mg/kg, 15 mg/kg or 20 mg/kg.
  • a lipid binding protein molecule e.g., ApoA-l
  • the induction regimen comprises eight doses of a lipid binding protein molecule (e.g., ApoA-l) administered over four days at a dose of 5 mg/kg, 10 mg/kg, 15 mg/kg or 20 mg/kg. In some embodiments, the induction regimen comprises ten doses of a lipid binding protein molecule (e.g., ApoA-l) administered over five days at a dose of 5 mg/kg, 10 mg/kg, 15 mg/kg or 20 mg/kg. In some embodiments, the induction regimen comprises twelve doses of a lipid binding protein molecule (e.g., ApoA-l) administered over six days at a dose of 5 mg/kg, 10 mg/kg, 15 mg/kg or 20 mg/kg.
  • a lipid binding protein molecule e.g., ApoA-l
  • a lipid binding protein molecule e.g., ApoA-l
  • the unit dosage used in the induction phase can vary from 300 mg to 4000 mg (e.g., 300 mg to 3000 mg) (on a protein weight basis) per administration by infusion.
  • the dosage of a lipid binding protein molecule used during the induction phase is 300 mg to 1500 mg, 400 mg to 1500 mg, 500 mg to 1200 mg, or 500 mg to 1000 mg (on a protein weight basis) per administration by infusion.
  • a lipid binding protein molecule e.g., ApoA-l
  • Consolidation regimens suitable for use in the methods of the disclosure entail administering one dose or multiple doses of a lipid binding protein molecule (e.g., ApoA-l) following an induction regimen.
  • the consolidation regimen comprises administering two doses of a lipid binding protein molecule (e.g., ApoA-l).
  • the two doses can be administered approximately 12 hours apart, or administered as a morning dose and an evening dose (which may be more or less than 12 hours apart).
  • the dose(s) of a lipid binding protein molecule (e.g., ApoA-l) in a consolidation regimen can in some embodiments be administered on day 6 of a dosing regimen that begins with an induction regimen on day 1.
  • the dose(s) of a lipid binding protein molecule (e.g., ApoA-l) in a consolidation regimen can in some embodiments be administered on day 4 of a dosing regimen that begins with an induction regimen on day 1.
  • the dose(s) of a lipid binding protein molecule (e.g., ApoA-l) in a consolidation regimen can in some embodiments be administered on day 5 of a dosing regimen that begins with an induction regimen on day 1.
  • the dose(s) of a lipid binding protein molecule (e.g., ApoA-l) in a consolidation regimen can in some embodiments be administered on day 7 of a dosing regimen that begins with an induction regimen on day 1 .
  • a consolidation regimen comprises once daily administration of a lipid binding protein molecule (e.g., ApoA-l) following an induction regimen that comprises twice daily administration of the lipid binding protein molecule (e.g., ApoA-l).
  • Each individual dose of the consolidation regimen can be the same or higher than each individual dose of the induction regimen.
  • the lipid binding protein molecule e.g., ApoA-l
  • the lipid binding protein molecule can be administered once daily for up to 15 days (e.g., up to seven days, up to 10 days, one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days).
  • a therapeutic dose of a lipid binding protein molecule (e.g., ApoA-l) administered by infusion in the consolidation regimen can range from 4 mg/kg to 40 mg/kg (e.g., 4 to 30 mg/kg) on a protein weight basis (e.g., 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, or 40 mg/kg, or any range bounded by any two of the foregoing values, e.g., 5 to 15 mg/kg, 10 to 20 mg/kg, or 15 to 25 mg/kg).
  • the dose of a lipid binding protein molecule (e.g., ApoA-l) used in the consolidation regimen is 5 mg/kg.
  • the dose of a lipid binding protein molecule (e.g., ApoA-l) used in the consolidation regimen is 10 mg/kg. In some embodiments, the dose of a lipid binding protein molecule (e.g., ApoA-l) in the consolidation regimen is 15 mg/kg. In some embodiments, the dose of a lipid binding protein molecule (e.g., ApoA-l) used in the consolidation regimen is 20 mg/kg. In some embodiments, the consolidation regimen comprises two doses of a lipid binding protein molecule (e.g., ApoA-l) administered on one day at a dose of 5 mg/kg, 10 mg/kg, 15 mg/kg or 20 mg/kg.
  • a lipid binding protein molecule (e.g., ApoA-l) can be administered on a unit dosage basis.
  • the unit dosage used in the consolidation phase can vary from 300 mg to 4000 mg (e.g., 300 mg to 3000 mg) (on a protein weight basis) per administration by infusion.
  • the dosage of a lipid binding protein molecule used during the consolidation phase is 300 mg to 1500 mg, 400 mg to 1500 mg, 500 mg to 1200 mg, or 500 mg to 1000 mg (on a protein weight basis) per administration by infusion.
  • a lipid binding protein molecule e.g., ApoA-l
  • the lipid binding protein molecule (e.g., ApoA-l) can be administered during the consolidation phase in the same manner as described in Section 6.3, e.g., as an IV infusion over a one-hour period.
  • a lipid binding protein molecule (e.g., ApoA-l) can be administered to a subject as described herein as a monotherapy or a part of a combination therapy regimen.
  • a combination therapy can comprise a lipid binding protein molecule (e.g., ApoA-l) in combination with a standard of care treatment for the condition from which the subject suffers or is at risk of suffering, e.g., sepsis and/or AKI. See, e.g., Rhodes et al., 2017, Intensive Care Med 43:304-377; Dugar et al., 2020, Cleveland Clinic Journal of Medicine 87(1 ):53-64; Singer et al., 2016, JAMA 315(8):801-810.
  • the subject is treated with a lipid binding protein molecule (e.g., ApoA-l) in combination with fluid replacement therapy.
  • a lipid binding protein molecule e.g., ApoA-l
  • an antimicrobial e.g., an antimicrobial.
  • the subject is treated with a lipid binding protein molecule (e.g., ApoA-l) in combination with an antibiotic (e.g., ceftriaxone, meropenem, ceftazidime, cefotaxime, cefepime, piperacillin and tazobactam, ampicillin and sulbactam, imipenem and cilastatin, levofloxacin, or clindamycin).
  • an antibiotic e.g., ceftriaxone, meropenem, ceftazidime, cefotaxime, cefepime, piperacillin and tazobactam, ampicillin and sulbactam, imipenem and cilastatin, levofloxacin, or clindamycin.
  • an antiviral e.g., ceftriaxone, meropenem, ceftazidime, cefotaxime, cefepime, piperacill
  • the subject is treated with a lipid binding protein molecule (e.g., ApoA-l) in combination with a medication that raises blood pressure (e.g., norepinephrine or epinephrine).
  • a medication that raises blood pressure e.g., norepinephrine or epinephrine.
  • the subject is treated with a lipid binding protein molecule (e.g., ApoA-l) in combination with an immunosuppressant such as tacrolimus or everolimus.
  • a combination therapy regimen can in some embodiments comprise one or more anti-IL-6 agents and/or one or more other agents for treating CRS such as corticosteroids (e.g., methylprednisolone and/or dexamethasone).
  • exemplary anti-IL6 agents include tocilizumab, siltuximab, olokizumab, elsilimomab, BMS-945429, sirukumab, levilimab, and CPSI-2364.
  • a lipid binding protein molecule e.g., ApoA-l is administered in combination with tocilizumab.
  • an antihistamine e.g., diphenhydramine, cetirizine, fexofenadine, or loratadine
  • a lipid binding protein molecule e.g., ApoA-l
  • the antihistamine can reduce the likelihood of allergic reactions.
  • Example 1 Lipid binding protein molecule therapy in a swine model of LPS- induced acute kidney injury
  • Sepsis was induced in pigs by intravenous infusion of a saline solution containing 300 pg/kg of LPS at TO.
  • Single dose CER-001 treated pigs and CER-001 multiple dose treated pigs received a 20 mg/kg dose of CER-001 at TO.
  • CER-001 multiple dose treated pigs received a second 20 mg/kg dose of CER-001 three hours later (T3).
  • Quinolinic acid, kynurenic acid, tryptophan (Trp), and kynurenine (Kyn) levels and Kyn:Trp ratios were monitored over time.
  • n 3. Animals were humanely sacrificed. Brain tissue was extracted and qPCR performed on mRNA encoding aromatic-L-amino-acid/L-tryptophan decarboxylase (DDC); indoleamine 2,3-dioxygenase 1 (IDO1); interleukin-6 (IL-6); kynurenine 3-monooxygenase (KMO); kynurenine formamidase isoform X1 (AFMID); and kynurenine-oxoglutarate transaminase 3 (KYAT3).
  • DDC aromatic-L-amino-acid/L-tryptophan decarboxylase
  • IDO1 indoleamine 2,3-dioxygenase 1
  • IL-6 interleukin-6
  • KMO kynurenine 3-monooxygenase
  • AFMID kynurenine formamidase isoform X1
  • KYAT3
  • FIG. 1A LPS injection led to a time-dependent increase of quinolinic acid in endotoxemic animals (FIG. 1A, FIG. 1B) compared to the basal condition (TO).
  • CER-001 treatment was able to reverse LPS effects, as shown by essentially unchanged quinolinic acid levels, both at one dose of 20 mg/kg (FIG. 1 A, “20 mg”) and two doses of 20 mg/kg each, 40 mg/kg total (FIG. 1B, “40 mg”).
  • One of the three pigs in the 20 mg/kg group had a time dependent increase in quinolinic acid at T3 and T6. All ten pigs in the experimental groups had quinolinic acid levels at the end of the experiment that were essentially at baseline.
  • FIG. 1A endotoxemic animals
  • TO basal condition
  • FIG. 2C graphically summarizes the results observed for the three groups. *, p ⁇ 0.05 vs LPS.
  • LPS injection also led to a time-dependent increase of kynurenic acid in two of three endotoxemic animals (FIG. 2A, FIG. 2B) compared to the basal condition (TO).
  • CER-001 treatment was able to reverse LPS effects, as shown by essentially unchanged kynurenic acid levels, both at one dose of 20 mg/kg (FIG. 2A, “20 mg”) and two doses of 20 mg/kg each, 40 mg/kg total (FIG. 2B, “40 mg”). All ten pigs in the experimental groups had kynurenic acid levels at the end of the experiment that were essentially at baseline.
  • FIG. 2C graphically summarizes the results observed for the three groups. *, p ⁇ 0.05 vs LPS for the 40 mg group.
  • FIG. 3A LPS injection also led to a time-dependent decrease of Trp in endotoxemic animals (FIG. 3A, FIG. 3B) compared to the basal condition (TO).
  • CER-001 treatment was able to reverse LPS effects, as shown by either no decrease or an increase in Trp levels, both for the 20 mg/kg group (FIG. 3A, “20 mg”) and the 2x20 mg/kg group (FIG. 3B, “40 mg”). All ten pigs in the experimental groups had Trp levels at the end of the experiment that were essentially at or above baseline.
  • FIG. 3C graphically summarizes the results observed for the three groups. *, p ⁇ 0.05 vs LPS for the 20 mg group; **, p ⁇ 0.-05 vs LPS for the 40 mg group.
  • FIG. 4A LPS injection also led to a time-dependent increase of Kyn in endotoxemic animals (FIG. 4A, FIG. 4B) compared to the basal condition (TO).
  • CER-001 treatment was able to reverse LPS effects, as shown by essentially unchanged Kyn levels for some pigs, both for the 20 mg/kg group (FIG. 4A, “20 mg,” 1 of 2 animals) and the 2x20 mg/kg group (FIG. 4B, “40 mg”, 5 of 6 animals).
  • FIG. 4C graphically summarizes the results observed for the three groups.
  • FIG. 5A summarizes Kyn:Trp ratios observed for the three groups. *, p ⁇ 0.05 vs the 40 mg group for the 20 mg group; **, p ⁇ 0.005 vs LPS.
  • FIG. 6A shows fold gene expression (2 -Aact ) of indoleamine 2,3-dioxygenase 1 (IDO1 ) in brain tissue relative to the housekeeping gene, as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg.
  • IDO1 catalyzes the conversion of tryptophan to formylkynurenine.
  • brain tissue from the LPS cohort roughly 2-fold greater expression of IDO1 was seen relative to the housekeeping gene, compared to essentially unchanged expression of IDO1 in the 20 mg and 40 mg CER-001 cohorts.
  • FIG. 6B shows relative fold gene expression of aromatic-L-amino-acid/L-tryptophan decarboxylase (DDC) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg. DDC catalyzes the conversion of tryptophan to serotonin. CER-001 led to increased expression of DDC in both test groups. *, p ⁇ 0.05 vs. LPS for 40 mg group.
  • FIG. 6B shows relative fold gene expression of aromatic-L-amino-acid/L-tryptophan decarboxylase (DDC) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg. DDC catalyzes the conversion of tryptophan to serotonin. CER-001 led to increased expression of DDC in both test groups. *, p ⁇ 0.05 vs. LPS for
  • 6C shows relative fold gene expression of kynurenine formamidase isoform X1 (AFMID) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg.
  • AFMID catalyzes the conversion of formylkynurenine to kynurenine.
  • CER-001 partially reversed an increase in AFMID expression induced by LPS.
  • FIG. 6D shows relative fold gene expression of kynurenine 3-monooxygenase (KMO) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg.
  • KMO catalyzes the conversion of kynurenine to 3-hydroxykynurenine.
  • CER-001 partially reversed an increase in KMO expression induced by LPS. . *, p ⁇ 0.05 vs. LPS for 40 mg group.
  • FIG. 6E shows relative fold gene expression of kynurenine-oxoglutarate transaminase 3 (KYAT3) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20 mg, and 40 mg.
  • KYAT3 catalyzes the conversion of 3-hydroxykynurenine to xanthurenic acid.
  • CER-001 partially reversed an increase in KYAT3 expression induced by LPS.
  • FIG. 6F shows relative fold gene expression of interleukin-6 (IL-6) in brain tissue as determined by qPCR for cohorts from all three groups of endotoxemic pigs: LPS, 20g, and 40 mg.
  • CER-001 partially reversed an increase in IL-6 expression induced by LPS.
  • Kynurenine pathway activity is associated with various diseases and conditions, including sepsis-induced cognitive deficiency (“brain fog”).
  • Example 2 Randomized pilot study comparing short-term CER-001 infusions at different doses to prevent sepsis-induced acute kidney injury
  • the study reported in this example included 20 patients with gram-negative sepsis who were at high risk for acute kidney injury due to high levels of endotoxin activity and decline in function of one or more organ systems. Patients received either standard of care treatment alone, or in combination with one of three dosage regimens of CER-001 (five patients per group), to investigate whether the use of CER-001 at different doses, in combination with standard of care (SOC) treatment, was safe and effective, providing a potential new strategy to treat septic patients, reducing the inflammatory response to endotoxin and preventing the progression to AKI according to KDIGO (Kidney Disease: Improving Global Outcomes) criteria, as well as safety and tolerability of the dosage regimens in order to select the optimal dose of CER-001 .
  • SOC standard of care
  • One of the metabolic characteristics of bacterial (like sepsis) or virus infections (like SARS-CoV- 2) is the strong decrease of circulating lipoprotein and particularly the High-Density Lipoprotein (HDL) with its main containing protein apolipoprotein A-l (ApoA-l).
  • HDL High-Density Lipoprotein
  • ApoA-l level was recently described as the biomarker predictive of long-term mortality after surgical sepsis.
  • Study population This was a single-center, randomized, dose-ranging (phase II) study including patients with sepsis due to intra-abdominal cavity infection or urosepsis, admitted at the Intensive Care Unit (ICU) of the participating center. The investigators ensured that all patients meeting the following inclusion and exclusion criteria were offered enrollment in the study.
  • ICU Intensive Care Unit
  • Endotoxin level (measured by Endotoxin Activity Assay (EEATM); Spectral Medical) >0.6 (see, Marshall et al., 2004, J Infect Dis. 190(3):527-34);
  • ALT/AST Alanine transaminase/aspartate transaminase
  • Stage 4 severe chronic kidney disease or requiring dialysis i.e. estimated glomerular filtration rate (eGFR) ⁇ 30 ml /min/1.73 m 2 );
  • the subject populations had the following baseline clinical and demographic characteristics. [0571] Duration of study. This study was completed in 24 weeks (6 months). The enrolment period was approximately 20 weeks (5 months) from the first subject enrolled. The end of the study was the last visit of the last subject.
  • Baseline is defined as the last measurements taken prior to dosing on Day 1 .
  • Intervention/exposure Twenty patients meeting the eligibility criteria, who signed and dated an ethical committee (EC)-approved informed consent form, were randomized and assigned (1 :1 :1 :1 ) ratio to conventional therapy (Group A), low dose CER-001 (Group B) or medium dose CER-001 (Group C) or high dose CER-001 (Group D). Conventional therapy was modulated according to the clinical conditions. All non-experimental treatments were allowed to be administered concomitantly during the patient’s participation in this study: any medication the patient took, other than study drugs specified per protocol, was considered a concomitant medication and was recorded in the study records.
  • Each patient was identified at the screening by a patient number. Once assigned to a patient, the patient number was not reused. The investigators that enrolled patients did not participate in randomization and allocation assignment. The randomization list divided into blocks was adequately concealed to prevent attempts at subversion of randomization.
  • Treatment group All patients received conventional therapy. Treated groups received an additional therapy with the study drugs. In particular:
  • Group A Conventional therapy (i.e., antibiotic treatments and hemodynamic support according to patient’s conditions).
  • Group B Conventional therapy + CER-001 5 mg/kg BID for 3 consecutive days, followed by 5 mg/kg BID on Day 6.
  • Group C Conventional therapy + CER-001 10 mg/kg BID for 3 consecutive days, followed by 10 mg/kg BID on Day 6.
  • Group D Conventional therapy + CER-001 20 mg/kg BID for 3 consecutive days, followed by 20 mg/kg BID on Day 6. [0577]
  • FIG. 7 summarizes the study regimen.
  • Procedures The following procedures were performed during the screening visit. Following randomization, subjects initiated treatment within 2 business days.
  • Medical history included: recording past and present illnesses and collection of the subject’s demographic data (birth date, sex, and race).
  • CBC Complete blood count
  • WBC white blood cell count
  • RBC red blood cell count
  • Hb hemoglobin
  • Het hematocrit
  • Fasting chemistry panel/electrolytes included sodium, potassium, chloride, blood urea nitrogen (BUN; or urea), serum creatinine, calculated clearance creatinine (CKD-EPI), glucose, calcium, phosphorus, total protein, uric acid, AST, ALT, y GT, ALP, total and direct bilirubin, albumin, total cholesterol, HDL, LDL, triglycerides, LDH, CPK,
  • ABG for assessing respiratory and/or metabolic disorders
  • PT prothrombin time
  • I NR international normalized ratio
  • PTT partial thromboplastin time
  • Urinalysis included specific gravity, pH, assessment of protein/albumin, glucose, ketones, and hemoglobin/blood.
  • Serum or urine pregnancy test for women of childbearing potential within 7 days before randomization.
  • Pharmacokinetic and pharmacodynamic assessment included ApoA-l and total cholesterol levels. Endotoxin levels were measured using the EAATM kit. AKI Biomarkers (TIMP-2 and IGFBP-7) are measured using the Nephrocheck® kit. Inflammatory markers include: CRP, D-dimer, Ferritin, IL-6, IL-8, GM-CSF, MOP 1 and TNF-a.
  • biological samples collected for the daily routine laboratory assessments performed at the Central laboratory were collected, including:
  • Treatment period was defined as from the start of treatment. The visit was planned at Day 3, Day 6 and Day 9. A final visit was planned on Day 30. The following procedures were performed during the therapy visits:
  • CBC Complete blood count
  • WBC white blood cell count
  • RBC red blood cell count
  • Hb hemoglobin
  • Het hematocrit
  • Fasting chemistry panel/electrolytes includes sodium, potassium, chloride, blood urea nitrogen (BUN; or urea), serum creatinine, calculated clearance creatinine (CKD-EPI), glucose, calcium, phosphorus, total protein, uric acid, AST, ALT, DGT, ALP, total and direct bilirubin, albumin, total cholesterol, HDL, LDL, triglycerides, LDH, CPK
  • ABG for assessing respiratory and/or metabolic disorders
  • PT prothrombin time
  • I NR international normalized ratio
  • PTT partial thromboplastin time
  • Urinalysis included specific gravity, pH, assessment of protein/albumin, glucose, ketones, and hemoglobin/blood.
  • Serum or urine pregnancy test for women of childbearing potential within 7 days before randomization.
  • Pharmacokinetic and pharmacodynamic assessment included ApoA-l and total cholesterol levels. Endotoxin levels were measured using the EAATM kit. AKI Biomarkers (TIMP-2 and IGFBP-7) were measured using the Nephrocheck® kit. Inflammatory markers included: CRP, D-dimer, Ferritin, IL- 6, IL-8, GM-CSF, MOP 1 and TNF-a.
  • Clinical scores included the SOFA score (Table 2) and the KDIGO criteria for AKI assessment and staging (Table 3). Individual components of each score were documented.
  • Table 4 provides a summary of the study protocol of this Example.
  • Safety evaluations were attained utilizing information collected from the following assessments: physical examination (including weight), vital signs (blood pressure, pulse, temperature), CBC with differential, platelet count, blood chemistries, and fasting lipid profiles [including HDL-cholesterol, LDL-cholesterol and Lipoprotein (a) ], urea, glucose, 24 hour urine protein determination, serum creatinine and calculated creatinine clearance (CKD-EPI) and adverse events monitoring. All women of childbearing potential had a qualitative serum pregnancy test during pre-study screening/baseline evaluation and subsequently, if clinically indicated. Patients were monitored throughout the study for the occurrence of adverse events, that were recorded.
  • Adverse events volunteered by the subject or discovered as a result of general questioning by the investigator or by physical examination were recorded. The duration (start and end dates), severity, cause and relationship to study medication, patient outcome, action taken, and an assessment of whether the event was serious were recorded for each reported adverse event.
  • FIG. 8A-FIG. 8F show lipopolysaccharide (LPS) changes for the standard of care group (Group A) and the experimental groups (Groups B-D).
  • FIG. 8A LPS changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 8B LPS changes from baseline for Group A, Group B, Group C, and Group D.
  • the treatment x study day effect relative to peak was p ⁇ 0.0005.
  • FIG. 8D LPS changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 8E LPS changes from baseline for each subject in Group A and aggregated Groups B-D.
  • FIG. 8F LPS changes from baseline for each subject in each of Groups A-D. LPS levels were measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 9A-FIG. 9D show endotoxin activity assay (EAA) changes for the standard of care group (Group A) and the experimental groups (Groups B-D).
  • FIG. 9A EAA changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 9B EAA changes from baseline for Group A, Group B, Group C, and Group D.
  • the treatment x study day effect relative to peak was p ⁇ 0.1769.
  • FIG. 9A-FIG. 9D show endotoxin activity assay (EAA) changes for the standard of care group (Group A) and the experimental groups (Groups B-D).
  • FIG. 9A EAA changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 9B EAA changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 9C EAA changes
  • FIG. 9D EAA changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 9E EAA changes from baseline for Group A and aggregated Groups B-D. EAA was performed at each timepoint using commercial kits (Spectral Medical, Toronto, Canada). Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 10A-FIG. 10F show TNF-a changes for the standard of care group (Group A) and the experimental groups (Groups B-D) measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 10A TNF-a changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 10B TNF-a changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 10A-FIG. 10F show TNF-a changes for the standard of care group (Group A) and the experimental groups (Groups B-D) measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 10A TNF
  • FIG. 10D TNF-a changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 10E TNF-a changes from baseline for each subject in Group A and aggregated Groups B-D.
  • FIG. 10F TNF-a changes from baseline for each subject in each of Groups A-D.
  • FIG. 11A-FIG. 11F show MCP-1 changes for the standard of care group (Group A) and the experimental groups (Groups B-D) measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 11 A MCP-1 changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 11B MCP-1 changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 11 A MCP-1 changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 11 D MCP-1 changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 11E MCP-1 changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 11F MCP-1 changes from baseline for each subject in each of Groups A-D.
  • FIG. 12A-FIG. 12F show IL-6 changes for the standard of care group (Group A) and the experimental groups (Groups B-D) measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 12A IL-6 changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 12B IL-6 changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 12A IL-6 changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 12D IL-6 changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 12E IL-6 changes from baseline for each subject in Group A and aggregated Groups B-D.
  • FIG. 12F IL-6 changes from baseline for each subject in each of Groups A-D.
  • FIG. 13A-FIG. 13F show IL-8 changes for the standard of care group (Group A) and the experimental groups (Groups B-D).
  • FIG. 13A IL-8 changes from baseline for Group A and aggregated Groups B-D measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 13B IL-8 changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 13A IL-8 changes from baseline for Group A and aggregated Groups B-D measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 13B IL-8 changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 13D IL-8 changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 13E IL-8 changes from baseline for each subject in Group A and aggregated Groups B-D.
  • FIG. 13F IL-8 changes from baseline for each subject in each of Groups A-D.
  • FIG. 14A-FIG. 14D show IL-10 changes for the standard of care group (Group A) and the experimental groups (Groups B-D) measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 14A IL-10 changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 14B IL-10 changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 14D IL-10 changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 15A-FIG. 15F show TREM-1 changes for the standard of care group (Group A) and the experimental groups (Groups B-D) measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 15A TREM-1 changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 15B TREM-1 changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 15A-FIG. 15F show TREM-1 changes for the standard of care group (Group A) and the experimental groups (Groups B-D) measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 15A TREM-1 changes from baseline for Group
  • FIG. 15E TREM-1 changes from baseline for each subject in Group A and aggregated Groups B-D.
  • FIG. 15F TREM-1 changes from baseline for each subject in each of Groups A-D.
  • FIG. 16A-FIG. 16F show VCAM changes for the standard of care group (Group A) and the experimental groups (Groups B-D) measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 16A VCAM changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 16B VCAM changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 16A-FIG. 16F show VCAM changes for the standard of care group (Group A) and the experimental groups (Groups B-D) measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 16A VCAM changes from baseline for Group A and aggregated Group
  • FIG. 16D VCAM changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 16E VCAM changes from baseline for each subject in Group A and aggregated Groups B-D.
  • FIG. 16F VCAM changes from baseline for each subject in each of Groups A-D.
  • FIG. 17A-FIG. 17F show ICAM changes for the standard of care group (Group A) and the experimental groups (Groups B-D) measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 17A ICAM changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 17B ICAM changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 17A-FIG. 17F show ICAM changes for the standard of care group (Group A) and the experimental groups (Groups B-D) measured by ELISA. Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 17A ICAM changes from baseline for Group A and aggregated Group
  • FIG. 17D ICAM changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 17E ICAM changes from baseline for each subject in Group A and aggregated Groups B-D.
  • FIG. 17F ICAM changes from baseline for each subject in each of Groups A-D.
  • FIG. 18A-FIG. 18D show ferritin changes for the standard of care group (Group A) and the experimental groups (Groups B-D).
  • FIG. 18A ferritin changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 18B ferritin changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 18D ferritin changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 19A-FIG. 19D show white blood cell count changes for the standard of care group (Group A) and the experimental groups (Groups B-D).
  • FIG. 19A white blood cell count changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 19B white blood cell count changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 19D white blood cell count changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 20A-FIG. 20F show CRP changes for the standard of care group (Group A) and the experimental groups (Groups B-D). Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 20A CRP changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 20B CRP changes from baseline for Group A, Group B, Group C, and Group D.
  • the treatment x study day effect relative to peak was p ⁇ 0.6446.
  • FIG. 20D CRP changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 20E CRP changes from baseline for each subject in Group A and aggregated Groups B-D.
  • FIG. 20F CRP changes from baseline for each subject in each of Groups A-D.
  • FIG. 21A-FIG. 21D show KIM-1 changes for the standard of care group (Group A) and the experimental groups (Groups B-D).
  • FIG. 21A KIM-1 changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 21 B KIM-1 changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 21 D KIM-1 changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 22A-FIG. 22D show serum albumin changes for the standard of care group (Group A) and the experimental groups (Groups B-D).
  • FIG. 22A serum albumin changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 22B serum albumin changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 22D serum albumin changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 22E serum albumin changes from baseline for each subject in Group A and aggregated Groups B-D.
  • treatment regimens providing CER-001 in addition to the SOC raised serum albumin more than the SOC alone.
  • FIG. 23A-FIG. 23F show serum creatinine changes for the standard of care group (Group A) and the experimental groups (Groups B-D).
  • FIG. 23A serum creatinine changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 23B serum creatinine changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 23D serum creatinine changes for Group A and aggregated Groups B-D, broken out by whether the subject was enrolled from the ICU or the nephrology department of the center.
  • FIG. 23E area under the curve (AUG) (mean ⁇ SEM) for serum creatinine for Group A and aggregated Groups B-D for all subjects, and subject populations from ICU and nephrology intake routes.
  • FIG. 23F AUC (95% confidence interval) for serum creatinine for Group A and aggregated Groups B-D, and subject populations from ICU and nephrology intake routes..
  • FIG. 24A-FIG. 24B show eGFR changes for all subjects in the standard of care group (Group A) and the experimental groups (Groups B-D). Estimated GFR was determined by CKD-EPI.
  • FIG. 24A eGFR changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 24B eGFR changes from baseline for Group A, Group B, Group C, and Group D.
  • FIG. 24C-FIG. 24D show eGFR changes for subjects entering the study with AKI.
  • FIG. 24C eGFR changes from baseline for Group A and aggregated Groups B-D.
  • FIG. 24D eGFR changes for Group A, Group B, Group C, and Group D.
  • FIG. 25 shows changes in the P/F ratio for all subjects in the standard of care group (Group A) and aggregated Groups B-D.
  • FIG. 26 shows survival proportions for all subjects after days in ICU for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”).
  • FIG. 27A shows 30-day survival proportions for all subjects for the standard of care group (Group A, “SOC’’) and aggregated Groups B-D (“CER-001”).
  • FIG. 27B shows 30-day survival proportions for all subjects who entered the study from the center's ICU, for the standard of care group (Group A, “SOC”) and aggregated Groups B-D (“CER-001”).
  • SOC standard of care group
  • CER-001 aggregated Groups B-D
  • FIG. 28A shows the evolution of AKI, as assessed by KDIGO staging criteria, for all subjects in the standard of care group (Group A, “SOC”).
  • AKI stages 0, serum creatinine ⁇ 1 .5x baseline or increased by less than 0.3 mg/dl within 48 hours, and urine volume > 0.5 ml/kg/h for 6-12 hours; 1, serum creatinine from 1 .5 to 1 .9x baseline or increased by more than 0.3 mg/dl, or urine volume ⁇ 0.5 ml/kg/h for 6-12 hours; 2, serum creatinine from 2.0 to 2.9x baseline or urine volume ⁇ 0.5 ml/kg/h for more than 12 hours; 3, serum creatinine 3.
  • FIG. 28B shows the evolution of AKI, as assessed by KDIGO staging criteria, for all subjects in aggregated Groups B-D (“CER-001”). About 60% of CER-001 subjects were at AKI 0 and 20% were at AKI 3 at Day 6.
  • FIG. 29 shows the number of days on mechanical ventilation for all subjects who entered into the study while in the center’s ICU, for the standard of care group (Group A, SOC) and aggregated Groups B-D (CER-001 ) CER-001 reduced the number of days on mechanical ventilation relative to SOC for 5/7 subject.
  • FIG. 30 shows the number of days on vasopressor for all subjects who entered into the study while in the center's ICU, for the standard of care group (Group A, SOC) and aggregated Groups B-D (CER-001).
  • FIG. 31 A shows the number of days on dialysis for all subjects who entered into the study while in the center’s ICU, for the standard of care group (Group A) and aggregated Groups B-D.
  • FIG. 31 B shows this result for all subjects. Both intermittent and continuous modalities were considered. A reduction in the number of days of dialysis is indicative of improved kidney function. 7.2.2.23. Days Alive Without Organ Support, Days Until ICU Discharge, and Changes in Hemodynamics
  • FIG. 32 shows the number of days alive without organ support for all subjects who entered into the study while in the center’s ICU, for the standard of care group (SOC) and aggregated Groups B-D (CER-001). Any use of vasopressors, mechanical ventilation, and/or renal support was considered organ support.
  • SOC standard of care group
  • CER-001 aggregated Groups B-D
  • FIG. 65 shows days until ICU discharge for all subjects who entered the study from the center’s ICU, for the standard of care group (SOC) and aggregated Groups B-D (CER-001).
  • SOC standard of care group
  • CER-001 aggregated Groups B-D
  • FIG. 33A and FIG. 33B show changes in daily average mean arterial pressure (MAP) for all subjects who entered into the study while in the center’s ICU, for the standard of care group (SOC) and aggregated Groups B-D (CER-001). Decreases in MAP are generally desirable for ICU subjects.
  • SOC standard of care group
  • CER-001 aggregated Groups B-D
  • FIG. 34 shows the change in daily average heart rate (HR) for all subjects who entered into the study while in the center’s ICU, for the standard of care group (SOC) and aggregated Groups B-D (CER- 001 ). Decreases in MAP are generally desirable for ICU subjects.
  • HR daily average heart rate
  • FIG. 35 shows the change in daily average P/F ratio for all subjects who entered into the study while in the center's ICU, for the standard of care group (SOC) and aggregated Groups B-D (CER-001). Increases in P/F ratio are generally desirable for ICU subjects.
  • SOC standard of care group
  • CER-001 aggregated Groups B-D
  • FIG. 56A shows mean ApoA-l levels for the control group and the aggregated study groups in the clinical study of Example 2.
  • FIG. 56 shows mean ApoA-l levels for the control group and the aggregated study group
  • FIG. 56B shows mean ApoA-l levels for the control group and each study group
  • FIG. 56 shows ApoA-l changes for each subject in the standard of care group (SOC) and the experimental groups (CER-001 )
  • FIG. 56 shows changes from baseline of ApoA-l levels for each subject broken out by study group, as measured by ELISA.
  • Serum ApoA-l levels rapidly increased in the first 3 days of treatment in patients receiving CER-001 , while the increase in the SOC group was delayed.
  • Statistically significant differences were assessed by using a mixed model ANOVA (ns: p>0.05).
  • FIG. 65 shows changes in serum quinolinic acid (QA) levels from baseline (day 1) for subjects in the standard of care (SOC) group (Group A) and aggregated Groups B-D in the clinical study of Example 2.
  • SOC standard of care
  • FIG. 67 shows changes in serum kynurenine/tryptophan ratios (Kyn/Trp) levels from baseline (day 1 ) for subjects in the standard of care (SOC) group (Group A) and aggregated Groups B-D in the clinical study of Example 2.
  • FIG. 68 shows changes in serum serotonin levels from baseline (day 1 ) for subjects in the standard of care (SOC) group (Group A) and aggregated Groups B-D in the clinical study of Example 2. 7.2.2.26. Additional Results
  • liver enzymes changed by anywhere from about 0.2-fold to about 4-fold of baseline levels after 9 days (FIG. 57A, FIG. 57B). Subjects receiving CER-001 had comparable changes to liver enzyme levels as the group receiving standard of care only.
  • CER-001 Recently the anti-inflammatory capacity of CER-001 was highlighted in a severe COVID-19 patient in ICU where assessment of serum amyloid A-1, inflammatory markers, and cytokines showed predominantly significant decreases during CER-001 infusion (Begue, et al., 2021 , Sci Rep 11 , 2291). Similarly, the data of this Example showed that CER-001 treatment induced a significant reduction of serum levels of MCP-1 , TNF-a, IL-6 and IL-8 in treated-patients compared to SOC group (FIG. 11E, FIG. 10E, FIG. 12E, and FIG. 13E), suggesting the immunomodulatory and anti-inflammatory effects of CER- 001 treatment and its ability to modulate the cytokine storm. Consistent with modulating the cytokine storm, a more pronounced reduction in C-reactive protein (CRP) in the first 9 days in the treated group compared to the SOC group was shown (FIG. 20E).
  • CRP C-reactive protein
  • Soluble triggering receptor expressed on myeloid cells- 1 has been suggested as a strong predictor for poor prognosis and poor survival in septic patients. Persistently high sTREM-1 levels during the first days following ICU admission are associated with mortality in human septic shock (Jolly, et al., 2021 , Cell Mol Immunol 18, 2054-2056). The results of this Example suggest that sTREM-1 rapidly decreases with CER-001 treatment within the first 3 days and remains low and stable through at least 30 days (FIG. 15E). More importantly, this decrease is accompanied by an amelioration of clinical signs and symptoms.
  • FIG. 68 shows serum serotonin levels remained near baseline for subjects in the CER-001 groups during the entire study period.
  • the SOC group experienced a sizable drop of about 40 ng/mL in serum serotonin at day 6 and did not return to baseline levels even by the end of the study period.
  • a subset of critically ill patients enrolled in the ICU were focused on to analyze the main clinical outcomes. Although the small sample size (7 treated patients; 2 SOC subjects) limits statistical evaluation, a reduced length of ICU stay among patients in the treatment group was observed (mean days of ICU stay 23.2 vs 29) (FIG. 65). In addition, the daily average mean arterial pressure (MAP) during the study period improved after the second day of treatment as compared to SOC subjects with an overall lower days on vasopressors (mean days 6.5 vs 8 in the SOC group) (FIG. 33B, FIG. 30).
  • MAP mean arterial pressure
  • Example 3 Lipid binding protein molecule therapy in a swine model of LPS- induced acute kidney injury
  • endotoxemia was induced by intravenous infusion of a saline solution containing 300pg/kg of LPS (lipopolysaccharide membrane of Escherichia coli). Left untreated, the swine progress to LPS-induced AKI, as previously described (Sallustio, et al., 2019, FASEB J 33, 10753-10766). The goal of the study was to determine if treatment with CER-001 could prevent AKI from developing.
  • LPS lipopolysaccharide membrane of Escherichia coli
  • the CER20 group was treated with CER-001 infusion through isolated venous access.
  • the CER20x2 group was treated by administering two doses of CER-001 (20mg/kg) through the previously isolated venous access. The first dose was administered a few minutes after the start of the LPS infusion (TO); the second dose was administered 3h after the start of the LPS infusion (T3/T0 bis).
  • the drug product was thawed and then diluted with normal saline to a volume of 250 mL containing 20 mg/kg of CER-001 , individualized for each animal based on weight, and was administered over a period of one hour using an infusion pump at a fixed rate of 250 nnL/hr.
  • the LPS group received 250 ml of normal saline solution at the same infusion rate.
  • CER-001 The dose of CER-001 is defined as the human ApoA-l concentration present in the dosing solution. Surviving animals were sacrificed after approximately 24 hours from LPS/saline infusion with an overdose of IV propofol, immediately followed by a 10-ml IV bolus of an oversaturated solution of potassium chloride (2 mEq/ml, Galenica Senese, srl, Italy).
  • kidneys and livers were collected from all animals and processed using standard procedures as previously described (Sallustio, et al., 2019, FASEB J 33, 10753-10766).
  • Urine samples were collected via catheter from all animals and urinary output was recorded every hour. Swine sera were collected at baseline (TO; before LPS infusion), and at intermediate time points up to 24h from an indwelling arterial blood catheter.
  • Bile samples were collected from all animals at sacrifice. LPS was extracted from bile samples using the phenol-water extraction method (Harada, et al., 2003, Lab Invest 83, 1657-1667), with an LPS extraction kit (Intron Biotechnology, Kyungki-Do, Korea) according to manufacturer’s instructions.
  • Serum IL-6 and TNF-a levels were measured by ELISA (R&D Systems, Minneapolis MN, USA) as well as s-VCAM, s-ICAM and MCP-1 (MyBioSource, San Diego CA, USA).
  • Serum/urine creatinine, serum/urine Kidney Injury Molecule-1 (KIM-1) and serum/urine Cystatin C measurements were performed with commercially available ELISA kits (MyBioSource, San Diego, USA) according to manufacturer's instructions. Liver function was assessed by serum measurements of ALT enzyme with commercially available ELISA (MyBioSource, San Diego, USA).
  • Renal and hepatic tissues were processed for histologic staining [hematoxylin and eosin (HE) (Millipore Sigma)].
  • Digital slides were acquired and analyzed using the AperioScanScope CS2 device (Aperio, Vista, CA, USA) as previously described (Stasi, et al., 2021 , Front Immunol 12, 605212;
  • HE staining was performed to evaluate histological injury in both kidneys and livers. Tubular and glomerular damage was scored semi-quantitatively by two blinded observers. The score index in each animal was expressed as a mean value of all scores obtained. Both tubular and glomerular pathological score for each group was expressed as mean ⁇ SEM.
  • Hepatic injury was defined as the amount of destruction of hepatic lobules, infiltration of inflammatory cells, hemorrhage, and hepatocyte necrosis (Baranova, et al., 2016, J Immunol 196, 3135-3147). The score, from 1 through 4, was assessed using criteria from a previously published study (Ibid.). Pathological score for each group was expressed as mean ⁇ SEM.
  • Liver tissues were homogenized and treated with RIPA lysis buffer (1 mM PMSF, 5 mM EDTA, 1 mM sodium orthovanadate, 150 mM sodium chloride, 8 pg/mL leupeptin, 1.5% Nonidet P-40, and 20 mM Tris-HCI, pH 7.4)) with phosphatase and protease inhibitors.
  • RIPA lysis buffer (1 mM PMSF, 5 mM EDTA, 1 mM sodium orthovanadate, 150 mM sodium chloride, 8 pg/mL leupeptin, 1.5% Nonidet P-40, and 20 mM Tris-HCI, pH 7.4
  • the samples (30 pg of proteins) were separated in 4-15% polyacrylamide gel and then transferred to PVDF membrane (0.2 mM) by Trans-Blot Turbo (BioRad, Hercules, CA, USA).
  • Nonspecific binding sites on the blots were blocked by incubation in 5% BSA for 1 h, and the membranes were then incubated overnight with primary antibodies and incubated with secondary antibodies for 1 h.
  • Immune complexes were detected by the ECL chemiluminescence system (Amersham Pharmacia, Little Chalfont, UK), according to the manufacturer’s instructions.
  • the primary antibodies used were anti-LPS (Abeam) and anti-£actin antibody (1 :20,000; Sigma).
  • the secondary antibodies used were HRP-conjugated anti-rabbit (Abeam) and anti-mouse antibodies (Abeam).
  • the chemiluminescent blots were acquired by Chemidoc and analyzed using Image J software. The protein expression levels were standardized relative to the level of 0-actin.
  • Urine output, tubular injury score, glomerular injury score and hepatic injury score were analyzed using one-way ANOVA, corrected for multiple comparison of pairwise treatment group differences using Tukey’s method.
  • ANOVA analyses pairwise treatments were also tested without correction for multiple comparisons. In general, both corrected and uncorrected tests were consistent in terms of statistically significant findings. The following table shows the significant findings from both methods and highlights any differences between Tukey’s correction and Uncorrected Fisher's LSD.
  • the gray bands show two infusions (from T0-T1 and T3-T4) of saline or CER-001 with flow rates 250 ml/hour. Results are presented as mean ⁇ SEM.
  • TNF-a typically released by monocytes/macrophages early in the inflammatory cascade, rapidly rises, followed in time by increasing MCP-1 and IL-6 in endotoxemic animals (LPS group, FIG. 39-FIG. 41).
  • Endothelial dysfunction is a key pathological feature of septic patients that is also prominent in the swine model of LPS-induced sepsis (Castellano, et al., 2014, Crit Care 18, 520; Stasi, A. et al., 2021 , Front Immunol 12, 605212).
  • sepsis contributes to endothelial dysfunction such as the hemodynamic instability, the direct interaction with bacterial components, the release of pro-inflammatory cytokines by pathogens-activated immune cells and pro-coagulant mediators (Boisrame-Helms, et al., 2013, Curr Vase Pharmacol 11 , 150-160).
  • endothelial cells induces the upregulation and expression of different adhesion molecules, such as ICAM and VCAM, that enhance leukocyte migration and homing, amplifying innate and adaptive immune response (de Pablo, R. et al.,
  • CER-001 infusion ameliorated systemic endothelial dysfunction by reducing VCAM (FIG. 37) and ICAM (FIG. 38) serum levels in both treated groups, with an increased effect of the two doses of ApoA-l complexes doses group as emphasized at T6.
  • liver dysfunction is a grave manifestation in the course of sepsis, principally caused by alterations and/or direct and indirect insult to hepatocytes (Yan, et al.,
  • the hepatic damage was resolved by CER-001 infusion (as measured by the modest increase in ALT (FIG. 45E) and a statistically significant decrease of liver histology score (FIG. 45D).
  • the gray bands show two infusions (0-1 h and 3-4 h) of saline or CER-001 with flow rates 250 ml/hour.
  • Kidney injury in the swine model was assessed by a time-dependent increase of serum creatinine (FIG. 47) with significant reduction in urinary output (mL/kg/h) (FIG. 48) compared to basal level (TO).
  • tubular damage biomarkers Cystatin C and KIM-1 both in serum (FIG. 49A and FIG. 50A, respectively) and urine samples (FIG. 49B and FIG. 50B, respectively), were increased compared with the basal level (TO).
  • urinary output ml/kg/h
  • FIG. 46A LPS group
  • FIG. 46B tubular pathology
  • FIG. 46C tubular pathology
  • FIG. 53 shows a dosedependent increase of endotoxin in the bile of CER-001 treated septic pigs, as determined by ELISA assay.
  • FIG. 54 the time course of human ApoA-l serum levels
  • FIG. 55 a dose-dependent increase of human ApoA-l in bile samples
  • Human ApoA-l levels were determined by ELISA.
  • Gray bands show two infusions (0-1 h and 3-4 h) of saline or CER-001 with flow rates 250ml/hour. The data are presented as the mean ⁇ SEM. Statistically significant differences were assessed by one-way ANOVA with Tukey correction (n.s.: p>0.05).
  • Example 4 Lipid binding protein molecule therapy in a model of LPS-induced vascular endothelial injury
  • HEVEC Human umbilical vein endothelial cells
  • ATCC-LGC Standards S.r.L, Sesto San Giovanni, Milan, Italy were purchased from American Type Culture Collection (ATCC-LGC Standards S.r.L, Sesto San Giovanni, Milan, Italy). EC were maintained in their recommended medium, EndGro (Merck Millipore, Darmstadt, Germany).
  • PBMCs Peripheral blood mononuclear cells
  • EC and PBMCs were incubated with LPS at 0.3 pg/ml and/or CER-001 at 50 and 500 pg/ml for 60 min and 24 hours.
  • Proliferation rate was measured by MTT Cell Proliferation Assay Kit, according to the manufacturer instructions (Sigma Aldrich). Briefly, 3> ⁇ 10 4 cells/well were seeded in a 96-well plate, and then cells were treated with LPS and CER-001 as indicated. Absorbance at 570 nm was then measured by a spectrophotometer. 7.4.1.3. Immunophenotypic analysis
  • EC were permeabilized with IntraPrep kit (Instrumentation Laboratory) and incubated with unconjugated primary antibody p-ENOS (Abeam) for 25 minutes at 4°C. Cells were then washed and labeled with secondary Antibody AlexaFluor 488 (Molecular Probes) for 25 minutes at 4°C. Finally, cells were washed twice and resuspended in FACS buffer for acquisition.
  • PBMCs were stained with the following monoclonal antibody, CD14 Monoclonal Antibody (61D3)-PE, (eBioscienceTM, Thermo Fisher Scientific, Italy), for 20 minutes in the dark at room temperature, washed twice, and resuspended in FACS buffer. Stained PBMCs were then acquired. [0689] Data were obtained by using a FC500 (Beckman Coulter) flow cytometer and analyzed with Kaluza software. Three independent studies were performed for both EC and PBMCs. The area of positivity was determined by using an isotype-matched mAb, and in total, 104 events for each sample were acquired.
  • Data shown are representative of three independent studies. Data are shown as mean ⁇ standard deviation (SD) and compared with the Student-t test.
  • CER-001 modulated the response of peripheral blood mononuclear cells (PBMC) stimulated with LPS at 0.3 pg/ml and/or CER-001 at 50 and 500 pg/ml for 24 hours, decreasing mCD14 expression and TNF-a secretion.
  • MTT assay showed no significant difference in cell viability with respect to the basal for the above conditions.
  • PBMC culture supernatants were analyzed by ELISA with results shown in FIG. 62. After 24 h from LPS stimulation, PBMCs increased TNF-a synthesis. Stimulation of PBMCs with CER-001 at 50 and 500 pg/m alone did not influence TNF-a production.
  • a clinical Phase 2B/3 trial is conducted to evaluate CER-001 plus the standard of care (SOC) versus placebo plus SOC on 90-day survival in subjects with septic shock.
  • Secondary objectives include observation of the effect of CER-001 on organ dysfunction and use of organ support, morbidity and mortality, and health-related quality of life; and the pharmacokinetics of CER-001 ; and further evaluation of a range of biomarkers in relation to the mode of action of CER-001 .
  • the primary endpoint is all-cause mortality (defined as the fraction of subjects that have died, regardless of cause) at Day 90. Secondary endpoints include days alive and not in ICU up to Day 90; days alive and not requiring mechanical ventilation up to Day 90; days alive and not on renal replacement therapy (RRT) up to Day 90; vasopressor-free days up to Day 30; days alive without organ support up to Day 90; and Sepsis Support Index through Day 90.
  • RRT renal replacement therapy
  • Secondary efficacy endpoints include:
  • Exploratory endpoints include:
  • the overall trial design includes two parts (Part 1 - Phase 2b and Part 2 - Phase 3) as represented in FIG. 69.
  • Part 1 has fixed randomization (1 :1 :1 ) to placebo, CER-001 10 mg/kg or CER-001 20 mg/kg.
  • Part 1 When 30-day survival data is available from approximately 150 randomized subjects, the results of Part 1 is used to determine whether the study transitions to Part 2 (with continuing enrollment into Part 1 during analysis) and whether one dosage arm will be eliminated with the participants from that arm being allocated to the remaining CER-001 arm.
  • the investigational medicinal products (IMPs) used are (1 ) CER-001 sterile solution for intravenous infusion and (2) Placebo: sterile 0.9% sodium chloride solution (250 ml_).
  • CER-001 is provided frozen in 20 ml_ vials containing approximately 18 mL of product at a concentration of 8 mg/mL (ApoA-l content).
  • CER-001 is dosed by weight. All doses are thawed and then diluted with normal saline to a volume of 250 mL and are administered using an infusion pump over a 1-hour period (250 mL/hour).
  • the infusion of IMP starts as early as possible and no later 48 hours after ICU admission. To ensure start of IMP treatment without delay, informed consent is obtained, in compliance with local regulations, as early as possible.
  • IMP is administered twice a day, twelve hours apart, for five consecutive days.
  • Subjects are at least 11 but less than 80 years of age; have proven or suspected infection; and have septic shock characterized by hypotension (systolic arterial pressure ⁇ 90mmHg or mean arterial pressure (MAP) ⁇ 65 mm Hg) requiring the use of vasopressors for more than 1 hour despite intravenous fluid resuscitation.
  • hypotension systolic arterial pressure ⁇ 90mmHg or mean arterial pressure (MAP) ⁇ 65 mm Hg
  • Exclusion criteria include inability to initiate IMP treatment within 24 hours from start of vasopressors for septic shock; previous severe sepsis with ICU admission within the last 12 months; hypotension secondary to causes other than sepsis (e.g.
  • the primary analysis compares all subjects treated with a CER-001 dosage regimen, from both parts of the trial (pooled together and treated as a single arm) to all subjects on the placebo arm from both parts of the trial.
  • the primary analysis evaluates CER-001 superiority using a one-sided 5% significance level test.
  • the analysis is based on both the modified intent-to-treat set (mITT) and the per protocol (PP) analysis set, with the mITT being considered the primary analysis to judge statistical significance and the PP analysis considered as supportive.
  • the mITT comprises all randomized subjects receiving at least one dose of blinded therapy.
  • the secondary endpoints are aimed at supporting primary efficacy by further demonstrating treatment effect accompanied by an acceptable safety profile. All secondary endpoints are analyzed using both the mITT and the PP analysis set.
  • All-cause mortality (at Days 30 and 180) is analyzed in the same manner as the primary endpoint. Mortality is presented graphically by a Kaplan-Meier plot. Parameters measured in days, as well as the Sepsis Support Index, are analyzed with a test of superiority using a two-sided 5% significance level test. Endpoints addressing changes in SOFA score, health-related quality of life, cytokine levels and endothelial dysfunction markers are analyzed by analysis of variance (ANOVA) or covariance (ANCOVA) methods as appropriate and presented graphically.
  • ANOVA analysis of variance
  • ANCOVA covariance
  • the safety profile including adverse events, vital signs, and safety laboratory variables, are summarized descriptively.
  • the safety analyses are performed using the safety analysis set.
  • the safety analysis set comprises all IMP-treated subjects and are analyzed according to the actual treatment received.
  • Table 5 provides the schedule of study procedures.
  • CER-001 therapy provides a therapeutic benefit to subjects with septic shock.
  • a method of treating a subject having or at risk of a condition, which is optionally an acute condition comprising administering a dose, e.g., a high dose, of a lipid binding protein molecule to the subject.
  • kynurenine pathway biomarker is 2-amino-3- carboxymuconate-semialdehyde. 19. The method of embodiment 12, wherein the kynurenine pathway biomarker is picolinic acid.
  • the dose comprises an amount of the lipid binding protein molecule which decreases a kynurenine/tryptophan ratio in the subject.
  • the dose comprises an amount of the lipid binding protein molecule which decreases a level of TNFa in the subject.
  • the dose comprises an amount of the lipid binding protein molecule which decreases a level of IL-6 in the subject.
  • the dose comprises an amount of the lipid binding protein molecule which decreases a level of VCAM-1 in the subject.
  • the dose comprises an amount of the lipid binding protein molecule which decreases a level of ICAM-1 in the subject.
  • bacterial infection is a Pseudomonas aeruginosa infection.
  • bacterial infection is a Acinetobacter baumanni infection.

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Abstract

L'invention concerne des méthodes de traitement de sujets présentant ou présentant un risque de développer une ou de plusieurs affections, telles que la sepsie (par exemple, un choc septique) à l'aide de molécules à protéine d'ancrage lipidique.
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