HK1223015B - Methods of treating and preventing endothelial dysfunction using bardoxolone methyl or analogs thereof - Google Patents

Description

Methods for treating and preventing endothelial dysfunction using bardoxolone methyl or its analogs

This application claims priority to U.S. Provisional Application No. 61/869,527, filed on August 23, 2013, which is hereby incorporated by reference in its entirety.

Background Art

1. Technical Field

The present invention relates generally to the fields of biology and medicine. More specifically, in some aspects, it relates to methods of using bardoxolone methyl and its analogs to treat and/or prevent endothelial dysfunction in patients diagnosed with or at risk of cardiovascular disease (including patients diagnosed with or at risk of pulmonary hypertension, other forms of pulmonary hypertension, atherosclerosis, restenosis, hyperlipidemia, hypercholesterolemia, metabolic syndrome, or obesity) and other diseases or conditions.

2. Existing Technology

Diseases of the cardiovascular system often involve oxidative stress and inflammation in affected tissues. Oxidative stress occurs in cells when the production of antioxidant proteins (such as glutathione, catalase, and superoxide dismutase) is insufficient to handle intracellular or localized levels of reactive oxygen or reactive nitrogen species (such as superoxide, hydrogen peroxide, and peroxynitrite). Although nitric oxide is an important signaling molecule, its excessive production can also promote oxidative stress. Inflammation is a biological process that provides resistance to infectious or parasitic organisms and repairs damaged tissues. Inflammation is typically characterized by local vasodilation, redness, swelling, and pain, recruitment of white blood cells to the site of infection or injury, production of inflammatory cytokines (such as TNF-α and IL-1), and the production of reactive oxygen or reactive nitrogen species. In the later stages of inflammation, tissue remodeling, angiogenesis, and scarring (fibrosis) may occur as part of the wound healing process. Under normal circumstances, the inflammatory response is regulated and temporary, and once the infection or injury has been appropriately addressed, it is resolved in a carefully orchestrated manner. However, if the regulatory mechanisms fail, acute inflammation can become excessive and life-threatening. Alternatively, inflammation can become chronic and cause cumulative tissue damage or systemic complications. Specialized cells activated by proinflammatory signaling pathways (e.g., macrophages) can be an important source of reactive oxygen and reactive nitrogen species, which produce or maintain oxidative stress in surrounding tissues. Inflammatory cytokines (e.g., TNFα, IL-6, and gamma interferon) also stimulate the production of reactive oxygen/reactive nitrogen species in various cells and thereby promote oxidative stress.

Endothelial dysfunction (the inability of vascular endothelial cells to perform their normal functions) is a common early feature of many cardiovascular diseases and related conditions, including atherosclerosis, hypertension, coronary artery disease, chronic kidney disease, pulmonary hypertension, vascular complications of diabetes, and cardiovascular complications of many chronic diseases. See, for example, Pepine, 1998. Under normal circumstances, the endothelium (a monolayer of cells lining essentially the entire vascular system) regulates the balance between vasoconstriction and vasodilation. It also exerts anticoagulant and antiplatelet properties and provides a physical barrier between the bloodstream and the rest of the body, regulating both cell trafficking and the delivery of fluids to tissues. Known risk factors for cardiovascular disease, including hyperlipidemia, smoking, and diabetes, are associated with endothelial dysfunction. Endothelial damage is believed to be a key early step in the development of atherosclerotic plaques. Endothelial dysfunction can be clinically detected by an increase in the number of circulating endothelial cells (CECs). See, for example, Burger (2012).

A hallmark of endothelial dysfunction is impaired endothelium-dependent vasodilation, which is mediated by nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS), the constitutive form of NOS expressed primarily in endothelial cells (e.g., Davignon, 2004). In healthy vasculature, NO produced by the endothelium diffuses to vascular smooth muscle cells (VSMCs), where it activates guanylate cyclase and stimulates the production of cyclic guanosine monophosphate (cGMP), thereby promoting VSMC relaxation and, therefore, vasodilation. Other endothelial functions (e.g., inhibition of platelet aggregation, inhibition of leukocyte adhesion, and inhibition of VSMC proliferation) are also mediated by NO. In dysfunctional endothelium, NO production is impaired. Oxidative stress is a major underlying factor in the development of endothelial dysfunction. Many risk factors associated with cardiovascular disease (e.g., hypertension, activation of the renin/angiotensin system, hypercholesterolemia, smoking, and diabetes) can activate NADPH oxidase (NOX) in endothelial cells, VSMCs, and other cells of the vessel wall. Activation of NOX increases the local concentration of superoxide. This excess superoxide is a direct source of oxidative stress and can also activate other enzymes that generate reactive oxygen species (e.g., xanthine oxidase; Forstermann, 2006). Excess superoxide can also react with NO to form peroxynitrite, which in turn can oxidize (and deplete) tetrahydrobiopterin (BH4), an essential cofactor for NO production by eNOS. When BH4 is depleted, eNOS becomes "uncoupled" and produces superoxide instead of NO, which increases the overall state of oxidative stress (e.g., Forstermann, 2006).

The clinical significance of endothelial dysfunction is very important. Endothelial dysfunction causes damage to the arterial wall and is considered to be an early marker of atherosclerosis that occurs before detectable atherosclerotic plaques (e.g., Davignon, 2004). As mentioned above, endothelial dysfunction causes VSMC contraction, leading to vasoconstriction and hypertension. More generally, endothelial dysfunction is involved in diseases involving the proliferation of VSMC (including restenosis after vascular surgery and pulmonary arterial hypertension (PAH)).

Atherosclerosis (the underlying defect that leads to many forms of cardiovascular disease) occurs when physical defects or damage to the lining of the arteries (endothelium) trigger endothelial dysfunction and an inflammatory response involving the proliferation of vascular smooth muscle cells and the infiltration of leukocytes into the affected area. Ultimately, a complex lesion called an atherosclerotic plaque can form. This plaque contains a combination of the above-mentioned cells and deposits of lipoproteins with cholesterol and other substances. These plaques can directly interfere with adequate blood circulation and can rupture to produce thrombi (blood clots), which contribute to heart attacks, strokes, or other ischemic events (e.g., Hansson et al., 2006).

Medical treatments for cardiovascular disease include preventive treatments (e.g., the use of drugs intended to lower blood pressure or circulating levels of cholesterol and lipoproteins) and treatments designed to reduce the adhesion tendency of platelets and other blood cells (thereby reducing the rate of plaque progression and the risk of thrombosis). Recently, drugs such as streptokinase and tissue plasminogen activator have been introduced and used to dissolve blood clots and restore blood flow. Surgical treatments include coronary artery bypass grafting to create an alternative blood supply, balloon angioplasty to compress plaque tissue and increase the diameter of the arterial lumen, and carotid endarterectomy to remove plaque tissue in the carotid artery. The treatments, especially balloon angioplasty, can be combined with stents and expandable sieves designed to support the arterial wall in the affected area and keep the blood vessels open. Recently, the use of drug-eluting stents has become common to prevent postoperative restenosis (restenosis of the artery) in the affected area. Restenosis is primarily driven by VSMC proliferation and endothelial dysfunction triggered by injury-driven inflammatory signaling. The device is a filamentous stent coated with a biocompatible polymer matrix containing a drug that inhibits cell proliferation (e.g., paclitaxel or rapamycin). The polymer allows for slow local release of the drug in the affected area while minimizing exposure to non-target tissues. Although the treatment provides significant benefits, the mortality rate of cardiovascular disease remains high and there is still a significant unmet need in the treatment of cardiovascular disease.

Pulmonary hypertension (PH) is a condition in which elevated blood pressure is found in the pulmonary arteries. Pulmonary hypertension (PH) is defined as a mean resting pulmonary hypertension greater than 25 mmHg. If not successfully treated, it can lead to right ventricular hypertrophy and right-sided heart failure. Endothelial dysfunction is often involved in the pathogenesis of PH (e.g., Gologanu et al., 2012; Bolignano et al., 2013; Dumitrascu et al., 2013; Kosmadakis et al., 2013; Guazzi and Galie, 2012). Pulmonary hypertension associated with a variety of conditions can be elevated. The World Health Organization recognizes five categories of PH (Bolignano et al., 2013): (I) idiopathic, familial, and associated pulmonary arterial hypertension or PAH; (II) PH associated with left-sided heart disease; (III) PH associated with lung disease (e.g., COPD) and/or hypoxia (e.g., hypoxia due to sleep apnea); (IV) chronic thromboembolic PH resulting from pulmonary artery vascular occlusion; and (V) PH with unknown or multifactorial etiology (e.g., dialysis-dependent chronic kidney disease).

Pulmonary arterial hypertension (PAH), a particularly severe subtype of pulmonary hypertension (category 1 in the WHO classification of PH), can be idiopathic, familial, secondary to congenital heart disease, secondary to connective tissue disease, secondary to portal hypertension and pulmonary veno-occlusive disease, or related to drug or toxin exposure (e.g., Bolignano et al., 2013). PAH is a disease of the small pulmonary arteries characterized by excessive vasoconstriction, fibrosis, thrombosis, pulmonary vascular remodeling, and right ventricular hypertrophy (RVH). Endothelial dysfunction is believed to play a key role in the pathogenesis of this disease (e.g., Humbert, 2004, which is incorporated herein by reference in its entirety). PAH causes a gradual increase in pulmonary vascular resistance, which ultimately leads to right ventricular failure and death. Although PAH does not metastasize or disrupt tissue boundaries, it shares some common features with cancer, including excessive proliferation and resistance to apoptosis of certain cells (e.g., VSMCs) and glycolysis by the proliferating cells (similar to the well-known Warburg effect in cancer). Activation of transcription factors involved in cancer, such as NF-κB and STAT3, has been reported in PAH (eg, Paulin et al., 2012; Hosokawa, 2013).

There are approximately 15,000-20,000 patients with PAH in the United States. Despite treatment with existing PAH therapies, PAH has a 1-year mortality rate of 15% and a 5-year survival rate of only 22%-38% (Thenappan, 2007). Clearly, there is a need for improved PAH therapies.

Summary of the Invention

In one aspect, the present invention provides methods of using bardoxolone methyl and its analogs to treat and/or prevent endothelial dysfunction in patients diagnosed with or at risk of cardiovascular disease, including patients diagnosed with or at risk of pulmonary hypertension, other forms of pulmonary hypertension, atherosclerosis, restenosis, hyperlipidemia, hypercholesterolemia, metabolic syndrome, or obesity, and other diseases or conditions.

In some embodiments, the present invention provides a method of treating or preventing endothelial dysfunction in a patient in need thereof, comprising administering to the patient a pharmaceutically effective amount of a compound of the formula:

in:

R ₁ is -CN, halo, -CF ₃ or -C(O)R _a , wherein R _a is -OH, alkoxy _(C1-4) , -NH ₂ , alkylamino _(C1-4) or -NH-S(O) ₂ -alkyl _(C1-4) ;

_R2 is hydrogen or methyl;

R ₃ and R ₄ are each independently hydrogen, hydroxy, methyl or any of these groups together with the group R _c are as defined below; and

Y is:

-H, -OH, -SH, -CN, -F, -CF ₃ , -NH ₂ or -NCO;

Alkyl _(C≤8) , alkenyl _(C≤8) , alkynyl _{(C≤8), aryl (C≤12)} , aralkyl _{(C≤12) ,} heteroaryl _{(C≤8), heterocycloalkyl (C≤12)} , alkoxy _{(C≤8) ,} aryloxy _(C≤12) , acyloxy _{(C≤8), alkylamino (C≤8)} , dialkylamino _(C≤8) , alkenylamino _{(C≤8), arylamino (C≤8) ,} aralkylamino (C≤8 ₎ , alkylthio _{(C≤8), acylthio (C≤8) ,} alkylsulfonylamino ( _{C≤8 )} , or a substituted form of any of these groups;

-alkanediyl _(C≤8) -R _b , -alkenediyl _(C≤8) -R _b , or a substituted form of any of these groups, wherein R _b is:

hydrogen, hydroxy, halo, amino or thiol; or

heteroaryl _(C≤8) , alkoxy _(C≤8) , alkenyloxy (C≤8), aryloxy _(C≤8) , aralkyloxy _(C≤8) , heteroaryloxy _(C≤8) , acyloxy _{(C≤8), alkylamino (C≤8) ,} dialkylamino _(C≤8) , alkenylamino _{(C≤8), arylamino (C≤8 ), aralkylamino (C≤8 ), heteroarylamino (} C≤8), alkylsulfonylamino _(C≤8 ), acylamino _{(C≤8) ,} -OC(O)NH-alkyl _{( C≤8)} , -OC(O) _CH2NHC (O)O-tert-butyl, _-OCH2 -alkylthio _(C≤8) , or a substituted form of any of these groups;

-(CH ₂ ) _m C(O)R _c , wherein m is 0-6 and R _c is:

hydrogen, hydroxy, halo, amino, -NHOH, or thiol; or

alkyl _(C≤8) , alkenyl _(C≤8) , alkynyl _{(C≤8), aryl (C≤8)} , aralkyl _(C≤8) , heteroaryl _{(C≤8) ,} heterocycloalkyl (C≤8), alkoxy _(C≤8) , alkenyloxy _(C≤8) , aryloxy ( _C≤8) , aralkyloxy _(C≤8) , heteroaryloxy _(C≤8) , acyloxy (C≤8 _{), alkylamino (C≤8)} , dialkylamino _{(C≤8) ,} arylamino _{(C≤8), alkylsulfonylamino ( C≤8)} , acylamino _(C≤8) , -NH-alkoxy _(C≤8) , -NH-heterocycloalkyl _{(C≤8) ,} -NHC(NOH)-alkyl _(C≤8) , -NH-acylamino _(C≤8) , or a substituted form of any of these groups;

R _c and R ₃ together are -O- or -NR _d -, wherein R _d is hydrogen or alkyl _(C≤4) ; or

R _c and R ₄ together are -O- or -NR _d -, wherein R _d is hydrogen or alkyl _(C≤4) ; or

-NHC(O) _Re , wherein _Re is:

hydrogen, hydroxyl, amino; or

Alkyl _(C≤8) , alkenyl _(C≤8) , alkynyl _{(C≤8), aryl (C≤8)} , aralkyl _{(C≤8) ,} heteroaryl _(C≤8) , heterocycloalkyl (C≤8), alkoxy _(C≤8) , aryloxy _{( C≤8)} , aralkyloxy (C≤8), heteroaryloxy _{(C≤8), acyloxy (C≤8), alkylamino (C≤8) , dialkylamino ( C≤8)} , arylamino _(C≤8) , or a substituted form of any of these groups;

or a pharmaceutically acceptable salt or tautomer thereof,

wherein the patient has been identified as not having at least one of the following characteristics:

(a) History of left-sided myocardial disease;

(b) Elevated B-type natriuretic peptide (BNP) levels;

(c) elevated albumin/creatinine ratio (ACR); and

(d) Chronic kidney disease (CKD).

In some embodiments, the patient has pulmonary hypertension or exhibits one or more symptoms of pulmonary hypertension. In some embodiments, the patient has been identified as not having at least two of the characteristics. In some embodiments, the patient has been identified as not having at least three of the characteristics. In some embodiments, the patient has been identified as not having all four of the characteristics.

In some embodiments, the compound is CDDO-Me. 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) comprising significant diffraction peaks at approximately 8.8, 12.9, 13.4, 14.2, and 17.4 degrees 2θ. In a non-limiting example, the X-ray diffraction pattern (CuKa) is substantially as shown in Figure 1A or Figure 1B. In other variations, at least a portion of the CDDO-Me is present as a polymorph, wherein the polymorph is an amorphous form having an X-ray diffraction pattern (CuKa) having a halo peak at approximately 13.5 degrees 2θ (substantially as shown in Figure 1C) and a _Tg . In some variations, the compound is amorphous. In some variations, the compound is in the form of a glassy solid of CDDO-Me having an X-ray powder diffraction pattern with a halo peak at about 13.5° 2θ (as shown in FIG1C ) and a T _g . In some variations, the T _g value is in the range of about 120° C. to about 135° C. In some variations, the T _g value is in the range of about 125° C. to about 130° C.

In some embodiments, the compound is administered locally. In some embodiments, the compound is administered systemically. In some embodiments, the compound is administered orally, intraadipose, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intracapsularly, intravitreally, liposomes, topically, mucosally, orally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, buccally, transdermally, vaginally, in a cream, in a lipid composition, via a catheter, via lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, via local perfusion, directly flushing the target cells, or any combination thereof. For example, in some variations, the compound is administered intravenously, intraarterially, or orally. For example, in some variations, the compound is administered orally.

In some embodiments, the compound is formulated as a hard capsule, soft capsule, tablet, syrup, suspension, solid dispersion, wafer, or elixir. In some variations, the soft capsule is a gelatin capsule. In some variations, the compound is formulated as a solid dispersion. In some variations, the hard capsule, soft capsule, tablet, or wafer further comprises a protective coating. In some variations, the formulated compound comprises a delayed absorption agent. In some variations, the formulated compound further comprises a solubility or dispersibility enhancer. In some variations, the compound is dispersed in liposomes, an oil-in-water emulsion, or a water-in-oil emulsion.

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

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

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

In some embodiments, the subject is a primate. In some embodiments, the primate is a human. In other variations, the subject is a cow, horse, dog, cat, pig, mouse, rat, or guinea pig.

In some variations of the above methods, the compound is substantially free of its optical isomers. In some variations of the above methods, the compound is in the form of a pharmaceutically acceptable salt. In other variations 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 organic polymer, (C) an organic acid salt, (D) a protein, polypeptide, or peptide, and (E) a high molecular weight polysaccharide. In some variations, the excipient is a synthetic organic polymer. In some variations, the excipient is selected from the group consisting of: hydroxypropyl methylcellulose, poly[1-(2-oxo-1-pyrrolidinyl)ethylene or a copolymer thereof, and methacrylic acid-methyl methacrylate copolymer. In some variations, the excipient is hydroxypropyl methylcellulose phthalate. In some variations, the excipient is PVP/VA. In some variations, the excipient is methacrylic acid-ethyl acrylate copolymer. In some variations, methacrylic acid and ethyl acrylate may be present in a ratio of about 1:1. In some variations, the excipient is copovidone.

Unless otherwise stated, any embodiment discussed herein with respect to one aspect of the invention is also applicable to other aspects of the invention.

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

Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific examples, while indicating specific embodiments of the present invention, are given by way of illustration only, as those skilled in the art will readily appreciate various changes and modifications within the spirit and scope of the present invention from this detailed description. It should be noted that simply 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 THE DRAWINGS

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

Figures 1A-C - X-ray powder diffraction (XRPD) spectra of Forms A and B of RTA 402. Figure 1A shows unmicronized Form A; Figure 1B shows micronized Form A; and Figure 1C shows Form B.

Figure 2 - Effect of bardoxolone methyl on circulating endothelial cells in diabetic CKD patients. Circulating endothelial cells (CECs) and inducible nitric oxide synthase (iNOS) were measured in diabetic CKD patients treated with bardoxolone methyl for 28 days (category 1; dose = 25, 75, or 150 mg/day) or 56 days (category 2; dose = 25 mg/day for 28 days and 75 mg/day on days 29-56). Values represent the mean change + SEM compared to baseline on day 28 (category 1) or day 56 (category 2). (402-C-0801). p < 0.05; *p < 0.01, relative to baseline. Not all baseline and post-treatment samples were available for all patients in any category.

Figures 3A-D - Reactive oxygen species (ROS) and nitric oxide (NO) levels in human endothelial cells after treatment with bardoxolone methyl (RTA 402) and RTA 403 (CDDO-Im). Confluent human umbilical vein endothelial (HUVEC) cells were treated with the indicated concentrations of bardoxolone methyl or RTA 403 for 48 hours. No toxicity was observed at the concentrations tested. ROS levels reflect mitochondrial superoxide levels as assessed by mitoSOX reagents. NO levels were measured using the DAF2-DA assay. AFU = arbitrary fluorescence units. Figure 3A shows ROS levels after treatment with RTA 402. Figure 3B shows ROS levels after treatment with RTA 403. Figure 3C shows NO levels after treatment with RTA 402. Figure 3D shows NO levels after treatment with RTA 403.

Figure 4 - Effects of bardoxolone methyl analogs on _ETA and _ETB receptors in a rat 5/6 nephrectomy model of stress-induced chronic renal failure (CRF). The bardoxolone methyl analog RTA dh404 inhibits ETA receptors and induces _ETB receptors in the kidneys of a rat 5/6 nephrectomy model of stress-induced chronic renal failure ( _CRF ). The bardoxolone methyl analog restored normal _ETA levels and partially restored ETB expression, which promoted vasodilation. Sprague-Dawley rats underwent sham surgery (control) or 5/6 nephrectomy to induce chronic renal failure (CRF). CRF rats were treated with RTA dh404 (2 mg/kg) or vehicle once daily for 12 weeks (N=9/group). **p<0.01, ***p<0.001 vs. control; p<0.05, p<0.01 vs. CRF.

Figure 5A-B - Effect of bardoxolone methyl on ETA expression in normal healthy non-human primates. Bardoxolone methyl downregulated ETA receptor expression in normal monkeys (approximately -65%); after a 14-day recovery period, ETA receptor levels returned to vehicle levels. No differences in ETB receptor expression were observed in the monkey kidneys after bardoxolone administration. BARD animals were orally dosed with 30/mg/kg/day BARD (in sesame oil) for 28 days. A subgroup of animals was treated with BARD for 28 days and then allowed to recover for 14 days without other treatment. **p<0.01 relative to vehicle control. Figure 5A shows _ETA immunohistochemistry. Figure 5B shows _ETA expression densitometry.

Figure 6 - Mean eGFR over time in BEACON (safety population). Mean observed eGFR over time (in treatment weeks) in patients receiving placebo versus bardoxolone methyl. Only eGFR assessments collected at or before the patient's last dose of study drug were included. Visits were performed relative to the patient's first dose of study drug. Data are mean ± SE.

Figure 7A-B - Percentage of eGFR decliners in patients receiving bardoxolone methyl versus placebo in BEACON (safety population). Percentage of patients whose eGFR changed from baseline by <-3, <-5, or <-7.5 mL/min/1.73 m ² (based on treatment weeks) in patients receiving placebo (Figure 7A) versus bardoxolone methyl (Figure 7B). Only the evaluation of eGFR collected at or before the patient's last dose of study drug was included. Visits were performed relative to the patient's first dose of study drug. Percentages were calculated relative to the number of patients with available eGFR data at each visit.

Fig. 8 - is in BEACON to the time of composite primary outcome event (ITT colony).Result is from the randomized, double-blind, placebo-controlled 3-phase study (BEACON, RTA402-C-0903) in the T2D patient suffering from the 4th stage CKD.Give patient placebo or 20mg methyl bardoxolone once a day.Analysis is only included in the ESRD or cardiovascular (CV) death event that occurs when or before the study drug termination date (October 18, 2012), and described event system is clearly judged by independent event adjudication committee (Event Adjudication Committee), as outlined in BEACON EAC charter.

Figure 9 - Time to first hospitalization for heart failure or death due to heart failure event in BEACON (ITT population). The analysis included only heart failure events that occurred on or before the study drug termination date (October 18, 2012) and were explicitly adjudicated by the Independent Event Adjudication Committee, as outlined in the BEACON EAC charter. The top line is BARD; the bottom line is placebo.

Figure 10 - Overall survival of patients receiving bardoxolone methyl versus placebo in BEACON. Results are from a randomized, double-blind, placebo-controlled Phase 3 study (BEACON, RTA402-C-0903) in patients with T2D and stage 4 CKD. Patients were given either placebo or 20 mg bardoxolone methyl once daily. The analysis included all deaths that occurred before the database lock (March 4, 2013). The top line is BARD; the bottom line is placebo.

Figure 11 - Mean serum magnesium levels in patients receiving bardoxolone methyl versus placebo in BEACON. Mean observed serum magnesium levels over time (in treatment weeks) in patients receiving placebo versus bardoxolone methyl. Only serum magnesium assessments before or after the patient's last dose of study drug are included. Visits were performed relative to the patient's first dose of study drug. Data are mean ± SE. Top line: placebo; bottom line: bardoxolone methyl.

Figures 12A-B - Changes in systolic blood pressure (Figure 12A) and diastolic blood pressure (Figure 12B) over time from baseline in patients receiving bardoxolone methyl versus placebo in BEACON (safety population). Data include only vital evaluations at or before the patient's last dose of study drug. Visits were performed relative to the patient's first dose of study drug.

FIG13A-B - 24-Hour Ambulatory Blood Pressure Monitoring (ABPM) Substudy: Change in Systolic Blood Pressure ( FIG13A ) and Diastolic Blood Pressure ( FIG13B ) from Baseline to Week 4 in Patients on Bardoxolone Methyl vs. Placebo. Data include only patients with baseline and Week 4 24-hour ABPM values. Change in systolic blood pressure was calculated using the mean of all valid measurements obtained from the patient's ambulatory blood pressure monitoring device during the entire 24-hour period, either daytime (6 AM to 10 PM) or nighttime (10 PM to 6 AM the following day).

Figures 14A-D - Placebo-corrected change in systolic blood pressure from baseline on study days 1 and 6 in healthy volunteers given bardoxolone methyl. Results are from a multiple-dose, randomized, double-blind, placebo-controlled, complete QT study in healthy volunteers (RTA402-C-1006). Patients were treated once daily with placebo, 20 mg or 80 mg bardoxolone methyl, or 400 mg moxifloxacin (active comparator) for 6 consecutive days. Data are mean changes (±SD) from baseline 0-24 hours post-dose on study days 1 and 6. Figure 14A shows the administration of 20 mg BARD on study day 1. Figure 14B shows the administration of 20 mg BARD on study day 6. Figure 14C shows the administration of 80 mg BARD on study day 1. Figure 14D shows the administration of 80 mg BARD on study day 6.

Figure 15A-B - Placebo-corrected change in QTcF from baseline in healthy volunteers given bardoxolone methyl. Results are from a multiple-dose, randomized, double-blind, placebo-controlled complete QT study in healthy volunteers (RTA402-C-1006). Changes in QTcF intervals in subjects given bardoxolone methyl (20 mg or 80 mg - Figures 15A and 15B, respectively) are shown relative to patients receiving placebo for 6 consecutive days. Data are mean ± 90% CI evaluated 0-24 hours after dosing on study day 6, with the upper limit of the 90% CI equivalent to a one-sided 95% confidence limit. The 10 ms threshold reference line is associated with the upper confidence limit.

Figure 16A-B - Kaplan-Meier plots of fluid overload events in ASCEND (Figure 16A) and heart failure events in BEACON (ITT population; Figure 16B). Analysis of the time to first event for fluid overload events in ASCEND and heart failure in BEACON. Fluid overload events in ASCEND were obtained from adverse event reports of local researchers. The signs and symptoms of fluid overload indicated on the adverse event form included: heart failure, edema, fluid overload, fluid retention, hypervolemia, dyspnea, pleural and pericardial effusion, ascites, weight gain, pulmonary rales, and pulmonary edema. The analysis included only heart failure events that occurred on or before the study drug termination date (October 18, 2012), which were clearly determined by an independent event adjudication committee as outlined in the BEACON EAC charter.

Figures 17A-B - Relationship between plasma and urinary endothelin and eGFR. Scatter plots of eGFR versus plasma ET-1 (Figure 17A) and urinary fractional excretion of ET-1 (Figure 17B). Blood and urine samples were collected from individuals with CKD (N=115) and without CKD (N=27) and evaluated for ET-1. Estimated GFR was calculated using the Cockcroft and Gault equation.

FIG18 - Effects of RTA dh404 on lung histology in a rat model of monocrotaline-induced pulmonary hypertension. Mean lung histology scores were assessed by board-certified veterinary pathologists on a 0 to 5 increasing severity scale. Histology scores were analyzed using Sigmaplot v12.5 (Systat, San Jose, CA) using a one-way ANOVA followed by Dunn's post hoc test, with significance set at p < 0.05.

Figure 19 - Effect of RTA dh404 on mRNA expression of Nrf2 target genes in rat lung. Data are normalized to the housekeeping gene Rpl19 and presented as mean ± S.E.M. relative to vehicle control. *p<0.05, **p<0.01, and ***p<0.001 relative to vehicle control.

Figure 20 - Effect of RTA dh404 on mRNA expression of NF-κB target genes in rat lung. Data are normalized to the mean of the housekeeping genes Ppib and Hprt and presented as mean ± S.E.M. relative to vehicle control. *p < 0.05 and **p < 0.01 relative to vehicle control. All values below the asterisk are significant.

DETAILED DESCRIPTION

In one aspect, the present invention provides novel methods for using bardoxolone methyl and its analogs for treating and/or preventing endothelial dysfunction and/or pulmonary hypertension in patients diagnosed with or at risk of cardiovascular disease (including patients diagnosed with or at risk of pulmonary hypertension, other forms of pulmonary hypertension, atherosclerosis, restenosis, hyperlipidemia, hypercholesterolemia, metabolic syndrome, or obesity), and other diseases or conditions. 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 improves 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 trial (BEACON) of bardoxolone methyl in patients with stage 4 CKD and type 2 diabetes. The primary endpoint in the BEACON trial was a composite of progression to end-stage renal disease (ESRD) and all-cause mortality. This trial was terminated due to excessive severe adverse events and deaths in the patient group treated with bardoxolone methyl.

As discussed below, subsequent analyses of data from the BEACON trial showed that most serious adverse events and deaths involved heart failure and were highly associated with the presence of one or more risk factors, including: (a) elevated baseline levels of B-type natriuretic peptide (BNP; e.g., >200 pg/mL); (b) baseline eGFR <20; (c) history of left-sided heart disease; (d) high baseline albumin-to-creatinine ratio (ACR; e.g., >300 mg/g, as defined by 3+ dipstick proteinuria); and (e) elderly age (e.g., >75 years). The analysis indicated that heart failure events were likely associated with the onset of acute fluid overload during the first three to four weeks of BARD treatment and that this was potentially due to inhibition of endothelin-1 signaling in the kidney. A previous trial of an endothelin receptor antagonist in stage 4 CKD patients was terminated due to a pattern of adverse events and mortality very similar to that found in the BEACON trial. Subsequent nonclinical studies have confirmed that BARD, at physiologically relevant concentrations, inhibits endothelin-1 expression in proximal renal tubular epithelial cells and endothelin receptor expression in human mesangial and endothelial cells. Therefore, patients at risk for adverse events from inhibition of endothelin signaling should be excluded from future clinical use of BARD.

The present invention relates to novel methods for treating conditions involving endothelial dysfunction as a significant contributing factor. The present invention also relates to the preparation of pharmaceutical compositions for treating these conditions. In the present invention, patients for treatment are selected based on several criteria: (1) diagnosis of a condition involving endothelial dysfunction as a significant contributing factor; (2) absence of elevated levels of B-type natriuretic peptide (BNP; e.g., BNP titer must be <200 pg/mL); (3) absence of chronic kidney disease (e.g., eGFR>60) or no advanced chronic kidney disease (e.g., eGFR>45); (4) no history of left-sided cardiomyopathy; and (5) absence of a high ACR (e.g., ACR must be <300 mg/g). In some embodiments of the present invention, patients diagnosed with type 2 diabetes are excluded. In some embodiments of the present invention, patients diagnosed with cancer are excluded. In some embodiments, elderly patients (e.g., >75 years old) are excluded. In some embodiments, patients are closely monitored for rapid weight gain indicative of fluid overload. For example, a patient may be instructed to weigh themselves daily during the first four weeks of treatment and to contact the prescribing physician if an increase of greater than 5 pounds is observed.

Pulmonary hypertension associated with non-dialysis-dependent CKD is WHO Class II, and pulmonary hypertension associated with dialysis-dependent CKD is WHO Class V (Bolignano et al., 2013). Only a small proportion of patients with stage 4-5 CKD present with WHO Class I pulmonary hypertension (i.e., pulmonary arterial hypertension), and it should be noted that these patients will be excluded based on the above criteria.

A. BEACON Study

1. Study Design

Study 402-C-0903, titled "Bardoxolone Methyl Evaluation in Patients with Chronic Kidney Disease and Type 2 Diabetes: The Occurrence of Renal Events" (BEACON), was a Phase 3, randomized, double-blind, placebo-controlled, parallel-group, multinational, multicenter study designed to compare the efficacy and safety of bardoxolone methyl (BARD) with placebo (PBO) in patients with stage 4 chronic kidney disease and type 2 diabetes. A total of 2,185 patients were randomized 1:1 to receive either bardoxolone methyl (20 mg) or placebo once daily. The primary efficacy endpoint of the study was the time to the first event of a composite endpoint defined as end-stage renal disease (ESRD; requirement for long-term dialysis, kidney transplantation, or renal death) or cardiovascular (CV) death. 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 due to heart failure; and (3) time to the first event of a composite endpoint consisting of nonfatal myocardial infarction, nonfatal stroke, hospitalization for heart failure, or cardiovascular death.

A subgroup of BEACON patients consented to an additional 24-hour assessment, including ambulatory blood pressure monitoring (ABPM) and 24-hour urine collection. An independent Event Adjudication Committee (EAC), blinded to study treatment assignment, assessed whether renal, cardiovascular, and neurological events met the pre-specified definitions of the primary and secondary endpoints. An IDMC, comprised of external clinical experts and supported by an independent statistical panel, reviewed the unblinded safety data from the entire study and made appropriate recommendations.

2. Demographics and baseline characteristics of the population

Table 1 presents summary statistics of selected demographic and baseline characteristics of patients enrolled in BEACON. Demographic characteristics were comparable between the two treatment groups. Across all treatment groups in the combination, the mean age was 68.5 years and 57% of patients were male. The bardoxolone methyl arm had slightly more patients in the age subgroup ≥75 years than the placebo arm (27% in the bardoxolone methyl arm vs. 24% in the placebo arm). The mean weight and BMI for the two treatment groups were 95.2 kg and 33.8 kg/m ² , respectively. Baseline renal function was generally similar in the two treatment groups; the mean baseline eGFR (as measured by the 4-variable Modification of Diet in Renal Disease (MDRD) equation) was 22.5 mL/min/1.73 m ² , and the geometric mean albumin/creatinine ratio (ACR) for the combined treatment group was 215.5 mg/g.

Table 1. Selected Demographics and Baseline Characteristics of Patients in Bardoxolone Methyl (BARD) vs. Placebo (PBO) in BEACON (ITT Population)

Patients were given either placebo or 20 mg of bardoxolone methyl once daily.

B.BEACON Results

1. Effect of bardoxolone methyl on eGFR

The mean eGFR values of patients treated with bardoxolone methyl and placebo are shown in Figure 6. On average, bardoxolone methyl patients have an expected eGFR increase, which occurs in the 4th week of treatment and remains higher than baseline until the 48th week. In contrast, placebo-treated patients showed no change or a slight decrease from baseline on average. The proportion of patients whose eGFR decreased was significantly reduced (Fig. 7) in bardoxolone methyl patients relative to placebo-treated patients. The observed eGFR trajectory and the ratio of the decliners after one year of treatment in BEACON were consistent with the modeled expected values and the results from the BEAM study (RTA402-C-0804). As shown in Table 2, the number of patients experiencing severe adverse events (SAEs) of kidney and urinary disorders in the bardoxolone methyl group was lower than that in the placebo group (52 to 71, respectively). In addition, and as discussed in the following sections, a slightly less ESRD event than the placebo group was observed in the bardoxolone methyl group. Overall, these data suggest that bardoxolone methyl treatment does not worsen renal status acutely or over time.

Table 2. Incidence of Treatment-Emergent Serious Adverse Events in Each Major System Organ Class in BEACON (Safety Population)

The table includes only serious adverse events with an onset greater than 30 days after the patient's last dose of study drug. The column header count and denominator are the number of patients in the safety population. Each patient is counted at most once per system organ class and preferred term.

2. Main composite results in BEACON

Table 3 provides a summary of the adjudicated primary endpoints that occurred on or before the day of study termination (October 18, 2012). Although the number of ESRD events was slightly lower in the bardoxolone methyl group compared to the placebo group, the number of composite primary endpoints was equal in the two treatment groups (HR = 0.98) due to a slight increase in cardiovascular death events, as shown in the time analysis to the first composite primary endpoint (Figure 8).

Table 3. Adjudicated Primary Endpoints in Patients Receiving Bardoxolone Methyl (BARD) vs. Placebo (PBO) in BEACON (ITT Population)

^aHazard ratios (bardoxolone methyl/placebo) and 95% confidence intervals (CIs) were estimated using a Cox proportional hazards model with treatment group, serial baseline eGFR, and serial baseline log ACR as covariates. Breslow's method of endpoints in the time-to-event treatment was used.

^b Treatment group comparisons were performed using the SAS type 3 chi-square test and two-sided p-values associated with treatment group variables in the Cox proportional hazards model.

C. Effects of bardoxolone methyl on heart failure and blood pressure

1. Diagnosed heart failure in BEACON

The data in Table 4 present post hoc analyses of demographics and selected laboratory parameters for BEACON patients categorized by treatment group and occurrence of adjudicated heart failure events. The number of patients with heart failure includes all events up to the date of last contact (ITT population).

To the comparison disclosure of the baseline characteristics of the patient with the heart failure event through judgment, the patient with heart failure of bardoxolone methyl treatment and placebo treatment is more likely to have the previous medical history of cardiovascular disease and heart failure and has the higher baseline value of the QTc interval (QTcF) corrected by B-type natriuretic peptide (BNP) and Fredericia. Although the risk of heart failure in bardoxolone methyl treatment patient is higher, these data show that the occurrence of heart failure in two groups seems to be relevant to the traditional risk factors of heart failure. The baseline ACR in the patient with heart failure event of bardoxolone methyl treatment is significantly higher than that without heart failure event. It should also be noted that the BNP mean baseline content of the patient experiencing heart failure in two treatment groups significantly increases, indicating that these patients may retain body fluid before randomization and are in subclinical heart failure.

Table 4. Selected Demographics and Baseline Characteristics of Bardoxolone Methyl Patients vs. Placebo Patients by Heart Failure Status

a BARD patients with HF vs. BARD patients without HF, p<0.05

b PBO patients with HF vs. PBO patients without HF, p < 0.05

cBARD patients with HF vs. PBO patients with HF, p<0.05

2. Evaluation of clinical parameters associated with increased BNP

As a surrogate for fluid retention, a post hoc analysis was performed on a subset of patients for whom BNP data were available at baseline and Week 24. Patients in the bardoxolone methyl arm experienced significantly higher increases in BNP than those in the placebo arm (mean ± SD: 225 ± 598 vs. 34 ± 209 pg/mL, p < 0.01). It was also noted that a higher proportion of patients treated with bardoxolone methyl experienced increases in BNP at Week 24 compared to patients treated with placebo (Table 5).

The increase in BNP at Week 24 did not appear to be associated with baseline BNP, baseline eGFR, change in eGFR, or change in ACR. However, baseline ACR was significantly correlated with the change from baseline in BNP at Week 24 only in patients treated with bardoxolone methyl, suggesting that the tendency to retain fluid may be related to the baseline severity of renal dysfunction (as defined by proteinuria status) and may not be related to overall changes in renal function (as assessed by eGFR) (Table 6).

Furthermore, these data suggest that increases in eGFR of glomerular origin are anatomically unique, as sodium and water regulation occurs in the renal tubules.

Table 5. Analysis of BNP and eGFR Values in Patients Receiving Bardoxolone Methyl vs. Placebo Categorized by Change from Baseline in BNP at Week 24

Post hoc analysis of changes in BNP at week 24 in BEACON.

Table 6. Relationship between the change from baseline in BNP at week 24 and baseline ACR in patients receiving bardoxolone methyl versus placebo in BEACON

treat N Correlation coefficient P-value PBO 216 0.05 0.5 BARD 211 0.20 <0.01

Post hoc analysis of BNP change in BEACON at Week 24. Only patients with baseline and Week 24 BNP values were included in the analysis.

3. Serum electrolytes

No clinically meaningful changes in serum potassium or serum sodium were noted in the subset of patients who underwent 24-hour urine collections (Table 7). Changes in serum magnesium levels in bardoxolone methyl-treated patients were consistent with those observed in previous studies (Figure 11).

Table 7. Changes from Baseline at Week 4 in Serum Electrolytes in Patients in the 24-Hour ABPM Substudy of Bardoxolone Methyl vs. Placebo

Data include only BEACON patients enrolled in the 24-hour ABPM substudy. Changes in serum electrolyte values were calculated only for patients with baseline and Week 4 data. *p < 0.05 for Week 4 changes relative to baseline within each treatment group; p < 0.05 for Week 4 changes in patients with BARD vs. PBO.

4. 24-hour urine collection

A subgroup of patients consented to additional 24-hour assessments with ambulatory blood pressure monitoring (ABPM) and 24-hour urine collection at selected visits (substudy). Data on urinary sodium excretion in patients in the BEACON substudy revealed clinically meaningful reductions in urine volume and sodium excretion relative to baseline at Week 4 in patients treated with bardoxolone methyl (Table 8). These reductions were significantly different from the Week 4 changes in urine volume and urinary sodium observed in patients treated with placebo. It should also be noted that the reduction in serum magnesium was not associated with renal magnesium loss.

Additionally, in a pharmacokinetic study in patients with type 2 diabetes and stage 3b/4 CKD who were administered bardoxolone methyl for 8 weeks (402-C-1102), patients with stage 4 CKD had significantly greater reductions in urinary sodium and water excretion compared to patients with stage 3b CKD (Table 9).

Table 9. Change from baseline at week 8 in 24-hour urine volume and 24-hour urine sodium in patients treated with bardoxolone methyl, grouped by CKD severity (from patient pharmacokinetic study)

Patients were treated with 20 mg of bardoxolone methyl once daily for 56 consecutive days; a post-treatment follow-up visit was conducted on study day 84. Data are mean values. Data include patients with baseline and week 8 data.

5. Hospital records from the EAC determination package

The first scheduled post-baseline evaluation in BEACON was conducted at Week 4. Because many heart failure events occurred before Week 4, the clinical database provided limited information to characterize these patients. A post-hoc review of the EAC case pack for heart failure cases occurring before Week 4 was performed to evaluate clinical, vital signs, laboratory, and imaging data collected at the time of the first heart failure event (Tables 10 and 11).

Examination of these records revealed rapid weight gain, dyspnea and orthopnea, peripheral edema, central/pulmonary edema on imaging, elevated blood pressure and heart rate, and general reports of preserved ejection fraction immediately after randomization. The data suggest that heart failure is caused by rapid fluid retention that occurs simultaneously with preserved ejection fraction and elevated blood pressure. Preserving ejection fraction is consistent with the clinical features of heart failure caused by diastolic dysfunction, which is due to ventricular stiffness and impaired diastolic relaxation. The clinical features of this collection of signs and symptoms are different from those of heart failure with a reduced ejection fraction, which occurs due to weakened cardiac pumping function or impaired contraction (Vasan et al., 1999). Therefore, rapid fluid accumulation in patients with blocked ventricles and minimal renal reserve volume may result in increased fluid reflux to the lungs and the mentioned clinical manifestations.

Baseline central laboratory values from the clinical database were compared with local laboratory values for heart failure obtained at admission, as included in the EAC package. There was no change in serum creatinine, sodium, and potassium concentrations in patients treated with bardoxolone methyl who experienced an event of heart failure within the first 4 weeks after randomization (Table 11), indicating that heart failure was not associated with acute renal failure or acute kidney injury. Overall, the clinical data suggest that the etiology of heart failure was not due to direct renal or cardiac toxic effects, but was more likely due to sodium and fluid retention.

Table 10. Post hoc analysis of cardiovascular parameters in patients receiving bardoxolone methyl versus placebo who experienced heart failure events within the first 4 weeks of treatment.

Post-hoc analysis of heart failure cases in BEACON. Vital signs at baseline were calculated based on the average of three standard cuff measurements. Vital signs for HF hospitalization were collected from admission records included in the EAC adjudication package and represent a single assessment using different BP monitoring devices. Only LVEF during HF hospitalization was evaluated. The time of HF hospitalization for each patient was calculated based on the event start date and treatment start date and varied between weeks 0 and 4.

Table 11. Post hoc analysis of serum electrolytes in patients receiving bardoxolone methyl versus placebo who experienced heart failure events within the first 4 weeks of treatment.

Post hoc analysis of heart failure cases in BEACON. Baseline clinical chemistries were assessed at a central laboratory. Clinical chemistries for HF hospitalizations were collected from admission records included in the EAC adjudication package and represent assessments performed at various local laboratories.

6. Blood pressure in BEACON

The mean change from baseline in systolic and diastolic blood pressure for patients treated with bardoxolone methyl and placebo, based on the mean of three standardized blood pressure cuff measurements collected at each visit, is shown in Figure 12. Blood pressure increased in the bardoxolone methyl group relative to the placebo group, with a mean increase of 1.9 mmHg in systolic blood pressure and 1.4 mmHg in diastolic blood pressure being noted in the bardoxolone methyl group by week 4 (the first randomized post-assessment). The increase in systolic blood pressure (SBP) decreased by week 32, while the increase in diastolic blood pressure (DBP) persisted.

The increase in SBP and DBP in patients treated with bardoxolone methyl was more obvious in ABPM measurements relative to placebo-treated patients at week 4 (Figure 13). This order of magnitude difference may be due to the different techniques used or due to differences in baseline characteristics of patients in the ABPM sub-study. Patients in the ABPM sub-study had a higher baseline ACR than the entire population. In any case, the data confirm that bardoxolone methyl increases blood pressure in the BEACON patient population.

7. Blood Pressure Changes in Previous CKD Studies

In an open-label, dose-ranging study (402-C-0902) in patients with type 2 diabetes and stage 3b-4 CKD, no dose-related trends in blood pressure changes or changes at any individual dose level were observed after 85 consecutive treatment days with doses ranging from 2.5 mg to 30 mg of bardoxolone methyl (amorphous dispersion formulation, as used in BEACON). Post hoc analyses of blood pressure data stratified by CKD stage showed that patients with stage 4 CKD treated with bardoxolone methyl tended to experience increases in blood pressure relative to baseline, with the effects being most pronounced in the three highest dose groups, whereas patients with stage 3b CKD treated with bardoxolone methyl had no significant changes (Table 12). Although the sample sizes in the dose groups stratified by CKD stage were small, these data suggest that the effect of bardoxolone methyl treatment on blood pressure may be related to CKD stage.

Blood pressure values from a Phase 2b study with bardoxolone methyl (BEAM, 402-C-0804), which used an earlier crystalline formulation of the drug and employed a titration design, were highly variable, and although increases were noted in some bardoxolone methyl treatment groups, no clear dose-related trend in blood pressure was observed.

Table 12. Change from Baseline in Systolic and Diastolic Blood Pressure in Patients with Type 2 Diabetes and Stage 3b-4 CKD Categorized by Baseline CKD Stage and Given Bardoxolone Methyl

Patients were given bardoxolone methyl at doses of 2.5 mg, 5 mg, 10 mg, 15 mg, or 30 mg once daily for up to 85 days.

8. Blood Pressure and QTcF in Healthy Volunteers

Intensive blood pressure monitoring was used in a separate, complete QT study conducted in healthy volunteers. In two bardoxolone methyl treatment groups, one given the therapeutic dose of 20 mg, also studied in BEACON, and one given a supratherapeutic dose of 80 mg, after six days of once-daily dosing, blood pressure changes were no different from those observed in placebo-treated patients ( FIG14 ). After six days of treatment with either 20 mg or 80 mg, bardoxolone methyl did not increase QTcF, as assessed by placebo-corrected QTcF change (ΔΔQTcF) ( FIG15 ).

Bardoxolone methyl has also been tested in the absence of CKD. In early clinical studies of bardoxolone methyl in tumor patients (RTA 402-C-0501, RTA 402-C-0702), no mean changes in blood pressure were observed in all treatment groups after 21 consecutive treatment days at doses ranging from 5 mg/day to 1300 mg/day (crystalline formulation). Similarly, in a randomized, placebo-controlled study in patients with hepatic dysfunction (RTA 402-C-0701), 14 consecutive days of treatment with bardoxolone methyl at doses of 5 mg/day and 25 mg/day (crystalline formulation) resulted in mean reductions in systolic and diastolic blood pressure (Table 13).

Overall, these data suggest that bardoxolone methyl does not prolong the QT interval or increase blood pressure in patients without baseline cardiovascular morbidity or stage 4 CKD.

Table 13. Changes from Baseline in Blood Pressure in Patients with Hepatic Dysfunction Treated with Bardoxolone Methyl

9. Overview and Analysis of Heart Failure

Comparison of the baseline characteristics of patients with heart failure events revealed that, although the risk of heart failure was higher in patients treated with bardoxolone methyl, both heart failure patients treated with bardoxolone methyl and placebo were more likely to have a previous history of cardiovascular disease and heart failure and, on average, had higher baseline ACR, BNP, and QTcF. Therefore, the occurrence of heart failure in these patients may be associated with traditional risk factors for heart failure. In addition, many patients with heart failure were in subclinical heart failure before randomization, as indicated by their high baseline BNP levels.

As a surrogate for fluid retention after randomization, a post hoc analysis was performed on the subset of patients for whom BNP data were available. At week 24, the increase in BNP was significantly higher in patients treated with bardoxolone methyl relative to those treated with placebo, with the increase in BNP in patients treated with bardoxolone methyl directly correlated with the baseline ACR. Urinary sodium excretion data from patients in the BEACON ABPM substudy revealed that only in patients treated with bardoxolone methyl were there clinically meaningful reductions in urine volume and sodium excretion relative to baseline at week 4. In another study, urine sodium levels and water excretion decreased in patients with stage 4 CKD, but not in patients with stage 3b CKD. In summary, these data suggest that bardoxolone methyl differentially affects sodium and water management, with retention of these components being more pronounced in patients with stage 4 CKD.

Consistent with this phenotype of fluid retention, descriptive descriptions of heart failure events provided in admission records, along with uncontrolled post-hoc assessments from investigators, indicate that heart failure events in bardoxolone methyl-treated patients were generally preceded by rapid fluid gain and were not associated with acute renal or cardiac decompensation.

In the bardoxolone methyl group, blood pressure changes indicating overall volume status were also increased relative to the placebo group, as measured by standardized blood pressure cuff monitoring in BEACON. Pre-specified blood pressure analyses in healthy volunteer studies confirmed no changes in either systolic or diastolic blood pressure. Although the intention-to-treat (ITT) analysis of Phase 2 CKD studies implemented with bardoxolone methyl showed no significant increase in blood pressure, post-hoc analysis of these studies showed that increases in both systolic and diastolic blood pressure depended on the CKD stage. Overall, these data suggest that the effect of bardoxolone methyl treatment on blood pressure may be associated with CKD disease severity.

Thus, urine electrolyte, BNP, and blood pressure data collectively support that bardoxolone methyl treatment may differentially affect volume status, with no clinically detectable effect in healthy volunteers or patients with early CKD, but more likely promoting fluid retention in patients with more advanced renal dysfunction at baseline and with traditional risk factors associated with heart failure. The increase in eGFR is likely due to glomerular effects, while the effects on sodium and water regulation originate in the renal tubules. Since changes in eGFR are not associated with heart failure, the data suggest that the effects on eGFR and sodium and water regulation are anatomically and pharmacologically distinct.

In previous studies, the risk (table 14) of the increase in heart failure and related adverse events utilizing bardoxolone methyl for treatment was not observed. However, because the previous study of bardoxolone methyl recruited one-tenth of the patients, the risk (if present) of increase may also be undetectable. In addition, BEACON limits recruitment of patients with stage 4 CKD, and the patient is a known colony in a higher risk of cardiovascular events relative to the patient with stage 3b CKD. Therefore, the late stage nature of the nephropathy of BEACON colony and significant cardiovascular risk burden (reflected by markers such as low baseline eGFR, high baseline ACR and high baseline BNP content) are the possible important factors of the pattern observed in cardiovascular events.

To further examine the relationship between the clinically significant thresholds of the key endpoints and traditional risk factors for fluid overload in BEACON, additional post-hoc analysis was performed. Various appropriate criteria relevant to these risk factors were used to exclude patients at maximum risk and to explore the results from BEACON. The combination of selected criteria (including excluding patients with eGFR of 20 mL/min/1.73 ^m or lower, significantly elevated proteinuria, and age over 75 years or BNP greater than 200 pg/mL) eliminated observed imbalances (Table 15). Applying these same criteria to SAEs also significantly improved or eliminated the imbalances noted (Table 16). Overall, these findings indicate the application of these and other kidney and cardiovascular risk markers in future selection criteria for clinical studies of bardoxolone methyl.

D. Potential mechanisms of fluid overload in BEACON

The data presented in the previous section indicate that bardoxolone methyl promotes fluid retention in the subset of patients at greatest risk for developing heart failure independent of drug administration. The data indicate that these effects are not associated with acute or chronic renal or cardiac toxicity. Therefore, a comprehensive list of well-established renal mechanisms affecting volume status (Table 17) was explored to determine whether any cause matched the clinical phenotype observed with bardoxolone methyl.

Initial studies focused on possible activation of the renin-angiotensin-aldosterone system. Activation of this pathway lowers serum potassium due to increased renal excretion. However, in the BEACON substudy, bardoxolone methyl did not affect serum potassium and slightly decreased urinary potassium (Table 7).

Another potential mechanism being investigated is whether altered transtubular ion gradients may lead to sodium reabsorption and, subsequently, water reabsorption, due to the effects of bardoxolone methyl on serum magnesium and other electrolytes. However, this mechanism also involves potassium regulation, and baseline serum magnesium does not appear to be associated with fluid retention or heart failure hospitalization.

After ruling out other etiologies for the reasons listed in Table 16, inhibition of endothelin signaling was the main remaining potential mechanism for volume regulation, which is consistent with the therapeutic effect of bardoxolone methyl in BEACON. Therefore, further investigation of modulation of the endothelin pathway as a possible explanation for the fluid retention observed in the BEACON study was performed.

1. Regulation of the endothelin system

The most directly comparable clinical data to the BEACON study for comparing the effects of known endothelin pathway modulators are those using the endothelin receptor antagonist (ERA) avosentan. Avosentan was studied in patients with stage 3-4 CKD and diabetic nephropathy in the ASCEND study, a large outcome study evaluating time to first doubling of serum creatinine, ESRD, or death (Mann et al., 2010). Although the baseline eGFR in this study was slightly higher than the mean baseline eGFR in BEACON, the mean ACR in patients in the ASCEND study was approximately 7 times that of BEACON (Table 18). Therefore, the overall cardiovascular risk profile between the two studies is likely similar.

As in BEACON, the ASCEND study was stopped early due to an early imbalance between heart failure hospitalizations and fluid overload events. Importantly, avosentan-induced fluid overload-related adverse events (both serious and minor) increased only within the first month of treatment (Figure 16).

Examination of the key endpoints in the ASCEND study revealed an approximately 3-fold increase in the risk of congestive heart failure (CHF) and a small, non-significant increase in death. In addition, a small numerical reduction in ESRD events was also observed. The BEACON study confirmed similar findings, although with a lower incidence of heart failure events. Regardless, the two studies showed striking similarities in the time to clinical manifestation and onset of heart failure and the impact on other key endpoints (Table 19).

2. Mechanism of fluid overload induced by endothelin receptor antagonists

The role of endothelin in fluid overload has been extensively studied. Using knockout models in mice, researchers have demonstrated that severe disruption of the endothelin pathway, followed by salt challenge, promotes fluid overload. Specific knockout of endothelin-1 (ET-1), endothelin receptor type A (ETA), endothelin receptor type B (ETB), or a combination of ETA and ETB have all been shown to promote fluid overload in animals, with clinical phenotypes consistent with ERA-mediated fluid overload in patients. These effects are caused by severe disruption of the epithelial sodium channel (ENaC), which is expressed in the collecting tubules of the kidney and reabsorbs sodium therein and promotes fluid retention (Vachiery and Davenport, 2009).

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

Evaluation of plasma and urine levels of endothelin-1 (ET-1) in humans with eGFR values ranging from stage 5 CKD to the supranormal range (8-131 mL/min/1.73 m ² ) has been previously reported (Dhaun et al., 2009). Plasma levels were significantly and negatively correlated with eGFR, but because the slope of the curve was not large, meaningful ET-1 differences were not apparent across the large eGFR range evaluated. As a surrogate for renal production of ET-1, the fractional excretion of ET-1 can be calculated by assessing ET-1 levels in plasma and urine. Urinary levels were relatively unchanged from eGFR >100 to approximately 30 mL/min/1.73 m ² ( FIG17 ). However, in patients with stages 4 and 5 CKD, ET-1 levels appear to increase exponentially with decreasing eGFR. These data suggest that renal ET-1 is fundamentally dysregulated in patients with advanced (stages 4 and 5) CKD. Based on these published data, the researchers hypothesized that the differential effects on fluid disposal by bardoxolone methyl, if due to endothelin modulation, could be due to differential endogenous production of ET-1 in the kidney, which is significantly increased in patients with stages 4 and 5 CKD.

4. Bardoxolone methyl regulates endothelin signaling

As mentioned above, bardoxolone methyl reduces ET-1 expression in human cell lines, including mesangial cells and endothelial cells found in the kidney. Furthermore, in vitro and in vivo data suggest that bardoxolone methyl and its analogs can modulate the endothelin pathway to promote a vasodilatory phenotype by inhibiting the vasoconstrictive ET _A receptor and restoring normal levels of the vasodilatory ET _B receptor. Therefore, robust activation of Nrf2-related genes by bardoxolone methyl is associated with inhibition of pathological endothelin signaling and may promote vasodilation by modulating ET receptor expression.

E. Basic principles of BEACON termination

1. Diagnosed with heart failure

Hospitalization due to heart failure or death due to heart failure belong to the cardiovascular disease events determined by EAC. It is determined that the imbalance system in heart failure and related disease events is due to the main findings of BEACON's early termination. In addition, heart failure-related AEs (such as edema) are due to a higher discontinuation rate than expected. The overall imbalance in the time to first heart failure seems to be the result of a large contribution of the events occurring in the first 3 to 4 weeks after treatment begins. Kaplan-Meyer (Kaplan-Meyer) analysis shows that after this initial period, the event rate between the treatment arms seems to maintain parallel tracks. The pattern reflected in Figure 9 shows the rapid physiological effects of being hospitalized due to heart failure relative to the cumulative toxic effects.

2. Mortality

At the end 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. Most of the fatal consequences (49 of 75 deaths) that occurred before the clinical database was locked (March 4, 2013) were confirmed to be cardiovascular (29 bardoxolone methyl patients versus 20 placebo patients). Based on the pre-specified definitions listed in the BEACON EAC chapter, most cardiovascular deaths were classified as "cardiac death-not otherwise specified". At the time of the final analysis, the Kaplan-Meier analysis of overall survival showed no significant separation until approximately the 24th week (Figure 10). There were three fatal heart failure events, all in bardoxolone methyl treated patients. In addition, as reflected in Table 16, the percentage of death occurring in patients over 75 years of age was higher in bardoxolone methyl treated patients than in placebo treated patients. It should be noted that if patients over 75 years of age are excluded, the number of fatal events in the bardoxolone methyl arm compared to the placebo arm was 20 and 23 respectively.

3. Summary of other safety data from BEACON

In addition to the effects of bardoxolone methyl treatment on eGFR and renal SAEs, the number of hepatobiliary SAEs was reduced in the bardoxolone methyl group compared to the placebo group (4 vs. 8, respectively; Table 2), and no Hy's Law cases were observed. The number of tumor-related SAEs was also balanced between the two groups. Finally, bardoxolone methyl treatment was not associated with QTc prolongation, as assessed by ECG evaluation at Week 24 (Table 20).

Table 20. Change from Baseline in QTcF at Week 24 in Patients Receiving Bardoxolone Methyl Versus Placebo in BEACON (Safety Population)

Data included only ECG evaluations on or before the patient's last dose of study drug. Visits were performed relative to the patient's first dose of study drug.

F.BEACON Conclusion

In summary, interrogation of data from studies conducted using bardoxolone methyl reveals that the drug can differentially regulate fluid retention, with no clinically detectable effect in healthy volunteers or early-stage CKD patients, while potentially promoting fluid retention pharmacologically in patients with advanced renal dysfunction. Since the occurrence of heart failure in both bardoxolone methyl and placebo-treated patients is associated with traditional risk factors for heart failure, this pharmacological effect in patients with baseline cardiac dysfunction may explain the increased risk of heart failure treated with bardoxolone methyl in BEACON. These data suggest that reducing the overall risk of heart failure in future clinical studies by selecting a patient population with a lower baseline risk of heart failure should avoid the increase in heart failure associated with bardoxolone methyl treatment. Importantly, available data show that fluid overload in BEACON is not caused by direct renal or cardiac toxicity. The clinical phenotype of fluid overload is similar to that observed using ERA in advanced CKD patients, and preclinical data demonstrate that bardoxolone methyl regulates the endothelin pathway. Since it is known that severe disruption of the endothelin pathway in patients with advanced CKD activates a specific sodium channel (ENaC) that can promote acute sodium and volume retention (Schneider, 2007), these mechanistic data, together with the clinical characteristics of patients with heart failure receiving bardoxolone methyl, provide a reasonable hypothesis for the mechanism of fluid retention in BEACON. Since impaired renal function may be an important factor that prevents patients from compensating for short-term fluid overload, and since the number of patients with early-stage CKD who have been treated to date is relatively limited, it may be prudent to exclude patients with CKD (e.g., patients with an eGFR <60) from treatment with BARD and other AIMs and is an element of the present invention.

II. Compounds for treating or preventing endothelial dysfunction, pulmonary hypertension, cardiovascular disease and related disorders.

In one aspect of the present invention, a method of reducing pulmonary artery pressure in a patient in need thereof is provided, comprising administering to the patient an amount of bardoxolone methyl or an analog thereof sufficient to reduce the patient's pulmonary artery pressure. Bardoxolone methyl analogs include compounds of the following formulae, or pharmaceutically acceptable salts or tautomers thereof:

in:

R ₁ is -CN, halo, -CF ₃ or -C(O)R _a , wherein R _a is -OH, alkoxy _(C1-4) , -NH ₂ , alkylamino _(C1-4) or -NH-S(O) ₂ -alkyl _(C1-4) ;

_R2 is hydrogen or methyl;

R ₃ and R ₄ are each independently hydrogen, hydroxy, methyl or any of these groups together with the group R _c are as defined below; and

Y is:

-H, -OH, -SH, -CN, -F, -CF ₃ , -NH ₂ or -NCO;

Alkyl _(C≤8) , alkenyl _(C≤8) , alkynyl _{(C≤8), aryl (C≤12)} , aralkyl _{(C≤12) ,} heteroaryl _{(C≤8), heterocycloalkyl (C≤12)} , alkoxy _{(C≤8) ,} aryloxy _(C≤12) , acyloxy _{(C≤8), alkylamino (C≤8)} , dialkylamino _(C≤8) , alkenylamino _{(C≤8), arylamino (C≤8) ,} aralkylamino (C≤8 ₎ , alkylthio _{(C≤8), acylthio (C≤8) ,} alkylsulfonylamino ( _{C≤8 )} , or a substituted form of any of these groups;

-alkanediyl _(C≤8) -R _b , -alkenediyl _(C≤8) -R _b , or a substituted form of any of these groups, wherein R _b is:

hydrogen, hydroxy, halo, amino or thiol; or

heteroaryl _(C≤8) , alkoxy _(C≤8) , alkenyloxy (C≤8), aryloxy _(C≤8) , aralkyloxy _(C≤8) , heteroaryloxy _(C≤8) , acyloxy _{(C≤8), alkylamino (C≤8) ,} dialkylamino _(C≤8) , alkenylamino _{(C≤8), arylamino (C≤8 ), aralkylamino (C≤8 ), heteroarylamino (} C≤8), alkylsulfonylamino _(C≤8 ), acylamino _{(C≤8) ,} -OC(O)NH-alkyl _{( C≤8)} , -OC(O) _CH2NHC (O)O-tert-butyl, _-OCH2 -alkylthio _(C≤8) , or a substituted form of any of these groups;

-(CH ₂ ) _m C(O)R _c , wherein m is 0-6 and R _c is:

hydrogen, hydroxy, halo, amino, -NHOH, or thiol; or

alkyl _(C≤8) , alkenyl _(C≤8) , alkynyl _{(C≤8), aryl (C≤8)} , aralkyl _(C≤8) , heteroaryl _{(C≤8) ,} heterocycloalkyl (C≤8), alkoxy _(C≤8) , alkenyloxy _(C≤8) , aryloxy ( _C≤8) , aralkyloxy _(C≤8) , heteroaryloxy _(C≤8) , acyloxy (C≤8 _{), alkylamino (C≤8)} , dialkylamino _{(C≤8) ,} arylamino _{(C≤8), alkylsulfonylamino ( C≤8)} , acylamino _(C≤8) , -NH-alkoxy _(C≤8) , -NH-heterocycloalkyl _{(C≤8) ,} -NHC(NOH)-alkyl _(C≤8) , -NH-acylamino _(C≤8) , or a substituted form of any of these groups;

R _c and R ₃ together are -O- or -NR _d -, wherein R _d is hydrogen or alkyl _(C≤4) ; or

R _c and R ₄ together are -O- or -NR _d -, wherein R _d is hydrogen or alkyl _(C≤4) ; or

-NHC(O) _Re , wherein _Re is:

hydrogen, hydroxyl, amino; or

Alkyl _(C≤8) , alkenyl _(C≤8) , alkynyl _{(C≤8), aryl (C≤8)} , aralkyl _{(C≤8) ,} heteroaryl _(C≤8) , heterocycloalkyl (C≤8), alkoxy _(C≤8) , aryloxy _{( C≤8)} , aralkyloxy (C≤8), heteroaryloxy _{(C≤8) ,} acyloxy _(C≤8 ), alkylamino _(C≤8) , dialkylamino _(C≤8) , arylamino _(C≤8) , or a substituted form of any of these groups.

These compounds are known as antioxidant inflammatory regulators. These compounds have shown the ability to activate Nrf2, as measured by elevated expression of one or more Nrf2 target genes (e.g., NQO1 or HO-1; Dinkova-Kostova et al., 2005). In addition, these compounds are able to indirectly and directly inhibit proinflammatory transcription factors, including NF-κB and STAT3 (Ahmad et al., 2006; Ahmad et al., 2008). In some aspects, a method for preventing pulmonary hypertension in an individual in need is provided, comprising administering to the individual an amount sufficient to prevent pulmonary hypertension in the individual, or an analog thereof. In some aspects, a method for preventing the progression of pulmonary hypertension in an individual in need is provided, comprising administering to the individual an amount sufficient to prevent the progression of pulmonary hypertension in the individual, or an analog thereof.

Triterpenoids, synthesized in plants by the cyclization of squalene, are used for medicinal purposes in many Asian countries; and some triterpenoids, such as ursolic acid and oleanolic acid, are known to have anti-inflammatory and anti-carcinogenic properties (Huang et al., 1994; Nishino et al., 1988). However, the biological activity of these naturally occurring molecules is relatively weak, so new analogs are synthesized to enhance their potency (Honda et al., 1997; Honda et al., 1998). Ongoing efforts to improve the anti-inflammatory and anti-proliferative activities of oleanolic acid and ursolic acid analogs have led to the discovery of 2-cyano-3,12-dioxooleanolic-1,9(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 potent derivatives of oleanolic acid have been identified, including methyl-2-cyano-3,12-dioxooleanolic-1,9-diene-28-oic acid (CDDO-Me; RTA 402; bardoxolone methyl). RTA 402 (Antioxidant Inflammatory Modulator (AIM)) suppresses the induction of several important inflammatory mediators (e.g., iNOS, COX-2, TNFα, and IFNγ) in activated macrophages, thereby restoring redox homeostasis in inflamed tissues. RTA 402 has also been reported to activate the Keap1/Nrf2/ARE signaling pathway, which leads to the production of several anti-inflammatory and antioxidant proteins (e.g., heme oxygenase-1 (HO-1)). It induces the cytoprotective transcription factor Nrf2 and suppresses the activation of the pro-oxidative and pro-inflammatory transcription factors NF-κB and STAT3. In vivo, RTA 402 has demonstrated significant single-agent anti-inflammatory activity in several animal models of inflammation, such as renal damage in the cisplatin model and acute kidney injury in the ischemia-reperfusion model. In addition, significant reductions in serum creatinine were observed in patients treated with RTA 402.

Thus, in pathologies involving oxidative stress alone or oxidative stress exacerbated by inflammation, treatment may comprise administering to the subject a therapeutically effective amount of a compound of the invention, such as those described above or throughout the specification. Treatment may be performed prophylactically prior to a predictable state of oxidative stress (e.g., organ transplantation or administration of therapy to a cancer patient), or may be performed therapeutically in settings involving established oxidative stress and inflammation.

Non-limiting examples of triterpenoid compounds that can be used in the methods of the present invention are shown herein.

Table 21 summarizes in vitro results for several of these compounds, in which RAW264.7 macrophages were pretreated with DMSO or various drug concentrations (nM) for 2 hours and then treated with 20 ng/mL IFNγ for 24 hours. NO concentration in the culture medium was determined using the Griess reagent system; cell viability was determined using the WST-1 reagent. NQO1CD represents the concentration required to induce a 2-fold increase in the expression of NQO1, an Nrf2-regulated antioxidant enzyme, in Hepa1c1c7 murine hepatoma cells (Dinkova-Kostova et al., 2005). All of these results are orders of magnitude more active than, for example, the parent oleanolic acid molecule. Due in part to the important protective effects against oxidative stress and inflammation afforded by the induction of antioxidant pathways resulting from Nrf2 activation, analogs of RTA 402 may also be useful in treating and/or preventing diseases such as pulmonary hypertension.

Table 21. Inhibition of IFNγ-induced NO production

Without being bound by theory, the potency of the compounds of the invention (e.g., RTA402) stems largely from the addition of an α,β-unsaturated carbonyl group. In in vitro assays, the activity of these compounds can be largely abolished by the introduction of dithiothreitol (DTT), N-acetylcysteine (NAC), or glutathione (GSH); thiols containing moieties that interact with α,β-unsaturated carbonyl groups (Wang et al., 2000; Ikeda et al., 2003; 2004; Shishodia et al., 2006). Biochemical analyses have established that RTA402 directly interacts with a key cysteine residue (C179) on IKKβ (see below) and inhibits its activity (Shishodia et al., 2006; Ahmad et al., 2006). IKKβ controls NF-κB activation via the "classical" pathway, which involves phosphorylation-induced IκB degradation, leading to the release of NF-κB dimers into the nucleus. In macrophages, this pathway is responsible for the production of numerous proinflammatory molecules in response to TNFα and other proinflammatory stimuli.

RTA 402 also inhibits the JAK/STAT signaling pathway at different levels. When activated by ligands such as interferon and interleukin, JAK proteins are recruited to transmembrane receptors (e.g., IL-6R). JAK then phosphorylates the intracellular portion of the receptor, which leads to the recruitment of STAT transcription factors. STAT is then phosphorylated by JAK, forms dimers, and translocates to the nucleus, where it activates the transcription of several genes involved in inflammation. RTA 402 inhibits constitutive and IL-6-inducible STAT3 phosphorylation and dimer formation and directly binds to cysteine residues in the kinase domain (C1077) of STAT3 (C259) and JAK1. Biochemical analysis has also established that triterpenoids directly interact with key cysteine residues on Keap1 (Dinkova-Kostova et al., 2005). Keap1 is an actin-tethered protein that, under normal conditions, keeps the transcription factor Nrf2 hidden in the cytoplasm (Kobayashi and Yamamoto, 2005). Oxidative stress leads to oxidation of regulatory cysteine residues on Keap1 and causes the release of Nrf2. Nrf2 then translocates to the nucleus and binds to the antioxidant response element (ARE), leading to the 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 plays many antioxidant and anti-inflammatory roles (Maines and Gibbs, 2005). HO-1 has recently been shown to be favorably induced by triterpenoids (including RTA 402) (Liby et al., 2005). RTA 402 and many structural analogs have also been shown to be potent inducers of expression of other phase 2 proteins (Yates et al., 2007). RTA 402 is a potent inhibitor of NF-κB activation. In addition, RTA 402 activates the Keap1/Nrf2/ARE pathway and induces HO-1 expression.

The compounds employed can be prepared using the methods described in Honda et al. (2000a); Honda et al. (2000b); Honda et al. (2002); and U.S. Patent Application Publication Nos. 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 techniques of organic chemistry employed by those skilled in the art. These principles and techniques are taught, for example, in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is also incorporated herein by reference.

The compounds employed in the methods of the present invention may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be separated in optically active or racemic forms. Therefore, unless a specific stereochemistry or isomeric form is explicitly indicated, all chiral, diastereomeric, racemic, epimeric, and geometric isomeric forms of the structure are intended to be encompassed. The compounds may exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the compounds of the present invention may have an S or R configuration.

Polymorphic forms of the compounds of the invention (e.g., Forms A and B of CDDO-Me) can be used according to the methods of the invention. Form B exhibits surprisingly better bioavailability than Form A. Specifically, when monkeys were orally administered equivalent doses of both forms (in gelatin capsules), the bioavailability of Form B was greater than the bioavailability of Form A CDDO-Me in monkeys. See U.S. Patent Application Publication No. 2009/0048204, the entire contents of which are incorporated herein by reference.

"Form A" of CDDO-Me (RTA 402) is unsolvated (anhydrous) and can be characterized by a unique crystal structure (space group P4 ₃ 2 ₁ 2 (number 96) with unit cell dimensions of 1 and 2) and a packing structure whereby three molecules pack in a helical pattern along the crystallographic b-axis. In some embodiments, Form A can also be characterized by an X-ray powder diffraction (XRPD) pattern (CuKa) comprising significant diffraction peaks at approximately 8.8, 12.9, 13.4, 14.2, and 17.4 degrees 2θ. In some variations, Form A has an X-ray powder diffraction pattern substantially as shown in FIG1A or FIG1B.

Unlike Form A, "Form B" of CDDO-Me is a single phase but lacks a defined crystalline structure. Samples of Form B exhibit no long-range molecular correlations, i.e., above approximately 100°C. Furthermore, thermal analysis of Form B samples reveals a glass transition temperature ( _Tg ) ranging from about 120°C to about 130°C. In contrast, disordered nanocrystalline materials do not exhibit a _Tg , but rather only a melting temperature ( _Tm ), above which the crystalline structure becomes liquid. Form B is characterized by an XRPD spectrum (Figure 1C) that differs from Form A (Figures 1A and 1B). Because it lacks a defined crystalline structure, Form B also lacks distinct XRPD peaks (such as those typical of Form A) and is instead characterized by a typical "halo" XRPD pattern. Specifically, non-crystalline Form B falls into the category of "X-ray amorphous" solids because its XRPD pattern exhibits three or fewer major diffraction halos. Within this category, Form B is a "glassy" material.

Forms A and B of CDDO-Me are readily prepared from various solutions of the compound. For example, Form B can be prepared by rapid or slow evaporation in MTBE, THF, toluene, or ethyl acetate. Form A can be prepared in several ways, including by rapid evaporation, slow evaporation, or slow cooling of a solution of CDDO-Me in ethanol or methanol. Preparation of CDDO-Me in acetone can use rapid evaporation to produce Form A or slow evaporation to produce Form B.

Various characterization methods can be used together to distinguish Form A and Form B CDDO-Me from each other and from other forms of CDDO-Me. Exemplary techniques suitable for this purpose are solid-state nuclear magnetic resonance (NMR), X-ray powder diffraction (compare Figures 1A and B with Figure 1C), X-ray crystallography, differential scanning calorimetry (DSC), dynamic vapor sorption/desorption (DVS), Karl Fischer analysis (KF), hot stage microscopy, amplitude-modulated differential scanning calorimetry, FT-IR, and Raman spectroscopy. In particular, analysis of XRPD and DSC data can distinguish Form A, Form B, and the hemibenzoate salt form of CDDO-Me. See U.S. Patent Application Publication No. 2009/0048204, the entire contents of which are incorporated herein by reference.

Additional details regarding polymorphs of CDDO-Me are described in U.S. Patent Application Publication No. 2009/0048204, PCT Publication WO 2009/023232, and PCT Publication WO 2010/093944, the entire contents of which are incorporated herein by reference.

Non-limiting specific formulations of the compounds disclosed herein include CDDO-Me polymer dispersions. See, for example, PCT Publication WO 2010/093944, the entire contents of which are incorporated herein by reference. Some formulations reported herein exhibited higher bioavailability than formulations of micronized Form A or nanocrystalline Form A. In addition, formulations based on polymer dispersions showed further surprising improvements in oral bioavailability relative to micronized Form B formulations. For example, methacrylic acid copolymer Type C and HPMC-P formulations exhibited the highest bioavailability in individual monkeys.

The compounds used in the methods of the present invention may also exist in the form of prodrugs. Since prodrugs enhance certain desired pharmaceutical properties, such as solubility, bioavailability, manufacturing, etc., the compounds used in some methods of the present invention may be delivered in the form of prodrugs if desired. Therefore, the present invention encompasses prodrugs of the compounds of the present invention and methods of delivering prodrugs. Prodrugs of the compounds used in the present invention may be prepared by modifying the functional groups present in the compounds in the following manner: the modifications are cleaved into the parent compound during conventional manipulations or in vivo. Therefore, prodrugs include, for example, compounds described herein in which a hydroxyl, amino, or carboxyl group is bonded to any group that, when administered to an individual, cleaves to form a hydroxyl, amino, or carboxyl group, respectively.

It will be appreciated that the specific anion or cation forming part of any salt of the invention is not critical so long as the salt as a whole is pharmacologically acceptable. Examples of pharmaceutically acceptable salts, methods for their preparation, and uses are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

The compounds used in the methods of the present invention may also have the following advantages over compounds known in the art for the indications described herein: they may be more effective, less toxic, have a longer duration of action, be more potent, produce fewer side effects, be more readily absorbed, have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance), and/or have other useful pharmacological, physical, or chemical properties.

PAH is a distinct disease from systemic hypertension. PAH is characterized by high pulmonary artery and right ventricular pressures due to increased pulmonary vascular resistance; systemic hypertension is characterized by elevated systemic circulatory pressures. Typically, patients with PAH do not suffer from systemic hypertension.

The symptoms of PAH are well known. In addition, animal models of these conditions have been used to optimize dosages (see, Bauer et al., 2007, which is incorporated herein by reference in its entirety). A skilled artisan should be able to determine the optimal dosage without undue experimentation.

Just because a drug may be effective as a treatment for systemic hypertension does not mean it will also be effective for treating PAH. For example, vasodilators that are effective for treating systemic hypertension (e.g., the ACE inhibitor captopril) can worsen pulmonary hypertension and RV failure in patients with PAH. Evidence for the potential deleterious effects of drugs used to treat systemic hypertension on PAH is provided by Packer (1985), which is incorporated by reference. One known exception to this limitation is that approximately 15%-20% of patients with idiopathic PAH respond to calcium channel blockers, agents that are also used to treat systemic hypertension. To determine whether a patient has so-called "responsive" PAH and may respond to therapy with calcium channel blockers, diagnostic evaluation of PAH includes pulmonary artery catheterization and an acute challenge with adenosine, prostacyclin, or inhaled nitric oxide. If a patient's mean pulmonary artery pressure decreases by more than 10 mm Hg with one of these agents and the mean pulmonary artery pressure decreases to less than or equal to 40 mm Hg, a test to determine whether the patient will respond to a calcium channel blocker can be performed (Rich et al., 1992; Badesch et al., 2004). Some clinicians consider PAH to be responsive if there is a 20% or greater decrease in mean pulmonary artery pressure in response to adenosine, prostacyclin, or inhaled nitric oxide. The reason for performing acute vasoreactivity testing with prostacyclin, adenosine, or inhaled nitric oxide before testing with calcium channel blockers is that some patients who were given calcium channel blockers and did not previously show acute vasoreactivity have died (Badesch et al., 2004). This complex assessment and treatment algorithm emphasizes that drugs used to treat systemic hypertension may not be suitable for patients with PAH.

III. Pulmonary Hypertension

Pulmonary hypertension is a life-threatening disease characterized by a significant and sustained increase in pulmonary artery pressure and an increase in pulmonary vascular resistance, which leads to right ventricular (RV) failure and death. The current therapeutic approaches for treating chronic pulmonary hypertension primarily provide symptom relief and some improvements in prognosis. Although all treatments are hypothesized, there is a lack of evidence for a direct antiproliferative effect in most approaches. In addition, the use of most currently used agents is hampered by undesirable side effects or inconvenient drug administration routes. The pathological changes in hypertensive pulmonary arteries include endothelial damage, proliferation, and excessive contraction of vascular smooth muscle cells (SMCs).

PAH without an obvious cause is called primary pulmonary hypertension ("PPH"). Recently, various pathophysiological changes associated with this condition have been characterized, including vasoconstriction, vascular remodeling (i.e., proliferation of both the media and intima in pulmonary resistance vessels), and in situ thrombosis (e.g., D'Alonzo et al., 1991; Palevsky et al., 1989; Rubin, 1997; Wagenvoort and Wagenvoort, 1970; Wood, 1958). Impairment of vascular and endothelial homeostasis is evidenced by decreased prostacyclin ( _PGI2 ) synthesis, increased thromboxane production, decreased nitric oxide formation, and increased endothelin-1 synthesis (Giaid and Saleh, 1995; Xue and Johns, 1995). Elevated intracellular free calcium concentrations in pulmonary artery vascular smooth muscle cells have been reported in PPH.

Pulmonary arterial hypertension (PAH) is defined as a pulmonary vascular disease affecting the pulmonary arterioles, resulting in elevated pulmonary artery pressure and pulmonary vascular resistance, but with normal or only slightly elevated left-sided filling pressures (McLaughlin and Rich, 2004). PAH is caused by a cluster of diseases affecting the pulmonary vasculature. PAH can be caused by or associated with collagen vascular disorders (e.g., systemic sclerosis (scleroderma)), uncorrected congenital heart disease, liver disease, portal hypertension, HIV infection, hepatitis C, certain toxins, splenectomy, hereditary hemorrhagic telangiectasia, and primary genetic abnormalities. Specifically, mutations in the bone morphogenetic protein type 2 receptor (TGF-β receptor) have been identified as a cause of familial primary pulmonary hypertension (PPH) (Lane et al., 2000; Deng et al., 2000). An estimated 6% of PPH cases are familial, with the remainder being "sporadic." The incidence of PPH is estimated to be approximately 1 case per 1 million people. Secondary causes of PAH have a much higher incidence. The pathological hallmark of PAH is plexiform lesions in the lungs, which consist of obstructive endothelial cell proliferation and vascular smooth muscle cell hypertrophy in small precapillary pulmonary arterioles. PAH is a progressive disease associated with a high mortality rate. Patients with PAH may develop right ventricular (RV) failure, the severity of which predicts outcome (McLaughlin et al., 2002).

The evaluation and diagnosis of PAH are reviewed by McLaughlin and Rich (2004) and McGoon et al. (2004). Clinical history (e.g., symptoms of shortness of breath), family history of PAH, presence of risk factors, and findings on physical examination, chest X-ray, and electrocardiogram (ECG) may lead to suspicion of PAH. The next step in the evaluation will usually include an echocardiogram. Echocardiography can be used to estimate pulmonary artery pressure by Doppler analysis of the regurgitant jet from the tricuspid valve. Echocardiography can also be used to assess right and left ventricular function and the presence of valvular heart disease (e.g., mitral stenosis and aortic stenosis). Echocardiography can also be used to diagnose congenital heart disease, such as uncorrected atrial septal defects or patent ductus arteriosus. Echocardiographic findings consistent with a diagnosis of PAH include: 1) Doppler evidence of elevated pulmonary artery pressure; 2) right atrial enlargement; 3) right ventricular enlargement and/or hypertrophy; 4) absence of mitral stenosis, pulmonary stenosis, and aortic stenosis; 5) normal-sized or small left ventricle; and 6) relatively preserved or normal left ventricular function. To confirm the diagnosis of PAH, cardiac catheterization to directly measure pressures in the right side of the heart and the pulmonary vasculature is mandatory. Accurate measurement of the pulmonary capillary wedge pressure (PCWP) is also necessary, as it provides an accurate estimate of left atrial and left ventricular end-diastolic pressures. If an accurate PCWP cannot be obtained, direct measurement of LV end-diastolic pressure via left heart catheterization is recommended. By definition, patients with PAH should have a low or normal PCWP. However, in advanced stages of PAH, the PCWP may become slightly elevated, but typically does not exceed 16 mm Hg (McLaughlin and Rich, 2004; McGoon et al., 2004). The upper limit of normal mean pulmonary artery pressure in adults is 19 mm Hg. Mean pulmonary artery pressure is commonly defined as one-third of the systolic pulmonary artery pressure plus two-thirds of the diastolic pulmonary artery pressure. Severe PAH can be defined as a mean pulmonary artery pressure greater than or equal to 25 mm Hg, a PCWP less than or equal to 15-16 mm Hg, and a pulmonary vascular resistance (PVR) greater than or equal to 240 dyne sec/ ^cm⁻¹ . Pulmonary vascular resistance is defined as mean pulmonary artery pressure minus PCWP, divided by cardiac output. This ratio is multiplied by 80 and expressed in dyne sec/ ^cm⁻¹ . PVR can also be expressed in millimeters of Hg/liter/minute, known as Wood units. The normal adult PVR is 67 ± 23 dyne sec/ ^cm⁻¹ , or 1 Wood unit (McLaughlin and Rich, 2004; McGoon et al., 2004; Galie et al., 2005). Patients with left-sided myocardial disease or valvular heart disease are often excluded from clinical trials testing the efficacy of drugs for PAH (Galie et al., 2005).

The patient's pulmonary hypertension status can be assessed according to the World Health Organization (WHO) classification (modified from the New York Association functional classification), as detailed below:

Category I - Patients have pulmonary hypertension that does not limit physical activity. Ordinary physical activity does not cause excessive dyspnea or fatigue, chest pain, or near syncope.

Class II - Patients have pulmonary hypertension that slightly limits physical activity. They are comfortable at rest. Ordinary physical activity causes excessive dyspnea or fatigue, chest pain, or near syncope.

Category III - Patients have pulmonary hypertension that significantly limits physical activity. They are comfortable at rest. More than mild ordinary activity causes excessive dyspnea or fatigue, chest pain, or near syncope.

Category IV - Patients have pulmonary hypertension and are unable to perform any physical activity without symptoms. These patients show signs of right-sided heart failure. They may experience dyspnea and/or fatigue even at rest. Any physical activity increases discomfort.

At one time, the only effective long-term treatment for PAH in combination with anticoagulant therapy was continuous intravenous administration of prostacyclin (also known as epoprostenol ( _PGI2 )) (Barst et al., 1996; McLaughlin et al., 1998). Later, the nonselective endothelin receptor antagonist bosentan showed efficacy in treating PAH (Rubin et al., 2002). Bosentan was a significant advance as the first orally bioavailable agent with efficacy in treating PAH. However, the mainstay of treatment for PAH currently utilizes selective endothelin type A receptor antagonists (Galie et al., 2005; Langleben et al., 2004). Phosphodiesterase type V (PDE-V) inhibitors, including sildenafil and tadalafil, are approved for the treatment of PAH (Lee et al., 2005; Kataoka et al., 2005). PDE-V inhibition results in an increase in cyclic GMP, which leads to vasodilation of the pulmonary vasculature. Treprostinil, an analog of PGI ₂ , can be administered subcutaneously to appropriately selected patients with PAH (Oudiz et al., 2004; Vachiery and Naeije, 2004). In addition, iloprost, another prostacyclin analog, can be administered by direct inhalation in a nebulized form (Galie et al., 2002). Riociguat, a stimulator of soluble guanylate cyclase (sGC), is also approved for the treatment of PAH. These agents are used to treat PAH of various etiologies, including PAH associated with or caused by familial PAH (primary pulmonary hypertension or PPH), idiopathic PAH, scleroderma, mixed connective tissue disease, systemic lupus erythematosus, HIV infection, toxins (e.g., phentermine/fenfluramine), congenital heart disease, hepatitis C, cirrhosis, chronic thromboembolic pulmonary hypertension (distal or inoperable), hereditary hemorrhagic telangiectasia, and splenectomy. All approved agents for PAH are essentially vasodilatory in effect. Therefore, they address only a portion of the overall pathology of PAH. In contrast, without being limited by theory, the compounds of the present invention have documented anti-inflammatory and antiproliferative effects in addition to their effects on vascular tone, potentially addressing PAH pathology in a more comprehensive manner. Additionally, the methods provided herein can be used to activate Nrf2 and have positive effects on mitochondrial function, thereby addressing metabolic and energetic aspects of PAH (Sutendra, 2014; Hayes and Dinkova-Kostova, 2014).

IV. Cardiovascular Disease

The compounds and methods of the present invention can be used to treat patients with cardiovascular disease. See U.S. Patent Application No. 12/352,473, which is incorporated herein by reference in its entirety. Cardiovascular (CV) disease is the most important cause of mortality worldwide and is the leading cause of death in many developed countries. The causes of CV disease are complex, but most involve inadequate or complete destruction of blood supply to critical organs or tissues. Frequently, this condition is caused by the rupture of one or more atherosclerotic plaques, which leads to the formation of thrombi that block blood flow to critical organs. Such thrombosis is the underlying cause of heart attacks, in which one or more coronary arteries become blocked and blood flow to the heart itself is interrupted. The resulting ischemia is highly damaging to cardiac tissue due to both the lack of oxygen during the ischemic event and the excessive formation of free radicals after blood flow is restored (a phenomenon known as ischemic reperfusion injury). Similar damage occurs in the brain during thrombotic strokes when cerebral arteries or other major blood vessels are blocked by thrombi. In contrast, hemorrhagic strokes involve the rupture of blood vessels and bleeding into surrounding brain tissue. This produces oxidative stress in the immediate area of the hemorrhage due to the presence of large amounts of free hemoglobin and other reactive species and causes ischemia in other parts of the brain due to impaired blood flow. Subarachnoid hemorrhage, which is often accompanied by cerebral vasospasm, also causes ischemia/reperfusion injury in the brain.

Alternatively, atherosclerosis may be so extensive that stenosis (narrowing of the arteries) occurs in key blood vessels and blood flow to key organs, including the heart, is chronically inadequate. This chronic ischemia can lead to many types of end-organ damage, including cardiac hypertrophy associated with congestive heart failure.

Atherosclerosis (the fundamental defect that leads to many forms of cardiovascular disease) occurs when physical defects or damage to the lining of the arteries (endothelium) trigger an inflammatory response involving the proliferation of vascular smooth muscle cells and the infiltration of leukocytes into the affected area. Ultimately, a complex lesion called an atherosclerotic plaque (comprising a combination of the above cells with deposits of cholesterol-carrying lipoproteins and other substances) can form (e.g., Hansson et al., 2006).

One study found that RTA dh404 reduced diabetes-associated atherosclerosis. In this study, an animal model was used with RTA dh404 as an alternative to RTA 402. Specifically, treatment with RTA dh404 was found to reduce plaques in the aortic arch, chest, and abdomen in an inversely proportional, dose-dependent manner, as well as attenuate focal deposits in the aortic sinus (Tam et al., 2014, which is incorporated herein by reference in its entirety).

Medical treatments for cardiovascular disease include preventive therapies (e.g., the use of drugs intended to lower blood pressure or circulating levels of cholesterol and lipoproteins) as well as therapies designed to reduce the adhesion tendency of platelets and other blood cells (thereby reducing the rate of plaque progression and the risk of thrombosis). More recently, drugs such as streptokinase and tissue plasminogen activator have been introduced and used to dissolve clots and restore blood flow. Surgical treatments include coronary artery bypass grafting to create an alternative blood supply, balloon angioplasty to compress plaque tissue and increase the diameter of the arterial lumen, and carotid endarterectomy to remove plaque tissue in the carotid artery. These treatments, particularly balloon angioplasty, can be achieved through the use of stents, expandable mesh tubes designed to support the arterial wall in the affected area and maintain blood vessel patency. More recently, the use of drug-eluting stents has become common to prevent postoperative restenosis (restoration of arteries) in the affected area. These devices are filament stents coated with a biocompatible polymer matrix containing drugs that inhibit cell proliferation (e.g., paclitaxel or rapamycin). The polymer allows for slow, localized release of the drug in the affected area while minimizing exposure to non-targeted tissues.Despite the significant benefits these treatments offer, cardiovascular disease mortality remains high and significant unmet needs exist in the treatment of cardiovascular disease.

As described above, HO-1 induction has been shown to be beneficial in many cardiovascular disease models, and low levels of HO-1 expression have been clinically associated with increased CV disease. Therefore, the compounds of the present invention are useful for treating or preventing a variety of cardiovascular disorders, including but not limited to atherosclerosis, hypertension, myocardial infarction, chronic heart failure, stroke, subarachnoid hemorrhage, and restenosis.

V. Pharmaceutical Preparations and Routes of Administration

Administration of the compounds of the present invention to patients should follow general protocols for pharmaceutical administration, taking into account drug toxicity, if any. It is anticipated that treatment cycles will be repeated as needed.

The compounds of the present invention can be administered by various methods, for example, orally or by injection (e.g., subcutaneously, intravenously, intraperitoneally, etc.). Depending on the route of administration, the active compound may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. They can also be administered by continuous perfusion/infusion at the site of disease or wound. Specific examples of formulations showing improved oral bioavailability, including polymer-based CDDO-Me dispersions, are provided in U.S. application Ser. No. 12/191,176, which is incorporated herein by reference in its entirety. One skilled in the art will recognize that other manufacturing methods can be used to produce dispersions of the present invention having equivalent properties and efficacy (see Repka et al., 2002 and references cited therein). These alternative methods include, but are not limited to, solvent evaporation, extrusion (e.g., hot melt extrusion), and other techniques.

To administer a therapeutic compound by a route other than parenteral administration, it may be necessary to coat the compound with a material or co-administer the compound to prevent its inactivation. For example, the therapeutic compound can be administered to the patient in an appropriate carrier (e.g., liposomes) or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., 1984).

The therapeutic compound can also be administered parenterally, intraperitoneally, intraspinal or intracerebrally. Dispersions can also be prepared in, for example, glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and fluid for easy syringability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the desired amount of the therapeutic compound in an appropriate solvent with one or a combination of the ingredients listed above, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the therapeutic compound into a sterile vehicle containing a basic dispersion medium and the required additional ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, which yield a powder of the active ingredient (i.e., therapeutic compound) and any additional desired ingredients from a previously sterile-filtered solution.

The therapeutic compound can be administered orally, for example, with an inert diluent or an absorbable edible carrier. The therapeutic compound and other ingredients can also be encapsulated in a hard or soft shell gelatin capsule, compressed into a tablet, or incorporated directly into the diet of the individual. For oral therapeutic administration, the therapeutic compound can be incorporated with excipients and utilized in the form of absorbable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the therapeutic compound in the compositions and formulations is of course variable. The amount of the therapeutic compound in these therapeutically useful compositions is such that a suitable dosage is obtained.

It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of the therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are dictated by and directly dependent on, for example, (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art of synthesis for treating the chosen condition in a patient.

The therapeutic compound may also be administered topically to the skin, eyes, or mucous membranes. Alternatively, if local delivery to the lungs is desired, the therapeutic compound may be administered by inhalation of a dry powder or aerosol formulation.

The therapeutic compound can be formulated in a biocompatible matrix for use in drug eluting stents.

The actual dosage of a compound of the invention or a composition comprising the compound of the invention administered to an individual can be determined by physical and physiological factors such as age, sex, weight, severity of the condition, type of disease being treated, prior or concurrent therapeutic interventions, individual morbidities, and route of administration. These factors can be determined by one skilled in the art. The physician administering the drug will typically determine the concentration of the active ingredient in the composition and the appropriate dosage for each individual. If any complications occur, the dosage can be adjusted by the individual physician.

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

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

In some embodiments, the pharmaceutically effective amount is a daily dose of 0.1-1000 mg of compound/kg body weight. In some variations, the daily dose is 0.15-20 mg of compound/kg body weight. In some variations, the daily dose is 0.20-10 mg of compound/kg body weight. In some variations, the daily dose is 0.40-3 mg of compound/kg body weight. In some variations, the daily dose is 0.50-9 mg of compound/kg body weight. In some variations, the daily dose is 0.60-8 mg of compound/kg body weight. In some variations, the daily dose is 0.70-7 mg of compound/kg body weight. In some variations, the daily dose is 0.80-6 mg of compound/kg body weight. In some variations, the daily dose is 0.90-5 mg of compound/kg body weight. In some variations, the daily dose is about 1 mg to about 5 mg of compound/kg body weight.

An effective amount will generally be from about 0.001 mg/kg to about 1,000 mg/kg, about 0.01 mg/kg to about 750 mg/kg, about 0.1 mg/kg to about 500 mg/kg, about 0.2 mg/kg to about 250 mg/kg, about 0.3 mg/kg to about 150 mg/kg, about 0.3 mg/kg to about 100 mg/kg, about 0.4 mg/kg to about 75 mg/kg, about 0.5 mg/kg to about 50 mg/kg, about 0.6 mg/kg to about 3 The dosage range may be from about 10,000 mg/kg to about 10,000 mg/kg, about 0.7 mg/kg to about 25 mg/kg, about 0.8 mg/kg to about 15 mg/kg, about 0.9 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 100 mg/kg to about 500 mg/kg, about 1.0 mg/kg to about 250 mg/kg, or about 10.0 mg/kg to about 150 mg/kg, with one or more doses administered daily for one or more days (depending, of course, on the mode of administration and the factors discussed above). Other suitable dosage ranges include 1 mg to 10,000 mg/day, 100 mg to 10,000 mg/day, 500 mg to 10,000 mg/day, and 500 mg to 1,000 mg/day. In some embodiments, the amount is less than 10,000 mg/day, ranging from, for example, 750 mg to 9,000 mg/day.

The effective amount can 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, less than 10 mg/kg/day or less than 5 mg/kg/day. Alternatively, it can be in the range of 1 mg/kg/day to 200 mg/kg/day. For example, for the treatment of diabetic patients, the unit dose can be an amount that reduces blood glucose by at least 40% compared to untreated individuals. In another embodiment, the unit dose is an amount that reduces blood glucose to a level within ±10% of the blood glucose level of a non-diabetic patient.

In other non-limiting examples, the dosage can also include 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, 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 to about 1000 mg/kg/body weight or more per administration and any range derived therefrom. In non-limiting examples of derived ranges from the values listed herein, based on the above values, a range of about 1 mg/kg/body weight to about 5 mg/kg/body weight, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 micrograms/kg/body weight to about 500 mg/kg/body weight, etc. can be administered.

In certain embodiments, the pharmaceutical compositions of the present invention may comprise, for example, at least about 0.1% of the compounds of the present invention. In other embodiments, the compounds of the present invention may comprise from about 2% to about 75% by weight of the unit, or for example, from about 25% to about 60% and any range derivable therefrom.

Single and multiple doses of the medicament are encompassed. The desired time interval for delivering multiple doses can be determined by those skilled in the art using only routine experimentation. As an example, two doses can be given to an individual daily at approximately 12-hour intervals. In some embodiments, the medicament is administered once a day.

The medicament can be administered on a conventional schedule. As used herein, a conventional schedule refers to a predetermined specified period. A conventional schedule can cover periods of the same or different lengths, as long as the schedule is predetermined. For example, a conventional schedule can involve administration twice a day, every day, every two days, every three days, every four days, every five days, every six days, every week, every month, or any set number of days or weeks in between. Alternatively, a predetermined conventional schedule can involve administration twice a day for the first week, followed by daily administration for several months, etc. In other embodiments, the present invention provides medicaments that can be taken orally and whose occurrence time is dependent on or independent of food intake. Thus, for example, the medicament can be taken every morning and/or every evening, regardless of whether the individual has eaten or is about to eat.

Non-limiting specific formulations include CDDO-Me polymer dispersions (see U.S. application Ser. No. 12/191,176, filed Aug. 13, 2008, which is incorporated herein by reference). Some of the formulations reported therein exhibited higher bioavailability than formulations of micronized Form A or nanocrystalline Form A. In addition, formulations based on polymer dispersions showed further surprising improvements in oral bioavailability relative to micronized Form B formulations. For example, methacrylic acid copolymer Type C and HPMC-P formulations showed the highest bioavailability in individual monkeys.

VI. Combination Therapy

In addition to use as monotherapy, the compounds of the present invention may also find use in combination therapies. Effective combination therapy can be achieved using a single composition or pharmacological formulation comprising both agents, or using two different compositions or formulations administered simultaneously, one comprising a compound of the present invention and the other comprising the second agent. Alternatively, therapy can be administered before or after treatment with the other agent, with intervals ranging from minutes to months.

Various combinations may be employed, for example, where the compound of the invention is "A" and "B" represents a second agent, non-limiting examples of which are described below:

It is contemplated that other anti-inflammatory agents may be used in conjunction with the treatment of the present invention. For example, other COX inhibitors can be used, including aryl carboxylic acids (salicylic acid, acetylsalicylic acid, diflunisal, cholinemagnesium trisalicylate, salicylates, benoylates, flufenamic acid, mefenamic acid, meclofenamic acid, and triflumic acid), aryl alkanoic acids (diclofenac, fenclofenac, alclofenac, fentiazac, ibuprofen, flurbiprofen, ketoprofen, naproxen, fenoprofen, fenbufen, suprofen, indoprofen, tiaprofenic acid, and fenprofenic acid). acid, benoxaprofen, pirprofen, tolmetin, zomepirac, clofenac, indomethacin, and sulindac) and enolic acids (phenylbutazone, oxyphenbutazone, azapropazone, feprazone, piroxicam, and isoxicam). See also U.S. Patent No. 6,025,395, which is incorporated herein by reference.

FDA-approved treatments for pulmonary hypertension include prostacyclins (epoprostenol, iloprost, and treprostinil), endothelin receptor antagonists (bosentan, ambrisentan, and macitentan), phosphodiesterase-5 inhibitors (sildenafil and tadalafil), and sGC stimulators (riocipapi). Any of these agents is contemplated for use in combination with the treatments of the present invention. When combined with the compounds of the present invention, these agents may be administered at standard recommended doses or within standard recommended dose ranges, or may be administered at doses lower than standard doses. In addition, the use of the following combination agents is contemplated: rho-kinase inhibitors, such as Y-27632, fasudil, and H-1152P; epoprostenol derivatives, such as prostacyclin, treprostinil, beraprost, and iloprost; serotonin blockers, such as sarpogrelate; endothelin receptor antagonists, such as bosentan, sitaxsentan, ambrisentan, and TBC3711; PDE inhibitors, such as sildenafil, tadalafil, udenafil, and vardenafil; calcium channel blockers, Examples include amlodipine, bepridil, clentiazem, diltiazem, fendiline, gallopamil, mibefradil, prenylamine, semotiadil, terodiline, verapamil, aranidipine, bamidipine, and benidipine , cilnidipine, efonidipine, elgodipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine nitrendipine, cinnarizine, flunarizine, lidoflazine, lomerizine, bencyclane, etafenone, and perhexiline; tyrosine kinase inhibitors, such as imatinib; inhaled nitric oxide and nitric oxide donors, such as inhaled nitrites; IκB inhibitors, such as IMD1041; prostacyclin receptor agonists, such as selexipag; hematopoietic stimulants, such as TXA 127 (angiotensin (1-7)), darbepoetin alfa, erythropoetin, and epoetin alfa; anticoagulants and platelet inhibitors; and diuretics.

Dietary and nutritional supplements with reported benefits for the treatment or prevention of Parkinson's, Alzheimer's, multiple sclerosis, amyotrophic lateral sclerosis, rheumatoid arthritis, inflammatory bowel disease, and all other diseases whose pathogenesis is believed to involve the overproduction of nitric oxide (NO) or prostaglandins (e.g., acetyl-L-carnitine, octacosanol, evening primrose oil, vitamin B6, tyrosine, phenylalanine, vitamin C, L-dopa, or a combination of several antioxidants) can be used in combination with the compounds of the present invention.

Other specific second therapies include immunosuppressants (for transplant and autoimmune-related RKD), antihypertensive drugs (for hypertension-related RKD, e.g., angiotensin-converting enzyme inhibitors and angiotensin receptor blockers), insulin (for diabetic RKD), lipid/cholesterol lowering agents (e.g., HMG-CoA reductase inhibitors such as atorvastatin or simvastatin), treatment of hyperphosphatemia or hyperthyroidism associated with CKD (e.g., sevelamer acetate, cinacalcet), dialysis, and dietary restrictions (e.g., protein, salt, fluid, potassium, phosphate).

VII. Diagnostic Tests

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

B-type natriuretic peptide (BNP) is a 32-amino acid neurohormone that is synthesized in the ventricular myocardium and released into the circulation in response to ventricular dilation and pressure overload. The functions of BNP include natriuresis, vasodilation, inhibition of the renin-angiotensin-aldosterone axis, and inhibition of sympathetic nerve activity. Plasma concentrations of BNP increase in patients with congestive heart failure (CHF) and increase in proportion to the degree of left ventricular dysfunction and the severity of CHF symptoms.

Those skilled in the art are familiar with many methods and devices for measuring BNP content in patient samples (including serum and plasma). For polypeptides such as BNP, immunoassay devices and methods are generally used. See, for example, U.S. Patents 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792. These devices and methods can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats to generate a signal related to the presence or amount of the analyte of interest. In addition, certain methods and devices (such as biosensors and optical immunoassays) can be used to determine the presence or amount of the analyte without the need for labeled molecules. See, for example, U.S. Patents 5,631,170 and 5,955,377. In a specific example, B-type natriuretic peptide (BNP) levels can be measured by protein immunoassay, as described in U.S. Patent Publication No. 2011/0201130, which is incorporated herein by reference in its entirety. In addition, there are several commercially available methods (e.g., Rawlins et al., 2005, which is incorporated herein by reference in its entirety).

B. Measurement of Albumin/Creatinine Ratio (ACR)

Proteinuria is routinely detected by a simple dipstick test. Traditionally, dipstick protein tests quantify the amount of total protein in a 24-hour urine collection.

Alternatively, the protein concentration in urine can be compared with the creatinine content (in the instant urine sample). This is called the protein/creatinine ratio (PCR). The UK Chronic Kidney Disease Guidelines (2005; incorporated herein by reference in its entirety) explain that PCR is a test that is superior to 24-hour urine protein measurement. Proteinuria is defined as a protein/creatinine ratio greater than 45 mg/mmol (which is equivalent to an albumin/creatinine ratio greater than 30 mg/mmol or approximately 300 mg/g, as defined by a 3+ test strip proteinuria), and for a PCR greater than 100 mg/mmol, it is very high proteinuria.

Protein dipstick measurements should not be confused with the amount of protein detected on a test for microalbuminuria, which represents the value of urine protein in mg/day, while urine protein dipstick values represent the value of protein in mg/dL. In other words, there is a baseline level of proteinuria that can be less than 30 mg/day that is considered non-pathological. Values between 30-300 mg/day are called microalbuminuria, which is considered pathological. Urine protein laboratory values of microalbumin>30 mg/day correspond to detected levels in the "trace" to "1+" range on a urine dipstick protein analysis. Therefore, a positive indication for any protein detected by a urine dipstick analysis eliminates any need for performing a urine microalbumin test because the upper limit for microalbuminuria has been exceeded.

C. Measurement of estimated glomerular filtration rate (eGFR)

Several formulas have been developed to estimate GFR values based on serum creatinine levels. A commonly used surrogate marker for estimating creatinine clearance (eC _Cr ) is the Cockcroft-Gault (CG) formula, which in turn estimates GFR (expressed in mL/min). It uses serum creatinine measurements and the patient's weight to predict creatinine clearance. The initially published formula is:

This formula requires weight to be measured in kilograms and creatinine to be measured in mg/dL, as is standard in the USA. If the patient is female, the resulting value is multiplied by a constant of 0.85. This formula is useful because it is simple and can usually be performed without the aid of a calculator.

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

The constant is 1.23 for males and 1.04 for females.

An interesting feature of the Cockcroft and Gault equation is that it shows a dependence of the estimate of _CCr on age. The age term is (140-age). This means that for the same serum creatinine level, the creatinine clearance of a 20-year-old (140-20=120) will be twice that of an 80-year-old (140-80=60). The Cockcroft and Gault equation assumes that the creatinine clearance of women is 15% lower than that of men at the same serum creatinine level.

Alternatively, eGFR values can be calculated using the Modification of Diet in Renal Disease (MDRD) formula. The four-variable formula is as follows:

eGFR = 175 × normalized serum creatinine ^{- 1.154} × age ^{- 0.203} × C

Where C is 1.212 if the patient is a black male, C is 0.899 if the patient is a black female, and C is 0.742 if the patient is a non-black female. Serum creatinine values are based on the IDMS-traceable creatinine assay (see below).

Chronic kidney disease is defined as a GFR less than 60 mL/min/1.73 ^m2 for three months or more.

D. Measurement of pulmonary artery pressure

There are two main methods for measuring pulmonary artery (PA) pressure: transthoracic echocardiography (TTE) and right heart catheterization.

An echocardiogram is an ultrasound of the heart; transthoracic means that the ultrasound probe is placed outside the chest (or "thorax"). Nothing is inserted into the body, so this test is called "non-invasive" and can be performed on an outpatient basis. On a TTE, both the right ventricle and the beginning of the pulmonary artery can be seen. TTE focuses on the right ventricle because it pumps blood into the pulmonary artery. Some blood from the right ventricle does not move forward into the pulmonary artery, but naturally leaks back into the right atrium through the tricuspid valve. When the PA pressure is higher than it should be, the right ventricle has difficulty pumping blood forward, so more blood will leak back through the tricuspid valve. TTE can measure the amount of leakage (or backflow) and use it to estimate the PA pressure. In some patients, PAH is only seen during exercise. In these cases, TTE can be performed after an exercise test (such as walking on a treadmill) to measure the PA pressure.

Right-sided heart catheterization is a more invasive test that requires the placement of a pressure monitor directly into the pulmonary artery. This technique allows for direct measurement of PA systolic and diastolic pressures and therefore generally yields more accurate measurements.

E. Measurement of serum creatinine levels

The serum creatinine test measures the amount of creatinine in the blood and provides an estimate of the glomerular filtration rate. The serum creatinine values in the BEACON and BEAM trials are based on isotope dilution mass spectrometry (IDMS)-traceable creatinine determination. Other commonly used creatinine analysis methods include (1) alkaline picric acid methods (e.g., Jaffe method [classic] and compensated [modified] Jaffe method), (2) enzymatic methods, (3) high performance liquid chromatography, (4) gas chromatography, and (5) liquid chromatography. The IDMS method is widely considered the most accurate analysis (Peake and Whiting, 2006, which is incorporated herein by reference in its entirety).

F. Measurement of Cystatin C Content

Cystatin C can be measured in random serum samples using immunoassays (e.g., turbidimetry or particle-enhanced turbidimetry). Reference values vary across many populations and by sex and age. Across studies, the average reference interval (as defined by the 5th and 95th percentiles) is 0.52 mg/L and 0.98 mg/L. For women, the average reference interval is 0.52 mg/L to 0.90 mg/L, with a mean of 0.71 mg/L. For men, the average reference interval is 0.56 mg/L to 0.98 mg/L, with a mean of 0.77 mg/L. Normal values decrease until the first year of life, remaining relatively stable until they increase again, especially after age 50. Creatinine levels increase until puberty and differ by sex thereafter, making their interpretation in pediatric patients problematic.

In a large study from the United States National Health and Nutrition Examination Survey (Kottgen et al., 2008), the reference interval (as defined by the 1st and 99th percentiles) was 0.57 mg/L and 1.12 mg/L. This range was 0.55-1.18 for women and 0.60-1.11 for men. Non-Hispanic blacks and Mexican Americans have lower normal cystatin C levels. Other studies have found that in patients with impaired kidney function, women have lower cystatin C levels and blacks have higher cystatin C levels for the same GFR. For example, for chronic kidney disease in a 60-year-old white woman, the cutoff value for cystatin C would be 1.12 mg/L, while in black men it would be 1.27 mg/L (a 13% increase). For serum creatinine values adjusted using the MDRD equation, these values would be 0.95 mg/dL to 1.46 mg/dL (54% increase).

G. Measurement of uric acid levels

Serum uric acid levels are usually determined by clinical chemistry methods, for example, spectrophotometric measurements based on the reaction of uric acid with a specific reagent to form a colored reaction product. Since uric acid determination is a standard clinical chemistry test, several products are commercially available for this purpose.

In human plasma, the reference range for uric acid is generally 3.4-7.2 mg/dL (200-430 μmol/L) (1 mg/dL=59.48 μmol/L) for men and 2.4-6.1 mg/dL (140-360 μmol/L) for women. However, blood test results should always be interpreted using the range provided by the laboratory performing the test. Uric acid concentrations above and below the normal range in plasma are referred to as hyperuricemia and hypouricemia, respectively. Similarly, uric acid concentrations above and below the normal range in urine are referred to as hyperuricemia and hypouricemia.

H. Measurement of circulating endothelial cells (CEC)

CECs were isolated from whole blood using CD146Ab (an antibody against the CD146 antigen expressed on endothelial cells and leukocytes). After CEC isolation, FITC (fluorescent isothiocyanate)-conjugated CD105 antibodies (specific antibodies for endothelial cells) were used to identify CECs using the CellSearch ^™ system. A fluorescent conjugate of the CD45 antibody was added to stain the leukocytes, and these leukocytes were then gated. For a general overview of this method, see Blann et al. (2005), which is incorporated herein by reference in its entirety. CEC samples were also analyzed for the presence of iNOS by immunostaining.

VIII. Definitions

When used in the context of chemical groups: "hydrogen" means -H; "hydroxy" means -OH; "oxo" means =O; "carbonyl" means -C(=O)-; "carboxy" means -C(=O)OH (also written as -COOH or _-CO2H ); "halo" independently means -F, -Cl, -Br, or -I; "amino" means _-NH2 ; "hydroxyamino" means -NHOH; "nitro" means -NO2; imino means = _NH ; "cyano" means -CN; "isocyanate" means -N=C=O; "azido" means _-N3 ; and in a monovalent context, "phosphate" means -OP(O)(OH) ₂ or its deprotonated form; in a divalent context, "phosphate" means -OP(O)(OH)O- or its deprotonated form; "thiol" means -SH; and "sulfanyl/thio" means ═S; "sulfonyl" means -S(O) ₂ -; and "sulfinyl" means -S(O)-.

In the context of chemical formulae, the symbol "-" means a single bond, "=" means a double bond, and "≡" means a triple bond. The symbol "----" represents an optional bond, which, if present, is a single bond or a double bond. The symbol represents a single bond or a double bond. Thus, for example, the formula includes and is understood to include that no such ring atom forms part of more than one double bond. Furthermore, it should be noted that the covalent bond symbol "-" does not indicate any preferred stereochemistry when connecting one or two stereogenic atoms. Instead, it encompasses all stereoisomers and mixtures thereof. The symbol "-" indicates the point of attachment to a group when drawn vertically through a bond (e.g., for a methyl group). It should be noted that the point of attachment of larger groups is generally identified in this manner only to help the reader clearly identify the point of attachment. The symbol "-" indicates a single bond where the group connected to the thick end of the wedge is "outside the page." The symbol "-" indicates a single bond where the group connected to the thick end of the wedge is "inside the page." The symbol "-" indicates a single bond where the geometry of the double bond (e.g., E or Z) is undefined. Therefore, both options and combinations thereof are contemplated. When any of the atoms connected by the bond is a metal atom (M), the above bond orders are not limiting. In these cases, it should be understood that the actual bonding may include significant multiple bonding and/or ionic character. Therefore, unless otherwise indicated, the formulas M-C, M=C, M----C and each refer to bonds of any type and order between metal atoms and carbon atoms. Undefined valences on atoms of the structures shown in this application imply hydrogen atoms bonded to the atoms. Dots on carbon atoms indicate that the hydrogen attached to the carbon is oriented outside the plane of the paper.

When the group "R" is depicted as a "floating group" on a ring system, for example in the following formula:

Then R can replace any hydrogen atom (including drawn, implied or explicitly defined hydrogen) attached to any of the ring atoms as long as a stable structure results. When the group "R" is depicted as a "floating group" of a fused ring system, such as (for example) in the following formula:

Unless otherwise indicated, R can replace any hydrogen attached to any of the ring atoms of any fused ring. Replaceable hydrogens include depicted hydrogens (e.g., hydrogen attached to nitrogen in the above formula), implied hydrogens (e.g., hydrogen not shown in the above formula but understood to be present), explicitly defined hydrogens, and optional hydrogens whose presence depends on the identity of the ring atoms (e.g., hydrogen attached to the group X when X is equal to -CH-), as long as a stable structure is formed. In the illustrated example, R can be located on a 5-membered or 6-membered ring of the fused ring system. In the above formula, the subscript letter "y" immediately following the group "R" and enclosed in brackets represents a numerical variable. Unless otherwise indicated, this variable can be 0, 1, 2, or any integer greater than 2, subject to the maximum number of replaceable hydrogen atoms in the ring or ring system.

For the groups and classes below, the subscripts in parentheses further define the group/class as follows: "(Cn)" defines the exact number of carbon atoms (n) in the group/class. "(C≤n)" defines the maximum number of carbon atoms (n) that can be present in the group/class, and the minimum number of carbon atoms in the group in question should be as small as possible. For example, it should be understood that the minimum number of carbon atoms in the group "alkenyl _(C≤8) " or the class "alkene _(C≤8) " is 2. For example, "alkoxy _(C≤10) " refers to those alkoxy groups having 1 to 10 carbon atoms. (Cn-n') defines the minimum number (n) and maximum number (n') of carbon atoms in the group. Similarly, "alkyl _(C2-10) " designates those alkyl groups having 2 to 10 carbon atoms.

As used herein, the term "saturated" means that the compound or group so modified has no carbon-carbon double bonds and no carbon-carbon triple bonds, except as noted below. In substituted forms of saturated groups, one or more carbon-oxygen double bonds or carbon-nitrogen double bonds may be present. When such bonds are present, carbon-carbon double bonds are not excluded as part of keto-enol tautomerism or imine/enamine tautomerism.

The term "aliphatic," when used without the "substituted" modifier, indicates that the compound/group so modified is a hydrocarbon compound or group that is acyclic or cyclic, but not aromatic. In an aliphatic compound/group, the carbon atoms may be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds/groups may be saturated (i.e., joined by single bonds) (alkanes/alkyls), or unsaturated, having one or more double bonds (alkenes/alkenyls) or one or more triple bonds (alkynes/alkynyls).

The term "alkyl," when used without the "substituted" modifier, refers to a monovalent saturated aliphatic radical having a carbon atom as the point of attachment, having a linear or branched, cyclic, cyclic, or acyclic structure, and having no atoms other than carbon and hydrogen present. Thus, as used herein, cycloalkyl is a subset of alkyl in which the carbon atom forming the point of attachment is also a member of one or more non-aromatic ring structures, wherein the cycloalkyl group consists solely of carbon and hydrogen atoms. As used herein, the term does not exclude the presence of one or more alkyl groups attached to a ring or ring system (carbon number limitations permitting). The groups _-CH3 (Me), _-CH2CH3 (Et), _-CH2CH2CH3 (n-Pr or propyl), _-CH (CH3 _{)2 (} i-Pr, ^iPr or isopropyl), -CH( _CH2 ) ₂ (cyclopropyl), _{-CH2CH2CH2CH3} (n- _Bu ), -CH ₍ CH3 _{) CH2CH3} ( _sec -butyl), _-CH2CH (CH3) ₂ (isobutyl), _-C ( _CH3 ) ₃ (tert-butyl, tert-butyl, t-Bu or ^t -Bu), _-CH2C ( _{CH3 ) 3} (neopentyl), cyclobutyl, cyclopentyl _, cyclohexyl and cyclohexylmethyl are _non -limiting examples of alkyl groups. The term "alkanediyl," when used without the "substituted" modifier, refers to a divalent saturated aliphatic radical having one or two saturated carbon atoms as points of attachment, a straight or branched chain, cyclic, cyclic, or acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups _-CH2- (methylene), _-CH2CH2- , _-CH2C ( _CH3 ) _2CH2- , _{-CH2CH2CH2- ,} and are non-limiting examples of _alkanediyl radicals. The _term "alkylene," when used without the "substituted" modifier, refers to the divalent radical =CRR', where R and R' are independently hydrogen, _{alkyl ,} or R and R' taken together represent an alkanediyl radical having at least two carbon atoms. Non-limiting examples of alkylene radicals include = _CH2 , =CH( _CH2CH3 ), and =C( _CH3 ) ₂ . "Alkane" refers to the compound HR in which R is alkyl, as that term is defined above. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms _{have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3 , -OCH2CH3 , -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2 , -C ( O ) NH2 , -OC ( O} )CH3, or _-S ( _{O)2NH2 .} The following groups are non-limiting examples of substituted alkyl groups: _-CH2OH , -CH2Cl, _-CF3 , _-CH2CN , _-CH2C (O)OH, _-CH2C (O) _OCH3 , _-CH2C (O)NH2, _-CH2C (O) _CH3 , _-CH2OCH3 , _-CH2OC (O)CH3, _-CH2NH2 , _-CH2N ( _CH3 ) _{2 ,} and _{-CH2CH2Cl .} The term "haloalkyl" is a subgroup of substituted alkyl groups in which one or _more hydrogen atoms have been replaced _by a halo group and no atoms other than carbon, _hydrogen , and _halogen are present. The group _-CH2Cl is a non-limiting example of a haloalkyl group. The term "fluoroalkyl" is a subgroup of substituted alkyl groups in which one or more hydrogen atoms have been replaced by a fluorine group and no atoms other than carbon, hydrogen, and fluorine are present. The groups _-CH2F , _-CF3 , and _-CH2CF3 are non-limiting examples of fluoroalkyl _groups .

The term "alkenyl," when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic radical having a carbon atom as the point of attachment, a straight or branched chain, cyclic, cyclic or acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples of alkenyl groups include: -CH= _CH2 (vinyl), -CH _{= CHCH3} , _-CH = _CHCH2CH3 , -CH2CH= _CH2 (allyl), _-CH2CH = _CHCH3 , _-CH =CHCH=CH2, and -CH=CH- _{C6H5 .} The term "alkenediyl," when used without the "substituted" modifier, refers to a divalent unsaturated aliphatic group having two carbon atoms as points of attachment, a linear or branched, cyclic, cyclic, or acyclic structure, having at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C( _CH3 ) _CH2- , -CH= _CHCH2- , and are non-limiting examples of alkenediyl groups. It should be noted that although alkenediyl is aliphatic, this does not preclude the group from forming an aromatic structure once attached at both ends. The term "alkene" or "olefin" is synonymous and refers to a compound of formula HR, where R is an alkenyl group, as that term is defined above. "Terminal olefin" refers to an olefin having only one carbon-carbon double bond, wherein the bond forms a vinyl group at one end of the molecule. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, _-CO2H , _-CO2CH3 , -CN, -SH, _-OCH3 , _-OCH2CH3 , -C(O) _CH3 , _{-NHCH3, -NHCH2CH3, -N(CH3)2 , -C} (O _{) NH2} , -OC( _O ) _CH3 , or _-S (O _{) 2NH2} . _The groups -CH=CHF, -CH=CHCl, and -CH=CHBr _are non-limiting examples of substituted _alkenyl groups.

The term "alkynyl," when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight or branched chain, cyclic, cyclic, or acyclic structure, having at least one carbon-carbon triple bond, and the absence of atoms other than carbon and hydrogen. As used herein, the term alkynyl does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, _-C≡CCH3 , and _-CH2C≡CCH3 are non-limiting examples of alkynyl _groups . "Alkyne" refers to the compound HR, where R is an alkynyl group. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have independently been replaced with -OH, -F, -Cl, -Br, -I, -NH2, _-NO2 , _-CO2H , -CO2CH3, -CN, -SH, _-OCH3 , _-OCH2CH3 , _-C (O _{) CH3} , _-NHCH3 , _-NHCH2CH3 , -N( _CH3 ) ₂ , -C ₍ O) _NH2 , _-OC (O) _CH3 , or -S( _{O ) 2NH2} .

The term "aryl," when used without the "substituted" modifier, refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as the point of attachment, said carbon atom forming part of one or more six-membered aromatic ring structures in which all of the ring atoms are carbon, and wherein the group consists solely of carbon and hydrogen atoms. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl or aralkyl groups attached to the first aromatic ring or any other aromatic rings present (carbon number limitations permitting). Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl) _{phenyl, -C6H4CH2CH3 ( ethylphenyl} ), naphthyl, and _monovalent groups derived from biphenyl. The term "aryldiyl" when used without the "substituted" modifier refers to a divalent aromatic group having two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more 6-membered aromatic ring structures in which all of the ring atoms are carbon and wherein the monovalent group consists solely of carbon and hydrogen atoms. As used herein, the term does not exclude the presence of one or more alkyl, aryl, or aralkyl groups (carbon number limitations permitting) attached to the first aromatic ring or any other aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected via one or more of the following: covalent bonds, alkanediyl, or alkenediyl (carbon number limitations permitting). Non-limiting examples of aryldiyl include:

"Aromatic" refers to the compound HR in which R is an aromatic group, as that term is defined above. Benzene and toluene are non-limiting examples of aromatics. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, _-F , _-Cl , -Br, _-I , _-NH2 , _-NO2 , -CO2H, -CO2CH3, -CN, -SH, _-OCH3 , _-OCH2CH3 , _-C (O) _CH3 , _-NHCH3 , -NHCH2CH3, -N( _{CH3 ) 2 ,} -C(O) _NH2 , -OC(O) _CH3 , or -S( _{O)2NH2 .}

The term "aralkyl," when used without the "substituted" modifier, refers to the monovalent radical -alkanediyl-aryl, wherein the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples of aralkyl are phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the "substituted" modifier, one or more hydrogen atoms from the alkanediyl and/or aryl groups are replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2 _{, -CO2H} , _-CO2CH3 , -CN, -SH, _-OCH3 , _-OCH2CH3 , _-C (O) _CH3 , _-NHCH3 , _-NHCH2CH3 , _-N (CH3) ₂ , -C(O) _NH2 , -OC( _O ) _CH3 , or -S(O) _2NH2 . Non _- limiting examples of substituted aralkyl groups are (3-chlorophenyl)-methyl and 2-chloro-2 _{- phenyl} -eth-1-yl.

The term "heteroaryl," when used without the "substituted" modifier, refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as the point of attachment, said carbon or nitrogen atom forming part of one or more aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and wherein the heteroaryl group consists solely of carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur atoms. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl, aryl, and/or aralkyl groups attached to an aromatic ring or aromatic ring system (carbon number limitations permitting). Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridyl, oxazolyl, phenylpyridyl, pyridyl, pyrrolyl, pyrimidinyl, pyrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as the point of attachment. The term "heteroaryldiyl" when used without the "substituted" modifier refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as two points of attachment, these atoms forming part of one or more aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and wherein the divalent group consists only of carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur atoms. If there is more than one ring, the rings may be fused or unfused. Unfused rings may be connected via one or more of the following: covalent bonds, alkanediyl, or alkenediyl (carbon number restrictions permitting). As used herein, the term does not exclude the presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number restrictions permitting) attached to an aromatic ring or aromatic ring system. Non-limiting examples of heteroaryldiyl include:

"Heteroarene" refers to the compound HR where R is a heteroaryl group. Pyridine and quinoline are non-limiting examples of heteroarene. When these terms are used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, _-NH2 , _-NO2 , _-CO2H , _-CO2CH3 , -CN, -SH _{, -OCH3} , _-OCH2CH3 , -C(O) _CH3 , _-NHCH3 , _-NHCH2CH3 , -N( _{CH3 ) 2} , -C(O _{) NH2} , -OC(O) _CH3 , or -S( _O ) _2NH2 .

The term "heterocycloalkyl," when used without the "substituted" modifier, refers to a monovalent non-aromatic group having a carbon or nitrogen atom as the point of attachment, wherein the carbon or nitrogen atom forms part of one or more non-aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and wherein the heterocycloalkyl group consists solely of carbon, hydrogen, nitrogen, oxygen, and sulfur atoms. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl groups attached to a ring or ring system (carbon number limitations permitting). Similarly, the term does not exclude the presence of one or more double bonds in a ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. The term "heterocycloalkanediyl" when used without the "substituted" modifier refers to a divalent cyclic group with two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as two points of attachment, these atoms forming part of one or more ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and wherein the divalent group consists solely of carbon, hydrogen, nitrogen, oxygen, and sulfur atoms. If there is more than one ring, the rings may be fused or unfused. Unfused rings may be connected via one or more of the following: covalent bonds, alkanediyl, or alkenediyl (carbon number restrictions permitting). As used herein, the term does not exclude the presence of one or more alkyl groups attached to the ring or ring system (carbon number restrictions permitting). Similarly, the term does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkanediyl include:

When these terms are used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, _-NH2 , -NO2, _-CO2H , _-CO2CH3 , -CN, -SH, _-OCH3 , _-OCH2CH3 , _{-C (} O) _CH3 , _-NHCH3 , -NHCH2CH3 _, -N( _CH3 ) ₂ , -C(O ₎ NH2, -OC(O) _CH3 , -S(O) _2NH2 , _or -C(O) _OC ( _CH3 ) ₃ (tert-butoxycarbonyl, BOC) _.

The term "acyl" when used without the "substituted" modifier refers to the group -C(O)R, in which R is hydrogen, alkyl, aryl, aralkyl, or heteroaryl, as those terms are defined above. The groups -CHO, -C(O) _CH3 (acetyl, Ac), -C(O) _CH2CH3 , -C(O) _{CH2CH2CH3 , -C} (O)CH ₍ CH3 _{) 2} , -C(O) _CH ( _CH2 ) ₂ , -C(O) _C6H5 , -C(O) _C6H4CH3 , -C ₍ O) _CH2C6H5 , -C(O)(imidazolyl ₎ are non-limiting examples of acyl groups. "Sulfacyl" is defined in a similar manner, except that the oxygen atom _of the group -C(O) _R has been replaced by a sulfur atom, -C(S)R. The term "aldehyde" corresponds to an alkane as defined above in which at least one hydrogen atom has been replaced by a -CHO group. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms (including the hydrogen atom directly attached to the carbonyl or thiocarbonyl group, if any) are replaced by -OH, -F, _-Cl , _-Br , -I, _-NH2 , _-NO2 , _-CO2H , -CO2CH3, _-CN , -SH, _-OCH3 , _-OCH2CH3 , -C(O) _CH3 , _-NHCH3 , _-NHCH2CH3 , -N(CH3 _{)2 ,} -C( _O ) _NH2 , -OC(O) _CH3 , or -S( _O ) _2NH2 . The groups -C(O) _CH2CF3 , _-CO2H (carboxy), _-CO2CH3 ( _{methylcarboxy} ), _-CO2CH2CH3 , -C(O _{) NH2 (} carbamoyl), and -CON( _CH3 ) ₂ are non _- limiting examples of substituted acyl groups.

The term "alkoxy," when used without the "substituted" modifier, refers to the radical -OR, in which R is alkyl, as that term is defined above. Non-limiting examples of alkoxy include: _-OCH3 (methoxy), _-OCH2CH3 (ethoxy), _-OCH2CH2CH3 , -OCH( _{CH3 ) 2} (isopropyloxy ₎ , -O(CH3) ₃ (tert-butyloxy), -OCH( _CH2 ) _{2 ,} -O-cyclopentyl, and -O- _cyclohexyl . The terms "alkenyloxy,""alkynyloxy,""aryloxy,""aralkyloxy,""heteroaryloxy,""heterocycloalkyloxy," and "acyloxy," when used without the "substituted" modifier, refer to radicals as defined for -OR, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term "alkoxydiyl" refers to the divalent radical -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio," when used without the "substituted" modifier, refer to the radical -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane, as defined above, in which at least one hydrogen atom has been replaced by a hydroxy group. The term "ether" corresponds to an alkane, as defined above, in which at least one hydrogen atom has been replaced by an alkoxy group. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have independently been replaced with -OH, -F, -Cl, -Br, -I, -NH2, _-NO2 , _-CO2H , -CO2CH3, -CN, -SH, _-OCH3 , _-OCH2CH3 , _-C (O _{) CH3} , _-NHCH3 , _-NHCH2CH3 , -N( _CH3 ) ₂ , -C ₍ O) _NH2 , _-OC (O) _CH3 , or -S( _{O ) 2NH2} .

The term "alkylamino" when used without the "substituted" modifier refers to the group -NHR, in which R is alkyl, as that term is defined above. Non-limiting examples of alkylamino include: _-NHCH3 and _-NHCH2CH3 . The term "dialkylamino" when used without the "substituted" modifier refers to the group -NRR', in which R and R' may be the same or different alkyl groups, or R and R' may be taken together to represent an alkanediyl group _. Non-limiting examples of dialkylamino include: -N( _CH3 ) ₂ , -N( _CH3 )( _CH2CH3 ), and _N -pyrrolidinyl. The terms "alkoxyamino,""alkenylamino,""alkynylamino,""arylamino,""aralkylamino,""heteroarylamino,""heterocycloalkylamino," and "alkylsulfonylamino" when used without the "substituted" modifier refer to groups defined as -NHR, in which R is alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is _-NHC6H5 . The term "acylamino" (acylamino) when used without the "substituted" modifier refers to the group -NHR, in which R is acyl, as that term is defined above. A non-limiting example of an acylamino group is _-NHC (O) _CH3 . The term "alkylimino" when used without the "substituted" modifier refers to the divalent group =NR, in which R is alkyl, as that term is defined above. The term "alkylaminodiyl" refers to the divalent radical -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, _-I , _-NH2 , -NO2, _-CO2H , _-CO2CH3 , -CN, -SH, -OCH3, _{-OCH2CH3 , -C} (O) _CH3 , _-NHCH3 , _-NHCH2CH3 , -N(CH3) ₂ , _-C (O) _NH2 , -OC( _O ) _CH3 , or _-S (O) _2NH2 . The groups -NHC(O) _OCH3 and -NHC(O) _NHCH3 are non-limiting examples of substituted amido _groups .

The terms "alkylsulfonyl" and "alkylsulfinyl" when used without the "substituted" modifier refer to the groups -S(O) _2R and -S(O)R, respectively, in which R is alkyl, as such terms are defined above. The terms "alkenylsulfonyl,""alkynylsulfonyl,""arylsulfonyl,""aralkylsulfonyl,""heteroarylsulfonyl," and "heterocycloalkylsulfonyl" are defined in a similar manner. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have independently been replaced with -OH, -F, -Cl, -Br, -I, -NH2, _-NO2 , _-CO2H , -CO2CH3, -CN, -SH, _-OCH3 , _-OCH2CH3 , _-C (O _{) CH3} , _-NHCH3 , _-NHCH2CH3 , -N( _CH3 ) ₂ , -C ₍ O) _NH2 , _-OC (O) _CH3 , or -S( _{O ) 2NH2} .

The term "alkylphosphate" when used without the "substituted" modifier refers to the group -OP(O)(OH)(OR), in which R is alkyl, as that term is defined above. Non-limiting examples of alkylphosphate groups include: -OP(O)(OH)(OMe) and -OP(O)(OH)(OEt). The term "dialkylphosphate" when used without the "substituted" modifier refers to the group -OP(O)(OR)(OR'), in which R and R' may be the same or different alkyl groups, or R and R' may be taken together to represent an alkanediyl group. Non-limiting examples of dialkylphosphate groups include: -OP(O)(OMe) ₂ , -OP(O)(OEt)(OMe), and -OP(O)(OEt) ₂ . When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have independently been replaced with -OH, -F, -Cl, -Br, -I, -NH2, _-NO2 , _-CO2H , -CO2CH3, -CN, -SH, _-OCH3 , _-OCH2CH3 , _-C (O _{) CH3} , _-NHCH3 , _-NHCH2CH3 , -N( _CH3 ) ₂ , -C ₍ O) _NH2 , _-OC (O) _CH3 , or -S( _{O ) 2NH2} .

In the claims and/or description, the word "a" or "an" when used in conjunction with the term "comprising" may mean "one", but is also consistent with the meaning of "one or more", "at least one" and "one or more than one".

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation in error for the device or method being employed to determine the value, or the variability that exists between the individual study subjects.

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

As used herein, "chiral auxiliary" refers to a removable chiral group that can affect the stereoselectivity of a reaction. Those skilled in the art are familiar with these compounds, and many are commercially available.

The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs (e.g., "comprises," "comprising," "has," "having," "includes," and "including") are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to possessing only those one or more steps and also encompasses other unlisted steps.

When the term "effective" is used in the specification and/or claims, the term means sufficient to accomplish a desired, expected, or intended result. "Effective amount,""therapeutically effective amount," or "pharmaceutically effective amount," when used in the context of treating a patient or individual with a compound, means the amount of the compound that, when administered to an individual or patient for treating a disease, is sufficient to achieve such treatment for the disease. In the case of PAH in humans, a medical response to a therapeutically effective amount may include any one or more of the following: 1) an improvement of 5-10 meters, 10-20 meters, 20-30 meters, or more in the 6-minute walk test, compared to a baseline study before initiation of therapy; 2) an improvement in World Health Organization functional class from Class IV to Class III, Class IV to Class II, Class IV to Class I, Class III to Class II, Class III to Class I, or Class II to Class I, wherein the previous class is the WHO class before initiation of therapy; 3) a decrease in mean pulmonary artery pressure of 2-4 mm Hg, 4-6 mm Hg, 6-10 mm Hg, or more, compared to a baseline study performed before initiation of therapy; 4) an increase in cardiac index of 0.05-0.1 liter/min/ ^m2 , 0.1-0.2 liter/min/ ^m2 , 0.2-0.4 liter/min/m2, compared to a baseline study performed before initiation of therapy. ² or more; 5) PVR improves (i.e., decreases) by 25-100 dyne sec/ ^cm5 , 100-200 dyne sec/ ^cm5 , 200-300 dyne sec/ ^cm5 , or more from a baseline value obtained before starting therapy; 6) right atrial pressure decreases by 0.1-0.2 mmHg, 0.2-0.4 mmHg, 0.4-1 mmHg, 1-5 mmHg, or more compared to a baseline study performed before starting therapy; 7) survival is improved compared to a group of patients not given therapy. The time between the baseline study before starting therapy and the time of efficacy assessment can vary, but will typically range from 4-12 weeks, 12-24 weeks, or 24-52 weeks. Examples of therapeutic efficacy endpoints are given in McLaughlin et al. (2002), Galie et al. (2005), Barst et al. (1996), McLaughlin et al. (1998), Rubin et al. (2002), Langleben et al. (2004) and Badesch et al. (2000).

The term "hydrate" when applied as a modifier to a compound means that the compound has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than one (e.g., dihydrate) molecule of water associated with each molecule of the compound (e.g., a compound in solid form).

As used herein, the term " _IC50 " refers to the inhibitory dose that is 50% of the maximal response obtained. This quantitative measurement indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical, or chemical process (or component of a process, i.e., an enzyme, cell, cell receptor, or microorganism) by half.

An "isomer" of a first compound is another compound wherein each molecule contains the same constituent atoms as the first compound, but differs in the three-dimensional configuration of those atoms.

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

As used generally herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and/or body fluids of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit/risk ratio.

"Pharmaceutically acceptable salts" means salts of the compounds of the present invention that are pharmaceutically acceptable as defined above and possess the desired pharmacological activity. These salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or acid addition salts formed with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(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 acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid , camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic 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 acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic 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. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It will be appreciated that the specific anion or cation forming part of any salt of the present invention is not critical, so long as the salt as a whole is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts, methods for their preparation, and uses are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl and C.G. Wermuth, eds., Verlag Helvetica Chimica Acta, 2002).

As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle involved in the carrying or delivery of a chemical agent, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.

"Prevention" or "preventing" includes: (1) inhibiting the onset of a disease in an individual or patient who may be at risk for and/or susceptible to a disease but who does not yet experience or display any or all of the pathology or symptoms of the disease, and/or (2) slowing the onset of a pathology or symptom of a disease in an individual or patient who may be at risk for and/or susceptible to a disease but who does not yet experience or display any or all of the pathology or symptoms of the disease.

"Prodrug" means a compound that can be metabolically converted into an inhibitor of the present invention in vivo. The prodrug itself may or may not be active relative to a given target protein. For example, a compound comprising a hydroxyl group can be administered as an ester that is converted into a hydroxyl compound by hydrolysis in vivo. Suitable esters that can be converted into hydroxyl compounds in vivo include acetate, citrate, lactate, phosphate, tartrate, malonate, oxalate, salicylate, propionate, succinate, fumarate, maleate, methylene-bis-β-hydroxynaphthoate, gentisate, isethionate, di-p-toluoyl tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylaminosulfonate, quinate, esters of amino acids, and the like. Similarly, a compound comprising an amine group can be administered as an amide that is converted into an amine compound by hydrolysis in vivo.

"Stereoisomers" or "optical isomers" are isomers of a given compound in which the same atoms are bonded to the same other atoms but the three-dimensional configuration of those atoms differs. "Enantiomers" are stereoisomers of a given compound that are mirror images of one another, like the left hand and the right hand. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also called a stereocenter or stereogenic center. This is any point in a molecule that carries groups, but not necessarily atoms, such that the exchange of any two groups produces a stereoisomer. In organic compounds, chiral centers are typically carbon, phosphorus, or sulfur atoms, but other atoms can also serve as stereocenters in both organic and inorganic compounds. A molecule can have multiple stereocenters, which gives rise to many stereoisomers. In compounds where stereoisomerism is due to tetrahedral stereogenic centers (e.g., tetrahedral carbon), it is assumed that the total number of possible stereoisomers will not exceed 2n, where n is the number of tetrahedral stereocenters. Molecules with symmetry often have fewer than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched, such that one enantiomer is present in an amount greater than 50%. Generally, enantiomers and/or diastereomers can be resolved or separated using techniques known in the art. This encompasses any stereocenter or chiral axis for which the stereochemistry has not been defined, where the stereocenter or chiral axis can exist in its R form, S form, or 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 another stereoisomer.

"Treatment" or "treating" includes: (1) inhibiting the disease in a subject or patient experiencing or exhibiting pathology or symptoms of the disease (e.g., arresting further development of the pathology and/or symptoms), (2) alleviating the disease in a subject or patient experiencing or exhibiting pathology or symptoms of the disease (e.g., reversing the pathology and/or symptoms), and/or (3) achieving any measurable reduction in the disease in a subject or patient experiencing or exhibiting pathology or symptoms of the disease.

The above definitions supersede any conflicting definitions in any reference incorporated herein by reference. However, the fact that certain terms are defined should not be construed as indicating that any undefined term is undefined. Rather, it is believed that all terms used will describe the present invention in a manner that allows those skilled in the art to understand the scope and practice the present invention.

IX. Examples

The present invention includes the following examples to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in practicing the invention and, therefore, can be considered to constitute preferred modes for its practice. However, those skilled in the art will appreciate, in light of this disclosure, that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

Example 1 - Bardoxolone Methyl Alleviates Endothelial Dysfunction

In a clinical trial of bardoxolone methyl (BARD) in patients with stage 3b or 4 chronic kidney disease and type 2 diabetes, significant improvements in the level of circulating endothelial cells were noted (Figure 2). Improvements were also noted in the number of CECs that tested positive for inducible nitric oxide synthase (iNOS), an inflammation-promoting enzyme. Nonclinical studies have shown that BARD and other AIMs can reduce ROS levels in endothelial cells and increase NO bioavailability therein (Figure 3).

Nonclinical studies have also shown that BARD can reduce the expression of endothelin-1 (ET-1) in mesangial cells (found in the kidney) and endothelial cells (Table 22). In this study, BARD also reduced the expression of the vasoconstrictive _ETA receptor while increasing the expression of the vasodilatory _ETB receptor (Table 22). ET-1 (a natural peptide) is the most potent endogenous vasoconstrictor and has been implicated in the pathogenesis of several cardiovascular diseases. ET-1 also acts as a mitogen and a proinflammatory signaling molecule. Excessive ET-1 activity is a source of endothelial dysfunction, partly due to inhibition of NO signaling (Sud and Black, 2009) and partly due to proinflammatory effects (Pernow, 2012). Therefore, inhibition of excessive ET-1 signaling is considered a potentially attractive therapeutic strategy for certain cardiovascular diseases. Endothelin receptor antagonists have been studied in many of these diseases and are approved for the treatment of pulmonary arterial hypertension. However, as discussed above, inhibition of ET-1 signaling may have adverse consequences in certain patient populations and patients in these groups should be excluded from treatment with agents that counteract ET-1 signaling.

Table 22. Effects of bardoxolone methyl on Nrf2 target and endothelin gene expression in human mesangial and endothelial cells

Human mesangial and endothelial cells were treated with bardoxolone methyl (50 or 250 nM) or vehicle. mRNA levels of Nrf2 target genes (NQO1, SRXN1, GCLC, and GCLM), as well as endothelin-1 and endothelin receptors A and B, were quantified. Values represent fold change relative to vehicle control; NC = no change.

In vivo treatment with AIM also altered the expression of endothelin receptors. The bardoxolone methyl analog RTA dh404 was tested in the 5/6 nephrectomy model of chronic renal failure in rats (a widely accepted model of ultrafiltration and pressure overload-induced kidney damage and failure). Increased oxidative stress and inflammation caused by increased NF-κB and decreased Nrf2 activity in the 5/6 nephrectomy model led to an approximately 30% increase in systolic and diastolic blood pressure (Kim, 2010). In addition, the 5/6 nephrectomy model is associated with intrarenal and extrarenal hypertension and endothelial dysfunction.

In the 5/6 nephrectomy model, in addition to providing histological protection and suppressing the expression of proinflammatory and profibrotic mediators, the bardoxolone methyl analog RTA dh404 promoted a vasodilatory endothelin receptor phenotype, whereby the induction of _ETA receptors was completely suppressed and the reduced expression of _ETB receptors was partially reversed (Figure 4). These data further provide evidence that bardoxolone methyl and analogs modulate the endothelin pathway to reverse endothelial dysfunction and promote vasodilation.

Example 2 - Effects of RTA dh404 on lung histology in a rat model of monocrotaline-induced pulmonary hypertension

The effects of orally administered bardoxolone methyl analog RTA dh404 were evaluated in a rat model of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). MCT is a macrolytic pyrrolizidine alkaloid that is activated in the liver by cytochrome P450 enzymes to a toxic metabolite (i.e., dehydromonoctaline), which then induces a syndrome characterized by PAH, pulmonary mononuclear vasculitis, and right ventricular hypertrophy (Gomez-Arroyo et al., 2012).

To evaluate RTA dh404, male Sprague-Dawley rats received a single injection of MCT on day 1 and then received vehicle (sesame oil), RTA dh404 (2, 10, or 30 mg/kg/day), or the positive control sildenafil (60 mg/kg/day) for 21 days. Lung tissue was then analyzed histopathologically for arterial hypertrophy, cellular infiltration, and pulmonary edema. Vehicle lungs appeared artificially more severe due to poor expansion during treatment, in part due to loss of compliance due to injury and edema. RTA dh404 inhibited MCT-induced microscopic changes in the lungs (i.e., arteriolar hypertrophy, pulmonary edema, and cellular and fibrin infiltration) at all dose levels, with the 10 mg/kg RTA dh404 dose being as effective as the positive control sildenafil. Treatment with 10 mg/kg/day RTA dh404 and 60 mg/kg/day sildenafil completely eliminated pulmonary edema ( FIG. 18 ).

Inhibition of MCT-induced PAH by injection of RTA dh404 was also associated with a dose-dependent increase in the mRNA expression of antioxidant Nrf2 target genes and a significant induction and decrease in the expression of pro-inflammatory NF-κB target genes (Figures 19 and 20).

Taken together, these data demonstrate that RTA dh404 administration is associated with improvements in lung histopathology in a rat model of MCT-induced PAH that are associated with induction of antioxidant Nrf2 target genes and repression of proinflammatory NF-κB target genes.

Example 3 - Dose-ranging study of efficacy and safety of bardoxolone methyl in patients with pulmonary hypertension

This two-part Phase 2 trial will investigate the safety, tolerability, and efficacy of bardoxolone methyl in patients with WHO Group 1 PAH. Part 1 will be a double-blind, randomized, dose-ranging, placebo-controlled treatment phase, and Part 2 will be an extension phase. Eligible patients must have been receiving oral, disease-specific PAH therapy consisting of an endothelin-receptor antagonist (ERA) and/or a phosphodiesterase type 5 inhibitor (PDE5i). The dose of the previous therapy must have been stable for at least 90 days prior to Day 1.

Part 1: Part 1 of the study will consist of two cohorts: dose escalation and expansion. Dose escalation cohorts will be enrolled, one at a time. Each cohort will include the next eight eligible patients randomized in a 3:1 ratio to receive either bardoxolone methyl or matching placebo, administered once daily for 16 weeks. The starting dose of bardoxolone methyl will be 2.5 mg, followed by doses of 5 mg, 10 mg, 20 mg, and 30 mg.

For each dose escalation group, after all 8 patients complete the Week 4 visit, the Protocol Safety Review Committee (PSRC) will use all available data from this study to evaluate the safety and tolerability of bardoxolone methyl to determine the appropriate dose for the next dose escalation group. The PSRC will choose to randomize the next higher dose, lower dose, or another dose escalation group at the current dose. Up to 8 dose escalation groups will be recruited to initially evaluate the safety and tolerability of bardoxolone methyl.

In each dose escalation assessment, the PSRC will also evaluate the data for pharmacodynamic activity and efficacy signs and may recommend adding an expansion group to further characterize the safety and efficacy of up to two bardoxolone methyl doses. The expansion groups will be recruited, one group at a time, and each will include at least 24 patients, who will be randomized at a 3:1 ratio to receive bardoxolone methyl or matching placebo. The two expansion groups will be randomized at the dose selected by the PSRC. The expansion group will be recruited only at a subgroup of the site selected by the trial sponsor for cardiopulmonary exercise test (CPET) evaluation, and all patients recruited in the expansion group will be required to undergo CPET evaluation. Additional near-infrared spectroscopy (NIRS) muscle testing and muscle biopsy are optional for the dose expansion group at the qualified site. The expansion group will be recruited in parallel with the dose escalation group, however, the randomization of these groups will be carried out independently. The size of the expansion cohorts may be increased by up to a total of 10 patients in both cohorts to ensure that at least 24 patients in each cohort have fully evaluable CPET assessments at baseline for comparison with the Week 16 assessments. Therefore, the two expansion cohorts combined will not exceed a total of 58 patients.

All patients in Part 1 of the study (i.e., both the dose-escalation and expansion groups) will follow the same visit schedule. Following randomization, patients will be evaluated on-site at Weeks 1, 2, 4, 8, 12, and 16 and by telephone on Days 3, 10, and 21 during the treatment period. Patients who discontinue study drug during Part 1 or who complete the planned 16-week treatment period but choose not to continue Part 2 of the study and are unable to enter Part 2 (i.e., the extension phase) will complete the end-of-treatment visit and a follow-up visit 4 weeks after the last dose of study drug.

Part 2 (Extension Phase): Patients who prematurely discontinued treatment in Part 1 of the study were ineligible to continue into Part 2 of the study. All patients from Part 1 who completed the planned 16-week treatment period will be eligible to continue directly into the extension phase to assess the intermediate and long-term safety and efficacy of bardoxolone methyl. Day 1 of the extension phase will coincide with the Week 16 treatment visit, and therefore, patients continuing in the extension phase will continue to take the same dose of study drug. Patients randomized to placebo in Part 1 of the study will be assigned to receive their group-specific dose of bardoxolone methyl during the extension phase. During the first four weeks of the extension phase, all patients will be evaluated using the same four-week visit schedule as in Part 1 and will be visited every 12 weeks thereafter for the duration of the extension phase, as long as they continue taking study drug. If study drug administration is discontinued during Part 2 of the study, patients will be evaluated on-site at the final follow-up visit, which will occur four weeks after the last dose of study drug. The extension phase is planned to last at least 12 weeks after the last patient enters the extension portion of the study.

A. Patient groups

Up to 122 patients (64 in the dose escalation cohort and 58 in the expansion cohort) will be enrolled in Part 1 of the study. All eligible patients from Part 1 will be included in Part 2, and no new patients will be randomized to Part 2 of the study.

The main criteria for inclusion will be:

1. Adult male and female patients aged ≥18 to ≤75 years old at the time of informed consent;

2. BMI>18.5kg/ ^m2 ;

3. Symptomatic pulmonary hypertension WHO/NYHA FC class II and III;

4. One of the following subtypes of WHO group 1 PAH:

a. Idiopathic or hereditary PAH;

b. PAH associated with connective tissue diseases;

c. PAH associated with simple, congenital systemic-to-pulmonary shunts at least 1 year after shunt repair;

d. Anorexigen-related PAH;

e. PAH associated with human immunodeficiency virus (HIV);

5. A diagnostic right heart catheterization has been performed and documented within 36 months prior to screening, confirming the diagnosis of PAH based on all of the following criteria:

a. Mean pulmonary artery pressure ≥ 25 mm Hg (at rest);

b. Pulmonary capillary wedge pressure (PCWP) ≤ 15 mmHg;

c. Pulmonary vascular resistance >240 dyn.sec/cm ⁵ or >3 mm Hg/liter (L)/minute;

6. BNP content ≤200 pg/mL;

7. Average 6-minute walk distance (6MWD) ≥ 150 meters and ≤ 450 meters on two consecutive tests performed on different days during the screening period, with the two test measurements within 15% of each other.

8. Already receiving oral, disease-specific PAH therapy consisting of an endothelin-receptor antagonist (ERA) and/or a phosphodiesterase type 5 inhibitor (PDE5i). PAH therapy must have been at a stable dose for at least 90 days prior to Day 1; (revised to include "at least one, but not more than two (2) disease-specific PAH therapies, including an endothelin-receptor antagonist (ERA), riocitabine, a phosphodiesterase type 5 inhibitor (PDE5i), or a prostacyclin (subcutaneous, oral, or inhaled)"

9. If receiving any of the following therapies that can affect PAH, the dose has been maintained at a stable level for 30 days prior to Day 1: vasodilators (including calcium channel blockers), digoxin, L-arginine supplements, or oxygen supplements;

10. If receiving prednisone, maintain a stable dose of ≤20 mg/day (or equivalent dose if other corticosteroids) for at least 30 days before Day 1. If receiving any other drug for connective tissue disease (CTD), the dose should remain stable for the duration of the study;

11. No evidence of significant parenchymal lung disease as determined by pulmonary function testing (PFT) performed within 90 days prior to Day 1 based on the following criteria:

a. Forced expiratory volume in one second (FEV1) ≥ 65% (estimated);

b. FEV1/forced vital capacity ratio (FEV1/FVC) ≥ 65%; or

c. Total vital capacity ≥ 65% (estimated), which must be measured in patients with connective tissue disease;

12. Ventilation-perfusion (V/Q) lung scan, spiral (spiral/helical/) electron beam computed tomography (CT), or pulmonary angiography prior to screening showed no signs of thromboembolic disease (i.e., normal or low-probability pulmonary embolism should be noted). If the V/Q scan is abnormal (i.e., results other than normal or low-probability), confirmatory CT or selective pulmonary angiography must be performed to rule out chronic thromboembolic disease;

13. Have adequate renal function defined as estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m ² using the Modification of Diet in Renal Disease (MDRD) 4-variable formula (note: this was subsequently reduced to ≥45 mL/min/1.73 m ² );

14. Willing and able to comply with scheduled visits, treatment plans, laboratory tests, and other study procedures;

15. Evidence of a signed and dated informed consent form indicating that the patient (or legal representative) has informed all relevant parties of the research before commencing any mandatory patient procedures.

The main criteria for exclusion will be:

1. Participation in other investigational clinical studies involving a medicinal product being tested or used in a manner different from the approved form or for an unapproved indication within the 30 days preceding Day 1;

2. Participate in an intensive exercise training program for pulmonary rehabilitation within 90 days prior to screening;

3. Receiving long-term treatment with prostacyclin/prostacyclin analogs within 60 days before Day 1. The use of prostacyclin for acute vasodilator testing during right heart catheterization is allowed;

4. Required intravenous inotropic drugs (inotrope) within 30 days before Day 1;

5. Uncontrolled systemic hypertension, as indicated by a sitting systolic blood pressure (BP) > 160 mm Hg or a sitting diastolic blood pressure > 100 mm Hg after a period of rest during the screening period;

6. Systolic BP < 90 mm Hg after a period of rest during the screening period;

7. Clinically significant left-sided heart disease and/or a history of clinically significant heart disease, including (but not limited to) any of the following:

a. Clinically significant congenital or acquired valvular heart disease, excluding tricuspid valve insufficiency due to pulmonary hypertension;

b. Pericardial constriction;

c. Restrictive or congestive cardiomyopathy;

d. Left ventricular ejection fraction <40% during screening echocardiography (ECHO);

e. Any current or previous history of symptomatic coronary heart disease (previous myocardial infarction, percutaneous coronary intervention, coronary artery bypass graft surgery, or anginal chest pain);

8. Acute decompensated heart failure within 30 days before Day 1, as assessed by the investigator;

9. Two or more of the following clinical risk factors for left ventricular diastolic dysfunction:

a. Age > 65 years old;

b.BMI ≥ 30 kg/m ² ;

c. History of systemic hypertension;

d. History of type 2 diabetes;

e. History of atrial fibrillation;

10. History of atrial septostomy within 180 days before Day 1;

11. History of untreated obstructive sleep apnea;

12. For patients with HIV-associated PAH, any of the following:

a. Active opportunistic infection within 180 days prior to screening;

b. Detectable viral load within 90 days prior to screening;

c. Cluster designation (CD+) T-cell count <200 mm ³ within 90 days before screening;

d. Changes in antiretroviral regimen within 90 days prior to screening;

e. Use of inhaled pentamidine;

13. Patients with a history of portal hypertension or chronic liver disease, including hepatitis B and/or hepatitis C (with evidence of recent infection and/or active viral replication), defined as moderate to severe liver damage (Child-Pugh Class A-C);

14. Serum transaminase (ALT or AST) level > upper limit of normal (ULN) at screening;

15. Hemoglobin (Hgb) concentration <8.5g/dL at screening;

16. Diagnosis of Down syndrome;

17. History of malignant tumor within 5 years before screening, except for local skin or cervical cancer;

18. Active bacterial, fungal or viral infection incompatible with the research;

19. Known or suspected active drug or alcohol abuse;

20. Underwent major surgery within 30 days before screening or planned to undergo major surgery during the study period;

21. Unwilling to use contraceptive methods during the screening period, while taking the study drug, and for at least 30 days after taking the last dose of the study drug (both males with partners of reproductive potential and females of reproductive potential);

22. Pregnant or breastfeeding women;

23. Any disability or injury that will prevent the implementation of the 6MWT;

24. Any abnormal laboratory value that, in the opinion of the investigator, would put the trial enrolled patients at risk;

25. According to the researcher's opinion, the patient is unable to comply with the requirements of the study protocol or is unsuitable for the study for any reason;

26. Known hypersensitivity to any component of the study drug;

27. Inability to communicate or cooperate with researchers due to language problems, poor mental development, or impaired brain function.

B. Procedure

During Part 1 of the study, bardoxolone methyl (2.5 mg, 5 mg, 10 mg, 20 mg, or 30 mg) or placebo will be administered orally once daily in the morning for 16 weeks. During Part 2 of the study, bardoxolone methyl (2.5 mg, 5 mg, 10 mg, 20 mg, or 30 mg) will be administered orally once daily in the morning for the duration of the extension period.

The sample size of eight patients randomized in each dose escalation group with a 3:1 (bardoxolone methyl:placebo) assignment ratio included six patients treated with bardoxolone methyl for the identification of the overall safety signal. It is expected that a small number of patients at each dose will not be adequately characterized for safety, so the identification of an issue of concern in only one of the six patients (16%) treated with bardoxolone methyl may suggest the need to collect additional information by adding another group at the current dose level or a lower dose as determined by the PSRC before dose escalation. At each of the two doses selected for expansion, the combined group size of 32 patients with a 3:1 assignment ratio (dose escalation group N=8; expansion group N=24) provides a minimum of 24 patients treated with bardoxolone methyl to characterize safety, tolerability, and efficacy.

C. Results

The objectives of this study will be to determine a recommended dose range for further studies of bardoxolone methyl, to evaluate the change from baseline in 6-minute walk distance (6MWD) in those patients treated with bardoxolone methyl for 16 weeks versus those given placebo for 16 weeks, and to evaluate the safety and tolerability of 16 weeks of treatment with bardoxolone methyl versus 16 weeks of placebo.

The following criteria will be evaluated:

Efficacy: Change from baseline in 6-minute walk distance (6MWD); N-terminal pro-B-type natriuretic peptide (NT-Pro BNP); Borg Dyspnea Index; WHO/NYHA Pulmonary Arterial Hypertension (PAH) Functional Class (FC); parameters collected during Doppler cardiac echocardiography (ECHO), cardiopulmonary exercise testing (CPET), optional cardiac magnetic resonance imaging (MRI), optional near-infrared spectroscopy (NIRS) muscle testing, and optional muscle biopsy at Week 16; and clinical worsening.

Safety: Frequency, intensity, and relationship of adverse events and serious adverse events to study drug, concomitant medications, and changes from baseline in the following assessments: physical examination, vital sign measurements, 24-hour ambulatory blood pressure monitoring (ABPM), 12-lead electrocardiogram (ECG), clinical laboratory measurements, and body weight.

Pharmacokinetics: Bardoxolone methyl plasma concentration-time data, metabolite concentration-time data, and estimated pharmacokinetic parameters for each analyte.

***

All of the methods disclosed and claimed herein can be made and performed without undue experimentation in accordance with the present invention. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be made to the steps or sequence of steps of these methods and the methods described herein without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically related reagents may be substituted for the reagents described herein while achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

X. References

The following references are expressly incorporated herein by reference to the extent that they provide exemplary procedural or other details supplementary to those set forth herein.

U.S. Patent No. 5,480,792

U.S. Patent No. 5,525,524

U.S. Patent No. 5,631,170

U.S. Patent No. 5,679,526

U.S. Patent No. 5,824,799

U.S. Patent No. 5,851,776

U.S. Patent No. 5,885,527

U.S. Patent No. 5,922,615

U.S. Patent No. 5,939,272

U.S. Patent No. 5,947,124

U.S. Patent No. 5,955,377

U.S. Patent No. 5,985,579

U.S. Patent No. 6,019,944

U.S. Patent No. 6,025,395

U.S. Patent No. 6,113,855

U.S. Patent No. 6,143,576

U.S. Patent Application No. 12/352,473

U.S. Patent Publication No. 2003/0232786

U.S. Patent Publication No. 2008/0261985

U.S. Patent Publication No. 2009/0048204

U.S. Patent Publication No. 2009/0326063

U.S. Patent Publication No. 2010/0041904

U.S. Patent Publication No. 2010/0048887

U.S. Patent Publication No. 2010/0048892

U.S. Patent Publication No. 2010/0048911

U.S. Patent Publication No. 2010/0056777

U.S. Patent Publication No. 2011/0201130

PCT Pub. WO 2009/023232

PCT Pub. WO 2010/093944

Badesch et al., Continuous intravenous epoprostenol for pulmonaryhypertension due to the scleroderma spectrum of disease. A randomized, controlled trial. Ann. Intern. Med., 132:425-434, 2000.

Badesch et al., Medical therapy for pulmonary arterial hypertension: ACCPevidence-based clinical practice guidelines. Chest, 126:35S-62S, 2004.

Barst et al., A comparison of continuous intravenous epoprostenol (prostacyclin) with conventional therapy for primary pulmonary hypertension. N. Engl. J. Med., 334: 296-301, 1996.

Blann et al., Thromb. Haemost., 93:228-235, 2005.

Bolignano et al., Pulmonary Hypertension in CKD, Am.J. Kidney Dis., 61:612-622, 2013.

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.

Deng et al., Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-IIgene. Am.J.Hum.Genet., 67:737-44, 2000.

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.

Dumitrascu et al., Oxid. Med. Cell. Longev., 2013:234631, 2013.

Forstermann, Biol. Chem., 387:1521-1533, 2006.

Galie et al., Ambrisentan therapy for pulmonary arterial hypertension. J. Am. Coll. Cardiol., 46:529-535, 2005.

Gologanu et al., Rom J Intern Med., 50:259-68, 2012.

Gomez-Arroyo et al., The monocrotaline model of pulmonary hypertension inperspective. Am. J. Physiol. Lung Cell. Mol. Physiol., 302: L363-L369, 2012.

Guazzi and Galie, Eur. Respir. Rev., 21:338-346, 2012.

Hansson et al., Annu. Rev. Pathol. Mech. Dis., 1:297-329, 2006.

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

Hayes and Dinkova-Kostova, The Nrf2regulatory network provides an interface between redox and intermediary metabolism. Trends in Biochem Sci., 39: 199-218, 2014.

Honda et al., J. Med. Chem., 43: 1866-1877, 2000a.

Honda et al., J. Med. Chem., 43:4233-4246, 2000b.

Honda et al., Bioorg. Med. Chem. Lett., 12:1027-1030, 2002.

Hosokawa, Cardiovascular Res., 99:35-43, 2013.

Humbert, New Engl. J. Med., 351:1425-1436, 2004.

Jones et al., Activation of thromboxane and prostacyclin receptor selicits opposing effects on vascular smooth muscle cell growth and mitogen-activated protein kinase signaling cascades. Mol. Pharm., 48:890-89, 1995.

Kataoka et al., Oral sildenafil improves primary pulmonary hypertension refractory to epoprostenol. Circ. J., 69:461-465, 2005.

Kim and Vaziri, Contribution of impaired Nrf2-Keap1 pathway to oxidativestress and inflammation in chronic renal failure. American Journal of Physiology-Renal Physiology, 298: F662-F671, 2010.

Kosmadakis et al., Ren. Fail., 35:514-20, 2013.

Kottgen et al., Serum cystatin C in the United States: the Third National Health and Nutrition Examination Survey, Am. J. Kidney Dis., 51:385-394, 2008.

Langleben et al., STRIDE 1: Effects of the Selective ETA Receptor Antagonist, Sitaxsentan Sodium, in a Patient Population with Pulmonary Arterial Hypertension that meets Traditional Inclusion Criteria of Previous Pulmonary Arterial Hypertension Trials. J. Cardiovasc. Pharmacol., 44: S80-S84.9, 2004.

Lee et al., Sildenafil for pulmonary hypertension. Ann. Pharmacother., 39:869-884, 2005.

Mann et al., Avosentan for overt diabetic nephropathy. Journal of the American Society of Nephrology, 21,: 527-535, 2010.

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

McGoon et al., Screening, early detection, and diagnosis of pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest, 126:14S-34S, 2004.

McLaughlin et al., Reduction in pulmonary vascular resistance with long-term epoprostenol (prostacyclin) therapy in primary pulmonary hypertension. N. Engl. J. Med., 338:273-277, 1998.

McLaughlin et al., Survival in primary pulmonary hypertension: the impact of epoprostenol therapy. Circulation, 106:1477-1482, 2002.

McLaughlin and Rich, Pulmonary hypertension. Curr. Probl. Cardiol., 29:575-634, 2004.

Oudiz et al., Treprostinil, a prostacyclin analogue, in pulmonary arterial hypertension associated with connective tissue disease. Chest, 126:420-427, 2004.

Packer, Vasodilator therapy for primary pulmonaryhypertension. Limitations and hazards. Ann. Intern. Med., 103: 258-270, 1985.

Paulin et al., STAT3 signaling in pulmonary arterial hypertension, JAK-STAT, 1:223-233, 2012.

Peake and Whiting, Clin. Biochem. Rev., 27:173-184, 2006.

Pergola et al., Bardoxolone methyl and kidney function in CKD with type2diabetes., New Engl. J. Med., 365, 327-336, 2011.

Rawlins et al., Am. J. Clin. Pathol., 123:439-445, 2005.

Rich et al., The effects of high doses of calcium-channel blockers onsurvival in primary pulmonary hypertension. N. Engl. J. Med., 327:76-81, 1992.

Rubin et al., Bosentan therapy for pulmonary arterial hypertension. N. Engl. J. Med., 346:896-903, 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.

Sussan et al., Targeting Nrf2 with the triterpenoid CDDO-imidazolideattenuate cigarette smoke-induced emphysema and cardiac dysfunction inmice. Proc. Natl. Acad. Sci. U.S.A., 106: 250-255, 2009.

Sutendra and Michelakis, The metabolic basis of pulmonary arterialhypertension. Cell Metabolism, 19:558-573, 2014.

Tam et al., A derivative of Bardoxolone methyl,dh404,in an inverse dose-dependent manner,lessens diabetes-associated atherosclerosis and improvesdiabetic kidney disease.Diabetes,doi:10.2337/db13-1743,2014.

The International PPH Consortium, Lane et al., Heterozygous germlinemutations in BMPR2, encoding a TGF-b receptor, cause familial primary pulmonaryhypertension. Nature Genetics, 26:81-84, 2000.

Thenappan et al., A USA-based registry for pulmonary arterial hypertension: 1982-2006. European Respiratory Journal, 30: 1103-1110, 2007.

Vachiéry and Davenport, The endothelin system in pulmonary and renalvasculopathy:les liaisons dangereuses. European Respiratory Review, 18: 260-271, 2009.

Vachiery and Naeije, Treprostinil for pulmonary hypertension. Expert Rev. Cardiovasc. Ther., 2:183-191, 2004.

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.

Claims (74)

1. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of pulmonary hypertension in patients of need. The patient in question has been identified as not having at least one of the following characteristics: (a) History of left-sided myocardial disease; (b) Increased levels of B-type natriuretic peptide (BNP); (c) An elevated albumin/creatinine ratio (ACR); and (d) Chronic kidney disease (CKD). 2. The use of claim 1, wherein the patient has been identified as not having chronic kidney disease. 3. The use of claim 1 or 2, wherein the patient has been identified as having pulmonary hypertension. 4. The use of claim 1 or 2, wherein the patient’s pulmonary artery pressure has been measured or will be measured. 5. The use of claim 4, wherein the patient's pulmonary artery pressure has been measured before administration of the compound and will be measured after administration of the compound. 6. The use of claim 1 or 2, wherein the patient does not have a history of left-sided myocardial disease. 7. The use of claim 1 or 2, wherein the patient does not have elevated BNP levels. 8. The use of claim 7, wherein the elevated BNP content is greater than 200 pg/mL. 9. The use of claim 1 or 2, wherein the patient does not have elevated ACR. 10. The use of claim 9, wherein the elevated ACR is greater than 300 mg/g. 11. The use of claim 1 or 2, wherein the estimated glomerular filtration rate (eGFR) of the said patient is greater than or equal to 45 mL/min/1.73 ^m² . 12. The use of claim 1 or 2, wherein the patient's eGFR is greater than or equal to 60 mL/min/1.73 ^m² . 13. The use of claim 1 or 2, wherein the patient is less than 75 years old. 14. The use of claim 13, wherein the patient is less than 70 years old. 15. The use of claim 14, wherein the patient is less than 65 years of age. 16. The use of claim 15, wherein the patient is less than 60 years old. 17. The use of claim 16, wherein the patient is less than 55 years old. 18. The use of claim 1 or 2, wherein the patient has been identified as not having COPD. 19. The use of claim 1 or 2, wherein the patient is not a smoker. 20. The use of claim 1 or 2, wherein the patient does not have kidney disease. 21. The use of claim 1 or 2, wherein the patient does not have elevated levels of kidney disease-related biomarkers. 22. The use of claim 21, wherein the biomarker is serum creatinine. 23. The use of claim 21, wherein the biomarker is cysteine protease inhibitor C. 24. The use of claim 21, wherein the biomarker is uric acid. 25. The use of claim 1 or 2, wherein the patient does not have stage 4 chronic kidney disease (CKD). 26. The use of claim 1 or 2, wherein the patient has been identified as not having cancer. 27. The use of claim 1 or 2, wherein the patient has been identified as not having type 2 diabetes. 28. The use of claim 1 or 2, wherein the patient is a human patient. 29. The use of claim 1 or 2, wherein at least a portion of said compound is presented in crystalline form as CuKα having an X-ray diffraction pattern containing prominent diffraction peaks at 8.8, 12.9, 13.4, 14.2 and 17.4°2θ. 30. The use of claim 29, wherein the X-ray diffraction pattern CuKα is shown in Figure 1A or Figure 1B. 31. The use of claim 1 or 2, wherein at least a portion of the compound is in an amorphous form having an X-ray diffraction pattern CuKα with a halo peak at 13.5°2θ as shown in FIG1C and a glass transition temperature ( _Tg ). 32. The use of claim 31, wherein the T _g value is in the range of 120°C to 135°C. 33. The use of claim 32, wherein the T _g value is in the range of 125°C to 130°C. 34. The use of claim 1 or 2, wherein the compound is administered to the patient in a pharmaceutically effective amount, the pharmaceutically effective amount being a daily dose of 0.1 mg to 300 mg of the compound. 35. The use of claim 34, wherein the daily dose is 0.5 mg to 200 mg of the compound. 36. The use of claim 35, wherein the daily dose is from 1 mg to 150 mg of the compound. 37. The use of claim 36, wherein the daily dose is 1 mg to 75 mg of the compound. 38. The use of claim 37, wherein the daily dose is 1 mg to 20 mg of the compound. 39. The use of claim 34, wherein the daily dose is 2.5 mg to 30 mg of the compound. 40. The use of claim 39, wherein the daily dose is 2.5 mg of the compound. 41. The use of claim 39, wherein the daily dose is 5 mg of the compound. 42. The use of claim 39, wherein the daily dose is 10 mg of the compound. 43. The use of claim 39, wherein the daily dose is 20 mg of the compound. 44. The use of claim 39, wherein the daily dose is 30 mg of the compound. 45. The use of claim 1 or 2, wherein the compound is administered to the patient in a pharmaceutically effective amount, the pharmaceutically effective amount being a daily dose of 0.01 mg of the compound to 100 mg of the compound per kilogram of body weight. 46. The use of claim 45, wherein the daily dose is from 0.05 mg of compound to 30 mg of compound per kilogram of body weight. 47. The use of claim 46, wherein the daily dose is from 0.1 mg of compound to 10 mg of compound per kilogram of body weight. 48. The use of claim 47, wherein the daily dose is from 0.1 mg of compound to 5 mg of compound per kilogram of body weight. 49. The use of claim 48, wherein the daily dose is from 0.1 mg of compound to 2.5 mg of compound per kilogram of body weight. 50. The use of claim 1 or 2, wherein the compound is given to the patient in a pharmaceutically effective amount, the pharmaceutically effective amount being given daily in a single dose. 51. The use of claim 1 or 2, wherein the compound is given to the patient in a pharmaceutically effective amount, the pharmaceutically effective amount being given in two or more doses daily. 52. The use of claim 1 or 2, wherein the compound is administered orally, intra-arterially, or intravenously. 53. The use of claim 1 or 2, wherein the compound is formulated into hard capsules, soft capsules, or tablets. 54. The use of claim 1 or 2, wherein the compound is formulated into a solid dispersion comprising (i) the compound and (ii) an excipient. 55. The use of claim 54, wherein the excipient is a copolymer of methacrylate and ethyl acrylate. 56. The use of claim 55, wherein the copolymer comprises methacrylic acid and ethyl acrylate in a 1:1 ratio. 57. The use of claim 1 or 2, wherein the medicament is prepared to be administered to the patient by means of a further second therapy. 58. The use of claim 57, wherein the second therapy comprises administering a pharmaceutically effective amount of the second drug to the patient. 59. The use of claim 58, wherein the second drug is a Rho kinase inhibitor, a tyrosine kinase inhibitor, an eprostol derivative, a serotonin receptor antagonist, an endothelin receptor antagonist, a phosphodiesterase (PDE) inhibitor, a calcium channel blocker, a soluble guanylate cyclase stimulator, or nitric oxide. 60. The use of claim 59, wherein the Rho kinase inhibitor is HA-1077, HA-1152P, or Y-27632. 61. The use of claim 59, wherein the tyrosine kinase inhibitor is Gleevec. 62. The use of claim 59, wherein the eprostol derivative is treprost, betaprost, or iloprost. 63. The use of claim 59, wherein the 5-hydroxytryptamine receptor antagonist is saxagrel ester. 64. The use of claim 59, wherein the endothelin receptor antagonist is bosentan, sitassentan, ambesentan, TBC3711, or macitentan. 65. The use of claim 59, wherein the PDE inhibitor is amrinone, sildenafil, tadalafil, vardenafil, enoximone, udenafil, or milrinone. 66. The use of claim 59, wherein the calcium channel blocker is amlodipine, diltiazem, iladipine, nicardipine, nifedipine, nimodipine, nisodipine, nifedipine, and verapamil. 67. The use of claim 59, wherein the soluble guanylate cyclase stimulant is riocipidate. 68. The use according to claim 1 or 2, wherein the pulmonary hypertension is pulmonary arterial hypertension. 69. The use according to claim 68, wherein the pulmonary hypertension is associated with connective tissue disease. 70. The use according to claim 68, wherein the pulmonary hypertension is idiopathic. 71. The use according to claim 1 or 2, wherein the pulmonary hypertension is associated with left-sided heart disease. 72. The use according to claim 1 or 2, wherein the pulmonary hypertension is associated with lung disease. 73. The use according to claim 1 or 2, wherein the pulmonary hypertension is chronic thromboembolic pulmonary hypertension. 74. The use according to claim 1 or 2, wherein the pulmonary hypertension is pulmonary hypertension with multifactorial etiology.