EA042615B1 - METHODS FOR TREATMENT OF ALPORT SYNDROME AND IMPROVEMENT OF KIDNEY FUNCTION USING BARDOXOLONE-METHYL - Google Patents

Description

This application claims priority over U.S. Provisional Application No. 62/580597 filed November 2, 2017, U.S. Provisional Application No. 62/535663 filed July 2017, and U.S. Provisional Application No. 62/419335 filed November 8, 2016 ., the full contents of each of which are incorporated herein by reference.

State of the art

The scope of the invention.

This invention relates generally to the fields of biology and medicine. More specifically, it relates, in certain aspects of the invention, to methods of treating or preventing Alport's syndrome and improving kidney function using bardoxolone-methyl.

Description of the Prior Art

Alport syndrome is a rare and serious hereditary disease caused by mutations in the genes encoding type IV collagen, the main structural component of the glomerular basement membrane (GBM) in the kidney. Patients with Alport syndrome show ultrastructural changes in the KBM due to abnormal expression of type IV collagen chains in the kidney. Loss of CBM integrity leads to abnormal leakage of proteins such as albumin, which are then overresorbed in the tubules and activate pro-inflammatory signaling pathways in glomerular endothelial cells, mesangial cells, and podocytes. Chronic activation of pro-inflammatory pathways in these kidney cells contributes to the loss of glomerular filtration rate (GFR) through several mechanisms: (a) in glomerular endothelial cells, inflammation-associated reactive oxygen species (ROS) induce the production of peroxynitrite, which depletes vasodilating nitric oxide levels and leads to endothelial dysfunction, vasoconstriction and a decrease in glomerular surface area for filtration; (b) inflammation-associated ROS induce a contractile response in mesangial cells, further reducing filtration; and (c) ROS-mediated activation of inflammatory pathways leads to fibrosis, causing structural changes in the mesangium and thickening of the CVD, leading to a decrease in GFR. A decrease in GFR as a result of these processes inevitably leads to end-stage renal disease (ESRD).

Bardoxolone-methyl has been shown to improve both estimated glomerular filtration rate (eGFR) and measured glomerular filtration rate (iGFR) in patients with CKD due to type 2 diabetes. Bardoxolone-methyl and some of its analogs have also been shown to inhibit profibrotic signaling pathways and reduce oxidative stress and inflammation in many models of CKD. These compounds have also been shown to inhibit inflammatory signaling and fibrosis in animal models of liver, skin and lung disease.

Despite the serious consequences of Alport syndrome, including ESRD, there are only limited treatment options for this pathological condition. Therefore, there remains a therapeutic need to develop new and effective methods for the treatment and/or prevention of Alport syndrome, its associated symptoms, as well as the prevention of the onset of such symptoms.

The essence of the invention

In one aspect, the invention provides methods for treating or preventing Alport syndrome in patients in need thereof, wherein the patients have been identified as having a B-type natriuretic peptide (BNP) level that is not greater than 200 pg/mL, the method comprising administering to patients a therapeutically effective amount of a compound of the formula

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides methods for improving kidney function in patients diagnosed with Alport syndrome, wherein the patients have been identified as having a B-type natriuretic peptide (BNP) level that is not greater than 200 pg/mL, the method comprising administering to patients a therapeutically effective amount of a compound of the formula:

or a pharmaceutically acceptable salt thereof.

Such methods are also described in the sections below, including, for example, the section of the claims, which is incorporated herein by reference.

In some embodiments, the method for improving kidney function is a method for treating chronic kidney disease in a patient diagnosed with Alport syndrome.

- 1 042615

In some embodiments of the invention, the patient does not have a cardiovascular disease. In such embodiments, the cardiovascular disease is left-sided myocardial disease, atherosclerosis, restenosis, thrombosis, or pulmonary hypertension.

In some embodiments of the invention, the patient does not have stage 4 or higher chronic kidney disease. In some embodiments, the patient does not have an estimated glomerular filtration rate (eGFR) of less than 45 ml/min/1.73 m ² . In some embodiments, the patient does not have an elevated albumin/creatinine ratio (ACR) that is greater than 2000 mg/g.

In some embodiments of the invention, the patient does not have diabetes or complications associated with diabetes. In some embodiments of the invention, the patient does not have insulin resistance. In some embodiments, the patient does not have fatty liver disease. In some embodiments of the invention, the patient does not have liver failure. In some embodiments of the invention, the patient is not overweight. In some embodiments of the invention, the patient does not have cancer. In some embodiments, the patient does not have chronic obstructive pulmonary disease (COPD). In some embodiments of the invention, the patient is not a smoker.

In some embodiments of the invention, the patient has elevated levels of at least one biomarker associated with kidney disease, which is selected from serum creatinine, cystatin C and uric acid. In some embodiments, the Alport syndrome is X-linked, autosomal recessive, or autosomal dominant. In some embodiments, the patient has a deletion or nonsense mutation in COL4A5, a splicing mutation in COL4A5, a missense mutation in COL4A5, a mutation in COL4A3, or a mutation in COL4A4. In some embodiments, the patient additionally has microhematuria. In some embodiments of the invention, the patient additionally has microalbuminuria. In some embodiments of the invention, the patient additionally has proteinuria. In some embodiments of the invention, the age of the patient is less than 75 years.

In some embodiments, the patient does not have at least three of the following characteristics:

(A) cardiovascular disease;

(B) baseline B-type natriuretic peptide (BNP) greater than 200 pg/mL;

(C) estimated glomerular filtration rate (eGFR) <45 ml/min/1.73 m ² ;

(D) elevated albumin/creatinine ratio (ACR) >2000 mg/g.

In some embodiments of the invention, the patient does not have all four of the characteristics (A)-(D).

In embodiments of the invention, the compound is bardoxolone-methyl (CDDO-Me or RTA 402). And in some of these embodiments, at least a portion of the CDDO-Me is present as a polymorph, wherein the polymorph is a crystalline form having an x-ray diffraction pattern (CuKa) containing characteristic diffraction peaks at about 8.8, 12.9, 13 .4, 14.2 and 17.4° 2θ. In non-limiting examples, the X-ray diffraction pattern (CuKa) is substantially as shown in FIG. 1A or 1B. In other embodiments, at least a portion of the CDDO-Me is present as a polymorph, the polymorph being an amorphous form having an X-ray diffraction pattern (CuKa) with a halo peak at approximately 13.5° 2θ, substantially as shown in FIG. 1C, and Tg. In some embodiments, the compound is in amorphous form. In some embodiments, the compound is a solid glassy form of CDDO-Me having an x-ray powder diffraction pattern with a halo peak at about 13.5° 2θ as shown in FIG. 1C, and T _g . In some embodiments, the value of T _g is in the range from about 120 to about 135°C. In some embodiments, the value of T _g is from about 125 to about 130°C.

In some embodiments, the compound is administered topically. In some embodiments, the compound is administered systemically. In some embodiments, the compound is administered orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostately, intrarectally, intrathecally, intratracheally, intratumorally, via the umbilical cord , intravaginally, intravenously, intravesically, intravitreally, liposomal, locally, through the mucosa, orally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, buccally, transdermally, vaginally, in cream, in lipid compositions, through a catheter, through lavage, via continuous infusion, by infusion, by inhalation, by injection, by local delivery, by local perfusion, by direct immersion of target cells, or by any combination thereof. For example, in some embodiments, the compound is administered

- 2 042615 intravenously, intra-arterially or orally. For example, in some embodiments, the compound is administered orally.

In some embodiments, the compound is formulated as a hard or soft capsule, tablet, syrup, suspension, solid dispersion, tablet, or infusion. In some embodiments, the softgel is a gelatin capsule. In embodiments, the compound is formulated as a solid dispersion. In some embodiments, the hard capsule, soft capsule, tablet, or plate further comprises a protective coating. In some embodiments, the compound formulated contains an agent that delays absorption. In some embodiments, the compound included in the composition contains an agent that increases solubility or dispersion. In some embodiments, the compound is dispersed in a liposome, an oil-in-water emulsion, or a water-in-oil emulsion.

In some embodiments, the pharmaceutically effective amount is about 0.1 mg to about 500 mg daily of the compound. In some embodiments, the daily dose is from about 1 to about 300 mg of the compound. In some embodiments, the daily dose is from about 10 to about 200 mg of the compound. In some embodiments, the daily dose is about 25 mg of the compound. In other embodiments, the daily dose is about 75 mg of the compound. In other embodiments, the daily dose is about 150 mg of the compound. In additional embodiments, the daily dose is from about 0.1 to about 30 mg of the compound. In some embodiments, the daily dose is from about 0.5 to about 20 mg of the compound. In some embodiments, the daily dose is from about 1 to about 15 mg of the compound. In some embodiments, the daily dose is from about 1 to about 10 mg of the compound. In some embodiments, the daily dose is from about 1 to about 5 mg of the compound. In some embodiments, the daily dose is from about 2.5 to about 30 mg of the compound. In some embodiments, the daily dose is about 2.5 mg of the compound. In other embodiments, the daily dose is about 5 mg of the compound. In other embodiments, the daily dose is about 10 mg of the compound. In other embodiments, the daily dose is about 15 mg of the compound. In other embodiments, the daily dose is about 20 mg of the compound. In other embodiments, the daily dose is about 30 mg of the compound.

In some embodiments of the invention, a pharmaceutically effective amount is a daily dose of 0.01-25 mg of the compound per 1 kg of body weight. In some embodiments, the daily dose is 0.05-20 mg of the compound per 1 kg of body weight. In some embodiments, the daily dose is 0.1-10 mg of the compound per 1 kg of body weight. In some embodiments, the daily dose is 0.1-5 mg of the compound per 1 kg of body weight. In some embodiments, the daily dose is 0.1-2.5 mg of the compound per 1 kg of body weight.

In some embodiments, the pharmaceutically effective amount is administered as a single dose per day. In some embodiments, a pharmaceutically effective amount is administered in two or more doses per day.

In some embodiments, the patient is a mammal, such as a primate. In some embodiments, the primate is a human. In other embodiments, the patient is a cow, horse, dog, cat, pig, mouse, rat, or guinea pig.

In some embodiments of the above methods, the compound is substantially free of its optical isomers. In some embodiments of the above methods, the compound is in the form of a pharmaceutically acceptable salt. In other embodiments of the above methods, the compound is not a salt.

In some embodiments, the compound is formulated as a pharmaceutical composition comprising (i) a therapeutically effective amount of the compound and (ii) an excipient selected from the group consisting of (A) a carbohydrate, carbohydrate derivative, or carbohydrate polymer, (B) a synthetic an organic polymer, (C) an organic acid salt, (D) a protein, polypeptide or peptide, and (E) a high molecular weight polysaccharide. In some embodiments, the adjuvant is a synthetic organic polymer. In some embodiments, the excipient is selected from the group consisting of hydroxypropyl methylcellulose, poly[1-(2-oxo-1-pyrrolidinyl)ethylene] or a copolymer thereof and a copolymer of methacrylic acid and methyl methacrylate. In some embodiments, the excipient is hydroxypropyl methylcellulose phthalate ester. In some embodiments, the excipient is PVP/VA. In some embodiments, the adjuvant is a copolymer of methacrylic acid and ethyl acrylate. In some embodiments, methacrylic acid and ethyl acrylate may be present in a ratio of about 1:1. In some embodiments, the excipient is copovidone.

Any embodiment of the invention discussed herein with respect to one aspect of the invention is also applicable to other aspects of the invention, unless specifically noted otherwise.

Additional aspects and embodiments of this invention are described in more detail, for example, in the section of the claims, which is incorporated herein by reference.

Other objects, features and advantages of this description will become apparent from the following.

- 3 042615 detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications will be apparent to those skilled in the art from this detailed description within the spirit and scope of the invention. Notably, just because a particular compound is assigned to one particular general formula does not mean that it cannot also belong to another general formula.

Brief description of graphic materials

The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description of specific embodiments provided herein.

Fig. 1A-C are X-ray powder diffraction (XRD) spectra of Forms A and B of RTA 402. A: illustrates non-micronised Form A; B: illustrates micronized Form A; C: illustrates form B.

Fig. 2 is the mean eGFR over time in BEACON (safety study group). Mean observed eGFR over time by week of treatment in patients treated with placebo (PBO) compared with patients treated with bardoxolone-methyl (BARD). Only eGFR estimates collected at or before the last dose of study drug to a patient are included. Visits occur relative to the first dose of study drug to the patient. Data are mean ± SE.

Fig. 3A, 3B is the percentage reduction in eGFR in patients treated with bardoxolone methyl compared with patients treated with placebo in BEACON (safety study group). Percentage of patients with changes in eGFR from baseline <-3, <-5, or <-7.5 ml/min/1.73 m ² by week of treatment for patients treated with placebo (PBO) (Fig. 3A), by compared with patients treated with bardoxolone-methyl (BARD) (Fig. 3B). Only eGFR estimates collected at or before the last dose of study drug to a patient are included. Visits occur relative to the first dose of study drug to the patient. Percentages are calculated relative to the number of patients with available eGFR data at each visit. The number of placebo patients with data for FIG. 3A was 1093 at 0 weeks, 1023 at 8 weeks, 885 at 16 weeks, 726 at 24 weeks, 547 at 32 weeks, 402 at 40 weeks, and 281 at 48 weeks. The number of placebo patients with data for FIG. 3B was 1092 at 0 weeks, 958 at 8 weeks, 795 at 16 weeks, 628 at 24 weeks, 461 at 32 weeks, 345 at 40 weeks, and 241 at 48 weeks.

Fig. 4 is the time to event, which is the combined primary efficacy endpoint, in BEACON (All Enrolled Patients (ITT) group). Results of a phase 3, randomized, double-blind, placebo study in patients with T2D and stage 4 CKD (BEACON, RTA402-C-0903). Patients received either placebo (PBO) or bardoxolone-methyl (BARD) 20 mg once daily. The analysis includes only deaths due to end-stage renal disease (ESRD) or cardiovascular (CV) events on or before the end of the study drug period (SDT) (October 18, 2012) that were positively assessed by an independent Event Review Committee as outlined in the EAC BEACON section.

Fig. 5 is mean serum magnesium levels in patients treated with bardoxolone-methyl compared with patients treated with placebo in BEACON. Mean observed serum magnesium levels over time by week of treatment in patients treated with placebo compared with patients treated with bardoxolone-methyl. Only serum magnesium estimates collected at or before the last dose of study drug in a patient are included. Visits occur relative to the first dose of study drug to the patient. Data are mean ± SE. The top line represents placebo; the bottom line is bardoxolone-methyl.

Fig. 6A, 6B are changes from baseline over time in systolic blood pressure (SBP) (FIG. 6A) and diastolic blood pressure (DBP) (FIG. 6B) in patients treated with bardoxolone-methyl (BARD) compared with patients treated with bardoxolone-methyl (BARD). placebo (PBO), in BEACON (safety study group). Data include only vital signs obtained at or before the last dose of study drug to the patient. Visits occur relative to the first dose of study drug to the patient. Data are mean ± SE.

Fig. 7A, 7B are a 24-hour ambulatory blood pressure monitoring (ABPM) satellite study: changes at week 4 in systolic blood pressure (SBP) (Fig. 7A) and diastolic blood pressure (DBP) (Fig. 7B) from baseline to 4 weeks in patients treated with bardoxolone-methyl (BARD) compared with patients treated with placebo (PBO). Data include only patients with baseline ABPM values and 24-hour ABPM values at week 4. Changes in systolic blood pressure are calculated using the average of all valid measurements taken from the patient's ambulatory blood pressure monitor over the entire 24-hour period, daytime (6 am to 10 pm) or night (10 pm before 6 a.m. the next day). Data are mean ± SE.

Fig. 8A-8D are placebo-adjusted changes in systolic blood pressure from baseline on study days 1 and 6 in bardoxolone-methyl (BARD) administered healthy volunteers. Results of a placebo, multi-dose, randomized, double-blind, comprehensive QT study in healthy volunteers (RTA402-C-1006). Patients were treated with placebo, bardoxolone-methyl 20 or 80 mg, or moxifloxacin 400 mg (active comparator) once daily for six consecutive days.

Data are mean changes (± SD) from baseline 0-24 hours post-dose on study day 1 and study day 6. Fig. 8A illustrates the administration of a 20 mg dose of BARD on day 1 of the study. Fig. 8B illustrates administration of a 20 mg BARD dose on day 6 of the study. Fig. 8C illustrates the administration of a dose of 80 mg BARD on day 1 of the study. Fig. 8D illustrates the administration of a dose of 80 mg BARD on day 6 of the study.

Fig. 9A, 9B are placebo-corrected QTcF changes from baseline in healthy volunteers administered bardoxolone-methyl (BM). Results of a placebo, multi-dose, randomized, double-blind, comprehensive QT study in healthy volunteers (RTA402-C-1006). Changes in the QTcF interval in subjects treated with bardoxolone-methyl (20 or 80 mg - Fig. 9A or 9B, respectively) are shown relative to changes in patients treated with placebo for six consecutive days. Data are means ± 90% CI estimated 0-24 hours post-dose on study day 6, where the upper limit of 90% CI is equivalent to the one-sided upper 95% confidence limit. The threshold comparison line of 10 ms corresponds to the upper confidence limits.

Fig. 10A, 10B are Kaplan-Meier plots for cases of hypervolemia in ASCEND (FIG. 10A) and cases of heart failure in BEACON (ITT group; FIG. 10B). Time to first analysis of avosentan-induced hypervolemia in ASCEND and heart failure in BEACON. Cases of avosentan-induced hypervolemia in ASCEND were taken from side-effect reports from local investigators. Some signs and symptoms in the form of side effects that indicate hypervolemia include: heart failure, edema, fluid volume increase, fluid retention, hypervolemia, dyspnea, pleural and pericardial effusions, ascites, weight gain, pulmonary rales and pulmonary edema. The analysis includes only cases of heart failure occurring on or before the study drug discontinuation date (October 18, 2012) that were positively assessed by an independent Event Evaluation Committee as specified in the EAC BEACON section.

Fig. 11 represents the time to first hospitalization for heart failure or heart failure death at BEACON (ITT group). The analysis includes only cases of heart failure (HF, HF) occurring on or before the study drug discontinuation date (SDT) (October 18, 2012) that were positively assessed by an independent Event Evaluation Committee as outlined in section EAC BEACON. The top line is bardoxolone-methyl (BARD); the bottom line is placebo (PBO).

Fig. 12 is the overall survival of patients treated with bardoxolone-methyl compared with patients treated with placebo in BEACON. Results of a phase 3, randomized, double-blind, placebo study in patients with T2D and stage 4 CKD (BEACON, RTA402-C-0903). Patients received placebo or 20 mg bardoxolone-methyl once daily. The analysis includes all deaths that occurred before the closure of the database (March 4, 2013). The top line is bardoxolone-methyl (BARD); the bottom line represents placebo.

Description of exemplary embodiments of the invention

In one aspect, the present invention provides novel methods for treating or preventing Alport syndrome or improving kidney function in patients using bardoxolone-methyl. These and other aspects of the invention are described in more detail below.

I. Characteristics of patients who should be excluded from treatment with bardoxolone-methyl.

Several clinical studies have shown that treatment with bardoxolone-methyl improved markers of renal function (including estimated glomerular filtration rate, or eGFR), insulin resistance, and endothelial dysfunction (Pergola et al., 2011). These observations led to the initiation of a large phase 3 (BEACON) study of bardoxolone-methyl in patients with stage 4 CKD and type 2 diabetes. The main endpoint in the BEACON study was a combination of progression to end-stage renal disease (ESRD) and all-cause mortality. This study was terminated due to excessively severe side effects and mortality in the bardoxolone-methyl group of patients.

As discussed below, post-analysis of data from the BEACON study showed that the majority of severe adverse events and mortality are associated with heart failure and are strongly associated with the presence of one or more risk factors, including (a) elevated baseline levels of B-type natriuretic peptide (BNP; for example, >200 pg/ml); (b) baseline eGFR <20; (c) history of left-sided heart disease; (d) high baseline albumin to creatinine ratio (ACR; eg >300 mg/g as determined by a 3+ proteinuria dipstick); and (e) advanced age (eg, >75 years). The analysis showed that the cases of heart failure were probably associated with the development of acute hypervolemia in the first 3-4 weeks of treatment with bardoxolone-methyl and that this could be due to inhibition of endothelin-1 signaling in the kidneys. A previous study of an endothelin receptor antagonist in patients with stage 4 CKD was discontinued due to a pattern of side effects and mortality very similar to those found in the BEACON study. Subsequent non-clinical studies have confirmed that bardoxolone-methyl at physiologically significant concentrations inhibits the expression of endothelin-1 in renal proximal tubular epithelial cells and inhibits the expression of endothelin receptors in human mesangial and endothelial cells. Accordingly, patients at risk of developing side effects from inhibition of endothelin signaling should be excluded from future clinical use of bardoxolone-methyl.

The present invention relates to novel treatments for Alport's syndrome that involve modification of the glomerular basement membrane as a significant contributing factor. It also concerns the preparation of pharmaceutical compositions for the treatment of such disorders. In some embodiments of the present invention, patients are selected for treatment based on several criteria:

(1) diagnosis of a disorder that includes endothelial dysfunction as a significant contributing factor;

(2) absence of elevated levels of B-type natriuretic peptide (BNP; for example, BNP titers should be <200 pg/mL);

(3) no chronic kidney disease (eg, eGFR >60) or no progressive chronic kidney disease (eg, eGFR >45);

(4) no history of left-sided myocardial disease; and (5) no high ACR (eg, ACR below 300 mg/g).

In some embodiments of the invention, patients diagnosed with type 2 diabetes are excluded. In some embodiments of the invention, patients diagnosed with cancer are excluded. Elderly patients (eg, >75 years of age) are excluded in some embodiments of the invention. In some embodiments, patients are closely monitored for rapid weight gain, indicative of hypervolemia. For example, patients may be instructed to weigh themselves daily for the first four weeks of treatment and contact their healthcare provider if there is an increase of more than five pounds.

A. The BEACON study.

1. Study design.

Study 402-C-0903, Evaluation of Bardoxolone-methyl in Patients with Chronic Kidney Disease and Type 2 Diabetes: Occurrence of Renal Adverse Effects (BEACON), was a phase 3, randomized, double-blind, multinational, multicenter, placebo, parallel group study designed to comparing the efficacy and safety of bardoxolone-methyl (BARD) and placebo (PBO) in patients with stage 4 chronic kidney disease and type 2 diabetes. A total of 2185 patients were randomized 1:1 to receive bardoxolone-methyl (20 mg) or placebo once daily. The primary efficacy endpoint of the study was the time to the first event in the composite endpoint, defined as end-stage renal disease (ESRD; need for chronic dialysis, kidney transplant, or death from kidney failure) or death from cardiovascular (CV) complications. The study had three secondary efficacy endpoints:

(1) change in estimated glomerular filtration rate (eGFR);

(2) time to first hospitalization for heart failure or death from heart failure; and (3) time to first event of the composite endpoint consisting of non-fatal myocardial infarction, non-fatal stroke, hospitalization for heart failure, or death from cardiovascular complications.

A subgroup of BEACON patients agreed to additional 24-hour assessments, including ambulatory blood pressure monitoring (ABPM) and 24-hour urine collection. An Independent Event Evaluation Committee (EAC) reviewed treatment intent in a masked manner, assessed whether renal events, cardiovascular events, and neurologic events met predefined definitions of primary and secondary endpoints. The IDMC (Independent Data Monitoring Committee), composed of external clinical experts supported by an independent statistical panel, reviewed the unmasked safety data throughout the study and made appropriate recommendations.

2. Demographic and baseline characteristics of the study group.

In table. 1 provides summary statistics on some of the demographic and baseline characteristics of patients involved in BEACON. Demographic characteristics were comparable between the two treatment groups. In all treatment groups, the mean age was 68.5 years and 57% of patients were male. There were slightly more patients in the age subgroup >75 years in the bardoxolone-methyl group than in the placebo group (27% in the bardoxolone-methyl group vs. 24% in the placebo group). The average weight and BMI in both treatment groups were 95.2 and 33.8 kg/m ² respectively. Baseline renal function was basically the same in the two treatment groups; mean baseline eGFR, as measured by the 4-variable dietary modification equation for kidney disease (MDRD), was 22.5 ml/min/1.73 m ² , and the geometric mean albumin/creatinine ratio (ACR) was 215.5 mg/ g for combined treatment groups.

Table 1

Some demographic and baseline characteristics of patients treated with bardoxolone-methyl (BARD) compared with patients treated with placebo (PBO), ________________ in BEACON (ITT group) ________________

BARD N=1088 RVO N=1097 Total N=2185 Sex, η (%) male 626 (58) 625 (57) 1251 (57) female 462 (42) 472 (43) 934 (43) Age at informed consent (years) η 1088 1097 2185 Average (SD) 68.9 (9.7) 68.2 (9.4) 68.5 (9.6) Range (min, max) 32, 92 29, 93 29, 93 Age subgroup, η (%) <75 786 (72) 829 (76) 1615 (74) >75 302 (27) 268 (24) 570 (26) Weight (kg) η 1087 1097 2184 Average (SD) 95.1 (22.0) 95.3 (21.1) 95.2 (21.5) Range (min, max) 46, 194 45, 186 45, 194 BMI (kg / m ² ) η 1087 1097 2184 Average (SD) 33.7 (7.1) 33.9 (7.2) 33.8 (7.1) Range (min, max) 19, 93 19, 64 19, 93 eGFR (ml/min/1.73 ^m2 ), mean (SD) η 1088 1097 2185 Average (SD) 22.4 (4.3) 22.5 (4.6) 22.5 (4.5) Range (min., max.) 13, 34 13.58 13.58 eGFR subgroup in according to MDRD, η (%) 15 - <20 325 (30) 347 (32) 672 (31) 20 - <25 399 (37) 366 (33) 765 (35) 25 - <30 311 (29) 318 (29) 629 (29) ACR (mg/g), mean geometric η 1088 1097 2185 geometric mean 210.4 220.7 215.5 (95% CI) (188, 236) (196, 249) (198, 234) Range (min., max.) <1,4581 <1.79466 <1.79466

- 7 042615

subgroup ACR, η (%) <30 200 (18) 211 (19) 411 (19) 30-300 348 (32) 308 (28) 656 (30) >300 540 (50) 578 (53) 1118 (51)

Patients received placebo or 20 mg bardoxolone-methyl once daily.

B. BEACON results.

1. Effect of bardoxolone-methyl on eGFR.

Mean eGFR values for patients treated with bardoxolone-methyl and placebo are shown in FIG. 2. On average, for patients treated with bardoxolone-methyl, an increase in eGFR was expected, which was observed at 4 weeks of treatment and remained above baseline until 48 weeks. In contrast, placebo-treated patients experienced, on average, a unchanged or non-significant decrease from baseline. The proportion of patients with decreased eGFR was markedly lower in patients treated with bardoxolone-methyl compared with patients treated with placebo (Figs. 3A, 3B). The eGFR curves and percentage reductions observed in BEACON after one year of treatment were in line with modeled expectations and the results of the BEAM study (RTA402-C-0804). As shown in Table. 2, the number of patients who experienced a serious side effect (SAE) in disorders of the kidneys and urinary system was lower in the bardoxolone-methyl group than in the placebo group (52 compared to 71, respectively). Additionally, as discussed in the next section, there were slightly fewer cases of ESRD in the bardoxolone-methyl group than in the placebo group. Taken together, these data suggest that treatment with bardoxolone-methyl did not worsen kidney function abruptly or over time.

Table 2 Prevalence of Treatment-Occurring Serious Adverse Events in BEACON in Each Major Organ System Class __________________(Safety Study Group)__________________

Organ system class in according to MedDRA placebo Ν=1093 η (%) Bardoxolone methyl N=1092 n (%)

- 8 042615

Patients with any serious side effect 295 (27) 363 (33) Number of serious side effects effects 557 717 Cardiac disorders 84(8) 124 (11) Infections and infections 63(6) 79(7) Kidney and urinary disorders 71(6) 52(5) Metabolic and nutritional disorders 42(4) 51(5) Gastrointestinal disorders 39(4) 46(4) Respiratory, thoracic and mediastinal disorders 32(3) 43(4) Nervous System Disorders 35(3) 37(3) General disorders and condition of the injection site 20(2) 29(3) Vascular disorders 18(2) 20(2) Injury, poisoning and complications caused by procedures 17(2) 19(2) Musculoskeletal and connective tissue disorders 13(1) 21(2) Blood and lymphatic system disorders 11(1) 20(2) Neoplasms benign, malignant and unspecified (including cysts and polyps) 10(1) 11(1)

- 9 042615

Liver and biliary tract disorders 8(1) 4 (<1) Mental disorders 3 (<1) 3 (<1) Eye disorders 2 (<1) 3 (<1) Laboratory and instrumental data 2 (<1) 3 (<1) Reproductive system and breast disorders 3 (<1) 2 (<1) Skin and subcutaneous tissue disorders 1 (<D 4 (<1) Ear disorders and labyrinth apparatus 1 (<1) 3 (<1) Endocrine Disorders 1 (<1) 1 (<D Immune disorders systems 0 2 (<1) Surgical and medical procedures 0 2 (<1)

Only serious adverse events that occurred more than 30 days after the last dose of study drug was administered to the patient are included in the table. The numbers in the column headings and denominators are the number of patients in the safety study group. Each patient is counted no more than once per organ system class and for the preferred term.

2. The main combined efficiency criterion in BEACON.

In table. Table 3 is a summary of the designated primary endpoints occurring on or before the study end date (October 18, 2012). Despite a small reduction in the incidence of ESRD in the bardoxolone-methyl treatment groups compared with the placebo treatment groups, the number of combined primary endpoints was similar in the two treatment groups (RR = 0.98) due to a small increase in cardiovascular deaths , as shown in the analysis plots of time to the first combined primary event (FIG. 4).

Table 3

Assigned primary endpoints in patients treated with bardoxolone-methyl (BARD) versus patients treated with placebo (PBO) in BEACON (ITT arm)

RVO N=1097n (%) BARD N=1088n (%) Hazard ratio (95% CI) ^{a value} Combined main efficiency criterion 69 (6) 69 (6) 0.98 (0.70, 1.37) 0.92 First patient event End stage renal disease (ESRD) 51(5) 43(4) Chronic dialysis 47(4) 40 (4) kidney transplant 3 (<1) 1 (<i) death due to kidney failure 1 (<1) 2 (<1) Death from CVD 18(2) 26(2)

- 10 042615 ^a Hazard ratio (bardoxolone-methyl/placebo) and 95% confidence interval (CI) were estimated using a Cox proportional hazards model with treatment group, continuous baseline eGFR, and continuous baseline log ACR as independent variables. The Breslow link processing method was used during the event.

^b Treatment groups were compared using a SAS type 3 chi-square test and a two-tailed p value associated with the treatment group variable in a Cox proportional hazards model.

C. Effect of bardoxolone-methyl on heart failure and blood pressure.

1. Recognized heart failure at BEACON.

The data in the table. 4 present a retrospective analysis of demographic and sample laboratory parameters of BEACON patients stratified by treatment group and occurrence of a recognized heart failure event. The number of patients with heart failure includes all events up to the last contact date (ITT group).

Comparison of baseline characteristics of patients with recognized cases of heart failure showed that patients with heart failure treated with both bardoxolone-methyl and placebo were more likely to have a history of cardiovascular disease and heart failure, and also had higher baseline natriuretic levels. B-type peptide (BNP) and Fridericia-corrected QTc interval (QTcF). Although the risk of heart failure was higher in patients treated with bardoxolone-methyl, these data suggest that the development of heart failure in both groups appears to be related to traditional risk factors for heart failure. Baseline ACR was significantly higher in bardoxolone-methyl treated patients with heart failure than in patients without it. It should also be noted that the mean baseline BNP in patients with heart failure in both treatment groups was significantly elevated, suggesting that these patients were likely to have fluid retention and subclinical heart failure prior to randomization.

Table 4

Some demographic and baseline characteristics of patients treated with bardoxolone-methyl or treated with placebo, stratified by the presence of heart failure

Patients With heart failure Without heart failure Total BARD (N=103) RVO (N=57) BARD (N=985 ) RVO (N=104 0) BARD (N=108 8) RVO (N=109 7) Age (years), mean ± SD 70.3±9 69.2± 8.2 68.7± 9, 8 68.1± 9.5 68.9± 9, 7 68.2± 9.4 History of CVD, N 80 47 529 572 609 619

- 11 042615

(%) (78) ^a (82) ^b (54) (55) (56) (56) History of HF, N (%) 36 (35) ^a 21 (37) ^b 130 (13) 133 (13) 166 (15) 154 (14) MI history, N (%) 33 (32) ^a 22 (3 9) ^b 185 (19) 188 (18) 218 (20) 210 (19) Α-FIB in history, N (%) 4 (4) 3(5) 46(5) 40 (4) 50 (5) 43 (4) Concomitant drug use, N (%) ACEi/ARB 35 (34) ^a 16 (2 8 ) ^b 659 (67) 701 (67) 694 (64) 717 (65) Diuretic 39 (38) ^a 15 (26) ^b 528 (54) 586 (56) 567 (52) 601 (55) Beta blocker 38 (37) ^a 23 (40) 482 (49) 506 (49) 520 (48) 529 (48) Statins 57 (55) 26 ( 4 6) ^b 640 (65) 721 (69) 697 (64) 747 (68) Calcium channel blocker 25 (24) ^a 17 (30) ^b 406 (41) 467 (45) 431 (40) 484 (44) eGFR (ml / min / 1.73 m ² ), Mean ± SD 21.7± 4.6 22.2± 4.7 22.5± 4.2 22.5± 4.6 22.4± 4.3 22.5± 4.6 ACR (mg/g) geometric mean 353.9 ^a 302.0 199.3 216.9 210.4 220.7 SBP (mm Hg), mean ± SD 139.5± 13.3 142.3± 11.2 139.5 ±11.6 139.6 ±11.8 139.5 ±11.7 139.8 ±11.8 DBP (mm Hg), mean ± SD 66.4 ± 9.1 ^a 69.1± 8.8 70.4± 8.7 70.8± 8.6 70.1± 8.8 70.7± 8.7 BNP (pg/ml) Mean ± SD 526.0 ± 549.4 ^a 429.8 ± ^434.3 223.1+ 257.5 232.3± 347.1 251.2± 309.1 242.7+ 354.7 >100, N (%) 78 (7 6) ^a 43 (75) ^b 547 (56) 544 (52) 625 (57) 587 (54) QTcF (ms) Mean ± SD 447.9 ± 31.2 ^{a 's} 432.5 ± 27.6 ^b 425.3 ±27.8 424.7 ±27.9 427.4 ±28.9 425.1 ±28 >450, N (%) 40 (39) ^a 14 (25) 170 (17) 167 (16) 210 (19) 181 (16)

^a p<0.05 for BARD-treated patients with HF versus BARD-treated patients without HF;

^b p<0.05 for patients treated with RVO with HF compared with patients treated with RVO without HF;

^with p<0.05 for patients treated with BARD and patients treated with RVO with HF.

2. Assessment of clinical parameters associated with an increase in BNP.

As an identifier for fluid retention, a retrospective analysis of the subset of patients for whom BNP data were available at baseline and at week 24 was performed. Patients in the bardoxolone-methyl group experienced a significantly greater increase in BNP levels than patients in the placebo group (mean ± SD: 225 ± 598 vs. 34 ± 209 pg/mL, p < 0.01). There was also a higher proportion of patients treated with bardoxolone-methyl, compared with patients treated with placebo, with elevated BNP levels at week 24 (Table 5).

The rise in BNP at week 24 does not appear to be related to baseline BNP, baseline eGFR, changes in eGFR, or changes in ACR. However, only in bardoxolone-methyl-treated patients did baseline ACR correlate markedly with changes in BNP at week 24 from baseline, suggesting that a tendency to fluid retention may be related to baseline severity of renal dysfunction as determined by grade albuminuria rather than with overall changes in kidney function as measured by eGFR (Table 6).

In addition, these data suggest that increases in eGFR that are of glomerular origin differ anatomically, as sodium and water are regulated in the renal tubules.

Table 5

Analysis of BNP and eGFR values in patients treated with bardoxolone-methyl and treated with placebo, stratified by changes in BNP levels from baseline at week 24

Level change BNP at week 24 Treatment N Median Baseline BNP Mean baseline eGFR Average ArSKF at 24 weeks Increase <25% RVO 131 119.0 23.5 -0.6 BARD 84 187.0 22.3 6, 1 Increase from 25% to 100% RVO 48 102.5 22.0 oh 4 BARD 45 119.0 22.7 5.5 Zoom ^100% RVO 37 143.5 23.1 0, 1 BARD 82 155.0 21.9 7.6

Retrospective analysis of BNP change in BEACON at 24 weeks.

Table 6

Correlations between change in BNP from baseline at week 24 and baseline ACR in bardoxolone-methyl versus placebo treated patients in BEACON

Treatment N Coefficient correlations p-value RVO 216 0.05 0.5 BARD 211 0.20 <0.01

Retrospective analysis of BNP change in BEACON at 24 weeks. Only patients with baseline BNP values and BNP values at week 24 were included in the analysis.

3. Serum electrolytes.

There were no clinically significant changes in serum potassium or serum sodium levels for a subset of patients on 24-hour urine collection (Table 7). The change in serum magnesium levels in patients treated with bardoxolone-methyl was consistent with changes observed in previous studies (Fig. 5).

Table 7

Changes at Week 4 from Baseline in Serum Electrolytes in Bardoxolone-Methyl vs. Placebo Patients at 24 Hours in the Ancillary ABPM Study

Kalin in serum (mmol/l) Serum sodium (mmol/L) Serum magnesium (mEq/L) Baseline Week 4 And at 4 weeks Baseline Week 4 And at 4 weeks Baseline Week 4 And at 4 weeks RVO P 88 87 87 88 87 87 88 87 87 Mean ± SE 4.8 i OD 47 ± OD -0.10±0.04 140.2 ± OD 139.7±0.3 -0.3±0.2 1.72 ± ODZ 1.69±0.03 -0.03±0.02 BARD P 83 77 77 83 77 77 83 77 77 Mean ± SE 4.7 ± OD 4.8±0.1 ODO ± 0.05 * 1 140.1±0.3 140.3±0.3 0.2±0.3 1.74±0.02 1.53±0.03 -0.21±0.02*1

Data include only BEACON patients enrolled in the 24-hour ABPM Ancillary Study. Changes in serum electrolyte levels are calculated for patients with baseline and week 4 data only;

* p<0.05 at 4 weeks compared to baseline in each treatment group;

- 13 042615 f p<0.05 for changes in patients treated with BARD compared to patients treated with PBO at week 4.

4. 24-hour urine collection.

A subset of patients agreed to additional 24-hour assessments (ancillary study) at outpatient blood pressure monitoring (ABPM) and 24-hour urine collection at some visits. Urinary sodium excretion data from a subgroup of patients from the BEACON study revealed a clinically significant decrease in urine volume and sodium excretion at week 4 from baseline in patients treated with bardoxolone-methyl (Table 8). These reductions were significantly different from the changes at week 4 in urine volume and urinary sodium observed in patients treated with placebo. It should also be noted that the decrease in serum magnesium levels was not associated with renal magnesium loss.

Additionally, in a pharmacokinetic study of patients with type 2 diabetes mellitus and stage 3b/4 CKD who were administered bardoxolone-methyl for eight weeks (402-C-1102), patients with stage 4 CKD had a significantly greater decrease in sodium and water excretion with urine than in patients with stage 3b CKD (Table 9).

Table 8

Changes at Week 4 from Baseline in Urinary Volume, Urinary Sodium and Urinary Potassium at 24 Hours in Bardoxolone-Methyl-Treated Patients Compared to Placebo-Treated Patients in the 24-Hour ABPM Ancillary Study

Urine volume (ml) Natrine in urine (mmol/24 h) Caline in urine (mmol/24 h) Magnesium in urine (mmol/24 h) Baseline Week 4 D at 4 weeks Baseline Week 4 D at 4 weeks Baseline Week 4 D at 4 weeks Baseline Week 4 D at 4 weeks RVO AND 87 72 71 81 68 62 81 68 62 59 53 46 Average ±SE 3053±82 1928 ±89 110 ±71 160 ±8 145 ±8 eleven ±9 55 ±3 52 ±3 3 ±3 7.5±0.5 6.0±0.5 0.6 ±0.4 BARD AND 82 64 63 77 61 57 77 61 57 56 43 40 Average ±SE 2024 ±83 1792 ±84 -247 ±7G 164 ±9 140 ±9 -27 ±9* 60 ±3 52 ±2 -7±3* 7.0±0.4 6.0±0.4 -0.9 ±0.5

Data include only BEACON patients enrolled in the 24-hour ABPM Ancillary Study. Changes at week 4 are calculated only for patients with baseline and week 4 data;

* p<0.05 at 4 weeks compared to baseline in each treatment group;

^f p<0.05 for change at week 4 in patients treated with BARD compared to patients treated with PBO.

Table 9

Changes at week 8 from baseline in 24-hour urine volume and 24-hour urine sodium in patients treated with bardoxolone-methyl grouped by severity of CKD (from a patient pharmacokinetic study)

CKD stage Urine volume (ml) Sodium in urine (mmol/24 h) N Δ at 8 weeks R- meaning Δ at 8 weeks p-value Stage 3b 9 355 0.04 -12 0.02 Stage 4 6 -610 -89

Patients received bardoxolone-methyl 20 mg once daily for 56 consecutive days; the follow-up visit after treatment was on study day 84. The data is averaged. Data includes patients at baseline and data at week 8.

5. Records of hospital records from packages of EAC expert opinions.

The first scheduled evaluation after the BEACON baseline was at week 4. Since many of the cases of heart failure occurred before 4 weeks, the clinical database provides limited information to characterize these patients. To evaluate the clinical, vital signs, laboratory, and imaging data collected during the first heart failure event, a retrospective review of EAS case packages was performed for heart failure events that occurred before week 4 (Tables 10 and 11).

Examination of these records revealed common reports of rapid weight gain immediately after randomization, dyspnea and orthopnea, peripheral edema, central/pulmonary edema on imaging, elevated blood pressure and heart rate, and fixed ejection fraction. Evidence suggests that heart failure was caused by rapid fluid retention concomitant with fixed ejection fraction and elevated blood pressure. The fixed ejection fraction is consistent with the clinical features of heart failure caused by diastolic dysfunction due to ventricular tension and impaired diastolic relaxation. This constellation of signs and symptoms differs in clinical characteristics from heart failure with reduced ejection fraction, which occurs due to impaired heart pumping or abnormal contractions (Vasan et al., 1999). Thus, the rapid accumulation of fluid in patients with tense ventricles and minimal renal reserve probably led to an increase in fluid outflow to the lungs and a marked clinical picture.

Baseline central laboratory values from the clinical database were compared with local laboratory values obtained at admission with heart failure, which were included in the EAC packages. Unchanged serum creatinine, sodium, and potassium concentrations in bardoxolone-methyl-treated patients with heart failure events that occurred within the first four weeks after randomization (Table 11) suggest that heart failure was not associated with acute decreased kidney function or acute kidney injury. In general, clinical evidence suggests that the etiology of heart failure is not determined directly by nephrotoxicity or cardiotoxicity, but rather by sodium and fluid retention.

Table 10

Retrospective analysis of cardiovascular parameters in bardoxolone-methyl versus placebo patients with heart failure occurring within the first four weeks of treatment

LVEF SBP (mm Hg) DBP (mm Hg) Heart rate (beats per minute) CH Baseline CH Λ Baseline CH Δ Baseline CH A RVO η 4 8 6 6 8 6 6 8 5 5 Mean ± SE 49%±6% 141±5 148 ± I 4.7±7 _; 2 65 zb 3 65±5 1.2±3 ₌ 6 70±3 65±3 -3.6±2.9 BARB η 23 42 33 33 42 34 34 42 32 32 Mean i SE 52%±2% 142zb2 154±4 10.5 ± ZD 67±2 75zb2 7.9zb2J 67±1 81±3 14.5± 2.7

Retrospective analyzes of heart failure cases at BEACON. Baseline vital signs are calculated from the mean of three standard cuffed pressure measurements. HF hospitalization vital signs are compiled from admission records included in EAC expert opinion packages and are single assessments using various BP monitoring equipment. LVEF was assessed only during hospitalization with HF. The timing of admission with HF is calculated from the date of onset of the disease to the dates of initiation of treatment and varies from 0 to 4 weeks for each patient.

Table 11 Retrospective analysis of serum electrolytes in patients treated with bardoxolone-methyl compared with patients treated with placebo with heart failure occurring during the first four weeks of treatment

Serum creatinine (mg/dl) Serum sodium (mmol/L) Serum potassium (mmol/l) Baseline CH Δ Baseline CH Δ Baseline CH Δ RVO η 8 8 8 8 8 8 8 8 8 Mean ± SE 3.4± 0.2 3.3± 0.2 -o, 1 ±0.2 140, 0 ± 10 137.0±1.0 -2.5 ±0.6 4.5± 0.2 4.4± 0.1 -o, 1 ±0.2 BAR D η 42 38 38 42 thirty thirty 42 34 34 Mean ± SE 2.8± 0.1 2.7 ± 0.1 -o, 1 ±0.1 140, 0 ± 0, 0 139.0±1.0 -10 ±0.5 4.7± 0, 1 4.8± 0.1 0.1 ± 0.1

Retrospective analyzes of heart failure cases at BEACON. Baseline clinical biochemical parameters were evaluated in the central laboratory. Clinical biochemical parameters obtained during hospitalization for HF,

- 15 042615 are compiled from hospital records included in EAC expert opinion packages and represent evaluations made at various local laboratories.

6. Blood pressure in BEACON.

Mean changes in systolic and diastolic blood pressure from baseline for patients treated with bardoxolone-methyl and placebo, based on the mean of three standardized blood pressure cuff measurements collected at each visit, are shown in FIG. 6A, 6B. Blood pressure was increased in the bardoxolone-methyl group compared to the placebo group, with a mean increase in systolic blood pressure of 1.9 mm Hg by week 4. Art., and diastolic blood pressure - 1.4 mm Hg. Art. in the bardoxolone-methyl group (after the first evaluation after randomization). The increase in systolic blood pressure (SBP) appeared to subside by week 32, while the increase in diastolic blood pressure (DBP) was continuous.

The increase in SBP and DBP at week 4 in patients treated with bardoxolone-methyl, compared with patients treated with placebo, was more pronounced in ABPM measurements (Fig. 7A, 7B). This difference in magnitude may be due to the different methods that were used or to differences in baseline characteristics among patients in the ABPM satellite study. Patients in the ABRM satellite study had a higher baseline ACR than the entire group. Despite this, the data show that bardoxolone-methyl increased blood pressure in the BEACON patient group.

7. Blood pressure changes in previous CKD studies.

In an unblinded dose-optimal study in patients with type 2 diabetes and stage 3b-4 CKD (402-C-0902), no dose-related changes or changes in blood pressure were observed at any individual dose level after 85 days of treatment. in consecutive doses of 2.5 to 30 mg of bardoxolone-methyl (amorphous particulate composition, as used in BEACON). A retrospective analysis of blood pressure data stratified by CKD stage shows that bardoxolone-methyl patients with stage 4 CKD tended to increase blood pressure relative to baseline levels, with the effect most pronounced in the three highest dose groups, while in no visible changes were observed in patients treated with bardoxolone methyl with stage 3b CKD (Table 12). Although sample sizes across dose groups stratified by CKD stage are small, these data suggest that the effect of bardoxolone-methyl treatment on blood pressure may be related to CKD stage.

Blood pressure values from the phase 2b study with bardoxolone-methyl (BEAM, 402-C0804), which used an earlier crystalline formulation and applied a titration design, were highly variable and despite a marked increase in some bardoxolone treatment groups -methyl, there was no clear dependence of blood pressure on the dose.

Table 12

Changes from baseline in systolic and diastolic blood pressure in patients with type 2 diabetes and CKD stage 3b-4, stratified by baseline CKD stage, treated with bardoxolone-methyl

Dose (mg) CKD stage N Assad Adad 2.5 3b/4 14 0.1 ± 4.2 0.2 ± 1.8 3b 10 0±4.4 1+2 4 4 0.3±11 -1.5 + 3.9 5 3b/4 24 -1.5±2.3 -1.4 + 1.5 3b 19 -2.1±2 -1.3±1.4 4 5 0.5 ± 9.1 -1.4 + 5.6 10 3b/4 24 -2.4±3.1 0.3 + 1.3 3b 20 -4.2+3.4 -0.3 + 1.3 4 4 6.1 ± 6.7 3.6 ± 4.5 15 3b/4 48 1.1+2.3 -1 + 1.2 3b 26 -2.2±3.3 -1.3 + 1.5 4 22 5+2.8 -0.6 + 1.9 thirty 3b/4 12 7.2 ± 6.2 3.2±2.2 3b 3 -0.4±13.8 -1.8 + 3.9 4 9 9.7 ± 7.3 4.7 + 2.5

- 16 042615

Patients were administered doses of 2.5, 5, 10, 15, or 30 mg of bardoxolone-methyl once daily for days.

8. Blood pressure and QTcF in healthy volunteers.

In a separate detailed study of QT, which was conducted in healthy volunteers, intensive monitoring of blood pressure was used. In both groups treated with bardoxolone-methyl, of which one received a therapeutic dose of 20 mg, which was also studied in BEACON, and the second received a dose of 80 mg, which exceeded the therapeutic dose, changes in blood pressure did not differ from those observed in patients who received placebo . (FIGS. 8A-8D) after 6 days of once daily dosing. Bardoxolone-methyl did not increase QTcF as assessed by placebo-corrected QTcF changes (ΔΔTTcF) after 6 days of treatment at 20 or 80 mg (FIGS. 9A, 9B).

Bardoxolone-methyl has also been tested in the absence of CKD disease. In early clinical studies of bardoxolone-methyl in cancer patients (RTA 402-C-0501, RTA 402-C-0702), after 21 consecutive days of treatment at doses that varied from 5 to 1300 mg/day (crystal composition), all no mean change in blood pressure was observed in the treatment groups. Similarly, in a placebo randomized trial in patients with hepatic impairment (RTA 402-C-0701), treatment with bardoxolone-methyl at doses of 5 and 25 mg/day (crystalline formulation) for 14 consecutive days resulted in a mean decrease in systolic and diastolic blood pressure (Table 13).

Taken together, these data suggest that bardoxolone-methyl does not prolong the QT interval and does not cause an increase in blood pressure in patients who do not have CVD or stage 4 CKD at baseline.

Table 13

Changes in blood pressure from baseline in patients with hepatic impairment treated with bardoxolone-methyl

Mean ASBP ± SE (mm Hg) Mean ADBP ± SE (mm Hg) Dose N Day 7 day 14 Day 7 day 14 RVO 4 -10+8.5 -1.3 + 5.5 -4.0 + 2.0 0.0 + 3.1 5 mg 6 -12.8 + 5.2 -8.8 + 5.1 -2.0 + 2.3 -1.7 + 3.2 25 mg 6 -11.5 + 5.2 -1.2 + 3.6 -4.0 +2.8 -1.5 + 4.1

9. Brief conclusions and analysis of heart failure.

A comparison of baseline characteristics of patients with cases of heart failure showed that although the risk of heart failure was higher in patients treated with bardoxolone-methyl, patients with heart failure treated with both bardoxolone-methyl and placebo were more likely to have cardiovascular disease and heart failure. had a history of failure and, on average, had higher baseline ACR, BNP, and QTcF. Thus, the development of heart failure in these patients was probably associated with traditional risk factors for heart failure. Additionally, many of the heart failure patients had underlying heart failure prior to randomization, as evidenced by their high baseline BNP levels.

As an indicator of fluid retention after randomization, a retrospective analysis was performed for the subgroup of patients for whom BNP levels were available, and the increase was significantly greater in patients treated with bardoxolone-methyl compared to patients treated with placebo at week 24, with an increase in BNP levels in patients treated with bardoxolone methyl was directly correlated with baseline ACR. Urinary sodium excretion data from patients in the ABPM BEACON satellite study showed a clinically significant decrease in urine volume and sodium excretion at week 4 from baseline only in patients treated with bardoxolone-methyl. In another study, urinary sodium levels and water excretion were reduced in patients with stage 4 CKD, but not in patients with stage 3b CKD. Together, these data suggest that bardoxolone-methyl affects sodium and water transport differently, with their retention being more pronounced in patients with stage 4 CKD.

Consistent with this phenotype for fluid retention, a retrospective review of the detailed description of heart failure cases presented in hospitalization records, together with individual investigator reports, shows that heart failure cases in patients treated with bardoxolone-methyl were often preceded by a rapid increase in fluid mass. and they were not associated with acute renal or cardiac decompensation.

Changes in blood pressure, indicative of the overall degree of volume change, were also greater in the bardoxolone-methyl group compared to the placen group.

- 17 042615 bo, as measured by standardized observation using a BEACON blood pressure cuff. Preset blood pressure analysis in healthy volunteer studies showed no change in either systolic or diastolic blood pressure. While an all-initiating patient (ITT) analysis from bardoxolone-methyl CKD phase 2 studies did not show clear changes in blood pressure, retrospective analyzes of these studies show that increases in both systolic and diastolic blood pressure pressure depends on the stage of CKD. Taken together, these data suggest that the effects of bardoxlon-methyl treatment on blood pressure may be related to disease severity in CKD.

Thus, data on urinary electrolytes, BNP, and blood pressure collectively support that treatment with bardoxolone-methyl may affect the degree of volume change differently, with no clinically detectable effect in healthy volunteers or early-stage CKD patients, although it is likely , contributes to fluid retention in patients with more advanced renal failure and with traditional risk factors associated with heart failure at baseline. The increase in eGFR is likely due to glomerular effects, while the effects on sodium and water regulation are related to the tubules. Since the change in eGFR did not correlate with heart failure, evidence suggests that the effects on eGFR and regulation of sodium and water are anatomically and pharmacologically distinct.

An increased risk of heart failure and related side effects with bardoxolone-methyl was not observed in previous studies (Table 14). However, since 10 times fewer patients were involved in previous studies with bardoxolone-methyl, the increased risk, if present, might not be detectable. Moreover, BEACON limited inclusion to patients with stage 4 CKD, who are known to have a higher risk of cardiovascular complications compared to patients with stage 3b CKD. Thus, the progressive nature of renal disease and the significant risks of cardiovascular events in the BEACON group (manifested, among other markers, by low baseline eGFR, high baseline ACR, and high baseline BNP levels) were probably important factors in the observed CV pattern. complications.

An additional retrospective analysis was performed to further explore the relationship between key endpoints in BEACON and clinically relevant thresholds for conventional risk factors for hypervolemia. Various selection criteria associated with these risk factors were applied to exclude patients at highest risk and examine the results obtained in BEACON. Combinations of selected criteria, including the exclusion of patients with an eGFR of 20 ml/min/ ^1.73 m Table 15). The application of the same criteria to the SPE also significantly improves or eliminates the noted imbalance (Table 16). Taken together, these results suggest the use of these and other markers of renal or cardiovascular risk in future selection criteria for clinical trials with bardoxolone-methyl.

- 18 042615

Table 14

Incidence of treatment-related heart failure1 side effects by major organ system class ⁽ SOC) observed in previous studies of chronic kidney disease using bardoxolone-methyl

Study 0804 (BEAM) 0902 VACT) (Crystalline form} BARD (SDD) SOC Presno shιa emyn term RVO (N = 57) 25 yg (N = 57) 75 mg (N=57) 150 mg (Ν = 56} 2.5 mg (n=14) 5 mg (n=25) L0 mg <N=23) 15 mg (n=50) 30 mg (N=14) PE Metabolism Edema perfErnchig ^ b. and and 3(5) 3(5) IP) 3(5) 0 0 0 0 ICO Hypervolemia 0 3(5) 2(4) 0 - - - - - fWinrwiB disorders Edema IERnfk^rncheskin And (19) 11(19) 10 (18) 11 (20) 0 3(12) 5(18) 3(6) 3(21) General edema 0 2(4) 0 0 - - - - - Respiratory disorders Difficulty breathing 5(9) 2(4) 6(11) 4(7) 0 0 0 0 1G0 Shortness of breath after exercise 0 1(2) 0 3(5) 1(7) 0 0 0 0 Orthopnea 1(2) 0 0 0 - - - - - Pulmonary edema 0 0 1(2) 0 - - - - - Laboratory and instrumental Mind 11 ifth rge fraction 0 1(2) 0 0 - - - - - paroxysm alnal shortness of breath spa Cardiac disorders Congestive heart failure 3(5) 2(4) 2(4) 2(4) 0 0 1(4) 0 1(7) General disorders Edema peripheral 0 0 0 1G) - - - - - Metabolism Hypervolemia 0 1(2) 1(2) 0 - - - - - Respiratory disorders Difficulty breathing 1(2) 0 0 0 - - - - - Pulmonary edema 0 0 1(2) 0 - - - - -

In 402-C-0804 patients were administered 25, 75, 150 mg bardoxolone-methyl (crystal formulation) or placebo once a day for 52 weeks. In RTA402-C-0903, patients were administered doses of 2.5, 5, 10, 15, or 30 mg bardoxolone-methyl (SDD formulation) once daily for 85 days.

¹ Adverse effects with preferred terms consistent with the standard MedDRA requests for heart failure set out in the EAC BEACON statutory document (Filing Serial No. 133 dated Feb. 2, 2012).

Table 15

Effect of Exclusion of Patients with Some Baseline Characteristics on Main Endpoints, Heart Failure, and Overall Mortality in BEACON

Event observable Selection criteria (N) N Baseline BMP <200 No history of HF Baseline ASA <1000 Baseline eGFR >20 Age <75 Baseline ACR < 1040, eGFR > 29, age <75 Baseline ACR < 300, eGFR > 20, BNP < 200 Heart failure EAKD 103 22 67 63 56 75 19 5 RVO 57 16 36 40 37 45 20 3 Death by all causes EAKD 44 14 35 32 27 20 eleven 5 RVO 31 8 24 21 18 23 eleven 4 TSPN EARD 47 12 35 21 18 38 9 1 RVO 55 22 44 27 14 46 6 1 randomized patients BARD 1088 559 922 798 735 786 368 209 RVO 1097 593 943 792 718 829 400 217

Retrospective analysis of results in BEACON. The observed total data for the number of patients with heart failure, total and cardiovascular deaths, and ESRD include all events up to the last contact date (ITT group).

- 19 042615

Table 16

Effect of exclusion of patients with some baseline characteristics on treatment-related serious adverse events by main SOC in BEACON (ITT group)

Primary SOC All patients Baseline ACR < 1000, eGFR > 20, age < 75 Baseline ACR < 300, eGFR > 20, BNP < 200 Treatment RVO (No. 1097) BARD (N=1088) RVO (No. 400) BARD (N= 368) RVO (No. 217) BARD (N= 209) Blood and lymphatic system disorders 11(1) 20 (2) 3 (<1) 4 (<1) 2 (<1) 0 Cardiac disorders activities 84(8) 124 (eleven) 32 (3) 35 (3) 10 (I) 16(1) Ear and labyrinth disorders 1 (<D 3 (<D 1 (<1) 1 (<1) 0 I (<1) Endocrine Disorders 1 (<D 1 (<1) 1 (<1) 1 (<1) 1 (<1) I (<1) Eye disorders 2 (<1) 3 (<1) 1 (<1) 1 (<1) 1 (<1) 0 Violations by gastrointestinal tract 39(4) 46 (4) 13 (I) 10 (I) 8 (1) 7(1) General disorders and condition of the injection site 20(2) 29 (3) 3 (<1) 2 (<1) 2 (<1) 3 (<1) Liver and biliary tract disorders 8(1) 4 (<1) 2 (<1) 1 (<1) 0 I (<1) Immune System Disorders 0 2 (<1) 0 0 0 0 Infections and infections 63(6) 79 (7) 20 (2) 20 (2) 12 (1) 9(1) Injury, poisoning and complications caused by procedures 17(2) 19 (2) 3 (<1) 4 (<1) 0 2 (<1)

- 20 042615

Laboratory and instrumental data 2 (<1) 3 (<1) 1 (<1) 2 (<1) 0 0 Metabolic disorders and nutrition 42(4) 51 (5) eleven (I) 14 (1) 9 (1) 5 (<1) Musculoskeletal and connective tissue disorders 13(1) 21 (2) 6(1) 9(1) 3 (<1) 6(1) Neoplasms benign, malignant and unspecified 10(1) eleven (1) 6(1) 3 (<1) 2 (<1) 1 (<D Nervous System Disorders 35(3) 37 (3) 13 (I) 6(1) 9 (1) 4 (<1) Mental disorders 3 (<1) 3 (<1) 1 (<1) 2 (<1) 1 (<1) 1 (<D kidney disorders and urinary system 71(6) 52 (5) 14 (I) 9(1) 2 (<1) 4 (<1) Reproductive system and breast disorders 3 (<1) 2 (<1) 0 0 0 0 Respiratory, thoracic and mediastinal disorders 32(3) 43 (4) eleven (I) 15 (1) 7 (1) 6(1) Skin and subcutaneous tissue disorders 1 (<D 4 (<1) 1 (<1) 1 (<1) 1 (<1) 1 (<D Surgical and medical procedures 0 2 (<1) 0 1 (<1) 0 1 (<D Vascular disorders 18(2) 20 (2) 5 (<1) 4 (<1) 2 (<1) 2 (<1)

Retrospective analysis of serious treatment-related adverse events in BEACON. Outcome events include only PETs that occur no more than 30 days after the last dose of study drug to the patient.

D. Possible mechanisms of hypervolemia in BEACON.

The data presented in the previous sections suggest that bardoxolone-methyl causes fluid retention in a subgroup of patients who are at highest risk of developing heart failure regardless of drug intake. The data suggest that the effects are not associated with acute or chronic nephro- or cardiotoxicity. Therefore, the full list of well-established renal mechanisms that influence fluid volume change was examined (Table 17) to determine if any of the etiologies corresponded to the clinical phenotype observed with bardoxolone-methyl.

Initial research focused on possible activation of the renin-angiotensin-aldosterone system. Activation of this pathway reduces serum potassium levels due to increased renal excretion. However, bardoxolone-methyl did not affect serum potassium levels and slightly reduced urinary potassium levels in the BEACON satellite study (Table 7).

Another potential mechanism that was investigated was whether changes in transtubular ionic gradients could lead to resorption of sodium, and consequently water, as bardoxolone-methyl affects magnesium and other serum electrolytes. However, this mechanism also involves potassium regulation, and baseline serum magnesium does not appear to be associated with fluid retention or hospitalization for heart failure.

After other causes were excluded for the reasons listed in Table. 16, inhibition of endothelin signaling was the main remaining potential mechanism for volume regulation, consistent with the effect of bardoxolone-methyl treatment in BEACON. Therefore, there has been extensive research into the modulation of the endothelin pathway as a possible explanation for the fluid retention observed in the BEACON study.

- 21 042615

Table 17

Established renal mechanisms affecting fluid volume

Mechanism Na ⁺ retention Hold K ⁺ Affects GFR Comments Bardoxolone-methyl ΐ No ΐ - t retention of Na ⁺ does not depend on K ⁺ in patients with CKD stage 4, t GFR endothelium No -Suppression of endothelin according to the properties of BARD Endothelial nitric oxide (NO) J. No ΐ -NO j. resorption of Na ⁺ and t GFR -BARD t and Na ⁺ , and GFR - BARD has been shown in vitro and in vivo to increase bioavailable endothelial NO, but the effect of Na ⁺ appears to be independent of changes in NO and GFR Antidiuretic hormone (ADH) ΐ ΐ 1 at ΐ level ADG -ADH t Na ⁺ and K ⁺ , and at the same time 1 GFR -BARD does not affect K ⁺ and t GFR Transtubular ion gradients T s ΐ GFR ΐ No direct effect -Ion gradients have a dual effect on Na ⁺ and K ⁺ ; C1' HCO3~ gradient, often generated as HCO3~ uptake, depends on Na ⁺ uptake -BARD does not affect K ⁺ or HCO3- Renin- Angiotensin Aldosterone (RAAS) Τ T - RAAS signaling t excretion of K in the urine and; Serum levels of -BARD do not affect K ⁺ levels and have been shown to; AP levels in CKD patients and suppresses AP signaling in vitro and in vivo Pressor natriuresis J. J. Nebol Shoe t -Volume increase leads to t medullary

- 22 042615

plasma flow and j. hypertonicity; ; absorption of water in the loop of Henle with j. Na ⁺ and K ⁺ - The amount of volume expansion mediated by BARD is unlikely to be sufficient to stimulate this effect; BARD t Na ⁺ and does not affect K ⁺ Prostaglandins (PGE ₂ , PGI ₂ ) J. small j. ΐ -PG t GFR and t urinary Na ⁺ excretion -BARD t Na ⁺ retention, not excretion Natriuretic peptides small j. ΐ -Natriuretic peptides have different effects on Na ⁺ and GFR and little effect on k+ -BNP and other natriuretic peptides t urinary Na ⁺ excretion -BARD t Na ⁺ retention rather than excretion -BARD does not affect natriuretic peptides because GFR is rather in total there will be j. Peritubular factors ΐ with ΐ GFR ΐ with ΐ GFR No -Na ⁺ and K ⁺ change with GFR -BARD does not affect K ⁺

Mechanisms and characteristics of fluid retention.

1. Modulation of the endothelin system.

The most direct analogous clinical data to compare the effects of known endothelin pathway modulators with the BEACON study is for the endothelin receptor antagonist (ERA) avocentan. Avosentan was studied in patients with stage 3-4 CKD with diabetic nephropathy in the ASCEND study, a large outcome evaluation study to assess time to first doubling of serum creatinine, ESRD, or death (Mann et al., 2010). Although baseline eGFR in this study was slightly higher than mean baseline eGFR in BEACON, patients in the ASCEND study had a mean ACR approximately seven times higher than in BEACON (Table 18). Therefore, the overall CV risk profile was likely similar between the two studies.

Like BEACON, the ASCEND trial was terminated prematurely due to an early imbalance in hospital admissions for heart failure and hypervolemia. Importantly, the side effects associated with avosentan-induced hypervolemia, including both serious and non-serious, increased only during the first month of treatment (FIGS. 10A, 10B).

The study of key endpoints in the ASCEND study shows an approximate three-fold increase in the risk of congestive heart failure (CHF) with an average non-significant increase in mortality. Additionally, there was also a small quantitative reduction in the number of cases of ESRD. The BEACON study showed similar results, albeit with a lower incidence of heart failure. However, the two studies showed striking similarities in the clinical presentation and timing of heart failure, as well as effects on other key endpoints (Table 19).

- 23 042615

Table 18

Some demographic and baseline characteristics for patients in ASCEND* and BEACON (ITT group)

Initial characteristic ASCEND BEACON RVO (No. 459) Avosentan 25 mg (#455) Avosentan 50 mg (#478) RVO (No. 1097) BARD 20 mg (#1088) Age 61 ± 9 61 ± 9 61 ± 9 68 ± 9 69 ± 10 History of CHF (% of patients) 13.5% 14.5% 14.4% 15% 14% systolic blood pressure (mmHg) Art.) 135±15 137+14 137+14 140+12 140+12 BMI (kg / m ² ) 30+6 30+6 30+7 34+7 34+7 eGFR (ml/min/1.73 ^m2 ) 33+11 34+11 33+11 22+5 22+4 Median ACR (mg/g) 1540 1416 1474 221 210 ACEi/ARB (% patients) 100% 100% 100% 84% 85% Diuretics (% patients) 65% 64% 65% 64% 64%

* Results of a randomized, double-blind, placebo study of 1392 patients with type 2 diabetes and severe nephropathy receiving avosentan (25 or 50 mg) or placebo in addition to continuous angiotensin-converting enzyme inhibition and/or angiotensin receptor blockade (ASCEND).

Table 19

Deaths, end-stage renal disease, or heart failure in ASCEND and BEACON (ITT group)

ASCEND BEACON Happening RVO (N=459) Avosentan 25 mg (N=455) Avosentan 50 mg (N=478) RVO (N=1097) BARD 20 mg (N=l088) CHF 2.2% 5.9%* 6.1%* 50% 8.8%* Death 2.6% 3.6% 4.6% 2.8% 4.0% TSPN 6.5% 4.4% 50% 4.6% 4.0%

Occurrence of established cases of CHF, death and ESRD in ASCEND and BEACON. In ASCEND, for a case to qualify as CHF, the patient must have had typical signs and/or symptoms of heart failure and be on new therapy for CHF and have been hospitalized for at least 24 hours; ESRD was defined as need for dialysis or kidney transplantation or eGFR <15 ml/min/1.73 m ² . Percentages for BEACON include all cases of CNS and ESRD up to the last contact date and total deaths at the time the database was closed (March 21, 2013). ESRD at BEACON was defined as need for chronic dialysis, kidney transplant, or death from kidney complications; additional details and definitions for heart failure are set out in the EAC BEACON bylaws;

* p<0.05 compared to placebo.

2. Mechanism of hypervolemia caused by an endothelin receptor antagonist.

The role of endothelin in hypervolemia has been extensively investigated. Using knockout models in mice, the researchers demonstrated that acute disruption of the endothelin pathway, accompanied by an increase in salts, contributes to hypervolemia. Specific knockout of endothelin-1 (ET-1), endothelin type A receptor (ETA), endothelin type B receptor (ETB), or a combination of ETA and ETB has been shown to induce hypervolemia in animals with a clinical phenotype consistent with ERA-mediated hypervolemia in sick people. These effects are caused by acute activation of the epithelial sodium channel (ENaC), which is expressed in the collecting ducts of the kidney, where it

- 24 042615 reabsorbs sodium and promotes fluid retention (Vachiery and Davenport, 2009).

3. Relationship between endothelin-1 in plasma and urine in humans.

Plasma and urinary endothelin-1 (ET-1) levels have been previously reported in humans with eGFR ranging from stage 5 CKD to abnormal (8 to 131 ml/min/1.73 m ² ) (Dhaun et al. , 2009). Plasma levels were significantly and inversely correlated with eGFR, but due to the slight slope of the curve, significant differences in ET-1 were not clearly seen across the large range of eGFR assessed. As an identifier for the production of ET-1 by the kidney, the organ where the highest amount of ET-1 is produced, the fractional excretion of ET-1 was calculated by assessing the levels of ET-1 in plasma and urine. From eGFR >100 to approximately 30 ml/min/1.73 m ² urinary levels were relatively unchanged (FIG. 17). However, ET-1 levels appear to increase exponentially with decreasing eGFR in patients with stage 4 and 5 CKD. These data suggest that the regulation of ET-1 in the kidneys is mainly impaired in patients with progressive (stages 4 and 5) CKD. Based on these published data, the inventors hypothesized that the differential effect of bardoxolone-methyl on fluid transport, if due to endothelin modulation, may be due to different endogenous production of ET-1 in the kidney, which is significantly increased in stages 4 and 5. patients with CKD.

4. Vardoxolone-methyl modulates endothelin signaling.

As described above, bardoxolone-methyl reduces ET-1 expression in human cell lines, including mesangial cells found in the kidneys, as well as endothelial cells. In addition, in vitro and in vivo data suggest that bardoxolone-methyl modulates the endothelin pathway to promote a vasodilating phenotype by downregulating the vasoconstrictive ETA receptor and restoring normal levels of the vasodilatory receptor ETB. Thus, efficient activation of Nrf2-related genes by bardoxolone-methyl is associated with suppression of pathological endothelin signaling and facilitates vasodilation by modulating ET receptor expression.

E. Rationale for Termination of BEACON.

1. Established heart failure.

Among the cases of cardiovascular complications identified by the EAC were hospitalizations for heart failure or death from heart failure. The imbalance in established heart failure and its associated complications was the main finding that contributed to the early discontinuation of BEACON. Additionally, heart failure-related AEs, such as edema, contributed to a higher than expected discontinuation rate. It appears that the overall imbalance in time to first established heart failure is the result of a large contribution of cases occurring within the first three to four weeks of treatment. The Kaplan-Meier analysis shows that after this initial period, the incidence between treatment groups appears to follow parallel curves. The nature of the relationship shown in Fig. 11 suggests an acute physiological effect that precipitated hospitalization for heart failure compared to a cumulative toxic effect.

2. Mortality.

At the time of termination of the study, more deaths occurred in the bardoxolone-methyl group than in the placebo group, and the relationship between mortality and heart failure was unclear. The majority of deaths (49 out of 75 deaths) that occurred prior to the closing of the clinical database (March 4, 2013) were confirmed as being of a cardiovascular nature (29 patients treated with bardoxolone-methyl compared with 20 patients treated with placebo) . The majority of deaths from cardiovascular disease were classified as irreversible cardiac arrest, unless otherwise noted, based on the pre-specified definitions set out in the EAC BEACON bylaws. Ultimately, Kaplan-Meier analysis for overall survival showed no apparent separation until approximately 24 weeks (FIG. 12). There were three deaths from heart failure, all in patients treated with bardoxolone-methyl. In addition, as shown in Table. 16, the percentage of deaths in patients over 75 years of age was higher in patients treated with bardoxolone-methyl compared with patients treated with placebo. Notably, if patients over 75 years of age are excluded, the number of deaths in the bardoxolone-methyl group compared to the placebo group is 20 and 23, respectively.

3. Compilation of other safety data from BEACON.

In addition to the effect of bardoxolone-methyl treatment on eGFR and renal PETs, the number of hepatobiliary PETs in the bardoxolone-methyl group was reduced compared to the placebo group (4 vs. 8, respectively; Table 2), and cases of Hay's law was not observed. The number of neoplasm-related PET was also commensurate in both groups. Finally, bardoxolone-methyl treatment was not associated with QTc prolongation, as determined by ECG assessments at 24 weeks (Table 20).

- 25 042615

Table 20

Change from baseline in QTcF at week 24 in patients treated with bardoxolone-methyl compared with patients treated with placebo in BEACON (safety study group)

Observable Change from baseline Moment of time/ QTcF Interval (ms) Placebo N=1093 Bardoxolone -methyl N=1092 Placebo N=1093 Bardoxolo n-methyl Ν=1092 η 719 639 719 637 Average (SD) 428.8 (29.2) 425.8 (26.5) 3, 6 (16.4) -0.9 (19.2) Range (min., Max.) 362, 559 355, 518 -59, 82 -88, 69 Quartiles (25th, median, 75th) 408, 426, 445 407, 425, 443 -7, 3, 13 -13, -1, 10

Data includes only ECG measurements collected at or before the last dose of study drug to the patient. Visits occur relative to the first dose of study drug to the patient.

F. BEACON Conclusions.

In conclusion, an analysis of data from studies conducted using bardoxolone-methyl showed that the drug can differentially regulate fluid retention without a clinically detectable effect in healthy volunteers or patients with early CKD, while at the same time, it probably pharmacologically promotes fluid retention in patients with progressive renal dysfunction. Since the development of heart failure in patients treated with bardoxolone-methyl and placebo has been associated with traditional risk factors for heart failure, this pharmacological effect in patients with underlying cardiac dysfunction may explain the increased risk of heart failure with bardoxolone-methyl treatment in BEACON. These data suggest that reducing the overall risk of developing heart failure in future clinical trials by selecting a group of patients with a lower baseline risk of developing heart failure should prevent an increase in the number of cases of heart failure associated with treatment with bardoxolone-methyl. Importantly, the available data indicate that the hypervolemia in BEACON was not caused by direct nephro- or cardiotoxicity. The clinical phenotype of hypervolemia is similar to that seen in ERA in patients with advanced CKD, and preclinical data demonstrate that bardoxolone-methyl modulates the endothelin pathway. Because acute disruption of the endothelin pathway in patients with advanced CKD is known to activate a specific sodium channel (ENaC), which may contribute to acute sodium and fluid retention (Schneider, 2007), these mechanistic findings, along with the clinical profile of heart failure patients treated with bardoxolone- methyl give a reasonable hypothesis for the mechanism of fluid retention in BEACON. Because impaired renal function may be an important factor that contributes to a patient's inability to compensate for transient hypervolemia, and because a relatively limited number of patients with earlier stages of CKD have been studied to date, excluding patients with CKD (eg, patients with eGFR <60) from treatment with BARD and other AIMs may be reasonable and may be included as part of this invention.

II. Compounds for the treatment or prevention of Alport's syndrome.

In one aspect of the present invention, methods are provided for treating or preventing Alport syndrome in a patient, comprising administering to the patient a therapeutically effective amount of bardoxolone-methyl.

Bardoxolone-methyl and its analogues are known as antioxidant modulators of inflammation. These compounds have demonstrated the ability to activate Nrf2 as measured by increased expression of one or more Nrf2 target genes (eg NQO1 or HO-1; Dinkova-Kostova et al., 2005). In addition, these compounds are capable of indirect and direct inhibition of pro-inflammatory transcription factors, including NF-κB and STAT3 (Ahmad et al., 2006; Ahmad et al., 2008).

Triterpenoids biosynthesized in plants by cyclization of squalene are used medicinally in many Asian countries; and some, such as ursolic and oleanolic acids, are known to be anti-inflammatory and anti-carcinogenic (Huang et al., 1994; Nishino et al., 1988). However, the biological activity of these naturally occurring molecules is relatively weak, and therefore the synthesis of new analogues has been undertaken to increase their activity (Honda et al., 1997;

- 26 042615

Honda et al., 1998). Continuous attempts to improve the anti-inflammatory and anti-proliferative activity of oleanolic and ursolic acid analogs have led to the discovery of 2-cyano-3,12-dioxoolean1,9(11)-diene-28-oic acid (CDDO) and related compounds (Honda et al., 1997, 1998, 1999, 2000a, 2000b, 2002; Suh et al., 1998; 1999; 2003; Place et al., 2003; Liby et al., 2005). Several active oleanolic acid derivatives have been identified, including methyl-2-cyano-3,12-dioxoolean-1,9diene-28-oic acid (CDDO-Me; RTA 402; bardoxolone-methyl). RTA 402, an antioxidant inflammatory modulator (AIM), suppresses the induction of several important inflammatory mediators such as iNOS, COX-2, TNFa and IFNy in activated macrophages, thereby restoring redox homeostasis in inflamed tissues. It has also been reported that RTA 402 activates the Keap1/Nrf2/ARE signaling pathway leading to the production of several anti-inflammatory and antioxidant proteins such as heme oxygenase-1 (HO-1). It induces the cytoprotective transcription factor Nrf2 and suppresses the activity of pro-oxidant and pro-inflammatory transcription factors NF-κB and STAT3. In vivo, RTA 402 has demonstrated significant anti-inflammatory activity as a single agent in several animal models of inflammation such as kidney injury in a cisplatin model and acute kidney injury in an ischemia-reperfusion model. In addition, patients treated with RTA 402 experienced a significant decrease in serum creatinine levels.

Accordingly, for pathologies involving only oxidative stress or oxidative stress exacerbated by inflammation, treatment may include administering to a subject or patient a therapeutically effective amount of a compound of this invention. Treatment may be administered prophylactically prior to a predictable state of oxidative stress (eg, organ transplantation or therapy in a cancer patient), or it may be administered therapeutically under conditions involving established oxidative stress and inflammation.

Some examples of triterpenoids are given in this document.

In table. 21 summarizes the in vitro results for these compounds in which RAW264.7 macrophages were pretreated with DMSO or drugs at various concentrations (nM) for 2 h and then treated with 20 ng/mL IFNy for 24 h.

The NO concentration in the medium was determined using the Griess reagent system; cell viability was determined using the WST-1 reagent. NQO1 CD represents the concentration required to induce a twofold increase in the expression of NQO1, a Nrf2-regulated antioxidant enzyme, in Hepa1c1c7 mouse hepatoma cells (Dinkova-Kostova et al., 2005). All these compounds are several orders of magnitude more active than, for example, the original oleanolic acid molecule.

Table 21

Suppression of IFNy-induced NO production

Work ID RAW264.7 (20 ng/ml IFNy) Hepa1c1c7 cells NO IC ₅₀ WST-1 1С ₅₀ NQO1 CD RTA 401 -10 nM > 200 nM 2.3 nM RTA 402 2.2 nM 8 0 nM 1.0 nM RTA 403 -0.6 nM 100 nM 3.3 nM RTA 404 5.8 nM 100 nM no data RTA 405 6 nM -2 0 0 nM no data TR-225 -0.4 nM 7 5 nM 0.28 nM

- 27 042615

Without wishing to be bound by any theory, the activity of the compound of this invention, RTA 402, is largely determined by the addition of α,β-unsaturated carbonyl groups. In in vitro assays, most of the activity of compounds can be abolished by administration of dithiothreitol (DTT), N-acetylcysteine (NAC), or glutathione (GSH); thiol-containing fragments that interact with α,β-unsaturated carbonyl groups (Wang et al., 2000; Ikeda et al., 2003; 2004; Shishodia et al., 2006). Biochemical analyzes have shown that RTA 402 interacts directly with a key cysteine residue (C179) on IKKe (see below) and inhibits its activity (Shishodia et al., 2006; Ahmad et al., 2006). IKKe controls NF-kB activation through a classical pathway that involves phosphorylation-induced cleavage of 1kB leading to the release of NF-kB dimers into the nucleus. In macrophages, this pathway is responsible for the production of many pro-inflammatory molecules in response to TNFa and other pro-inflammatory stimuli.

RTA 402 also inhibits JAK/STAT signaling at several levels. JAK proteins are recruited by transmembrane receptors (eg, IL-6R) after activation by ligands such as interferons and interleukins. The JAKs then phosphorylate the intracellular portion of the receptor, causing the recruitment of STAT transcription factors. STATs are then phosphorylated by JAKs, form dimers, and travel to the nucleus, where they activate the transcription of several genes involved in inflammation. RTA 402 inhibits constitutive and IL-6 induced STAT3 phosphorylation and dimer formation and binds directly to cysteine residues in STAT3 (C259) and in the kinase domain of JAK1 (C1077). Biochemical analyzes have also demonstrated that triterpenoids interact directly with key cysteine residues on Keap1 (Dinkova-Kostova et al., 2005). Keap1 is an actin-associated protein that retains the transcription factor Nrf2 sequestered in the cytoplasm under normal conditions (Kobayashi and Yamamoto, 2005). Oxidative stress leads to the oxidation of regulatory cysteine residues on Keap1 and causes the release of Nrf2. Nrf2 then moves to the nucleus and binds to antioxidant response elements (ARE), resulting in transcriptional activation of many antioxidant and anti-inflammatory genes. Another target of the Keap1/Nrf2/ARE pathway is heme oxygenase 1 (HO-1). HO-1 breaks down heme into bilirubin and carbon monoxide and has many antioxidant and anti-inflammatory functions (Maines and Gibbs, 2005). Recently, HO-1 has been shown to be strongly induced by triterpenoids (Liby et al., 2005), including RTA 402. RTA 402 and many structural analogs have also been shown to be strong inducers of the expression of other phase 2 proteins (Yates et al., 2007). RTA 402 is a strong inhibitor of NF-kB activation. In addition, RTA 402 activates the Keap1/Nrf2/ARE pathway and induces HO-1 expression.

The compound used can be obtained using the methods described in Honda et al. (2000a); Honda et al. (2000b); Honda et al. (2002) and in US Patent Application Publications 2009/0326063, 2010/0056777, 2010/0048892, 2010/0048911, 2010/0041904, 2003/0232786, 2008/0261985 and 2010/0048887, all of which are incorporated herein by reference. . These methods can be further modified and optimized using the principles and methods of organic chemistry used by a person skilled in the art. Such principles and methods are set forth, for example, in Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2013), which is incorporated herein by reference. In addition, synthetic methods can be further modified and optimized for preparative, pilot or large scale production, either batch or continuous, using the principles and methods of process chemistry employed by a person skilled in the art. Such principles and methods are set forth, for example, in Anderson, Practical Process Research & Development - A Guide for Organic Chemists (2012), which is incorporated herein by reference.

The compound of this invention may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic forms, epimeric forms, and all geometric isomeric forms of a chemical formula are meant unless a particular stereochemistry or isomeric form is specifically indicated.

Regardless of which tautomer is depicted for a given compound, and regardless of which one is the most common, all tautomers of a given chemical formula are meant.

Furthermore, the atoms constituting the compound of this invention are intended to include all isotopic forms of such atoms. As used herein, isotopes include those atoms that have the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include ¹³ C and ¹⁴ C.

Polymorphic forms of the compound of this invention, for example forms A and B of CDDO-Me, can be used in accordance with the methods of this invention. Form B exhibits a bioavailability that is surprisingly better than Form A. In particular, the bioavailability of Form B was greater than that of CDDO-Me Form A in monkeys when the monkeys received equivalent oral dosages of the two forms in gelatin capsules. see US Patent Application Publication 2009/0048204, which is hereby incorporated by reference in its entirety.

Form A of CDDO-Me (RTA 402) is non-solvated (non-aqueous) and may have a characteristic crystal structure with space group P4 ₃ 212 (No. 96), unit cell dimensions a=14.2 A, b=14.2 A, and c= 81.6 A, and a packing structure in which three molecules are packed in spirals down the b crystallographic axis. In some embodiments, Form A may also have an X-ray powder diffraction (XRD) pattern (CuKa) containing characteristic diffraction peaks at 8.8, 12.9, 13.4, 14.2, and 17.4°θ. In some embodiments, the Form A powder X-ray diffraction pattern is substantially the same as shown in FIG. 1A or B.

Unlike form A, form B of CDDO-Me is in a single phase but does not have such a defined crystal structure. Form B samples show that there is no long-range molecular correlation, i.e. more than about 20 A. In addition, thermal analysis of samples of Form B shows a glass transition temperature (Tg) in the range from about 120 to about 130°C. In contrast, a disordered nanocrystalline material does not exhibit T _g , but only a melting point (T _g ), above which the crystal structure becomes liquid. Form B has a different TX spectrum (FIG. 1C) from that of Form A (FIG. 1A or B). Because form B does not have a distinct crystal structure, it also does not have clear XRD peaks, such as those that characterize form A, and instead has a general XRD halo. In particular, the non-crystalline Form B falls into the category of X-ray amorphous solids because its XRD pattern shows three or fewer primary diffraction halos. In this category Form B is a glassy material.

Form A and form B of CDDO-Me are readily prepared from various solutions of the compound. For example, Form B can be made by flashing or slow flashing in MTBE, THF, toluene, or ethyl acetate. Form A can be obtained in several ways, including flash evaporation, slow evaporation, or slow cooling of a solution of CDDO-Me in ethanol or methanol. CDDO-Me preparations in acetone can give either Form A using fast evaporation or Form B using slow evaporation.

In order to distinguish CDDO-Me Forms A and B from each other and from other forms of CDDO-Me, various characterization methods can be used together. Illustrative methods suitable for this purpose are solid state nuclear magnetic resonance (NMR), X-ray powder diffraction (cf. Fig. 1A and B with Fig. 1C), X-ray crystallography, differential scanning calorimetry (DSC), dynamic sorption/desorption vapor analysis (DSP), Karl Fischer (KF) analysis, high temperature microscopy, modulated differential scanning calorimetry, Fourier IR and Raman spectroscopy. In particular, analysis of VRD and DSC data makes it possible to distinguish Form A, Form B, and the hemibenzeate form CDDO-Me. See US Patent Application Publication 2009/0048204, which is hereby incorporated by reference in its entirety.

Additional details regarding CDDO-Me polymorphs are described in US Patent Application Publication 2009/0048204, PCT Publication WO 2009/023232, and PCT Publication WO 2010/093944, all of which are incorporated herein by reference in their entirety.

Non-limiting specific formulations of the compounds disclosed herein include CDDO-Me polymer dispersions. See, for example, PCT Publication WO 2010/093944, which is incorporated herein by reference in its entirety. Some of the formulations reported therein show higher bioavailability than micronized form A or nanocrystalline form A formulations. based on a copolymer of methacrylic acid type C and HPMC-R showed the highest bioavailability in experimental monkeys.

It should be noted that the particular anion or cation that forms part of any salt of this invention is not critical as long as the salt as a whole is pharmacologically acceptable. Examples of pharmaceutically acceptable salts and methods for their preparation and use are provided in the Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

III. Doses and routes of administration.

Administration of a compound of this invention to a patient will be in accordance with general pharmaceutical administration protocols, taking into account drug toxicity, if any. Treatment cycles are expected to be repeated as needed.

The compound of this invention can be administered in various ways, such as orally or by injection (eg, subcutaneous, intravenous, intraperitoneal, etc.). Depending on the

The active compound may be coated with a material to protect the compound from acids and other environmental conditions that may inactivate the compound. It can also be administered by continuous perfusion/infusion to the affected or wound site.

The actual dosage of a compound of this invention administered to a subject or patient may be determined by physical and physiological factors such as age, sex, body weight, severity of condition, type of disease being treated, prior or concurrent therapeutic interventions, subject's idiopathic or patient and route of administration. These factors can be determined by the skilled person. The practitioner responsible for administration will generally determine the concentration of the active ingredient(s) and the appropriate dose(s) for the individual subject or patient. The dosage may be adjusted by the subject's physician in the event of any complication.

In some embodiments, the pharmaceutically effective amount is about 0.1 mg to about 500 mg daily of the compound. In some embodiments, the daily dose is from about 1 to about 300 mg of the compound. In some embodiments, the daily dose is from about 10 to about 200 mg of the compound. In some embodiments, the daily dose is about 25 mg of the compound. In other embodiments, the daily dose is about 75 mg of the compound. In other embodiments, the daily dose is about 150 mg of the compound. In additional embodiments, the daily dose is from about 0.1 to about 30 mg of the compound. In some embodiments, the daily dose is from about 0.5 to about 20 mg of the compound. In some embodiments, the daily dose is from about 1 to about 15 mg of the compound.

In some embodiments, the daily dose is from about 1 to about 10 mg of the compound.

In some embodiments, the daily dose is from about 1 to about 5 mg of the compound.

In some embodiments of the invention, a pharmaceutically effective amount is a daily dose of 0.01-25 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is 0.05-20 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is 0.1-10 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is 0.1-5 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is 0.1-2.5 mg of the compound per 1 kg of body weight.

In some embodiments of the invention, the pharmaceutically effective amount is a daily dose of 0.1-1000 mg of the compound per 1 kg of body weight. In some embodiments, the daily dose is 0.15-20 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is 0.20-10 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is 0.40-3 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is 0.50-9 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is 0.60-8 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is 0.70-7 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is 0.80-6 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is 0.90-5 mg of the compound per 1 kg of body weight.

In some embodiments, the daily dose is from about 1 to about 5 mg of the compound per 1 kg of body weight.

An effective amount will generally range from about 0.001 to about 1000 mg/kg, from about 0.01 to about 750 mg/kg, from about 0.1 to about 500 mg/kg, from about 0.2 to about 250 mg/kg, about 0.3 to about 150 mg/kg, about 0.3 to about 100 mg/kg, about 0.4 to about 75 mg/kg, about 0.5 to about 50 mg/kg kg, about 0.6 to about 30 mg/kg, about 0.7 to about 25 mg/kg, about 0.8 to about 15 mg/kg, about 0.9 to about 10 mg/kg, about 1 to about 5 mg/kg, about 100 to about 500 mg/kg, about 1.0 to about 250 mg/kg, or about 10.0 to about 150 mg/kg in one or more doses daily within one or more days (depending, of course, on the route of administration and the factors discussed above). Other suitable dosage ranges include 1 to 10,000 mg per day, 100 to 10,000 mg per day, 500 to 10,000 mg per day, and 500 to 1,000 mg per day. In some specific embodiments of the invention, the amount is less than 10,000 mg per day in the range, for example, from 750 to 9000 mg per day.

An effective amount may be less than 1 mg/kg/day, less than 500 mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than 50 mg/kg/day, less than 25 mg/kg/day day, less than 10 mg/kg/day or less than 5 mg/kg/day. Alternatively, it may be in the range of 1 mg/kg/day to 200 mg/kg/day. For example, with regard to the treatment of patients with Alport's syndrome, a single dosage may be an amount that reduces the protein concentration in the urine by at least 40% compared to an untreated subject or patient. In another embodiment, the unit dosage is an amount that reduces the urinary protein concentration to a level that is within ±10% of the urinary protein level of a healthy subject or patient.

In other non-limiting examples, the dose may also be from about 1 µg/kg/body weight, about 5 µg/kg/body weight, about 10 µg/kg/body weight, about 50 µg/kg/body weight, about 100 µg/kg /body weight, about 200 µg/kg/body weight, about 350 µg/kg/body weight, about 500 µg/kg/body weight,

- 30 042615 about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500 mg/kg/body weight up to about 1000 mg/kg/body weight or more per administration, and any range within these ranges. In non-limiting examples of the range derived from the numbers listed herein, a range of about 1 to about 5 mg/kg/body weight, a range of about 5 to about 100 mg/kg/body weight, about 5 µg/kg /body weight up to about 500 mg/kg/body weight, etc., based on the numbers described above.

Single or multiple doses of agents are contemplated. Desired time intervals for multiple dose delivery can be determined by one of ordinary skill in the art using no more than routine experimentation. For example, subjects or patients may be administered two doses per day approximately 12 hours apart. In some embodiments, the agent is administered once per day.

The agent(s) can be administered on a regular schedule. As used herein, a regular schedule refers to a predetermined designated time period. A regular schedule may cover time periods that are identical or differ in length, provided that the schedule is predetermined. For example, a typical schedule may include administration twice a day, every day, every two days, every three days, every four days, every five days, every six days, weekly, monthly, or after any set number of days or weeks. Alternatively, a predetermined routine schedule may include twice daily administration for the first week, followed by daily administration for several months, and so on. In other embodiments, the invention provides that the agent(s) can be taken orally and that the time of administration depends or does not depend on food intake. Thus, for example, the agent can be taken every morning and/or every evening, regardless of when the subject or patient has eaten or will eat.

IV. diagnostic tests.

A. Measurement of levels of natriuretic peptide type B (BNP).

Type B natriuretic peptide (BNP) is a 32 amino acid neurohormone that is synthesized in the ventricular myocardium and released into the bloodstream in response to ventricular expansion and excess pressure. The functions of BNP include natriuresis, vasodilation, inhibition of the renin-angiotensin-aldosterone system, and inhibition of sympathetic nerve activity. Plasma BNP concentration is increased in patients with congestive heart failure (CHF, CHF) and increases in proportion to the degree of left ventricular dysfunction and severity of CHF symptoms.

Numerous methods and devices are known to those skilled in the art for measuring BNP levels in patient samples, including serum and plasma. With respect to polypeptides such as BNP, immunoassay devices and methods are often used. See, for example, US Pat. Nos. 6,143,576; 6113855; 6019944; 5985579; 5947124; 5939272; 5922615; 5885527; 5851776; 5824799; 5679526; 5525524; and 5,480,792. These devices and methods can use labeled molecules in a variety of sandwich, competitive, or non-competitive assay formats to generate a signal that is associated with the presence or amount of an analyte of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, can be used to determine the presence or amount of analytes without the need for a labeled molecule. See, for example, US Pat. document in its entirety by reference. In addition, there are a number of commercially available methods (eg, Rawlins et al., 2005, which is incorporated herein in its entirety by reference).

B. Measurement of the albumin/creatinine ratio (ACR).

Proteinuria is usually diagnosed with a simple dipstick test. Traditionally, quantitative protein assays with test strips have been performed by measuring the total amount of protein in a 24-hour urine collection.

Alternatively, the protein concentration in the urine can be compared to the creatinine level in a single urine sample. This is called the protein/creatinine ratio (PCR). The UK Chronic Kidney Disease guidelines (2005; incorporated herein in their entirety by reference) indicate that PCR is a better test than 24-hour urine protein measurement. Proteinuria is defined as a protein/creatinine ratio greater than 45 mg/mmol (equivalent to an albumin/creatinine ratio greater than 30 mg/mmol or approximately 300 mg/g as measured by a 3+ proteinuria test strip), with very high proteinuria levels correspond to a PCR greater than 100 mg/mmol.

The dipstick protein measurements should not be confused with the amount of protein found in the microalbuminuria test, which indicates urinary protein levels in mg/day, and the values obtained in the urine protein dipstick test indicate protein levels. in mg/dl. That is

- 31 042615 there is an initial level of proteinuria, which may be below 30 mg/day, which is not considered a pathology. Values between 30-300 mg/day are called microalbuminuria, which is considered pathological. Urine protein laboratory values for microalbuminuria >30 mg/day correspond to a detection level ranging from trace amounts to 1+ in urine protein dipstick testing. Therefore, a positive indication for any protein found in a urinalysis with a dipstick eliminates the need for a urinalysis for microalbuminuria, since the upper limit for microalbuminuria has already been exceeded.

C. Measurement of estimated glomerular filtration rate (eGFR). A number of formulas have been developed to estimate GFR values based on serum creatinine levels. A commonly used surrogate marker for creatinine clearance (eCCr) is the Cockcroft-Gault formula (CG), which in turn estimates GFR in ml/min. It uses measurement of serum creatinine and patient weight to predict creatinine clearance. The formula, as originally published, is:

(140 - Age) x Weight (in kg) eCCg = ----------------------------- mg 72 x serum creatinine (in —)

This formula assumes that weight is measured in kilograms and creatinine is measured in mg/dl, as is customary in the United States. The resulting value is multiplied by a constant of 0.85 if the patient is a woman. This formula is useful because the calculations are simple and can often be done without the aid of a calculator.

When serum creatinine is measured in µmol/L, then:

(140 - Age) x Mass (in kg) x Constant ^- µmol

Serum creatinine (at—---) where the constant is 1.23 for men and 1.04 for women.

One interesting feature of the Cockcroft and Gault equation is that it shows how dependent the CCr estimate is on age. The age term is (140 - age). This means that a 20 year old person (140-20=120) will have twice the creatinine clearance compared to an 80 year old person (140-80=60) at the same serum creatinine level. The CG equation assumes that a woman will have 15% lower creatinine clearance than a man with the same serum creatinine level.

Alternatively, eGFR values can be calculated using the Renal Disease Diet Modification Formula (MDRD). The 4-variable formula is as follows: eGFR = 175 x Standardized Serum Creatinine” ¹¹⁵⁴ x Age” ^0.203 x C where C is 1.212 if the patient is a black male, 0.899 if the patient is a black woman, and 0.742 if the patient is not black woman. Serum creatinine values are based on IDMS creatinine determination (see below).

Chronic kidney disease is defined as GFR less than 60 ml/min/1.73 m ² for three or more months.

D. Serum creatinine measurement.

The serum creatinine test measures the level of creatinine in the blood and gives an estimate of the glomerular filtration rate. Serum creatinine levels in the BEACON and BEAM studies were based on the determination of creatinine by isotope dilution mass spectrometry (IDMS). Other commonly used methods for creatinine determination include (1) alkaline picrate methods (e.g., Jaffe [classic] and compensated [modified] Jaffe methods), (2) enzymatic methods, (3) high performance liquid chromatography, (4) gas chromatography; and (5) liquid chromatography. It is widely recognized that the IDMS method is the most accurate analysis (Peake and Whiting, 2006, incorporated herein in its entirety by reference).

V. Definitions.

In the context of chemical formulas, the symbol - means a single bond, = means a double bond, and = ' means a triple bond. In addition, it should be noted that the symbol of a covalent bond - when connecting one or two stereogenic atoms does not indicate any preferred stereochemistry. Instead, it covers all stereoisomers as well as mixtures of them. The symbol -^ denotes a single bond when the group attached to the thick end of the wedge is located out of the page. Symbol * ^,| H denotes a single bond when the group attached to the thick end of the wedge is located on the page. The symbol άλλ denotes a single bond when the geometry around the double bond (eg E or Z) is not defined. Therefore, both options are implied, as well as their combinations. Any undefined valence on an atom of the structure shown in this document implicitly represents the hydrogen atom associated with that atom. A bold dot on a carbon atom indicates that the hydrogen attached to this carbon is oriented out of the plane of the paper.

The use of words in the singular together with the term containing in the claims and/or description may mean one, but it is also consistent with the meaning of one or more, at least one and one or more than one.

Throughout this document, the term about is used to indicate that a value includes the variation in error inherent in the device, the method used to determine the value, or the variation that exists among study subjects or patients.

An active ingredient (AI) (also called an active compound, active substance, active agent, pharmaceutical agent, agent, biologically active molecule, or therapeutic compound) is an ingredient in a pharmaceutical or pesticide that is biologically active. The similar terms active pharmaceutical ingredient (API) and bulk active ingredient are also used in medicine, and the term active ingredient can be used for pesticide formulations.

As used herein, average molecular weight refers to weight average molecular weight (Mw) as determined by static light scattering.

The terms contain, have, and include are non-exhaustive linking verbs. Any forms or tenses of one or more of these verbs, such as contains, containing, has, having, includes, and including, are also non-exhaustive. For example, any method that contains, has or includes one or more steps is not limited to having only these one or more steps and also covers other steps not listed.

The term effective, as this term is used in the description and/or in the claims, means adequate to achieve the desired, expected or intended result. An effective amount, a therapeutically effective amount, or a pharmaceutically effective amount, when used in the context of treating a patient or subject with a compound, means that amount of the compound which, when administered to a patient or subject for the treatment or prevention of a disease, is sufficient to effect treatment or prevention of the disease. For example, in the context of Alport's syndrome, one measure of treatment success is a decrease in one or more urinary biomarkers, such as the presence of blood or protein in the urine, or an improvement in glomerular filtration rate. In a specific embodiment of the invention, the indicator of treatment success is a decrease in the concentration of protein in the urine to a level of less than 300 mg/dL. In a preferred embodiment, the therapy is sufficient to reduce the urinary protein concentration to less than 100 mg/dl, or in a more preferred embodiment, less than 30 mg/dl. When the presence of blood is used as a marker of therapeutic efficacy, effective therapy results in the absence of blood in the urine macroscopically, while microscopically blood may still be present. In a preferred embodiment of the invention, effective therapy results in the complete absence of blood, including blood microscopically, that would only be visible with a microscope or urinalysis. Finally, effective therapy would lead to an improvement in glomerular filtration rate. Glomerular filtration rate can be assessed using a variety of creatinine-based methods, including the Cockcroft-Gault formula, the Renal Disease Diet Modification Formula (MDRD), the Chronic Kidney Disease Epidemiology Research Group (CKDEPI) formula, the Mayo quadratic formula, or the Schwartz formula. . In general, the Schwartz formula can be used for children under 12 years of age. These methods are discussed in more detail in the sections above and in the examples below. For example, effective treatment may result in a glomerular filtration rate (or estimated glomerular filtration rate) greater than 60 ml/min/1.73 m ² . More preferably, effective treatment can result in a glomerular filtration rate greater than 90 ml/min/1.73 m ² .

An excipient is a pharmaceutically acceptable substance that is incorporated with the active ingredient(s) into a drug product, pharmaceutical composition, formulation, or drug delivery system. Excipients can be used, for example, to stabilize the composition, to bulk up the composition (which is why they are often referred to as bulking agents, excipients or diluents when used for this purpose), or to provide a therapeutic benefit to the active ingredient in the finished dosage form, such as to facilitate absorption. drug, reducing viscosity or increasing solubility. Excipients include pharmaceutically acceptable variants of release agents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and carriers. The main excipient which serves as the vehicle for the delivery of the active ingredient is commonly referred to as a carrier. Excipients can also be used during the manufacturing process, for example, to facilitate the processing of the active substance, for example by improving powder flow or non-sticking properties, in addition to increasing in vitro stability, such as preventing denaturation or aggregation during the estimated shelf life. The suitability of an excipient generally varies depending on the route of administration, dosage form, active ingredient, as well as other factors.

As used herein, the term IC ₅₀ refers to an inhibitory dose that is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to half inhibit a given biological, biochemical or chemical process (or component of the process, i.e. enzyme, cell, cell receptor or microorganism).

An isomer of the first compound is a single compound in which each molecule contains the same constituent atoms as the first compound, but the configuration of these atoms in three dimensions is different.

As used herein, the term patient or subject refers to a living mammal, such as a human, monkey, bovine, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments of the invention, the patient or subject is a primate. Non-limiting examples of human patients are adults, adolescents, infants, and fetuses.

As commonly used herein, pharmaceutically acceptable refers to those compounds, substances, compositions and/or dosage forms that, based on careful medical evaluation, are suitable for use in contact with tissues, organs and/or body fluids of humans and animals without undue toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable salts means salts of the compounds of this invention which are pharmaceutically acceptable as defined above and which have the desired pharmacological activity. Non-limiting examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylene-bis-(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentane propionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid , succinic acid, tartaric acid, tert-butylacetic acid and trimethylacetic acid.

Pharmaceutically acceptable salts also include base addition salts, which can be formed when acid protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Non-limiting examples of acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, and N-methylglucamine. It should be noted that the particular anion or cation that forms part of any salt of this invention is not critical as long as the salt as a whole is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and methods for their preparation and use are provided in the Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

A pharmaceutically acceptable carrier, drug carrier, or simply carrier, is a pharmaceutically acceptable substance administered along with the active ingredient of a formulation that is involved in the transfer, delivery, and/or transport of a chemical agent. Drug carriers can be used to improve drug delivery and efficacy, including, for example, a controlled release method to alter drug bioavailability, reduce drug metabolism, and/or reduce drug toxicity. Some drug carriers can improve the efficiency of drug delivery to specific target sites. Examples of carriers include liposomes, microspheres (eg, made from poly(lactic-coglycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers.

A pharmaceutical agent (also referred to as a pharmaceutical product, pharmaceutical agent, pharmaceutical product, pharmaceutical composition, pharmaceutical composition, pharmaceutical product, medicinal product, drug, medication, medicament, or simply drug) is a drug used for diagnosis, therapy , treatment or prevention of disease. An active ingredient (AI) (defined above) is an ingredient in a pharmaceutical or pesticide that is biologically active. The similar terms active pharmaceutical ingredient (API) and bulk active ingredient are also used in medicine, and the term active ingredient can be used for pesticide formulations. Some medicines and pesticide products may contain more than one active ingredient. In contrast to active ingredients, inactive ingredients in a pharmaceutical context are usually referred to as excipients (defined above).

Prevention or prevention includes (1) inhibiting the onset of a disease in a subject or patient who may be at risk for and/or predisposed to the disease but does not yet experience or exhibit partial or complete pathology or symptoms of the disease, and/or (2) slowing the onset of pathology or symptoms of a disease in a subject or patient who may be at risk and/or predisposed to the disease but does not yet experience or exhibit partial or complete pathology or symptoms of the disease.

A stereoisomer or optical isomer is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but the three-dimensional configuration of those atoms is different. Enantiomers are stereoisomers of a given compound that are mirror images of each other, like left and right hands.

Diastereomers are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also known as a stereocenter or stereogenic center, which is any point, and optionally an atom, in a molecule bearing groups such that the interchange of any two groups results in a stereoisomer. In organic compounds, the chiral center is usually a carbon, phosphorus, or sulfur atom, although other atoms can also be stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, giving it many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers (eg, tetrahedral carbon), the total number of hypothetically possible stereoisomers will not exceed ²ⁿ , where n is the number of tetrahedral stereocenters. Molecules with symmetry often have fewer stereoisomers than the maximum possible number. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, the mixture of enantiomers may be enantiomerically enriched such that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and/or diastereomers can be isolated or separated using techniques known in the art. It is contemplated that for any stereocenter or axis of chirality for which no stereochemistry has been determined, the stereocenter or axis of chirality may be present in R-form, S-form, or as a mixture of R- and S-forms, including racemic and non-racemic mixtures. As used herein, the phrase substantially free of other stereoisomers means that the composition contains <15%, more preferably <10%, even more preferably <5%, or most preferably <1% of the other stereoisomer(s).

Treating or treating includes (1) inhibiting a disease in a subject or patient experiencing or exhibiting pathology or symptoms of the disease (eg, stopping further development of the pathology and/or symptoms); (2) amelioration of a disease in a subject or patient experiencing or exhibiting pathology or symptoms of the disease (eg, reversal of pathology and/or symptoms); and/or (3) effecting any measurable reduction in the severity of the disease in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease.

The above definitions supersede any conflicting definition in any source, which is incorporated herein by reference. However, the fact that some terms are defined should not be taken as evidence that any undefined term is fuzzy. Rather, all terms used are believed to describe the invention in such a way that a person skilled in the art can appreciate the scope and practice of the invention.

VI. Examples.

The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques developed by the inventor to operate effectively in the practice of the invention, and thus may be considered as constituting preferred modes of practice. However, those skilled in the art will appreciate in light of this disclosure that many changes can be made to the specific embodiments of the invention that are disclosed and still achieve a similar or similar result without departing from the spirit and scope of the invention.

- 35 042615

Example 1 Bardoxolone-methyl improves renal function.

Bardoxolone-methyl has been studied in seven CKD studies involving approximately 2600 patients with type 2 diabetes and CKD. Improvements in renal function, including inulin clearance, creatinine clearance, and eGFR, have been observed with treatment with bardoxolone methyl in a number of clinical studies. A recent study in CKD patients in Japan showed that treatment with bardoxolone methyl resulted in a significant improvement in measured GFR, as measured by inulin clearance, after 16 weeks of treatment compared with placebo. In addition, increases in measured GFR were significantly and positively correlated with improvements in eGFR. Two separate studies (studies 402-C-0804 and 402-C-0903) showed that the increase in eGFR in CKD patients treated with bardoxolone-methyl was maintained for at least one year. In addition, after one year of treatment, patients treated with bardoxolone-methyl showed a residual increase in eGFR from baseline after discontinuation of the drug for four weeks, while patients treated with placebo experienced a decrease in eGFR from baseline. In other studies of CKD, bardoxolone-methyl has been shown to significantly reduce the levels of solutes accompanying uremia (BUN, uric acid and phosphate), inversely with an increase in eGFR, and reduce the incidence of PET and ESRD.

Data from these studies suggest that bardoxolone-methyl has the potential to prevent decline in renal function, which may ultimately prevent or delay ESRD. In patients with Alport's syndrome, who have an average decrease in eGFR of 4.0 ml/min/1.73 m ² per year, the potential impact of a sustained increase in eGFR with bardoxolone methyl treatment is clinically significant and may delay disease progression to ESRD for many years.

The main characteristics of the effect of bardoxolone-methyl on renal function are summarized below.

A. Bardoxolone-methyl improves renal function as measured by measured GFR (inulin clearance), creatinine clearance and eGFR.

In a study in patients with stage 3 CKD in Japan, treatment with bardoxolone-methyl resulted in a significant improvement in measured GFR, as measured by inulin clearance, after 16 weeks of treatment compared with placebo (Table 22).

Table 22 Bardoxolone-methyl increases GFR as measured by inulin clearance

1.1. Change by 1.3. compared to the original. _{about π} . 1.2. Placebo N=23 level at week 16 (ml/min/1.73 m ² ) Bardoxolone methyl (N=17) 1.3.1. Mean ± SD 1.3.2. -0.42 ± 7.52 1.3.3. 5.58 ± 7.90 1.3.4. Average, 1.3.5. -0.69 obtained with the help of MNC 1.3.6. 5.95 1.3.7. 95% CI (NP, VP) 1.3.8. -3.83, 2.45 1.3.9. 2.29, 9, 60 1.3.10. p-value over 1 3 11 placebo ' ' ' 1.3.12. 0.008

In addition, increases in measured GFR were significantly and positively correlated with improvements in eGFR (p=0.002). In two studies, bardoxolone-methyl significantly increased creatinine clearance. Importantly, this increase was not associated with a change in total 24-hour creatinine excretion, indicating that bardoxolone-methyl does not affect creatinine metabolism. In other studies of CKD, bardoxolone-methyl has been shown to significantly reduce the levels of solutes accompanying uremia (BUN, uric acid and phosphate), inversely with an increase in eGFR, and reduce the incidence of PET and ESRD. These data demonstrate that the increase in eGFR observed in the seven CKD studies with bardoxolone-methyl treatment reflects the true increase in GFR and supports the use of eGFR as a reliable marker of renal function (Table 23).

- 36 042615

Table 23

Comparison between studies of increase in eGFR, inulin clearance, creatinine clearance with bardoxolone-methyl treatment

1.4. Study Phase/Country 1.5. Group, patients Placebo-adjusted mean FgFR (ml/min/1.73&/) ^(1> 402-С-0801 (Group 1) 1.5.1. 2a/USA 1.5.2. diabetic nephropathy 6.1 (p<0.001) ^<2> 402-С-0801 (group 2) 1.5.3. 2b/USA 1.5.4. diabetic nephropathy 7.2 (p < 0.001) (2) <h> 402-S-0804 1.5.6. 8.6 (p < 0.001 in (BEAM) CKD/Diabetes compared with RVO) 1.5.7. 402-C0902 1.5.8. 2/US 1.5.9. CKD/Diabetes 6.5 (p < 0.001) (2) 402-С-0903 (BEACON) 1.5.10. 3/International 1.5.11. CKD/Diabetes 6.4 (p < 0.001 vs RVO) ^s 1.5.12. 402-S- 1/US 1.5.13. 9.0 (p < 1102 CKD/Diabetes 0.05) (2) 6, 6 (GFR by 1.5.14. RTA402- 005 (TSUBAKI) 2/Japan 1.5.15. CKD/Diabetes inulin) 1.5.16. (p=0.008 vs. RVO) 1.5.17. 402-C0501 1/US 1.5.18. Cancer 18.2 (p < 0.0001) (2) 1.5.19. 402-C0702 1/2/US 1.5.20. Cancer 32.2 (p=0.001)(2) 1.5.21. 402-S- 1302 (LARIAT) 2/US 1.5.22. Pulmonary hypertension 14.7 (p < 0.001 vs RVO)

1.5.22.1.1.1. (1) Error! Reference source not found. Unless otherwise noted, data are differences between mean changes in eGFR from baseline for bardoxolone-methyl versus placebo groups, and calculated p-values compare mean differences between bardoxolone-methyl and placebo.

1.5.22.1.1.2. (2) Data are mean changes in eGFR from baseline for patients treated with bardoxolone-methyl, and p-values are calculated from two-tailed paired t-tests comparing the change in eGFR with 0.

1.5.22.1.1.3. (3) The study also showed a significant increase in creatinine clearance.

B. Bardoxolone-methyl gives a long-term increase in eGFR after drug withdrawal.

After one year of treatment with bardoxolone-methyl, there is a residual increase in eGFR from baseline after stopping treatment for four weeks, while patients treated with placebo show a decrease in eGFR from baseline. Subtherapeutic drug concentrations are reached within approximately 10 days after drug withdrawal. In studies 402-C-0804 and 402-C-0903, patients treated with bardoxolone-methyl maintained approximately 20-25% increase in eGFR on treatment (or an improvement of 1-4 ml/min/1.73 ^m2) after discontinuation of the drug. relative to baseline). The increase above baseline was statistically significant in both studies. Placebo patients in these studies lost approximately 1 ml/min/1.73 m ² during the year, and in study 402-C-0903 this decrease from baseline was statistically significant.

The sustained increase in eGFR over one year of treatment and the existence of a sustained improvement in eGFR after drug withdrawal suggest that maladaptive structural abnormalities that contribute to decline in kidney function (such as mesangial enlargement and interstitial fibrosis) can be improved over a long course of treatment. treatment with bardoxolone methyl.

Example 2. Clinical study of Alport's syndrome.

CARDINAL is an international, multicentre, phase 2/3 study that began in February 2017 and is enrolling patients aged 12 to 60 years with a genetically or histologically confirmed diagnosis of Alport syndrome. Patients should have a baseline eGFR of 30 to 90 mL/min/1.73 m ² and should be receiving stable blockade of the renin-angiotensin-aldosterone system unless contraindicated. The phase 2 CARDINAL portion was unmasked and included 27 patients. The primary endpoint of part of the Phase 2 study is change in eGFR from baseline at 12 weeks.

The Phase 3 portion of CARDINAL is intended to secure regulatory approval of bardoxolone-methyl for the treatment of Alport syndrome. It will be double blind using

- 37 042615 placebo, and will randomize 150 patients 1:1 to receive once daily oral bardoxolone or placebo. The change in eGFR at 48 weeks will be measured while the patient is on treatment and after stopping the drug for four weeks (maintenance of eGFR). After withdrawal, patients will be re-treated with study drug with their original treatment prescriptions and will continue to receive study drug for an additional 48 weeks. The change in eGFR from baseline in patients treated with bardoxolone methyl compared with patients treated with placebo will be measured again at the end of this second 48-week period (100 weeks in total). Change in eGFR will also be measured after drug withdrawal for four weeks (maintenance of eGFR; 104 weeks total). If the study is successful, first-year eGFR retention data may facilitate accelerated FDA Subpart H or FDA approval, and second-year eGFR retention data may facilitate full FDA approval .

Inclusion criteria include a genetically confirmed diagnosis of Alport syndrome and evidence of impaired renal function as assessed by eGFR. Patients with pre-existing clinically significant heart disease and elevated baseline BNP levels, as well as patients with severely impaired renal function (eGFR <45 ml/min/1.73 m ² and ACR >2000 mg/g), will be excluded from the study. Patients will be carefully monitored for possible hypervolemia.

A. Development of exclusion criteria for a clinical trial.

Study 402-C-0903, Evaluation of Bardoxolone-methyl in Patients with Chronic Kidney Disease and Type 2 Diabetes: Occurrence of Renal Adverse Effects (BEACON), was a phase 3, randomized, double-blind, multinational, multicenter, placebo, parallel group study designed to comparing the efficacy and safety of bardoxolone-methyl (BARD) and placebo (PBO) in patients with stage 4 chronic kidney disease and type 2 diabetes. A total of 2185 patients were randomized 1:1 to receive bardoxolone-methyl (20 mg) or placebo once daily. The primary efficacy endpoint of the study was the time to the first event in the composite endpoint, defined as end-stage renal disease (ESRD; need for chronic dialysis, kidney transplant, or death from kidney failure) or death from cardiovascular (CV) complications. The study had three secondary efficacy endpoints: (1) change in estimated glomerular filtration rate (eGFR); (2) time to first hospitalization for heart failure or death from heart failure; and (3) time to first event of the composite endpoint consisting of non-fatal myocardial infarction, non-fatal stroke, hospitalization for heart failure, or death from cardiovascular complications.

The BEACON study was terminated in October 2012 due to excess mortality and cases of heart failure in the treatment group. Subsequent analysis showed that the increased risk was associated with hypervolemia due to changes in sodium processing by the kidneys and was largely limited to patients with (a) a history of left-sided myocardial disease or (b) elevated levels of B-type natriuretic peptide (BNP) . Elevated albumin-creatinine ratio and progressive (stage 4) CKD have also been found to contribute to the risk of hypervolemia.

Based on this analysis, a study of bardoxolone-methyl in patients with pulmonary arterial hypertension, known as LARIAT, was proposed and approved by the US FDA. The exclusion criteria included risk factors noted in the BEACON analysis. Based on initial safety and efficacy indications in patients with PAH, the LARIAT study was expanded to include other forms of pulmonary hypertension. Based on further indications of safety and efficacy in patients with PAH, the FDA has proposed and approved a phase 3 trial of bardoxolone-methyl in patients with PAH associated with connective tissue disease (CTD-PAH), known as CATALYST.

Patients with Alport syndrome usually do not have other cardiovascular or metabolic comorbidities (eg, diabetes), unlike most patients with diabetes-induced CKD. Thus, patients with Alport syndrome are unlikely to be affected by the two major risk factors identified in the BEACON analysis.

B. Results of the CARDINAL Phase 2 study.

Part of the phase 2 study included 30 patients, and the available data demonstrated that bardoxolone significantly improved kidney function in patients with Alport syndrome, as measured by the estimated glomerular filtration rate (eGFR). All patients completed the treatment period without any interruption.

The mean baseline eGFR (± SD) was 54.7 ± 24 ml/min/1.73 m ² .

For the first eight patients reaching week 12, available data showed a mean improvement of 6.9 ml/min/1.73 m ² at week 4 (n=19; p<0.0005), increasing by 12.7 ml/min /1.73 m ² in 12 non- 38 042615 cases (n=8; p<0.00005). More than 80% of patients showed a clinically significant improvement in eGFR of at least 3.0 ml/min/1.73 m ² by week 8, and the 95% confidence interval at week 12 was 7.9 to 17.5 ml / min / 1.73 m ² . The observed effect of treatment exceeded the threshold

3.0 ml/min/1.73 m ² , which was the minimum effect size required to progress to the phase 3 part of the study.

For a full cohort of 30 patients, mean baseline eGFR (±SD) was 54±24 ml/min/1.73 m ² . Bardoxolone increased eGFR by 13.4 ml/min/1.73 m ² (n=3-; p<1x10 ^-9 ; 95% CI 10.54 to 16.3) after 12 weeks of treatment. All patients improved from baseline and 87% experienced an increase of at least 4 ml/min/1.73 m ² , which is the approximate annual rate of decline in renal function in patients with Alport syndrome. Improvement in eGFR led to reduction in CKD stage in 22/30 (73%) patients.

No serious side effects or discontinuations were reported in the study, and reported side effects were generally mild to moderate in intensity. An independent data monitoring committee reviewed all available safety data and voted to recommend that part of the Phase 3 study be initiated.

All methods disclosed and claimed herein can be performed and implemented without undue experimentation in light of this description. While the compositions and methods of this invention have been described in terms of the preferred embodiments of the invention, it should be apparent to those skilled in the art that changes can be made to the methods and steps or sequence of steps of the method described herein without deviating from concept, essence and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can replace the agents described herein and the same or similar results will be achieved. All such similar substitutions and modifications obvious to those skilled in the art are considered to be within the spirit, scope and concept of the invention as defined in the appended claims.

VII. Links.

The following references, to the extent that they provide illustrative procedural or other details in addition to those set forth herein, are expressly incorporated herein by reference.

US Patent No. 5480792

US Patent No. 5525524

US Patent No. 5631170

US Patent No. 5679526

US Patent No. 5824799

US Patent No. 5851776

US Patent No. 5885527

US Patent No. 5922615

US Patent No. 5939272

US Patent No. 5947124

US Patent No. 5955377

US Patent No. 5985579

US Patent No. 6019944

US Patent No. 6025395

US Patent No. 6113855

US Patent 6143576

US Patent Publication 2003/0232786

US Patent Publication 2008/0261985

US Patent Publication 2009/0048204

US Patent Publication 2009/0326063

US Patent Publication 2010/0041904

US Patent Publication 2010/0048887

US Patent Publication 2010/0048892

US Patent Publication 2010/0048911

US Patent Publication 2010/0056777

US Patent Publication 2011/0201130

PCT Publication WO 2009/023232

PCT Publication WO 2009/048204

PCT Publication WO 2010/093944

Ahmad et al., Triterpenoid CDDO-Me blocks the NF-kB pathway

- 39 042615 by direct inhibition of ΙΚΚβ on Cys-179, J. Biol. Chem. 281:35764-35769, 2006

Ahmad et al., Triterpenoid CDDO-Methyl Ester Inhibits the Janus-Activated Kinase-1 (JAKI)^Signal Transducer and Activator of Transciption-3 (STAT3) Pathway by Direct Inhibition of JAKI and STAT3, Cancer Res., 68(8) :2920-2926, 2008.

Anderson, Practical Process Research & Development - A Guide for Organic Chemists, ^2nd ed., Academic Press, New York, 2012.

Dhaun et al., Urinary endothelin-1 in chronic kidney disease and as a marker of disease activity in lupus nephritis, American Journal of Physiology - Renal Physiology, 296:F1477-F1483, 2009.

Dinkova-Kostova et al., Extremely Potent Triterpenoid Inducers of the Phase 2 Response: Correlations of Protection Against Oxidant and Inflammatory Stress, Proc. Natl. Acad. Sci., 102(12):4584-4589, 2005.

Honda et al., New Enone Derivatives of Oleanolic Acid and Ursolic Acid as Inhibitors of Nitric Oxide Production in Mouse Macrophages, 1997.

Honda et al., Design and Synthesis of 2-cyano-3,12dioxoolean-1,9-dien-28-oic acid, a Novel and Highly Active Inhibitor of Nitric Oxide Production in Mouse Macrophages, Bioorg. Med. Chem. Lett., 8(19):2711-2714, 1998.

Honda et al., Novel Synthetic Oleanane Triterpenoids: A Series of Highly Active Inhibitors of Nitric Oxide Production in Mouse Macrophages, Bioorg. Med. Chem. Lett., 9(24):3429-3434, 1999.

Honda et al., Novel Synthetic Oleanane and Ursane Triterpenoids with Various Enone Functionalities in Ring A as Inhibitors of Nitric Oxide Production in Mouse Macropahges, J. Med. Chem., 43:1866-1877, 2000a.

Honda et al., Synthetic Oleanane and Ursane Triterpenoids with Modified Rings A and C: A Series of Highly Active Inhibitors of Nitric Oxide Production in Mouse Macrophages, J. Med. Chem.,

- 40 042615

43:4233-4246, 2000b.

Honda et al., A novel dicyanotriterpenoid, 2-cyano-3,12dioxooleana-1,9(11)-dien-28-onitrile, active at picomolar concentrations for Inhibition of Nitric Oxide Production, Bloorg. Med. Chem. Lett., 12:1027-1030, 2002.

Handbook of Pharmaceutical Salts: Properties, and Use, Stahl and Wermuth Eds.), Verlag Helvetica Chimica Acta, 2002.

Huang et al., Inhibition of Skin Tumorigenesis by Rosemary and its Constituents Carnosol and Ursolic Acid, Cancer Res., 54:701-708, 1994.

Ikeda et al., The Novel Triterpenoid CDDO and its Derivatives Induce Apoptosis by Disruption of Intracellular Redox Balance, Cancer Res., 63:5551-5558, 2003.

Ikeda et al., Induction of Redox Imbalance and Apoptosis in Multiple Myeloma Cells by the Novel Triterpenoid 2-cyano-3,12dioxoolean-1,9-dien-28-oic acid, Mol. Cancer Ther., 3:39-45, 2004 .

Joint Specialty Committee on Renal Medicine of the Royal College of Physicians and the Renal Association, and the Royal College of General Practitioners. Chronic kidney disease in adults: UK guidelines for identification, management and referral. London: Royal College of Physicians, 2006.

Kobayashi and Yamamoto, Molecular Mechanism Activating the Nrf2-Keap 1 Pathway of Antioxidant Gene Regulation, Antioxid. redox. Signal., 7:385-394, 2005.

Liby et al., The Synthetic Triterpenoids, CDDO and CDDOimidazolide, are Potent Inducers of Heme Oxygenase-1 and Nrf2/ARE signaling, Cancer Res., 65:4789-4798, 2005.

Maines and Gibbs, 30 Some Years of Heme Oxygenase: From a 'molecular wrecking ball' to a 'mesmerizing' Trigger of Cellular Events, Biochem. Biophys. Res. Commun., 338:568-577, 2005.

March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 2007.

-

Claims (27)

Nishino et al., Inhibition of the Tumor-Promoting Action of 12-0 tetradecanoylphorbol-13-acetate by some Oleanane-type Triterpenoid Compounds, Cancer Res., 48:5210-5215, 1988 Peake and Whiting, Measurement of Serum Creatinine-Current Status and Future Goals, Clin. Biochem. Rev. 27:173-184, 2006. Pergola et al. , Bardoxolone methyl and kidney function in CKD with type 2 diabetes, New Engl. J. Med., 365, 327-336, 2011. Place et al., The Novel Synthetic Triterpenoid, CDDOImidazolide, Inhibits Inflammatory Response and Tumor Growth In Vivo,” Clin. Cancer Res. 9:2798-2806, 2003. Rawlins et al., Performance Characteristics of Four Automated Natriuretic Peptide Assays, Am. J. Clin. Pathol., 123:439-445, 2005. Reagan-Shaw et al., Dose Translation From Animal to Human Studies Revisited, FASEB J., 22(3):659-661, 2008 Repka et al., Encyclopedia of Pharmaceutical Technology, New York: Marcel Dekker, 2002. Schneider et al., Contrasting actions of endothelin ETA and ETB receptors in cardiovascular disease, Annual Review of Pharmacology and Toxicology, 47:731-759, 2007. Shishodia et al., A Synthetic Triterpenoid, CDDO-Me, Inhibits ΙκΒα Kinase and Enhances Apoptosis Induced by TNF and Chemotherapeutic Agents Through Down-Regulation of Expression of Nuclear Factor kV-Regulated Gene Products in Human Leukemic Cells, Clin. Cancer Res., 12(6):1828-1838, 2006. Suh et al., Novel Triterpenoids Suppress Inducible Nitric Oxide Synthase (iNOS) and Inducible Cyclooxygenase (COX-2) in Mouse Macrophages, Cancer Res., 58:717-723, 1998. Suh et al., A Novel Synthetic Oleanane Triterpenoid, 2-cyano3, 12-dioxoolean-l,9-dien-28-oic acid, with potent differentiating, antiproliferative, and anti-inflammatory activity, Cancer Res., 59(2) :336-341, 1999. Suh et al., Synthetic Triterpenoids Enhance Transforming Growth Factor β/Smad Signaling, Cancer Res., 63:1371-1376, 2003. Vachiery and Davenport, The endothelin system in pulmonary and renal vasculopathy: les liaisons dangereuses, European Respiratory Review, 18:260-271, 2009. Vasan et al., Congestive heart failure in subjects with normal versus reduced left ventricular ejection fraction: Prevalence and mortality in a population-based cohort, Journal of the American College of Cardiology, 33:1948-1955, 1999. Wang et al., A Synthetic Triterpenoid, 2-cyano-3,12dioxooleana-1,9-dien-28-oic acid (CDDO), is a Ligand for the Peroxisome Proliferator-Activated Receptor γ, Mol. Endocrin., 14(10):1550-1556, 2000. Yates et al., Pharmacodynamic Characterization of Chemopreventive Triterpenoids as Exceptionally Potent Inducers of Nrf2-regulated Genes, Mol. cancer. Ther. 6:154-162, 2007. CLAIM 1. A method of treating or preventing Alport's syndrome in a patient in need thereof, wherein the patient has been identified as having a B-type natriuretic peptide (BNP) level that is not greater than 200 pg/mL, the method comprising administering to the patient a therapeutically effective amount of a compound of formula or a pharmaceutically acceptable salt thereof. 2. A method for improving the kidney function of a patient diagnosed with Alport's syndrome, wherein a patient has been identified as having a B-type natriuretic peptide (BNP) level that is not greater than 200 pg/ml, the method comprising administering to the patient a therapeutically effective amount of a compound of formula or a pharmaceutically acceptable salt thereof. 3. The method of claim 2, which is a method of treating chronic kidney disease in a patient diagnosed with Alport's syndrome. 4. The method according to any one of claims 1 to 3, characterized in that the patient does not have a cardiovascular disease. 5. The method of claim 4, wherein the cardiovascular disease is left-sided myocardial disease, atherosclerosis, restenosis, thrombosis, or pulmonary hypertension. 6. The method according to any one of claims 1 to 5, characterized in that the patient does not have stage 4 chronic kidney disease or higher. 7. The method according to any one of claims 1 to 5, characterized in that the patient does not have an estimated glomerular filtration rate (eGFR) of less than 45 ml/min/1.73 m ² . 8. The method according to any one of claims 1 to 5, characterized in that the patient does not have an elevated albumin/creatinine ratio (ACR) that is greater than 2000 mg/g. 9. The method according to any one of claims 1 to 8, wherein the patient does not have diabetes or complications associated with diabetes. 10. The method according to any one of claims 1 to 9, characterized in that the patient does not have insulin resistance. 11. The method according to any one of claims 1 to 10, characterized in that the patient does not have fatty liver disease. 12. The method according to any one of claims 1 to 10, characterized in that the patient does not have liver failure. 13. The method according to any one of claims 1 to 12, characterized in that the patient is not overweight. 14. The method according to any one of claims 1 to 13, characterized in that the patient does not have cancer. 15. The method according to any one of claims 1 to 14, characterized in that the patient does not have chronic obstructive pulmonary disease (COPD). 16. The method according to any one of claims 1 to 15, characterized in that the patient is not a smoker. 17. The method of any one of claims 1 to 16, wherein the patient has an elevated level of at least one biomarker associated with kidney disease, which is selected from serum creatinine, cystatin C, and uric acid. 18. The method according to any one of claims 1 to 17, characterized in that Alport syndrome is X-linked, autosomal recessive or autosomal dominant. 19. A method according to any one of claims 1 to 17, wherein the patient has a COL4A5 deletion or nonsense mutation, a COL4A5 splicing mutation, a COL4A5 missense mutation, a COL4A3 mutation, or a COL4A4 mutation. 20. The method according to any one of claims 1 to 19, characterized in that the patient additionally has microhematuria. 21. The method according to any one of claims 1 to 20, characterized in that the patient additionally has microalbuminuria. 22. The method according to any one of claims 1 to 21, characterized in that the patient additionally has proteinuria. 23. The method according to any one of claims 1 to 22, characterized in that the age of the patient is less than 75 years. 24. The method according to any one of claims 1 to 23, characterized in that the patient does not have at least three of the following characteristics: (A) cardiovascular disease; (B) baseline B-type natriuretic peptide (BNP) greater than 200 pg/mL; (C) estimated glomerular filtration rate (eGFR) <45 ml/min/1.73 m ² ; (D) elevated albumin/creatinine ratio (ACR) >2000 mg/g. 25. The method according to claim 24, characterized in that the patient does not have all four of the characteristics (A)-(L). 26. The method according to any one of claims 1 to 25, characterized in that the patient is a human. 27. The method according to any one of claims 1 to 26, characterized in that at least part of the compound is present in the form of a crystalline form having an X-ray diffraction pattern (CuKa) containing characteristic diffraction peaks at about 8.8, 12.9, 13, 4, 14.2 and 17.4°2θ. -