Methods for Treating Pulmonary Hypertension

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 62/728,973, filed September 10, 2018, the entire contents of which are incorporated herein by reference.

BACKGROUND

[0002] Pulmonary hypertension is a progressive disease of various origins associated with a poor prognosis and results in right heart dysfunction. In all its variant presentations, it is estimated to affect up to 100 million people worldwide. Pulmonary hypertension is defined as a lung disorder characterized by increased pressure in the pulmonary artery. The pulmonary artery carries oxygen-poor blood from the lower chamber on the right side of the heart (right ventricle) to the lungs where it picks up oxygen. Pulmonary hypertension is present when the blood pressure in the circulation of the lungs is measured at greater than 25 mm of mercury (Hg) at rest or 30 mm Hg during exercise.

[0003] Pulmonary arterial hypertension (PAH), a category of chronic pulmonary hypertension, includes a variety of diseases that share several pathophysiological, histological, and prognostic features. PAH is a life-threatening disease that is characterized by increased pulmonary vascular resistance owing to progressive vascular remodeling, which can ultimately lead to right heart failure and death (Schermuly RT. et al., Nature Reviews Cardiology, 2011; 8: 443-455). PAH is characterized by excessive pulmonary vasoconstriction and abnormal vascular remodeling where inflammation plays a major role. Specifically, the vasoconstriction, remodeling of the pulmonary vessel wall marked by intimal fibrosis and increased medial thickness, pulmonary arteriolar occlusion,

thrombosis, and plexiform lesions are all key features of PAH (Humbert M. et ah, NEJM, 2004, 351: 1425-1436). There is growing evidence that release of local mediators in the lung is responsible for the inflammation and vascular remodeling that characterize PAH. The right ventricular response to increased pressure is recognized as critical to survival in patients with PAH (Schermuly RT. et ah, Nature Reviews Cardiology, 2011; 8: 443-455). [0004] Current treatments include phosphodiesterase type 5 inhibitors, prostanoids, endothelin receptor antagonists, and Riociguat, a member of a novel therapeutic class known as soluble guanylate cyclase stimulators (Ghofrani HA. et ah, NEJM, 2013, 369: 330-340). Riociguat has a dual mode of action, acting in synergy with endogenous nitric oxide and also directly stimulating soluble guanylate cyclase independently of nitric oxide availability. In several phase 1 and 2 clinical studies, Riociguat improved hemodynamic variables and exercise capacity in patients with pulmonary arterial hypertension

(Grimminger F. et al., The Eur. Resp. ., 2009, 33: 785-792). Results of the phase 3 Pulmonary Arterial Hypertension Soluble Guanylate Cyclase Stimulator Trial 1 (PATENT - 1). investigated the efficacy and side-effect profile of Riociguat in patients with

symptomatic pulmonary arterial hypertension, both those who were receiving no other treatment for the disease and those who were receiving treatment with endothelin receptor antagonists or nonintravenous prostanoids. Riociguat significantly improved exercise capacity in patients with pulmonary arterial hypertension. This benefit was consistent in patients who were receiving endothelin-receptor antagonists or prostanoids and in those who were receiving no other treatment for the disease. Riociguat also significantly and consistently improved a range of secondary efficacy end points, including pulmonary hemodynamics, WHO functional class, and time to clinical worsening (Ghofrani HA. et al., NEJM , 2013, 369: 330-340). Despite treatment, mortality remains high and there is a considerable unmet medical need in the management of this disorder.

SUMMARY OF THE DISCLOSURE

[0005] An aspect of this disclosure is directed to a method for treating pulmonary hypertension (PAH) comprising administering to a subject in need of such treatment an effective amount of a mast cell stabilizer.

[0006] In some embodiments, the mast cell stabilizer is selected from the group consisting of cromoglicic acid, ketotifen, olopatadine, rupatadine, mepolizumab, omalizumab, pemirolast, quercetin, nedocromil, azelastine, tranilast, palmitoylethanolamide, and vitamin

D.

[0007] In some embodiments, the mast cell stabilizer is nedocromil. [0008] In some embodiments, the mast cell stabilizer is administered systemically. In some embodiments, the mast cell stabilizer is administered locally to the lung.

[0009] Another aspect of this disclosure is directed to a method for treating pulmonary hypertension comprising administering to a subject in need of such treatment an effective amount of a HIF prolyl hydroxylase domain inhibitor.

[0010] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is selected from the group consisting of Roxadustat (RXD) (FG-4592), Vadadustat (AKB-6548),

Daprodustat (GSK- 1278863), and Molidustat (BAY 85-3934).

[0011] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered systemically. In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered locally to the lung.

[0012] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is RXD.

[0013] Another aspect of this disclosure is directed to a method for treating pulmonary hypertension comprising administering to a subject in need of such treatment a

combination therapy comprising an effective amount of a mast cell stabilizer and an effective amount of a HIF prolyl hydroxylase domain inhibitor.

[0014] In some embodiments, the mast cell stabilizer and the HIF prolyl hydroxylase domain inhibitor are administered as one composition. In some embodiments, the mast cell stabilizer and the HIF prolyl hydroxylase domain inhibitor are administered separately. In some embodiments, the mast cell stabilizer and the HIF prolyl hydroxylase domain inhibitor are administered consecutively. In some embodiments, the mast cell stabilizer and the HIF prolyl hydroxylase domain inhibitor are administered simultaneously.

[0015] In some embodiments, the mast cell stabilizer is selected from the group consisting of cromoglicic acid, ketotifen, olopatadine, rupatadine, mepolizumab, omalizumab, pemirolast, quercetin, nedocromil, azelastine, tranilast, palmitoylethanolamide, and vitamin

D.

[0016] In some embodiments, the mast cell stabilizer is nedocromil. [0017] In some embodiments, the mast cell stabilizer is administered systemically. In some embodiments, the mast cell stabilizer is administered locally to the lung.

[0018] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is selected from the group consisting of Roxadustat (RXD) (FG-4592), Vadadustat (AKB-6548),

Daprodustat (GSK- 1278863), and Molidustat (BAY 85-3934).

[0019] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is RXD.

[0020] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered systemically. In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered locally to the lung.

[0021] Another aspect of this disclosure is directed to a use of a mast cell stabilizer for the manufacture of a medicament for use in the treatment of pulmonary hypertension.

[0022] Another aspect of this disclosure is directed to a use of a HIF prolyl hydroxylase domain inhibitor for the manufacture of a medicament for use in the treatment of pulmonary hypertension.

[0023] Another aspect of this disclosure is directed to a use of a mast cell stabilizer and a HIF prolyl hydroxylase domain inhibitor for the manufacture of a medicament for use in the treatment of pulmonary hypertension.

[0024] In some embodiments, the mast cell stabilizer is selected from the group consisting of cromoglicic acid, ketotifen, olopatadine, rupatadine, mepolizumab, omalizumab, pemirolast, quercetin, nedocromil, azelastine, tranilast, palmitoylethanolamide, and vitamin

D.

[0025] In some embodiments, the mast cell stabilizer is nedocromil.

[0026] In some embodiments, the mast cell stabilizer is administered systemically. In some embodiments, the mast cell stabilizer is administered locally to the lung. [0027] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is selected from the group consisting of Roxadustat (RXD) (FG-4592), Vadadustat (AKB-6548),

Daprodustat (GSK- 1278863), and Molidustat (BAY 85-3934).

[0028] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is Roxadustat (RXD) (FG-4592).

[0029] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered systemically. In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered locally to the lung.

[0030] In some embodiments, the pulmonary hypertension is pulmonary arterial hypertension (PAH).

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The patent or application file contains at least one drawing executed in color.

Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0032] FIGS. 1A - IB. Mast cells are increased in pulmonary arteries with PAH. (A)

PAH (from monocrotaline (MCT)-injected rats) leads to an increase in the # of mast cells (MCs) in pulmonary artery (PA) (***p< 0.001; n=4 rats, control (CON (untreated)) and n=4 rats, MCT). (B) MCs are found in the PA wall in PAH. Toluidene-blue stained cross- sections of control (untreated) (CON) and MCT-rat (MCT) PA. PA from MCT-injected rat has MCs in the adventitia (a) and media (m) layers of the PA wall (white arrows), not observed in the CON PA. Notice the increased wall thickness in PA from MCT-injected rat compared to untreated CON as indicated by the black line. Viewed at 40x. Intima =

CD-

[0033] FIGS. 2A - 2C. Stabilizing mast cells prevents remodeling of the pulmonary artery (PA) wall associated with PAH (MCT-injected rats). Cross-sections of PA stained with Gomori's trichrome; collagen is stained blue. (A) Untreated control (CON), (B) MCT- injected, and (C) MCT-injected + nedocromil (a mast cell stabilizer) -treated PAs. Line in each image shows thickness of PA wall. Viewed at 15c. [0034] FIGS. 3A - 3B. Treating PAH (monocrotaline (MCT) rats) with the mast cell stabilizer nedocromil (+ treatment) reduced interstitial pulmonary fibrosis as evidenced by its reversed effect on elastance (A) and compliance (B).

[0035] FIGS. 4A - 4C. Stabilizing Mast Cells Prevents Abnormal Flow Velocity in PAH. Representative Doppler flow traces of pulmonary artery (PA) flow velocities from (A) Untreated control (CON), (B) PAH monocrotoline (MCT)-treated rats, and (C) MCT+mast cell stabilizer (nedocromil) -treated rats. Mast cell stabilization (C) prevents PA wave reflection typical of PAH (white arrows, B). Mast-cell stabilizer treated samples more closely resemble untreated controls (CON, A).

[0036] FIGS. 5A - 5D. Association of Increased Mast Cell Number with Excess Collagen Deposition in Right Ventricle of MCT-Treated Rats. Representative consecutive sections of right ventricle (RV) from (A) Control (CON) rat stained for mast cells (MSc). (B) PAH (monocrotaline (MCT)-injected ) rat stained for mast cells. (C) Control (CON) rat stained for collagen. (D) PAH (monocrotaline (MCT)-injected) rat stained for collagen. There is an increase in the number of mast cells populating the RV in MCT-injected rat compared to CON (B(PAH) vs A (CON)). The MCT-injected rat also has abundant collagen deposition as shown by the blue staining in (D) whereas there was minimal collagen staining in CON (C). The spatial distribution of mast cells correlated with the collagen stained regions of the RV in the MCT-rat (consecutive sections C and D).

[0037] FIGS. 6A - 6C. Treatment with a mast cell stabilizer (nedocromil) prevents right ventricular dilation (right ventricular overload) and its effect on the interventricular septum in PAH (monocrotaline (MCT))-injected rat (28d). (A) Echocardiogram in B-mode on control (CON) rats. (B) Echocardiogram in B-mode on MCT-injected (MCT) rats. (C) Echocardiogram in B-mode on PAH (monocrotaline (MCT)) and mast cell stabilizer (nedocromil) -injected rat.

[0038] FIG. 7. Blocking the ANG II ATiR with the active metabolite of losartan (ATiR- B) inhibits the mast cell-induced contraction of PA from PAH (monocrotaline (MCT))-rats. The contraction is expressed as percentage of the response to K+ (60 mM) in PA rings from CON and MCT-injected rats. ** p<0.0l. [0039] FIGS. 8A - 8B. (A) PAH (from neonatal mice exposed to hyperoxia (High Oxygen)) leads to an increase in the number of mast cells (MCs) in pulmonary arteries (** p<0.0l between room air and high oxygen, N=3 mice/group. (B) Echocardiography was used to monitor the development of PAH (5-week time point). Doppler flow shows typical PA wave reflection for PAH as shown by the arrow in the right panel.

[0040] FIG. 9. The HIF Stabilizer Roxadustat (RXD) prevents mast cells from

accumulating in the pulmonary artery (PA) in neonatal PAH. Therapeutic dosing with the hypoxia inducible factor (HIF) stabilizer roxadustat (10 mg/kg), prevents the increase in pulmonary artery (PA) mast cell number in neonatal hyperoxia- induced PAH. **P<0.0l between room air and high oxygen, N=3 mice/group. All other comparisons to room air were not significant. RA = room air, HO = High Oxygen, RXD = Roxadustat (a HIF prolyl hydroxylase domain inhibitor and HIF stabilizer).

[0041] FIG. 10. HIF stabilization with Roxadustat (RXD) (10 mg/kg) decreases the magnitude of PA contraction in rings from mice with PAH (exposed to neonatal high oxygen (HO)). Contractile response to C48/80 (300pg/ml) expressed in mg in PA rings from age-matched room air (RA), high oxygen (HO)-exposed, and HO-exposed and RXD- treated (HO + RXD), mice.

[0042] FIG. 11. The HIF Stabilizer roxadustat prevents mast cells from accumulating in the right ventricle (RV) in Hyperoxia-lnduced Neonatal Fung Disease. Treating mice with the HIF stabilizer roxadustat (10 mg/kg) prevents mast cells from accumulating in the right ventricle (RV). N= 4 mice/group. **P<0.0l between PAH and Room Air. There was no significant difference between PAH + HIF Stabilizer and Room Air.

[0043] FIGS. 12A - 12C. HIF Stabilization Inhibits Right Ventricle Free Wall Thickness, a Marker of Hypertrophy, in Neonatal PAH. (A) Room Air, (B) High Oxygen, and (C) High Oxygen + HIF stabilizer.

[0044] FIG. 13. HIF Stabilization Inhibits Right Ventricular Remodeling Due to Neonatal PAH. HIF stabilizer Roxadustat inhibits right ventricular remodeling, as measured by right ventricular (RV) weight normalized to tibia length in neonatal PAH. Treating mice with the HIF stabilizer roxadustat (10 mg/kg) prevents RV hypertrophy. N= 4 mice/group. **R<0.01 between PAH and Room Air. There was no significant difference between PAH + HIF Stabilizer and Room Air.

[0045] FIG. 14. Mast Cells are Abundant in the Pulmonary Arteries of Premature Babies with Bronchopulmonary Dysplasia (BPD) and PAH. Representative sections of pulmonary artery from an infant who died from BPD-associated PAH were stained for mast cells with HRP-avidin. Mast cells are stained brown as seen at 20x and in the inlay, 40x, where granules and nucleus are visible.

DETAILED DESCRIPTION

General Disclosure

Methods for treating pulmonary hypertension

[0046] One aspect of the present disclosure is directed to a method for treating pulmonary hypertension by administering to a subject in need of such treatment an effective amount of a mast cell stabilizer. In some embodiments, administering to a subject an effective amount of a mast cell stabilizer prevents or reduces the blood pressure in the arteries of lungs and the right side of the heart.

[0047] In some embodiments, the mast cell stabilizer is administered locally to the lungs.

In some embodiments, the mast cell stabilizer is administered as an aerosol. In a specific embodiment, the aerosol comprising the mast cell stabilizer is administered using a dose metered inhaler or a nebulizer. In some embodiments, the mast cell stabilizer is administered systemically.

[0048] In some embodiments, an effective amount of a mast cell stabilizer is about 0.2 mg/kg to 100 mg/kg. In other embodiments, the effective amount of a mast cell stabilizer is about 0.2mg/kg, 0.5mg/kg, lmg/kg, 8mg/kg, lOmg/kg, 20mg/kg, 30mg/kg, 40mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, 80mg/kg, 90mg/kg, lOOmg/kg, l50mg/kg, l75mg/kg or 200mg/kg of mast cell stabilizer. As used in this disclosure, the term "about" refers to a variation within approximately ±10% from a given value. [0049] Another aspect of the present disclosure is directed to a method for treating pulmonary hypertension by administering to a subject in need of such treatment an effective amount of a HIF prolyl hydroxylase domain inhibitor. In some embodiments, administering to a subject an effective amount of a HIF prolyl hydroxylase domain inhibitor reduces the blood pressure in the arteries of lungs and the right side of the heart.

[0050] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered locally. In some embodiments, the HIF prolyl hydroxylase domain inhibitor is

administered as an aerosol. In a specific embodiment, the aerosol comprising the HIF prolyl hydroxylase domain inhibitor is administered using a dose metered inhaler or a nebulizer. In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered systemically.

[0051] In some embodiments, an effective amount of a HIF prolyl hydroxylase domain inhibitor is about 0.2 mg/kg to 100 mg/kg. In other embodiments, the effective amount of a HIF prolyl hydroxylase domain inhibitor is about 0.2mg/kg, 0.5mg/kg, lmg/kg, 8mg/kg, lOmg/kg, 20mg/kg, 30mg/kg, 40mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, 80mg/kg, 90mg/kg, lOOmg/kg, l50mg/kg, l75mg/kg or 200mg/kg of HIF prolyl hydroxylase domain inhibitor.

[0052] A third aspect of the present disclosure is directed to a method for treating pulmonary hypertension by administering to a subject in need of such treatment a combination therapy comprising an effective amount of a mast cell stabilizer and an effective amount of a HIF prolyl hydroxylase domain inhibitor. In some embodiments, administering to a subject a combination therapy comprising an effective amount of a mast cell stabilizer and an effective amount of a HIF prolyl hydroxylase domain inhibitor prevents or reduces high blood pressure in the arteries of lungs and the right side of the heart.

[0053] In some embodiments, the mast cell stabilizer and the HIF prolyl hydroxylase domain inhibitor are administered in one composition. In some embodiments, the mast cell stabilizer and the HIF prolyl hydroxylase domain inhibitor are administered consecutively. In some embodiments, the mast cell stabilizer and the HIF prolyl hydroxylase domain inhibitor are administered separately. In some embodiments, the mast cell stabilizer and the HIF prolyl hydroxylase domain inhibitor are administered simultaneously.

[0054] In some embodiments, the mast cell stabilizer and the HIF prolyl hydroxylase domain inhibitor are administered in one composition. In one embodiment, a composition comprising a mast cell stabilizer and a HIF prolyl hydroxylase domain inhibitor is not administered to the subject continuously; rather it is administered intermittently. In a specific embodiment, intermittent administration is performed once every other day, every three days, every four days, every five days, or once a week. In another specific embodiment, intermittent administration is performed once every hour, every two hours, every three hours, every six hours, every ten hours, or every twelve hours.

[0055] In some embodiments, the subject in need of treatment is a neonatal (newborn) subject.

Mast Cell Stabilizers

[0056] In some embodiments, the phrase "mast cell stabilizer" refers to an agent which prevents mast cells from breaking open and releasing chemicals (e.g., histamine, proteoglycans, serotonin, and serine proteases) that help cause inflammation (aka.

"degranulation"). In some embodiments, the mast cell stabilizer is a small molecule. As used in this disclosure, the phrase "small molecule compound" refers to small organic chemical compound, generally having a molecular weight of less than 2000 daltons, 1500 daltons, 1000 daltons, 800 daltons, or 600 daltons.

[0057] In some embodiments, the mast cell stabilizer comprises compounds including, but not limited to

Cromoglycic acid (cromolyn) with the chemical formula C ₂₃ H ₁₆ O ₁₁ and the chemical structure ketotifen with the chemical formula C ₁₉ H ₁₉ NOS and the chemical structure

pemirolast with the chemical formula C ioHsNeO and the chemical structure

and nedocromil with the chemical formula Ci ₉ Hi ₇ N0 ₇ and the chemical structure

[0058] In some embodiments, the mast stabilizer is selected from the group consisting of Cromoglicic acid, Ketotifen, Olopatadine, Rupatadine, Mepolizumab, Omalizumab, Pemirolast, Quercetin, Nedocromil, Azelastine, Tranilast, Palmitoylethanolamide, and Vitamin D.

[0059] In some embodiments, the mast cell stabilizer is nedocromil.

[0060] In some embodiments, the mast cell stabilizer is ketotifen.

[0061] In some embodiments, the mast cell stabilizer is administered locally. In some embodiments, the mast cell stabilizer is administered systemically.

HIF prolyl hydroxylase domain inhibitors

[0062] In some embodiments, the phrase "HIF prolyl hydroxylase domain inhibitor" refers to an agent that inhibits the activity of HIF hydroxylase or other enzymes in the 2-OG (2- oxoglutarate)-dependent dioxygenase enzyme superfamily having structural similarity and a common reaction mechanism with HIF hydroxylase. An agent that inhibits HIF prolyl hydroxylase activity is any agent that reduces or otherwise modulates the activity of a HIF prolyl hydroxylase or related enzyme, such as C-P4H. In some embodiments, the HIF prolyl hydroxylase domain inhibitor is a small molecule.

[0063] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is selected from the group consisting of Roxadustat (RXD) (FG-4592), Vadadustat (AKB-6548),

Daprodustat (GSK- 1278863), and Molidustat (BAY 85-3934).

[0064] In some embodiments, the general structure of HIF prolyl hydroxylase domain inhibitors is

wherein:

Rl is selected from the group consisting of hydroxyl, alkoxy, substituted alkoxy, aryloxy, and substituted aryloxy; R2 and R3 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocycloalkyl, substituted heterocycloalkyl, heteroaryl, and substituted heteroaryl; or R2 and R3 together with the carbon atom to which they are attached form a cycloalkyl, substituted cycloalkyl, hererocycloalkyl, or substituted hererocycloalkyl;

R4 is selected from the group consisting of hydrogen, alkyl, and substituted alkyl;

R5 is selected from the group consisting of hydroxyl, alkoxy, and substituted alkoxy;

R6 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, cyano, halo, hydroxyl, alkoxy, substituted alkoxy, amino, substituted amino, arylozy, substituted arylozy, aminoacyl, substituted aminoacyl, cycloalkoxy, substituted cycloalkoxy, aryl, substituted aryl, aryloxy, substituted aryloxy, heterocycloalkyl, substituted heterocycloalkyl, heteroaryloxy, substituted heteroaryloxy, heteroaryl, and substituted heteroaryl; and

R7, R8, R9 and R10 are independently selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, aryloxy, and substituted aryloxy.

[0065] In some embodiments, R6 is alkyl. In another embodiment, the alkyl at R6 is methyl.

[0066] In some embodiments, R5 is hydroxyl.

[0067] In some embodiments, R8 is aryloxy.

[0068] In some embodiments, wherein R6 is methyl and R5 is hydroxyl.

[0069] As used herein, the term "alkyl" refers to a straight or branched, saturated hydrocarbon group having 1 to 10 carbon atoms, more particularly from 1 to 5 carbon atoms, and even more particularly 1 to 3 carbon atoms. Representative alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, n-pentyl, and the like.

[0070] The term "substituted alkyl" refers to an alkyl group of from 1 to 10 carbon atoms, more particularly 1 to 5 carbon atoms, and having from 1 to 5 substituents, preferably 1 to 3 substituents, each of which substituents is independently selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy, aryloxyaryl, substituted aryloxyaryl, cyano, halogen, hydroxyl, nitro, oxo, thioxo, carboxyl, carboxyl esters, cycloalkyl, substituted cycloalkyl, thio, alkylthio, substituted alkylthio, arylthio, substituted arylthio, cycloalkylthio, substituted cycloalkylthio, heteroarylthio, substituted heteroarylthio, heterocyclicthio, substituted heterocyclicthio, sulfonyl, substituted sulfonyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino, oxythiocarbonylamino,— OS(0)2-alkyl,— OS(0)2- substituted alkyl,— OS(0)2-aryl,— OS(0)2-substituted aryl,— OS(0)2-heteroaryl,— OS(0)2-substituted heteroaryl,— OS(0)2-heterocyclic,— OS(0)2-substituted heterocyclic, and— OS 02— NR11R11,— NR1 lS(0)2— NR1 l-alkyl,— NR1 lS(0)2— NR1 l-substituted alkyl,— NRl lS(0)2— NRl l-aryl,— NRl lS(0)2— NR1 l-substituted aryl,—

NR1 lS(0)2— NR1 l-heteroaryl,— NR1 lS(0)2— NR1 l-substituted heteroaryl,—

NR1 lS(0)2— NR11 -heterocyclic, and— NR1 lS(0)2— NR1 l-substituted heterocyclic, wherein each Rl 1 is independently selected from hydrogen or alkyl. Representative substituted alkyl groups include trifluoromethyl, benzyl, pyrazol-l-ylmethyl and the like.

[0071] The term "alkoxy" refers to the group "alkyl-0— ," which includes, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy, sec-butoxy, n- pentoxy, and the like.

[0072] The term "substituted alkoxy" refers to the group "substituted alkyl-0— ". [0073] The term "acyl" refers to the groups H— C(O)— , alkyl-C(O)— , substituted alkyl- C(O)— , alkenyl-C(O)— , substituted alkenyl-C(O)— , alkynyl-C(O)— , substituted alkynyl- C(O)— , cycloalkyl-C(O)— , substituted cycloalkyl-C(O)— , aryl-C(O)— , substituted aryl- C(O)— , heteroaryl-C(O)— , substituted heteroaryl-C(O), heterocyclic-C(O)— , and substituted heterocyclic-C(O)— , provided that a nitrogen atom of the heterocyclic or substituted heterocyclic is not bound to the— C(O)— group, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0074] The term "aminoacyl" or "amide", or the prefix "carbamoyl," "carboxamide," "substituted carbamoyl" or "substituted carboxamide" refers to the group— C(0)NRl2Rl2, wherein each R12 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; or wherein each R12 is joined to form together with the nitrogen atom a heterocyclic or substituted heterocyclic, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0075] The term "acyloxy" refers to the groups alkyl-C(0)0— , substituted alkyl-C(0)0— , alkenyl-C(0)0— , substituted alkenyl-C(0)0— , alkynyl-C(0)0— , substituted alkynyl- C(0)0— , aryl-C(0)0— , substituted aryl-C(0)0— , cycloalkyl-C(0)0— , substituted cycloalkyl-C(0)0— , heteroaryl-C(0)0— , substituted heteroaryl-C(0)0— , heterocyclic- C(0)0— , and substituted heterocyclic-C(0)0— , wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted

heterocyclic are as defined herein.

[0076] The term "alkenyl" refers to a vinyl unsaturated monovalent hydrocarbyl group having from 2 to 6 carbon atoms, and preferably 2 to 4 carbon atoms, and having at least 1, and preferably from 1 to 2 sites of vinyl (>C=C<) unsaturation. Representative alkenyl groups include vinyl (ethen-l-yl), allyl, but-3-enyl and the like.

[0077] The term "substituted alkenyl" refers to alkenyl groups having from 1 to 3 substituents, and preferably 1 to 2 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic. This term includes both E (trans) and Z (cis) isomers as appropriate. It also includes mixtures of both E and Z components.

[0078] The term "alkynyl" refers to acetylenic unsaturated monovalent hydrocarbyl groups having from 2 to 6 carbon atoms, and preferably 2 to 3 carbon atoms, and having at least 1, and preferably from 1 to 2 sites of acetylenic (— CºC— ) unsaturation. Representative alkynyl groups include ethyn-l-yl, propyn-l-yl, propyn-2-yl, and the like.

[0079] The term "amino" refers to the group— NH2.

[0080] The term "substituted amino" refers to the group— NR13R13, wherein each R13 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted

heterocyclic, sulfonyl, and substituted sulfonyl, provided that both R13 groups are not hydrogen; or the R13 groups can be joined together with the nitrogen atom to form a heterocyclic or substituted heterocyclic ring. Representative substituted amino groups include phenylamino, methylphenylamino, and the like. Representative substituted amino groups include (ethanic acid-2-yl)amino, and the like.

[0081] The term "acylamino" refers to the groups— NRl4C(0)alkyl,—

NRl4C(0)substituted alkyl,— NRl4C(0)cycloalkyl,— NRl4C(0)substituted cycloalkyl,

— NRl4C(0)alkenyl,— NRl4C(0)substituted alkenyl,— NRl4C(0)alkynyl,—

NRl4C(0)substituted alkynyl,— NRl4C(0)aryl,— NRl4C(0)substituted aryl,—

NRl4C(0)heteroaryl,— NRl4C(0)substituted heteroaryl,— NRl4C(0)heterocyclic, and — NRl4C(0)substituted heterocyclic, wherein R14 is hydrogen or alkyl, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,

heterocyclic, and substituted heterocyclic are defined herein.

[0082] The term "oxycarbonylamino" refers to the groups— NRl5C(0)0-alkyl,—

NRl5C(0)0-substituted alkyl,— NRl5C(0)0-alkenyl,— NRl5C(0)0-substituted alkenyl,— NRl5C(0)0-alkynyl,— NRl5C(0)0-substituted alkynyl,— NRl5C(0)0- cycloalkyl,— NRl5C(0)0-substituted cycloalkyl,— NRl5C(0)0-aryl,— NRl5C(0)0- substituted aryl,— NRl5C(0)0-heteroaryl,— NRl5C(0)0-substituted heteroaryl,— NRl5C(0)0-heterocyclic, and— NRl5C(0)0-substituted heterocyclic, wherein R15 is hydrogen or alkyl, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0083] The term "oxythiocarbonylamino" refers to the groups— NRl6C(S)0-alkyl,— NRl6C(S)0-substituted alkyl,— NRl6C(S)0-alkenyl,— NRl6C(S)0-substituted alkenyl, — NRl6C(S)0-alkynyl,— NRl6C(S)0-substituted alkynyl,— NRl6C(S)0-cycloalkyl,— NRl6C(S)0-substituted cycloalkyl,— NRl6C(S)0-aryl,— NRl6C(S)0-substituted aryl,

— NRl6C(S)0-heteroaryl,— NRl6C(S)0-substituted heteroaryl,— NRl6C(S)0- heterocyclic, and— NRl6C(S)0-substituted heterocyclic, wherein R16 is hydrogen or alkyl, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0084] The term "aminocarbonyloxy," or the prefix "carbamoyloxy" or "substituted carbamoyloxy," refers to the groups— 0C(0)NRl7Rl7, wherein each R17 is

independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; or wherein each R17 is joined to form, together with the nitrogen atom, a heterocyclic or substituted heterocyclic, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted

heterocyclic are as defined herein.

[0085] The term "aminocarbonylamino" refers to the group— NRl8C(0)— NR18R18, wherein each R18 is independently selected from the group consisting of hydrogen and alkyl.

[0086] The term "aminothiocarbonylamino" refers to the group— NRl9C(S)— NR19R19, wherein each R19 is independently selected from the group consisting of hydrogen and alkyl.

[0087] The term "aryl" refers to a monovalent aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl) which condensed rings may or may not be aromatic (e.g., 2-benzoxazolinone, 2H-l,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the point of attachment is the aryl group. Preferred aryls include phenyl and naphthyl.

[0088] The term "substituted aryl" refers to aryl groups, as defined herein, which are substituted with from 1 to 4, particularly 1 to 3, substituents selected from the group consisting of hydroxyl, acyl, acylamino, acyloxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amidino (— C(=NH)-amino or substituted amino), amino, substituted amino, aminoacyl,

aminocarbonyloxy, aminocarbonylamino, aminothiocarbonylamino, aryl, substituted aryl, aryloxy, substituted aryloxy, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy, carboxyl, carboxyl esters, cyano, thio, alkylthio, substituted alkylthio, arylthio, substituted arylthio, heteroarylthio, substituted heteroarylthio, cycloalkylthio, substituted cyclo alkylthio, heterocyclicthio, substituted heterocyclicthio, cycloalkyl, substituted cycloalkyl, guanidino (— NH— C(=NH)-amino or substituted amino), halo, nitro, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, oxycarbonylamino, oxythiocarbonylamino, sulfonyl, substituted sulfonyl,— 0S(0) ₂ -alkyl,— 0S(0) ₂ - substituted alkyl,— 0S(0) ₂ -aryl,— OS(0) ₂ -substituted aryl,— OS(0)2-heteroaryl,— OS(0) ₂ -substituted heteroaryl,— OS(0) ₂ -heterocyclic,— OS(0) ₂ -substituted heterocyclic, and— 0S0 _2— NR20R20,— NR20S(O) _2— NR20-alkyl,— NR20S(O) _2— NR20-substituted alkyl,— NR20S(O) _2— NR20-aryl,— NR20S(O) _2— NR20-substituted aryl,— NR20S(O) _2— NR20-heteroaryl,— NR20S(O) _2— NR20-substituted heteroaryl,— NR20S(O) _2— NR20- heterocyclic,— NR20S(O) _2— NR20-substituted heterocyclic, wherein each R20 is independently selected from hydrogen or alkyl, and wherein each of the terms is as defined herein. Representative substituted aryl groups include 4-fluorophenyl, 3-methoxyphenyl, 4-t-butylphenyl, 4-trifluoromethylphenyl, 2-trifluoromethoxyphenyl, 3- trifluoromethoxyphenyl, 4-trifluoromethoxyphenyl, 2-chlorophenyl, 3-chlorophenyl, 4- chlorophenyl, 2-chloro-6-fluorophenyl, 2,4-dichlorophenyl, 4-methoxyphenyl, 3- cyanophenyl, 4-cyanophenyl, 4-phenoxyphenyl, 4-methanesulfonylphenyl, biphenyl-4-yl, and the like.

[0089] The term "aryloxy" refers to the group aryl-0— that includes, by way of example, phenoxy, naphthoxy and the like.

[0090] The term "substituted aryloxy" refers to substituted aryl-0— groups.

[0091] The term "aryloxyaryl" refers to the group -aryl-O-aryl.

[0092] The term "substituted aryloxyaryl" refers to aryloxyaryl groups substituted with from 1 to 3 substituents on either or both aryl rings as defined above for substituted aryl.

[0093] The term "carboxyl" refers to— COOH or salts thereof.

[0094] The term "carboxyl ester" refers to the groups— C(0)0-alkyl,— C(0)0-substituted alkyl,— C(0)0-alkenyl,— C(0)0-substituted alkenyl,— C(0)0-alkynyl,— C(0)0- substituted alkynyl,— C(0)P-cycloalkyl,— C(0)0-substituted cycloalkyl,— C(0)0-aryl, — C(0)0 -substituted aryl,— C(0)0-heteroaryl,— C(0)0-substituted heteroaryl,— C(0)0-heterocyclic, and— C(0)0-substituted heterocyclic. [0095] The term "cyano" refers to the group— CN.

[0096] The term "cycloalkyl" refers to a saturated or an unsaturated but nonaromatic cyclic alkyl groups of from 3 to 10, 3 to 8 or 3 to 6 carbon atoms having single or multiple cyclic rings including, by way of example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, cyclohexenyl, and the like.

[0097] The term "substituted cycloalkyl" refers to a cycloalkyl group, having from 1 to 5 substituents selected from the group consisting of oxo (=0), thioxo (=S), alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxy, nitro, carboxyl, carboxyl esters, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic.

[0098] The term "cycloalkylene" and "substituted cycloalkylene" refer to divalent cycloalkyl and substituted cycloalkyl groups as defined above.

[0099] The term "cycloalkoxy" refers to— O-cycloalkyl groups.

[00100] The term "substituted cycloalkoxy" refers to— O-substituted cycloalkyl groups.

[00101] The term "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[00102] The term "hydroxy" or "hydroxyl" refers to the group— OH.

[00103] The term "heteroaryl" refers to an aromatic ring of from 1 to 15 carbon atoms, preferably from 1 to 10 carbon atoms, and 1 to 4 heteroatoms within the ring selected from the group consisting of oxygen, nitrogen, and sulfur. Such heteroaryl groups can have a single ring (e.g., pyridinyl, furyl, or thienyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl) provided the point of attachment is through a ring containing the heteroatom and that ring is aromatic. The nitrogen and/or sulfur ring atoms can optionally be oxidized to provide for the N-oxide or the sulfoxide, and sulfone derivatives. Representative heteroaryl groups include pyridinyl, pyrimidinyl, pyrrolyl, pyrazolyl, indolyl, thiophenyl, thienyl, furyl, and the like.

[00104] The term "substituted heteroaryl" refers to heteroaryl groups that are substituted with from 1 to 3 substituents selected from the same group of substituents defined for substituted aryl. Representative substituted heteroaryl groups include 5-fluoro- pyridin-3-yl, l-benzyl-lH-[l,2,3]triazol-4-yl, 5-bromo-furan-2-yl, trifluoromethyl-2H- pyrazol-3-yl, and the like.

[00105] The term "heteroaryloxy" refers to the group— O-heteroaryl, and

"substituted heteroaryloxy" refers to the group— O-substituted heteroaryl.

[00106] The term "heterocyclyl" or "heterocyclic" refers to a saturated or unsaturated

(but not aromatic) group having a single ring or multiple condensed rings, from 1 to 10 carbon atoms, and from 1 to 4 hetero atoms selected from the group consisting of nitrogen, sulfur or oxygen within the ring, wherein in fused ring systems, one or more of the rings can be aryl or heteroaryl provided that the point of attachment is at the heterocycle. The nitrogen and/or sulfur ring atoms can optionally be oxidized to provide for the N-oxide or the sulfoxide, and sulfone derivatives.

[00107] The term "substituted heterocyclyl" or "substituted heterocyclic" refers to heterocycle groups that are substituted with from 1 to 3 of the same substituents as defined for substituted cycloalkyl.

[00108] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, l,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b] thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like. [00109] The term "nitro" refers to the group— N0 ₂ .

[00110] The term "oxo" refers to the atom (=0) or to the atom (— 0-).

[00111] The term "sulfonyl" refers to the group— S(0) ₂ H.

[00112] The term "substituted sulfonyl" refers to the group— S0 ₂ -alkyl,— S0 ₂ - substituted alkyl,— S0 ₂ -alkenyl,— S0 ₂ -substituted alkenyl,— S0 ₂ -alkynyl,— S0 ₂ - substituted alkynyl,— S0 ₂ -cycloalkyl,— S0 ₂ -substituted cycloalkyl,— S0 ₂ -cycloalkenyl, — S0 ₂ -substituted cycloalkenyl,— S0 ₂ -aryl,— S0 ₂ -substituted aryl,— S0 ₂ -heteroaryl,— S0 ₂ -substituted heteroaryl,— S0 ₂ -heterocyclic,— S0 ₂ - substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein. Representative sulfonyl groups include methyl-S0 _2— , phenyl-S0 _2— , 4- methylphenyl-S0 _2— , and the like.

[00113] The term "heterocyclyloxy" refers to the group— O-heterocyclic, and "substituted heterocyclyloxy" refers to the group— O-substituted heterocyclic.

[00114] The term "thio" refers to the group— SH.

[00115] The term "alkylsulfanyl," "alkylthio," or "thioether" refers to the groups— S-alkyl, wherein alkyl is as defined above.

[00116] The term "substituted alkylthio," "substituted alkylsulfanyl," or "substituted alkylthio" refers to the group— S-substituted alkyl, wherein substituted alkyl is as defined above.

[00117] The term "cycloalkylthio" or "cycloalkylsulfanyl" refers to the groups— S- cycloalkyl wherein cycloalkyl is as defined above.

[00118] The term "substituted cycloalkylthio" refers to the group— S-substituted cycloalkyl wherein substituted cycloalkyl is as defined above. [00119] The term "arylthio" or "arylsulfanyl" refers to the group— S-aryl, and "substituted arylthio" refers to the group— S-substituted aryl, wherein aryl and substituted aryl are as defined above.

[00120] The term "heteroarylthio" or "heteroarylsulfanyl" refers to the group— S- heteroaryl, and "substituted heteroarylthio" refers to the group— S-substituted heteroaryl, wherein heteroaryl and substituted heteroaryl are as defined above.

[00121] The term "heterocyclicthio" or "heterocyclicsulfanyl" refers to the group—

S-heterocyclic, and "substituted heterocyclicthio" refers to the group— S-substituted heterocyclic wherein heterocyclic, and substituted heterocyclic are as defined above.

[00122] The term "ester" refers to the group— C(0)OR2l, wherein R21 is alkyl, substituted alkyl, aryl, or substituted aryl.

[00123] In one embodiment, in Formula (I), Rl and R5 are hydroxyl; R2, R3, R4, R7, R9, and R10 are hydrogen; R6 is methyl; and R8 is phenox, and the compound has a structure shown below in formula (II). This molecule is also known as FG-4592 (aka. Roxadustat), which is an isoquinolone having the chemical name, N- [(4-hydroxy- l-methyl- 7-phenoxyisoquinolin-3-yl)carbonyl]glycine)].

[00124] FG-4592 (Roxadustat) is in phase 3 clinical trials for the treatment of anemia in chronic kidney disease with no untoward effects reported.

[00125] In a specific embodiment, an effective amount of FG-4592 (Roxadustat) is an amount between 0.2 mg/kg and 20 mg/kg. In another embodiment an effective dosage of FG-4592 (Roxadustat) is 0.2mg/kg, 0.5mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 18 mg/kg, or 20mg/kg.

[00126] In some embodiments, a FG-4592 (Roxadustat) analogue is used. FG-4592 analogues are described in US Patents Nos: 9,701,647; 9,439,888; 7,863,292; and US Patent Application Nos: 13/186351 and 11/549571, which are all incorporated by reference in their entirety.

[00127] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is RXD (roxadustat).

[00128] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered locally. In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered systemically.

Pharmaceutical compositions comprising mast cell stabilizer or a HIF prolyl hydroxylase domain inhibitor

[00129] In some embodiments, a mast cell stabilizer or a HIF prolyl hydroxylase domain inhibitor can be combined with a pharmaceutically acceptable carrier prior to administration. For the purposes of this disclosure, "pharmaceutically acceptable carriers" means any of the standard pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include, but are not limited to, any of the standard

pharmaceutical carriers such as a phosphate buffered saline solution and various wetting agents. Other carriers may include additives used in tablets, granules and capsules, and the like. Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gum, glycols or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well-known conventional methods.

[00130] A mast cell stabilizer or a HIF prolyl hydroxylase domain inhibitor can be admixed with a pharmaceutically acceptable carrier to make a pharmaceutical preparation in any conventional form including, inter alia, a solid form such as tablets, capsules (e.g. hard or soft gelatin capsules), pills, cachets, powders, granules, and the like; a liquid form such as solutions, suspensions; or in micronized powders, sprays, aerosols and the like.

[00131] The pharmaceutical compositions of the present disclosure can be used in liquid, solid, tablet, capsule, pill, ointment, cream, nebulized or other forms as explained below. In some embodiments, the composition of the present disclosure may be administered by different routes of administration such as oral, oronasal, parenteral or topical.

[00132] Oral" or "peroral" administration refers to the introduction of a substance into a subject's body through or by way of the mouth and involves swallowing or transport through the oral mucosa (e.g., sublingual or buccal absorption) or both.

[00133] Oronasal" administration refers to the introduction of a substance into a subject's body through or by way of the nose and the mouth, as would occur, for example, by placing one or more droplets in the nose. Oronasal administration involves transport processes associated with oral and intranasal administration.

[00134] Parenteral administration" refers to the introduction of a substance into a subject's body through or by way of a route that does not include the digestive tract.

Parenteral administration includes subcutaneous administration, intramuscular

administration, transcutaneous administration, intradermal administration, intraperitoneal administration, intraocular administration, and intravenous administration.

[00135] “Topical administration” means the direct contact of a substance with tissue, such as skin or membrane, particularly the oral or buccal mucosa.

[00136] The pharmaceutical preparations of the present disclosure can be made up in any conventional form including, inter alia ,: (a) a solid form for oral administration such as tablets, capsules (e.g. hard or soft gelatin capsules), pills, sachets, powders, granules, and the like; (b) preparations for topical administrations such as solutions, suspensions, ointments, creams, gels, micronized powders, sprays, aerosols and the like. The

pharmaceutical preparations may be sterilized and/or may contain adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, salts for varying the osmotic pressure and/or buffers.

[00137] For topical administration to the skin or mucous membrane the

aforementioned composition is preferably prepared as ointments, tinctures, creams, gels, solution, lotions, sprays; aerosols and dry powder for inhalation, suspensions, shampoos, hair soaps, perfumes and the like. In fact, any conventional composition can be utilized in this invention. Among the preferred methods of applying the composition containing the agents of this invention is in the form of an ointment, gel, cream, lotion, spray; aerosol or dry powder for inhalation. The pharmaceutical preparation for topical administration to the skin can be prepared by mixing the aforementioned active ingredient with non-toxic, therapeutically inert, solid or liquid carriers customarily used in such preparation. These preparations generally contain 0.01 to 5.0 percent by weight, or 0.1 to 1.0 percent by weight, of the active ingredient, based on the total weight of the composition.

[00138] In preparing the topical preparations described above, additives such as preservatives, thickeners, perfumes and the like conventional in the art of pharmaceutical compounding of topical preparation can be used. In addition, conventional antioxidants or mixtures of conventional antioxidants can be incorporated into the topical preparations containing the aforementioned active agent. Among the conventional antioxidants which can be utilized in these preparations are included N-methyl-a-tocopherolamine, tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, ethoxyquin and the like.

[00139] Cream-based pharmaceutical formulations containing the active agent, used in accordance with this invention, are composed of aqueous emulsions containing a fatty acid alcohol, semi- solid petroleum hydrocarbon, ethylene glycol and an emulsifying agent.

[00140] Ointment formulations containing the active agent in accordance with this invention comprise admixtures of a semi- solid petroleum hydrocarbon with a solvent dispersion of the active material. Cream compositions containing the active ingredient for use in this invention preferably comprise emulsions formed from a water phase of a humectant, a viscosity stabilizer and water, an oil phase of a fatty acid alcohol, a semi-solid petroleum hydrocarbon and an emulsifying agent and a phase containing the active agent dispersed in an aqueous stabilizer-buffer solution. Stabilizers may be added to the topical preparation. Any conventional stabilizer can be utilized in accordance with this invention. In the oil phase, fatty acid alcohol components function as a stabilizer. These fatty acid alcohol components function as a stabilizer. These fatty acid alcohol components are derived from the reduction of a long-chain saturated fatty acid containing at least- 14 carbon atoms.

[00141] Also, conventional perfumes and lotions generally utilized in topical preparation for the hair can be utilized in accordance with this invention. Furthermore, if desired, conventional emulsifying agents can be utilized in the topical preparations of this invention.

[00142] In some embodiments, compositions comprising a mast cell stabilizer or a HIF prolyl hydroxylase domain inhibitor, or a combination thereof, can be administered by aerosol. For example, this can be accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing a composition comprising a mast cell stabilizer or a HIF prolyl hydroxylase domain inhibitor preparation. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers can also be used. An aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound.

Use of mast stabilizers and/or HIF prolyl hydroxylase domain inhibitors for the manufacture of a medicament

[00143] Another aspect of this disclosure is directed to a use of a mast cell stabilizer for the manufacture of a medicament for use in the treatment of pulmonary hypertension.

[00144] Another aspect of this disclosure is directed to a use of a HIF prolyl hydroxylase domain inhibitor for the manufacture of a medicament for use in the treatment of pulmonary hypertension. [00145] Another aspect of this disclosure is directed to a use of a mast cell stabilizer and a HIF prolyl hydroxylase domain inhibitor for the manufacture of a medicament for use in the treatment of pulmonary hypertension.

[00146] In some embodiments, the mast cell stabilizer is selected from the group consisting of cromoglicic acid, ketotifen, olopatadine, rupatadine, mepolizumab, omalizumab, pemirolast, quercetin, nedocromil, azelastine, tranilast,

palmitoylethanolamide, and vitamin D.

[00147] In some embodiments, the mast cell stabilizer is nedocromil.

[00148] In some embodiments, the mast cell stabilizer is administered systemically.

In some embodiments, the mast cell stabilizer is administered locally to the lung.

[00149] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is selected from the group consisting of Roxadustat (RXD) (FG-4592), Vadadustat (AKB- 6548), Daprodustat (GSK- 1278863), and Molidustat (BAY 85-3934).

[00150] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is Roxadustat (RXD) (FG-4592).

[00151] In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered systemically. In some embodiments, the HIF prolyl hydroxylase domain inhibitor is administered locally to the lung.

[00152] In some embodiments, the pulmonary hypertension is pulmonary arterial hypertension (PAH).

[00153] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. [00154] The specific examples listed below are only illustrative and by no means limiting.

EXAMPLES

Example 1: Materials and Methods

Animal Models of Pulmonary Arterial Hypertension (PAH)

[00155] Rat model - PAH chemically induced with monocrotaline (MCT) and 2. Mouse model - via exposure to neonatal hyperoxia resulting in bronchopulmonary dysplasia and persistent PAH. The inventors demonstrate in these murine models of PAH that mast cells, part of the inflammatory response, play a crucial role in PAH and therapeutically targeting their activity diminishes severity of the disease.

[00156] Rat model - PAH chemically induced with monocrotaline (MCT) (FIGS. 1 - 7). Sprague Dawley rats (6-8 weeks old) were injected with MCT- or vehicle (CON) and analyzed 28 days later. The mast cell stabilizer nedocromil, was administered by pellet implanted subcutaneously (s.c.), prior to the MCT-injection and for the duration of the experiment.

[00157] Mouse model - PAH induced by neonatal hyperoxia (FIGS. 8 - 13).

Neonatal CD1 mice were continuously exposed to 80% oxygen for 2 weeks followed by return to room air for an additional 3 weeks (High Oxygen, HO). Controls were age matched (5 weeks) in room air (RA or control). The HIF stabilizer roxadustat (RXD) was administered during exposure to hyperoxia as previously reported (Hoppe G. et ah, PNAS, 2016, 113: E2516-2525). Tissues were analyzed at the 5-week time point.

Example 2: Rat model Experiments

Mast Cells Are Increased in the Pulmonary Arteries in PAH

[00158] Using the MCT rat model, experiments were performed to evaluate the contribution of mast cells to pulmonary artery (PA) remodeling. It was observed that PA from MCT-injected rats contain markedly more mast cells than PA from vehicle-injected (CON) rats (FIG. 1A). Mast cells were not found in these layers of the PA wall in the control rats (FIG. 1B).

Stabilizing Mast Cells Inhibits Remodeling of the Pulmonary Arteries in PAH

[00159] To determine whether mast cells contribute to remodeling and thickening of the PA associated with PAH as in the MCT-injected rats, rats were treated with the mast cell stabilizer, nedocromil (FIG. 2C). Microdissected PAs from control, MCT-injected, and MCT-injected treated with nedocromil were fixed, sectioned and analyzed for wall thickness and collagen. Representative cross-sections of size-matched PAs stained for collagen with Gomori trichrome are shown in FIGS. 2A-2C.

[00160] PA from control (CON) rat had minimal collagen staining in the wall measuring (60 mm) (FIG. 2A). PA from MCT-rat was significantly different and remodeled with a wall thickness measuring 150 mm and plenty of collagen (FIG. 2B). In contrast the PA from MCT rat treated with the mast cell stabilizer nedocromil had minimal collagen staining and remodeling with a wall thickness of 40 mm (FIG. 2C).

Stabilizing Mast Cells Reduces Pulmonary Fibrosis in PAH

[00161] Respiratory mechanics were measured on the rats. PAH is associated with interstitial fibrosis. Tissue elastance and compliance were determined by applying pressure wave impedance values to a constant phase model. Tissue elastance was increased in the rats with PAH compared to the controls (FIG. 3A). Treatment with nedocromil reversed the elastance almost to baseline values. Consistently, compliance was decreased in the rats with PAH and increased in the treated rats again (FIG. 3B).

Stabilizing Mast Cells Prevents Abnormal Flow Velocity in PAH

[00162] Echocardiography was used to monitor the development of PAH at 28 days

(28d) post-MCT-injection. FIG. 4A - 4C compares the echocardiographic recordings of

PA flow velocities from CON, MCT-injected and MCT -injected nedocromil-treated rats.

As shown in the representative traces, the MCT-rats exhibit wave reflection (white arrows), typical of PAH (FIG. 4B) which is absent in MCT-rats + mast cell stabilizer (FIG. 4C) and control (CON) rats (FIG. 4A).

Remodeling of the Right Ventricle is Associated with Mast Cells in PAH

[00163] Fixed sections of the right ventricle (RV) from +MCT -injected rats were next analyzed for fibrosis and mast cells. FIGS. 5 A - 5D show representative consecutive sections of RV from MCT-injected (FIG. 5B, FIG. 5D) and control (FIG. 5A, FIG. 5C) rats, stained for mast cells (FIG. 5A and FIG. 5B) and collagen (FIG. 5C and FIG. 5D). There is a noticeable increase in the number of mast cells populating the RV in MCT- injected rats compared to CON (FIG. 5B vs. FIG. 5A). The MCT -injected rat also has abundant collagen as shown by the blue staining seen in FIG. 5D whereas there was minimal collagen staining in control rat (FIG. 5C). The spatial distribution of mast cells correlated with the collagen stained region of the RV in the MCT-injected rat.

Stabilizing Mast Cells Prevents PAH Right Ventricular Hypertrophy

[00164] Echocardiography was also used to document right ventricular overload in the MCT-injected rats. Because protection against PA remodeling was observed with nedocromil-treatment in the MCT-injected rats, it was checked to see whether mast-cell- stabilization also protects against RVH. FIGS. 6A - 6C compares representative rat echocardiograms from control (CON) (FIG. 6A), MCT-injected (FIG. 6B) and MCT- injected treated with nedecromil (FIG. 6C). The typical sign of right ventricular overload were observed in the MCT-injected rat (FIG. 6B), namely a flattening of the

interventricular septum (Dshaped left ventricle). In contrast, MCT-injected rats treated with nedocromil, the mast cell stabilizer, prevented RVH as shown by the donut- shaped left ventricle (FIG. 6C) that was similar to CON (FIG. 6A).

Blocking the Angiotensin ATI Receptor Inhibits PAH Pulmonary Artery Vasoconstriction (Mast cell de granulation and release of mast cell renin leading to locally formed ANG II in the pulmonary artery )

[00165] Vasoconstriction is a key feature of PAH. In that vascular smooth muscle expresses Angiotensin II receptor, type 1 (ATiR) which when activated causes vasoconstriction. Whether release of mast cell renin (5, 6) and the ensuing ANG II lead to vasoconstriction of the PA was tested. Mast cell-dependent isometric constriction was measured by myograph in isolated rings of PA excised from ± MCT-injected rats (PAH). Individual PA rings are first exposed to high extracellular K ⁺ (60 mM) to induce a constriction response. Mast cell-dependent constrictions are then measured in response to chemically-induced mast cell degranulation using compound 48/80 (C48/80) (300 J,l9/ml). A significant mast cell-dependent induced constriction was measured in PA from MCT- injected rats, amounting to 21% ±7 SEM of the response to K ⁺ (n= 10 rings MCT-injected rats (PAH) and 10 PA rings) (FIG. 7). This response was due to the release of mast cell renin and the ensuing ANG II, in that it was abolished in the presence of EXP3174, the active metabolite of losartan, an ANG II receptor blocker (0.5% ±4 (n= 4) 2 MCT-injected rats and 4 PA rings). There was no mast cell-specific constriction in PA from vehicle- injected rats (0.4 ±1 % n= 11 PA rings). These results are consistent with the findings showing increased mast cell density in PA from MGT- injected rats (FIGS. 1A - 1B).

[00166] FIGS. 1A - 7 show that mast cells and their products make a significant contribution to MGT-induced PAH. Inhibiting mast cell activity in vivo with a mast cell stabilizer (nedocromil) or inhibiting the ANG II ATiR ex vivo in pulmonary artery rings, attenuates PAH.