Anticoagulants for treating nephrotic syndrome

Abstract

A method of treating or preventing glomerular disease or chronic kidney disease in a subject is described. The method includes administering to the subject a therapeutically effective amount of a thrombin inhibitor or a pharmaceutically acceptable salt thereof.

Description

ANTICOAGULANTS FOR TREATING NEPHROTIC SYNDROMESTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under Grant Numbers DK103982 and DK124549, awarded by the National Institutes of Health. The Government has certain rights in this invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 612,157, filed on December 19, 2023, which is incorporated herein by reference.BACKGROUND

[0003] Kidney disease is the 8th leading cause of death in the U.S. and cardiovascular disease is the leading cause of mortality in patients with chronic kidney disease (CKD). Glomerular diseases are a leading cause of CKD and end-stage kidney disease (ESKD). The glomerular filtration barrier is composed of three layers: (1) fenestrated endothelial cells, (2) collagen basement membrane, and (3) podocytes which interdigitate to form the slit diaphragm (a specialized adherence junction). Proteinuria, glomerular podocyte injury (podocytopathy) leading to podocyte loss (podocytopenia), and glomerulosclerosis are hallmarks of glomerular disease-mediated CKD. Breyer, M. and Susztak, K., Nat Rev Drug Discov., 15: 568-588 (2016). Podocytes are terminally differentiated epithelial cells that maintain glomerular endothelial cell phenotype via paracrine signaling, synthesize basement membrane components, and mechanically resist pulsatile intracapillary blood pressure. Podocyte function is thus critical to glomerular filtration barrier maintenance and podocyte injury and / or death leads to urinary loss of plasma proteins (proteinuria). Nagata M., Kidney Int., 89: 1221-1230 (2016). While proteinuria due to acute podocyte dysfunction is potentially reversible, it becomes irreversible with >20% podocyte depletion and glomerulosclerotic lesions form on the extraluminal surface of glomerular capillaries in the areas of podocyte loss. Proteinuria, in turn, is a key driver of CKD progression, leading to renal tubular inflammation and interstitial fibrosis. Thus, podocyte preservation and proteinuriaconstraint is crucial to delay or prevent both CKD progression and CKD-related cardiovascular disease.

[0004] Nephrotic syndrome (NS) is comprised of a group of primary glomerular diseases that collectively are a leading cause of ESKD, the most severe form of CKD. NS is clinically characterized by massive proteinuria, hypoalbuminemia, edema, and dyslipidemia. The monogenic, familial forms of NS are predominantly due to mutations in podocyte- specific genes and idiopathic NS is typified by podocytopathy. Rodent models of podocytopathy-mcdiatcd proteinuria have thus become important tools for the investigation of NS pathogenesis and CKD progression. Brinkkoetter et al., Am J Physiol Renal Physiol., 296: F213-229 (2009). The massive proteinuria of NS includes urinary loss of albumin and other plasma proteins, including coagulation system zymogens, cofactors, and inhibitors with variable synthetic compensation that manifests as an acquired hypercoagulopathy. Andersen et al., Proteomics Clin Appl., 6: 382-393 (2012). Thus, NS is itself a profoundly prothrombotic condition with increased risk for both venous and arterial thrombosis, even before CKD onset. Go et al., J Am Soc Nephrol 2021; 32: 2303- 2314.

[0005] The inventors and others have recently shown that prothrombin (F2) may directly injure podocytes during NS. Sharma et al., J Am Soc Nephrol., 28: 2618-2630 (2017). Thrombin injured conditionally immortalized rat podocytes via protease-activated receptor (PAR) 1 in a PAR4- dependent manner and thrombin inhibition reduced proteinuria in two mechanistically distinct rat NS models. Yamashita et al., Eur J Pharmacol., 589: 239-244 (2008). Moreover, prothrombin colocalization to podocytes in vivo was proportional to proteinuria, strongly suggesting that prothrombin, originating from the plasma compartment, interacts with podocytes to drive podocytopathy, podocytopenia, and proteinuria during NS.SUMMARY OF THE INVENTION

[0006] The inventors aimed to directly determine the influence of circulating prothrombin on glomerular pathobiology. They hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria. Glomerular proteinuria was induced with puromycin aminonucleoside (PAN) in Wistar rats. Circulating prothrombin was either knocked down using a rat- specific antisense oligonucleotide or elevated by serial intravenous infusions of prothrombinprotein, which are previously established methods to model hypo- (LoPT) and hyperprothrombinemia (HiPT), respectively. After 10 days (peak proteinuria in this model) plasma prothrombin levels were determined, kidneys were examined for (pro)thrombin co-localization to podocytes and podocytopathy, podocytopenia, proteinuria, and plasma albumin were measured. LoPT significantly reduced podocyte exposure to prothrombin, podocytopathy, and proteinuria with improved plasma albumin. In contrast, HiPT significantly increased podocytopathy and proteinuria. Podocytopenia was significantly improved in LoPT vs. HiPT rats. In summary, prothrombin knockdown ameliorated podocytopathy, podocytopenia, and proteinuria in rats with PAN-induced glomerular proteinuria. Thus, (pro)thrombin antagonism may be a viable strategy to simultaneously provide thromboprophylaxis and prevent podocytopathy-mediated CKD progression.BRIEF DESCRIPTION OF THE FIGURES

[0007] The present invention may be more readily understood by reference to the following drawings, wherein:

[0008] Figure 1 provides an experimental schema showing that puromycin aminonucleoside (PAN) was delivered intravenously to induce nephrosis on day 0 (purple arrow), control rats received intravenous saline instead. Hyperprothrombinemia (HiPT) was modeled using intravenously delivered human prothrombin on days 0, 3, 6, and 9 (green arrows). Hypoprothrombinemia (LoPT) was modeled by twice weekly delivery of rat prothrombin- specific antisense oligonucleotide (ASO) 401027 beginning on day -17 (blue arrows). Urine was collected on days 0 and 10 (yellow arrows). On day 10, after urine collection, the rats were exsanguinated (red arrow) and their kidneys were collected (pink arrow).

[0009] Figures 2A-2E provide graphs showing antisense oligonucleotide-mediated hypoprothrombinemia in healthy rats. (A)Twice weekly treatment with ASO 401027 for 17.5 days reduced plasma prothrombin enzymatic activity in a dose-dependent manner that appeared to plateau at -60 mg / kg / dose (relative to rat pooled normal plasma prothrombin activity; n=2 per dose level). Twice weekly ASO 401027 at 60 mg / kg / dose significantly reduced plasma prothrombin enzymatic activity (B) and endogenous thrombin potential (ETP, C, D) measured at day 27.5 (n=3). (E) RT-qPCR demonstrated that twice weekly ASO 401027 at 60 mg / kg / dosesignificantly reduced day 27.5 hepatic prothrombin (F2) gene expression (relative to |3-actin (Actb)) (n=3). ***P<0.001

[0010] Figures 3A-3D provide graphs showing serial prothrombin infusion-mediated hyperprothrombinemia in healthy rats. (A) A single dose of intravenous prothrombin (31.25 mg / kg) increased plasma prothrombin enzymatic activity to a peak of -207% at 1-hour post-dose with an apparent half-life of -61 hours (relative to rat pooled normal plasma prothrombin activity; n=2 per time point). This 31.25 mg / kg loading dose was then followed by maintenance doses of 16.67 mg / kg on days 3, 6, and 9 resulting in significantly increased plasma prothrombin enzymatic activity (B) and endogenous thrombin potential (ETP, C, D) on day 10 (n=3). ***P<0.001

[0011] Figures 4A-4E provide graphs showing prothrombin modulation during Rat PAN-NS. On day 10, (A) RT-qPCR revealed that ASO 401027 significantly reduced hepatic prothrombin (F2) transcript levels (relative to (3-actin (Actb)) whereas F2 expression was not significantly altered by PAN-induced proteinuria or prothrombin infusions (n=3-12). Plasma prothrombin protein quantity (B), enzymatic activity (C), and endogenous thrombin potential (ETP, D, E) were significantly reduced in LoPT rats and significantly increased in HiPT rats in comparison to both control rats and PAN-NS (Sham) rats. n=3-12; *P<0.05, **P<0.01, ***P<0.001; o; Control; ▲ : LoPT (ASO- mediated hypopro thrombinemia); •: Sham; ■: HiPT (prothrombin infusion-mediated hyperprothrombinemia)

[0012] Figures 5A & 5B provide a graphs and image showing Plasma Prothrombin Levels Dictated Thrombin-Podocyte Interactions during Rat PAN-NS. (A) Representative immunofluorescence histology of glomeruli on day 10 (original magnification, x60). (B) Relative proportion of synaptopodin-positive pixels with colocalized thrombin calculated using the colocalization coefficient feature of ZEN Black software (Zeiss USA). n=3- 12 rats per group; each point represents averaged data taken from 20 random glomeruli per rat, *P<0.05, **P<0.01 ; o: Control; ▲ : LoPT (ASO-mcdiatcd hypoprothrombincmia); •: Sham; ■: HiPT (prothrombin infusion-mediated hyperprothrombinemia)

[0013] Figures 6A-6C provide graphs showing hypoprothrombinemia diminished in situ podocyte injury. (A) Representative dot plot and subsequent gating strategy used to identify the proportion of synaptopodin-positive podocyte nuclei that were TUNEL-positive, indicating DNA nicking (amarker of terminal podocyte injury). (B) Representative histograms of TUNEL-positive podocyte nuclei by treatment group. (C) On day 10, LoPT PAN-NS rats had significantly reduced TUNEL- positive podocyte nuclei, in contrast HiPT PAN-NS rats had significantly increased TUNEL- positive podocyte nuclei. n=3-12 rats per group; each point represents data derived from 100,000 events per rat, *P<0.05, **P<0.01, ***P<0.001; SSC-A: side scatter-area; FSC-A: forward scatterarea; DAPLUV: 4',6-diamidino-2-phenylindole; Synaptopodin-APC: allophycocyanin-labeled anti-synaptopodin antibody; TUNEL-FITC: Fluorescein isothiocyanate-terminal deoxynucleotidyl transferase dUTP nick end labeling; o; Control; A : LoPT (ASO-mediated hypopro thrombinemia); •: Sham; ■: HiPT (prothrombin infusion-mediated hyperprothrombinemia)

[0014] Figures 7A-7C provide graphs showing hyperprothrombinemia reduced in situ podocyte survival. (A) Representative dot plot and subsequent gating strategy used to count synaptopodin- positive podocytes. (B) Representative histograms of podocytes by treatment group. (C) On day 10, HiPT rats had significantly reduced podocyte counts in comparison to control and LoPT rats. n=3-12 rats per group; each point represents data derived from 100,000 events per rat; *P<0.05; SSC-A: side scatter-area; FSC-A: forward scatter-area; DAPI-UV: 4',6-diamidino-2-phenylindole; Synaptopodin-FITC: Fluorescein isothiocyanate-labeled synaptopodin; o; Control; ▲ : LoPT (ASO-mediated hypopro thrombinemia); •: Sham; ■: HiPT (prothrombin infusion-mediated hyperprothrombinemia)

[0015] Figures 8A-8H provide graphs showing prothrombin regulated proteinuria and plasma albumin during Rat PAN-NS. (A) Day 10 proteinuria was significantly reduced in LoPT rats whereas HiPT rats had significantly more proteinuria than any other group. (B) Plasma albumin was significantly improved in LoPT rats. Proteinuria was significantly correlated with plasma prothrombin protein quantity (C), enzymatic activity (D), and endogenous thrombin potential (ETP, E). Proteinuria correlation with podocyte-prothrombin colocalization, TUNEL-positive podocytes, and podocyte counts per glomerulus are shown in panels F-H, respectively. n=3-12 rats per group; *P<0.05, **P<0.01; o; Control; ▲ : LoPT (ASO-mediated hypoprothrombinemia); •: Sham; ■: HiPT (prothrombin infusion-mediated hyperprothrombinemia)DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides a method of treating or preventing glomerular disease in a subject, comprising administering to the subject a therapeutically effective amount of a thrombin inhibitor or a pharmaceutically acceptable salt thereof.Definitions

[0017] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting of the invention as a whole. As used in the description of the invention and the appended claims, the singular forms “a”, “an”, and “the” are inclusive of their plural forms, unless contraindicated by the context surrounding such.

[0018] Treat", "treating", and "treatment", etc., as used herein, refer to any action providing a benefit to a subject at risk for or afflicted with a condition or disease such as chronic kidney disease, including improvement in the condition through lessening or suppression of the major symptom of proteinuria such as proteinuria and / or lessening of associated complications such as hypercoagulopathy, delay in progression of the disease, etc. The subject may be at risk due to the presence of a risk factor such as heart failure, hepatitis, diabetes, being genetically predisposed to chronic kidney disease, and so on.

[0019] The term "in need of treatment" as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a disease or condition that is treatable by a method or compound of the disclosure.

[0020] Prevention, as used herein, refers to any action providing a benefit to a subject at risk of being afflicted with a condition or disease such as chronic kidney disease, including avoidance of the development of glomerular or chronic kidney disease or a decrease of one or more symptoms of the disease should chronic kidney disease develop.

[0021] Within the present invention, a "therapeutically effective amount" of a composition is that amount which is sufficient to show a benefit (e.g., a reduction in a symptom associated with the disorder, disease, or condition being treated) while avoiding adverse side effects such as thosetypically associated with alternative therapies. The therapeutically effective amount may be administered in one or more doses.

[0022] As used herein, the term "pharmaceutically acceptable carrier" refers to carriers that do not negatively affect the biological activity of the therapeutic molecule or compound to be placed therein. The characteristics of the delivery vehicle will depend on the route of administration. Therapeutic compositions may contain, in addition to the active compound, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. A pharmaceutically acceptable carrier can deliver the active agent without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

[0023] A subject, as defined herein, is an animal, preferably a mammal such as a domesticated farm animal (e.g., cow, horse, pig) or a pet (e.g. , dog, cat). More preferably, the subject is a human. The subject may also be a subject in need of treatment of a chronic kidney disease or glomerular disease.Treating or Preventing Glomerular Disease or Chronic Kidney Disease

[0024] In one aspect, the present invention provides a method of treating or preventing glomerular disease or chronic kidney disease in a subject. The method includes administering to the subject a therapeutically effective amount of a thrombin inhibitor or a pharmaceutically acceptable salt thereof.Chronic Kidney Disease

[0025] The method includes treating or preventing chronic kidney disease in a subject. Chronic kidney disease is a type of kidney disease in which there is a gradual loss of kidney function over a period of months to years. Chronic kidney disease can be caused by a variety of different factors, with the most common being diabetes mcllitus, hypertension, and glomerulonephritis. Symptoms of chronic kidney disease include increased blood pressure, accumulation of urea, hyperkalemia, edema, hyperphosphatemia, hypocalcemia, metabolic acidosis, and anemia. Types of chronic kidney disease include vascular kidney disease (e.g., kidney artery stenosis), glomerular disease, tubulointerstitial disease (e.g., toxin-induced tubulointerstitial nephritis), and obstructive nephropathy. Chronic kidney disease can be diagnosed by measurement of serum creatininelevels, examination of the patient, and / or the use of a urine dipstick. Signs of chronic kidney disease and the resulting kidney damage are seen in blood, urine, or imaging studies which includes lab albumin / creatinine ratio (ACR) > 30. Humans with a glomerular filtration rate <60 ml / min / 1.73 nr for 3 months are defined as having chronic kidney disease.

[0026] Treatment of chronic kidney disease can include reduction of a variety of symptoms associated with chronic kidney disease. In some embodiments, treatment decreases proteinuria in the subject. Proteinuria is a disorder in which an excess amount of protein is found in the urine. Proteinuria is defined as urine having a protein / creatinine ratio greater than 45 mg / mmol. In other embodiments, treatment decreases hypercoagulopathy in the subject. Hypercoagulopathy, also known as thrombophilia, is an abnormality of blood coagulation that increases the risk of thrombosis. Hypercoagulopathy can be determined by measuring one or more blood coagulation factors such as blood count, thrombin time, or prothrombin time.

[0027] In some embodiments, the method is used to prevent glomerular disease or chronic kidney disease in a subject in need thereof. Prevention refers to preventing the disease or a symptom of a disease from occurring in a subject. A subject who is in need of prevention is one who has been identified as being predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease). Prevention may include completely or partially preventing a disease or symptom. In some embodiments, prevention also includes reducing the risk that a subject will develop the disease, while not guaranteeing that the disease will be prevented. For example, prevention can reduce the likelihood that a subject will develop the disease or one or more symptoms of the disease by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared with a subject who has not been administered a PPARy agonist to prevent occurrence of glomerular disease or chronic kidney disease.Glomerular Disease

[0028] In some embodiments, the method can be used to treat or prevent glomerular disease. Glomerular diseases affect the function of the kidneys and the glomeruli, which are small units within the kidney where blood is cleaned. Glomerular diseases include many conditions with a variety of genetic and environmental causes, but they fall into two major categories:Glomerulonephritis, which describes the inflammation of the membrane tissue in the kidney that serves as a filter, separating wastes and extra fluid from the blood, and glomerulosclerosis, which describes the scarring or hardening of the tiny blood vessels within the kidney. In some cases, glomerular disease results in chronic kidney disease, though this is not always the case. Although glomerulonephritis and glomerulosclerosis have different causes, they can both lead to kidney failure. Examples of glomerular disease include nephrotic syndrome, minimal change disease, diabetic nephropathy, and other conditions known to those skilled in the art.

[0029] Glomerular diseases damage the glomeruli, letting protein and sometimes red blood cells leak into the urine. Glomerular disease can also interfere with the clearance of waste products by the kidney, so they begin to build up in the blood. Furthermore, loss of blood proteins like albumin in the urine can result in a fall in their level in the bloodstream. When albumin leaks into the urine, the blood loses its capacity to absorb extra fluid from the body. Fluid can accumulate outside the circulatory system in the face, hands, feet, or ankles and cause swelling. Symptoms of glomerular disease include albuminuria, hematuria, reduced glomerular filtration rate, proteinuria, and edema. In some embodiments, the subject has been diagnosed as having glomerular disease. Glomerular disease can be diagnosed if a patient is identified as having glomerular hematuria and / or glomerular proteinuria. Hebert et al., Am J Nephrol., 38(3):253-66 (2013).

[0030] In some embodiments, the glomerular disease is nephrotic syndrome. Nephrotic syndrome (NS) is a general term that refers to the loss of protein in the urine (proteinuria), hyperlipidemia (hypercholesterolemia and hypertriglyceridemia), dyslipidemia, and edema. Nephrotic syndrome involves changes in the pathology of cells in the kidney, such as podocytes. Many conditions are categorized as nephrotic syndromes, including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN) (also called membranous glomerulonephritis, MGN), and membranoproliferative glomerulonephritis (MPGN). For years pathologists found no changes in MCD tissue when viewing specimens under light microscopy, hence the name minimal change disease. With the advent of electron microscopy, the changes now known as the hallmarks for the disease include diffuse loss of podocyte foot processes, vacuolation of the podocyte foot processes, and growth of microvilli on the visceral epithelial cells. Diabetic nephropathy is the most common cause of nephrotic syndrome.

[0031] A variety of signs and symptoms are known to be associated with nephrotic syndrome. These include respiratory tract infection, allergy, macrohematuria, symptoms of infection, hypotension, respiratory distress, tachypnea, seizure, anorexia, irritability, fatigue, and diarrhea. Nephrotic syndrome can be diagnosed using urinalysis, urine protein quantification, serum albumin quantification, and a lipid panel. With the advent of electron microscopy, the changes now known as the hallmarks for the disease include diffuse loss of podocyte foot processes, vacuolation of the podocyte foot processes, and growth of microvilli on the visceral epithelial cells. Diabetic nephropathy is the most common cause of nephrotic syndrome.

[0032] Nephrotic syndrome, or nephrosis, is defined by the presence of nephrotic-range proteinuria, edema, hyperlipidemia, and hypoalbuminemia. While nephrotic-range proteinuria in adults is characterized by protein excretion of 3.5 g or more per day, in children it is defined as protein excretion of more than 40 mg / m2 / h or a first-morning urine protein / creatinine of 2-3 mg / mg creatinine or greater. In some embodiments, the nephrotic syndrome is pediatric nephrotic syndrome, which is the occurrence of pediatric nephrotic syndrome in a child.

[0033] In some embodiments, the subject has been diagnosed with nephrotic syndrome, and treatment prevents the development of end-stage kidney disease (ESKD), also known as end-stage renal disease. ESKD is the result of a gradual loss of kidney disease reaching an advanced state, where the kidneys no longer function enough to remove waste and excess fluid from the body, leading to the buildup of excessive levels of electrolytes and waste. A subject having ESKD will typically need either dialysis or a kidney transplant in order to stay alive.Thrombin Inhibitors

[0034] The method described herein includes administering to the subject a therapeutically effective amount of a thrombin inhibitor or a pharmaceutically acceptable salt thereof. Thrombin inhibitors include direct thrombin inhibitors, and compounds that inhibit thrombin indirectly such as factor X inhibitors. A variety of direct thrombin inhibitors arc known to those skilled in the art, including dabigatran, lepirudin, desirudin, bivalirudin and argatroban. See Lee, C., and Ansell, J., Br J Clin Pharmacol., 72(4):581-92 (2011). In some embodiments, the thrombin inhibitor is an indirect thrombin inhibitor. Indirect thrombin inhibitors work by binding to antithrombin and heparin cofactor II, which increases the inactivation of factor Xa and other serine proteases.Examples of indirect thrombin inhibitors include warfarin, heparin, low molecular weight heparins, and fondaparinux. In some embodiments, the thrombin inhibitor is a factor X inhibitor. Examples of factor X inhibitors include apixaban, edoxaban, fondaparinux, betrixaban, and rivaroxaban. In some embodiments, the thrombin inhibitor is rivaroxaban or dabigatran. When referring to the agents (i.e., thrombin inhibitors), known prodrugs are also included. For example, a common prodrug for dabigatran is dabigatran etexilate.

[0035] In some embodiments, the method further includes administering an additional therapeutic agent for treating or preventing glomerular disease or chronic kidney disease to the subject. Preferably, the thrombin inhibitor is administered concomitantly with the additional therapeutic agent. Concomitantly, as used herein, means that the two drugs are administered either in combination or that the thrombin inhibitor and the additional therapeutic agent are administered within a short period of time (e.g., a day or an hour) of each other such that their therapeutic effects overlap.

[0036] Therapeutic agents that are useful for treating chronic kidney disease include agents to manage blood pressure, blood sugar, and lower cholesterol. Management of blood sugar and lowering cholesterol are important because diabetes and heart disease are both cause and are exacerbated by chronic kidney disease. Angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists are recommended as first-line agents since they have been found to slow the decline of kidney function.

[0037] Agents for treating glomerular disease include endothelin antagonists (Raina et al., Kidney Dis (Basel), 6(1 ):22-34 (2020)), agents that target the complement system (Andrighetto et al., Int J Mol Sci., 20(24) (2019)), and sodium-glucose co-transporter 2 (SGLT2) inhibitors (Davidson, JA, Postgrad Med., 131(4):251-260 (2019), the disclosures of which are incorporated herein by reference.

[0038] Glucocorticoids are the primary therapy for nephrotic syndrome, although they have serious side effects and are ineffective in -20-50% of patients. However, a variety of non-steroid methods for treating nephrotic syndrome are also known. See Tune B. and Mendoa S., J Am Soc Nephrol., 8(5), 824-32 (1997), the disclosure of which is incorporated herein by reference. Nonsteroidal immunosuppressants suitable for treatment of nephrotic syndrome include cytotoxicdrugs such as cyclophosphamide and chlorambucil, calcineurin inhibitors such as cyclosporine and tacrolimus, inosine monophosphate dehydrogenase (IMPDH) inhibitors such as mycophenolate mofetil and mizoribine, and the anti-CD20 antibody rituximab.Administration and Formulation

[0039] The pharmaceutical compositions used in the present invention comprise a thrombin inhibitor, or pharmaceutically acceptable salts thereof, as the active ingredient. The pharmaceutical compositions may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.

[0040] The term "composition", as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.

[0041] The compositions include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration, although the most suitable route in any given case will depend on the nature and severity of the conditions being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy. Because of its convenience, in some embodiments oral administration is used.

[0042] The active agents(s) (e.g., thrombin inhibitor) can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agentsand the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.

[0043] Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers arc obviously employed. If desired, tablets may be coated by standard aqueous or non-aqueous techniques. Such compositions and preparations should contain at least 0.1 percent of active compound. The percentage of active compound in these compositions may, of course, be varied and may conveniently be between about 2 percent to about 60 percent of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that an effective dosage will be obtained. The active compounds can also be administered intranasally as, for example, liquid drops or spray.

[0044] The tablets, pills, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin. When a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

[0045] The thrombin inhibitor may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0046] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0047] The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts in the solid form may exist in more than one crystal structure, and may also be in the form of hydrates. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethypiperideine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0048] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0049] As defined herein, a therapeutically effective amount of the thrombin inhibitor (i.e., an effective dosage) ranges from 0.001 to 40 mg / kg body weight, preferably 0.01 to 25 mg / kg body weight, more preferably 0.1 to 20 mg / kg body weight, and even more preferably 1 to 10 mg / kg, 2 to 9 mg / kg, 3 to 8 mg / kg, 4 to 7 mg / kg, or 5 to 6 mg / kg body weight. The active compounds can be administered once a day or one time per week for between 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between 3 to 7 weeks, and even more preferably for 4, 5, or 6 weeks.The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a mammal including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the mammal, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a PPARy agonist can include a single treatment or, preferably, can include a series of treatments.

[0050] The present invention is illustrated by the following example. It is to be understood that the particular example, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.EXAMPLE Example 1: Prothrombin Knockdown Protects Podocytes and Reduces Proteinuria in Glomerular Disease

[0051] Prothrombin colocalization to podocytes in vivo was proportional to proteinuria, strongly suggesting that prothrombin, originating from the plasma compartment, interacts with podocytes to drive podocytopathy, podocytopenia, and proteinuria during NS. The objective of this study was to test this hypothesis in the well-established puromycin aminonucleoside (PAN)-induced rat NS model. Kerlin et al., J Am Soc Nephrol., 26: 3009-3019 (2015).

[0052] Transgenic rat F2 hypo- and hyper-expressing models are not yet available to facilitate examination of this phenomenon. Sun et al., Thromb Haemost., 88: 984-991 (2002). However, antisense oligonucleotides (ASOs) have been used to successfully knock-down mouse prothrombin expression to levels comparable to those seen in F2lox / ‘ knockdown mice. Crosby et al., Nucleic Acid Ther., 25: 297-305 (2015). Thus, ASOs provide an alternative means to achieve in vivo prothrombin knockdown. Intravenous administration of human prothrombin protein has been used to model hyperprothrombinemia in mice. Aleman et al., Arterioscler Thromb Vase Biol., 33: 1829-1836 (2013). The inventors utilized ASO-mediated prothrombin knockdown and serial prothrombin infusions to mimic hypo- and hyper-prothrombinemia (LoPT and HiPT), respectively. Using this strategy, we demonstrated that plasma prothrombin levels modulate both podocyte health and proteinuria in experimental rat NS.METHODSAnimals

[0053] All experimental rat protocols were approved by the Institutional Animal Care and Use Committee at the Abigail Wexner Research Institute, in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Wistar rats (body weight -150 g, age -45-50 d) were used for all experiments. Subcutaneous ASO or intravenous prothrombin was administered at the indicated doses and frequencies to achieve LoPT and HiPT conditions, respectively (Figure 1). Proteinuria was induced with a single tail vein injection of PAN (MilliporeSigma, Burlington, MA; 50 mg / kg diluted in saline) on Day 0.10, 34, 39 Anti-F2 ASO (401027) treatment (60 mg / kg subcutaneous twice weekly) was begun on Day -17.5 to induce LoPT prior to onset of podocyte injury and continued through Day +9. Prothrombin infusions were begun on Day 0, immediately following PAN (or saline) administration. Control rats received twice weekly scrambled ASO (141923) on Days -17.5 to +9, saline tail vein injections on Days 0, 3, 6, and 9 (instead of PAN and / or prothrombin). Sham rats were treated identically to control rats but did receive PAN on Day 0. Morning spot urine samples were collected on Days 0 (before PAN or saline) and 10 to determine urinary protein-to-creatinine ratio. Following Day 10 urine collection, the rats were anesthetized with 3% isoflurane and blood was collected from the inferior vena cava through a 23- G needle into final concentration 0.32% NaCitrate / 1.45 pM Corn Trypsin Inhibitor (CTI; Prolytix, Essex Junction, VT) and processed to Platelet Poor Plasma (PPP), as previously described. Kerlin et al., J Am Soc Nephrol., 26: 3009-3019 (2015). Urine and plasma were stored at -80°C until analyzed. Following exsanguination, both kidneys were removed, decapsulated, and placed in ice cold PBS. One quarter kidney was set aside for immunohistology and glomeruli were isolated from the remainder of both kidneys.Glomerular Isolation and Podocyte Flow Cytometry

[0054] Glomeruli were isolated from renal cortex using a standard sequential sieving method, washed and resuspended in ice cold PBS, then manually counted in a hemocytometer, as previously described. Agrawal et al., Kidney Int., 86: 1150-1160 (2014). An aliquot of the glomerular suspension (-2,500 glomeruli) was dissociated to a single cell suspension in Digestion Buffer at 37°C on a 1400 / min shaker with intermittent shearing through an 18-G needle for 26 minutes. Tissue debris was removed with a 40-micron filter, the cells were washed with 10 mLHBSS, centrifuged at 1500 rpm for 5 min, then resuspended in 1 mL HBSS supplemented with 0.1% BSA and 4’,6-diamidino-2-phenylindole (DAPI; 1 pg / mL). The cells were then prepared for flow cytometry analysis by staining with Live / Dead near IR dye and FITC-conjugated synaptopodin antibody (Fitzgerald, North Acton, MA; 1:5) in Perm / Wash buffer. The cells were analyzed on a LSR II flow cytometer (BD Biosciences) and synaptopodin expression was gated only on cells with a DAPI-positive intact nucleus. Isotype controls, unstained cells, and an open channel were used to identify and calibrate for autofluoresence. Synaptopodin-positive podocytes were normalized to bead standards to estimate podocyte counts which were then divided by the starting number of glomeruli to enumerate podocytes / glomerulus. Reassuringly, a direct linear relationship was observed between podocyte counts determined by flow cytometry and a histologic method. Lemley et al., J Am Soc Nephrol 2013; 24: 1193-1202 (2013). A second aliquot of the glomerular cell suspension was stained with FITC-TUNEL (1:5) and APC-conjugated synaptopodin antibody (1:4) to determine % TUNEL-positive podocytes. Flow cytometry data points for each individual rat represents an analysis of 100,000 events.Immunofluorescence Histology

[0055] Paraffin-embedded rat kidney sections were deparaffinized and processed for synaptopodin and thrombin co-immunofluorescence, as previously described. Sharma et al., J Am Soc Nephrol., 28: 2618-2630 (2017). However, the sections were photobleached for 72 hours prior to deparaffinization using a custom-built lightbox to reduce background autofluorescence. Lightbox construction was adapted from previous descriptions using mirrored acrylic panels and a broad-spectrum LED panel. Neumann el al., J Histochem Cytochem 2002; 50: 437-439 (2002). Images were captured with a Leica DMI 4000B inverted fluorescence microscope (Deerfield, IL) and analyzed with ZEN Black software (Zeiss USA, Thornwood, NY). Colocalization analysis was performed on a pixel-by-pixel basis, wherein every pixel within the defined region of interest is plotted in a scattergram based on fluorescence intensity of each channel. The software calculates the colocalization coefficient as the proportion of colocalizcd pixels (positive on both channels) within the total population of a single channel, green (synaptopodin-positive) for the purposes of this study. Manders et al., J Microsc 1993; 169: 375-382.Coagulation Parameters

[0056] Plasma prothrombin concentration was measured with rat- and human- specific ELISA (MyBioSource Inc, San Diego, California). Prothrombin activity was determined chromogenically using a commercially available assay (Rox Prothrombin; DiaPharma, West Chester, OH), as previously described, (Waller et al., Physiol Rep., 8: el4515 (2020)) and are expressed as a percentage of rat pooled normal plasma (rPNP). Endogenous thrombin potential (ETP) was determined on a 1:1 dilution of PPP with the Technothrombin TGA kit (Technoclone, Vienna, Austria) using TGA RC low reagent, and read on a Spectramax M2 fluorescent plate reader (Molecular Devices, Sunnyvale, California), as previously described. Waller et al., Physiol Rep., 8: el4515 (2020).Reagents and Resources

[0057] Antisense oligonucleotides 401027 (100% complementary to both mouse and rat F2 mRNA), 401025 (100% complementary to mouse F2 mRNA, 75% to rat F2 mRNA), and 141923 (scrambled control) were a kind gift from lonis Pharmaceuticals, Carlsbad, CA. Human prothrombin was from Prolytix (Essex Junction, VT). Digestion Buffer: Dispase II (2.8 units / mL; Sigma- Aldrich, St. Louis, MO), type 4 collagenase (300 units / mL) and DNase I (50 units / mL; both from Worthington Biochemical Corp., Lakewood, NJ) in HBSS (ThermoFisher, Waltham, MA). Antibodies were fluorescently conjugated using FITC and APC kits from Abeam (Cambridge, MA), as indicated. Mouse monoclonal synaptopodin antibody (10R-S125A) was purchased from Fitzgerald Industries International (Acton, MA, USA). Rabbit polyclonal thrombin antibody (bs- 1914R; raised against the thrombin heavy chain) was from Bioss (Woburn, MA). Secondary antibodies (Alexa Fluor 488 anti-mouse IgG and Alexa Fluor 594 antirabbit IgG) were from ThermoFisher.Cell Culture

[0058] Primary rat hepatocytes from male Wistar rats were from Life Technologies (Waltham, MA). In brief, rat hepatocytes were grown on plates coated with collagen type 1 at 37 °C in Williams’ E medium (Invitrogen, Waltham, MA) with 5% Fetal Bovine Serum (Fisher, USA). For experiments, hepatocytes were seeded at 106cells / well in 6- well plates and allowed to expand to -80% confluence over 48 hours. Cells were washed with PBS and then incubated with media plusantisense oligonucleotide (control ASO, ASO 401025, or ASO 401027) at 5 pM. After 72 hours, the cells were washed with PBS and lysed with RLT buffer (Qiagen, Valencia, CA) containing 1% P-mercaptoethanol.Healthy Control Animals

[0059] Hypo- (LoPT) and hyper-prothrombinemia (HiPT) was modeled without PAN to exclude podocyte effects in the absence of proteinuria. LoPT rats received anti-F2 ASO (401027) on Days -17.5 to +9 and saline (instead of PAN) on Day 0. HiPT rats received prothrombin infusions on Days 0 to +9 and saline (instead of PAN) on Day 0.RNA Extraction and Reverse Transcriptase - Quantitative Polymerase Chain Reaction

[0060] Whole rat liver was stored at -80C in RNAlater (ThermoFisher, Waltham, MA) until lysed in RLT buffer (Qiagen, Valencia, CA) containing 1% -mercaptoethanol.1 Total RNA was isolated from primary rat hepatocytes or whole rat liver using the RNeasy kit (Qiagen, USA). cDNA was made from 0.5 pg total RNA using the Protoscript First Strand cDNA Synthesis kit (New England Biolabs; Ipswich, MA), according to the manufacturer’s instructions. Purity and yield of RNA and cDNA were confirmed by measuring the absorbance at 260 and 280 nm. cDNA was amplified by quantitative polymerase chain reaction (qPCR); 0.5 pL cDNA (diluted in a total volume of 11 pL RNase-free H2O), 0.5 uL forward (fwd) and reverse (rvs) primers (below), and 12.5 uL iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). Primers were obtained from Invitrogen. qPCR was performed as follows; 1 cycle at 95 °C for 5 min, 40 cycles (melting at 94 °C for 30 sec; annealing gradient 55^72 °C for 30 sec; extension at 72 °C for 30 sec), with a final extension of 72 °C for 10 min using a CFX96 Real-Time System thermal cycler (Bio-Rad, Hercules, CA). All RT-qPCR samples were run in triplicate and reported as mean F2 expression relative to mean housekeeping gene (Actb) expression.Immunofluorescent Three-Dimensional Podocyte Counts

[0061] Aliquots of the glomerular suspensions (3000-6000 glomeruli) were prepared for whole mount immunofluorescence histology using an adaptation of previously reported methods for whole mount analysis of D. melanogaster organs. Thomas etal., Development, 125(11):2125-2134 (1998). Glomeruli were fixed in 4% paraformaldehyde for 35 minutes, washed in IncubationBuffer (0.5% Triton X-100 in PBS supplemented with 5% FBS) for 10 minutes x3, then stored in 70% ethanol at -20°C until ready for analysis. The glomeruli were then washed once in Incubation Buffer, transferred to an optically pure 96 well plate, sealed with an evaporation barrier, and photobleached. The glomeruli were then transferred to 600 pL Eppendorf tubes, permeabilized with 1% Triton X-100 in PBS supplemented with 5% FBS for 60 minutes, washed x3 in Incubation Buffer, and blocked with 0.5% Triton X-100 in SuperBlock for 30 minutes. Glomeruli were then stained with anti-WT-1 primary antibody (mouse IgGl (Novus Biologicals, Centennial, CO) at 1:200 in Incubation Buffer) at 4°C for 4 days on a vertical rotator, washed in Incubation Buffer x3, followed by secondary anti-mouse IgGl (Invitrogen, Waltham, MA) at 4°C for 4 days, washed in Incubation Buffer x3, then equilibrated in 30 pL Prolong Gold with DAPI (Invitrogen) for 30 minutes. Stained glomeruli (15 pL; 1500-3000 glomeruli) were then whole mounted on glass slides under a coverslip and random glomeruli (n=15 / rat) were selected for imaging as 1 pm z- stacks using a Zeiss LSM 700 microscope (Carl Zeiss Microscopy, LLC, White Plains, NY) with Alexa Fluor 488 and DAPI 355 filter settings. Z-stacks were then 3D reconstructed and analyzed for WT- 1-positive nuclei using ZEN Blue (Zeiss USA, Thornwood, NY) software. Individual podocyte nuclei in healthy control glomeruli were manually analyzed to determine their average z-dimcnsion (~8 pm). WT-l-positivc nuclei in every 8th Z-stack (at least 8 pm apart in depth) were then counted as podocytes using an automated Zen Blue software algorithm.Other

[0062] Plasma albumin concentrations were determined using bromocresol purple (BCP) assay (QuantiChrom BCP; BioAssay Systems, Hayward, CA), as described previously. Waller el al., Physiol Rep., 8(15):el4515 (2020). Proteinuria was determined by urinary protein-to-creatinine (UPC) ratio both of which were quantified by Antech Diagnostics (Morrisville, NC), using standard techniques.Statistical Analyses

[0063] One- or two-way ANOVA (analysis of variance) for multiple group comparisons, using SigmaStat software (Systat, San Jose, CA). When a significant difference was identified by ANOVA, post hoc tests were performed using the Student-Newman-Keuls technique. Statisticalsignificance was defined as P<0.05. Figures were prepared using GraphPad Prism (Boston, MA). Data are presented as mean + SE.RESULTSAntisense Oligonucleotide-Mediated Hypoprothrombinemia in Healthy Rats

[0064] ASO 401027, which is 100% complementary to a 3’ segment of rat F2 mRNA, effectively abolished F2 mRNA expression in primary rat hepatocytes vs. control ASO (0.00+0.00 vs. 1.0±0.01 relative fold change; P<0.001), whereas ASO 401025 (75% complementary to rat F2 mRNA) less effectively suppressed F2 expression (0.63+0.14 relative fold change; P=0.024). ASO 401027 also reduced prothrombin activity in primary rat hepatocyte protein lysates to 1.41+0.16 vs. 9.13±0.13 mIU / mL in control ASO lysates (P<0.001), whereas ASO 401025 less efficiently reduced activity to 2.31+0.10 mlU / mL (P<0.001). In dose finding studies, ASO 401027 was administered subcutaneously to healthy rats twice weekly for 17.5 days (Figure 2). These data suggested that maximal plasma prothrombin activity reduction (-32% of rPNP) was achieved with 60 mg / kg / dose. To mimic the planned full-length experiment, this dose (60 mg / kg twice weekly) was then continued for 27.5 days after which plasma prothrombin activity was reduced to 31.6±3.52% (vs. 100.3±1.45% in rPNP; P<0.001) and endogenous thrombin potential (ETP) was reduced to 514.3+58.7 nM*min (vs. 2451.0+140.5 nM*min; P<0.001). Thus, whereas prothrombin activity was reduced by -68%, ETP was reduced by -71% suggesting that circulating prothrombin reduction impaired thrombin-dependent coagulation cascade amplification. Wolberg AS, Campbell RA. Transfus Apher Sci., 38: 15-23 (2008). Hepatic synthesis is the major source of circulating prothrombin and hepatic F2 mRNA expression was essentially abolished by this 27.5 day ASO treatment regimen (P<0.01). Stenberg et al., Biochem Biophys Res Commun 2001; 280: 1036-1041. Thus, ASO 401027 effectively induced sustained LoPT in otherwise healthy rats.Serial Prothrombin Infusion-Mediated Hyperprothrombinemia in Healthy Rats

[0065] The hemostatic system is highly conserved amongst vertebrate species, including mammals. Kaye S, Stokol T., Vet Clin North Am Exot Anim Pract, 25: 613-630 (2022). Thus, rPNP thrombin generation was augmented by addition of human prothrombin as expected. Moreover, rat thrombomodulin effectively regulated the spiked-in human prothrombin. These observations are similar to those previously observed using human prothrombin added to mousePNP and indicate that human prothrombin is compatible with rat pro- and anti-coagulant systems. In preliminary experiments, intravenously administered human prothrombin (31.25 mg / kg) generated peak plasma prothrombin activity of -207% 1-hour post-dose with an apparent half-life of -61 hours (Figure 3), which is similar to that previously reported in humans. Monroe DM, Hoffman M, Roberts HR. Molecular Biology and Biochemistry of the Coagulation Factors and Pathways of Hemostasis. In: K. K, Lichtman MA, Beutler E, Kipps TJ, et al. (eds). Williams Hematology. McGraw-Hill Education: NY, NY, 2010. To mimic the planned full-length experiment, 31.25 mg / kg prothrombin was administered as a loading dose on day 0 with maintenance doses of 16.67 mg / kg on days 3, 6, and 9. On day 10, 24 hours after the day 9 dose, prothrombin activity was 186.8+10.4% (vs. 100.3+1.45% in rPNP; P<0.001). Similarly, day 10 ETP was elevated to 5766.0+207.0 (vs. 2451.0+140.5 nM*min; P<0.001). Thus, whereas prothrombin activity was enhanced by -86%, ETP was enhanced by -135% suggesting that elevated circulating prothrombin enhanced thrombin-dependent coagulation cascade amplification. These data demonstrate that human prothrombin induces hyperprothrombinemia (HiPT) in otherwise healthy rats for up to 10 days.Prothrombin Modulation during Puromycin Aminonucleoside-Induced Rat Nephrotic Syndrome

[0066] Peak NS disease activity occurs at day 8-11 following PAN administration, we therefore evaluated prothrombin levels at day 10 to ensure that the manipulations observed above in otherwise healthy rats were sustained during PAN-mediated NS (PAN-NS). Pippin et al., Am J Physiol Renal Physiol, 296: F213-229 (2009). Similar to the primary rat hepatocyte and in vivo dose finding studies described above, ASO 401027 treated rats (LoPT) had significantly reduced hepatic F2 expression (P<0.05 vs Control) whereas F2 expression was not significantly affected by PAN (Sham; P=0.35 vs Control) or prothrombin infusions (HiPT; P=0.28 and P=0.98 vs. Control and Sham, respectively; Figure 4). PAN-NS rats treated with ASO 401027 (LoPT) exhibited significantly reduced day 10 plasma prothrombin concentrations (0.92+0.10 pM) vs. Control (4.07±0.17 pM) and Sham (4.20±0.15 pM) whereas prothrombin infusions (HiPT) resulted in significantly elevated concentrations (8.16+0.38 pM; Figure 4). The LoPT and HiPT changes in prothrombin concentrations translated into significant changes in chromogenic prothrombin activity: LoPT 29.5±3.9% and HiPT 222.3±12.8% vs. Control (105.3±1.2%) and Sham (123.6+7.7%; Figure 4). Congruently, ETP was reduced to 306.7+62.9 nM*min in LoPT(vs. 2893.0±122.0 and 3417.0±235.8 nM*min in Control and Sham rats respectively; P<0.001) whereas HiPT rats had significantly elevated ETP (4406.0+407.2 nM*min; P<0.05). These data demonstrate successful manipulation of plasma prothrombin levels during rat PAN-NS.Plasma Prothrombin Levels Dictated Thrombin-PodocyteInteractions during Puromycin Aminonucleoside-Induced Rat Nephrotic Syndrome

[0067] The inventors previously demonstrated that thrombin colocalizcd to podocytes during rat PAN-NS. Sharma et al., J Am Soc Nephrol, 28: 2618-2630 (2017). They thus predicted that thrombin-podocyte interactions would fluctuate with plasma prothrombin levels. As expected, thrombin-podocyte colocalization was significantly reduced in LoPT rats (7.8±1.2% vs. 18.9+1.8% in Sham; P<0.01) to levels similar to Control (5.2+1.5%; P=0.30; Figure 5). Meanwhile, thrombin-podocyte colocalization was significantly higher than Control in both Sham (P<0.01) and HiPT (18.1±3.4; P<0.05) rat PAN-NS. The latter data suggest that thrombin interactions with podocyte-expressed protease-activated receptors may be saturated at physiologic prothrombin concentrations. Nonetheless, these data strongly suggest that plasma prothrombin levels dictate podocyte exposure to thrombin during glomerular proteinuria.Hypoprothrombinemia Diminished in situ Podocyte Injury

[0068] The inventors and others previously demonstrated that in vitro thrombin-exposure leads to DNA nicking that is consistent with terminal podocyte injury. Sharma et al., J Am Soc Nephrol., 28: 2618-2630 (2017). Importantly, podocytes may be terminally injured prior to detachment from the glomerular capillary surface. Mundel P, Shankland SJ., J Am Soc Nephrol., 13: 3005-3015 (2002). Thus, to evaluate the health status of in situ podocytes we performed TUNEL assays on dissociated cells from isolated glomeruli. Importantly, neither LoPT nor HiPT altered podocyte DNA-nicking in otherwise healthy rats. However, HiPT significantly increased TUNEL-positive podocytes (17.4±0.5%) in PAN-NS (11.7±1.8% vs Sham; P<0.05; Figure 6). In contrast, LoPT reduced TUNEL-positivity (6.9+1.5%; P<0.05 vs. Sham) to levels not significantly different from Control (6.5±1.5%; P=0.90). Podocyte DNA-nicking was correlated with prothrombin levels, and thrombin-colocalization during PAN-NS.Hyperprothrombinemia Reduced in situ Podocyte Survival

[0069] Glomerular filtration barrier function and CKD progression are highly dependent on glomerular podocyte loss. The inventors thus evaluated in situ podocyte survival by counting remaining podocytes in dissociated glomerular cells. Whereas LoPT and HiPT did not alter podocyte counts in otherwise healthy rats, HiPT PAN-NS rats had significantly lower podocyte counts (134.2±21.5 podocytes / glomerulus) vs. either LoPT (350.9±65.3; P<0.01) or Control (389.0+41.8; P<0.001; Figure 7). However, the LoPT and HiPT conditions did not translate into significantly improved or diminished podocyte counts vs. Sham. Meanwhile, podocyte counts were correlated with prothrombin levels, thrombin-colocalization, and podocyte injury in PAN- NS rats. These data suggest that hyperprothrombinemia may accelerate podocyte loss during PAN- NS.Prothrombin Regulated Proteinuria and PlasmaAlbumin during Puromycin Aminonucleoside-Induced Rat Nephrotic Syndrome

[0070] Podocyte injury and loss leads to glomerular filtration barrier dysfunction resulting in proteinuria and hypoalbuminemia. Nagata M., Kidney Int., 89: 1221-1230 (2016). Consistent with preserved podocyte function, LoPT PAN-NS rats had significantly lower day 10 proteinuria values in comparison to Sham (6.6±0.7 vs. 15.0±3.7 mg / mg creatinine; P<0.05) that was not significantly different than Control (1.0+0.1 mg / mg; Figure 8). Meanwhile, HiPT rats had significantly worse proteinuria (34.9+9.3 mg / mg; P<0.05). Consistent with improved overall protein homeostasis, plasma albumin was significantly improved in LoPT vs. Sham (3.32±0.03 vs. 3.07±0.08 g / dL; P<0.05) to values that were not significantly different from Control (3.41+0.02 g / dL). However, HiPT did not significantly worsen hypoalbuminemia (3.05+0.1 g / dL). Importantly, neither LoPT nor HiPT altered proteinuria or plasma albumin in otherwise healthy rats. In PAN-NS proteinuria was significantly correlated with prothrombin levels and podocyte injury (Figure 8). Similarly, plasma albumin was correlated with prothrombin levels, podocyte injury, podocyte survival, and proteinuria.DISCUSSION

[0071] Proteinuria, podocytopathy, and podocytopenia all contribute to glomerular dysfunction and progression of chronic kidney disease. Wanner etal., J Am Soc Nephrol., 25: 707-716 (2014).The data presented in this study demonstrate that circulating levels of prothrombin (the zymogen precursor of thrombin) modulated in vivo podocyte thrombin exposure, injury, survival, and function in the setting of proteinuric glomerular disease. We and others have previously shown that thrombin injures cultured podocytes in vitro and that in vivo thrombin inhibition preserves glomerular function as determined by proteinuria reduction. Benchetrit et al., Nephron., 87: 155- 160 (2001). In contrast to the previous studies which utilized anticoagulant agents which may have off-target effects, this study directly manipulated prothrombin levels in vivo, providing direct evidence that (pro)thrombin is involved in glomerular disease pathogenesis. The inventors previously demonstrated that thrombin injures in vitro podocytes via its cognate protease-activated receptors. Sharma et al., J Am Soc Nephrol., 28: 2618-2630 (2017). Collectively, these data suggest that thrombin originates as circulating prothrombin that is pathologically filtered during proteinuria leading to intraglomerular thrombin generation and direct podocytopathic thrombin signaling. Thus, interruption of this signaling pathway may represent a novel therapeutic target to slow or halt glomerular disease progression toward CKD / ESKD.

[0072] Mounting indirect evidence from in vitro studies and in vivo pharmacologic manipulation strongly suggest that thrombin is a pathological driver of podocyte injury in the setting of proteinuria. Wang et al., Blood, 117: 5231-5242 (2011). The present study provides direct evidence supporting this hypothesis. HiPT rats exhibiting prothrombin levels -195% of normal developed worse proteinuria along with increased podocyte injury. The human thrombophilic single nucleotide polymorphism, F2 G20210A (rs 1799963), is associated with prothrombin levels -115-170% of normal. Aleman et al., Arterioscler Thromb Vase Biol 2013; 33: 1829-1836. Rsl799963 has not been associated with enhanced CKD progression or incidence, but most studies were not directly looking for such a signal, were likely underpowered, or were investigating CKD- related thrombotic risk. Bauer et al., Hamostaseologie, 36: 103-107 (2016). It is also possible that since the reference interval for prothrombin extends to 150% that the mildly increased levels associated with this polymorphism do not lead to meaningfully increased CKD risk. Mann et al., J Thromb Haemost 2003; 1: 1504-1514. However, the present data suggest that rsl799963 should be more carefully analyzed as a potential driver of proteinuria-mediated CKD progression. Protein overload, achieved with >3 g / kg cumulative albumin dose, is another commonly employed method to induce transient proteinuria in animal models. Wagner et al., J Am Soc Nephrol., 27: 482-494(2016). However, it is unlikely that HiPT mimics protein overload since the cumulative prothrombin protein dose (81.26 mg / kg) used in these studies is only 2.7% of the minimum albumin dose required to induce proteinuria and did not induce proteinuria in the absence of PAN.

[0073] In contrast, highly specific ASO-mediated prothrombin knockdown (to -24% of normal) resulted in diminished podocyte thrombin exposure, podocytopathy, podocytopenia, proteinuria, and improved plasma albumin. Prothrombin is predominantly synthesized in the liver but several other organs, including the kidney, also produce prothrombin. Moreover, ASO therapy has been demonstrated to reduce target mRNA in many tissues, including both liver and kidney. Hung et al., Nucleic Acid Ther., 23: 369-378 (2013). Thus, while it is clear from these experiments that ASO-mediated prothrombin knockdown improves podocyte function and survival, the origin of podocytopathic prothrombin remains ill-defined. However, manipulation of circulating prothrombin did not induce proteinuria in healthy animals, strongly suggesting that proteinuria is a prerequisite for (pro)thrombin-mediated podocyte injury and that podocytopathic prothrombin thus likely originates from the plasma compartment. Alternatively, podocyte injury or proteinuria may stimulate glomerular cells or adjacent tissues to upregulate local prothrombin synthesis. A previous study identified prothrombin protein in tubular epithelial cells, but not glomeruli, suggesting that juxtaglomerular tubules could be a relevant local source of prothrombin synthesis. Stenberg et al., Biochem Biophys Res Commun., 280: 1036-1041 (2001).

[0074] The inventors previously demonstrated that in vitro thrombin-mediated podocytopathy is dependent upon activation of podocyte-expressed protease-activated receptors. Sharma et al., J Am Soc Nephrol., 28: 2618-2630 (2017). Activation of these receptors is dependent upon enzymatic cleavage and exposure of a cryptic tethered ligand by thrombin or other enzymes. Once generated, thrombin has a <1 minute half-life in vivo, suggesting that prothrombin is most likely converted to thrombin locally in the glomerulus, perhaps even on the podocyte surface. Lane et al., Blood, 106: 2605-2612 (2005). However, the mechanism by which prothrombin is converted to intraglomerular thrombin during proteinuria remains undefined. The canonical hemostatic mechanism is cleavage of prothrombin by the factor Xa / Va prothrombinase complex. While prothrombin and cofactor V have been found in the urine of nephrotic patients, factor X has not. Additionally, a proteomic study has demonstrated that neither factor X nor V protein are expressed by healthy murine podocytes. Rinschen et al., Cell Rep., 23: 2495-2508 (2018). Thus, whilepodocytes do express tissue factor, the complete complement of canonical prothrombinase components do not appear to be available. Apostolopoulos etal., Nephron Exp Nephrol., 116: e72- 83 (2010). Alternatively, adjacent kidney cells have been reported to synthesize both factor X and V. Liu et al., Kidney Int., 58: 598-606 (2000). Moreover, these components may originate from the plasma during proteinuria. It is thus possible that the canonical prothrombinase mechanism is at work in the diseased glomerulus. Alternatively, one or more putative tissue prothrombinases may explain glomerular thrombin activity. Amongst the latter, fibrinogen-like protein 2 (Fgl2) is expressed by podocytes and is thus a leading candidate. Rinschen etal., Cell Rep., 23: 2495-2508 (2018). This possibility is particularly intriguing as it would provide a potential target to ameliorate (pro)thrombin-mediated podocytopathy without increasing the risk of CKD-related bleeding.

[0075] There are several limitations to consider when interpreting these experiments. Although the mammalian coagulation system is highly conserved, it is possible that the use of human prothrombin in these studies could have resulted in subtle species-dependent differences in prothrombin activation efficiency or protease-activated receptor signaling responses in the rat glomerulus. The persistent hyperprothrombinemia after multiple human prothrombin injections argues against xenoimmunity in this model for up to 10 days. Nonetheless, the use of rat prothrombin or creation of a gene duplication-mediated hyperprothrombinemia model would provide more specific evidence. Longer term experiments should be conducted to determine if these short-term improvements translate into slowed or halted CKD progression such as improvements in glomerular filtration rate loss. Although PAN is a relatively podocyte-specific toxin, it is possible that there are off-target PAN effects on the coagulation system. However, PAN reportedly does not alter protein synthesis in isolated rat hepatocytes, making it unlikely that PAN directly altered coagulation protein synthesis. Kovacs AL, Seglen PO., Biochim Biophys Acta 1981; 676: 213-220. Finally, these data, generated in a rat NS model, may not be generalizable to other forms of glomerular disease that lead to CKD such as diabetic nephropathy, lupus nephritis, or hypertensive nephropathy and thus, should be examined in other animal models of glomerular disease.

[0076] In summary, prothrombin is able to modulate both podocyte function and survival in the PAN model of nephrotic syndrome. To determine the importance of prothrombin-mediated podocyte injury and its potential as a novel therapeutic target to slow CKD progression will requireadditional studies. Important next steps include defining the relevance of this mechanism in CKD models, determining the prothrombinase mechanism driving thrombin formation in the glomerulus, and evaluating its suitability as a druggable target using thrombin inhibitors, especially those that are already approved by relevant regulatory agencies for use in humans with kidney disease and may thus be suitable for rapid clinical translation. Derebail et al., Kidney Int 2020; 97: 664-675 (2020), Because proteinuria imparts increased thrombotic risk, renally excreted anticoagulant agents that may inhibit glomerular thrombin should be investigated as a novel means to simultaneously reduce thrombotic complications and slow proteinuria-mediated CKD progression.

[0077] The complete disclosure of all patents, patent applications, and publications, and electronically available materials cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

CLAIMSWhat is claimed is:

1. A method of treating or preventing glomerular disease or chronic kidney disease in a subject, comprising administering to the subject a therapeutically effective amount of a thrombin inhibitor or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the thrombin inhibitor is a factor X inhibitor.

3. The method of claim 1, wherein the thrombin inhibitor is rivaroxaban or dabigatran.

4. The method of claim 1, wherein the method is used to prevent glomerular disease in a subject in need thereof.

5. The method of claim 4, wherein the glomerular disease is nephrotic syndrome.

6. The method of claim 5, wherein the subject has been diagnosed with nephrotic syndrome, and treatment prevents the development of end-stage kidney disease.

7. The method of claim 4, wherein the subject has been diagnosed with glomerular disease.

8. The method of claim 1, wherein treatment decreases hypercoagulopathy in the subject.

9. The method of claim 1, wherein the subject is human.

10. The method of claim 1, wherein the method further includes administering an additional therapeutic agent for treating or preventing glomerular disease or chronic kidney disease.

11. The method of claim 1, wherein the thrombin inhibitor is administered together with a pharmaceutically acceptable carrier.

12. The method of claim 1 , wherein the thrombin inhibitor is administered orally.

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