Method for treating an acute condition using a complex based on a lipid-binding protein

Abstract

A method for treating acute conditions (e.g., acute conditions including acute inflammation), such as cytokine release syndrome, sepsis, and acute kidney injury, using a complex based on a lipid-binding protein.

Description

Technical Field

[0001] 1. Cross - reference to related applications This application claims the benefit of priority of U.S. Application No. 63 / 351,125, filed on June 10, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] 2. Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML sequence listing created on June 1, 2023 is named CRN - 049WO_SL.xml and has a size of 3,263 bytes.

Background Art

[0003] 3. Background Art Various acute conditions, such as those associated with acute inflammation, such as sepsis, acute kidney injury (AKI), and cytokine release syndrome (CRS), are common and can be life - threatening. Current treatment methods for such conditions are often inappropriate or sub - optimal.

[0004] 3.1. Sepsis Sepsis is a life-threatening systemic response of the immune system caused by infection and can also cause damage to tissues and organs (Singer et al., 2016, JAMA. 315(8):801-10). Common signs and symptoms of sepsis include fever, increased heart rate, increased respiratory rate, and altered mental status. Symptoms associated with specific infections may also be present, such as cough with pneumonia or dysuria with kidney infection (Jui et al., 2011, ”Ch. 146: septic shock.” In Tintinalli JE, et al. (eds.). Tintinalli's Emergency Medicine: A Comprehensive Study Guide (7th ed.). New York: McGraw-Hill. pp. 1003-14). Severe sepsis can be associated with organ dysfunction or poor blood flow (Dellinger et al., 2013, Critical Care Medicine. 41(2):580-637). Hypotension, increased blood lactate levels, or decreased urine output, if present, may suggest poor blood flow. Sepsis can progress to septic shock (characterized by hypotension that does not improve even after fluid resuscitation) (Dellinger et al., 2013, Critical Care Medicine. 41(2):580-637).

[0005] Bacterial infections are the most common cause of sepsis; however, fungal, viral, and protozoal infections can also cause sepsis (Jui et al., 2011, ”Ch. 146: Septic Shock.” In Tintinalli JE, et al. (eds.). Tintinalli's Emergency Medicine: A Comprehensive Study Guide (7th ed.). New York: McGraw-Hill. pp. 1003-14). Common sites of primary infection include the lung, brain, urinary tract, skin, and abdominal organs (Jui et al., 2011, ”Ch. 146: Septic Shock.” In Tintinalli JE, et al. (eds.). Tintinalli's Emergency Medicine: A Comprehensive Study Guide (7th ed.). New York: McGraw-Hill. pp. 1003-14). Risk factors include extreme youth, advanced age, immunosuppression due to cancer or diabetes, major trauma, or burns (cdc.gov / sepsis / what-is-sepsis.html). The diagnosis of sepsis can be made based on the sequential organ failure assessment score (SOFA score) (Vincent et al., 1996, Intensive Care Med, 22:707-710). It is also possible to diagnose sepsis based on a shortened version of the SOFA score, also known as the quick SOFA (qSOFA) score, in which case at least two of the following three criteria must be met: increased respiratory rate, change in level of consciousness, low blood pressure (Singer et al., 2016, JAMA. 315(8):801-10). For example, sepsis can be diagnosed based on an increase in the patient's total SOFA score or qSOFA score.

[0006] In sepsis, prompt treatment with intravenous fluids and antibiotics may be required (Rhodes et al., 2017, Intensive Care Medicine. 43(3):304-377). Continuous care is often provided in the intensive care unit. If attempts to adequately resuscitate the patient are not sufficient to maintain blood pressure, the use of drugs to raise blood pressure may be necessary. Mechanical ventilation and dialysis may be required to support the function of the lungs and kidneys, respectively. Other useful parameters include cardiac output and oxygen saturation in the superior vena cava (Dellinger et al., 2013, Critical Care Medicine. 41(2):580-637).

[0007] The risk of death due to sepsis is up to 30%, while in severe sepsis it is up to 50% and in septic shock it is up to 80% (Jawad et al., 2012, J Glob Health. 2(1):010404). Early detection and treatment are essential for survival and disability prevention.

[0008] 3.2. Acute Kidney Injury Acute kidney injury (AKI) commonly occurs in ICU patients, with an estimated incidence of over 50% (Hoste et al., 2015, Intensive Care Med; 41:1411-1423). Furthermore, an increase in the severity of AKI is associated with an increase in mortality. Sepsis is the main cause of AKI, accounting for 45% - 70% of cases, and approximately 25% of sepsis cases originate from the intra-abdominal cavity (Seymour et al., 2016, JAMA, 315:762-774; Bagshaw et al., 2007, Clin J Am Soc Nephrol, 2:431-439). Ischemia / reperfusion injury (IRI) may cause AKI and is a common complication in organ transplant recipients, with an incidence of 50 - 75% after lung and heart transplantation (Gueler et al., 2014, Transplantation 98:337-338). Cardiac surgery-related AKI (CSA AKI) has been reported to occur in up to 30% of cardiac surgery patients (Rosner and Okusa, 2006, Clin J Am Soc Nephrol. 1(1):19-323). The onset and outcome of AKI can be predicted from the levels of IL6 and IL10 after surgery (Zhang et al., 2015, J Am Soc Nephrol. 26(12):3123-32), and there are no good treatment options other than dialysis (Kullmar et al., 2020, Crit Care Clin. 36(4):691-704).

[0009] Early diagnosis of AKI in sepsis is important to provide optimal treatment and avoid further kidney injury (Peerapornratana et al., 2019, Kidney International 2019, 96:1083-1099). Treatment options for sepsis-related AKI are limited to supportive care. The use of blood filtration devices, including high-volume hemofiltration and polymyxin B hemoperfusion, has not shown significant benefits (Joannes-Boyau et al., 2013, Intensive care medicine, 39:1535-1546; Zhang et al., 2012, Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association - European Renal Association 27:967-973; Vincent et al., 2005, Shock, 23:400-405; Cruz et al., 2009, JAMA, 301:2445-2452; Payen et al., 2015, Intensive care medicine, 41:975-98; Dellinger et al., 2018, JAMA, 320:1455-463).

[0010] Experimental pharmacological treatments usually target AKI itself, rather than sepsis-induced AKI, with the exceptions of alkaline phosphatase (AP), angiotensin II (ATII), levocarnitine, and lesinurad (AB103). In recent clinical trials, recombinant AP did not reduce endogenous creatinine clearance, which is a primary clinical endpoint, but did improve mortality, which is a secondary endpoint (Pickkers et al., 2018, JAMA, 320:1998-2009). ATII has shown some effect in a post hoc analysis of AKI patients in the high-output shock trial (ATHOS-3), and a trial is currently ongoing for sepsis-related AKI in the ASK-IT trial (NCT00711789), but there has been no updated information since 2011. Levocarnitine did not show improvement in organ dysfunction occurring in septic shock in the RACE trial (Jones et al., 2018, JAMA network open, 1:e186076), but is currently being tested in the Carnisave trial (NCT02664753) as an adjunctive treatment for septic shock patients with AKI. Lesinurad was tested in a phase 3 placebo-controlled trial (NCT03403751) for patients with sepsis-related AKI, but the trial was recently terminated due to low enrollment (clinicaltrials.gov / ct2 / show / NCT03403751).

[0011] During the infection period, changes occur in the metabolism of lipids and lipoproteins, which have been reported to cause the redelivery of nutrients to cells important in host defense or tissue repair (Khovidhunkit et al., 2004, J Lipid Res, 45(7):1169-96). Furthermore, lipoproteins and lipids play important roles in host defense against infection and protect the host from the toxic effects of microorganisms (Feingold and Grunfeld, 2012, J Lipid Res. 53(12):2487-248). High-density lipoprotein (HDL) is a major component of circulating blood and contains mainly phospholipids, free cholesterol, cholesteryl esters, triglycerides, apolipoproteins (ApoA-I, ApoA-II), and other proteins. HDL is regarded as an anti-inflammatory lipoprotein and regulates vascular endothelial function and immunity (Singh et al., 2007, JAMA, 298(7):786-798; Navab et al., 2011, Nat Rev Cardiol 8(4):222-32). Indeed, HDL plays an important protective role at all stages of endothelial dysfunction, including the suppression of inflammatory signaling in immune effector cells and the direct inhibition of endothelial activation. Clinical trials have demonstrated that HDL levels decrease by 40-70% during systemic inflammation, which is associated with a poor prognosis in subjects with sepsis (van Leeuwen et al., 2003, Critical care medicine, 31:1359-1366; Chien et al., 2005, Critical care medicine, 33:1688-1693; Tsai et al., Journal of hepatology, 50:906-915; Eggesbo et al., 1996, Cytokine, 8(2):152-160; Morin et al., 2015, Frontiers in Pharmacology, doi.org / 10.3389 / fphar.2015.00244).Furthermore, reduced HDL levels are associated with an increased risk of acute kidney injury (AKI) in sepsis (Roveran et al., 2017, Journal of internal medicine, 281:518-529; Zhang et al., 2009, Am J Physiol Heart Circ Physiol 297:H866-H873). Since the kidney is involved in the recycling of aged HDL particles and its filtration function is related to HDL levels and content, renal function and plasma HDL are strongly interrelated (Yang et al., 2016, Current opinion in nephrology and hypertension, 25:174-179).

[0012] HDL-based treatment methods have been proposed for sepsis-induced systemic inflammatory response syndrome (SIRS) (Morin et al., 2015, Frontiers in Pharmacology, doi.org / 10.3389 / fphar.2015.00244; Tanaka et al., 2020, Crit Care 24:134). Several studies have suggested that correction of dyslipoproteinemia by recombinant HDL (rHDL) may offer a strategy for the prevention and treatment of systemic inflammatory responses (Morin et al., 2015, Frontiers in Pharmacology, doi.org / 10.3389 / fphar.2015.00244; Roveran et al., 2017, Journal of internal medicine, 281:518-529; Pajkrt et al., 1996, Journal of Experimental Medicine, 184(5):1601-1608; Pajkrt et al., 1997, Thrombosis and Haemostasis, 77(2):303-7; Guo et al., 2013, J. Biol. Chem. 288(25):17947-53;Li et al., 2008, European journal of pharmacology 590:417-422; McDonald et al., 2003, Shock 20(6):551-7).CSL-111, an rHDL originally generated for treating atherosclerosis (Tardif et al., 2007, JAMA, 297(15):1675-82), has been shown to be effective in suppressing the inflammatory response during the LPS-induced endotoxemia period in vitro and in rabbit models (Casas et al., 1995, The Journal of surgical research, 59:544-552), as well as in human models (Pajkrt et al., 1996, Journal of Experimental Medicine, 184(5):1601-8; Pajkrt et al., 1997, Thrombosis and Haemostasis, 77(2):303-7). In human models, it has been shown that infusion of CSL-111 reduces the procoagulant state induced by endotoxin exposure, suppresses monocyte activation and cytokine production, and improves clinical symptoms (Pajkrt et al., 2016, Journal of Experimental Medicine, 184(5):1601-1608; Pajkrt et al., 1997, Thrombosis and Haemostasis, 77(2):303-7). ApoA1 Milano, a natural variant of ApoA1, has been extensively tested in phase I trials (Casas et al., 1995, The Journal of surgical research, 59:544-552) and other additional clinical trials in the context of cardiovascular disease (CVD). In recent years, Zhang and his co-researchers demonstrated that ApoA1 is also effective against inflammation in an endotoxemia rat model (Zhang et al., 2015, Biological Chemistry, 396(1):53-60).Among HDL-mimicking peptides, L-4F has been employed in several preclinical models of sepsis, and has been shown to block cytokine production, reverse sepsis-induced hypotension, prevent organ injury, restore kidney, liver, and heart function, and improve survival (Zhang et al., 2009, Am J Physiol Heart Circ Physiol 297:H866-H873). Changes in serum lipid levels, particularly cholesterol levels, have also been reported to occur during infection with viruses including human immunodeficiency virus (HIV) and hepatitis C virus (HCV) (Meher et al., 2019, J. Phys. Chem. B, 123(50):10654-10662). Despite the growing interest in HDL and HDL therapy, regulatory approval for HDL or HDL mimetics for the treatment of sepsis or AKI (including sepsis-related AKI, ischemia / reperfusion AKI, and CSA AKI) has not yet been obtained.

[0013] 3.3. Cytokine Release Syndrome Cytokine release syndrome (CRS), also known as cytokine storm syndrome (CSS), is a systemic inflammatory response that can be caused by various factors, such as infection or treatment with some types of immunotherapy (e.g., monoclonal antibodies and adoptive T cell therapy) (Shimabukuro-Vornhagen, et al., 2018, J. Immunotherapy Cancer, 6:56). Symptoms of CRS include fever, nausea, headache, rash, palpitations, hypotension, and dyspnea. Most patients with CRS have mild reactions, but CRS can sometimes be severe and life-threatening (NCI Dictionary of Cancer Terms (cancer.gov / publications / dictionaries / cancer-terms / def / cytokine-release-syndrome)).

[0014] Since the second half of 2019, the novel coronavirus COVID-19 (SARS-CoV-2) has spread around the world. Data suggests that in severely affected patients, mild or severe cytokine storms with high expression of interleukin-6 (IL-6) are observed. CRS may contribute to the death of these patients (Zhang et al., 2020, International Journal of Antimicrobial Agents, doi.org / 10.1016 / j.ijantimicag.2020.105954; Mehta et al., 2020, The Lancet, 395(10229):1033-1034).

[0015] Therefore, there is still a need for new treatment methods for acute conditions such as sepsis, AKI (including sepsis-related AKI, ischemia / reperfusion AKI, and CSA AKI), and CRS (such as CRS related to immunotherapy and CRS following infections such as COVID-19).

Summary of the Invention

[0016] 4. Summary The present disclosure provides a method for treating a subject having an acute condition, such as a condition associated with acute inflammation, while using a high-dose lipid-bound protein-based complex (e.g., a lipid-bound protein such as ApoA-I, and a formulation containing one or more lipids). The high dose is typically higher than the dose that would be used to treat chronic conditions such as familial hypercholesterolemia. The high dose is typically administered over a relatively short period, e.g., over a period of 1 day to 2 weeks, and typically includes multiple administrations of the lipid-bound protein-based complex, e.g., 2 to 10 individual doses. The individual doses may be separated by less than 1 day (e.g., twice-daily administration) or by more than 1 day (e.g., once-daily administration).

[0017] In some embodiments of the methods of the present disclosure, lipid-binding protein-based complexes include sphingomyelin and / or charged lipids, such as CER-001. CER-001 is a charged lipoprotein complex that includes recombinant human ApoA-I, sphingomyelin (SM), and 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (dipalmitoyl phosphatidyl-glycerol, DPPG). This mimics native nascent discoidal pre-beta HDL, which is the form that HDL particles take before acquiring cholesterol. Without being bound by theory, CER-001 treatment is thought to lower serum levels of inflammatory cytokines, such as IL-6, and thereby provide a clinical benefit to subjects having or at risk of an acute condition, such as subjects having or at risk of an acute inflammatory condition.

[0018] In some aspects, the present disclosure provides methods for treating a subject having an infectious disease, such as a coronavirus infection, such as a COVID-19 infection, with a lipid-binding protein-based complex (e.g., CER-001).

[0019] In some aspects, the present disclosure provides methods for treating a subject having sepsis, and for treating a subject having or at risk of AKI with a lipid-binding protein-based complex (e.g., CER-001).

[0020] In one aspect, the present disclosure provides a method for treating a subject having sepsis, the method comprising administering to the subject a lipid-binding protein-based complex (e.g., CER-001).

[0021] In another aspect, the present disclosure provides a method of treating a subject having acute kidney injury (AKI) or at risk of AKI (e.g., a subject having sepsis that has not yet caused AKI, an organ transplant recipient, or a subject who has undergone heart surgery, or a subject having acute or chronic liver disease, and a subject at risk of hepatorenal syndrome (HRS)), the method comprising administering to the subject a lipid-bound protein-based complex (e.g., CER-001).

[0022] In some aspects, the present disclosure provides a method for treating cytokine release syndrome (CRS) and / or reducing one or more inflammatory markers in a subject in need thereof using a lipid-bound protein-based complex (e.g., CER-001).

[0023] In one aspect, the present disclosure provides a method of treating a subject having CRS or at risk of CRS, e.g., a subject having CRS subsequent to COVID-19, or a subject having CRS caused by immunotherapy, the method comprising administering to the subject a therapeutically effective amount of a lipid-bound protein-based complex (e.g., CER-001).

[0024] In another aspect, the present disclosure provides a method of reducing the serum levels of one or more inflammatory markers, e.g., one or more markers associated with CRS, e.g., IL-6, in a subject in need thereof. The subject can be, for example, a subject having CRS or at risk of CRS, e.g., a subject infected with a virus, e.g., COVID-19, or a subject undergoing immunotherapy.

[0025] In some embodiments, the present disclosure provides a dosing regimen for lipid - bound protein - based therapy (e.g., CER - 001 therapy) for subjects having an acute condition (e.g., associated with acute inflammation), such as sepsis, AKI (e.g., AKI caused by sepsis, ischemia / reperfusion, cardiac surgery, or hepatorenal syndrome), or for subjects having a risk of an acute condition, such as AKI (e.g., a subject having sepsis that has not yet caused AKI) or CRS.

[0026] The dosing regimen of the present disclosure generally requires multiple administrations of CER - 001 to the subject (e.g., administered daily or twice a day). The CER - 001 treatment can be continued for a pre - determined period, such as within one week (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days), or for a period longer than one week (e.g., 2 weeks). Alternatively, the administration of CER - 001 to the subject can be continued until one or more symptoms of the acute condition (e.g., acute inflammation or CRS) decrease, or until the serum levels of one or more inflammatory markers decrease, e.g., to normal levels, or decrease as compared to baseline measurements taken prior to the start of CER - 001 treatment. In the case of a subject having a risk of CRS or AKI due to an infection, or a risk of CRS due to immunotherapy, the treatment can, in some embodiments, be continued until the subject recovers from the infection or discontinues the immunotherapy.

[0027] The dosing regimen of the present disclosure may involve administering a lipid - bound protein - based complex (e.g., CER - 001) to the subject according to an initial "induction" regimen, optionally followed by administering a lipid - bound protein - based complex to the subject according to a "consolidation therapy" regimen.

[0028] The induction regimen typically includes administering multiple doses of a lipid - bound protein - based complex (e.g., CER - 001) to the subject, such as 6 doses over 3 days.

[0029] The consolidation therapy regimen typically involves administering one or more doses of a lipid-bound protein-based complex (e.g., CER-001) to a subject, for example, one day or several days after the final dose of the induction regimen. In some embodiments, the first dose of the consolidation therapy regimen is administered on the third day after the final dose of the induction regimen. For example, the dosing regimen can include administering a lipid-bound protein-based complex (e.g., CER-001) to the subject according to the induction regimen on days 1, 2, and 3, and administering a lipid-bound protein-based complex to the subject according to the consolidation therapy regimen on day 6. In some embodiments, the consolidation therapy regimen includes two doses of a lipid-bound protein-based complex.

[0030] In certain embodiments, the present disclosure - on days 1, 2, and 3, two doses per day (induction regimen), optionally followed by - after day 4, two subsequent doses (consolidation therapy regimen) provides a method of treating a subject having CRS, sepsis, or AKI, or a subject at risk of CRS or AKI (e.g., a subject having COVID-19) with a lipid-bound protein-based complex (e.g., CER-001) according to a dosing regimen comprising. In some embodiments, the regimen - on days 1, 2, and 3, two doses per day (induction regimen), followed by - on day 6, two doses (consolidation therapy regimen) comprises.

[0031] In certain aspects, the lipid-bound protein-based complex (e.g., CER-001) is administered in combination with standard treatment for sepsis, such as antibiotic treatment and / or hemodynamic support.

[0032] In certain embodiments, an antihistamine (e.g., dexchlorpheniramine, hydroxyzine, diphenhydramine, cetirizine, fexofenadine, or loratadine) can be administered prior to the administration of a complex based on a lipid-binding protein (e.g., CER-001). The antihistamine can reduce the likelihood of an allergic reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0034]

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Modes for Carrying Out the Invention

[0035] 6. Detailed Description The present disclosure provides a method for treating a subject having an acute condition, such as an acute condition including acute inflammation, with a high-dose lipid-binding protein-based complex (e.g., administered as a formulation comprising a lipid-binding protein, such as ApoA-I, and one or more lipids).

[0036] In one aspect, the present disclosure provides a method for treating a subject having an infectious disease, such as a coronavirus infection, such as COVID-19, with a lipid-binding protein-based complex (e.g., CER-001).

[0037] In one aspect, the present disclosure provides a method for treating a subject having sepsis with a lipid-binding protein-based complex (e.g., CER-001).

[0038] In other aspects, the present disclosure provides a method for treating a subject having acute kidney injury (AKI) or at risk of AKI (e.g., due to sepsis, viral infection, ischemia / reperfusion, cardiac surgery, or hepatorenal syndrome) with a lipid-binding protein-based complex (e.g., CER-001).

[0039] In other aspects, the present disclosure provides a method of treating a subject having CRS, or a subject at risk of CRS, such as a subject having CRS subsequent to COVID-19 or a subject having CRS caused by immunotherapy.

[0040] In some embodiments, the lipid-binding protein-based complex is an Apomer, a Cargomer, an HDL-based complex, or an HDL mimetic-based complex. In a specific embodiment, the lipid-binding protein-based complex is CER-001.

[0041] Exemplary features of lipid-binding protein-based complexes that can be used in the methods and compositions of the present disclosure are described in Section 6.1. Exemplary populations of subjects that can be treated using the compositions of the present disclosure by the methods of the present disclosure are described in Section 6.2.

[0042] In some embodiments, the method of the present disclosure comprises administering a lipid-binding protein-based complex (such as CER-001) to a subject in two stages. First, the lipid-binding protein-based complex (such as CER-001) is administered in an initial high-intensity "induction" regimen. A lower-intensity "consolidation therapy" regimen follows the induction regimen. Alternatively, the lipid-binding protein-based complex (such as CER-001) can be administered to the subject in a single stage, for example, according to an administration regimen corresponding to the dosage and frequency of administration of the induction or consolidation therapy regimens described herein.

[0043] Induction regimens that can be used in the methods of the present disclosure are described in Section 6.3, and consolidation therapy regimens that can be used in the methods of the present disclosure are described in Section 6.3.2. The dosing regimens of the present disclosure involve administering a lipid-binding protein-based complex (e.g., CER-001) as monotherapy or as part of combination therapy with one or more agents, for example, in combination with standard treatments for sepsis such as antibiotic treatment and / or hemodynamic support. Combination therapies are described in Section 6.4.

[0044] 6.1. Complexes based on lipid-binding proteins 6.1.1. Complexes based on HDL and HDL mimetics In one aspect, the lipid-binding protein-based complex comprises a complex based on HDL or an HDL mimetic. For example, the complex can comprise a lipoprotein complex described in U.S. Patent No. 8,206,750, International Publication No. 2012 / 109162, International Publication No. 2015 / 173633 (e.g., CER-001), or U.S. Patent Application Publication No. 2004 / 0229794, the entire contents of each of which are incorporated herein by reference. The terms "lipoprotein" and "apolipoprotein" are used interchangeably herein, and unless the context requires otherwise, the term "lipoprotein" encompasses lipoprotein mimetics. The terms "lipid-binding protein" and "lipid-binding polypeptide" are also used interchangeably herein, and unless the context requires otherwise, these terms do not imply a particular length of amino acid sequence.

[0045] The lipoprotein complex can comprise a protein fraction (e.g., an apolipoprotein fraction) and a lipid fraction (e.g., a phospholipid fraction). Examples of the protein fraction include one or more lipid-binding protein molecules such as apolipoproteins, peptides, or peptide analogs or mimetics of apolipoproteins, for example, one or more lipid-binding protein molecules described in Section 6.1.2.

[0046] The lipid fraction typically includes one or more phospholipids, which can be neutral, negatively charged, positively charged, or a combination thereof. Exemplary phospholipids and other amphiphilic molecules that can be included in the lipid fraction are described in Section 6.1.3.

[0047] In certain embodiments, the lipid fraction contains at least one neutral phospholipid (e.g., sphingomyelin (SM)) and optionally one or more negatively charged phospholipids. In lipoprotein complexes containing both neutral and negatively charged phospholipids, the neutral and negatively charged phospholipids can have fatty acid chains with the same or different numbers of carbons and the same or different degrees of saturation. In some examples, the neutral and negatively charged phospholipids have the same acyl tail, e.g., an acyl chain of C16:0 or palmitoyl. In a specific embodiment, particularly when egg SM is used as the neutral lipid, the weight ratio of the apolipoprotein fraction to the lipid fraction ranges from about 1:2.7 to about 1:3 (e.g., 1:2.7).

[0048] Any phospholipid that is at least partially negatively charged at physiological pH can be used as the negatively charged phospholipid. Non-limiting examples include phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, and negatively charged forms of phosphatidic acid, such as salts. In a specific embodiment, the negatively charged phospholipid is 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], or DPPG, phosphatidylglycerol. Preferred salts include potassium and sodium salts.

[0049] In some embodiments, the lipoprotein complex used in the methods of the present disclosure is the lipoprotein complex described in U.S. Patent No. 8,206,750 or International Publication No. 2012 / 109162 (and its U.S. counterpart, U.S. Patent Application Publication No. 2012 / 0232005), the entire contents of each of which are incorporated herein by reference in their entirety. In certain embodiments, the protein component of the lipoprotein complex is described in Section 6.1 and preferably Section 6.1.1 of International Publication No. 2012 / 109162 (and U.S. Patent Application Publication No. 2012 / 0232005), the lipid component is described in Section 6.2 of International Publication No. 2012 / 109162 (and U.S. Patent Application Publication No. 2012 / 0232005), and these can be complexed together, optionally, in the amounts described in Section 6.3 of International Publication No. 2012 / 109162 (and U.S. Patent Application Publication No. 2012 / 0232005). The entire contents of these sections are incorporated herein by reference. In certain aspects, as described in Section 6.4 of International Publication No. 2012 / 109162 (and U.S. Patent Application Publication No. 2012 / 0232005), the entire contents of which are incorporated herein by reference, the lipoprotein complexes of the present disclosure are in a population of complexes that are at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% homogeneous.

[0050] In a specific embodiment, the lipoprotein complex that can be used in the methods of the present disclosure comprises 2 - 4 ApoA-I equivalents, 2 charged phospholipid molecules, 50 - 80 lecithin molecules, and 20 - 50 SM molecules.

[0051] In another specific embodiment, the lipoprotein complex that can be used in the methods of the present disclosure comprises 2 - 4 ApoA-I equivalents, 2 charged phospholipid molecules, 50 lecithin molecules, and 50 SM molecules.

[0052] In yet another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure comprises 2 to 4 ApoA-I equivalents, 2 charged phospholipid molecules, 80 lecithin molecules, and 20 SM molecules.

[0053] In yet another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure comprises 2 to 4 ApoA-I equivalents, 2 charged phospholipid molecules, 70 lecithin molecules, and 30 SM molecules.

[0054] In yet another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure comprises 2 to 4 ApoA-I equivalents, 2 charged phospholipid molecules, 60 lecithin molecules, and 40 SM molecules.

[0055] In a specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure consists essentially of 2 to 4 ApoA-I equivalents, 2 charged phospholipid molecules, 50 to 80 lecithin molecules, and 20 to 50 SM molecules.

[0056] In another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure consists essentially of 2 to 4 ApoA-I equivalents, 2 charged phospholipid molecules, 50 lecithin molecules, and 50 SM molecules.

[0057] In yet another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure consists essentially of 2 to 4 ApoA-I equivalents, 2 charged phospholipid molecules, 80 lecithin molecules, and 20 SM molecules.

[0058] In yet another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure consists essentially of 2 to 4 ApoA-I equivalents, 2 charged phospholipid molecules, 70 lecithin molecules, and 30 SM molecules.

[0059] In yet another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure consists essentially of 2-4 ApoA-I equivalents, 2 charged phospholipid molecules, 60 lecithin molecules, and 40 SM molecules.

[0060] In a specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure contains a lipid component comprising about 90-99.8 wt% SM and about 0.2-10 wt% 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 charged phospholipid. In another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure contains a lipid component comprising about 90-99.8 wt% lecithin and about 0.2-10 wt% 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 charged phospholipid.

[0061] In a specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure contains a lipid component consisting essentially of about 90-99.8 wt% SM and about 0.2-10 wt% 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 charged phospholipid. In another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure consists essentially of about 90-99.8 wt% lecithin and about 0.2-10 wt% 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 charged phospholipid.

[0062] In yet another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure comprises about 9.8 to 90 wt% SM, about 9.8 to 90 wt% lecithin, and about 0.2 to 10 wt% charged phospholipid, for example, a lipid fraction containing a total charged phospholipid from about 0.2 to 1 wt%, 0.2 to 2 wt%, 0.2 to 3 wt%, 0.2 to 4 wt%, 0.2 to 5 wt%, 0.2 to 6 wt%, 0.2 to 7 wt%, 0.2 to 8 wt%, 0.2 to 9 wt%, 0.2 to 10 wt%.

[0063] In yet another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure comprises a lipid fraction consisting essentially of about 9.8 to 90 wt% SM, about 9.8 to 90 wt% lecithin, and about 0.2 to 10 wt% charged phospholipid, for example, a total charged phospholipid from about 0.2 to 1 wt%, 0.2 to 2 wt%, 0.2 to 3 wt%, 0.2 to 4 wt%, 0.2 to 5 wt%, 0.2 to 6 wt%, 0.2 to 7 wt%, 0.2 to 8 wt%, 0.2 to 9 wt%, 0.2 to 10 wt%.

[0064] In another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure comprises ApoA-I apolipoprotein and a lipid fraction, the lipid fraction contains sphingomyelin and about 3 wt% charged phospholipid, the molar ratio of the lipid fraction to ApoA-I apolipoprotein is about 2:1 to 200:1, and the complex is small or large discoidal particles containing 2 to 4 ApoA-I equivalents.

[0065] In another specific embodiment, the lipoprotein complex that can be used in the method of the present disclosure comprises ApoA-I apolipoprotein and a lipid fraction, the lipid fraction consists essentially of sphingomyelin and about 3 wt% charged phospholipid, the molar ratio of the lipid fraction to ApoA-I apolipoprotein is about 2:1 to 200:1, and the complex is small or large discoidal particles containing 2 to 4 ApoA-I equivalents.

[0066] Complexes based on HDL or HDL mimetics can comprise a single type of lipid-binding protein, or a mixture of two or more different lipid-binding proteins that can be derived from the same or different species. Optionally but preferably, the complex comprises a lipid-binding protein that is derived from the animal species being treated or has an amino acid sequence corresponding thereto, in order to avoid inducing an immune response to the treatment. Thus, for the treatment of human patients, lipid-binding proteins of human origin are preferably used. The use of peptidomimetic apolipoproteins can also reduce or avoid an immune response.

[0067] In some embodiments, the lipid component comprises two phospholipids, namely sphingomyelin (SM) and a charged phospholipid. Exemplary SM and charged lipids are described in Section 6.1.3.1.

[0068] The lipid component comprising SM can optionally contain a small amount of additional lipid. Virtually any type of lipid can be used, including but not limited to lysophospholipids, galactocerebrosides, gangliosides, cerebrosides, glycerides, triglycerides, and cholesterol and its derivatives.

[0069] When included, such optional lipids typically comprise less than about 15 wt% of the lipid fraction, although in some instances, more optional lipid can be included. In some embodiments, the optional lipid comprises 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 lipid.

[0070] In a specific embodiment, the phospholipid fraction contains egg SM or palmitoyl SM or phytosphingomyelin and DPPG in a weight ratio (SM:charged phospholipid) in the range of 90:10 to 99:1, more preferably in the range of 95:5 to 98:2. In one embodiment, the weight ratio is 97:3.

[0071] The molar ratio of the lipid component to the protein component of the complex of the present disclosure may vary and depends, among other factors, on what the apolipoprotein containing the protein component is, what the lipid containing the lipid component is and its amount, and the desired size of the complex. Since the biological activity of apolipoproteins such as ApoA-I is thought to be mediated by amphipathic helices containing the apolipoprotein, it is convenient to use the equivalent of the ApoA-I protein to represent the apolipoprotein fraction of the lipid:apolipoprotein molar ratio. ApoA-I generally contains 6 to 10 amphipathic helices depending on the method used to calculate the helices. Other apolipoproteins can be represented in terms of ApoA-I equivalents based on the number of amphipathic helices they contain. For example, ApoA-I M which typically exists as a disulfide-bridged dimer M can be represented as 2ApoA-I equivalents because each of its molecules contains twice the number of amphipathic helices of an ApoA-I molecule. Conversely, a peptide apolipoprotein containing a single amphipathic helix can be represented as 1 / 10 to 1 / 6 ApoA-I equivalents because each of its molecules contains 1 / 10 to 1 / 6 the number of amphipathic helices of an ApoA-I molecule. Generally, the molar ratio of lipid:ApoA-I equivalents of the lipoprotein complex (defined herein as "Ri") ranges from about 105:1 to 110:1. In some embodiments, Ri is about 108:1. The weight ratio can be obtained using an approximate MW of 650 - 800 for the phospholipid.

[0072] In some embodiments, the molar ratio of lipid:ApoA-I equivalents ("RSM") ranges from about 80:1 to about 110:1, such as from about 80:1 to about 100:1. In one specific example, the RSM of the complex can be about 82:1.

[0073] In some embodiments, the lipoprotein complex used in the methods of the present disclosure is a charged complex comprising a protein fraction that is preferably the mature full-length ApoA-I, and a lipid fraction comprising neutral lipids, sphingomyelin (SM), and charged lipids.

[0074] In a specific embodiment, the lipid component contains SM (e.g., egg SM, palmitoyl SM, phytosphingosine SM, or a combination thereof) and charged lipids (e.g., DPPG) in a weight ratio (SM:charged lipid) in the range of 90:10 to 99:1, more preferably in the range of 95:5 to 98:2, such as 97:3.

[0075] In a specific embodiment, the ratio of the protein component to the lipid component can range from about 1:2.7 to about 1:3, with 1:2.7 being preferred. This corresponds to a molar ratio of ApoA-I protein to lipid in the range of 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.

[0076] In certain embodiments, the complex comprises CER-001, CSL-111, CSL-112, CER-522, or ETC-216. In a preferred embodiment, the complex is CER-001.

[0077] CER-001 as used in the literature and in the following examples refers to the complex described in Example 4 of International Publication No. WO 2012 / 109162. International Publication No. WO 2012 / 109162 refers to CER-001 as a complex having a lipoprotein weight:total lipid weight ratio of 1:2.7 and a weight:weight ratio of SM:DPPG of 97:3. Example 4 of International Publication No. WO 2012 / 109162 also describes its manufacturing method.

[0078] When used in the context of the methods of the present disclosure and / or the CER-001 dosing regimen, CER-001 refers to a lipoprotein complex in which its individual components can vary by up to 20% from the CER-001 described in Example 4 of International Publication No. WO 2012 / 109162. In certain embodiments, the components of the lipoprotein complex vary by up to 10% from the CER-001 described in Example 4 of International Publication No. WO 2012 / 109162. Preferably, the components of the lipoprotein complex are those described in Example 4 of International Publication No. WO 2012 / 109162 (± variations within acceptable manufacturing tolerances). The SM in CER-001 can be natural or synthetic. In some embodiments, the SM is natural SM, such as the natural SM described in International Publication No. WO 2012 / 109162, such as chicken egg SM. In some embodiments, the SM is synthetic SM, such as the synthetic SM described in International Publication No. WO 2012 / 109162, such as synthetic palmitoyl sphingomyelin, such as that described in International Publication No. WO 2012 / 109162. Methods for synthesizing palmitoyl sphingomyelin are known in the art, such as those described in International Publication No. WO 2014 / 140787. The apolipoprotein A-I (ApoA-I), which is a lipoprotein included in CER-001, preferably has an amino acid sequence corresponding to amino acids 25 to 267 of SEQ ID NO: 2 (previously published as SEQ ID NO: 1 in International Publication No. WO 2012 / 109162). ApoA-I can be purified from an animal source (particularly from a human source) or recombinantly produced. In a preferred embodiment, the ApoA-I in CER-001 is recombinant ApoA-I. CER-001 used in the dosing regimen of the present disclosure is preferably highly homogeneous, such as 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, for example, Section 6.4 of International Publication No. WO 2012 / 109162.

[0079] In a particular embodiment, the ApoA-I in CER-001 is recombinant ApoA-I produced by a mammalian host cell. The host cell can be obtained from any mammalian cell line. The polynucleotide encoding ApoA-I can be codon-optimized for expression in a recombinant host cell. Preferred host cells are mammalian host cells, including, but not limited to, Chinese hamster ovary cells (e.g., CHO-K1; ATCC number CCL61; CHO-S (GIBCO Life Technologies Inc., Rockville, MD, catalog number 11619012)), Vero cells, BHK (ATCC number CRL1632), BHK570 (ATCC number CRL10314), HeLa cells, COS-1 (ATCC number CRL1650), COS-7 (ATCC number CRL1651), MDCK cells, 293 cells (ATCC number CRL1573; Graham et al. J. Gen. Virol. 36:59-72, 1977), 3T3 cells, myeloma cells (especially mouse-derived), PC12 cells, and W138 cells. In certain embodiments, mammalian cells, such as CHO-S cells (Invitrogen™, Carlsbad CA), are configured for growth in serum-free media. Additional suitable cell lines are known in the art and are available from public depositories such as the American Type Culture Collection (ATCC, Manassas, VA).

[0080] In a preferred embodiment, the recombinant ApoA-I is produced by CHO cells. Recombinant ApoA-I expressed by a mammalian host cell, such as a CHO cell, may undergo post-translational modifications (e.g., glycosylation). The resulting recombinant ApoA-I may have one or more structural features (e.g., glycosylation patterns) that differ from ApoA-I purified from human plasma.

[0081] In the case of recombinant expression of ApoA-I, a polynucleotide encoding ApoA-I is operably linked to one or more control sequences, such as a promoter or terminator, that regulate the expression of ApoA-I in the host cell of interest. The control sequence(s) may be native or foreign to the ApoA-I coding sequence and may also be native or foreign to the host cell in which ApoA-I is expressed. Control sequences include, but are not limited to, a promoter, ribosome binding site, leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, and transcription termination sequence. In some embodiments, the control sequence includes a promoter, ribosome binding site, and transcription and translation stop signals. The control sequence also includes one or more linkers for introducing specific restriction sites and can facilitate the binding of the control sequence to the coding region of the nucleotide sequence encoding ApoA-I.

[0082] The promoter driving the recombinant expression of ApoA-I can be a constitutive promoter, a regulated promoter, or an inducible promoter. Suitable promoter sequences can be obtained from genes encoding extracellular or intracellular polypeptides that are endogenous or heterologous to the host cell. Methods for isolating, identifying, and manipulating promoters of various strengths are available or can be readily applied from the art. See, for example, Nevoigt et al. (2006) Appl. Environ. Microbiol. 72:5266-5273, the disclosure of which is incorporated herein by reference in its entirety.

[0083] One or more control arrays can be derived from a viral source. For example, in certain embodiments, the promoter is derived from the major late promoter of polyomavirus or adenovirus. In other embodiments, the promoter is from Simian virus 40 (SV40) (Fiers et al., 1978, Nature, 273:113-120), which can be obtained as a fragment that also includes the SV40 virus origin of replication, or from cytomegalovirus, such as the simian cytomegalovirus immediate early promoter (see U.S. Patent No. 4,956,288). Other suitable promoters include those derived from the metallothionein gene (see U.S. Patent Nos. 4,579,821 and 4,601,978).

[0084] Recombinant ApoA-I expression vectors are also provided herein. The recombinant expression vector can be any vector, such as a plasmid or a virus, which is operable by recombinant DNA techniques to promote the expression of heterologous ApoA-I in a recombinant host cell. The expression vector is integratable into the chromosome of the recombinant host cell and contains one or more heterologous genes operably linked to one or more control sequences useful for ApoA-I production. In other embodiments, the expression vector is an extrachromosomally replicable DNA molecule, such as a linear or circular plasmid, found in a low copy number (e.g., about 1 to about 10 copies per genome equivalent) or a high copy number (e.g., more than 10 copies per genome equivalent) state. In various embodiments, the expression vector contains a selectable marker, such as a gene conferring antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol, or tetracycline resistance) to the recombinant host organism containing the vector. In a particular embodiment, the DNA construct, vector, and polynucleotide are suitable for ApoA-I expression in mammalian cells. A vector for expressing ApoA-I in mammalian cells can contain an origin of replication, a promoter, and any required ribosome binding site, RNA splice site, polyadenylation site, and transcription termination sequence that are compatible with the host cell line. In some embodiments, the origin of replication is heterologous to the host cell and is, for example, of viral origin (SV40, polyoma virus, adenovirus, VSV, BPV). In other embodiments, the origin of replication is provided by the chromosomal replication machinery of the host cell.

[0085] Methods, reagents, and tools for introducing foreign DNA into mammalian host cells are known in the art and include, but are not limited to, calcium phosphate-mediated transfection (Wigler et al., 1978, Cell 14:725; Corsaro et al., 1981, Somatic Cell Genetics 7:603; Graham et al., 1973, Virology 52:456), electroporation (Neumann et al., 1982, EMBO J. 1:841-5), DEAE-dextran-mediated transfection (Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3rd Edition (John Wiley & Sons 1995)), and liposome-mediated transfection (Hawley-Nelson et al., 1993, Focus 15:73; Ciccarone et al., 1993, Focus 15:80).

[0086] In the case of high-yield production, stable expression of ApoA-I is preferred. For example, after introducing foreign DNA into a host cell, the host cell can be grown in an enriched medium for 1-2 days and then switched to a selective medium. Instead of using an expression vector containing a viral origin of replication, the host cell can be transformed with a vector containing a nucleotide sequence comprising an ApoA-I coding sequence controlled by appropriate expression control elements and a selectable marker. The selectable marker in the vector confers resistance to selection and allows the cell to stably integrate the vector into its chromosome and grow to form foci, resulting in the ability to clone and expand into a cell line. Several selection systems can be used, including but not limited to those containing the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, although they are also tk - , hgprt - , or aprt -They can each be employed intracellularly. Also, for example, dhfr that confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527), gpt that confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072), neo that confers resistance to aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1), and / or hyg that confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147) can be used to use anti-metabolite resistance as a criterion for selection.

[0087] Stable high-yield expression can also be achieved by using a retroviral vector that integrates into the host cell genome (see, for example, U.S. Patent Publication Nos. 2008 / 0286779 and 2004 / 0235173). Alternatively, stable high-yield expression of ApoA-I can be achieved by a gene activation method, for example, activating the expression of the endogenous ApoA-I gene in the genomic DNA of a selected mammalian cell and requiring its amplification as described in International Publication No. 1994 / 012650. Increasing the copy number of the ApoA-I gene (including the ApoA-I coding sequence and one or more control elements) can promote high-yield expression of ApoA-I. Preferably, the mammalian host cell in which ApoA-I is expressed has at least 2, at least 3, at least 4, or at least 5 ApoA-I gene copy indices. In a specific embodiment, the mammalian host cell in which ApoA-I is expressed has at least 6, at least 7, at least 8, at least 9, or at least 10 ApoA-I gene copy indices.

[0088] In certain embodiments, the mammalian cells are configured to produce ApoA-I in an amount of at least 0.5 g / L, at least 1 g / L, at least 1.5 g / L, at least 2 g / L, at least 2.5 g / L, at least 3 g / L, at least 3.5 g / L, and optionally, up to 4 g / L, up to 4.5 g / L, up to 5 g / L, up to 5.5 g / L, or up to 6 g / L. The mammalian host cells preferably have the ability to produce at least about 0.5, 1, 2, or 3 g / L of ApoA-I in culture and / or up to about 20 g / L of ApoA-I in culture, such as up to 4, 5, 6, 7, 8, 9, 10, 12, or 15 g / L of ApoA-I in culture.

[0089] In certain embodiments, the mammalian cells are configured to grow in serum-free medium. In these embodiments, ApoA-I is secreted from the cells. In other embodiments, ApoA-I is not secreted from the cells.

[0090] The mammalian host cells provided herein can be used to produce ApoA-I. Generally, the method includes culturing the mammalian host cells described herein under conditions in which ApoA-I is expressed. Further, the method can include recovering mature ApoA-I from the supernatant of the mammalian cell culture and optionally purifying it.

[0091] The culture conditions (including culture medium, temperature, pH) can be adapted to the mammalian host cells being cultured and the selected culture mode (shake flask, bioreactor, roller bottle, etc.). The mammalian cells can grow in large-scale batch culture methods or in continuous or semi-continuous culture methods.

[0092] Also provided herein are mammalian cell cultures comprising a plurality of ApoA-I-producing mammalian host cells as described herein. In some embodiments, the mammalian cell culture comprises at least 0.5 g / L, at least 1 g / L, at least 1.5 g / L, at least 2 g / L, at least 2.5 g / L, at least 3 g / L, at least 3.5 g / L, and optionally up to 4 g / L, up to 4.5 g / L, up to 5 g / L, up to 5.5 g / L, or up to 6 g / L of ApoA-I. The culture can be at any scale ranging from about 150 mL to about 500 mL, 1 L, 10 L, 15 L, 50 L, 100 L, 200 L, 250 L, 300 L, 350 L, 400 L, 500 L, 750 L, 1000 L, 1500 L, 2000 L, 2500 L, 3000 L, 5000 L, 7500 L, 10000 L, 15000 L, 20000 L, 25000 L, 50000 L, or more. In some cases, the culture is a large-scale culture, such as 15 L, 20 L, 25 L, 30 L, 50 L, 100 L, 200 L, 300 L, 500 L, 1000 L, 5000 L, 10000 L, 15000 L, 20000 L, 25000 L, up to 50000 L, or more.

[0093] The mammalian host cells of the disclosure can grow in culture. Accordingly, the disclosure further provides for mammalian cell cultures comprising a plurality of mammalian host cells as described above. The cell culture has the following characteristics: (a) The culture (optionally, a large-scale batch culture of at least 10 liters, at least 20 liters, at least 30 liters, at least 50 liters, at least 100 liters, 300 L, 500 L, 1000 L, 5000 L, 10,000 L, 15,000 L, 20,000 L, 25,000 L, up to 50,000 L, or a continuous culture of at least 10 liters, at least 20 liters, at least 30 liters, at least 50 liters, at least 100 liters, 300 L, 500 L, 1000 L, 5000 L, or up to 10,000 L) contains or consists of a mature ApoA-I protein comprising the amino acid sequence corresponding to amino acids 25 - 267 of SEQ ID NO:2 at at least about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 g / L or more; (b) At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the protein in the culture medium is ApoA-I protein lacking a signal sequence; (c) At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the protein in the culture medium is mature ApoA-I protein lacking a signal sequence and a propeptide sequence; and (d) At least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the mature ApoA-I is not truncated, oxidized, or deamidated and may include one or more of the above characteristics.

[0094] CSL - 111 is a complex of reconstituted human ApoA-I purified from plasma with soybean phosphatidylcholine (SBPC) (Tardif et al., 2007, JAMA 297:1675 - 1682).

[0095] CSL-112 is a formulation of ApoA-I that is purified from plasma, reconstituted, and forms HDL suitable for intravenous injection (Diditchenko et al., 2013, DOI 10.1161 / ATVBAHA.113.301981).

[0096] ETC-216 (also known as MDCO-216) is a lipid-depleted form of HDL containing recombinant ApoA-I ミラノ See Nicholls et al., 2011, Expert Opin Biol Ther. 11(3):387-94. doi: 10.1517 / 14712598.2011.557061.

[0097] In another embodiment, the complex that can be used in the methods of the present disclosure is CER-522. CER-522 is a lipoprotein complex comprising a combination of three phospholipids and a 22-amino acid peptide CT80522:

[0098]

Chemical formula

[0099] The phospholipid component of CER-522 consists of egg sphingomyelin, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dipalmitoyl phosphatidylcholine, DPPC), and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (dipalmitoyl phosphatidyl-glycerol, DPPG) in a weight ratio of 48.5:48.5:3. The ratio of peptide to total phospholipid in the CER-522 complex is 1:2.5 (w / w).

[0100] In some embodiments, the lipoprotein complex is delipidated HDL. Most HDL in plasma is cholesterol-rich. The lipids in HDL can be depleted, e.g., partially and / or selectively depleted, e.g., to reduce its cholesterol content. In some embodiments, delipidated HDL may resemble the small α, preβ-1, and other preβ forms of HDL. The process of selective depletion of HDL is described in Sacks et al., 2009, J Lipid Res. 50(5): 894-907.

[0101] In certain embodiments, the lipoprotein complex comprises bioactive agent delivery particles described in U.S. Patent Application Publication No. 2004 / 0229794.

[0102] The bioactive agent delivery particles can include a lipid-binding polypeptide (e.g., an apolipoprotein already described in this section or in Section 6.1.2), a lipid bilayer (e.g., one containing one or more phospholipids already described 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 contains a hydrophobic region and the bioactive agent is associated with the hydrophobic region of the lipid bilayer. In some embodiments, the bioactive agent delivery particles described in U.S. Patent Application Publication No. 2004 / 0229794.

[0103] In some embodiments, the bioactive agent delivery particles do not include a hydrophilic core.

[0104] In some embodiments, the bioactive agent delivery particles are disk-shaped (e.g., having a diameter of about 7 to about 29 nm).

[0105] The bioactive agent delivery particles include bilayer-forming lipids, such as phospholipids (e.g., those already described in this section or in Section 6.1.3.1). In some embodiments, the bioactive agent delivery particles include both bilayer-forming and non-bilayer-forming lipids. In some embodiments, the lipid bilayer of the bioactive agent delivery particles includes phospholipids. In one embodiment, examples of phospholipids incorporated into the delivery particles include dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG). In one embodiment, the lipid bilayer includes DMPC and DMPG in a molar ratio of 7:3.

[0106] In some embodiments, the lipid-binding polypeptide is an apolipoprotein (e.g., those already described in this section or in Section 6.1.2). The predominant interaction between a lipid-binding polypeptide, such as an apolipoprotein molecule, and the lipid bilayer is generally a hydrophobic interaction between residues on the hydrophobic surface of the amphiphilic structure, such as the α-helix of the lipid-binding polypeptide and the fatty acid acyl chains of the lipids on the outer surface of the particle periphery. Examples of bioactive agent delivery particles can include exchangeable and / or non-exchangeable apolipoproteins. In one embodiment, the lipid-binding polypeptide is ApoA-I.

[0107] In some embodiments, examples of bioactive agent delivery particles include lipid-binding polypeptide molecules, such as apolipoprotein molecules, modified to increase the stability of the particles. In one embodiment, examples of the modification include the introduction of cysteine residues to form intramolecular and / or intermolecular disulfide bonds.

[0108] In another embodiment, examples of bioactive agent delivery particles include chimeric lipid-binding polypeptide molecules, such as chimeric apolipoprotein molecules, having one or more attached functional moieties that can enhance or act synergistically with the activity of the bioactive agent incorporated into the delivery particles, such as one or more targeting moieties and / or one or more moieties having a desired biological activity, such as antimicrobial activity.

[0109] 6.1.2. Lipid-binding protein molecules Examples of lipid-binding protein molecules that can be used in the complexes described herein include apolipoproteins such as those described in Section 6.1.2.1, and apolipoprotein mimetic peptides such as those described in Section 6.1.2.2. In some embodiments, the complex comprises a mixture of lipid-binding protein molecules. In some embodiments, the complex comprises a mixture of one or more lipid-binding protein molecules and one or more apolipoprotein mimetic peptides.

[0110] In some embodiments, the complex comprises 1 to 8 ApoA-I equivalents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-8, 2-6, 2-4, 4-6, or 4-8 ApoA-I equivalents). Lipid-binding proteins can be expressed in terms of ApoA-I equivalents based on the number of amphipathic helices they contain. For example, ApoA-I, which typically exists as a disulfide-bridged dimer, can be expressed as 2 ApoA-I equivalents because each molecule of ApoA-I contains twice the number of amphipathic helices as an ApoA-I molecule. Conversely, a peptide mimetic containing a single amphipathic helix can be expressed as 1 / 10 to 1 / 6 ApoA-I equivalents because each molecule contains 1 / 10 to 1 / 6 the number of amphipathic helices as an ApoA-I molecule. M is M such that each molecule of ApoA-I contains twice the number of amphipathic helices as an ApoA-I molecule and can thus be expressed as 2 ApoA-I equivalents. Conversely, a peptide mimetic containing a single amphipathic helix can be expressed as 1 / 10 to 1 / 6 ApoA-I equivalents because each molecule contains 1 / 10 to 1 / 6 the number of amphipathic helices as an ApoA-I molecule.

[0111] 6.1.2.1. Apolipoproteins Suitable apolipoproteins that can be included in a complex based on a lipid-binding protein include apolipoprotein ApoA-I, ApoA-II, ApoA-IV, ApoA-V, ApoB, ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE, ApoJ, ApoH, and any combination of two or more of the foregoing. Polymorphic forms, isoforms, variants, and mutants of the foregoing apolipoproteins, as well as truncated forms, can also be used, the most common of which are apolipoprotein A-I Milano (ApoA-IM), apolipoprotein A-I Paris (ApoA-IP), and apolipoprotein A-I Zaragoza (ApoA-IZ). Apolipoprotein mutants containing cysteine residues are also known and can also be used (see, for example, U.S. Patent Publication No. 2003 / 0181372). The apolipoprotein can be in monomeric form, or in dimeric form which can be homodimeric or heterodimeric. For example, homodimers and heterodimers (where possible) of ApoA-I (Duverger et al., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29), ApoA-IM (Franceschini et al., 1985, J. Biol. Chem. 260:1632-35), ApoA-IP (Daum et al., 1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol. Chem. 260(14):8637-46, Shelness et al., 1984, J. Biol. Chem. 259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem. 201(2):373-83), ApoE (McLean et al., 1983, J. Biol. Chem. 258(14):8993-9000), ApoJ, and ApoH can be used.

[0112] Apolipoproteins can have their primary sequences modified to be less susceptible to oxidation, as described, for example, in U.S. Patent Publication Nos. 2008 / 0234192 and 2013 / 0137628, and U.S. Patent Nos. 8,143,224 and 8,541,236. Apolipoproteins can contain residues corresponding to elements that facilitate their isolation, such as His tags, or other elements designed for other purposes. Preferably, the apolipoprotein in the complex is soluble in biological fluids (e.g., lymph, cerebrospinal fluid, vitreous humor, aqueous humor, blood, or blood fractions such as serum or plasma).

[0113] In some embodiments, the complex comprises a covalently bound lipid-binding protein monomer, such as the dimeric apolipoprotein A-I Milano, a variant of ApoA-I containing cysteine. Cysteine enables the formation of disulfide bridges, which can lead to the formation of homodimers or heterodimers (e.g., ApoA-I Milano - ApoA-II).

[0114] In some embodiments, the apolipoprotein molecule comprises an ApoA-I, ApoA-II, ApoA-IV, ApoA-V, ApoB, ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE, ApoJ, or ApoH molecule, or a combination thereof.

[0115] In some embodiments, the apolipoprotein molecule comprises or consists of an ApoA-I molecule. In some embodiments, the ApoA-I molecule is a human ApoA-I molecule. In some embodiments, the ApoA-I molecule is recombinant. In some embodiments, the ApoA-I molecule is not ApoA-I Milano.

[0116] In some embodiments, the ApoA-I molecule is an apolipoprotein A-I Milano (ApoA-IM), apolipoprotein A-I Paris (ApoA-IP), or apolipoprotein A-I Zaragoza (ApoA-IZ) molecule.

[0117] Apolipoproteins can be purified from animal sources (especially human sources) or recombinantly produced, as is well known in the art. See, for example, 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. Patent Publication Nos. 2002 / 0156007, 2004 / 0067873, 2004 / 0077541, and 2004 / 0266660, as well as International Publication Nos. 2008 / 104890 and 2007 / 023476. Other purification methods are possible, such as those described in International Publication No. 2012 / 109162, the disclosure of which is hereby incorporated by reference in its entirety. ApoA-I molecules can be produced recombinantly, for example, in CHO cells, as described elsewhere herein.

[0118] Apolipoproteins can be in a prepro form, a pro form, or a mature form. For example, the complex can contain ApoA-I (e.g., human ApoA-I), wherein the ApoA-I is prepro ApoA-I, pro ApoA-I, or mature ApoA-I. In some embodiments, the complex contains ApoA-I having at least 90% sequence identity to SEQ ID NO:1.

[0119]

Chemical formula

[0120] In other embodiments, the complex comprises ApoA-I having at least 95% sequence identity to SEQ ID NO: 1. In other embodiments, the complex comprises ApoA-I having at least 98% sequence identity to SEQ ID NO: 1. In other embodiments, the complex comprises ApoA-I having at least 99% sequence identity to SEQ ID NO: 1. In other embodiments, the complex comprises ApoA-I having 100% sequence identity to SEQ ID NO: 1.

[0121] In some embodiments, the complex is SEQ ID NO: 2:

[0122]

Chemical formula

[0123] In other embodiments, the complex comprises ApoA-I having at least 95% sequence identity to amino acids 25-267 of SEQ ID NO: 2. In other embodiments, the complex comprises ApoA-I having at least 98% sequence identity to amino acids 25-267 of SEQ ID NO: 2. In other embodiments, the complex comprises ApoA-I having at least 99% sequence identity to amino acids 25-267 of SEQ ID NO: 2. In other embodiments, the complex comprises ApoA-I having 100% sequence identity to amino acids 25-267 of SEQ ID NO: 2.

[0124] In some embodiments, the complex comprises from 1 to 8 apolipoprotein molecules (e.g., from 1 to 6, from 1 to 4, from 1 to 2, from 2 to 8, from 2 to 6, from 2 to 4, from 4 to 8, from 4 to 6, or from 6 to 8 apolipoprotein molecules). In some embodiments, the complex comprises 1 apolipoprotein molecule. In some embodiments, the complex comprises 2 apolipoprotein molecules. In some embodiments, the complex comprises 3 apolipoprotein molecules. In some embodiments, the complex comprises 4 apolipoprotein molecules. In some embodiments, the complex comprises 5 apolipoprotein molecules. In some embodiments, the complex comprises 6 apolipoprotein molecules. In some embodiments, the complex comprises 7 apolipoprotein molecules. In some embodiments, the complex comprises 8 apolipoprotein molecules.

[0125] The apolipoprotein molecule can comprise a chimeric apolipoprotein comprising an apolipoprotein and one or more attached functional moieties, such as one or more CRN-001 complexes, one or more targeting moieties, a moiety having a desired biological activity, an affinity tag for aiding purification, and / or a reporter molecule for characterization or localization studies. The attached moiety having biological activity can have an activity capable of enhancing and / or synergizing with the biological activity of a compound incorporated into the complex of the present disclosure. For example, the moiety having biological activity can have antimicrobial (e.g., antifungal, antibacterial, antiprotozoal, bacteriostatic, fungistatic, or antiviral) activity. In one embodiment, the attached functional moiety of the chimeric apolipoprotein is not in contact with the hydrophobic surface of the complex. In another embodiment, the attached functional moiety is in contact with the hydrophobic surface of the complex. In some embodiments, the functional moiety of the chimeric apolipoprotein can be endogenous to the native protein. In some embodiments, the chimeric apolipoprotein comprises a ligand or sequence that can be recognized by or interact with a cell surface receptor or other cell surface moiety.

[0126] In one embodiment, the chimeric apolipoprotein comprises a targeting moiety that is not endogenous to the native apolipoprotein, such as, for example, the budding yeast (S. cerevisiae) α - mating factor peptide, folic acid, transferrin, or lactoferrin. In another embodiment, the chimeric apolipoprotein comprises a moiety having a desired biological activity that enhances and / or synergizes with the activity of the compound incorporated into the complex of the present disclosure. In one embodiment, the chimeric apolipoprotein may comprise a functional moiety that is endogenous to the apolipoprotein. An example of an apolipoprotein - endogenous functional moiety is an endogenous targeting moiety substantially formed by amino acids 130 - 150 of human ApoE, including a receptor - binding region recognized by a member of the low - density lipoprotein receptor family. Other examples of apolipoprotein - endogenous functional moieties include the region of ApoB - 100 that interacts with the low - density lipoprotein receptor and the region of ApoA - I that interacts with the scavenger receptor B1. In other embodiments, the functional moiety may be added synthetically or recombinantly to yield a chimeric apolipoprotein. Another example is an apolipoprotein having a prepro or pro sequence from another preproapolipoprotein (e.g., one in which the prepro sequence of preproapolipoprotein A - I is replaced with the prepro sequence from preproproapolipoprotein A - II). Another example is an apolipoprotein in which a portion of an amphipathic sequence segment is replaced by another amphipathic sequence segment from another apolipoprotein.

[0127] As used herein, "chimera" refers to two or more molecules that can exist separately and combine with each other to form a single molecule having all the desired functionality of its constituent molecules. The constituent molecules of a chimeric molecule can be synthetically joined together by chemical conjugation, or, when all the constituent molecules are polypeptides or analogs thereof, the polynucleotides encoding the polypeptides can be recombinantly fused together such that a single continuous polypeptide is expressed. Such chimeric molecules are referred to as fusion proteins. A "fusion protein" is a chimeric molecule in which all the constituent molecules are polypeptides and are attached (fused) to each other so as to form a continuous single chain. The various components can be attached directly to each other or can be coupled through one or more linkers. One or more segments of the various components can be inserted, for example, into the sequence of an apolipoprotein, or, as another example, added to the N-terminus or C-terminus of the sequence of an apolipoprotein. For example, a fusion protein can include an antibody light chain, an antibody fragment, a heavy chain antibody, or a single domain antibody.

[0128] In some embodiments, chimeric apolipoproteins are prepared by chemically conjugating a functional moiety to be attached to the apolipoprotein. Means for chemically conjugating molecules are well known to those of skill in the art. Such means will vary depending on the structure of the moiety to be attached, but will be readily ascertainable by those of skill in the art. Polypeptides typically contain various functional groups, such as carboxylic acid (-COOH), free amino (-NH2), or sulfhydryl (-SH) groups, that can be reacted with appropriate functional groups on the functional moiety or on a linker to attach the moiety thereto. The functional moiety can be attached to a functional group on the N-terminus, C-terminus, or internal residue (i.e., one residue intermediate the N-terminus and C-terminus) of the apolipoprotein molecule. Alternatively, the apolipoprotein and / or the tagging moiety can be derivatized to expose or attach additional reactive functional groups.

[0129] In some embodiments, the fusion protein comprising the polypeptide functional portion is synthesized using a recombinant expression system. Typically, this involves creating a nucleic acid (e.g., DNA) sequence that encodes the apolipoprotein and the functional portion such that the two polypeptides are 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.

[0130] The nucleic acid encoding the chimeric apolipoprotein can be incorporated into a recombinant expression vector in a form suitable for expression in a host cell. As used herein, an "expression vector" is a nucleic acid that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. The vector can include regulatory sequences such as a promoter, enhancer, or other expression control elements (e.g., polyadenylation signal). Such regulatory sequences are known to those of skill in the art (see, e.g., Goeddel, 1990, Gene Expression Technology: Meth. Enzymol. 185, Academic Press, San Diego, Calif., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, Calif., Sambrook et al., 1989, Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, etc.).

[0131] In some embodiments, the apolipoprotein is modified such that when the apolipoprotein is incorporated into the complex of the present disclosure, the modification increases the stability of the complex and confers or increases the targeting ability. In one embodiment, the modification includes, for example, the introduction of cysteine residues into the apolipoprotein molecule to enable the formation of intramolecular or intermolecular disulfide bonds, such as by site-directed mutagenesis. In another embodiment, an intermolecular linkage is formed between apolipoprotein molecules using a chemical crosslinking agent to enhance the stability of the complex. The intermolecular crosslinking bond prevents or reduces the dissociation of apolipoprotein molecules from the complex and / or prevents the replacement of endogenous apolipoprotein molecules in an individual administered with the complex. In other embodiments, the apolipoprotein is modified either by chemical derivatization or site-directed mutagenesis of one or more amino acid residues to confer a targeting ability or recognition thereof to cell surface receptors.

[0132] The complex can be targeted to specific cell surface receptors by engineering receptor recognition specificity into the apolipoprotein. For example, the complex can be targeted to specific cell types known to carry certain types of infectious agents by, for example, modifying the apolipoprotein to enable it to interact with receptors on the surface of the cell type to which it is targeted. For example, the complex can be targeted to macrophages by modifying the apolipoprotein to confer recognition by the macrophage endocytosis class A scavenger receptor (SR-A). SR-A binding ability can be conferred on the complex by modifying the apolipoprotein by site-directed mutagenesis to replace one or more positively charged amino acids with neutral or negatively charged amino acids. SR-A recognition can also be conferred by preparing chimeric apolipoproteins that contain an N- or C-terminal extension with a ligand recognized by SR-A or an amino acid sequence with a high density of negatively charged residues. Complexes containing apolipoproteins can also interact with apolipoprotein receptors such as, but not limited to, the ABCA1 receptor, the ABCG1 receptor, Megalin, Cubulin, and HDL receptors such as SR-B1.

[0133] 6.1.2.2. Apolipoprotein mimetics Peptides, peptidomimetics, and agonists that mimic the activity of apolipoproteins (collectively referred to herein as "apolipoprotein peptidomimetics") can also be used in the complexes described herein, either alone or in combination with one or more other lipid-binding proteins. Peptides and peptidomimetics corresponding to apolipoproteins, as well as ApoA-I, ApoA-I, that are suitable for inclusion in the complexes and compositions described herein M, Non-limiting examples of agonists that mimic the activities of ApoA-II, ApoA-IV, and ApoE are described in U.S. Patent Nos. 6,004,925, 6,037,323, and 6,046,166 (issued to Dasseaux et al.), U.S. Patent No. 5,840,688 (issued to Tso), U.S. Patent No. 6,743,778 (issued to Kohno), U.S. Patent Publication Nos. 2004 / 0266671, 2004 / 0254120, 2003 / 0171277, and 2003 / 0045460 (Fogelman), U.S. Patent Publication No. 2006 / 0069030 (Bachovchin), U.S. Patent Publication No. 2003 / 0087819 (Bielicki), U.S. Patent Publication No. 2009 / 0081293 (Murase et al.), and International Publication No. 2010 / 093918 (Dasseux et al.), the disclosures of which are incorporated herein by reference in their entireties. These peptides and peptide analogs may be composed of L-amino acids or D-amino acids or mixtures of L- and D-amino acids. They may also include one or more non-peptide or amide bonds, such as one or more well-known peptide / amide isosteres. Such apolipoprotein peptide mimetics can be synthesized or manufactured using any technique of peptide synthesis known in the art, including, for example, the techniques described in U.S. Patent Nos. 6,004,925, 6,037,323, and 6,046,166.

[0134] In some embodiments, the lipid-binding protein molecule includes an apolipoprotein peptide mimetic molecule and optionally one or more apolipoprotein molecules, such as those described above.

[0135] In some embodiments, the apolipoprotein peptide mimetic molecule includes an ApoA-I peptide mimetic, an ApoA-II peptide mimetic, an ApoA-IV peptide mimetic, or an ApoE peptide mimetic, or a combination thereof.

[0136] 6.1.3. Amphiphilic Molecules An amphiphilic molecule is a molecule that possesses both hydrophobic (nonpolar) and hydrophilic (polar) elements. Amphiphilic molecules that can be used in the 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 nonpolar molecules and sterols (e.g., as described in Section 6.1.3.4) that are covalently attached to polar molecules such as, but not limited to, sugars or nucleic acids.

[0137] The complex can include a single class of amphiphilic molecules (e.g., a single species of phospholipid or a mixture of phospholipids), or can contain a combination of classes of amphiphilic molecules (e.g., phospholipids and detergents). The complex can contain one type of amphiphilic molecule or a combination of amphiphilic molecules that are configured to facilitate solubilization of lipid-binding protein molecules.

[0138] In some embodiments, the amphiphilic molecules included include phospholipids, detergents, fatty acids, nonpolar moieties or sterols covalently attached to sugars, or combinations thereof (e.g., selected from the types of amphiphilic molecules described above).

[0139] In some embodiments, the amphiphilic molecule comprises or consists of phospholipid molecules. In some embodiments, the phospholipid molecules include negatively charged phospholipids, neutral phospholipids, positively charged phospholipids, or combinations thereof. In some embodiments, the phospholipid molecules contribute from 1 to 3 effective charges per apolipoprotein molecule in the complex. In some embodiments, the effective charge is a negative effective charge. In some embodiments, the effective charge is a positive effective charge. In some embodiments, the phospholipid molecules consist of a combination of negatively charged and neutral phospholipids. In some embodiments, the molar ratio of negatively charged phospholipids to neutral phospholipids ranges from 1:1 to 1:3. In some embodiments, the molar ratio of negatively charged phospholipids to neutral phospholipids is about 1:1 or about 1:2.

[0140] In some embodiments, the amphiphilic molecule comprises a neutral phospholipid and a charged phospholipid in a weight ratio of 95:5 to 99:1.

[0141] 6.1.3.1. Lipids Complexes based on lipid-binding proteins can comprise one or more lipids. In various embodiments, the one or more lipids can be saturated and / or unsaturated, natural and / or synthetic, charged or uncharged, zwitterionic or not. In some embodiments, lipid molecules (e.g., phospholipid molecules) can together contribute an effective charge of 1 to 3 (e.g., 1 to 3, 1 to 2, 2 to 3, 1, 2, or 3) per lipid-binding protein molecule in the complex. In some embodiments, the effective charge is negative. In other embodiments, the effective charge is positive.

[0142] In some embodiments, the lipid comprises a phospholipid. The phospholipid can have two same or different acyl chains (e.g., chains having a different number of carbon atoms, a different degree of saturation between acyl chains, a 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 avantilipids.com / product-category / products / fluorescent-lipids / ). Preferably, the lipid comprises at least one phospholipid.

[0143] The phospholipid can have an unsaturated or saturated acyl chain with about 6 to about 24 carbon atoms (e.g., 6 to 20, 6 to 16, 6 to 12, 12 to 24, 12 to 20, 12 to 16, 16 to 24, 16 to 20, or 20 to 24). In some embodiments, the phospholipid used in the complexes of the present 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 lengths).

[0144] Non-limiting examples of acyl chains present in commonly occurring fatty acids that may be contained in phospholipids are provided in Table 1 below.

[0145] [Table 1]

[0146] Lipids that can be present in the complexes of the present disclosure include, but are not limited to, small alkyl chain phospholipids, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-myristoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dilauroyl phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, for example, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, brain phosphatidylserine, brain sphingomyelin, palmitoyl sphingomyelin, dipalmitoyl sphingomyelin, egg sphingomyelin, milk sphingomyelin, phytosphingomyelin, distearoyl sphingomyelin, dipalmitoyl phosphatidylglycerol salt, phosphatidic acid, galactosylceramide, ganglioside, cerebroside, dilauryl phosphatidylcholine, (1,3)-D-mannosyl-(1,3) diglyceride, aminophenyl glycoside, 3-cholesteryl-6'-(glycosylthio)hexyl ether glycolipid, and cholesterol and its derivatives. Lipid oxidation can be minimized using synthetic lipids such as synthetic palmitoyl sphingomyelin or N-palmitoyl-4-hydroxysphinganine-1-phosphocholine (a form of phytosphingomyelin).

[0147] In some embodiments, the lipid-binding protein-based complex comprises two types of phospholipids, namely neutral lipids such as lecithin and / or sphingomyelin (abbreviated as SM), and charged phospholipids (e.g., negatively charged phospholipids). "Neutral" phospholipids have an effective charge of approximately zero at physiological pH. In many embodiments, the neutral phospholipids are zwitterionic, although other types of net-neutral phospholipids are known and can be used. In some embodiments, the molar ratio of charged phospholipids (e.g., negatively charged phospholipids) to neutral phospholipids ranges from 1:1 to 1:3, e.g., about 1:1, about 1:2, or about 1:3.

[0148] The neutral phospholipids can include, for example, one or both of lecithin and / or SM, and optionally can include other neutral phospholipids. In some embodiments, the neutral phospholipids include lecithin but not SM. In other embodiments, the neutral phospholipids include SM but not lecithin. In still other embodiments, the neutral phospholipids include both lecithin and SM. All of these specific exemplary embodiments can include neutral phospholipids in addition to lecithin and / or SM, although in many embodiments such additional neutral phospholipids are not included.

[0149] As used herein, the expression "SM" includes sphingomyelin derived from or obtained from natural sources, as well as analogs and derivatives of naturally occurring SM that are not affected by hydrolysis by LCAT as naturally occurring SM is. SM is a phospholipid with a structure very similar to lecithin, but unlike lecithin, it does not have a glycerol backbone and thus does not have an ester linkage to attach acyl chains. Instead, SM has a ceramide backbone and an amide bond links the acyl chains. SM can be obtained, for example, from milk, eggs, or the brain. Analogs or derivatives of SM can also be used. Non-limiting examples of useful SM analogs and derivatives include, but are not limited to, palmitoyl sphingomyelin, N-palmitoyl-4-hydroxysphinganine-1-phosphocholine (a form of phytosphingomyelin), palmitoyl sphingomyelin, stearoyl sphingomyelin, D-erythro-N-16:0-sphingomyelin and its dihydro isomer, D-erythro-N-16:0-dihydro-sphingomyelin. Synthetic SM such as synthetic palmitoyl sphingomyelin or N-palmitoyl-4-hydroxysphinganine-1-phosphocholine (phytosphingomyelin) can be used to produce a more homogeneous complex with fewer contaminants and / or oxidation products than sphingolipids of animal origin. Methods for synthesizing SM are described in U.S. Patent Publication No. 2016 / 0075634.

[0150] Sphingomyelin isolated from natural sources can be artificially enriched for one specific saturated or unsaturated acyl chain. For example, milk sphingomyelin (Avanti Phospholipid, Alabaster, Alabama) is characterized by long saturated acyl chains (i.e., acyl chains having 20 or more carbon atoms). In contrast, egg sphingomyelin is characterized by short saturated acyl chains (i.e., acyl chains having fewer than 20 carbon atoms). For example, only about 20% of milk sphingomyelin contains the C16:0 (16 carbons, saturated) acyl chain, while about 80% of egg sphingomyelin contains the C16:0 acyl chain. Using solvent extraction, the composition of milk sphingomyelin can be enriched to have an acyl chain composition comparable to that of egg sphingomyelin, or vice versa.

[0151] SM can be semi-synthetic to have a specific acyl chain. For example, milk sphingomyelin can first be purified from milk and then one specific acyl chain, such as the C16:0 acyl chain, can be cleaved and replaced by another acyl chain. SM can also be fully synthesized, for example, by large-scale synthesis. See, for example, U.S. Patent No. 5,220,043 to Dong et al., issued June 15, 1993, entitled Synthesis of D-erythro-sphingomyelins, and Weis, 1999, Chem. Phys. Lipids 102 (1-2):3-12. SM can be fully synthetic, for example, as described in U.S. Patent Publication No. 2014 / 0275590.

[0152] The length and saturation level of the acyl chains, including semi-synthetic or synthetic SMs, 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 a different number of carbon atoms. In some embodiments, the semi-synthetic or synthetic SMs include 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. In other embodiments, the acyl chains of the semi-synthetic or synthetic SMs are either both saturated or both unsaturated. Again, the chains can contain the same or different numbers of carbon atoms. In some embodiments, both acyl chains, including the semi-synthetic or synthetic SMs, are identical. In specific embodiments, the chains correspond to acyl chains of naturally occurring fatty acids such as oleic acid, palmitic acid, or stearic acid. In another embodiment, SMs with saturated or unsaturated functionalized chains are used. In another specific embodiment, both acyl chains are saturated and contain 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.

[0153] In some embodiments, the SM is palmitoyl SM, such as synthetic palmitoyl SM having a C16:0 acyl chain, or egg SM containing palmitoyl SM as a major component.

[0154] In specific embodiments, functionalized SMs such as phytosphingomyelin are used.

[0155] Lecithin can be derived from or isolated from natural sources, or it can be obtained synthetically. Examples of suitable lecithins isolated from natural sources include, but are not limited to, egg phosphatidylcholine and soybean phosphatidylcholine. Further non-limiting examples of suitable lecithins include dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-myristoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1-oleoyl-2-palmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, and ether derivatives or analogs thereof.

[0156] Lecithin derived from or isolated from natural sources can be enriched to contain a specified acyl chain. In embodiments using semi-synthetic or synthetic lecithin, as described above in connection with SM, the acyl chain can be selectively varied as to what it is. In some embodiments of the complexes described herein, both acyl chains on the lecithin are the same. In some embodiments of complexes containing both SM and lecithin, all of the acyl chains of SM and lecithin are the same. In specific embodiments, the acyl chain corresponds to the acyl chain of myristic acid, palmitic acid, oleic acid, or stearic acid.

[0157] The complexes of the present disclosure can include one or more negatively charged phospholipids (e.g., alone or in combination with one or more neutral phospholipids). As used herein, "negatively charged phospholipid" refers to a phospholipid that has a net negative charge at physiological pH. The negatively charged phospholipid can include a single type of negatively charged phospholipid, or a mixture of two or more different negatively charged phospholipids. In some embodiments, the charged phospholipid is a negatively charged glycerophospholipid. Specific examples of suitable negatively charged phospholipids include, but are not limited to, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and salts thereof (e.g., sodium or potassium salts). In some embodiments, the negatively charged phospholipid includes one or more of phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, and / or phosphatidic acid. In a specific embodiment, the negatively charged phospholipid comprises, or consists of, a salt of phosphatidylglycerol or a salt of phosphatidylinositol. In another specific embodiment, the negatively charged phospholipid comprises, or consists of, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] or DPPG, or a salt thereof.

[0158] Charged phospholipids can be obtained from natural sources or prepared by chemical synthesis. In embodiments using synthetic charged phospholipids, as described above in connection with SM, the acyl chain can be selectively varied as to what it is. In some embodiments of the complexes of the present disclosure, both acyl chains on the charged phospholipid are the same. In some embodiments, the acyl chains of all types of phospholipids included in the complexes of the present disclosure are all the same. In a specific embodiment, the complex comprises a charged phospholipid and / or SM having all C16:0 or C16:1 acyl chains. In a specific embodiment, the fatty acid moiety of SM is mainly C16:1 palmitoyl. In a specific embodiment, the acyl chains of the charged phospholipid, lecithin and / or SM correspond to the acyl chains of palmitic acid. In yet another specific embodiment, the acyl chains of the charged phospholipid, lecithin and / or SM correspond to the acyl chains of oleic acid.

[0159] Examples of positively charged phospholipids that can be included in the complexes of the present disclosure include N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleoyloxy]-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-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-3-dimethylammonium-propane 1,2-dimyristoyl-3-dimethylammonium-propane, 1,2-dipalmitoyl-3-dimethylammonium-propane, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium, 1,2-dioleoyl-3-trimethylammonium-propane, 1,2-dioleoyl-3-trimethylammonium-propane, 1,2-stearoyl-3-trimethylammonium-propane, 1,2-dipalmitoyl-3-trimethylammonium-propane, 1,2-dimyristoyl-3-trimethylammonium-propane, N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide, N,N,N-trimethyl-2-bis[(1-oxo-9-octadecenyl)oxy]-(Z,Z)-1 propanaminium methyl sulfate, and salts thereof (e.g., chloride or bromide salts).

[0160] The lipid to be used is preferably at least 95% pure and / or has a reduced level of oxidizing agents (including but not limited to peroxides, etc.). Lipids obtained from natural sources preferably have fewer polyunsaturated fatty acid moieties and / or fatty acid moieties that are not sensitive to oxidation. The level of oxidation in the sample can be determined using an iodine titration method that provides a peroxide value, which is expressed as the milliequivalent number of isolated iodine per kg of sample and abbreviated as meq O / kg. See, for example, Gray, 1978, Measurement of Lipid Oxidation: A Review, Journal of the American Oil Chemists Society 55:539-545, Heaton, F.W. and Ur, Improved Iodometric Methods for the Determination of Lipid Peroxides, 1958, Journal of the Science of Food and Agriculture 9:781-786. Preferably, the level of oxidation, or peroxide level, is low, for example, 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.

[0161] In some embodiments, the complex can contain a small amount of additional lipid. Virtually any type of lipid can be used and is not limited thereto, including, but not limited to, lysophospholipids, galactocerebrosides, gangliosides, cerebrosides, glycerides, triglycerides, and sterols and sterol derivatives (e.g., plant sterols, animal sterols such as cholesterol, or sterol derivatives such as cholesterol derivatives). For example, the complexes of the present disclosure can contain cholesterol or a cholesterol derivative, such as a cholesterol ester. The cholesterol derivative can also be a substituted cholesterol or a substituted cholesterol ester. The complexes of the present disclosure can also contain oxidized sterols, such as, but not limited to, oxidized cholesterol or oxidized sterol derivatives (such as, but not limited to, oxidized cholesterol esters). In some embodiments, the complex does not contain cholesterol and / or its derivatives (cholesterol esters or oxidized cholesterol esters).

[0162] 6.1.3.2. Detergent The complex can contain one or more detergents. The detergents 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). Exemplary 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 sulfate, and alkyl sulfonate.

[0163] 6.1.3.3. Fatty Acids The complex can contain one or more fatty acids. The one or more fatty acids can include a monounsaturated fatty acid having an aliphatic tail of 5 or fewer carbons (e.g., butyric acid, isobutyric acid, valeric acid, or isovaleric acid), a medium-chain fatty acid having an aliphatic tail of 6 to 12 carbons (e.g., caproic acid, caprylic acid, capric acid, or lauric acid), a long-chain fatty acid having an aliphatic tail of 13 to 21 carbons (e.g., myristic acid, palmitic acid, stearic acid, or arachidic acid), a very-long-chain fatty acid having an aliphatic tail of 22 or more carbons (e.g., behenic acid, lignoceric acid, or cerotic acid), or a combination thereof. 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, linolelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, or docosahexaenoic acid), or a combination thereof. The unsaturated fatty acid can be a cis or trans fatty acid. In some embodiments, the unsaturated fatty acid used in the complexes of the present disclosure is a cis fatty acid.

[0164] 6.1.3.4. Sugar and Attached Nonpolar Molecules and Sterols The complex can contain one or more amphiphilic molecules including a nonpolar molecule or moiety (e.g., a hydrocarbon chain, 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). The sugar can be a modified sugar or a substituted sugar. Exemplary amphiphilic molecules including a sugar and an attached nonpolar molecule include dodecan-2-yl-oxy-β-D-maltoside, tridecan-3-yl-oxy-β-D-maltoside, tridecan-2-yl-oxy-β-D-maltoside, n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-glucoside, n-nonyl-β-D-glucoside, n-decyl-β-D-maltoside, n-dodecyl-β-D-maltopyranoside, 4-n-dodecyl-α,α-trehalose, 6-n-dodecyl-α,α-trehalose, and 3-n-dodecyl-α,α-trehalose.

[0165] In some embodiments, the nonpolar moiety is an acyl or diacyl chain.

[0166] In some embodiments, the sugar is a modified sugar or a substituted sugar.

[0167] 6.1.4. Formulations Complexes based on lipidated proteins 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).

[0168] CER-001, intended for administration by infusion, can be formulated in a phosphate buffer containing sucrose and mannitol excipients, for example, as described in International Publication No. WO 2012 / 109162.

[0169] 6.2. Target Population Subjects that can be treated according to the methods described herein are preferably mammals, most preferably humans.

[0170] In some embodiments, the subject has an acute condition including acute inflammation.

[0171] In some embodiments, the subject has an infectious disease, such as a coronavirus infection, such as COVID-19.

[0172] In some embodiments, the subject may be a subject in need of treatment for sepsis and / or AKI.

[0173] In some embodiments, the subject has sepsis (e.g., associated with a gram-negative bacterial infection). Sepsis can, in some embodiments, be caused by an intra-abdominal infection or urosepsis. Sepsis is a risk factor for AKI. Thus, in some embodiments, the subject may be at risk of AKI, for example, due to sepsis. In some embodiments, the subject has sepsis associated with a gram-negative bacterial infection. In other embodiments, the subject has sepsis associated with a gram-positive bacterial infection.

[0174] In some embodiments, the subject has a total SOFA score of 2 - 24, such as a total score of 2 - 5, 2 - 10, 5 - 10, 5 - 15, 10 - 15, 10 - 20, or 15 - 24, prior to treatment with a lipid - binding protein - based complex. The SOFA scoring system is described in Vincent et al. 1996, Intensive Care Med, 22:707 - 710 and uses a 0 - 4 scale to assess the function of six different body systems (respiratory, cardiovascular, liver, coagulation, kidney, and neurology). The sum of the individual scores for the six different body systems provides the total SOFA score for the subject. In some embodiments, the subject has a total SOFA score of 2 - 5 prior to treatment with a lipid - binding protein - based complex. In other embodiments, the subject has a total SOFA score of 2 - 10 prior to treatment with a lipid - binding protein - based complex. In other embodiments, the subject has a total SOFA score of 5 - 15 prior to treatment with a lipid - binding protein - based complex. In other embodiments, the subject has a total SOFA score of 8 - 13 prior to treatment with a lipid - binding protein - based complex.

[0175] In some embodiments, the subject has an endotoxin activity level measured by an endotoxin activity assay (EAA™) (Spectral Medical) >0.6 prior to administration of a lipid - binding protein - based complex (see Marshall et al., 2004, J Infect Dis. 190(3):527 - 34).

[0176] In some embodiments, the subject has or is at risk of AKI. For example, the AKI can be sepsis-related AKI, ischemia / reperfusion AKI, CSA-AKI, or hepatorenal syndrome (HRS) AKI. In some embodiments, the AKI is sepsis-related AKI. In other embodiments, the AKI is ischemia / reperfusion AKI. In other embodiments, the AKI is CSA AKI. In other embodiments, the AKI is HRS AKI. Subjects at risk of HRS include those having a liver disease (e.g., chronic liver disease or acute liver disease). In some embodiments, the subject has a chronic liver disease. In some embodiments, the subject has an acute liver disease. In some embodiments, the subject has an alcoholic liver disease. Historically, HRS has been classified as type 1 HRS (where renal function rapidly deteriorates over days to weeks) and type 2 HRS (where deterioration occurs over months). Thus, in some embodiments, the subject treated according to the dosing regimen of the present disclosure has type 1 HRS. In other embodiments, the subject treated according to the dosing regimen of the present disclosure has type 2 HRS. Newer criteria for the diagnosis and classification of HRS have been developed, such as the ICA diagnostic criteria for acute kidney injury (AKI) in HRS. See, for example, Amin et al., 2019, Seminars in Nephrology 39(1):17-30. Thus, in some embodiments, the subject having HRS meets the ICA diagnostic criteria for HRS AKI.

[0177] In some embodiments, the subject can be any subject having or at risk of CRS and / or any subject in need of a decrease in the serum level of one or more inflammatory markers, such as IL-6. In some embodiments, the subject has CRS. In some embodiments, the subject has CRS subsequent to an infectious disease, such as a viral infectious disease, such as an infection due to COVID-19 or influenza. In some embodiments, the subject has CRS subsequent to a COVID-19 infection. In other embodiments, the subject has CRS caused by an immunotherapy, such as an antibody or chimeric antigen receptor (CAR) T cell therapy. In still other embodiments, the subject is at risk of CRS due to, for example, an infectious disease, such as COVID-19 or influenza. In other embodiments, the subject is at risk of CRS due to an immunotherapy.

[0178] In another embodiment, the subject is a subject in need of a decrease in the serum level of one or more inflammatory markers, e.g., a subject having an elevated level of one or more inflammatory markers as 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, and tumor necrosis factor alpha (TNFα). In some embodiments, the one or more cytokines include IL-6. In some embodiments, the one or more cytokines include a combination of the foregoing, e.g., two, three, four, five, six, seven, or all eight 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, and tumor necrosis factor alpha (TNFα).

[0179] 6.3. Dosage Regimen The methods of the present disclosure generally require multiple administrations, e.g., 2 to 10 individual doses, of a lipid-binding protein-based complex (e.g., CER-001), although single doses may be used in some embodiments. In some embodiments, the dosing regimen may include two or more, three or more, or four or more doses, e.g., 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 doses of a lipid-binding protein-based complex (e.g., CER-001). In some embodiments, the dosing regimen comprises or consists of a single dose. In some embodiments, the dosing regimen comprises or consists of two individual doses. In some embodiments, the dosing regimen comprises or consists of three individual doses. In some embodiments, the dosing regimen comprises or consists of four individual doses.

[0180] In some embodiments, lipid-binding protein-based complexes are administered according to the derivatization and optional tempering regimens described in Sections 6.3.1 and 6.3.2, respectively. In some embodiments, lipid-binding protein-based complexes can be administered in a single step, e.g., according to the dosing regimens described in this section. In some embodiments, the subject does not undergo treatment with a lipid-binding protein-based complex according to a maintenance regimen, e.g., a regimen that includes administration of a lipid-binding protein-based complex for an extended period (e.g., more than one month).

[0181] The dosing regimen of the lipid-binding protein-based complex (e.g., CER-001) of the present disclosure can last up to one week, one week, or longer than one week (e.g., two weeks).

[0182] For example, the dosing regimen of a lipid-binding protein-based complex (e.g., CER-001) can include administering: - Five doses of CER-001 over one week, - Six doses of CER-001 over one week, - Seven doses of CER-001 over one week, - 10 doses of CER-001 over a 2-week period, - 12 doses of CER-001 over a 2-week period, - 14 doses of CER-001 over a 2-week period.

[0183] In one embodiment, the method of the present disclosure (e.g., a method for treating a subject having CRS or at risk of CRS) includes administering 7 doses of CER-001 over a 1-week period, e.g., on days 1, 2, 3, 4, 5, 6, and 7.

[0184] In some embodiments of the methods of the present disclosure, multiple doses of a lipid-binding protein-based complex (e.g., CER-001) are administered at intervals of 1 day or less. For example, in some embodiments, two or more individual doses are administered at approximately 12-hour intervals. In some embodiments, two individual doses are administered at approximately 12-hour intervals. In other embodiments, three individual doses are administered at approximately 12-hour intervals. In other embodiments, two individual doses are administered at approximately 12-hour intervals, and the third individual dose is administered approximately 1 day later. In other embodiments, three individual doses are administered at approximately 12-hour intervals, and the fourth individual dose is administered approximately 1 day later.

[0185] In some embodiments of the methods of the present disclosure, a lipid-binding protein-based complex (e.g., CER-001) is administered to a subject at 0 and 12 hours, e.g., at a dose of 10 mg / kg or 15 mg / kg (e.g., over 0.5 - 1 hour). In some embodiments of the methods of the present disclosure, a lipid-binding protein-based complex (e.g., CER-001) is administered to a subject at 0 hours and at 12, 24, and 48 hours, e.g., at a dose of 10 mg / kg or 15 mg / kg (e.g., over 0.5 - 1 hour).

[0186] In some embodiments of the methods of the present disclosure, a lipid-binding protein-based complex (e.g., CER-001) is administered daily, for example, for at least 5 days, at least 6 days, at least 7 days, or longer than 7 days daily (e.g., daily for up to 1 week or daily for up to 2 weeks). In other embodiments, a lipid-binding protein-based complex (e.g., CER-001) is administered at a lower frequency, for example, every other day, twice a week, three times a week, or once a week.

[0187] In practice, an administration window can be provided to account for minor variations in a dosing schedule, for example, multiple times per week. For example, a window of ±2 days or ±1 day around the dosing day can be used.

[0188] A lipid-binding protein-based complex (e.g., CER-001) can be administered in the methods of the present disclosure for a pre-determined period, for example, for 1 week. Alternatively, administration of a lipid-binding protein-based complex (e.g., CER-001) can be continued until one or more symptoms of acute signs (e.g., CRS) decrease, or until the serum levels of one or more inflammatory markers decrease, for example, to normal levels, or decrease compared to a baseline value in the subject, for example, a baseline value measured prior to initiation of lipid-binding protein-based complex (e.g., CER-001) therapy. Reference or "normal" levels of various inflammatory markers are known in the art. For example, the Mayo Clinic Laboratories test catalog (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 (male), 11 - 307 mcg / L (female), IL-8 <57.8 pg / mL, TNF-α <5.6 pg / mL.

[0189] When administering a lipid-binding protein-based complex (e.g., CER-001) to a subject having CRS resulting from immunotherapy or at risk of CRS resulting from immunotherapy, the lipid-binding protein-based complex (e.g., CER-001) can be administered before initiating immunotherapy, concurrently with immunotherapy, after completion of immunotherapy, or in combination thereof. For example, the lipid-binding protein-based complex (e.g., CER-001) may be administered before and concurrently with immunotherapy, concurrently with and after immunotherapy, or before, concurrently with, and after immunotherapy. Concurrent administration is not limited to administering the lipid-binding protein-based complex (e.g., CER-001) and immunotherapy completely simultaneously, but also includes administering one agent during the course of treatment with the other.

[0190] The methods of the present disclosure (e.g., methods for treating acute conditions described herein) generally include administering a high dose of a lipid-binding protein-based complex (e.g., CER-001). The high dose may be a collection of multiple individual doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 individual doses) and can be administered, for example, over one day or multiple days (e.g., a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days). In some embodiments, the individual doses of the high dose are administered daily, twice a day, or at 2- to 3-day intervals.

[0191] In some embodiments, the high dose is an amount effective to increase the subject's HDL and / or ApoA-I blood levels and / or to improve the subject's vascular endothelial function as measured, for example, by circulating vascular cell adhesion molecule 1 (VCAM-1) and / or intercellular adhesion molecule 1 (ICAM-1) levels. In some embodiments, the high dose or individual dose is an amount that increases the subject's HDL and / or ApoA-I levels by at least 25%, at least 30%, or at least 35% 2 to 4 hours after administration.

[0192] In some embodiments, the high dose is an amount effective to reduce the serum level of one or more inflammatory markers, such as one or more of IL-6, C-reactive protein, D-dimer, ferritin, IL-8, GM-CSF, and MCP1 TNF-α. In some embodiments, the serum level of one or more inflammatory markers decreases from an elevated range to a normal range and / or decreases by at least 20%, at least 40%, or at least 60%.

[0193] The dose of the lipid-bound protein-based complex (e.g., CER-001) administered to a subject (e.g., an individual dose that forms a high dose when combined with one or more other individual doses) is, in some embodiments, in the range of 4 - 40 mg / kg (e.g., 10 - 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 - 20 mg / kg, 15 - 25 mg / kg, 20 - 40 mg / kg, 25 - 35 mg / kg, or 30 - 40 mg / kg). As used herein, the expression "on a protein weight basis" means that the dose of the lipid-bound protein-based complex (e.g., CER-001) administered to a subject is calculated based on the amount of ApoA-I in the lipid-bound protein-based complex (e.g., CER-001) being administered and the body weight of the subject. For example, a subject weighing 70 kg and receiving a dose of CER-001 of 20 mg / kg would receive an amount of CER-001 that provides 1400 mg of ApoA-I (70 kg × 20 mg / kg).

[0194] In yet other aspects, the lipid-bound protein-based complex (e.g., CER-001) can be administered on a unit dosage basis. The unit dosage used in the methods of the present disclosure can, in some embodiments, vary from 300 mg to 4000 mg (e.g., 600 mg to 4000 mg) / administration (on a protein weight basis).

[0195] In certain embodiments, the dosage of the lipid-bound protein-based complex (e.g., CER-001) is 600 mg to 3000 mg, 800 mg to 3000 mg, 1000 mg to 2400 mg, or 1000 mg to 2000 mg / dose (protein weight basis).

[0196] In some aspects, a high dose of a lipid-bound protein-based complex (e.g., CER-001), e.g., the aggregate of a plurality of individual doses, is 600 mg to 40 g (protein weight basis). In certain embodiments, the high dose is 3 g to 35 g or 5 g to 30 g (protein weight basis).

[0197] The lipid-bound protein-based complex (e.g., CER-001) is preferably administered as an IV infusion. For example, the stock solution of CER-001 can be diluted with a standard saline such as normal saline (0.9% NaCl) to a total volume of 125 - 250 ml. In some embodiments, subjects weighing less than 80 kg will have a total volume of 125 ml, while subjects weighing at least 80 kg will have a total volume of 250 ml. In some embodiments, the dose of CER-001 is administered at a total volume of 250 ml. The lipid-bound protein-based complex (e.g., CER-001) can be administered over a period ranging from 1 hour to 24 hours. Depending on the needs of the subject, the administration can be by a slow infusion over a period longer than 1 hour (e.g., up to 2 hours or up to 24 hours), by a rapid infusion of less than 1 hour, or by a single bolus injection. In one embodiment, the lipid-bound protein-based complex (e.g., CER-001) is administered over a 1-hour period, for example, using an infusion pump at a fixed rate of 125 ml / hr or 250 ml / hr. In one embodiment, the dose of the lipid-bound protein-based complex (e.g., CER-001) is administered as an infusion over a 24-hour period.

[0198] 6.3.1. Induction Regimen In one embodiment, an induction regimen suitable for use in the methods of the present disclosure involves administering multiple doses of a lipid-bound protein-based complex (e.g., CER-001) over a number of consecutive days, such as three consecutive days.

[0199] In some embodiments, an induction regimen suitable for use in the methods of the present disclosure involves administering a lipid-bound protein-based complex (e.g., CER-001) twice a day, such as twice a day for a number of consecutive days. Twice-daily administration can include, for example, two doses at approximately 12-hour intervals or a morning dose and an evening dose (which can be at intervals longer or shorter than 12 hours).

[0200] In one embodiment, the induction regimen includes twice-daily doses of a lipid-bound protein-based complex (e.g., CER-001) for three consecutive days.

[0201] In the induction regimen, the therapeutic dose of a lipid-bound protein-based complex (e.g., CER-001) administered by infusion 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). In some embodiments, the dose of the lipid-bound protein-based complex (e.g., CER-001) used in the induction regimen is 5 mg / kg. In some embodiments, the dose of the lipid-bound protein-based complex (e.g., CER-001) used in the induction regimen is 10 mg / kg. In some embodiments, the dose of the lipid-bound protein-based complex (e.g., CER-001) used in the induction regimen is 15 mg / kg. In some embodiments, the dose of the lipid-bound protein-based complex (e.g., CER-001) used in the induction regimen is 20 mg / kg. In some embodiments, the induction regimen comprises 6 doses of a lipid-bound protein-based complex (e.g., CER-001) administered over 3 days at a dose of 5 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg.

[0202] In yet other embodiments, the lipid-bound protein-based complex (e.g., CER-001) can be administered on a unit dosage basis. The unit dosage used in the induction phase can vary for infusion administration from 300 mg to 4000 mg (e.g., 300 mg to 3000 mg) (protein weight basis).

[0203] In certain embodiments, the dosage of the lipid-bound protein-based complex (e.g., CER-001) 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 (protein weight basis) / infusion administration.

[0204] 6.3.2. Soil Consolidation Therapy Regimen A soil consolidation therapy regimen suitable for use in the methods of the present disclosure involves administering one or more doses of a lipid - bound protein - based complex (e.g., CER - 001) following an induction regimen.

[0205] In one embodiment, the soil consolidation therapy regimen includes administering two doses of a lipid - bound protein - based complex (e.g., CER - 001). For example, the two doses can be administered at approximately 12 - hour intervals, or as a morning dose and an evening dose (which can be at intervals longer or shorter than 12 hours).

[0206] The dose of the lipid - bound protein - based complex (e.g., CER - 001) in the soil consolidation therapy regimen can, in some embodiments, be administered on the 6th day of the dosing regimen starting from the induction regimen on the 1st day. The dose of the lipid - bound protein - based complex (e.g., CER - 001) in the soil consolidation therapy regimen can, in some embodiments, be administered on the 4th day of the dosing regimen starting from the induction regimen on the 1st day. The dose of the lipid - bound protein - based complex (e.g., CER - 001) in the soil consolidation therapy regimen can, in some embodiments, be administered on the 5th day of the dosing regimen starting from the induction regimen on the 1st day. The dose of the lipid - bound protein - based complex (e.g., CER - 001) in the soil consolidation therapy regimen can, in some embodiments, be administered on the 7th day of the dosing regimen starting from the induction regimen on the 1st day.

[0207] In a consolidation therapy regimen, the therapeutic dose of a lipid-binding protein-based complex (e.g., CER-001) administered by infusion 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). In some embodiments, the dose of the lipid-binding protein-based complex (e.g., CER-001) used in a consolidation therapy regimen is 5 mg / kg. In some embodiments, the dose of the lipid-binding protein-based complex (e.g., CER-001) used in a consolidation therapy regimen is 10 mg / kg. In some embodiments, the dose of the lipid-binding protein-based complex (e.g., CER-001) in a consolidation therapy regimen is 15 mg / kg. In some embodiments, the dose of the lipid-binding protein-based complex (e.g., CER-001) used in a consolidation therapy regimen is 20 mg / kg. In some embodiments, the consolidation therapy regimen comprises a lipid-binding protein-based complex (e.g., CER-001) at a dose of 5 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg, administered as two doses in one day.

[0208] In yet other aspects, the lipid-binding protein-based complex (e.g., CER-001) can be administered on a unit dosage basis. The unit dosage used in the consolidation therapy phase can vary for infusion-based administration from 300 mg to 4000 mg (e.g., 300 mg to 3000 mg) (protein weight basis).

[0209] In certain embodiments, the dosage of the lipid-binding protein-based complex (e.g., CER-001) used during the consolidation therapy phase is 300 mg to 1500 mg, 400 mg to 1500 mg, 500 mg to 1200 mg, or 500 mg to 1000 mg (protein weight basis) / infusion-based administration.

[0210] Complexes based on lipid-binding proteins (e.g., CER-001) can be administered, during the consolidation therapy phase, in the same manner as described in Section 6.3, for example, as an IV infusion over a period of 1 hour.

[0211] 6.4. Combination Therapy Complexes based on lipid-binding proteins (e.g., CER-001) can be administered to the subjects described herein as part of a monotherapy or combination therapy regimen. For example, the combination therapy can include a complex based on a lipid-binding protein (e.g., CER-001) in combination with standard treatments for sepsis and / or AKI. See, for example, Rhodes et al., 2017, Intensive Care Med 43:304-377, Dugar et al., 2020, Cleveland Clinic Journal of Medicine 87(1):53-64.

[0212] In some embodiments, the subject is treated using a lipid-binding protein-based complex (e.g., CER-001) in combination with fluid replacement therapy. In some embodiments, the subject is treated using a lipid-binding protein-based complex (e.g., CER-001) in combination with an antimicrobial. In some embodiments, the subject is treated with a lipid-binding protein-based complex (e.g., CER-001) in combination with an antibiotic (e.g., ceftriaxone, meropenem, ceftazidime, cefotaxime, cefepime, piperacillin and tazobactam, ampicillin and sulbactam, imipenem and cilastatin, levofloxacin, clindamycin, or an antibiotic of the oxazolidinone class, such as linezolid or tedizolid). In some embodiments, the subject is treated using a lipid-binding protein-based complex (e.g., CER-001) in combination with an antiviral agent. In some embodiments, the subject is treated using a lipid-binding protein-based complex (e.g., CER-001) in combination with an agent that raises blood pressure (e.g., norepinephrine or epinephrine). In some embodiments, the subject is treated with a lipid-binding protein-based complex (e.g., CER-001) in combination with an immunosuppressant, such as tacrolimus or everolimus.

[0213] In some embodiments, the combination therapy regimen can include 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, olaratumab, elsilimomab, BMS-945429, sirukumab, rilonacept, and CPSI-2364. In some embodiments, a lipid-bound protein-based complex (e.g., CER-001) is administered in combination with tocilizumab. Subjects who have had or have COVID-19 infection can be treated with a lipid-bound protein-based complex (e.g., CER-001) in combination with one or more additional therapies, such as antibodies from recovered COVID-19 patients, antibodies against the spike protein of COVID-19 (e.g., casirivimab, imdevimab), one or more antiviral agents (e.g., lopinavir, remdesivir, danoprevir, galidesivir, darunavir, ritonavir), chloroquine, hydroxychloroquine, azithromycin, interferon (e.g., interferon alpha or interferon beta, which may each be pegylated), or combinations thereof.

[0214] In certain embodiments, an antihistamine (e.g., diphenhydramine, cetirizine, fexofenadine, or loratadine) can be administered prior to administration of a lipid-bound protein-based complex (e.g., CER-001). The antihistamine can reduce the likelihood of an allergic reaction.

Example

[0215] 7. Example [Example 1] 7.1. CER-001 Treatment for COVID-19 The SARS-CoV-2 virus may promote life-threatening hyperinflammatory states in at-risk patients. Remodeling of the lipid profile, including a dramatic decrease in serum levels of apolipoprotein A-I (ApoA-I), is characteristic of severe COVID-19. ApoA-I can reduce lung inflammation, modulate innate and adaptive immunity, and prevent endothelial dysfunction and blood coagulation. In this example, we describe a compassionate access trial conducted in four subjects with progressive COVID-19 cytokine storm despite standard treatment. To raise ApoA-I to normal levels, the subjects received 2 - 4 infusions of CER-001 (10 mg / kg each). The injections were well tolerated and no serious adverse events were observed. Three patients improved rapidly and were discharged 3 - 4 days after the CER-001 infusion. The fourth patient, who received CER-001 while on mechanical ventilation, showed a transient improvement but then experienced a worsening related to bacterial pneumonia. This trial provides initial safety data and proof-of-concept data for treating patients with virus-induced cytokine storm while using lipid-binding protein-based complexes such as CER-001.

[0216] 7.1.1. Materials and Methods 7.1.1.1. Medical History Subject 1 was a 52-year-old male with a history of IgA vasculitis, diabetes, and ischemic heart disease, who had received a kidney transplant in 2018. He had received three doses of the mRNA COVID-19 vaccine but developed only weak anti-SARS-CoV-2 immunity (anti-spike antibody 15.5 BAU / mL). The subject developed symptoms of COVID-19 (fever, diarrhea, and dyspnea) and was admitted to the transplant ward 8 days later. Oxygen saturation in room air was 92%, and oxygen supplementation (1 L / min) was initiated. Chest CT scan revealed bilateral interstitial lung disease consistent with COVID-19 (25% of the parenchymal infiltration), and nasopharyngeal PCR detected the SARS-CoV-2 Delta strain (variant of concern (VOC)). Blood tests revealed a hyperinflammatory state (ferritin 5,037 μg / L, C-reactive protein 34 mg / L), abnormal liver tests (AST and ALT were 2.5 and 3.5 times the upper limit of normal (ULN), respectively), and thrombocytopenia. Tacrolimus was administered, mycophenolate mofetil was discontinued, and dexamethasone (6 mg / day) was introduced along with antibiotics. Blood tests on the second day revealed pancytopenia and progression of the hyperinflammatory state (ferritin 6,870 μg / L, C-reactive protein 55 mg / L). The subject received one infusion of the monoclonal antibody anti-IL-6R antibody tocilizumab (8 mg / kg, i.v.) and one infusion of the neutralizing monoclonal anti-SARS-CoV-2 antibody (casirivimab / imdevimab). On the fourth day, the subject's ferritin increased to 19,219 μg / L, the subject's AST and ALT increased to 17 and 14 times the ULN values, respectively, and the subject's arterial lactate level was 2.7 mmol / L. Bone marrow aspiration revealed findings of hemophagocytosis. Blood PCR for SARS-CoV-2 was weakly positive. The worsening of hypoxemia required an increase in the oxygen supply (4 L / min; PaO2 62 mmHg), and CT scan revealed progressive lung lesions typical of COVID-19 (50% of the parenchyma). Despite increasing dexamethasone to 10 mg / day, on the sixth day, serum triglycerides and ferritin increased to 3.2 mmol / L and 27,394 μg / L, respectively.

[0217] Subject 2 was a 38-year-old woman with a history of systemic lupus erythematosus (SLE) and obesity, who had received a kidney transplant in 2011. She had received three doses of the mRNA COVID-19 vaccine but did not acquire anti-SARS-CoV-2 immunity. The subject developed symptoms of COVID-19 (cough, chills, diarrhea, fever) and was admitted to the transplant ward 10 days later. The SARS-CoV-2 VOC Omicron strain was identified by nasopharyngeal PCR testing. At admission, SaO2 was 94% while receiving oxygen inhalation at 9 L / min while wearing a face mask. Chest CT scan revealed typical lesions of COVID-19 (infiltration range 50%). Blood tests revealed hepatitis with cytolysis and cholestasis (7- to 10-fold the ULN value respectively), acute kidney injury (KDIGO stage 1), and a hyperinflammatory state (ferritin 2,000 μg / L, C-reactive protein 107 mg / L). High-flow oxygen supplementation, awake pronation, dexamethasone (10 mg / day), tocilizumab (8 mg / kg as a single dose), and antibiotics were initiated. Everolimus was discontinued and tacrolimus was administered. On the 4th day, despite maximal treatment, high-flow oxygen supplementation was still required and the subject's hyperinflammatory state worsened (ferritin 2,800 μg / L).

[0218] Subject 3 was a 47-year-old woman with a history of diabetes, adrenal Cushing's syndrome, hypertension, and end-stage renal disease requiring chronic renal replacement therapy since 2020. The subject had not received anti-SARS-CoV-2 vaccination and had no anti-SARS-CoV-2 immunity at the time of admission. She developed symptoms of COVID-19 (cough, dyspnea, abdominal pain, fever) and was admitted to the hospital 4 days later. SARS-CoV-2 VOC Omicron strain was identified by nasopharyngeal PCR. Chest CT scan revealed mild to moderate lung lesions (10 - 25%) typical of COVID-19. Oxygen supplementation was not required. Blood tests revealed a hyperinflammatory syndrome (ferritin 4,350 μg / L, C-reactive protein 55 mg / L), moderate elevation of AST and ALT (2 times and 1.5 times the ULN value respectively), as well as mild thrombocytopenia and anemia. Dexamethasone (6 mg / day) was introduced. On the 3rd day, hyperferritinemia (ferritin 4,142 μg / L) and abnormal liver test items persisted, and the subject developed encephalopathy and was transferred to the intensive care unit.

[0219] Subject 4 was a 59-year-old man with a history of hepatitis B, liver transplantation in 2006, HHV8-negative Kaposi's sarcoma (complete remission), and end-stage renal disease requiring chronic renal replacement therapy since 2020. The subject had received three doses of mRNA COVID-19 vaccine but did not express anti-SARS-CoV-2 antibodies. Due to SARS-CoV-2 exposure within the family, a nasopharyngeal PCR test was performed and the VOC Omicron strain was identified. The subject developed symptoms of COVID-19 (malaise) but initially had no respiratory symptoms. Chest CT scan revealed mild to moderate lung lesions (10 - 25%) typical of COVID-19. Two days later, he developed dyspnea, cough, and fever. At the time of admission, PaO2 was 54 mmHg in room air, respiratory rate was 30 breaths / min, and body temperature was 38.5°C. Blood tests revealed hyperferritinemia (ferritin 1,223 μg / L) and leukopenia (1,080 cells / mm 3) became apparent. In the CT scan, the progression of the lung lesion (25 - 50%) became apparent. Oxygen supplementation, dexamethasone (10 mg / day), tocilizumab (administered once at 8 mg / kg), antibiotics, and fresh frozen plasma obtained from convalescent patients were administered. Mycophenolate mofetil was discontinued and tacrolimus was administered. On the 5th day, acute respiratory failure developed and mechanical ventilation with oral endotracheal intubation and neuromuscular blockade was required. Blood tests revealed a hyperinflammatory state (ferritin 4,535 μg / L) accompanied by an increase in AST and ALT (3 times the ULN value). At that time, the culture test of the bronchoalveolar lavage fluid was negative, suggesting only severe COVID-19. The PaO2 / FiO2 ratio was 150 - 180. Antibiotics were administered.

[0220] 7.1.1.2. Administration Method CER-001 was intravenously administered to Subject 1 at a dose of 10 mg / kg at 0 hours and 12 hours over a period of 0.5 - 1 hour. CER-001 was intravenously administered to Subjects 2 - 4 at a dose of 10 mg / kg at 0, 12, 24, and 48 hours over a period of 0.5 - 1 hour. Before each administration of CER-001, prophylactic administration of an antihistamine using hydroxyzine (50 mg i.v.) preceded. Dexamethasone was also administered to all subjects.

[0221] 7.1.2. Results and Discussion 7.1.2.1. General Safety Subjects 1, 2, and 3 did not exhibit any serious adverse events. Subject 4 exhibited two episodes of ventilator-associated pneumonia (VAP; Klebsiella pneumoniae and Aspergillus fumigatus + mucormycosis), and one episode of bacteremia (Staphylococcus haemolyticus).

[0222] 7.1.2.2. Biological Efficacy: Lipid Profile When CER-001 was first administered, in all 4 subjects, the serum levels of ApoA-I (in the range of 0.74 - 0.79 mg / L, normal value > 1.1 g / L) (Figures 2A - 2D) and HDL (in the range of 0.26 - 0.35 g / L, normal value > 0.45 g / L) (Figures 3A - 3D) were very low, while the serum level of triglyceride was high (in the range of 2.16 - 3.4 g / L, normal value < 1.5 g / L). After treatment with CER-001, on the 2nd day, the ApoA-I and HDL levels normalized in all subjects, but remained slightly below the normal range in the most inflammatory subject. In subject 4 who developed ventilator-associated pneumonia 3 days after the start of CER-001, ApoA-I then decreased below the normal value.

[0223] 7.1.2.3. Dynamics of Inflammation At baseline, IL-1β was normal in all individuals, but for IL-6, it increased in 3 subjects who had received tocilizumab previously, and was normal in the 4th subject (3.3 - 1,295 pg / mL). On the other hand, TNF-α increased moderately (9.7 - 42.1 pg / mL). IL-8 was the only inflammatory cytokine that increased in all cases (> 10 pg / mL; 14.8 - 64.5 pg / mL). After administration of CER-001, IL-8 normalized in subjects 1, 2, and 3. In subject 4, IL-8 decreased rapidly after injection but increased again at the time of onset of ventilator-associated pneumonia. The serum ferritin level decreased from 6,616 ± 8,696 μg / L to 1,712 ± 815 μg / L 6 days after the start of CER-001. In 3 out of 4 subjects, the analysis of C-reactive protein was excluded because an anti-IL-6R antibody had been administered before CER-001. The body temperature remained below 37.5°C in all subjects.

[0224] 7.1.2.4. Clinical Outcome After administration of CER-001, the clinical conditions of Subjects 1, 2, and 3 improved rapidly, and they were able to be discharged 3 to 4 days after the infusion of CER-001 (Figures 1A to 1D). In Subjects 1 and 2, oxygen supplementation was discontinued 2 and 3 days after administration, respectively. In Subject 3, the confused state dissipated within 2 days. In these 3 subjects, the inflammatory parameters, liver test values, and blood cell counts improved by the time of discharge. Subject 4 had been receiving mechanical ventilation for 3 days at the time of CER-001 introduction. After 3 days of initial improvement (discontinuation of neuromuscular blocking agents and reduction of sedation), the subject developed several ventilator-associated pneumonias and ultimately died 1 month later.

[0225] 7.1.2.5. Discussion CER-001 not only showed very good tolerance in the acute phase, but also rapid improvement in the respiratory state, reduction of inflammatory parameters, and normalization of blood cell counts were observed in 3 out of 4 subjects, in parallel with the normalization of ApoA-I levels after administration of CER-001. Although these subjects developed severe COVID-19-related cytokine storms, they were able to be discharged without oxygen assistance just 3 to 4 days after the infusion of CER-001. In the 3 subjects who had a favorable outcome after administration of CER-001, a rapid decrease in IL-8 was observed in parallel with clinical and biological improvement. In Subject 4, after initial clinical improvement accompanied by normalization of ApoA-I and decrease in IL-8, ventilator-associated pneumonia and clinical deterioration were accompanied by increases in C-reactive protein (CRP) and IL-8, as well as a decrease in ApoA-I. In this study, the subjects received 4 infusions of CER-001 (10 mg / kg), but it is thought that using a higher dose (e.g., 15 mg / kg) in the first injection would help ApoA-I and non-oxidized HDL reach optimal concentrations more rapidly and achieve the maximum therapeutic effect.

[0226] 8. Specific Embodiments Various aspects of the present disclosure are described in the embodiments set forth in the following numbered paragraphs. 1. A method of treating a subject in an acute condition, comprising administering to a subject in need thereof a high-dose lipid-bound protein-based complex, optionally wherein the acute condition comprises acute inflammation. 2. The method according to embodiment 1, wherein the high dose is administered over a period of 1 day to approximately 2 weeks, optionally wherein the high dose is administered over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. 3. The method according to embodiment 1 or 2, wherein the high dose is a collection of 2 to 10 individual doses, optionally wherein the high dose is a collection of 3, 4, 5, 6, 7, 8, 9, or 10 individual doses. 4. The method according to embodiment 3, wherein the plurality of individual doses are administered daily or twice a day. 5. The method according to embodiment 3 or 4, wherein the plurality of individual doses are administered at intervals of 2 to 3 days. 6. The method according to embodiment 3, wherein the plurality of individual doses are administered at intervals within 1 day. 7. The method according to embodiment 6, comprising administering two or more individual doses at intervals of approximately 12 hours. 8. The method according to embodiment 7, comprising administering two individual doses at intervals of approximately 12 hours. 9. The method according to embodiment 7, comprising administering three individual doses at intervals of approximately 12 hours. 10. The method according to embodiment 8 or 9, further comprising administering an individual dose approximately 1 day later. 11. The method according to embodiment 3, comprising administering three individual doses at intervals of approximately 12 hours and a fourth individual dose approximately 1 day later. 12. The method according to embodiment 1, wherein the high dose is administered as a single individual dose. 13. The method according to embodiment 1, wherein the high dose is a collection of two individual doses administered within 1 day. 14. The method according to embodiment 13, wherein the two individual doses are administered at intervals of approximately 12 hours. 15. The method according to any one of embodiments 3 to 14, wherein each individual dose is effective to increase the HDL level of the subject. 16. The method according to embodiment 15, wherein the high dose is effective to increase the serum HDL level of the subject to a normal value (e.g., > 0.45 g / L). 17. The method according to embodiment 15, wherein each individual dose is effective to increase the HDL level of the subject by at least 25%, at least 30%, or at least 35% 2 to 4 hours after administration. 18. The method according to embodiment 17, wherein each individual dose is effective to increase the HDL level of the subject by at least 25%, at least 30%, or at least 35% 2 hours after administration. 19. The method according to embodiment 17, wherein each individual dose is effective to increase the HDL level of the subject by at least 25%, at least 30%, or at least 35% 3 hours after administration. 20. The method according to embodiment 17, wherein each individual dose is effective to increase the HDL level of the subject by at least 25%, at least 30%, or at least 35% 4 hours after administration. 21. The method according to any one of embodiments 3 to 20, wherein each individual dose is effective to increase the ApoA-I level of the subject. 22. The method according to embodiment 21, wherein the high dose is effective to increase the serum ApoA-I level of the subject to a normal value (e.g., > 1.1 g / L). 23. The method according to embodiment 21, wherein each individual dose is effective to increase the ApoA-I level of the subject by at least 25%, at least 30%, or at least 35% 2 to 4 hours after administration. 24. The method according to embodiment 22, wherein each individual dose is effective to increase the ApoA-I level of the subject by at least 25%, at least 30%, or at least 35% 2 hours after administration. 25. The method according to embodiment 22, wherein each individual dose is effective to increase the subject's ApoA-I level by at least 25%, at least 30%, or at least 35% three hours after administration. 26. The method according to embodiment 22, wherein each individual dose is effective to increase the subject's ApoA-I level by at least 25%, at least 30%, or at least 35% four hours after administration. 27. The method according to any one of embodiments 1 to 26, wherein the high dose is effective to improve the subject's vascular endothelial function, and optionally, the vascular endothelial function is measured by circulating VCAM-1 and / or ICAM-1. 28. The method according to any one of embodiments 1 to 27, wherein the high dose is effective to reduce the serum level of one or more inflammatory markers in the subject. 29. The method according to embodiment 28, wherein the high dose is effective to reduce the serum level of interleukin-6 ("IL-6"). 30. The method according to embodiment 28 or embodiment 29, wherein the high dose is effective to reduce the serum level of C-reactive protein. 31. The method according to any one of embodiments 28 to 30, wherein the high dose is effective to reduce the serum level of D-dimer. 32. The method according to any one of embodiments 28 to 31, wherein the high dose is effective to reduce the serum level of ferritin. 33. The method according to any one of embodiments 28 to 32, wherein the high dose is effective to reduce the serum level of interleukin 8 (IL-8). 34. The method according to any one of embodiments 28 to 32, wherein the high dose is effective to normalize the serum level of interleukin 8 (IL-8). 35. The method according to any one of embodiments 28 to 34, wherein the high dose is effective to reduce the serum level of granulocyte macrophage colony-stimulating factor (GM-CSF). 36. The method according to any one of embodiments 28 to 35, wherein the high dose is effective for reducing the serum level of monocyte chemoattractant protein (MCP) 1. 37. The method according to any one of embodiments 28 to 36, wherein the high dose is effective for reducing the serum level of tumor necrosis factor α (TNF-α). 38. The method according to any one of embodiments 28 to 37, wherein the high dose is effective for reducing the serum level of one or more inflammatory markers from an elevated range to a normal range. 39. The method according to any one of embodiments 28 to 38, wherein the high dose is effective for reducing the serum level of one or more inflammatory markers by at least 20%, at least 40%, or at least 60%. 40. The method according to any one of embodiments 1 to 39, wherein the subject has a viral infection. 41. The method according to embodiment 40, wherein the viral infection is a coronavirus infection. 42. The method according to embodiment 41, wherein the coronavirus infection is COVID-19. 43. The method according to any one of embodiments 1 to 42, wherein the subject has or is at risk of having CRS. 44. The method according to embodiment 43, wherein the subject has CRS. 45. The method according to embodiment 44, wherein the subject has CRS subsequent to an infection. 46. The method according to embodiment 45, wherein the infection is a viral infection. 47. The method according to embodiment 46, wherein the viral infection is a coronavirus infection. 48. The method according to embodiment 47, wherein the coronavirus is COVID-19. 49. The method according to embodiment 46, wherein the viral infection is an influenza infection. 50. The method according to embodiment 44, wherein the subject has CRS caused by immunotherapy. 51. The method according to embodiment 50, wherein the immunotherapy includes antibody therapy. 52. The method according to embodiment 50, wherein the immunotherapy comprises chimeric antigen receptor (CAR) T cell therapy. 53. The method according to any one of embodiments 50 to 52, wherein the lipid-binding protein-based complex is administered before initiation of the immunotherapy. 54. The method according to any one of embodiments 50 to 53, wherein the lipid-binding protein-based complex is administered concomitantly with the immunotherapy. 55. The method according to any one of embodiments 50 to 54, wherein the lipid-binding protein-based complex is administered after completion of the immunotherapy. 56. The method according to embodiment 43, wherein the subject is at risk of CRS. 57. The method according to embodiment 56, wherein the subject is at risk of CRS caused by an infectious disease. 58. The method according to embodiment 57, wherein the infectious disease is a viral infection. 59. The method according to embodiment 58, wherein the viral infection is a coronavirus infection. 60. The method according to embodiment 59, wherein the coronavirus is COVID-19. 61. The method according to embodiment 58, wherein the viral infection is an influenza infection. 62. The method according to embodiment 56, wherein the subject is at risk of CRS caused by the immunotherapy. 63. The method according to embodiment 62, wherein the immunotherapy comprises antibody therapy. 64. The method according to embodiment 62, wherein the immunotherapy comprises chimeric antigen receptor (CAR) T cell therapy. 65. The method according to any one of embodiments 62 to 64, wherein the lipid-binding protein-based complex is administered before initiation of the immunotherapy. 66. The method according to any one of embodiments 62 to 65, wherein the lipid-binding protein-based complex is administered concomitantly with the immunotherapy. 67. The method according to any one of embodiments 62 to 66, wherein the lipid-binding protein-based complex is administered after completion of the immunotherapy. 68. The method according to any one of embodiments 1 to 42, wherein the subject has or is at risk of developing sepsis. 69. The method according to embodiment 68, wherein the sepsis is associated with a Gram-negative bacterial infection. 70. The method according to embodiment 68, wherein the sepsis is associated with a Gram-positive bacterial infection. 71. The method according to any one of embodiments 68 to 70, wherein the subject has an intra-abdominal infection. 72. The method according to any one of embodiments 68 to 70, wherein the subject has urosepsis. 73. The method according to any one of embodiments 68 to 72, wherein the high dose is effective in reducing the severity of sepsis. 74. The method according to any one of embodiments 1 to 73, wherein the high dose is effective in reducing the likelihood that the subject will develop acute kidney injury (AKI). 75. The method according to any one of embodiments 1 to 74, wherein the high dose is effective in delaying the onset of AKI. 76. The method according to any one of embodiments 1 to 74, wherein the high dose is effective in preventing AKI. 77. The method according to any one of embodiments 1 to 73, wherein the subject has or is at risk of developing acute kidney injury (AKI). 78. The method according to embodiment 77, wherein the AKI is sepsis-related AKI. 79. The method according to embodiment 77, wherein the AKI is ischemia / reperfusion AKI. 80. The method according to embodiment 77, wherein the AKI is cardiac surgery-related AKI. 81. The method according to embodiment 77, wherein the AKI is hepatorenal syndrome (HRS) AKI. 82. The method according to embodiment 81, wherein the HRS is type 1 HRS. 83. The method according to embodiment 81, wherein the HRS is type 2 HRS. 84. The method according to any one of embodiments 77 to 83, wherein the subject has AKI. 85. The method according to embodiment 84, wherein the AKI follows a viral infection, and optionally, the viral infection is COVID-19. 86. The method according to embodiment 84 or embodiment 85, wherein the high dose is effective in reducing the severity of AKI. 87. The method according to any one of embodiments 77 to 81, wherein the subject has a risk of AKI. 88. The method according to embodiment 87, wherein the subject has sepsis. 89. The method according to embodiment 88, wherein the sepsis is associated with a gram-negative bacterial infection. 90. The method according to embodiment 88, wherein the sepsis is associated with a gram-positive bacterial infection. 91. The method according to any one of embodiments 88 to 90, wherein the subject has an intra-abdominal infection. 92. The method according to any one of embodiments 88 to 90, wherein the subject has urosepsis. 93. The method according to embodiment 87, wherein the subject has a viral infection, and optionally, the viral infection is COVID-19. 94. The method according to embodiment 87, wherein the subject has undergone cardiac surgery. 95. The method according to embodiment 87, wherein the subject has acute liver disease. 96. The method according to embodiment 87, wherein the subject has chronic liver disease. 97. The method according to any one of embodiments 87 to 96, wherein the high dose is effective in reducing the likelihood that the subject will develop AKI. 98. The method according to any one of embodiments 87 to 97, wherein the high dose is effective in delaying the onset of AKI. 99. The method according to any one of embodiments 87 to 97, wherein the high dose is effective in preventing AKI. 100. The method according to any one of embodiments 87 to 98, wherein if the subject develops AKI, the high dose is effective in reducing the severity of AKI. 101. The method according to any one of embodiments 68 to 100, wherein the subject has a total SOFA score of 2 to 24 prior to administration of the lipid-bound protein-based complex. 102. The method according to embodiment 101, wherein the subject has a total SOFA score of 2 - 5 before administration of the complex based on a lipid-binding protein. 103. The method according to embodiment 101, wherein the subject has a total SOFA score of 2 - 10 before administration of the complex based on a lipid-binding protein. 104. The method according to embodiment 101, wherein the subject has a total SOFA score of 5 - 15 before administration of the complex based on a lipid-binding protein. 105. The method according to embodiment 101, wherein the subject has a total SOFA score of 8 - 13 before administration of the complex based on a lipid-binding protein. 106. The method according to embodiment 101, wherein the subject has a total SOFA score of 10 - 15 before administration of the complex based on a lipid-binding protein. 107. The method according to embodiment 101, wherein the subject has a total SOFA score of 10 - 20 before administration of the complex based on a lipid-binding protein. 108. The method according to embodiment 101, wherein the subject has a total SOFA score of 15 - 24 before administration of the complex based on a lipid-binding protein. 109. The method according to any one of embodiments 1 to 108, wherein the subject has an endotoxin activity level > 0.6 before administration of the complex based on a lipid-binding protein. 110. The method according to any one of embodiments 1 to 109, wherein the high dose is effective to reduce the endotoxin activity level of the subject. 111. The method according to any one of embodiments 1 to 110, wherein the complex based on a lipid-binding protein is reconstituted HDL or an HDL mimetic. 112. The method according to any one of embodiments 1 to 110, wherein the complex based on a lipid-binding protein is an Apomer or a Cargomer. 113. The method according to any one of embodiments 1 to 112, wherein the complex based on a lipid-binding protein contains sphingomyelin. 114. The method according to any one of embodiments 1 to 113, wherein the complex based on a lipid-binding protein contains a negatively charged lipid. 115. The method according to embodiment 114, wherein the charged lipid is 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) or a salt thereof. 116. The method according to embodiment 111, wherein the complex based on the lipid-binding protein is CER-001, CSL-111, CSL-112, CER-522, or ETC-216. 117. The method according to embodiment 116, wherein the complex based on the lipid-binding protein is CER-001. 118. The method according to any one of embodiments 1 to 117, wherein the complex based on the lipid-binding protein is administered systemically, optionally by infusion. 119. The method according to any one of embodiments 1 to 118, wherein the complex based on the lipid-binding protein is administered until the serum level of one or more inflammatory markers decreases. 120. The method according to embodiment 119, wherein the complex based on the lipid-binding protein is administered until the serum level of one or more inflammatory markers decreases to the normal range(s). 121. The method according to embodiment 119, wherein the complex based on the lipid-binding protein is administered until the serum level of one or more inflammatory markers decreases below the baseline level(s) for the one or more inflammatory markers measured prior to administration of the complex based on the lipid-binding protein. 122. The method according to any one of embodiments 1 to 121, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 4 - 40 mg / kg (protein weight basis). 123. The method according to embodiment 122, wherein each individual dose of the complex based on the lipid-binding protein is 4 - 30 mg / kg (protein weight basis). 124. The method according to embodiment 122, wherein each individual dose of the complex based on the lipid-binding protein is 15 - 25 mg / kg (protein weight basis). 125. The method according to embodiment 122, wherein the individual dose of each complex based on the lipid-binding protein is 10 to 30 mg / kg (protein weight basis). 126. The method according to embodiment 122, wherein the individual dose of each complex based on the lipid-binding protein is 10 to 20 mg / kg (protein weight basis). 127. The method according to embodiment 122, wherein the individual dose of each complex based on the lipid-binding protein is 5 mg / kg (protein weight basis). 128. The method according to embodiment 122, wherein the individual dose of each complex based on the lipid-binding protein is 10 mg / kg (protein weight basis). 129. The method according to embodiment 122, wherein the individual dose of each complex based on the lipid-binding protein is 15 mg / kg (protein weight basis). 130. The method according to embodiment 122, wherein the individual dose of each complex based on the lipid-binding protein is 20 mg / kg (protein weight basis). 131. The method according to embodiment 122, wherein the individual dose of each complex based on the lipid-binding protein is 5 to 15 mg / kg (protein weight basis). 132. The method according to embodiment 122, wherein the individual dose of each complex based on the lipid-binding protein is 10 to 20 mg / kg (protein weight basis). 133. The method according to embodiment 122, wherein the individual dose of each complex based on the lipid-binding protein is 15 to 25 mg / kg (protein weight basis). 134. The method according to any one of embodiments 1 to 133, wherein the high dose is administered according to an induction regimen, optionally followed by a consolidation therapy regimen. 135. The method according to embodiment 134, wherein the induction regimen comprises administering the complex based on the lipid-binding protein once or twice a day. 136. The method according to embodiment 134 or embodiment 135, wherein the consolidation therapy regimen comprises administering the complex based on the lipid-binding protein once a day or once every two days. 137. The method according to any one of embodiments 1 to 136, wherein the subject is not treated with a maintenance regimen. 138. The method according to any one of embodiments 134 to 137, wherein the consolidation therapy regimen comprises administering to the subject one or more doses of a lipid-binding protein-based complex one or more days after the administration of the final dose of the induction regimen. 139. The method according to embodiment 138, wherein the first dose of the lipid-binding protein-based complex administered during the consolidation therapy regimen is administered more than 2 days after the administration of the final dose of the induction regimen. 140. The method according to embodiment 138, wherein the first dose of the lipid-binding protein-based complex administered during the consolidation therapy regimen is administered more than 3 days after the administration of the final dose of the induction regimen. 141. The method according to embodiment 140, wherein the first dose of the lipid-binding protein-based complex administered during the consolidation therapy regimen is administered 3 days after the administration of the final dose of the induction regimen. 142. An induction regimen comprising administering the lipid-binding protein-based complex twice a day on days 1, 2, and 3, and a consolidation therapy regimen comprising two doses of the lipid-binding protein-based complex on day 6, according to any one of embodiments 134 to 141. 143. The method according to any one of embodiments 134 to 142, wherein each individual dose of the lipid-binding protein-based complex administered in the induction regimen is 4 to 40 mg / kg (protein weight basis). 144. The method according to any one of embodiments 134 to 143, wherein each individual dose of the lipid-binding protein-based complex administered in the induction regimen is 4 to 30 mg / kg (protein weight basis). 145. The method according to any one of embodiments 134 to 143, wherein each individual dose of the lipid-binding protein-based complex administered in the induction regimen is 15 to 25 mg / kg (protein weight basis). 146. The method according to any one of embodiments 134 to 143, wherein the individual dose of each lipid-binding protein-based complex administered in the induction regimen is 10 to 30 mg / kg (protein weight basis). 147. The method according to any one of embodiments 134 to 143, wherein the individual dose of each lipid-binding protein-based complex administered in the induction regimen is 10 to 20 mg / kg (protein weight basis). 148. The method according to any one of embodiments 134 to 143, wherein the individual dose of each lipid-binding protein-based complex administered in the induction regimen is 5 mg / kg (protein weight basis). 149. The method according to any one of embodiments 134 to 143, wherein the individual dose of each lipid-binding protein-based complex administered in the induction regimen is 10 mg / kg (protein weight basis). 150. The method according to any one of embodiments 134 to 143, wherein the individual dose of each lipid-binding protein-based complex administered in the induction regimen is 15 mg / kg (protein weight basis). 151. The method according to any one of embodiments 134 to 143, wherein the individual dose of each lipid-binding protein-based complex administered in the induction regimen is 20 mg / kg (protein weight basis). 152. The method according to any one of embodiments 134 to 151, wherein the dose of the lipid-binding protein-based complex administered in the consolidation therapy regimen is 5 to 15 mg / kg (protein weight basis). 153. The method according to any one of embodiments 134 to 151, wherein the dose of the lipid-binding protein-based complex administered in the consolidation therapy regimen is 10 to 20 mg / kg (protein weight basis). 154. The method according to any one of embodiments 134 to 151, wherein the dose of the lipid-binding protein-based complex administered in the consolidation therapy regimen is 15 to 25 mg / kg (protein weight basis). 155. The method according to any one of embodiments 134 to 151, wherein the dose of the complex based on the lipid-binding protein administered in the consolidation therapy regimen is 5 mg / kg (protein weight basis). 156. The method according to any one of embodiments 134 to 151, wherein the dose of the complex based on the lipid-binding protein administered in the consolidation therapy regimen is 10 mg / kg (protein weight basis). 157. The method according to any one of embodiments 134 to 151, wherein the dose of the complex based on the lipid-binding protein administered in the consolidation therapy regimen is 15 mg / kg (protein weight basis). 158. The method according to any one of embodiments 1 to 157, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 300 mg to 4000 mg (protein weight basis). 159. The method according to embodiment 158, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 300 mg to 3000 mg (protein weight basis). 160. The method according to embodiment 158, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 300 mg to 1500 mg (protein weight basis). 161. The method according to embodiment 158, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 400 mg to 4000 mg (protein weight basis). 162. The method according to embodiment 158, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 400 mg to 1500 mg (protein weight basis). 163. The method according to embodiment 158, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 500 mg to 1200 mg (protein weight basis). 164. The method according to embodiment 158, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 500 mg to 1000 mg (protein weight basis). 165. The method according to embodiment 158, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 600 mg to 3000 mg (protein weight basis). 166. The method according to embodiment 158, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 800 mg to 3000 mg (protein weight basis). 167. The method according to embodiment 158, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 1000 mg to 2400 mg (protein weight basis). 168. The method according to embodiment 158, wherein each individual dose of the complex based on the lipid-binding protein to be administered is 1000 mg to 2000 mg (protein weight basis). 169. The method according to any one of embodiments 1 to 168, wherein the high dose of the complex based on the lipid-binding protein is 600 mg to 40 g (protein weight basis). 170. The method according to any one of embodiments 1 to 168, wherein the high dose of the complex based on the lipid-binding protein is 3 g to 35 g (protein weight basis). 171. The method according to any one of embodiments 1 to 168, wherein the high dose of the complex based on the lipid-binding protein is 5 g to 30 g (protein weight basis). 172. The method according to any one of embodiments 1 to 171, wherein the complex based on the lipid-binding protein is administered by infusion. 173. The method according to embodiment 172, wherein each individual dose is administered over a period of 1 to 24 hours. 174. The method according to embodiment 173, wherein each individual dose is administered over a period of 24 hours. 175. The method according to embodiment 172, wherein each individual dose is administered over a period of 1 hour or less. 176. The method according to embodiment 172, wherein each individual dose is administered over a period of 0.5 hour to 1 hour. 177. The method according to any one of embodiments 1 to 176, further comprising administering an antihistamine to the subject before each individual dose. 178. The method according to embodiment 177, wherein the antihistamine comprises dechlorpheniramine or hydroxyzine. 179. The method according to any one of embodiments 1 to 178, wherein the subject is a person who has received or is receiving one or more additional treatments, and / or further comprising administering to the subject one or more additional treatments. 180. The method according to embodiment 179, wherein the one or more additional treatments comprise one or more anti-IL-6 agents. 181. The method according to embodiment 180, wherein the one or more anti-IL-6 agents comprise tocilizumab, siltuximab, ocrelizumab, elsilimomab, BMS-945429, sirukumab, revlimomab, CPSI-2364, or a combination thereof. 182. The method according to embodiment 181, wherein the one or more anti-IL-6 agents comprise tocilizumab. 183. The method according to any one of embodiments 179 to 182, wherein the one or more additional treatments comprise one or more antibiotics. 184. The method according to embodiment 183, wherein the one or more antibiotics comprise one or more antibiotics of the oxazolidinone class. 185. The method according to embodiment 183 or 184, wherein the one or more antibiotics comprise linezolid. 186. The method according to any one of embodiments 183 to 185, wherein the one or more antibiotics comprise tedizolid. 187. The method according to any one of embodiments 179 to 186, wherein the one or more additional treatments comprise one or more corticosteroids. 188. The method according to embodiment 187, wherein the one or more corticosteroids comprise methylprednisolone, dexamethasone, or a combination thereof. 189. The method according to any one of embodiments 179 to 188, wherein the subject is a person who has or had COVID-19 infection, and the one or more additional treatments comprise antibodies from recovered COVID-19 patients. 190. The method according to any one of embodiments 179 to 189, wherein the subject is a person who has or had COVID-19 infection, and the one or more additional treatments comprise antibodies against the spike protein of COVID-19 (e.g., casirivimab and / or imdevimab). 191. The method according to any one of embodiments 179 to 190, wherein the subject is a person who has or had COVID-19 infection, and the one or more additional treatments comprise one or more antiviral agents. 192. The method according to embodiment 191, wherein the one or more antiviral agents comprise lopinavir. 193. The method according to embodiment 191 or embodiment 192, wherein the one or more antiviral agents comprise remdesivir. 194. The method according to any one of embodiments 191 to 193, wherein the one or more antiviral agents comprise danoprevir. 195. The method according to any one of embodiments 191 to 194, wherein the one or more antiviral agents comprise galidesivir. 196. The method according to any one of embodiments 191 to 195, wherein the one or more antiviral agents comprise darunavir. 197. The method according to any one of embodiments 191 to 196, wherein the one or more antiviral agents comprise ritonavir. 198. The method according to any one of embodiments 179 to 197, wherein the subject is a person who has or had COVID-19 infection, and the one or more additional treatments comprise chloroquine or hydroxychloroquine. 199. The method according to any one of embodiments 179 to 198, wherein the subject is a person who has or had COVID-19 infection, and the one or more additional treatments comprise azithromycin. 200. The method according to any one of embodiments 179 to 199, wherein the subject has or had COVID-19 infection, and one or more additional treatments include interferon. 201. The method according to embodiment 200, wherein the interferon is interferon alpha. 202. The method according to embodiment 200, wherein the interferon is interferon beta. 203. The method according to any one of embodiments 200 to 202, wherein the interferon is pegylated. 204. The method according to any one of embodiments 179 to 203, wherein the subject has or had COVID-19 infection, and one or more additional treatments include tacrolimus. 205. The method according to any one of embodiments 179 to 204, wherein the subject has or had COVID-19 infection, and one or more additional treatments include dexamethasone. 206. The method according to any one of embodiments 179 to 205, wherein the subject has or had COVID-19 infection, and one or more additional treatments include an antibiotic. 207. The method according to any one of embodiments 179 to 206, wherein the subject has or had COVID-19 infection, and one or more additional treatments include supplemental oxygen. 208. The method according to any one of embodiments 1 to 207, wherein the lipid-binding protein-based complex is CER-001. 209. The method according to embodiment 208, wherein CER-001 is a lipoprotein complex comprising ApoA-I and phospholipids at an ApoA-I weight:total phospholipid weight ratio of 1:2.7 ± 20%, and sphingomyelin phospholipid and DPPG at a sphingomyelin weight:DPPG weight ratio of 97:3 ± 20%. The method according to embodiment 208, wherein CER-001 is a lipoprotein complex comprising ApoA-I and phospholipids at a ratio of ApoA-I weight: total phospholipid weight of 1:2.7 ± 10%, and sphingomyelin phospholipid and DPPG at a ratio of sphingomyelin weight: DPPG weight of 97:3 ± 10%. The method according to embodiment 208, wherein CER-001 is a lipoprotein complex comprising ApoA-I and phospholipids at a ratio of ApoA-I weight: total phospholipid weight of 1:2.7, and sphingomyelin phospholipid and DPPG at a weight: weight ratio of sphingomyelin: DPPG of 97:3. The method according to any one of embodiments 209 to 211, wherein ApoA-I has the amino acid sequence of amino acids 25 to 267 of SEQ ID NO: 2. The method according to any one of embodiments 209 to 212, wherein ApoA-I is recombinantly expressed. The method according to any one of embodiments 209 to 213, wherein CER-001 contains natural sphingomyelin. The method according to embodiment 214, wherein the natural sphingomyelin is chicken egg sphingomyelin. The method according to any one of embodiments 209 to 213, wherein CER-001 contains synthetic sphingomyelin. The method according to embodiment 216, wherein the synthetic sphingomyelin is palmitoyl sphingomyelin. The method according to any one of embodiments 208 to 217, wherein CER-001 is administered in the form of a formulation in which CER-001 is at least 95% homogeneous. The method according to embodiment 218, wherein CER-001 is administered in the form of a formulation in which CER-001 is at least 97% homogeneous. The method according to embodiment 218, wherein CER-001 is administered in the form of a formulation in which CER-001 is at least 98% homogeneous. The method according to embodiment 218, wherein CER-001 is administered in the form of a formulation in which CER-001 is at least 99% homogeneous. 222. The method according to any one of embodiments 1 to 221, wherein the subject is a human. 223. The method according to any one of embodiments 1 to 222, wherein the subject is not receiving mechanical ventilation when CER-001 is first administered. 224. The method according to any one of embodiments 116, 118 to 207, 222, or 223, wherein the complex based on the lipid-binding protein is CSL-112. 225. The method according to any one of embodiments 209 to 223, wherein ApoA-I is produced by a mammalian host cell. 226. The method according to embodiment 225, wherein the mammalian host cell is a Chinese hamster ovary (CHO) cell. 227. The method according to embodiment 226, wherein the CHO cell is a CHO-S cell. 228. The method according to any one of embodiments 225 to 227, wherein ApoA-I has undergone post-translational processing (e.g., glycosylation) such that ApoA-I has one or more structural features (e.g., glycosylation pattern) different from human ApoA-I purified from human plasma. 229. A method of treating a subject having an acute condition, comprising administering to a subject in need thereof a high-dose apolipoprotein A-I (''ApoA-I'') formulation comprising ApoA-I and one or more lipids, wherein the ApoA-I and lipids are in the form of a lipoprotein complex, and optionally wherein the acute condition comprises acute inflammation. 230. The method according to embodiment 229, wherein the high dose is administered over a period of 1 day to approximately 2 weeks, and optionally wherein the high dose is administered over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. 231. The method according to embodiment 229 or embodiment 230, wherein the high dose is a collection of 2 to 10 individual doses, and optionally wherein the high dose is a collection of 3, 4, 5, 6, 7, 8, 9, or 10 individual doses. The method according to embodiment 231, wherein a plurality of individual doses are administered daily or twice a day. The method according to embodiment 231 or embodiment 232, wherein a plurality of individual doses are administered at intervals of 2 to 3 days. The method according to embodiment 231, wherein a plurality of individual doses are administered at intervals within one day. The method according to embodiment 234, comprising administering two or more individual doses at intervals of approximately 12 hours. The method according to embodiment 235, comprising administering two individual doses at intervals of approximately 12 hours. The method according to embodiment 235, comprising administering three individual doses at intervals of approximately 12 hours. The method according to embodiment 236 or embodiment 237, further comprising administering an individual dose approximately one day later. The method according to embodiment 231, comprising administering three individual doses at intervals of approximately 12 hours and administering a fourth individual dose approximately one day later. The method according to embodiment 229, wherein the high dose is administered as a single individual dose. The method according to embodiment 229, wherein the high dose is a set of two individual doses administered within one day. The method according to embodiment 241, wherein the two individual doses are administered at intervals of approximately 12 hours. The method according to any one of embodiments 231 to 242, wherein each individual dose is effective to increase the HDL level of the subject. The method according to embodiment 243, wherein the high dose is effective to increase the serum HDL level of the subject to a normal value (for example, >0.45 g / L). The method according to embodiment 243, wherein each individual dose is effective to increase the HDL level of the subject by at least 25%, at least 30%, or at least 35% 2 to 4 hours after administration. 246. The method according to embodiment 245, wherein each individual dose is effective to increase the subject's HDL level by at least 25%, at least 30%, or at least 35% two hours after administration. 247. The method according to embodiment 245, wherein each individual dose is effective to increase the subject's HDL level by at least 25%, at least 30%, or at least 35% three hours after administration. 248. The method according to embodiment 245, wherein each individual dose is effective to increase the subject's HDL level by at least 25%, at least 30%, or at least 35% four hours after administration. 249. The method according to any one of embodiments 231 to 248, wherein each individual dose is effective to increase the subject's ApoA-I level. 250. The method according to embodiment 249, wherein the high dose is effective to increase the subject's serum ApoA-I level to a normal value (e.g., > 1.1 g / L). 251. The method according to embodiment 249, wherein each individual dose is effective to increase the subject's ApoA-I level by at least 25%, at least 30%, or at least 35% two to four hours after administration. 252. The method according to embodiment 251, wherein each individual dose is effective to increase the subject's ApoA-I level by at least 25%, at least 30%, or at least 35% two hours after administration. 253. The method according to embodiment 251, wherein each individual dose is effective to increase the subject's ApoA-I level by at least 25%, at least 30%, or at least 35% three hours after administration. 254. The method according to embodiment 251, wherein each individual dose is effective to increase the subject's ApoA-I level by at least 25%, at least 30%, or at least 35% four hours after administration. 255. The method according to any one of embodiments 229 to 254, wherein the high dose is effective to improve the endothelial function of the target blood vessel, and optionally, the endothelial function of the blood vessel is measured by circulating VCAM-1 and / or ICAM-1. 256. The method according to any one of embodiments 229 to 255, wherein the high dose is effective to reduce the serum level of one or more inflammatory markers in the subject. 257. The method according to embodiment 256, wherein the high dose is effective to reduce the serum level of interleukin-6 ("IL-6"). 258. The method according to embodiment 256 or embodiment 257, wherein the high dose is effective to reduce the serum level of C-reactive protein. 259. The method according to any one of embodiments 256 to 258, wherein the high dose is effective to reduce the serum level of D-dimer. 260. The method according to any one of embodiments 256 to 259, wherein the high dose is effective to reduce the serum level of ferritin. 261. The method according to any one of embodiments 256 to 260, wherein the high dose is effective to reduce the serum level of interleukin 8 (IL-8). 262. The method according to any one of embodiments 256 to 260, wherein the high dose is effective to normalize the serum level of interleukin 8 (IL-8). 263. The method according to any one of embodiments 256 to 262, wherein the high dose is effective to reduce the serum level of granulocyte macrophage colony-stimulating factor (GM-CSF). 264. The method according to any one of embodiments 256 to 263, wherein the high dose is effective to reduce the serum level of monocyte chemoattractant protein (MCP) 1. 265. The method according to any one of embodiments 256 to 264, wherein the high dose is effective to reduce the serum level of tumor necrosis factor α (TNF-α). 266. The method according to any one of embodiments 256 to 265, which is effective to lower the high dose from the elevated range of the serum level of one or more inflammatory markers to the normal range. 267. The method according to any one of embodiments 256 to 266, which is effective to lower the high dose of the serum level of one or more inflammatory markers by at least 20%, at least 40%, or at least 60%. 268. The method according to any one of embodiments 229 to 267, wherein the subject has a viral infection. 269. The method according to embodiment 268, wherein the viral infection is a coronavirus infection. 270. The method according to embodiment 269, wherein the coronavirus infection is COVID-19. 271. The method according to any one of embodiments 229 to 270, wherein the subject has or is at risk of CRS. 272. The method according to embodiment 271, wherein the subject has CRS. 273. The method according to embodiment 272, wherein the subject has CRS following an infection. 274. The method according to embodiment 273, wherein the infection is a viral infection. 275. The method according to embodiment 274, wherein the viral infection is a coronavirus infection. 276. The method according to embodiment 275, wherein the coronavirus is COVID-19. 277. The method according to embodiment 276, wherein the viral infection is an influenza infection. 278. The method according to embodiment 272, wherein the subject has CRS caused by immunotherapy. 279. The method according to embodiment 278, wherein the immunotherapy comprises antibody therapy. 280. The method according to embodiment 278, wherein the immunotherapy comprises chimeric antigen receptor (CAR) T cell therapy. 281. The method according to any one of embodiments 278 to 280, wherein the formulation is administered before the initiation of immunotherapy. The method according to any one of embodiments 278 to 281, wherein the formulation is administered concurrently with immunotherapy. The method according to any one of embodiments 278 to 282, wherein the formulation is administered after completion of immunotherapy. The method according to embodiment 271, wherein the subject is at risk of CRS. The method according to embodiment 284, wherein the subject is at risk of CRS caused by an infectious disease. The method according to embodiment 285, wherein the infectious disease is a viral infectious disease. The method according to embodiment 286, wherein the viral infectious disease is a coronavirus infectious disease. The method according to embodiment 287, wherein the coronavirus is COVID-19. The method according to embodiment 286, wherein the viral infectious disease is an influenza infectious disease. The method according to embodiment 284, wherein the subject is at risk of CRS caused by immunotherapy. The method according to embodiment 290, wherein the immunotherapy includes antibody therapy. The method according to embodiment 290, wherein the immunotherapy includes chimeric antigen receptor (CAR) T cell therapy. The method according to any one of embodiments 290 to 292, wherein the formulation is administered before the start of immunotherapy. The method according to any one of embodiments 290 to 293, wherein the formulation is administered concurrently with immunotherapy. The method according to any one of embodiments 290 to 294, wherein the formulation is administered after completion of immunotherapy. The method according to any one of embodiments 229 to 270, wherein the subject has or is at risk of developing sepsis. The method according to embodiment 296, wherein the sepsis is associated with a gram-negative bacterial infection. The method according to embodiment 296, wherein the sepsis is associated with a gram-positive bacterial infection. The method according to any one of embodiments 296 to 298, wherein the subject has an intra-abdominal infectious disease. 300. The method according to any one of embodiments 296 to 298, wherein the subject has urosepsis. 301. The method according to any one of embodiments 296 to 300, wherein the high dose is effective in reducing the severity of sepsis. 302. The method according to any one of embodiments 229 to 301, wherein the high dose is effective in reducing the likelihood that the subject will develop acute kidney injury (AKI). 303. The method according to any one of embodiments 229 to 302, wherein the high dose is effective in delaying the onset of AKI. 304. The method according to any one of embodiments 229 to 302, wherein the high dose is effective in preventing AKI. 305. The method according to any one of embodiments 229 to 301, wherein the subject has or is at risk of developing acute kidney injury (AKI). 306. The method according to embodiment 305, wherein the AKI is sepsis-related AKI. 307. The method according to embodiment 305, wherein the AKI is ischemia / reperfusion AKI. 308. The method according to embodiment 305, wherein the AKI is cardiac surgery-related AKI. 309. The method according to embodiment 305, wherein the AKI is hepatorenal syndrome (HRS) AKI. 310. The method according to embodiment 309, wherein the HRS is type 1 HRS. 311. The method according to embodiment 309, wherein the HRS is type 2 HRS. 312. The method according to any one of embodiments 305 to 311, wherein the subject has AKI. 313. The method according to embodiment 312, wherein the AKI follows a viral infection, and optionally, the viral infection is COVID-19. 314. The method according to embodiment 312 or embodiment 313, wherein the high dose is effective in reducing the severity of AKI. 315. The method according to any one of embodiments 305 to 311, wherein the subject is at risk of AKI. 316. The method according to embodiment 315, wherein the subject has sepsis. 317. The method according to embodiment 316, wherein the sepsis is associated with a gram-negative bacterial infection. 318. The method according to embodiment 316, wherein the sepsis is associated with a gram-positive bacterial infection. 319. The method according to any one of embodiments 316 to 318, wherein the subject has an intra-abdominal infection. 320. The method according to any one of embodiments 316 to 318, wherein the subject has urosepsis. 321. The method according to embodiment 315, wherein the subject has a viral infection, and optionally, the viral infection is COVID-19. 322. The method according to embodiment 315, wherein the subject has undergone cardiac surgery. 323. The method according to embodiment 315, wherein the subject has acute liver disease. 324. The method according to embodiment 315, wherein the subject has chronic liver disease. 325. The method according to any one of embodiments 315 to 324, wherein the high dose is effective in reducing the likelihood that the subject will develop AKI. 326. The method according to any one of embodiments 315 to 325, wherein the high dose is effective in delaying the onset of AKI. 327. The method according to any one of embodiments 315 to 325, wherein the high dose is effective in preventing AKI. 328. The method according to any one of embodiments 315 to 326, wherein when the subject develops AKI, the high dose is effective in reducing the severity of AKI. 329. The method according to any one of embodiments 296 to 328, wherein the subject has a total SOFA score of 2 to 24 before administration of the formulation. 330. The method according to embodiment 329, wherein the subject has a total SOFA score of 2 to 5 before administration of the formulation. 331. The method according to embodiment 329, wherein the subject has a total SOFA score of 2 to 10 before administration of the formulation. 332. The method according to embodiment 329, wherein the subject has a total SOFA score of 5 to 15 before administration of the formulation. 333. The method according to embodiment 329, wherein the subject has a total SOFA score of 8 to 13 before administration of the formulation. 334. The method according to embodiment 329, wherein the subject has a total SOFA score of 10 to 15 before administration of the formulation. 335. The method according to embodiment 329, wherein the subject has a total SOFA score of 10 to 20 before administration of the formulation. 336. The method according to embodiment 329, wherein the subject has a total SOFA score of 15 to 24 before administration of the formulation. 337. The method according to any one of embodiments 229 to 336, wherein the subject has an endotoxin activity level > 0.6 before administration of the formulation. 338. The method according to any one of embodiments 229 to 337, wherein the high dose is effective in reducing the endotoxin activity level of the subject. 339. The method according to any one of embodiments 229 to 338, wherein the formulation is reconstituted HDL or an HDL mimetic. 340. The method according to any one of embodiments 229 to 339, wherein the formulation contains sphingomyelin. 341. The method according to any one of embodiments 229 to 340, wherein the formulation contains a negatively charged lipid. 342. The method according to embodiment 341, wherein the negatively charged lipid is 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) or a salt thereof. 343. The method according to any one of embodiments 229 to 342, wherein the formulation is optionally administered systemically by infusion. 344. The method according to any one of embodiments 229 to 343, wherein the formulation is administered until the serum level of one or more inflammatory markers decreases. 345. The method according to embodiment 344, wherein the formulation is administered until the serum level of one or more inflammatory markers decreases to the normal range(s). 346. The method according to embodiment 344, wherein the formulation is administered until the serum level of one or more inflammatory markers decreases to less than the baseline level(s) for the one or more inflammatory markers measured prior to administration of the complex based on the lipid-binding protein. 347. The method according to any one of embodiments 229 to 346, wherein each individual dose of the administered formulation is 4 - 40 mg / kg (protein weight basis). 348. The method according to embodiment 347, wherein each individual dose of the formulation is 4 - 30 mg / kg (protein weight basis). 349. The method according to embodiment 347, wherein each individual dose of the formulation is 15 - 25 mg / kg (protein weight basis). 350. The method according to embodiment 347, wherein each individual dose of the formulation is 10 - 30 mg / kg (protein weight basis). 351. The method according to embodiment 347, wherein each individual dose of the formulation is 10 - 20 mg / kg (protein weight basis). 352. The method according to embodiment 347, wherein each individual dose of the formulation is 5 mg / kg (protein weight basis). 353. The method according to embodiment 347, wherein each individual dose of the formulation is 10 mg / kg (protein weight basis). 354. The method according to embodiment 347, wherein each individual dose of the formulation is 15 mg / kg (protein weight basis). 355. The method according to embodiment 347, wherein each individual dose of the formulation is 20 mg / kg (protein weight basis). 356. The method according to embodiment 347, wherein each individual dose of the formulation is 5 - 15 mg / kg (protein weight basis). 357. The method according to embodiment 347, wherein each individual dose of the formulation is 10 - 20 mg / kg (protein weight basis). 358. The method according to embodiment 347, wherein each individual dose of the formulation is 15 - 25 mg / kg (protein weight basis). 359. The method according to any one of embodiments 229 to 358, wherein the high dose is administered according to an induction regimen and optionally followed by a consolidation therapy regimen. 360. The method according to embodiment 359, wherein the induction regimen comprises administering the formulation once or twice a day. 361. The method according to embodiment 359 or 360, wherein the consolidation therapy regimen comprises administering the formulation once a day or once every two days. 362. The method according to any one of embodiments 229 to 361, wherein the subject is not treated with a maintenance regimen. 363. The method according to any one of embodiments 359 to 362, wherein the consolidation therapy regimen comprises administering one or more doses of the formulation to the subject one day or several days after administering the final dose of the induction regimen. 364. The method according to embodiment 363, wherein the first dose of the formulation administered during the consolidation therapy regimen is administered more than two days after administering the final dose of the induction regimen. 365. The method according to embodiment 363, wherein the first dose of the formulation administered during the consolidation therapy regimen is administered more than three days after administering the final dose of the induction regimen. 366. The method according to embodiment 365, wherein the first administration of the formulation administered during the consolidation therapy regimen is administered three days after administering the final dose of the induction regimen. 367. The method according to any one of embodiments 359 to 366, comprising an induction regimen that includes administering the formulation twice a day on days 1, 2, and 3, and a consolidation therapy regimen that includes two doses of the formulation on day 6. 368. The method according to any one of embodiments 359 to 367, wherein each individual dose of the formulation administered in the induction regimen is 4 - 40 mg / kg (protein weight basis). 369. The method according to any one of embodiments 359 to 368, wherein each individual dose of the formulation administered in the induction regimen is 4 - 30 mg / kg (protein weight basis). 370. The method according to any one of embodiments 359 to 368, wherein the individual dose of each formulation administered in the induction regimen is 15 to 25 mg / kg (protein weight basis). 371. The method according to any one of embodiments 359 to 368, wherein the individual dose of each formulation administered in the induction regimen is 10 to 30 mg / kg (protein weight basis). 372. The method according to any one of embodiments 359 to 368, wherein the individual dose of each formulation administered in the induction regimen is 10 to 20 mg / kg (protein weight basis). 373. The method according to any one of embodiments 359 to 368, wherein the individual dose of each formulation administered in the induction regimen is 5 mg / kg (protein weight basis). 374. The method according to any one of embodiments 359 to 368, wherein the individual dose of each formulation administered in the induction regimen is 10 mg / kg (protein weight basis). 375. The method according to any one of embodiments 359 to 368, wherein the individual dose of each formulation administered in the induction regimen is 15 mg / kg (protein weight basis). 376. The method according to any one of embodiments 359 to 368, wherein the individual dose of each formulation administered in the induction regimen is 20 mg / kg (protein weight basis). 377. The method according to any one of embodiments 359 to 376, wherein the dose of the formulation administered in the consolidation therapy regimen is 5 to 15 mg / kg (protein weight basis). 378. The method according to any one of embodiments 359 to 376, wherein the dose of the formulation administered in the consolidation therapy regimen is 10 to 20 mg / kg (protein weight basis). 379. The method according to any one of embodiments 359 to 376, wherein the dose of the formulation administered in the consolidation therapy regimen is 15 to 25 mg / kg (protein weight basis). The method according to any one of embodiments 359 to 376, wherein the dose of the formulation administered in the soil consolidation therapy regimen is 5 mg / kg (protein weight basis). The method according to any one of embodiments 359 to 376, wherein the dose of the formulation administered in the soil consolidation therapy regimen is 10 mg / kg (protein weight basis). The method according to any one of embodiments 359 to 376, wherein the dose of the formulation administered in the soil consolidation therapy regimen is 15 mg / kg (protein weight basis). The method according to any one of embodiments 229 to 382, wherein each individual dose of the formulation administered is 300 mg to 4000 mg (protein weight basis). The method according to embodiment 383, wherein each individual dose of the formulation administered is 300 mg to 3000 mg (protein weight basis). The method according to embodiment 383, wherein each individual dose of the formulation administered is 300 mg to 1500 mg (protein weight basis). The method according to embodiment 383, wherein each individual dose of the formulation administered is 400 mg to 4000 mg (protein weight basis). The method according to embodiment 383, wherein each individual dose of the formulation administered is 400 mg to 1500 mg (protein weight basis). The method according to embodiment 383, wherein each individual dose of the formulation administered is 500 mg to 1200 mg (protein weight basis). The method according to embodiment 383, wherein each individual dose of the formulation administered is 500 mg to 1000 mg (protein weight basis). The method according to embodiment 383, wherein each individual dose of the formulation administered is 600 mg to 3000 mg (protein weight basis). The method according to embodiment 383, wherein each individual dose of the formulation administered is 800 mg to 3000 mg (protein weight basis). The method according to embodiment 383, wherein the individual dose of each formulation administered is 1000 mg to 2400 mg (protein weight basis). The method according to embodiment 383, wherein the individual dose of each formulation administered is 1000 mg to 2000 mg (protein weight basis). The method according to any one of embodiments 229 to 393, wherein the high dose of the formulation is 600 mg to 40 g (protein weight basis). The method according to any one of embodiments 229 to 393, wherein the high dose of the formulation is 3 g to 35 g (protein weight basis). The method according to any one of embodiments 229 to 393, wherein the high dose of the formulation is 5 g to 30 g (protein weight basis). The method according to any one of embodiments 229 to 396, wherein the formulation is administered by infusion. The method according to embodiment 397, wherein each individual dose is administered over 1 to 24 hours. The method according to embodiment 398, wherein each individual dose is administered over 24 hours. The method according to embodiment 397, wherein each individual dose is administered over a period of 1 hour or less. The method according to embodiment 397, wherein each individual dose is administered over a period of 0.5 hour to 1 hour. The method according to any one of embodiments 229 to 401, further comprising administering an antihistamine to the subject prior to each individual dose. The method according to embodiment 402, wherein the antihistamine comprises dechlorpheniramine or hydroxyzine. The method according to any one of embodiments 229 to 403, wherein the subject has received or is receiving one or more additional treatments, and further comprising administering one or more additional treatments to the subject. The method according to embodiment 404, wherein the one or more additional treatments comprise one or more anti-IL-6 agents. 406. The method according to embodiment 405, wherein the one or more anti-IL-6 agents comprise tocilizumab, siltuximab, olokizumab, elsilimomab, BMS-945429, sirukumab, levilimomab, CPSI-2364, or a combination thereof. 407. The method according to embodiment 406, wherein the one or more anti-IL-6 agents comprise tocilizumab. 408. The method according to any one of embodiments 404 to 407, wherein the one or more additional treatments comprise one or more antibiotics. 409. The method according to embodiment 408, wherein the one or more antibiotics comprise one or more antibiotics of the oxazolidinone class. 410. The method according to embodiment 408 or embodiment 409, wherein the one or more antibiotics comprise linezolid. 411. The method according to any one of embodiments 408 to 410, wherein the one or more antibiotics comprise tedizolid. 412. The method according to any one of embodiments 404 to 410, wherein the one or more additional treatments comprise one or more corticosteroids. 413. The method according to embodiment 412, wherein the one or more corticosteroids comprise methylprednisolone, dexamethasone, or a combination thereof. 414. The method according to any one of embodiments 404 to 413, wherein the subject has or has had COVID-19 infection, and the one or more additional treatments comprise antibodies obtained from recovered COVID-19 patients. 415. The method according to any one of embodiments 404 to 414, wherein the subject has or has had COVID-19 infection, and the one or more additional treatments comprise antibodies against the spike protein of COVID-19 (e.g., casirivimab and / or imdevimab). 416. The method according to any one of embodiments 404 to 415, wherein the subject has or has had COVID-19 infection, and the one or more additional treatments comprise one or more antiviral agents. 417. The method according to embodiment 416, wherein the one or more antiviral agents comprise lopinavir. 418. The method according to embodiment 416 or embodiment 417, wherein the one or more antiviral agents comprise remdesivir. 419. The method according to any one of embodiments 416 to 418, wherein the one or more antiviral agents comprise danoprevir. 420. The method according to any one of embodiments 416 to 419, wherein the one or more antiviral agents comprise galidesivir. 421. The method according to any one of embodiments 416 to 420, wherein the one or more antiviral agents comprise darunavir. 422. The method according to any one of embodiments 416 to 421, wherein the one or more antiviral agents comprise ritonavir. 423. The method according to any one of embodiments 416 to 422, wherein the subject has or has had COVID-19 infection, and the one or more additional treatments comprise chloroquine or hydroxychloroquine. 424. The method according to any one of embodiments 404 to 423, wherein the subject has or has had COVID-19 infection, and the one or more additional treatments comprise azithromycin. 425. The method according to any one of embodiments 404 to 424, wherein the subject has or has had COVID-19 infection, and the one or more additional treatments comprise interferon. 426. The method according to embodiment 425, wherein the interferon is interferon alpha. 427. The method according to embodiment 425, wherein the interferon is interferon beta. 428. The method according to any one of embodiments 425 to 427, wherein the interferon is pegylated. 429. The method according to any one of embodiments 404 to 428, wherein the subject has or has had COVID-19 infection, and the one or more additional treatments comprise tacrolimus. 430. The method according to any one of embodiments 404 to 429, wherein the subject has or has had COVID-19 infection and one or more additional treatments include dexamethasone. 431. The method according to any one of embodiments 404 to 430, wherein the subject has or has had COVID-19 infection and one or more additional treatments include antibiotics. 432. The method according to any one of embodiments 404 to 431, wherein the subject has or has had COVID-19 infection and one or more additional treatments include supplemental oxygen. 433. The method according to embodiment 432, wherein the formulation comprises ApoA-I and phospholipids at an ApoA-I weight:total phospholipid weight ratio of 1:2.7 ± 20%, and sphingomyelin phospholipid and DPPG at a sphingomyelin weight:DPPG weight ratio of 97:3 ± 20%. 434. The method according to embodiment 432, wherein the formulation comprises ApoA-I and phospholipids at an ApoA-I weight:total phospholipid weight ratio of 1:2.7 ± 10%, and sphingomyelin phospholipid and DPPG at a sphingomyelin weight:DPPG weight ratio of 97:3 ± 10%. 435. The method according to embodiment 432, wherein the formulation comprises ApoA-I and phospholipids at an ApoA-I weight:total phospholipid weight ratio of 1:2.7, and sphingomyelin phospholipid and DPPG at a sphingomyelin weight:DPPG weight ratio of 97:3. 436. The method according to any one of embodiments 433 to 435, wherein ApoA-I has the amino acid sequence of amino acids 25 to 267 of SEQ ID NO: 2. 437. The method according to any one of embodiments 433 to 436, wherein ApoA-I is expressed by recombinant methods. 438. The method according to any one of embodiments 433 to 437, wherein the formulation comprises natural sphingomyelin. 439. The method according to embodiment 438, wherein the natural sphingomyelin is chicken egg sphingomyelin. 440. The method according to any one of embodiments 433 to 437, wherein the formulation comprises synthetic sphingomyelin. 441. The method according to embodiment 440, wherein the synthetic sphingomyelin is palmitoyl sphingomyelin. 442. The method according to any one of embodiments 433 to 441, wherein the formulation is at least 95% homogeneous. 443. The method according to embodiment 442, wherein the formulation is at least 97% homogeneous. 444. The method according to embodiment 442, wherein the formulation is at least 98% homogeneous. 445. The method according to embodiment 442, wherein the formulation is at least 99% homogeneous. 446. The method according to any one of embodiments 443 to 445, wherein the subject is a human. 447. The method according to any one of embodiments 443 to 446, wherein the subject is not receiving mechanical ventilation when the formulation is first administered. 448. The method according to any one of embodiments 437 to 447, wherein the ApoA-I is produced by a mammalian host cell. 449. The method according to embodiment 448, wherein the mammalian host cell is a Chinese hamster ovary (CHO) cell. 450. The method according to embodiment 449, wherein the CHO cell is a CHO-S cell. 451. The method according to any one of embodiments 448 to 450, wherein the ApoA-I has undergone post-translational processing (e.g., glycosylation) such that the ApoA-I has one or more structural features (e.g., glycosylation pattern) that are different from human ApoA-I purified from human plasma.

[0227] Although various specific embodiments have been illustrated and described, it is recognized that various modifications can be made without departing from the spirit and scope of the present disclosure(s).

[0228] 9. Incorporation by reference All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were specifically and individually indicated and incorporated by reference for every purpose.

[0229] Any description of documents, acts, materials, devices, articles, etc. contained in this specification is for the sole purpose of providing the content of this disclosure. None of these items, either individually or in combination, should be taken as an admission that any of them existed anywhere prior to the priority date of this application, formed part of the prior art base, or was common general knowledge in the relevant field of this disclosure.

Claims

1. An ApoA-I formulation for use in a method of treating an acute condition including acute inflammation, comprising apolipoprotein A-I ("ApoA-I") in the form of a lipoprotein complex, and the phospholipids sphingomyelin and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol) ("DPPG"), The method includes administering a high dose of the ApoA-I preparation to a human subject in need in two to four individual doses administered at intervals of no more than one day. The aforementioned high dose is a collection of the two to four individual doses. Each individual dose of the ApoA-I preparation administered is 4 to 40 mg / kg (protein weight basis) or 300 mg to 4000 mg (protein weight basis), An ApoA-I preparation comprising the ApoA-I and the phospholipid in a weight ratio of 1:2.7 for ApoA-I to total phospholipids and a weight ratio of 97:3 for sphingomyelin to DPPG.

2. The ApoA-I formulation for use according to claim 1, wherein the high dose is a collection of two, three, or four individual doses.

3. An ApoA-I formulation for use according to claim 2, comprising administering two or more separate doses at intervals of approximately 12 hours.

4. An ApoA-I formulation for use according to claim 3, comprising administering two separate doses approximately 12 hours apart.

5. An ApoA-I formulation for use according to claim 3, comprising administering three separate doses at intervals of approximately 12 hours.

6. An ApoA-I formulation for use according to claim 4 or 5, further comprising administering individual doses approximately one day apart.

7. An ApoA-I formulation for use according to claim 2, comprising administering three individual doses at intervals of approximately 12 hours, and a fourth individual dose approximately one day later.

8. ApoA-I formulation for use according to any one of claims 1 to 5 or 7, wherein the subject has a viral infection, and the viral infection may be a coronavirus infection including COVID-19.

9. An ApoA-I formulation for use according to any one of claims 1 to 5 or 7, wherein the subject has CRS or is at risk of CRS.

10. The ApoA-I formulation for use according to claim 9, wherein the subject may have a risk of CRS, and the subject may have a risk of CRS due to an infection.

11. An ApoA-I preparation for use according to any one of claims 1 to 5 or 7, wherein the subject has sepsis or is at risk of developing sepsis.

12. An ApoA-I formulation for use according to any one of claims 1 to 5 or 7, wherein the subject has or is at risk of developing acute kidney injury (AKI).

13. The ApoA-I preparation for use according to any one of claims 1 to 5 or 7, wherein the method comprises administering the ApoA-I preparation systemically, possibly by intravenous infusion.

14. The ApoA-I preparation for use according to any one of claims 1 to 5 or 7, wherein the method comprises administering the ApoA-I preparation over a period of one hour or less.

15. The ApoA-I preparation for use according to any one of claims 1 to 5 or 7, wherein the method comprises administering the ApoA-I preparation over a period of 0.5 hours to 1 hour.

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