US 20130224259A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0224259 A1 Ghebremariam et al. (43) Pub. Date: Aug. 29, 2013

(54) DMETHYLARGINNE Publication Classification DMETHYLAMINOHYDROLASE INHIBITORS AND METHODS OF USE (51) Int. Cl. THEREOF A63/542 (2006.01) A613 L/437 (2006.01) (71) Applicant: The Board of Trustees of the Leland A6M 16/14 (2006.01) Stanford Junior University, (US) A6M II/04 (2006.01) A6M I5/00 (2006.01) (72) Inventors: Yohannes T. Ghebremariam, Santa A613 L/4439 (2006.01) Clara, CA (US); John P. Cooke, Palo A613 L/428 (2006.01) Alto, CA (US) (52) U.S. Cl. CPC ...... A6 IK3I/542 (2013.01); A61 K3I/4439 (2013.01); A61 K3I/437 (2013.01); A61 K (73) Assignee: The Board of Trustees of the Leland 3 1/428 (2013.01); A61M II/04 (2013.01): Stanford Junior University, Palo Alto, A61M 15/00 (2013.01); A61M 16/14 (2013.01) CA (US) USPC ...... 424/400: 514/338: 514/303; 424/43; 514/224.5; 514/301; 514/373; 506/11; (21) Appl. No.: 13/766,336 128/200.23; 128/203.15; 128/203.12 (57) ABSTRACT (22) Filed: Feb. 13, 2013 The present disclosure provides DDAH modulators. Thus, the present disclosure provides a method of treating a patient suffering from a disorder characterized by excessive NO pro Related U.S. Application Data duction and/or elevated DDAH activity, the method compris (60) Provisional application No. 61/598.758, filed on Feb. ing administering to said patient an effective amount of a 14, 2012, provisional application No. 61/645,383, compound of one of formulae I-X. The present disclosure filed on May 10, 2012, provisional application No. also provides a pharmaceutical composition comprising a 61/701,990, filed on Sep. 17, 2012. compound of one of formulae I-X. Patent Application Publication Aug. 29, 2013 Sheet 1 of 34 US 2013/0224259 A1

isKiis

$388 is , Patent Application Publication Aug. 29, 2013 Sheet 2 of 34 US 2013/0224259 A1

$38.8 s. Patent Application Publication Aug. 29, 2013 Sheet 3 of 34 US 2013/0224259 A1

Re-Validation of PPS as DDAH inhibitors. Usin

100Z ROC, a 3 - i.

i. &

Compounds (50puM each)

FIG. 2 Patent Application Publication Aug. 29, 2013 Sheet 4 of 34 US 2013/0224259 A1

Orthodonal ASSaV To Validate PPS as DDAH inhibitors

p40.0001 R2 = 0.9955 2% i.

Compounds (50puM each) FIG. 3 Patent Application Publication Aug. 29, 2013 Sheet 5 of 34 US 2013/0224259 A1

Production of L-Citrulline from ADMA is Reduced by PPIs

20 p<0.0001 R2 = 0.9922

1 5

s t

Compounds (50M each) FIG. 4 Patent Application Publication Aug. 29, 2013 Sheet 6 of 34 US 2013/0224259 A1

INTRACELLULARADMA

O2O

S 3 0.15 O 0.10 O C O

s? 0.05 C

O.OO Vehicle L-257 Esomeprazole Groups

FIG. 5 Patent Application Publication Aug. 29, 2013 Sheet 7 of 34 US 2013/0224259 A1

DIRECT INHIBITION OF DDAH ACTIVITY BY Small Molecule PD 404, 182

s Vehicle O.78 1.56 3.13 6.25 12.5 25 50 PD 404 (uM) FIG. 6 Patent Application Publication Aug. 29, 2013 Sheet 8 of 34 US 2013/0224259 A1

Re-validation of PD 404 as a DDAH Inhibitor using CPM Assay

120 p=0.0003 1OO R2 = 0.9922 8O

60

40

20

Vehicle PD 404 Groups FIG. 7 Patent Application Publication Aug. 29, 2013 Sheet 9 of 34 US 2013/0224259 A1

Orthogonal Assay to Validate PD 404 as a DDAH inhibitor 120

1OO

Š 80 : 60 p40.0001 R2 = 0.9990 s 40 ?

2O

O Vehicle PD 404 Patent Application Publication Aug. 29, 2013 Sheet 10 of 34 US 2013/0224259 A1

INTRACELLULAR ADMA *p-0.05

s 3t 0.15 C 9 0.10 O C O O C > 0.05 ? C

O.OO Vehicle L-257 PD 404 182 Groups FIG. 9 Patent Application Publication Aug. 29, 2013 Sheet 11 of 34 US 2013/0224259 A1

kDa

97

64 56.5 51

39

28

19 14 Patent Application Publication Aug. 29, 2013 Sheet 12 of 34 US 2013/0224259 A1

PLY FTW1 W2. W3. E1 E2 E3 kDa

FIG. 1 1A Patent Application Publication Aug. 29, 2013 Sheet 13 of 34 US 2013/0224259 A1

kDa

64 DDAH1/GST 51

39 s -C-DDAH1

Š M 1 2 3 4 5 6 7 8 9

FIG. 11B Patent Application Publication Aug. 29, 2013 Sheet 14 of 34 US 2013/0224259 A1

ADMA METABOLISM IS LINEAR WITH DDAH CONCENTRATION 2.4

O) 18

C Vs 2 i 1.2 C g O.6

O.O O 2 4 6 8 10 DDAH Conc. (uM)

ADMA METABOLISM IS LINEAR WITH TIME 2.O

31.5 s O 2 3.S 1.O C s ? O5

O.O O 2 4 6 8 10 Time (hours) FIG. 12 Patent Application Publication Aug. 29, 2013 Sheet 15 of 34 US 2013/0224259 A1

DDAH Activity is Temperature, Time & Conc. Dependent

15 p<0.05 R2 = 0.88OO

3 1.0 V

O O C g 0.5

4h at RT 2h at 37 deg 4h at 37 deg 4h at RT2X enzyme Temperature/Time FIG. 12 (Cont.) Patent Application Publication Aug. 29, 2013 Sheet 16 of 34 US 2013/0224259 A1

34OO 33OO 32OO 3100 3OOO 29OO 28OO 2700 26OO Vehicle L-Arginine Groups

FIG. 13 Patent Application Publication Aug. 29, 2013 Sheet 17 of 34 US 2013/0224259 A1

:: SS 25.8-07, V F.G. (A Patent Application Publication Aug. 29, 2013 Sheet 18 of 34 US 2013/0224259 A1

i i

is: 25-8-073 is S$388 is FG 43 Patent Application Publication Aug. 29, 2013 Sheet 19 of 34 US 2013/0224259 A1

i ris --- is issississists: G C Patent Application Publication Aug. 29, 2013 Sheet 20 of 34 US 2013/0224259 A1

IC50 = 13.25 uM Hillslope = 2.1

1 10 Final SCH-202676 uM ConCentration FIG. 14D Patent Application Publication Aug. 29, 2013 Sheet 21 of 34 US 2013/0224259 A1

LGOZZIVO‘ONUOISSÐOO\/>|ueguê5) LHWC1C]SueldesOuOH Patent Application Publication Aug. 29, 2013 Sheet 22 of 34 US 2013/0224259 A1

IntraCellular Proteins

PRMTS NCE inhibitOr

SDMA, ADMA, L-NMMA

DDAH1 Hydrolysis ADMA —>DMA + Citruline

L-Arginine NO + Citruline NOS

FG 16 Patent Application Publication Aug. 29, 2013 Sheet 23 of 34 US 2013/0224259 A1

1 10 Final COncentration FIG. 17 Patent Application Publication Aug. 29, 2013 Sheet 24 of 34 US 2013/0224259 A1

IPF Lung Fibroblasts: Collagen Production

D1 D2 D3 D4

Seed Cell Serum Stimulation Collagen (4 x 104 cells) Synchronization + Vehicle/DDAH assay + protein normalization

80 5 O 60 Sl 2. 5 40 95. t o 20 C) O TGFB1 -- --

Vehicle H

PPI (20 uM) H Patent Application Publication Aug. 29, 2013 Sheet 25 of 34 US 2013/0224259 A1

a.

S

(uu Ogiy OO) uoeuoduopu npu . Patent Application Publication Aug. 29, 2013 Sheet 26 of 34 US 2013/0224259 A1

090Wri9?OWri9Wri [??OZeudeuuO] (Wri£70)HVOICI

Ogºf O?$3 03ºg 09È ****|00|- 3. ~~ U Patent Application Publication Aug. 29, 2013 Sheet 27 of 34 US 2013/0224259 A1 s3s S g9

CN O OO CO r CN O r Y d d d d o O O O O O O O (Wr) uoeupueouoo WWOW Patent Application Publication Aug. 29, 2013 Sheet 28 of 34 US 2013/0224259 A1

5.

3

3

S g9

9 OO CO V CN O (Nri) uoe Juegouoo XON Patent Application Publication Aug. 29, 2013 Sheet 29 of 34 US 2013/0224259 A1

Collagen Production by IPF Fibroblasts: Effect of PP

D1 D2 D3 D4

Seed Cell Synchronization Serum Stimulation Collagen Assay + (6 x 104 cells) + Vehicle Protein Normalization /DDAHit TGF-31

Colladen Production by PF Fibroblasts: Effect of PP

75

40 DDDs

10 O TGF-B1 ------

Vehicle -- Omeprazole - 50 100 L-257 50 A83-O1 2O Patent Application Publication Aug. 29, 2013 Sheet 30 of 34 US 2013/0224259 A1

PPI Ameliorates Lund Alveolar Epithelial Cell Proliferation

D1 D2 D3 D4

Seed Cell Synchronization Serum Stimulation Brd J Cell (5 x 103 cells) + vehicle Proliferation Assay /DDAH BrdUIncorporation (4 h)

PPl Ameliorates Human Lund Alveolar Epithelial Cell Proliferation

1.O

O. 8

O.6

O 4

O 2

O.O FBS ------

Vehicle -- L-257 50 100 Omeprazole - 3 10 2O 5O 1 OO Patent Application Publication Aug. 29, 2013 Sheet 31 of 34 US 2013/0224259 A1

PPI Ameliorates Mouse Lund Alveolar Epithelial 15 Cell Proliferation

1 O

O 5

O.O FBS ------Vehicle -- L-257 5O 1 OO - Omeprazole - 3 1 O 20 50 1 OO Patent Application Publication Aug. 29, 2013 Sheet 32 of 34 US 2013/0224259 A1

Apoptosis ASSav (HistOne-associated DNA Fradmentation 22

2O

18

16

14

in in

s + 1 uMA231.87 &

FIG. 26 Patent Application Publication Aug. 29, 2013 Sheet 33 of 34 US 2013/0224259 A1

In Vitro ToxicolodV Test (LDHASSav: A-549 Cells 120

1OO

8 O 46 OO

2 O

O Veh 50 100 200 20 50 100 200 300 Lysed L-257 Omeprazole

FIG. 27 Patent Application Publication Aug. 29, 2013 Sheet 34 of 34 US 2013/0224259 A1

in Vitro ToxicolodV Test (LDHASSaV: MLE-12 Cells 120

1OO

8 O 46 OO

2 O

O Veh 50 100 200 20 50 100 200 300 Lysed L-257 Omeprazole

FIG. 28 US 2013/0224259 A1 Aug. 29, 2013

DMETHYLARGINNE DMETHYLAMINOHYDROLASE (Ia) INHIBITORS AND METHODS OF USE THEREOF

CROSS-REFERENCE 0001. This application claims the benefit of U.S. Provi R5 R4 sional Patent Application Nos. 61/598.758, filed Feb. 14, 2012, 61/645,383, filed May 10, 2012, and 61/701,990, filed 0007 wherein Sep. 17, 2012, each of which applications are incorporated 0008 Q is N or CH: herein by reference in their entirety. I0009) R' is selected from alkyl, substituted alkyl, hydroxy, alkoxy, Substituted alkoxy, amino, Substituted amino, car INTRODUCTION boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl: 0002 Nitric oxide (NO) is a potent signaling molecule that (0010 R. R. R', and Rare independently selected from needs to be tightly regulated to maintain metabolic and car hydrogen, alkyl, Substituted alkyl, hydroxy, alkoxy, Substi diovascular homeostasis. The nitric oxide synthase (NOS)/ tuted alkoxy, amino, Substituted amino, carboxyl, carboxyl dimethylarginine dimethylaminohydrolase (DDAH)/Asym ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substi metric Dimethylarginine (ADMA) pathway is central to this tuted alkenyl, alkynyl, and Substituted alkynyl; and regulation. The small molecule ADMA competitively inhib 00.11 m is an integer from Zero to four; and its NOS, thus lowering NO levels. The majority of ADMA is 0012 a flowable formulation suitable for delivery by inha physiologically metabolized by DDAH, thus maintaining NO lation. levels at physiological concentration. However, under patho 0013 The present disclosure provides a pharmaceutical physiological conditions, NO synthesis and/or DDAH activ formulation comprising a DDAH inhibitor of the formula: ity may be pathologically increased. Such states include sep sis; fibrosis interstitial, e.g., pulmonary fibrosis; migraine (Ib) headaches; and some inflammatory and autoimmune dis O CaSCS. N y- N 0003. There is a need in the art for compounds that modu late DDAH activity, e.g., compounds that inhibit DDAH A-4NNH enzymatic activity. (R), R5 R4 LITERATURE 0014 wherein 0004 U.S. Pat. No. 8,063,104; Johnson et al. (2011) J. I0015I R' is selected from alkyl, substituted alkyl, hydroxy, Am. Chem. Soc. 133(5):1553-62; Johnson et al. (2011) J. alkoxy, Substituted alkoxy, amino, Substituted amino, car Am. Chem. Soc. 133(28):10951-9: Kotthaus et al. (2012) J boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, Enzyme Inhib Med. Chem. 27(1):24-8; Kotthaus et al. (2008) alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl: Bioorg Med. Chem. 16(24):10205-9: Lluis et al. (2011) 0016 R. R. R', and Rare independently selected from Chem Med Chem 6:81-88: Wang et al. (2009) Biochemistry is selected from alkyl, Substituted alkyl, hydroxy, alkoxy, 48(36):8624-35; U.S. Patent Publication No. 2011/0294878: Substituted alkoxy, amino, Substituted amino, carboxyl, car Knippet al. (2005) JAm Chem. Soc. 127(8):2372-3; Linsky boxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, et al. (2011) ACS Med Chem. Lett. 2(8):592-596: Hartzou Substituted alkenyl, alkynyl, and Substituted alkynyl; and lakis et al. (2007) Bioorg Med Chem. Lett. 17(14):3953-6. 0017 m is an integer from Zero to four; and 0018 a flowable formulation suitable for delivery by inha SUMMARY lation. 0019. The present disclosure provides a pharmaceutical 0005. The present disclosure provides DDAH inhibitors, formulation comprising a DDAH inhibitor of the formula: and compositions, including pharmaceutical compositions, comprising the inhibitors. DDAH inhibitors of the present disclosure are useful in treating disorders associated with (Ic) excessive NO production and/or elevated DDAH activity. The present disclosure provides methods of treating disorders associated with excessive NO production and/or elevated DDAH activity, the methods generally involving administer ing to an individual in need thereof an effective amount of a subject DDAH inhibitor. 0006. The present disclosure provides a pharmaceutical formulation comprising a DDAH inhibitor of the formula: US 2013/0224259 A1 Aug. 29, 2013

0020 wherein -continued I0021) R' is selected from alkyl, substituted alkyl, hydroxy, Ne alkoxy, Substituted alkoxy, amino, Substituted amino, car N boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl: y (V Y 0022 R. R. R', and Rare independently selected from is selected from alkyl, Substituted alkyl, hydroxy, alkoxy, O-. Substituted alkoxy, amino, Substituted amino, carboxyl, car (Rabeprazole) boxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl; and 0026. In any one of a subject pharmaceutical formulation 0023 m is an integer from Zero to four; and (e.g., a Subject pharmaceutical formulation comprising a 0024 a flowable formulation suitable for delivery by inha DDAH inhibitor of Formula Ia, Ib, or Ic), the DDAH inhibitor lation. can be formulated with a fluid carrier and a propellant. In any one of a subject pharmaceutical formulation (e.g., a subject 0025. In some cases, in a subject pharmaceutical for pharmaceutical formulation comprising a DDAH inhibitor of mulation, the DDAH inhibitor is selected from: Formula Ia, Ib, or Ic), the DDAH inhibitor can be in a dry powder formulation. 0027. The present disclosure provides a package for use in OCH treating a disorder associated with excessive NO production H3C and/or elevated DDAH activity, where the package comprises e CH: a container having therein a Subject pharmaceutical formula HCO tion (e.g., a Subject pharmaceutical formulation comprising a N N h DDAH inhibitor of Formula Ia, Ib, or Ic). In some cases, the N package is a metered dose inhaler, and the DDAH inhibitor is HX O formulated with a propellant. In some cases, the package is a dry powder inhaler, and the DDAH inhibitor is formulated in (Omeprazole) a dry powder formulation. In some cases, the package is a nebulizer, and the DDAH inhibitor is in an aqueous or etha nolic solution. 0028. The present disclosure provides a metered dose F O N inhaler having therein a subject pharmaceutical formulation, wherein the DDAH inhibitor is formulated with a propellant. O)-( - The present disclosure provides a dry powder inhaler having therein a Subject pharmaceutical formulation, wherein the DDAH inhibitor is formulated in a dry powder formulation. (Pantoprazole) The present disclosure provides a nebulizer having therein a Na subject pharmaceutical formulation, wherein the DDAH inhibitor is in an aqueous or ethanolic Solution. N N h 0029. The present disclosure provides anaerosol compris N ing a pharmaceutically active DDAH inhibitor. In some cases, N)- O o the DDAH inhibitor is contained within aerosolized particles CF: having a diameter in a range of from about 0.25um to about (Lansoprazole) 12 um. In some cases, the aerosol is in the form of a solution. OCH In some cases, the aerosol is in the form of a suspension. In Some cases, the aerosol is in the form of a powder. In some cases, the aerosol is in the form of a semi-solid preparation. N-N / \ 0030 The present disclosure provides a pharmaceutical N S NR formulation comprising a dimethylarginine dimethylamino 2 - \, hydrolase (DDAH) inhibitor of the formula: (Tenatoprazole) OCH3 (III)

HCO N Xu'sV \ / N O (Esomeprazole) US 2013/0224259 A1 Aug. 29, 2013

0031 wherein boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, 0032 Q' is S, O, NH or CH: alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl: 0033 Q is N or CH: and 0034. Q is N or CH: a pharmaceutically acceptable excipient. I0035) R' is selected from alkyl, substituted alkyl, hydroxy, 0048 alkoxy, Substituted alkoxy, amino, Substituted amino, car 0049. The present disclosure provides a pharmaceutical boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, formulation comprising a dimethylarginine dimethylamino alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl: hydrolase (DDAH) inhibitor of the formula: I0036) R is selected from alkyl, substituted alkyl, hydroxy, alkoxy, Substituted alkoxy, amino, Substituted amino, car boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, (IIb) alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl: CH O O R 0037 m is an integer from Zero to four; and \ 0038 n is an integer from Zero to three; and N V 0039 a flowable formulation suitable for delivery by inha N R2 lation. HC N2Ns/ 0040. In some cases, the DDAH inhibitor is: 0050 wherein I0051) R' is selected from hydrogen, alkyl, substituted alkyl, alkoxy, Substituted alkoxy, cycloalkyl, Substituted ON cycloalkyl, aryl, Substituted aryl, heterocyclyl, and Substi tuted heterocyclyl: S ls NH. I0052 R is selected from hydrogen, alkyl, and substituted alkyl; and 0041. In some cases, the DDAH inhibitor of Formula III is 0053 a pharmaceutically acceptable excipient. formulated with a fluid carrier and a propellant; or the DDAH inhibitor of Formula III is in a dry powder formulation. 0054. In some cases, the DDAH inhibitor is selected from: 0042. The present disclosure provides a package for use in treating a disorder associated with excessive NO production and/or elevated DDAH activity, the package comprising a CH3 O O container having therein a Subject pharmaceutical formula H tion comprising a DDAH inhibitor of Formula III. In some cases, the package is a metered dose inhaler, and the DDAH N N )- CH: inhibitor is formulated with a propellant; or the package is a 2 M dry powder inhaler, and the DDAH inhibitor is formulated in H3C N S a dry powder formulation; or the package is a nebulizer, and the DDAH inhibitor is in an aqueous or ethanolic solution. 0043. The present disclosure provides a pharmaceutical CH O O formulation comprising a dimethylarginine dimethylamino hydrolase (DDAH) inhibitor of the formula: N CH 3

2 N (IIa) HC N S CH: R3 O O R1 CH O O N \M N R2 N R4 4Ns 2 S X-/ \ S HC N 0044 wherein I0045) R' is selected from hydrogen, alkyl, substituted alkyl, alkoxy, Substituted alkoxy, cycloalkyl, Substituted cycloalkyl, aryl, Substituted aryl, heterocyclyl, and Substi tuted heterocyclyl: CH O O 0046 R is selected from hydrogen, alkyl, and substituted alkyl: N 0047 R and R are independently selected from is N CH: selected from hydrogen, alkyl, Substituted alkyl, hydroxy, HC N O-/S \ O -ch alkoxy, Substituted alkoxy, amino, Substituted amino, car US 2013/0224259 A1 Aug. 29, 2013

-continued -continued CH3 O O CH O O H N N N )- N CH: 2 M HC N S H3C N 2Ns/ s

O-CH3; and CH3 O O -/

CH3 O O N

H3C N2Ns/ O CH O O H3C N2Ns/ O N N CH3 O O HC N 2Ns/ N C N H3C N2Ns/ 0055. In some cases, in a subject pharmaceutical formu lation (e.g., a pharmaceutical formulation comprising a DDAH inhibitor of Formula IIa or Formula IIb, or any of the above-listed specific DDAH inhibitors of Formula IIa or IIb) Cl; the DDAH inhibitor is formulated with a fluid carrier and a propellant. In some cases, in a Subject pharmaceutical formu CH3 O O lation (e.g., a pharmaceutical formulation comprising a DDAH inhibitor of Formula IIa or Formula IIb, or any of the N above-listed specific DDAH inhibitors of Formula IIa or IIb) N the DDAH inhibitor is in a dry powder formulation. H3C N 2Ns/ 0056. The present disclosure provides a package for use in treating a disorder associated with excessive NO production and/or elevated DDAH activity, the package comprising a container having therein a Subject pharmaceutical formula tion (e.g., a pharmaceutical formulation comprising a DDAH inhibitor of Formula IIa or Formula IIb, or any of the above o listed specific DDAH inhibitors of Formula IIa or IIb). In CH: Some cases, the package is a metered dose inhaler, and the DDAH inhibitor is formulated with a propellant. In some cases, the package is a dry powder inhaler, and the DDAH inhibitor is formulated in a dry powder formulation. In some cases, the package is a nebulizer, and the DDAH inhibitor is CH3 O O in an aqueous or ethanolic solution. N 0057 The present disclosure provides a method of treating an individual suffering from a disorder characterized by 2 N excessive NO production and/or elevated DDAH activity, the H3C N S yCH is method comprising administering to the individual an effec O O / tive amount of a subject pharmaceutical formulation. In some HC cases (e.g., where the pharmaceutical formulation comprises a DDAH inhibitor of Formula IIa, IIb, a compound of any of CH O O Formulas IV-IX, or a compound of Formula X), the formula tion is administered by injection. In some cases (e.g., where N the pharmaceutical formulation comprises a DDAH inhibitor N of Formula IIa, IIb, a compound of any of Formulas IV-IX, or HC N2Ns/ a compound of Formula X), the formulation administered with a carrier in the form of normal saline solution. In some cases (e.g., where the pharmaceutical formulation comprises a DDAH inhibitor of Formula Ia, Ib, Ic, or III, or where the formulation comprises a DDAH inhibitor of Formula IIa, IIb, a compound of any of Formulas IV-IX, or a compound of US 2013/0224259 A1 Aug. 29, 2013

Formula X), the DDAH inhibitor is administered locally to 0070 FIG. 12 shows a citrulline assay showing that the the airways of the patient. In some cases (e.g., where the conversion of ADMA to L-citrulline by DDAH1 is propor pharmaceutical formulation comprises a DDAH inhibitor of tional to time, temperature and enzyme concentration. Formula Ia, Ib, Ic, or III, or where the formulation comprises (0071 FIG. 13 shows measurement of L-citrulline from a DDAH inhibitor of Formula IIa, Ilb, a compound of any of primary human endothelial cell lysate. Formulas IV-IX, or a compound of Formula X), the DDAH 0072 FIG. 14A-D show curve fit data showing inhibition inhibitor is administered by inhalation. In some cases (e.g., of human DDAH-1 activity by selected small molecules where the pharmaceutical formulation comprises a DDAH using the CPM assay. inhibitor of Formula Ia, Ib, Ic, or III, or where the formulation 0073 FIG. 15 provides an amino acid sequence of a comprises a DDAH inhibitor of Formula IIa, IIb, a compound DDAH polypeptide. of any of Formulas IV-IX, or a compound of Formula X), the (0074 FIG. 16 depicts the NO/ADMA/DDAH pathway. DDAH inhibitor is administered by insufflating an aerosol DMA: dimethylamine; ADMA: asymmetric dimethylargin comprising the DDAH inhibitor. In some cases, the DDAH ine: SDMA: symmetric dimethyl arginine: L-NMMA: inhibitor is in a dry powder formulation. In some cases, the monomethyl arginine: PRMTS: protein arginine methyltrans DDAH inhibitor is administered using a nebulizer. In some ferases; NO: nitric oxide: NCE: new chemical entity. cases, the DDAH inhibitor is in an aqueous or ethanolic (0075 FIG. 17 shows curve fit data showing inhibition of Solution. In some cases, the individual being treated is a DDAH activity by a compound of Formula X. human. In some cases, the individual being treated is a non (0076 FIG. 18 depicts the effect of a proton pump inhibitor human mammal. In some cases, the disease being treated is on collagen production by lung fibroblasts from patients with fibrosis. In some cases, the disease being treated is pulmonary late-stage idiopathic pulmonary fibrosis. fibrosis, e.g., IPF. (0077 FIG. 19 depicts the effect of PPIs on lung alveolar 0058. The present disclosure provides an in vitro method epithelial cell proliferation. of identifying an agent that inhibits enzymatic activity of a (0078 FIG. 20 depicts reversible inhibition of DDAH by DDAH polypeptide, the method comprising: a) contacting PPIs. the DDAH polypeptide and a DDAH substrate with a test (0079 FIG. 21 is a graph showing the effect of PPI (Ome agent; and b) determining the effect, ifany, of the testagent on prazole) on cellular ADMA. DDAH enzymatic activity, wherein the substrate is asymmet 0080 FIG.22 is a graph showing the effect of PPI on nitric ric dimethylarginine, and the determining step is a colorimet oxide production. ric assay for L-citrulline. A test agent that decreases DDAH I0081 FIG. 23 depicts the effect of a proton pump inhibitor activity, compare to a control, is an agent that inhibits DDAH on collagen production by lung fibroblasts from patients with activity. An agent that inhibits DDAH activity can be consid idiopathic pulmonary fibrosis (IPF). ered a candidate agent for treating a disorder characterized by I0082 FIG. 24 is a graph showing the effect of PPI on excessive NO production and/or elevated DDAH activity lev human lung alveolar epithelial cell proliferation. els. In some cases, the substrate is ADMA, and said deter I0083 FIG. 25 is a graph showing the effect of PPI on mining step comprises reacting L-citrulline with 2,3-Dim mouse lung alveolar epithelial cell proliferation. ethyl-1-phenyl-3-pyrazolin-5-one and 2,3-butanedione I0084 FIG. 26 is a graph showing the effect of PPI on oXime. apoptosis in an Endoplasmic Reticulum (ER)-stressed human alveolar epithelial cell-line (A-549). BRIEF DESCRIPTION OF THE DRAWINGS I0085 FIG. 27 is a graph showing an in vitro toxicology 0059 FIG. 1 provides graphs showing inhibition of test of human alveolar epithelial cell-line (A-549) tested with DDAH activity by various compounds. PPIs. 0060 FIG. 2 is a graph showing re-validation of proton I0086 FIG. 28 is a graph showing an in vitro toxicology pump inhibitors (PPIs) as DDAH inhibitors using CPMassay. test of mouse alveolar epithelial cell-line (A-549) tested with 0061 FIG. 3 is a graph showing an orthogonal assay to PPIs. validate PPIs as DDAH inhibitors. 0062 FIG. 4 is a graph showing production of L-citrulline DEFINITIONS from ADMA as a result of administration of various PPIs. I0087. The following terms have the following meanings 0063 FIG. 5 is a graph that shows concentration of intra unless otherwise indicated. Any undefined terms have their cellular ADMA as a result of administration of various com art recognized meanings. pounds. I0088 Alkyl refers to monovalent saturated aliphatic 0064 FIG. 6 is a graph showing direct inhibition of DDAH hydrocarbyl groups having from 1 to 10 carbon atoms, e.g., activity by small molecule PD 404. from 1 to 6 carbon atoms. This term includes, by way of 0065 FIG. 7 is a graph showing re-validation of PD 404 as example, linear and branched hydrocarbyl groups such as a DDAH inhibitor using CPM assay. methyl (CH ), ethyl (CHCH ), n-propyl 0066 FIG. 8 is a graph showing an orthogonal assay to (CHCHCH ), isopropyl ((CH),CH ), n-butyl validate PD 404 as a DDAH inhibitor. (CHCHCHCH ), isobutyl ((CH) CHCH ), sec-bu 0067 FIG.9 is a graph showing production of L-citrulline tyl ((CH)(CHCH2)CH ), t-butyl ((CH)C ), n-pentyl from ADMA as a result of administration of PD 404. (CHCHCHCHCH ), and neopentyl (CH),CCH ). 0068 FIG. 10 shows a Western blot analysis showing the I0089. The term “substituted alkyl refers to an alkyl group production of GST-DDAH (56.5 kDa). as defined herein wherein one or more carbon atoms in the 0069 FIG. 11A shows SDS-PAGE analysis of purified alkyl chain have been optionally replaced with a heteroatom human DDAH1. FIG. 11B shows Western blot showing puri Such as —O— —N— —S——S(O) = (where n is 0 to 2), fied (after GST cleavage) recombinant human DDAH1 (-37 —NR— (where R is hydrogen or alkyl) and having from 1 to kDa). 5 substituents selected from the group consisting of alkoxy, US 2013/0224259 A1 Aug. 29, 2013

Substituted alkoxy, cycloalkyl, Substituted cycloalkyl, wherein alkyl, substituted alkyl, alkylene and substituted cycloalkenyl, Substituted cycloalkenyl, acyl, acylamino, acy alkylene are as defined herein. loxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, 0101 The term “alkylthioalkoxy” refers to the group azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, -alkylene-5-alkyl, alkylene-S-substituted alkyl, substituted carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocy alkylene-5-alkyl and substituted alkylene-5-substituted alkyl clooxy, thiol, thioalkoxy, Substituted thioalkoxy, aryl, ary loxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocy wherein alkyl, substituted alkyl, alkylene and substituted clooxy, hydroxyamino, alkoxyamino, nitro. —SO-alkyl, alkylene are as defined herein. —SO-aryl, -SO-heteroaryl, -SO-alkyl, -SO-aryl, 0102 Alkenyl refers to straight chain or branched —SO-heteroaryl, and - NR'R'', wherein Rand R" may be hydrocarbyl groups having from 2 to 6 carbon atoms or from the same or different and are chosen from hydrogen, option 2 to 4 carbon atoms, and having at least 1 site of double bond ally Substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alky unsaturatio, e.g., from 1 to 2 sites of double bond unsatura nyl, aryl, heteroaryland heterocyclic. tion. This term includes, by way of example, bi-vinyl, allyl, 0090. “Alkylene' refers to divalent aliphatic hydrocarbyl and but-3-en-1-yl. Included within this term are the cis and groups, e.g., having from 1 to 6 carbon atoms or from 1 to 3 trans isomers or mixtures of these isomers. carbonatoms that are either straight-chained or branched, and 0103) The term “substituted alkenyl refers to an alkenyl which are optionally interrupted with one or more groups group as defined herein having from 1 to 5 substituents, or selected from -O-, - NR' , – NR'C(O) , —C(O) from 1 to 3 substituents, selected from alkoxy, substituted NR'' and the like. This term includes, by way of example, alkoxy, cycloalkyl, Substituted cycloalkyl, cycloalkenyl, Sub methylene (-CH ), ethylene (-CH2CH2—), n-propy stituted cycloalkenyl, acyl, acylamino, acyloxy, amino, Sub lene ( CHCHCH ), iso-propylene (—CHCH stituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, (CH)—), (-C(CH)2CHCH ), (-COCH)2CHC azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, (O) ), ( C(CH),CHC(O)NH ), (-CH(CH)CH ), carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocy and the like. clooxy, thiol, thioalkoxy, Substituted thioalkoxy, aryl, ary 0091 “Substituted alkylene' refers to an alkylene group loxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocy having from 1 to 3 hydrogens replaced with Substituents as clooxy, hydroxyamino, alkoxyamino, nitro. —SO-alkyl, described for carbons in the definition of “substituted' below. —SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, 0092. The term "alkane' refers to alkyl group and alkylene —SO-alkyl, -SO-substituted alkyl, -SO-aryl and group, as defined herein. —SO-heteroaryl. 0093. The term “alkylaminoalkyl”, “alkylaminoalkenyl 0104 Alkynyl refers to straight or branched monovalent and “alkylaminoalkynyl refers to the groups R'NHR" hydrocarbyl groups having from 2 to 6 carbon atoms or from where R is alkyl group as defined herein and R" is alkylene, 2 to 3 carbon atoms and having at least 1 site of triple bond alkenylene or alkynylene group as defined herein. unsaturation, e.g., from 1 to 2 sites of triple bond unsatura 0094. The term “alkaryl” or “aralkyl refers to the groups tion. Examples of Such alkynyl groups include acetylenyl -alkylene-aryland -substituted alkylene-aryl where alkylene, (—C=CH), and propargyl (—CH2C=CH). substituted alkylene and aryl are defined herein. 0105. The term “substituted alkynyl refers to an alkynyl 0095 “Alkoxy” refers to the group -O-alkyl, wherein group as defined herein having from 1 to 5 substituents, or alkyl is as defined herein. Alkoxy includes, by way of from 1 to 3 substituents, selected from alkoxy, substituted example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, alkoxy, cycloalkyl, Substituted cycloalkyl, cycloalkenyl, Sub t-butoxy, Sec-butoxy, n-pentoxy, and the like. The term stituted cycloalkenyl, acyl, acylamino, acyloxy, amino, Sub “alkoxy” also refers to the groups alkenyl-O cycloalkyl stituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, O—, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein. carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocy 0096. The term “substituted alkoxy' refers to the groups clooxy, thiol, thioalkoxy, Substituted thioalkoxy, aryl, ary substituted alkyl-O substituted alkenyl-O substituted loxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocy cycloalkyl-O-, Substituted cycloalkenyl-O-, and Substi clooxy, hydroxyamino, alkoxyamino, nitro. —SO-alkyl, tuted alkynyl-O where substituted alkyl, substituted alk —SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, enyl, Substituted cycloalkyl, Substituted cycloalkenyl and —SO-alkyl, -SO-substituted alkyl, -SO-aryl, and substituted alkynyl are as defined herein. —SO-heteroaryl. 0097. The term “alkoxyamino” refers to the group - NH alkoxy, wherein alkoxy is defined herein. 0106 “Alkynyloxy' refers to the group —O-alkynyl, 0098. The term “haloalkoxy” refers to the groups alkyl wherein alkynyl is as defined herein. Alkynyloxy includes, by O— wherein one or more hydrogenatoms on the alkyl group way of example, ethynyloxy, propynyloxy, and the like. have been substituted with a halo group and include, by way 0107 “Acyl” refers to the groups H C(O) , alkyl-C of examples, groups such as trifluoromethoxy, and the like. (O)—, substituted alkyl-C(O)—, alkenyl-C(O)—, substi 0099. The term “haloalkyl refers to a substituted alkyl tuted alkenyl-C(O)—, alkynyl-C(O)—, substituted alkynyl group as described above, wherein one or more hydrogen C(O)—, cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—, atoms on the alkyl group have been substituted with a halo cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—, group. Examples of Such groups include, without limitation, aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl, substituted heteroaryl-C(O)—, heterocyclyl-C(O)—, and trifluoroethyl and the like. substituted heterocyclyl-C(O)—, wherein alkyl, substituted 0100. The term “alkylalkoxy' refers to the groups -alky alkyl, alkenyl, Substituted alkenyl, alkynyl, Substituted alky lene-O-alkyl, alkylene-f-substituted alkyl, substituted alky nyl, cycloalkyl, Substituted cycloalkyl, cycloalkenyl, Substi lene-O-alkyl, and substituted alkylene-O-substituted alkyl tuted cycloalkenyl, aryl, Substituted aryl, heteroaryl, Substi US 2013/0224259 A1 Aug. 29, 2013 tuted heteroaryl, heterocyclic, and substituted heterocyclic 0114 “Sulfonylamino” refers to the group are as defined herein. For example, acyl includes the “acetyl - NRSOR’, wherein R and R’ independently are group CHC(O)—. selected from the group consisting of hydrogen, alkyl, Sub 0108 “Acylamino” refers to the groups - NRC(O) stituted alkyl, alkenyl, Substituted alkenyl, alkynyl, Substi alkyl, - NRC(O)substituted alkyl, N RC(O)cycloalkyl, tuted alkynyl, aryl, Substituted aryl, cycloalkyl, Substituted -NRC(O)substituted cycloalkyl, - NRC(O)cycloalk cycloalkyl, cycloalkenyl, Substituted cycloalkenyl, het enyl, - NRC(O)substituted cycloalkenyl, - NRC(O) eroaryl, substituted heteroaryl, heterocyclic, and substituted alkenyl, - NRC(O)substituted alkenyl, - NRC(O)alky heterocyclic and where R'' and R’ are optionally joined nyl, - NRC(O)substituted alkynyl, - NRC(O)aryl, together with the atoms bound thereto to form a heterocyclic - NRC(O)substituted aryl, - NRC(O)heteroaryl, or Substituted heterocyclic group, and wherein alkyl, Substi -NRC(O)substituted heteroaryl, - NRC(O)heterocy tuted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted clic, and NRC(O)substituted heterocyclic, wherein R' alkynyl, cycloalkyl, Substituted cycloalkyl, cycloalkenyl, is hydrogen or alkyl and wherein alkyl, Substituted alkyl, Substituted cycloalkenyl, aryl, Substituted aryl, heteroaryl, alkenyl, Substituted alkenyl, alkynyl. Substituted alkynyl, substituted heteroaryl, heterocyclic, and substituted hetero cycloalkyl, Substituted cycloalkyl, cycloalkenyl, Substituted cyclic are as defined herein. cycloalkenyl, aryl. Substituted aryl, heteroaryl, Substituted 0115 Aryl or 'Ar' refers to a monovalent aromatic car heteroaryl, heterocyclic, and Substituted heterocyclic are as bocyclic group of from 6 to 18 carbon atoms having a single defined herein. ring (Such as is present in a phenyl group) or a ring system 0109) “Aminocarbonyl' or the term “aminoacyl” refers to having multiple condensed rings (examples of Such aromatic the group - C(O)NR'R'', wherein R and R’ indepen ring systems include naphthyl, anthryl and indanyl) which dently are selected from the group consisting of hydrogen, condensed rings may or may not be aromatic, provided that alkyl, Substituted alkyl, alkenyl, Substituted alkenyl, alkynyl, the point of attachment is through an atom of an aromatic Substituted alkynyl, aryl, Substituted aryl, cycloalkyl, Substi ring. This term includes, by way of example, phenyl and tuted cycloalkyl, cycloalkenyl, Substituted cycloalkenyl, het naphthyl. Unless otherwise constrained by the definition for eroaryl, substituted heteroaryl, heterocyclic, and substituted the aryl Substituent, such aryl groups can optionally be Sub stituted with from 1 to 5 substituents, or from 1 to 3 substitu heterocyclic and where R'' and R’ are optionally joined ents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, together with the nitrogen bound thereto to form a heterocy alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, Substi clic or Substituted heterocyclic group, and wherein alkyl, tuted alkyl, substituted alkoxy, substituted alkenyl, substi substituted alkyl, alkenyl, Substituted alkenyl, alkynyl, Sub tuted alkynyl, substituted cycloalkyl, substituted cycloalk stituted alkynyl, cycloalkyl, Substituted cycloalkyl, cycloalk enyl, amino, Substituted amino, aminoacyl, acylamino, enyl, Substituted cycloalkenyl, aryl, Substituted aryl, het alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, eroaryl, substituted heteroaryl, heterocyclic, and substituted halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, het heterocyclic are as defined herein. erocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, Sub 0110 "Aminocarbonylamino” refers to the group stituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO NRC(O)NR'R' where R', R, and Rare indepen alkyl, -SO-substituted alkyl, -SO-aryl, —SO-heteroaryl, dently selected from hydrogen, alkyl, aryl or cycloalkyl, or —SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO where two R groups are joined to form a heterocyclyl group. heteroaryl and trihalomethyl. 0111. The term “alkoxycarbonylamino” refers to the 0116 'Aryloxy' refers to the group —O-aryl, wherein group - NRC(O)OR where each R is independently hydro aryl is as defined herein, including, by way of example, phe gen, alkyl, Substituted alkyl, aryl, heteroaryl, or heterocyclyl noxy, naphthoxy, and the like, including optionally Substi wherein alkyl, substituted alkyl, aryl, heteroaryl, and hetero tuted aryl groups as also defined herein. cyclyl are as defined herein. 0117 “Amino” refers to the group -NH. 0112 The term “acyloxy' refers to the groups alkyl-C(O) 0118. The term “substituted amino” refers to the group O— substituted alkyl-C(O)O—, cycloalkyl-C(O)O— sub —NRR where each R is independently selected from the stituted cycloalkyl-C(O)O—, aryl-C(O)O—, heteroaryl-C group consisting of hydrogen, alkyl, Substituted alkyl, (O)O—, and heterocyclyl-C(O)O— wherein alkyl, cycloalkyl, Substituted cycloalkyl, alkenyl, Substituted alk substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, het enyl, cycloalkenyl, Substituted cycloalkenyl, alkynyl, Substi eroaryl, and heterocyclyl are as defined herein. tuted alkynyl, aryl, heteroaryl, and heterocyclyl provided that 0113 Aminosulfonyl refers to the group at least one R is not hydrogen. —SONR'R'', wherein R and R’ independently are 0119) The term “azido” refers to the group - N. selected from the group consisting of hydrogen, alkyl, Sub I0120 “Carboxyl,” “carboxy” or “carboxylate” refers to stituted alkyl, alkenyl, Substituted alkenyl, alkynyl, Substi —COH or salts thereof. tuted alkynyl, aryl, Substituted aryl, cycloalkyl, Substituted I0121 “Carboxyl ester” or “carboxy ester” or the terms cycloalkyl, cycloalkenyl, Substituted cycloalkenyl, het “carboxyalkyl or “carboxylalkyl refers to the groups eroaryl, substituted heteroaryl, heterocyclic, substituted het —C(O)C-alkyl, —C(O)O-substituted alkyl, —C(O)O-alk erocyclic and where R'' and R’ are optionally joined enyl, —C(O)O-substituted alkenyl, —C(O)O-alkynyl, together with the nitrogen bound thereto to form a heterocy —C(O)C)-substituted alkynyl, —C(O)O-aryl, —C(O)O-sub clic or Substituted heterocyclic group and alkyl, Substituted stituted aryl, —C(O)f-cycloalkyl, —C(O)O-substituted alkyl, alkenyl, Substituted alkenyl, alkynyl, Substituted alky cycloalkyl, —C(O)(3-cycloalkenyl, —C(O)O-substituted nyl, cycloalkyl, Substituted cycloalkyl, cycloalkenyl, Substi cycloalkenyl, —C(O)O-heteroaryl, —C(O)O-substituted tuted cycloalkenyl, aryl, Substituted aryl, heteroaryl, Substi heteroaryl, —C(O)O-heterocyclic, and —C(O)O-substituted tuted heteroaryl, heterocyclic and substituted heterocyclic are heterocyclic, wherein alkyl, substituted alkyl, alkenyl, sub as defined herein. stituted alkenyl, alkynyl. Substituted alkynyl, cycloalkyl, Sub US 2013/0224259 A1 Aug. 29, 2013

stituted cycloalkyl, cycloalkenyl, Substituted cycloalkenyl, I0131 “Halo' or “halogen” refers to fluoro, chloro, bromo, aryl, substituted aryl, heteroaryl, substituted heteroaryl, het and iodo. erocyclic, and Substituted heterocyclic are as defined herein. (0132) “Hydroxy” or “hydroxyl refers to the group –OH. 0122) “(Carboxyl ester)oxy” or “carbonate” refers to the 0.133 “Heteroaryl” refers to an aromatic group of from 1 groups —O C(O)O-alkyl, —O C(O)O-substituted alkyl, to 15 carbon atoms, such as from 1 to 10 carbonatoms and 1 —O C(O)O-alkenyl, —O C(O)O-substituted alkenyl, to 10 heteroatoms selected from the group consisting of oxy —O C(O)O-alkynyl, —O C(O)O-substituted alkynyl, gen, nitrogen, and Sulfur within the ring. Such heteroaryl —O C(O)O-aryl, —O C(O)O-substituted aryl, —O C groups can have a single ring (such as, pyridinyl, imidazolyl (O)O-cycloalkyl, O—C(O)O-substituted cycloalkyl, or furyl) or multiple condensed rings in a ring system (for —O C(O)3-cycloalkenyl, —O—C(O)O-substituted example as in groups such as, indolizinyl, quinolinyl, benzo cycloalkenyl, —O—C(O)O-heteroaryl, —O—C(O)O-sub furan, benzimidazolyl or benzothienyl), wherein at least one stituted heteroaryl, —O C(O)O-heterocyclic, and —O C ring within the ring system is aromatic and at least one ring (O)O-substituted heterocyclic, wherein alkyl, substituted within the ring system is aromatic, provided that the point of alkyl, alkenyl, Substituted alkenyl, alkynyl, Substituted alky attachment is through an atom of an aromatic ring. In certain nyl, cycloalkyl, Substituted cycloalkyl, cycloalkenyl, Substi embodiments, the nitrogen and/or Sulfur ring atom(s) of the tuted cycloalkenyl, aryl, Substituted aryl, heteroaryl, Substi heteroaryl group are optionally oxidized to provide for the tuted heteroaryl, heterocyclic, and substituted heterocyclic N-oxide (N->O), sulfinyl, or sulfonyl moieties. This term are as defined herein. includes, by way of example, pyridinyl, pyrrolyl, indolyl, (0123 “Cyano” or “nitrile” refers to the group –CN. thiophenyl, and furanyl. Unless otherwise constrained by the 0.124 “Cycloalkyl refers to cyclic alkyl groups of from 3 definition for the heteroaryl substituent, such heteroaryl to 10 carbon atoms having single or multiple cyclic rings groups can be optionally substituted with 1 to 5 substituents, including fused, bridged, and spiroring systems. Examples of or from 1 to 3 substituents, selected from acyloxy, hydroxy, Suitable cycloalkyl groups include, for instance, adamantyl, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. cycloalkenyl, substituted alkyl, substituted alkoxy, substi Such cycloalkyl groups include, by way of example, single tuted alkenyl, substituted alkynyl, substituted cycloalkyl, ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, Substituted cycloalkenyl, amino, Substituted amino, aminoa cyclooctyl, and the like, or multiple ring structures Such as cyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, car adamantanyl, and the like. boxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, 0.125. The term “substituted cycloalkyl” refers to heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, cycloalkyl groups having from 1 to 5 substituents, or from 1 thioalkoxy, substituted thioalkoxy, thioaryloxy, thiohet to 3 substituents, selected from alkyl, substituted alkyl, eroaryloxy, —SO-alkyl, - SO-substituted alkyl, -SO-aryl, alkoxy, Substitutedalkoxy, cycloalkyl, Substituted cycloalkyl, —SO-heteroaryl, -SO-alkyl, -SO-substituted alkyl, cycloalkenyl, Substituted cycloalkenyl, acyl, acylamino, acy —SO-aryland—SO-heteroaryl, and trihalomethyl. loxy, amino, Substituted amino, aminoacyl, aminoacyloxy, I0134. The term "heteroaralkyl refers to the groups -alky oxyaminoacyl, azido, cyano, halogen, hydroxyl, OXo, lene-heteroaryl where alkylene and heteroaryl are defined thioketo, carboxyl, carboxylalkyl, thioaryloxy, thiohet herein. This term includes, by way of example, pyridylm eroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, Substituted ethyl, pyridylethyl, indolylmethyl, and the like. thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, hetero I0135) “Heteroaryloxy” refers to O-heteroaryl. cyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, (0.136) “Heterocycle,” “heterocyclic,” “heterocycloalkyl.” —SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-het and "heterocyclyl refer to a saturated or unsaturated group eroaryl, -SO-alkyl, -SO-substituted alkyl, -SO-aryl having a single ring or multiple condensed rings, including and —SO-heteroaryl. fused bridged and spiroring systems, and having from 3 to 20 0126 “Cycloalkenyl refers to non-aromatic cyclic alkyl ring atoms, including 1 to 10 hetero atoms. These ring atoms groups of from 3 to 10 carbonatoms having single or multiple are selected from the group consisting of nitrogen, Sulfur, or rings and having at least one double bond, e.g., from 1 to 2 oxygen, wherein, in fused ring systems, one or more of the double bonds. rings can be cycloalkyl, aryl, or heteroaryl, provided that the 0127. The term “substituted cycloalkenyl refers to point of attachment is through the non-aromatic ring. In cer cycloalkenyl groups having from 1 to 5 substituents, or from tain embodiments, the nitrogen and/or Sulfur atom(s) of the 1 to 3 substituents, selected from alkoxy, substituted alkoxy, heterocyclic group are optionally oxidized to provide for the cycloalkyl, Substituted cycloalkyl, cycloalkenyl, Substituted N-oxide. —S(O)—, or —SO - moieties. cycloalkenyl, acyl, acylamino, acyloxy, amino, Substituted 0.137 Examples of heterocycles and heteroaryls include, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, but are not limited to, aZetidine, pyrrole, imidazole, pyrazole, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxy pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoin lalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, dole, indole, dihydroindole, indazole, purine, quinolizine, thiol, thioalkoxy, Substituted thioalkoxy, aryl, aryloxy, het isoquinoline, quinoline, phthalazine, naphthylpyridine, qui eroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, noxaline, quinazoline, cinnoline, pteridine, carbazole, carbo hydroxyamino, alkoxyamino, nitro. —SO-alkyl, -SO-Sub line, phenanthridine, acridine, phenanthroline, isothiazole, stituted alkyl, —SO-aryl, -SO-heteroaryl, -SO-alkyl, phenazine, isoxazole, phenoxazine, phenothiazine, imidazo —SO-substituted alkyl, -SO-aryland—SO-heteroaryl. lidine, imidazoline, piperidine, piperazine, indoline, phthal 012.8 “Cycloalkynyl refers to non-aromatic cycloalkyl imide, 1.2.3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahy groups of from 5 to 10 carbonatoms having single or multiple drobenzobthiophene, thiazole, thiazolidine, thiophene, rings and having at least one triple bond. benzobthiophene, morpholinyl, thiomorpholinyl (also 0129. “Cycloalkoxy” refers to O-cycloalkyl. referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl, 0130 “Cycloalkenyloxy' refers to —O-cycloalkenyl. piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like. US 2013/0224259 A1 Aug. 29, 2013

0.138. Unless otherwise constrained by the definition for 0151. The term “thioaryloxy' refers to the group aryl-S- the heterocyclic Substituent, Such heterocyclic groups can be wherein the aryl group is as defined herein including option optionally substituted with 1 to 5, or from 1 to 3 substituents, ally Substituted aryl groups also defined herein. selected from alkoxy, Substituted alkoxy, cycloalkyl, Substi 0152 The term “thioheteroaryloxy' refers to the group tuted cycloalkyl, cycloalkenyl, Substituted cycloalkenyl, heteroaryl-S wherein the heteroaryl group is as defined acyl, acylamino, acyloxy, amino, Substituted amino, aminoa herein including optionally Substituted aryl groups as also cyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, defined herein. hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioary 0153. The term “thioheterocyclooxy' refers to the group loxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thio heterocyclyl-S— wherein the heterocyclyl group is as alkoxy, Substituted thioalkoxy, aryl, aryloxy, heteroaryl, het defined herein including optionally substituted heterocyclyl eroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, groups as also defined herein. alkoxyamino, nitro. —SO-alkyl, - SO-substituted alkyl, 0154. In addition to the disclosure herein, the term “sub —SO-aryl, -SO-heteroaryl, -SO-alkyl, -SO-substi stituted, when used to modify a specified group or radical, tuted alkyl, - SO-aryl, —SO-heteroaryl, and fused hetero can also mean that one or more hydrogen atoms of the speci cycle. fied group or radical are each, independently of one another, 0139) “Heterocyclyloxy' refers to the group —O-hetero replaced with the same or different Substituent groups as cyclyl. defined below. 0140. The term "heterocyclylthio’ refers to the group het 0.155. In addition to the groups disclosed with respect to erocyclic-S-. the individual terms herein, substituent groups for substitut 0141. The term "heterocyclene' refers to the diradical ing for one or more hydrogens (any two hydrogens on a single group formed from a heterocycle, as defined herein. carbon can be replaced with =O, —NR', =N-OR', 0142. The term “hydroxyamino” refers to the group —Nor=S) on Saturated carbonatoms in the specified group NHOH. or radical are, unless otherwise specified, R', halo, =O, 0143 “Nitro” refers to the group - NO. OR7, SR 7, NR'R'', trihalomethyl, CN, OCN, 0144) “Oxo” refers to the atom (=O). —SCN, NO, NO. —N - N - SOR', -SOO (0145 "Sulfonyl refers to the group SO-alkyl, SO-sub M", SOOR7, OSOR7, OSOOM", stituted alkyl, SO-alkenyl, SO substituted alkenyl, SO OSO,OR7, -P(O)(O)(Mt), -P(O)(OR7)OM", cycloalkyl, SO-substituted cylcoalkyl, SO-cycloalkenyl, P(O)(OR7), C(O)R7, C(S)R7, C(NR7)R’, SO-substituted cylcoalkenyl, SO-aryl, SO-substituted C(O)OM", C(O)OR7), C(S)OR7), C(O)NR'R'', aryl, SO-heteroaryl, SO-substituted heteroaryl, SO-het C(NR7)NR'R'', OC(O)R7)R79, OC(O)OM", erocyclic, and SO-substituted heterocyclic, wherein alkyl, OC(O)OR79, OC(S)OR79, NR7C(O)R79, NR7C substituted alkyl, alkenyl, substituted alkenyl, alkynyl, sub (S)R79, NR7COM", NR7COR70, NR7C(S) stituted alkynyl, cycloalkyl, Substituted cycloalkyl, cycloalk OR79, NR7C(O)NR'R89, NR7C(NR70)R70 and enyl, Substituted cycloalkenyl, aryl, Substituted aryl, het NR7°C(NR7)NR'R'', where R is selected from the eroaryl, substituted heteroaryl, heterocyclic, and substituted group consisting of optionally Substituted alkyl, cycloalkyl, heterocyclic are as defined herein. Sulfonyl includes, by way heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, ary of example, methyl-SO , phenyl-SO , and 4-meth lalkyl, heteroaryl and heteroarylalkyl, each R" is indepen ylphenyl-SO "Sulfonyloxy” refers to the group —OSO dently hydrogen or R'; each R is independently R' or alkyl, OSO-substituted alkyl, OSO-alkenyl, OSO-substi alternatively, two R's, taken together with the nitrogen tuted alkenyl, OSO-cycloalkyl, OSO-substituted atom to which they are bonded, form a 5-, 6- or 7-membered cylcoalkyl, OSO-cycloalkenyl, OSO-substituted cylcoalk heterocycloalkyl which may optionally include from 1 to 4 of enyl, OSO-aryl, OSO-substituted aryl, OSO-heteroaryl, the same or different additional heteroatoms selected from the OSO-substituted heteroaryl, OSO-heterocyclic, and OSO group consisting of O, N and S. of which N may have —H or substituted heterocyclic, wherein alkyl, substituted alkyl, alk C-C alkyl substitution; and each M is a counter ion with a enyl, Substituted alkenyl, alkynyl, Substituted alkynyl, net single positive charge. Each M may independently be, cycloalkyl, Substituted cycloalkyl, cycloalkenyl, Substituted for example, an alkali ion, Such as K", Na', Li; an ammo cycloalkenyl, aryl. Substituted aryl, heteroaryl, Substituted nium ion, such as)"N(R"), or an alkaline earth ion, such as heteroaryl, heterocyclic, and Substituted heterocyclic are as Calos, Mg"los, or Balos (“subscript 0.5 means that defined herein. one of the counter ions for Such divalent alkali earth ions can be an ionized form of a compound of the invention and the 0146 The term "aminocarbonyloxy” refers to the group other a typical counter ion Such as chloride, or two ionized —OC(O)NRR where each R is independently hydrogen, compounds disclosed herein can serve as counter ions for alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic Such divalent alkali earth ions, or a doubly ionized compound wherein alkyl, substituted alkyl, aryl, heteroaryland hetero of the invention can serve as the counterion for such divalent cyclic are as defined herein. alkali earth ions). As specific examples, NR'R' is meant 0147 “Thiol” refers to the group - SH. to include - NH, —NH-alkyl, N-pyrrolidinyl, N-piperazi 0148 “Thioxo' or the term “thioketo refers to the atom nyl, 4N-methyl-piperazin-1-yl and N-morpholinyl. (—S). 0156. In addition to the disclosure herein, substituent 0149 “Alkylthio” or the term “thioalkoxy” refers to the groups for hydrogens on unsaturated carbon atoms in “sub group—S-alkyl, wherein alkyl is as defined herein. In certain stituted alkene, alkyne, aryl and heteroaryl groups are, embodiments, sulfur may be oxidized to —S(O)—. The sul unless otherwise specified, R', halo, OM', -OR', foxide may exist as one or more stereoisomers. - SR", NR'R'', trihalomethyl, CF, CN, OCN, 0150. The term “substituted thioalkoxy” refers to the SCN, NO, NO, N, SOR7, SOM, group —S-Substituted alkyl. SOR7, OSOR7, OSOM, OSOR7, PO, US 2013/0224259 A1 Aug. 29, 2013

2(M), P(O)(OR7)OM', -P(O)(OR7), C(O)R7, molecule contains a basic functionality, salts of organic or C(S)R79, C(NR7)R79, COM", COR7), C(S) inorganic acids, such as hydrochloride, hydrobromide, for OR7), C(O)NR'R, CONR70)NR'R89, OC(O)R70, mate, tartrate, besylate, meSylate, acetate, maleate, oxalate, OC(S)R79, OCOM, OCOR79, OC(S)OR79, and the like. NR7C(O)R79, NR7C(S)R79, NR7COM", 0163 The term "salt thereof means a compound formed NR7COR70, NR7C(S)OR70, NR7C(O)NR'R80, when a proton of an acid is replaced by a cation, such as a NR7C(NR7)R70 and NR7C(NR7)NR'R'', where metal cation or an organic cation and the like. Where appli R', R', Rand Mare as previously defined, provided that cable, the salt is a pharmaceutically acceptable salt, although in case of substituted alkene oralkyne, the Substituents are not this is not required for salts of intermediate compounds that OM, OR7, - SR", or SMt. are not intended for administration to a patient. By way of 0157. In addition to the groups disclosed with respect to example, salts of the present compounds include those the individual terms herein, Substituent groups for hydrogens wherein the compound is protonated by an inorganic or on nitrogen atoms in “substituted heteroalkyl and cyclohet organic acid to form a cation, with the conjugate base of the eroalkyl groups are, unless otherwise specified, -R', inorganic or organic acid as the anionic component of the salt. OR70, SR70, SM", NR'R'', trihalomethyl, 0164 “Solvate” refers to a complex formed by combina CF, CN, NO, NO, -S(O)R’, S(O).OM, tion of solvent molecules with molecules or ions of the solute. S(O),OR', OS(O).R', OS(O),OM", OS(O) The solvent can be an organic compound, an inorganic com OR', P(O)(O)(M) - P(O)(OR7)OM', -P(O) pound, or a mixture of both. Some examples of solvents (OR70)(OR7), C(O)R70, C(S)R70, C(NR70)R70, include, but are not limited to, methanol, N,N-dimethylfor C(O)OR7), C(S)OR7), C(O)NR'R'', C(NR7) mamide, tetrahydrofuran, dimethylsulfoxide, and water. NR8R89, OC(O)R70, OC(S)R79, OC(O)OR79, OC When the solvent is water, the solvate formed is a hydrate. (S)OR7, NR7C(O)R79, NR7°C(S)R79, NR7C(O) 0.165 “Stereoisomer' and “stereoisomers’ refer to com OR70, NR7C(S)OR70, NR7C(O)NR8R89, NR7C pounds that have same atomic connectivity but different (NR7)R79 and NR7°C(NR7)NR'R'', where R, R79, atomic arrangement in space. Stereoisomers include cis-trans R" and M" are as previously defined. isomers, E and Z isomers, enantiomers, and diastereomers. 0158. In addition to the disclosure herein, in a certain (0166 “Tautomer refers to alternate forms of a molecule embodiment, a group that is substituted has 1, 2, 3, or 4 that differ only in electronic bonding of atoms and/or in the substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 position of a proton, such as enol-keto and imine-enamine substituent. tautomers, or the tautomeric forms of heteroaryl groups con 0159. It is understood that in all substituted groups defined taining a —N=C(H)—NH- ring atom arrangement. Such above, polymers arrived at by defining substituents with fur as pyrazoles, imidazoles, benzimidazoles, triazoles, and tet ther substituents to themselves (e.g., Substituted aryl having a razoles. A person of ordinary skill in the art would recognize substituted aryl group as a substituent which is itself substi that other tautomeric ring atom arrangements are possible. tuted with a substituted aryl group, which is further substi 0167. It will be appreciated that the term “or a salt or tuted by a Substituted aryl group, etc.) are not intended for solvate or stereoisomer thereof is intended to include all inclusion herein. In Such cases, the maximum number of such permutations of salts, Solvates and Stereoisomers, such as a substitutions is three. For example, serial substitutions of Solvate of a pharmaceutically acceptable salt of a stereoiso Substituted aryl groups specifically contemplated herein are mer of Subject compound. limited to substituted aryl-(substituted aryl)-substituted aryl. 0.168. As used herein, a “pharmaceutical composition' is 0160 Unless indicated otherwise, the nomenclature of meant to encompass a composition Suitable for administra substituents that are not explicitly defined herein are arrived tion to a Subject, Such as a mammal, especially a human. In at by naming the terminal portion of the functionality fol general a “pharmaceutical composition' is sterile, and is free lowed by the adjacent functionality toward the point of of contaminants that are capable of eliciting an undesirable attachment. For example, the substituent “arylalkyloxycarbo response within the Subject (e.g., the compound(s) in the nyl refers to the group (aryl)-(alkyl)-O C(O)—. pharmaceutical composition is pharmaceutical grade). Phar 0161. As to any of the groups disclosed herein which maceutical compositions can be designed for administration contain one or more Substituents, it is understood, of course, to subjects or patients in need thereof via a number of differ that such groups do not contain any Substitution or Substitu ent routes of administration including oral, buccal, rectal, tion patterns which are sterically impractical and/or syntheti parenteral, intraperitoneal, intradermal, intratracheal, inhala cally non-feasible. In addition, the Subject compounds tional, and the like. In some embodiments a Subject compo include all stereochemical isomers arising from the Substitu sition is formulated with an excipient other than dimethylsul tion of these compounds. foxide (DMSO). In other embodiments, the pharmaceutical 0162 The term “pharmaceutically acceptable salt' means compositions are suitable for administration by a route other a salt which is acceptable for administration to a patient, Such than transdermal administration. A pharmaceutical composi as a mammal (salts with counterions having acceptable mam tion will in some embodiments include a subject compound malian safety for a given dosage regime). Such salts can be and a pharmaceutically acceptable excipient. In some derived from pharmaceutically acceptable inorganic or embodiments, a pharmaceutically acceptable excipient is organic bases and from pharmaceutically acceptable inor other than DMSO. ganic or organic acids. "Pharmaceutically acceptable salt 0169. As used herein, “pharmaceutically acceptable refers to pharmaceutically acceptable salts of a compound, derivatives” of a compound of the invention include salts, which salts are derived from a variety of organic and inor esters, enol ethers, enol esters, acetals, ketals, orthoesters, ganic counter ions well known in the art and include, by way hemiacetals, hemiketals, acids, bases, Solvates, hydrates or of example only, sodium, potassium, calcium, magnesium, prodrugs thereof. Such derivatives may be readily prepared ammonium, tetraalkylammonium, and the like; and when the by those of skill in this art using known methods for such US 2013/0224259 A1 Aug. 29, 2013

derivatization. The compounds produced may be adminis and is not intended to be limiting, since the scope of the tered to animals or humans without substantial toxic effects present invention will be limited only by the appended claims. and are either pharmaceutically active or are prodrugs. 0176 Where a range of values is provided, it is understood (0170 “Pharmaceutically effective amount” and “thera that each intervening value, to the tenth of the unit of the lower peutically effective amount” refer to the amount of a com limit unless the context clearly dictates otherwise, between pound that, when administered to a mammal or other subject the upper and lower limit of that range and any other stated or for treating a disease, is sufficient to effect such treatment for intervening value in that stated range, is encompassed within the disease. The “therapeutically effective amount” will vary the invention. The upper and lower limits of these smaller depending on the compound or the cell, the disease and its ranges may independently be included in the Smaller ranges, severity and the age, weight, etc., of the Subject to be treated. and are also encompassed within the invention, Subject to any 0171 The terms “co-administration' and “in combination specifically excluded limit in the stated range. Where the with include the administration of two or more therapeutic stated range includes one or both of the limits, ranges exclud agents either simultaneously, concurrently or sequentially ing either or both of those included limits are also included in within no specific time limits. In one embodiment, the agents the invention. are present in the cellor in the subjects body at the same time 0177. Unless defined otherwise, all technical and scien or exert their biological ortherapeutic effect at the same time. tific terms used herein have the same meaning as commonly In one embodiment, the therapeutic agents are in the same understood by one of ordinary skill in the art to which this composition or unit dosage form. In other embodiments, the invention belongs. Although any methods and materials simi therapeutic agents are in separate compositions or unit dosage lar or equivalent to those described herein can also be used in forms. In certain embodiments, a first agent can be adminis the practice or testing of the present invention, the preferred tered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 methods and materials are now described. All publications minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, mentioned herein are incorporated herein by reference to 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 disclose and describe the methods and/or materials in con weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), nection with which the publications are cited. concomitantly with, or Subsequent to (e.g., 5 minutes, 15 0.178 It must be noted that as used herein and in the minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 appended claims, the singular forms “a,” “an and “the hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 include plural referents unless the context clearly dictates week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, otherwise. Thus, for example, reference to “a DDAH antago or 12 weeks after) the administration of a second therapeutic nist” includes a plurality of such antagonists, reference to “a agent. PPI includes a plurality of proton pump inhibitors, and ref (0172. The terms “individual,” “subject,” “host” and erence to “the inhalational formulation' includes reference to "patient used interchangeably herein, refer to a mammal, one or more inhalational formulations and equivalents thereof including, but not limited to, murines (rats, mice), non-human known to those skilled in the art, and so forth. It is further primates, humans, canines, felines, ungulates (e.g., equines, noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as ante bovines, Ovines, porcines, caprines), etc. cedent basis for use of such exclusive terminology as “solely.” (0173 “Fibrosis” or “fibrotic disorder” refers to a patho “only' and the like in connection with the recitation of claim logical condition resulting from an overproduction or aber elements, or use of a “negative' limitation. rant production of fibrous tissue (e.g., fibrous connective 0179. It is appreciated that certain features of the inven tissue) in an organ or tissue, e.g., in a reparative or reactive tion, which are, for clarity, described in the context of separate process. Fibrotic disorders include, but are not limited to, embodiments, may also be provided in combination in a pulmonary fibrosis, including idiopathic pulmonary fibrosis single embodiment. Conversely, various features of the (IPF) and pulmonary fibrosis from a known etiology; liver invention, which are, for brevity, described in the context of a fibrosis; and renal fibrosis. Other exemplary fibrotic condi single embodiment, may also be provided separately or in any tions include musculoskeletal fibrosis, cardiac fibrosis, vas suitable sub-combination. All combinations of the embodi cular fibrosis, post-Surgical adhesions, Scleroderma, glau ments pertaining to the invention are specifically embraced coma, and skin lesions such as keloids. by the present invention and are disclosed herein just as if 0.174. The term “treating” or “treatment” as used herein each and every combination was individually and explicitly means the treating or treatment of a disease or medical con disclosed. In addition, all sub-combinations of the various dition in a patient, Such as a mammal (e.g., a human) that embodiments and elements thereof are also specifically includes: (a) preventing the disease or medical condition from embraced by the present invention and are disclosed herein occurring, such as, prophylactic treatment of a Subject; (b) just as if each and every such sub-combination was individu ameliorating the disease or medical condition, Such as, elimi ally and explicitly disclosed herein. nating or causing regression of the disease or medical condi 0180. The publications discussed herein are provided tion in a patient; (c) Suppressing the disease or medical con solely for their disclosure prior to the filing date of the present dition, for example by, slowing or arresting the development application. Nothing herein is to be construed as an admission of the disease or medical condition in a patient; or (d) allevi that the present invention is not entitled to antedate such ating a symptom of the disease or medical condition in a publication by virtue of prior invention. Further, the dates of patient. publication provided may be different from the actual publi (0175 Before the present invention is further described, it cation dates which may need to be independently confirmed. is to be understood that this invention is not limited to par 0181 Except as otherwise noted, the methods and tech ticular embodiments described, as such may, of course, vary. niques of the present embodiments are generally performed It is also to be understood that the terminology used herein is according to conventional methods well known in the art and for the purpose of describing particular embodiments only, as described in various general and more specific references US 2013/0224259 A1 Aug. 29, 2013

that are cited and discussed throughout the present specifica Inhalational Formulations tion. See, e.g., Loudon, Organic Chemistry, Fourth Edition, 0188 The present disclosure provides pharmaceutical New York: Oxford University Press, 2002, pp. 360-361, compositions comprising a DDAH inhibitor and a pharma 1084-1085; Smith and March, March's Advanced Organic ceutically acceptable excipient, in a formulation Suitable for Chemistry: Reactions, Mechanisms, and Structure, Fifth Edi administration by inhalation, e.g., inhalation into the lungs. tion, Wiley-Interscience, 2001; or Vogel, A Textbook of Prac The present disclosure provides a pharmaceutical composi tical Organic Chemistry, Including Qualitative Organic tion comprising a compound of any one of Formulae I-X. Analysis, Fourth Edition, New York: Longman, 1978. below, and a pharmaceutically acceptable excipient, where 0182. The nomenclature used herein to name the subject the compound is in a formulation suitable for delivery by compounds is illustrated in the Examples herein. This nomen inhalation. clature has generally been derived using the commercially 0189 For therapeutic use in diseases characterized by available AutoNom software (MDL, San Leandro, Calif.). excessive NO production and/or elevated DDAH activity, local delivery to the lung can be carried out. Delivery by DETAILED DESCRIPTION inhalation or insufflating aerosols provide high level concen 0183 The present disclosure provides DDAH modulators, trations of drug compared to the concentration absorbed sys e.g., DDAH inhibitors, and compositions, including pharma temically. ceutical compositions (e.g., inhalational formulations) com 0190. Administration by inhalation can provide for prising Such inhibitors. smaller doses delivered locally to the specific cells in the lung which are most in need of treatment. By delivering smaller 0184 The NO/ADMA/DDAH pathway is depicted in doses, any adverse side effects are eliminated or Substantially FIG. 16. ADMA and monomethyl-L-arginine (L-NMMA) reduced. By delivering directly to the cells which are most in are endogenous competitive inhibitors of nitric oxide Syn need of treatment, the effect of the treatment will be realized thase (NOS). These methylarginines are generated by the more quickly. methylation of arginine residues on histones and other pro 0191 The compound of Formulae I-X may be adminis teins by a family of enzymes known as Protein Arginine tered to the afflicted patient by means of a pharmaceutical Methyl Transferases (PRMTs). During the hydrolysis of pro delivery system for the inhalation route. The compounds may teins containing methylarginine residues, free ADMA and be formulated in a form suitable for administration by inha L-NMMA (monomethyl arginine) are released. The present lation. The pharmaceutical delivery system is one that is disclosure provides Small molecules and formulation that suitable for respiratory therapy by administration via inhala regulate NO by controlling the activity of DDAH. Pharma tion of a compound of any one of Formulae I-Xthereof to lung cological inhibition of DDAH1 leads to higher ADMA levels tissue, e.g., the bronchi. and regulation of NO production. 0.192 The pharmaceutical compositions of the embodi 0185. A subject DDAH is useful for treating a patient ments can be prepared by thoroughly and intimately mixing suffering from a disorder characterized by excessive NO pro or blending a compound of any one of Formulae I-X with a duction, and/or elevated DDAH activity. Thus, the present pharmaceutically acceptable carrier and one or more optional disclosure provides a method of treating a patient Suffering ingredients. If necessary or desired, the resulting uniformly from a disorder characterized by excessive NO production, blended mixture can then be shaped or loaded into tablets, and/or elevated DDAH activity, the method comprising capsules, pills, canisters, cartridges, dispensers and the like administering to said patient an effective amount of a com using conventional procedures and equipment. pound of formulae I-X, or an inhalational formulation com 0193 The disclosure provides a system that depends on prising Such compound. The present disclosure also provides the power of a compressed gas to expel a compound of any a pharmaceutical composition comprising a compound of the one of Formulae I-X from a container. An aerosol or pressur formulae I-X and a pharmaceutically acceptable excipient, ized package can be employed for this purpose. As used wherein the compound is in a formulation suitable for deliv herein, the term “aerosol is used in its conventional sense as ery by inhalation. referring to very fine liquid or Solid particles carries by a 0186 The following substituents and values are intended propellant gas under pressure to a site of therapeutic applica to provide representative examples of various aspects and tion. When a pharmaceutical aerosol is employed in this embodiments. These representative values are intended to invention, the aerosol contains the therapeutically active further define and illustrate such aspects and embodiments compound, which can be dissolved, Suspended, or emulsified and are not intended to exclude other embodiments or to limit in a mixture of a fluid carrier and a propellant. The aerosol can the scope of this invention. In this regard, the representation be in the form of a solution, Suspension, emulsion, powder, or that a particular value or substituent is preferred is not semi-solid preparation. Aerosols employed in the present intended in any way to exclude other values or Substituents embodiments are intended for administration as fine, Solid from this invention unless specifically indicated. particles or as liquid mists via the respiratory tract of a patient. 0187. These compounds may contain one or more chiral Various types of propellants known to one of skill in the art centers and therefore, the embodiments are directed to race can be utilized. Examples of suitable propellants include, but mic mixtures; pure stereoisomers (i.e., enantiomers or dias are not limited to, hydrocarbons or other Suitable gas. In the tereomers); Stereoisomer-enriched mixtures and the like case of the pressurized aerosol, the dosage unit may be deter unless otherwise indicated. When a particular stereoisomer is mined by providing a value to deliver a metered amount. shown or named herein, it will be understood by those skilled 0194 In certain embodiments, the pharmaceutical com in the art that minor amounts of other Stereoisomers may be positions are suitable for inhaled administration. Suitable present in the compositions unless otherwise indicated, pro pharmaceutical compositions for inhaled administration will vided that the desired utility of the composition as a whole is typically be in the form of an aerosol or a powder. Such not eliminated by the presence of Such other isomers. compositions are generally administered using well-known US 2013/0224259 A1 Aug. 29, 2013

delivery devices, such as a nebulizer inhaler, a metered-dose pellant gas. Accordingly, pharmaceutical compositions inhaler (MDI), a dry powder inhaler (DPI) or a similar deliv administered using a metered-dose inhaler can comprise a ery device. Solution or Suspension of the active agent in a liquefied pro 0.195. In certain embodiments, the pharmaceutical com pellant. Any Suitable liquefied propellant may be employed position comprising the active agent (e.g., a compound of any including chlorofluorocarbons, such as CC1F, and hydrof one of Formulae I-X) is administered by inhalation using a luoroalkanes (HFAs), such as 1,1,1,2-tetrafluoroethane (HFA nebulizer inhaler. Such nebulizer devices typically produce a 134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227). stream of high Velocity air that causes the pharmaceutical Due to concerns about chlorofluorocarbons affecting the composition comprising the active agent to spray as a mist oZone layer, formulations containing HFAS can be used. that is carried into the patient’s respiratory tract. Accordingly, Additional optional components of HFA formulations when formulated for use in a nebulizer inhaler, the active include co-solvents, such as ethanol or pentane, and Surfac agent is typically dissolved in a Suitable carrier to form a tants, such as Sorbitan trioleate, oleic acid, lecithin, and glyc Solution. Alternatively, the active agent can be micronized erin. See, for example, U.S. Pat. No. 5.225,183, EP 0717987 and combined with a suitable carrier to form a suspension of A2, and WO92/22286. micronized particles of respirable size, where micronized is 0202 A representative pharmaceutical composition for typically defined as having about 90% or more of the particles use in a metered-dose inhaler comprises from about 0.01% to with a diameter of less than about 10 um. Suitable nebulizer about 5% by weight of a compound of any one of Formulae devices are provided commercially, for example, by PARI I-X, or a pharmaceutically acceptable salt or Solvate or stere GmbH (Starnberg, German). Other nebulizer devices include oisomer thereof; from about 0% to about 20% by weight Respimat (Boehringer Ingelheim) and those disclosed, for ethanol; and from about 0% to about 5% by weight surfactant; example, in U.S. Pat. No. 6,123,068 and WO 97/12687. with the remainder being an HFA propellant. 0196. A representative pharmaceutical composition for 0203 Such compositions are typically prepared by adding use in a nebulizer inhaler comprises an isotonic aqueous chilled or pressurized hydrofluoroalkane to a suitable con solution comprising from about 0.05 ug/mL to about 10 tainer containing the active agent, ethanol (if present) and the mg/mL of a compound of any one of Formulae I-X or a Surfactant (if present). To prepare a suspension, the active pharmaceutically acceptable salt or Solvate or Stereoisomer agent is micronized and then combined with the propellant. thereof. The formulation is then loaded into anaerosol canister, which 0197) In certain embodiments, the pharmaceutical com forms a portion of a metered-dose inhaler device. Examples position comprising the active agent is administered by inha of metered-dose inhaler devices developed specifically for lation using a dry powder inhaler. Such dry powder inhalers use with HFA propellants are provided in U.S. Pat. Nos. typically administer the active agent as a free-flowing powder 6,006,745 and 6,143,277. Alternatively, a suspension formu that is dispersed in a patient’s air-stream during inspiration. In lation can be prepared by spray drying a coating of surfactant order to achieve a free-flowing powder, the active agent is on micronized particles of the active agent. See, for example, typically formulated with a suitable excipient Such as lactose WO99/53901 and WOOOf 61108. or starch. 0204 For additional examples of processes of preparing 0198 A representative pharmaceutical composition for respirable particles, and formulations and devices suitable for use in a dry powder inhaler comprises dry lactose having a inhalation dosing see U.S. Pat. Nos. 6,268,533, 5,983,956, particle size between about 1 um and about 100 um and 5,874,063, and 6,221,398, and WO 99/55319 and WO micronized particles of a compound of any one of Formulae 00/30614. I-X, or a pharmaceutically acceptable salt or Solvate or stere 0205 With respect to each of the patents recited above, oisomer thereof. applicants point out that these patents cite other publications 0199 Such a dry powder formulation can be made, for in intrapulmonary drug delivery and Such publications can be example, by combining the lactose with the active agent and referred to for specific methodology, devices and formula then dry blending the components. Alternatively, if desired, tions which could be used in connection with the delivery of the active agent can be formulated without an excipient. The agonists of the present embodiments. Further, each of the pharmaceutical composition is then typically loaded into a patents are incorporated herein by reference in their entirety dry powder dispenser, or into inhalation cartridges or cap for purposes of disclosing formulations, devices, packaging sules for use with a dry powder delivery device. and methodology for the delivery of agonist formulations of 0200 Examples of dry powder inhaler delivery devices the present embodiments. include Diskhaler (GlaxoSmithKline, Research Triangle 0206. Any conventional carrier or excipient may be used Park, N.C.) (see, e.g., U.S. Pat. No. 5,035,237); Diskus in the pharmaceutical compositions of the embodiments. The (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 6,378,519: Tur choice of a particular carrier or excipient, or combinations of buhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S. Pat. carriers or excipients, will depend on the mode of adminis No. 4,524,769); Rotahaler (GlaxoSmithKline) (see, e.g., U.S. tration being used to treat a particular patient or type of Pat. No. 4,353.365) and Handihaler (Boehringer Ingelheim). medical condition or disease state. In this regard, the prepa Further examples of suitable DPI devices are described in ration of a suitable pharmaceutical composition for a particu U.S. Pat. Nos. 5,415,162, 5,239,993, and 5,715,810 and ref lar mode of administration is well within the scope of those erences cited therein. skilled in the pharmaceutical arts. Additionally, the ingredi 0201 In certain embodiments, the pharmaceutical com ents for Such compositions are commercially available from, position comprising an active agent (e.g., a compound of any for example, Sigma, P.O. Box 14508, St. Louis, Mo. 63178. one of Formulae I-X) is administered by inhalation using a By way of further illustration, conventional formulation tech metered-dose inhaler. Such metered-dose inhalers typically niques are described in Remington: The Science and Practice discharge a measured amount of the active agent or a phar of Pharmacy, 20th Edition, Lippincott Williams & White, maceutically acceptable salt thereof using compressed pro Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical US 2013/0224259 A1 Aug. 29, 2013

Dosage Forms and Drug Delivery Systems, 7th Edition, Lip pincott Williams & White, Baltimore, Md. (1999). (Ib) 0207 Representative examples of materials which can N N / serve as pharmaceutically acceptable carriers or excipients N / N include, but are not limited to, the following: (1) Sugars. Such as lactose, glucose and Sucrose; (2) starches, such as corn (R),- starch and potato starch; (3) cellulose, and its derivatives, Such as Sodium carboxymethyl cellulose, ethyl cellulose and R5 R4 cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gela tin; (7) talc.; (8) excipients, such as cocoa butter and Supposi tory waxes; (9) oils, such as peanut oil, cottonseed oil, saf 0219 wherein flower oil, sesame oil, olive oil, corn oil and soybean oil: (10) (0220) R' is selected from alkyl, substituted alkyl, hydroxy, glycols, such as propylene glycol, (11) polyols, such as glyc alkoxy, Substituted alkoxy, amino, Substituted amino, car erin, Sorbitol, mannitol and polyethylene glycol; (12) esters, boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, Such as ethyl oleate and ethyl laurate; (13) agar, (14) buffering alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl: agents, such as magnesium hydroxide and aluminum hydrox 0221) R. R. R', and Rare independently selected from ide; (15) alginic acid; (16) pyrogen-free water, (17) isotonic alkyl, Substituted alkyl, hydroxy, alkoxy, Substituted alkoxy, saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phos amino, Substituted amino, carboxyl, carboxyl ester, cyano, phate buffer Solutions; (21) compressed propellant gases, halogen, acyl, aminoacyl, nitro, alkenyl, Substituted alkenyl, Such as chlorofluorocarbons and hydrofluorocarbons; and alkynyl, and Substituted alkynyl; and (22) other non-toxic compatible Substances employed in 0222 m is an integer from Zero to four; pharmaceutical compositions. 0223 or a pharmaceutical salt thereof. 0224. According to one aspect, the embodiments include 0208 DDAH Inhibitors for Inhalational Formulations pharmaceutical compositions formulated for inhalational 0209. A subject pharmaceutical formulation suitable for delivery, which include a compound of Formula Ic: inhalation comprises a DDAH inhibitor. The following are suitable DDAH inhibitors. In the formulae herein, a formula number is meant to encompass all forms of the formula num (Ic) ber. Thus, reference to formula (I) is meant to include com pounds of formula (Ia), (Ib), and (Ic). 0210 Formula I 0211. According to one aspect, the embodiments include pharmaceutical compositions formulated for inhalational delivery, which compositions a compound of Formula Ia: 0225 wherein (Ia) 10226) R' is selected from alkyl, substituted alkyl, hydroxy, Q O R alkoxy, Substituted alkoxy, amino, Substituted amino, car boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl: CO-A-4NN N R3, 0227. R. R. R', and Rare independently selected from (R), alkyl, Substituted alkyl, hydroxy, alkoxy, Substituted alkoxy, amino, Substituted amino, carboxyl, carboxyl ester, cyano, R5 R4 halogen, acyl, aminoacyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl; and 0212 wherein 0228 m is an integer from Zero to four; 0229 or a pharmaceutical salt thereof. 0213 Q is N or CH: 0230. In Formula Ia, Q is N or CH. In certain embodi 0214) R' is selected from alkyl, substituted alkyl, hydroxy, ments, Q" is N. In certain embodiments, Q" is CH. alkoxy, Substituted alkoxy, amino, Substituted amino, car 0231. In Formula I, R' is selected from alkyl, substituted boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alkyl, hydroxy, alkoxy, Substituted alkoxy, amino, Substituted alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl: amino, carboxyl, carboxyl ester, cyano, halogen, acyl, ami 0215 R. R. R', and Rare independently selected from noacyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Sub hydrogen, alkyl, Substituted alkyl, hydroxy, alkoxy, Substi stituted alkynyl. tuted alkoxy, amino, Substituted amino, carboxyl, carboxyl 0232. In certain embodiments, R' is alkyl or substituted ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substi alkyl. In certain embodiments, R is hydroxy. In certain tuted alkenyl, alkynyl, and Substituted alkynyl; and embodiments, R' is alkoxy or substituted alkoxy. In certain embodiments, R is alkoxy. For example, R' may be a C-C, 0216 m is an integer from Zero to four, alkoxy, such as a C-C alkoxy. In certain embodiments, R is 0217 or a pharmaceutical Salt thereof. —OCH. In certain embodiments, R is substituted alkoxy. 0218. According to one aspect, the embodiments include For example, in embodiments where R' is a substituted pharmaceutical compositions formulated for inhalational alkoxy, the alkoxy group may be substituted with one or more delivery, which compositions include a compound of For groups, such as, but not limited to, alkyl, hydroxy, alkoxy, mula Ib: amino, carboxyl, carboxyl ester, cyano, halogen, acyl, ami US 2013/0224259 A1 Aug. 29, 2013 noacyl, nitro, alkenyl, alkynyl, and the like. The Substituents 0238. In certain embodiments, R is hydrogen. In certain on the substituted alkoxy may in turn be substituted with one embodiments, R is alkyl or substituted alkyl. In certain or more groups as described above. In certain embodiments, embodiments, R is alkyl. For example, R may be a C-C, the alkoxy is Substituted with one or more halogen groups alkyl, such as a C-C alkyl. In certain embodiments, R is (e.g., F. Cl, Br, I). In certain embodiments, the alkoxy is methyl. In certain embodiments, R is hydroxy, alkoxy, or Substituted with one or more fluoro groups. In certain substituted alkoxy. In certain embodiments, R is alkoxy. For embodiments, R' is —OCHF. In certain embodiments, R' is example, R may be a C-C alkoxy, such as a C-C alkoxy. amino or substituted amino. In certain embodiments, R' is carboxyl or carboxyl ester. In certain embodiments, R is In certain embodiments, R is —OCH. In certain embodi cyano, halogen, acyl, aminoacyl, or nitro. In certain embodi ments, R is amino or substituted amino. In certain embodi ments, R' is alkenyl, Substituted alkenyl, alkynyl, or Substi ments, R is carboxyl or carboxyl ester. In certain embodi tuted alkynyl. ments, R is cyano, halogen, acyl, aminoacyl, or nitro. In 0233. In Formula I, m is an integer from Zero to four. In certain embodiments, Risalkenyl, substituted alkenyl, alky certain embodiments, m is Zero. In certain embodiments, mis nyl, or Substituted alkynyl. one. In certain embodiments, m is two. In certain embodi 0239 Particular compounds of interest, and salts or sol ments, m is three. In certain embodiments, m is four. vates or stereoisomers thereof for formulation for inhala 0234. In Formula I, R. R. R. and Rare independently tional delivery according to the embodiments, include: selected from alkyl, substituted alkyl, hydroxy, alkoxy, sub stituted alkoxy, amino, Substituted amino, carboxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substi OCH tuted alkenyl, alkynyl, and Substituted alkynyl. H3C 0235. In certain embodiments, R is hydrogen. In certain e embodiments, R is alkyl or substituted alkyl. In certain CH3: embodiments, R is hydroxy, alkoxy, or substituted alkoxy. In H3CO h certain embodiments, R is amino or substituted amino. In N NN certain embodiments, R is carboxyl or carboxyl ester. In y v certain embodiments, R is cyano, halogen, acyl, aminoacyl, N O or nitro. In certain embodiments, R is alkenyl, substituted (Omeprazole) alkenyl, alkynyl, or substituted alkynyl. 0236. In certain embodiments, R is hydrogen. In certain embodiments, R is alkyl or substituted alkyl. In certain embodiments, R is alkyl. For example, R may be a C-C, F O alkyl, such as a C-C alkyl. In certain embodiments, R is methyl. In certain embodiments, R is hydroxy, alkoxy, or substituted alkoxy. In certain embodiments, R is amino or O)-( - substituted amino. In certain embodiments, R is carboxyl or carboxyl ester. In certain embodiments, R is cyano, halogen, (Pantoprazole) acyl, aminoacyl, or nitro. In certain embodiments, R is alk Nie enyl, Substituted alkenyl, alkynyl, or Substituted alkynyl. 0237. In certain embodiments, R is hydrogen. In certain N N h embodiments, R is alkyl or substituted alkyl. In certain N embodiments, R is hydroxy, alkoxy, or substituted alkoxy. In N)-( O o certain embodiments, Risalkoxy. For example, R may be a CF: C-C alkoxy, such as a C-C alkoxy. In certain embodi (Lansoprazole) ments, R is —OCHs. In certain embodiments, R is substi tuted alkoxy. For example, in embodiments where R is a OCH3: substituted alkoxy, the alkoxy group may be substituted with one or more groups, such as, but not limited to, alkyl, HCO hydroxy, alkoxy, amino, carboxyl, carboxyl ester, cyano, N-N / \ halogen, acyl, aminoacyl, nitro, alkenyl, alkynyl, and the like. Y-s NR The substituents on the substituted alkoxy may in turn be l- y substituted with one or more groups as described above. In certain embodiments, the alkoxy is substituted with one or (Tenatoprazole) more halogen groups (e.g., F. Cl, Br, I). In certain embodi OCH3; and ments, the alkoxy is substituted with one or more fluoro groups. In certain embodiments, Ris—OCHCF. In certain embodiments, the alkoxy is Substituted with analkoxy group, HCO N \ / such as a C-C alkoxy, or a C-C alkoxy. In certain embodi Yu. ments, R is —O(CH), OCHs. In certain embodiments, Ris y V N amino or substituted amino. In certain embodiments, R is H O carboxyl or carboxyl ester. In certain embodiments, R is (Esomeprazole) cyano, halogen, acyl, aminoacyl, or nitro. In certain embodi ments, R is alkenyl, substituted alkenyl, alkynyl, or substi tuted alkynyl. US 2013/0224259 A1 Aug. 29, 2013

-continued boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, Ne alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl. (0254. In certain embodiments, R is hydrogen. In certain NN N embodiments, R is alkyl or substituted alkyl. In certain embodiments, R is alkyl. For example, R may be a C-C, )-( alkyl, such as a C-C alkyl. In certain embodiments, R is N O methyl. In certain embodiments, R is hydroxy, alkoxy, or O- . substituted alkoxy. In certain embodiments, R is amino or (Rabeprazole) substituted amino. In certain embodiments, R is carboxyl or carboxyl ester. In certain embodiments, R is cyano, halogen, 0240. In some embodiments, one or more of omeprazole, acyl, aminoacyl, or nitro. In certain embodiments, R is alk pantoprazole, lanSoprazole, tenatoprazole, esomeraprazole, enyl, Substituted alkenyl, alkynyl, or Substituted alkynyl. and rabeprazole is specifically excluded. (0255. In certain embodiments, R is hydrogen. In certain embodiments, R is alkyl or substituted alkyl. In certain 0241. Formula II embodiments, R is alkyl. For example. R* may be a C-C, 0242. According to one aspect, the present disclosure pro alkyl, such as a C-C alkyl. In certain embodiments, R is vides a compound of Formula IIa. methyl. In certain embodiments, R is hydroxy, alkoxy, or substituted alkoxy. In certain embodiments, R is amino or substituted amino. In certain embodiments, R is carboxyl or (IIa) carboxyl ester. In certain embodiments, R is cyano, halogen, acyl, aminoacyl, or nitro. In certain embodiments, R is alk N M enyl, Substituted alkenyl, alkynyl, or Substituted alkynyl. N )-(R2 0256 In the formulae herein, a formula number is meant to encompass all forms of the formula number. Thus, reference R4 4. s/ to formula (II) is meant to include compounds of formula (IIa) and (Ilb). In Formula II, R' is selected from hydrogen, alkyl, 0243 wherein Substituted alkyl, alkoxy, Substituted alkoxy, cycloalkyl, Sub 0244) R' is selected from hydrogen, alkyl, substituted stituted cycloalkyl, aryl, Substituted aryl, heterocyclyl, and alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted substituted heterocyclyl. cycloalkyl, aryl, Substituted aryl, heterocyclyl, and Substi 0257. In certain embodiments, R' is hydrogen. In certain tuted heterocyclyl: embodiments, R' is alkyl or substituted alkyl. In certain 0245) R is selected from hydrogen, alkyl, and substituted embodiments, R is alkyl. For example, R' may be a C-C, alkyl: alkyl, such as a C-C alkyl. In certain embodiments, R' is 0246 R and Rare independently selected from hydro substituted alkyl. For example, in embodiments where R' is a gen, alkyl, Substituted alkyl, hydroxy, alkoxy, Substituted substituted alkyl, the alkyl group may be substituted with one alkoxy, amino, Substituted amino, carboxyl, carboxyl ester, or more groups, such as, but not limited to, alkyl, hydroxy, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substituted alkoxy, amino, carboxyl, carboxyl ester, cyano, halogen, thio. alkenyl, alkynyl, and Substituted alkynyl: acyl, aminoacyl, nitro, alkenyl, alkynyl, cycloalkyl, hetero 0247 or a pharmaceutical salt thereof. cycloalkyl, aryl, heteroaryl, and the like. In certain embodi ments, the alkyl is substituted with an alkyl group. Such as a 0248. According to one aspect, the embodiments include C-C alkyl, or a C-C alkyl. In certain embodiments, the pharmaceutical compositions, which include a compound of alkyl is substituted with an alkoxy group, Such as a C-C, Formula IIb: alkoxy, or a C-C alkoxy. In certain embodiments, the alkyl group is substituted with a cycloalkyl group, such as a satu (IIb) rated or unsaturated cycloalkyl group. In certain embodi CH ments, the alkyl group is Substituted with an aryl. Combina O O R tions of the above substituents may also be included. The N \V substituents on the substituted alkyl may in turn be substituted N R2 with one or more groups as described above, such as, but not 2 M limited to, alkyl, hydroxy, alkoxy, amino, carboxyl, carboxyl HC N S ester, cyano, halogen (e.g., F. Cl, Br, I), thio, acyl, aminoacyl, nitro, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and the like. In certain embodiments, R is alkoxy 0249 wherein or substituted alkoxy. In certain embodiments, R' is (0250) R' is selected from hydrogen, alkyl, substituted cycloalkyl or Substituted cycloalkyl. In certain embodiments, alkyl, alkoxy, Substituted alkoxy, cycloalkyl, Substituted R" is cycloalkyl, such as Cs-Co cycloalkyl, or a C-C, cycloalkyl, aryl, Substituted aryl, heterocyclyl, and Substi cycloalkyl. In certain embodiments, R' is cyclohexyl. In cer tuted heterocyclyl: tain embodiments, R' is a substituted cycloalkyl, where the (0251 R is selected from hydrogen, alkyl, and substituted cycloalkyl group is Substituted with one or more groups as alkyl: described above, such as, but not limited to, alkyl, hydroxy, 0252 or a pharmaceutical salt thereof. alkoxy, amino, carboxyl, carboxyl ester, cyano, halogen (e.g., 0253) In Formula IIa, R and R are independently F. Cl, Br, I), thio, acyl, aminoacyl, nitro, alkenyl, alkynyl, selected from hydrogen, alkyl, Substituted alkyl, hydroxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and the like. alkoxy, Substituted alkoxy, amino, Substituted amino, car For example, the cycloalkyl group may be substituted with US 2013/0224259 A1 Aug. 29, 2013 17 one or more alkyl groups, such as a C-C alkyl, or a C-C, gen. In certain embodiments, R is selected from alkyl. In alkyl (e.g., methyl). In certain embodiments, R' is aryl or certain embodiments, R is substituted alkyl. Substituted aryl. In certain embodiments, R" is heterocyclyl or 0259 Particular compounds of interest, and salts or sol substituted heterocyclyl. In certain embodiments, R' is Vates or stereoisomers thereof for formulation according to selected from the following: the embodiments, include:

CH3 O O N )-H s s 2N's/N CH3:

0258. In Formula II, R is selected from hydrogen, alkyl, Cl; and substituted alkyl. In certain embodiments, R is hydro US 2013/0224259 A1 Aug. 29, 2013

-continued CH O O (III) H

N N )- J-R . 2 M HC N S r (s2 Q 1s NH

0262 wherein O 10263 Q' is S, O, NH or CH: 0264. Q is N or CH: CH: 0265 Q is N or CH: CH O O 0266) R' is selected from alkyl, substituted alkyl, hydroxy, H alkoxy, Substituted alkoxy, amino, Substituted amino, car N )- boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, N alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl: 2 S CH 10267 R’ is selected from alkyl, substituted alkyl, hydroxy, HC3 N / 3. alkoxy, Substituted alkoxy, amino, Substituted amino, car O O / boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, HC alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl: CH O O 0268 m is an integer from Zero to four; and H 0269 n is an integer from Zero to three: 0270 or a pharmaceutical salt thereof. N N )- (0271. In Formula III, Q is S. O. NH or CH. In certain 2 M embodiments, Q' is S. In certain embodiments, Q' is O. In HC N S certain embodiments, Q is NH. In certain embodiments, Q' is CH H3C (0272. In Formula III, Q is N or CH. In certain embodi ments, Q is N. In certain embodiments, Q is CH. CH3 O O (0273. In Formula III, Q is N or CH. In certain embodi H ments, Q is N. In certain embodiments, Q is CH. N N )- (0274. In Formula III, R' is selected from alkyl, substituted 2 M alkyl, hydroxy, alkoxy, Substituted alkoxy, amino, Substituted H3C N S amino, carboxyl, carboxyl ester, cyano, halogen, acyl, ami noacyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Sub stituted alkynyl. (0275. In certain embodiments, R' is alkyl or substituted alkyl. In certain embodiments, R' is hydroxy, alkoxy, or sub stituted alkoxy. In certain embodiments, R' is amino, Substi tuted amino, carboxyl, or carboxyl ester. In certain embodi O-CH3; and ments, R' is cyano, halogen, acyl, aminoacyl, nitro, alkenyl, CH3 O O substituted alkenyl, alkynyl, or substituted alkynyl. (0276. In Formula III, R is selected from alkyl, substituted alkyl, hydroxy, alkoxy, Substituted alkoxy, amino, Substituted N amino, carboxyl, carboxyl ester, cyano, halogen, acyl, ami 2 M noacyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Sub H3C N S stituted alkynyl. CH3 O O (0277. In certain embodiments, R is alkyl or substituted H alkyl. In certain embodiments, R is hydroxy, alkoxy, or sub N )- stituted alkoxy. In certain embodiments, R is amino, Substi N tuted amino, carboxyl, or carboxyl ester. In certain embodi 2 M ments, R is cyano, halogen, acyl, aminoacyl, nitro, alkenyl, H3C N S substituted alkenyl, alkynyl, or substituted alkynyl. C 0278. In Formula III, m is an integer from Zero to four. In certain embodiments, m is Zero. In certain embodiments, mis one. In certain embodiments, m is two. In certain embodi 0260 Formula III ments, m is three. In certain embodiments, m is four. 0261 According to one aspect, the embodiments include 0279. In Formula III, n is an integer from Zero to three. In pharmaceutical compositions formulated for inhalational certain embodiments, n is Zero. In certain embodiments, n is delivery, which compositions include a compound of For one. In certain embodiments, n is two. In certain embodi mula III: ments, n is three. US 2013/0224259 A1 Aug. 29, 2013

0280 A particular compound of interest, and salts or sol -continued Vates or stereoisomers thereof for formulation according to (IX) the embodiments, is: / \ OH.

O H3C O

0283 Formula X 0284. In the formulae herein, a formula number is meant to S ls NH. encompass all forms of the formula number. Thus, reference to formula (X) is meant to include compounds of formula 0281 Formula IV-IX (Xa) and (Xb). 0282 Particular compounds of interest, and salts or sol 0285 According to one aspect, the present disclosure pro Vates or stereoisomers thereof for formulation according to vides a pharmaceutical formulation that includes a compound the embodiments, include: of Formula Xa:

(Xa) (IV) RI O R2 N N O NR' R3 4 S

N 0286 wherein (0287 R', R and R are independently selected from hydrogen, alkyl, substituted alkyl, hydroxy, alkoxy, substi tuted alkoxy, amino, Substituted amino, carboxyl, carboxyl (V) ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substi O tuted alkenyl, alkynyl, and Substituted alkynyl: 0288 R is selected from hydrogen, alkyl, substituted HO alkyl, alkoxy, Substituted alkoxy, cycloalkyl, Substituted cycloalkyl, aryl, Substituted aryl, heterocyclyl, and Substi tuted heterocyclyl; and 0289 Q is NorCR, where R, if present, is selected from hydrogen, alkyl, Substituted alkyl, hydroxy, alkoxy, Substi tuted alkoxy, amino, Substituted amino, carboxyl, carboxyl (VI) ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substi tuted alkenyl, alkynyl, and Substituted alkynyl; and 0290 a flowable formulation suitable for delivery by inha lation. (VII) 0291. According to one aspect, the embodiments include H3C pharmaceutical formulations that include a compound of For mula Xb:

NH (Xb) R1 O R2 NH2: NH M R3 S (VIII) HN R5

0292 wherein 0293 R', R, R and Rare independently selected from hydrogen, alkyl, Substituted alkyl, hydroxy, alkoxy, Substi O S; and tuted alkoxy, amino, Substituted amino, carboxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substi tuted alkenyl, alkynyl, and Substituted alkynyl; and US 2013/0224259 A1 Aug. 29, 2013 20

0294 a flowable formulation suitable for delivery by inha 0303 Particular compounds of interest, and salts or sol lation. Vates or stereoisomers thereof for formulation according to 0295). In Formula X, R, R and R are independently the embodiments, include: selected from hydrogen, alkyl, Substituted alkyl, hydroxy, alkoxy, Substituted alkoxy, amino, Substituted amino, car boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl. NH. 0296) In certain embodiments, R' is hydrogen. In certain M embodiments, R' is alkyl or substituted alkyl. In certain S embodiments, R' is hydroxy, alkoxy, or substituted alkoxy. In certain embodiments, R' is amino or substituted amino. In benzodisothiazol-3(2H)-one certain embodiments, R' is carboxyl or carboxyl ester. In certain embodiments, R' is cyano, halogen, acyl, aminoacyl, or nitro. In certain embodiments, R' is alkenyl, substituted DDAH Inhibitors alkenyl, alkynyl, or Substituted alkynyl. In certain embodi ments, R' is alkyl, such as a C-C alkyl, or a C-C alkyl. In 0304. The present disclosure provides DDAH inhibitors, certain embodiments, R' is methyl. as well as compositions, including pharmaceutical composi 0297. In certain embodiments, R is hydrogen. In certain tions, comprising a subject DDAH inhibitor. embodiments, R is alkyl or substituted alkyl. In certain 0305. In some embodiments, a DDAH inhibitor has a half embodiments, R is hydroxy, alkoxy, or substituted alkoxy. In maximal inhibitor concentration of from about 1 nM to about certain embodiments, R is amino or substituted amino. In 1 mM, e.g., from about 1 nM to about 10 nM, from about 10 certain embodiments, R is carboxyl or carboxyl ester. In nM to about 15 nM, from about 15 nM to about 25 nM, from certain embodiments, R is cyano, halogen, acyl, aminoacyl, about 25 nM to about 50 nM, from about 50 nM to about 75 or nitro. In certain embodiments, R is alkenyl, substituted nM, from about 75 nM to about 100 nM, from about 100 nM alkenyl, alkynyl, or Substituted alkynyl. In certain embodi to about 150 nM, from about 150 nM to about 200 nM, from ments, R is alkyl, such as a C-C alkyl, or a C-C alkyl. In about 200 nM to about 250 nM, from about 250 nM to about certain embodiments, R is methyl. 300 nM, from about 300 nM to about 350 nM, from about 350 0298. In certain embodiments, R is hydrogen. In certain nM to about 400 nM, from about 400 nM to about 450 nM, embodiments, R is alkyl or substituted alkyl. In certain from about 450 nM to about 500 nM, from about 500 nM to embodiments, R is hydroxy, alkoxy, or substituted alkoxy. In about 750 nM, from about 750 nM to about 1 uM, from about certain embodiments, R is amino or substituted amino. In 1 M to about 10 uM from about 10 uM to about 25uM, from certain embodiments, R is carboxyl or carboxyl ester. In about 25uM to about 50 uM, from about 50 uM to about 75 certain embodiments, R is cyano, halogen, acyl, aminoacyl, uM, from about 75uM to about 100 uM from about 100 uM or nitro. In certain embodiments, R is alkenyl, substituted to about 250 uM from about 250 uM to about 500 uM, or from alkenyl, alkynyl, or Substituted alkynyl. In certain embodi about 500 uM to about 1 mM. ments, R is alkyl, such as a C-C alkyl, or a C-C alkyl. In 0306. In some embodiments, a DDAH inhibitor inhibits certain embodiments, R is methyl. enzymatic activity of a DDAH polypeptide by at least about 0299. In Formula X, R is selected from hydrogen, alkyl, 10%, at least about 15%, at least about 20%, at least about Substituted alkyl, alkoxy, Substituted alkoxy, cycloalkyl, Sub 25%, at least about 30%, at least about 40%, at least about stituted cycloalkyl, aryl, Substituted aryl, heterocyclyl, and 50%, or more than 50%, compared to the activity of the substituted heterocyclyl. DDAH polypeptide in the absence of the inhibitor. 0300. In certain embodiments, R is hydrogen. In certain 0307. In some cases, a DDAH inhibitor is selective, e.g., embodiments, R is alkyl or substituted alkyl. In certain the DDAH inhibitor inhibits DDAH enzymatic activity, but embodiments, R is alkoxy or substituted alkoxy. In certain does not substantially inhibit enzymes other than DDAH. embodiments, R is cycloalkyl or substituted cycloalkyl. In 0308. In the formulae herein, a formula number is meant to certain embodiments, R is aryl or substituted aryl. In certain encompass all forms of the formula number. Thus, reference embodiments, R is heterocyclyl or substituted heterocyclyl. to formula (II) is meant to include compounds of formula (IIa) 0301 In Formula X, R, if present, is selected from hydro and (IIb). gen, alkyl, Substituted alkyl, hydroxy, alkoxy, Substituted 0309 Formula II alkoxy, amino, Substituted amino, carboxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substituted 0310. According to one aspect, the present disclosure pro alkenyl, alkynyl, and Substituted alkynyl. vides a compound of Formula IIa. (0302) In certain embodiments, R is hydrogen. In certain embodiments, R is alkyl or substituted alkyl. In certain (IIa) embodiments, R is hydroxy, alkoxy, or substituted alkoxy. In R3 certain embodiments, R is amino or substituted amino. In O O R1 certain embodiments, R is carboxyl or carboxyl ester. In N \V certain embodiments, R is cyano, halogen, acyl, aminoacyl, N R2 or nitro. In certain embodiments, R is alkenyl, substituted alkenyl, alkynyl, or Substituted alkynyl. In certain embodi R4 % S ments, R is alkyl, such as a C-C alkyl, or a C-C alkyl. In certain embodiments, R is methyl. US 2013/0224259 A1 Aug. 29, 2013

0311 wherein 0325 In certain embodiments, R' is hydrogen. In certain 0312) R' is selected from hydrogen, alkyl, substituted embodiments, R' is alkyl or substituted alkyl. In certain alkyl, alkoxy, Substituted alkoxy, cycloalkyl, Substituted embodiments, R' is alkyl. For example, R' may be a C-C, cycloalkyl, aryl, Substituted aryl, heterocyclyl, and Substi alkyl, such as a C-C alkyl. In certain embodiments, R' is tuted heterocyclyl: substituted alkyl. For example, in embodiments where R' is a 0313 R’ is selected from hydrogen, alkyl, and substituted substituted alkyl, the alkyl group may be substituted with one alkyl: or more groups, such as, but not limited to, alkyl, hydroxy, 0314) R and Rare independently selected from hydro alkoxy, amino, carboxyl, carboxyl ester, cyano, halogen, thio. gen, alkyl, Substituted alkyl, hydroxy, alkoxy, Substituted acyl, aminoacyl, nitro, alkenyl, alkynyl, cycloalkyl, hetero alkoxy, amino, Substituted amino, carboxyl, carboxyl ester, cycloalkyl, aryl, heteroaryl, and the like. In certain embodi cyano, halogen, acyl, aminoacyl, nitro, alkenyl, Substituted ments, the alkyl is substituted with an alkyl group. Such as a alkenyl, alkynyl, and Substituted alkynyl: C-C alkyl, or a C-C alkyl. In certain embodiments, the 0315 or a pharmaceutical salt thereof. alkyl is substituted with an alkoxy group, such as a C-C, 0316. According to one aspect, the embodiments include alkoxy, or a C-C alkoxy. In certain embodiments, the alkyl pharmaceutical compositions, which comprise a compound group is substituted with a cycloalkyl group, such as a satu of Formula IIb: rated or unsaturated cycloalkyl group. In certain embodi ments, the alkyl group is Substituted with an aryl. Combina tions of the above substituents may also be included. The (IIb) substituents on the substituted alkyl may in turn be substituted CH3 O O R M with one or more groups as described above, such as, but not N N limited to, alkyl, hydroxy, alkoxy, amino, carboxyl, carboxyl N R2 ester, cyano, halogen (e.g., F. Cl, Br, I), thio, acyl, aminoacyl, 2Ns/ nitro, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, H3C N heteroaryl, and the like. In certain embodiments, R' is alkoxy or substituted alkoxy. In certain embodiments, R is 0317 wherein cycloalkyl or Substituted cycloalkyl. In certain embodiments, 0318 R' is selected from hydrogen, alkyl, substituted R" is cycloalkyl, such as Cs-Co cycloalkyl, or a C-C, alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl. In certain embodiments, R' is cyclohexyl. In cer cycloalkyl, aryl, Substituted aryl, heterocyclyl, and Substi tain embodiments, R' is a substituted cycloalkyl, where the tuted heterocyclyl: cycloalkyl group is Substituted with one or more groups as 0319 R is selected from hydrogen, alkyl, and substituted described above, such as, but not limited to, alkyl, hydroxy, alkyl: alkoxy, amino, carboxyl, carboxyl ester, cyano, halogen (e.g., 0320 or a pharmaceutical salt thereof. F. Cl, Br, I), thio, acyl, aminoacyl, nitro, alkenyl, alkynyl, 0321. In Formula IIa, R and R are independently cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and the like. selected from hydrogen, alkyl, Substituted alkyl, hydroxy, For example, the cycloalkyl group may be substituted with alkoxy, Substituted alkoxy, amino, Substituted amino, car one or more alkyl groups, such as a C-C alkyl, or a C-C, boxyl, carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alkyl (e.g., methyl). In certain embodiments, R' is aryl or alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl. substituted aryl. In certain embodiments, R' is heterocyclylor 0322. In certain embodiments, R is hydrogen. In certain substituted heterocyclyl. In certain embodiments, R is embodiments, R is alkyl or substituted alkyl. In certain selected from the following: embodiments, R is alkyl. For example, R may be a C-C, alkyl, such as a C-C alkyl. In certain embodiments, R is methyl. In certain embodiments, R is hydroxy, alkoxy, or substituted alkoxy. In certain embodiments, R is amino or substituted amino. In certain embodiments, R is carboxyl or carboxyl ester. In certain embodiments, R is cyano, halogen, acyl, aminoacyl, or nitro. In certain embodiments, R is alk enyl, Substituted alkenyl, alkynyl, or Substituted alkynyl. 0323. In certain embodiments, R is hydrogen. In certain embodiments, R is alkyl or substituted alkyl. In certain to / embodiments, R is alkyl. For example. R* may be a C-C, alkyl, such as a C-C alkyl. In certain embodiments, R is methyl. In certain embodiments, R is hydroxy, alkoxy, or substituted alkoxy. In certain embodiments, R is amino or substituted amino. In certain embodiments, R is carboxyl or carboxyl ester. In certain embodiments, R is cyano, halogen, acyl, aminoacyl, or nitro. In certain embodiments, R is alk enyl, Substituted alkenyl, alkynyl, or Substituted alkynyl. - 0324) In Formula II, R' is selected from hydrogen, alkyl, Substituted alkyl, alkoxy, Substituted alkoxy, cycloalkyl, Sub stituted cycloalkyl, aryl, Substituted aryl, heterocyclyl, and substituted heterocyclyl. US 2013/0224259 A1 Aug. 29, 2013 22

-continued -continued CH3 O O N )-H CH s O, N - H3C N 2 S CH3:

CH O O N )-H 2 HC N SX-/ \ S

CH O O

Cl, O- s N N HC N S O CH O O H / s <-- N N )- CH3: O O 2 s/ / HC N

s -- , and CH3 O O H

N N )- 2 M C. H3C N S l O CH3 O O N N 0326 In Formula II, R,2 is selected from hydrogen, alkyl, H3C N 2 S M and substituted alkyl. In certain embodiments, R is hydro gen. In certain embodiments, R is selected from alkyl. In certain embodiments, R is substituted alkyl. 0327 Particular compounds of interest, and salts or sol- Cl; Vates or stereoisomers thereof for formulation according to CH3 O O the embodiments, include: N

2 N CH O O H3C N S H

N N )- CH3: 2 M HC N S

CH: US 2013/0224259 A1 Aug. 29, 2013 23

-continued -continued (V) CH O O O H

N )- HO O; 2 N HC N S CH3: N O / H3C O CH3 O O (VI) S pi N N X- P-CH: 2Ns/ / O H3C N (VII) H3C H3C CH O O NH N N O

HC N 2N's/ NH2:

O (VIII) HN

O CH3; and CH3 O O /

N c. C. and (IX) H3C N2Ns/ W \ OH.

CH O O O N O-O-, N

HC N2N's/ C Pharmaceutical Formulations Comprising a Compound of Formula II, a Compound of any One of Formulae IV-IX or a Compound of Formula X 0328. Formula IV-IX 0330. The present disclosure provides pharmaceutical 0329 Particular compounds of interest, and salts or sol compositions comprising a compound of Formula II, com Vates or stereoisomers thereof for formulation according to prising a compound of one of Formulas IV-IX, or a compound the embodiments, include: of Formula X. A compound of Formula II, a compound of one of Formulas IV-IX, or a compound of Formula X, is prepared in a pharmaceutically acceptable composition(s) for delivery (IV) to a host. In the context of pharmaceutical formulations, below, the terms “active agent,” “drug.” “agent,” “therapeutic agent, and the like are used interchangeably hereinto refer to a compound of Formula II, a compound of one of Formulas IV-IX, or a compound of Formula X. 0331 In some instances, a composition comprising an active agent (e.g., a compound of Formula II, a compound of one of Formulas IV-IX, or a compound of Formula X) can comprise a pharmaceutically acceptable excipient, a variety of which are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, US 2013/0224259 A1 Aug. 29, 2013 24 for example, A. Gennaro (1995) “Remington: The Science mined quantity of an active agent calculated in an amount and Practice of Pharmacy”, 19th edition, Lippincott, Will sufficient to produce the desired effect in association with a iams, & Wilkins. pharmaceutically acceptable diluent, carrier or vehicle. The 0332 Formulations specifications for a Suitable dosage form depend, e.g., on the 0333. In the subject methods, the active agent(s) may be particular active agent employed and the effect to be administered to the host using any convenient means capable achieved, and the pharmacodynamics associated with each of resulting in the desired therapeutic effect or clinical out compound in the host. come. Thus, an active agent can be incorporated into a variety 0341. Other modes of administration will also find use of formulations for therapeutic administration. More particu with the Subject invention. For instance, an active agent can be larly, an active agent can be formulated into pharmaceutical formulated in Suppositories and, in Some cases, aerosol and compositions by combination with appropriate, pharmaceu intranasal compositions. For Suppositories, the vehicle com tically acceptable carriers or diluents, and may be formulated position can include traditional binders and carriers such as, into preparations in Solid, semi-solid, liquid orgaseous forms, polyalkylene glycols, or triglycerides. Such Suppositories Such as tablets, capsules, powders, granules, ointments, solu may be formed from mixtures containing the active ingredi tions, Suppositories, injections, inhalants and aerosols. ent in the range of about 0.5% to about 10% (w/w), e.g., about 0334. In pharmaceutical dosage forms, an active agent 1% to about 2%. may be administered in the form of its pharmaceutically 0342 Intranasal formulations will usually include acceptable salt, or an active agent may also be used alone or vehicles that neither cause irritation to the nasal mucosa nor in appropriate association, as well as in combination, with significantly disturb ciliary function. Diluents such as water, other pharmaceutically active compounds. The following aqueous saline or other known Substances can be employed methods and excipients are merely exemplary and are in no with the Subject invention. The nasal formulations may also way limiting. contain preservatives such as, but not limited to, chlorobu 0335 For oral preparations, an active agent can be used tanol and benzalkonium chloride. A surfactant may be present alone or in combination with appropriate additives to make to enhance absorption of an active agent by the nasal mucosa. tablets, powders, granules or capsules, for example, with 0343 An active agent can be administered in a composi conventional additives, such as lactose, mannitol, corn starch tion Suitable for injection. Typically, injectable compositions or potato starch; with binders, such as crystalline cellulose, are prepared as liquid Solutions or Suspensions; Solid forms cellulose derivatives, acacia, corn starch or gelatins; with Suitable for Solution in, or Suspension in, liquid vehicles prior disintegrators, such as corn starch, potato starch or sodium to injection may also be prepared. The preparation may also carboxymethylcellulose; with lubricants, such as talc or mag be emulsified or the active ingredient encapsulated in lipo nesium stearate; and if desired, with diluents, buffering Some vehicles. agents, moistening agents, preservatives and flavoring agents. 0344 Suitable excipient vehicles are, for example, water, 0336 An active agent can be formulated into preparations saline, dextrose, glycerol, ethanol, or the like, and combina for injection by dissolving, Suspending or emulsifying them tions thereof. In addition, if desired, the vehicle may contain in an aqueous or nonaqueous solvent, such as Vegetable or minor amounts of auxiliary Substances such as wetting or other similar oils, syntheticaliphatic acid glycerides, esters of emulsifying agents or pH buffering agents. Actual methods of higheraliphatic acids or propylene glycol; and if desired, with preparing Such dosage forms are known, or will be apparent, conventional additives such as solubilizers, isotonic agents, to those skilled in the art. See, e.g., Remington’s Pharmaceu Suspending agents, emulsifying agents, stabilizers and pre tical Sciences, Mack Publishing Company, Easton, Pa., 17th servatives. edition, 1985. The composition or formulation to be admin 0337. An active agent can be utilized in aerosol formula istered will, in any event, contain a quantity of the agent tion to be administered via inhalation. The compounds of the adequate to achieve the desired State in the Subject being present invention can be formulated into pressurized accept treated. able propellants such as dichlorodifluoromethane, propane, 0345 The pharmaceutically acceptable excipients, such nitrogen and the like. as vehicles, adjuvants, carriers or diluents, are readily avail 0338 Furthermore, an active agent can be made into sup able to the public. Moreover, pharmaceutically acceptable positories by mixing with a variety of bases such as emulsi auxiliary Substances, such as pH adjusting and buffering fying bases or water-soluble bases. An active agent can be agents, tonicity adjusting agents, stabilizers, wetting agents administered rectally via a Suppository. The Suppository can and the like, are readily available to the public. include vehicles Such as cocoa butter, carbowaxes and poly 0346 Oral Formulations ethylene glycols, which melt at body temperature, yet are 0347 In some embodiments, an active agent is formulated solidified at room temperature. for oral delivery to an individual in need of Such an agent. 0339 Unit dosage forms for oral or rectal administration 0348 For oral delivery, a formulation comprising an Such as syrups, elixirs, and Suspensions may be provided active agent will in some embodiments include an enteric wherein each dosage unit, for example, teaspoonful, table soluble coating material. Suitable enteric-soluble coating spoonful, tablet or suppository, contains a predetermined material include hydroxypropyl methylcellulose acetate Suc amount of the composition containing one or more inhibitors. cinate (HPMCAS), hydroxypropyl methyl cellulose phtha Similarly, unit dosage forms for injection or intravenous late (HPMCP), cellulose acetate phthalate (CAP), polyvinyl administration may comprise an active agentina composition phthalic acetate (PVPA), EudragitTM, and shellac. as a solution in Sterile water, normal saline or another phar 0349. As one non-limiting example of a suitable oral for maceutically acceptable carrier. mulation, an active agent is formulated with one or more 0340. The term “unit dosage form,” as used herein, refers pharmaceutical excipients and coated with an enteric coating, to physically discrete units suitable as unitary dosages for as described in U.S. Pat. No. 6,346.269. For example, a solu human and animal Subjects, each unit containing a predeter tion comprising an active agent and a stabilizer is coated onto US 2013/0224259 A1 Aug. 29, 2013

a core comprising pharmaceutically acceptable excipients, to 0352 Also suitable for use are formulations such as those form an active agent-coated core; a Sub-coating layer is described in U.S. Pat. No. 6.296,842 (Alkermes Controlled applied to the active agent-coated core, which is then coated Therapeutics, Inc.); U.S. Pat. No. 6,187,330 (Scios, Inc.); and with an enteric coating layer. The core generally includes the like. pharmaceutically inactive components such as lactose, a 0353 Also suitable for use herein are formulations com starch, mannitol, Sodium carboxymethyl cellulose, sodium prising an intestinal absorption enhancing agent. Suitable starch glycolate, sodium chloride, potassium chloride, pig intestinal absorption enhancers include, but are not limited to, ments, salts of alginic acid, talc, titanium dioxide, Stearic calcium chelators (e.g., citrate, ethylenediamine tetracetic acid, Stearate, micro-crystalline cellulose, glycerin, polyeth acid); Surfactants (e.g., sodium dodecyl sulfate, bile salts, ylene glycol, triethyl citrate, tributyl citrate, propanyl triac palmitoylcarnitine, and sodium salts of fatty acids); toxins etate, dibasic calcium phosphate, tribasic sodium phosphate, (e.g., Zonula occludens toxin); and the like. calcium Sulfate, cyclodextrin, and castor oil. Suitable sol 0354 Controlled Release Formulations 0355. In some embodiments, an active agent is formulated vents for an active agent include aqueous solvents. Suitable in a controlled release formulation. stabilizers include alkali-metals and alkaline earth metals, 0356 Controlled release within the scope of this invention bases of phosphates and organic acid salts and organic can be taken to mean any one of a number of extended release amines. The Sub-coating layer comprises one or more of an dosage forms. The following terms may be considered to be adhesive, a plasticizer, and an anti-tackiness agent. Suitable substantially equivalent to controlled release, for the pur anti-tackiness agents include talc, Stearic acid, Stearate, poses of the present invention: continuous release, controlled Sodium Stearyl fumarate, glyceryl behenate, kaolin and aero release, delayed release, depot, gradual release, long-term sil. Suitable adhesives include polyvinyl pyrrolidone (PVP), release, programmed release, prolonged release, proportion gelatin, hydroxyethyl cellulose (HEC), hydroxypropyl cellu ate release, protracted release, repository, retard, slow lose (HPC), hydroxypropyl methyl cellulose (HPMC), vinyl release, spaced release, Sustained release, time coat, timed acetate (VA), polyvinyl alcohol (PVA), methyl cellulose release, delayed action, extended action, layered-time action, (MC), ethyl cellulose (EC), hydroxypropyl methyl cellulose long acting, prolonged action, repeated action, slowing act phthalate (HPMCP), cellulose acetate phthalates (CAP), xan ing, Sustained action, Sustained-action medications, and than gum, alginic acid, salts of alginic acid, EudragitTM, extended release. Further discussions of these terms may be copolymer of methyl acrylic acid/methyl methacrylate with found in Lesczek Krowczynski, Extended-Release Dosage polyvinyl acetate phthalate (PVAP). Suitable plasticizers Forms, 1987 (CRC Press, Inc.). include glycerin, polyethylene glycol, triethylcitrate, tributyl 0357 The various controlled release technologies cover a citrate, propanyl triacetate and castor oil. Suitable enteric very broad spectrum of drug dosage forms. Controlled release soluble coating material include hydroxypropyl methylcellu technologies include, but are not limited to physical systems lose acetate succinate (HPMCAS), hydroxypropyl methyl and chemical systems. cellulose phthalate (HPMCP), cellulose acetate phthalate 0358 Physical systems include, but are not limited to, (CAP), polyvinyl phthalic acetate (PVPA), EudragitTM and reservoir Systems with rate-controlling membranes, such as shellac. microencapsulation, macroencapsulation, and membrane systems; reservoir systems without rate-controlling mem 0350 Suitable oral formulations also include an active branes, such as hollow fibers, ultra microporous cellulose agent formulated with any of the following: microgranules triacetate, and porous polymeric Substrates and foams; mono (see, e.g., U.S. Pat. No. 6,458,398); biodegradable mac lithic systems, including those systems physically dissolved romers (see, e.g., U.S. Pat. No. 6,703,037); biodegradable in non-porous, polymeric, or elastomeric matrices (e.g., non hydrogels (see, e.g., Graham and McNeill (1989) Biomateri erodible, erodible, environmental agent ingression, and als 5:27-36); biodegradable particulate vectors (see, e.g., U.S. degradable), and materials physically dispersed in non-po Pat. No. 5,736,371); bioabsorbable lactone polymers (see, rous, polymeric, or elastomeric matrices (e.g., nonerodible, e.g., U.S. Pat. No. 5,631,015); slow release protein polymers erodible, environmental agent ingression, and degradable); (see, e.g., U.S. Pat. No. 6,699,504; Pelias Technologies, Inc.); laminated structures, including reservoir layers chemically a poly(lactide-co-glycolide/polyethylene glycol block similar or dissimilar to outer control layers; and other physi copolymer (see, e.g., U.S. Pat. No. 6,630, 155; Atrix Labora cal methods, such as osmotic pumps, or adsorption onto ion tories, Inc.); a composition comprising a biocompatible poly exchange resins. mer and particles of metal cation-stabilized agent dispersed 0359 Chemical systems include, but are not limited to, chemical erosion of polymer matrices (e.g., heterogeneous, within the polymer (see, e.g., U.S. Pat. No. 6,379,701: Alk or homogeneous erosion), or biological erosion of a polymer ermes Controlled Therapeutics, Inc.); and microspheres (see, matrix (e.g., heterogeneous, or homogeneous). Additional e.g., U.S. Pat. No. 6,303,148; Octoplus, B.V.). discussion of categories of systems for controlled release may 0351 Suitable oral formulations also include an active be found in Agis F. Kydonieus, Controlled Release Technolo agent formulated with any of the following: a carrier Such as gies: Methods. Theory and Applications, 1980 (CRC Press, Emisphere.R. (Emisphere Technologies, Inc.); TIMERX, a Inc.). hydrophilic matrix combining Xanthan and locust bean gums 0360. There are a number of controlled release drug for which, in the presence of dextrose, form a strong binder gel in mulations that are developed for oral administration. These water (Penwest); GeminexTM (Penwest); ProciseTM (Glaxo include, but are not limited to, osmotic pressure-controlled SmithKline); SAVITTM (Mistral Pharma Inc.); RingCapTM gastrointestinal delivery systems; hydrodynamic pressure (Alza Corp.); Smartrix R(Smartrix Technologies, Inc.); SQZ controlled gastrointestinal delivery systems; membrane per gelTM (MacroMed, Inc.); GeomatrixTM (Skye Pharma, Inc.); meation-controlled gastrointestinal delivery systems, which Oros(R Tri-layer (Alza Corporation); and the like. include microporous membrane permeation-controlled gas US 2013/0224259 A1 Aug. 29, 2013 26 trointestinal delivery devices; gastric fluid-resistant intestine drug from the matrix. The solubility of an active agent may targeted controlled-release gastrointestinal delivery devices; also be increased owing to Some interaction with the carriers. gel diffusion-controlled gastrointestinal delivery systems; 0366 Examples of carriers useful in solid dispersions and ion-exchange-controlled gastrointestinal delivery sys include, but are not limited to, water-soluble polymers such as tems, which include cationic and anionic drugs. Additional polyethylene glycol, polyvinylpyrrolidone, and hydroxypro information regarding controlled release drug delivery sys pylmethyl-cellulose. Alternative carriers include phosphati tems may be found in Yie W. Chien, Novel Drug Delivery dylcholine. Phosphatidylcholine is an amphoteric but water Systems, 1992 (Marcel Dekker, Inc.). Some of these formu insoluble lipid, which may improve the solubility of lations will now be discussed in more detail. otherwise insoluble active agents in an amorphous state in 0361 Enteric coatings are applied to tablets to prevent the phosphatidylcholine Solid dispersions. release of drugs in the stomach either to reduce the risk of 0367. Other carriers include polyoxyethylene hydroge unpleasant side effects or to maintain the stability of the drug nated castor oil. Poorly water-soluble active agents may be which might otherwise be subject to degradation of expose to included in a solid dispersion system with an enteric polymer the gastric environment. Most polymers that are used for this Such as hydroxypropylmethylcellulose phthalate and car purpose are polyacids that function by virtue or the fact that boxymethylethylcellulose, and a non-enteric polymer, their solubility in aqueous medium is pH-dependent, and they hydroxypropylmethylcellulose. Another solid dispersion require conditions with a pH higher than normally encoun dosage form includes incorporation of an active agent with tered in the stomach. ethyl cellulose and stearic acid in different ratios. 0362. One exemplary type of oral controlled release struc 0368. There are various methods commonly known for ture is enteric coating of a Solid or liquid dosage form. The preparing Solid dispersions. These include, but are not limited enteric coatings are designed to disintegrate in intestinal fluid to, the melting method, the solvent method and the melting for ready absorption. Delay of absorption of the active agent solvent method. that is incorporated into a formulation with an enteric coating 0369. Another controlled release dosage form is a com is dependent on the rate of transfer through the gastrointesti plex between an ion exchange resin and an active agent. Ion nal tract, and so the rate of gastric emptying is an important exchange resin-drug complexes have been used to formulate factor. In one exemplary embodiment, an active agent can be Sustained-release products of acidic and basic drugs. In one contained in an enterically coated multiple-unit dosage form. exemplary embodiment, a polymeric film coating is provided In an exemplary embodiment, a dosage form comprising an to the ion exchange resin-drug complex particles, making active agent is prepared by spray-coating granules of the drug release from these particles diffusion controlled. See Y. active agent-enteric coating agent solid dispersion on an inert Raghunathan et al., Sustained-release drug delivery system I: core material. These granules can result in prolonged absorp Coded ion-exchange resin Systems for phenylpropanolamine tion of the active agent with good bioavailability. and other drugs, J. Pharm. Sciences 70:379-384 (1981). 0363 Typical enteric coating agents include, but are not 0370. Injectable microspheres are another controlled limited to, hydroxypropylmethylcellulose phthalate, meth release dosage form. Injectable micro spheres may be pre acryclic acid-methacrylic acid ester copolymer, polyvinyl pared by non-aqueous phase separation techniques, and acetate-phthalate and cellulose acetate phthalate Akihiko spray-drying techniques. Microspheres may be prepared Hasegawa, Application of Solid dispersions of Nifedipine using polylactic acid or copoly(lactic/glycolic acid). with enteric coating agent to prepare a Sustained-release dos Shigeyuki Takada, Utilization of an Amorphous Form of a age form, Chem. Pharm. Bull.33:1615-1619 (1985). Various Water-Soluble GPIb/IIIa Antagonist for Controlled Release enteric coating materials may be selected on the basis of From Biodegradable Micro spheres, Pharm. Res. 14:1146 testing to achieve an enteric coated dosage form designed ab 1150 (1997), and ethylcellulose, Yoshiyuki Koida, Studies on initio to have an optimal combination of dissolution time, Dissolution Mechanism of Drugs from Ethyl Cellulose coating thicknesses and diametral crushing strength. S. C. Microcapsules, Chem. Pharm. Bull.35:1538-1545 (1987). Porter et al., The Properties of Enteric Tablet Coatings Made 0371 Other controlled release technologies that may be From Polyvinyl Acetate-phthalate and Cellulose acetate used include, but are not limited to, SODAS (Spheroidal Oral Phthalate, J. Pharm. Pharmacol. 22:42p (1970). Drug Absorption System), INDAS (Insoluble Drug Absorp 0364 Another type of useful oral controlled release struc tion System), IPDAS (Intestinal Protective Drug Absorption ture is a Solid dispersion. A Solid dispersion may be defined as System), MODAS (Multiporous Oral Drug Absorption Sys a dispersion of one or more active ingredients in an inert tem), EFVAS (Effervescent Drug Absorption System), PRO carrier or matrix in the solid state prepared by the melting DAS (Programmable Oral Drug Absorption System), and (fusion), Solvent, or melting-solvent method. Akihiko Hase DUREDAS (Dual Release Drug Absorption System) avail gawa, Super Saturation Mechanism of Drugs from Solid Dis able from Elan Pharmaceutical Technologies. SODAS are persions with Enteric Coating Agents, Chem. Pharm. Bull. multi particulate dosage forms utilizing controlled release 36: 4941-4950 (1998). The solid dispersions may be also beads. INDAS are a family of drug delivery technologies called solid-state dispersions. The term “coprecipitates' may designed to increase the solubility of poorly soluble drugs. also be used to refer to those preparations obtained by the IPDAS are multi particulate tablet formation utilizing a com solvent methods. bination of high density controlled release beads and an 0365. The selection of the carrier may have an influence on immediate-release granulate. MODAS are controlled release the dissolution characteristics of the dispersed active agent single unit dosage forms. Each tablet consists of an inner core because the dissolution rate of a component from a Surface Surrounded by a semipermeable multiparous membrane that may be affected by other components in a multiple compo controls the rate of drug release. EFVAS is an effervescent nent mixture. For example, a water-soluble carrier may result drug absorption system. PRODAS is a family of multi par in a fast release of the drug from the matrix, or a poorly ticulate formulations utilizing combinations of immediate soluble or insoluble carrier may lead to a slower release of the release and controlled release mini-tablets. DUREDAS is a US 2013/0224259 A1 Aug. 29, 2013 27 bilayer tablet formulation providing dual release rates within the body is controlled. Because of the minimal use of excipi the one dosage form. Although these dosage forms are known ents, MODAS can readily accommodate Small dosage size to one of skill, certain of these dosage forms will now be forms. Each MODAS tablet begins as a core containing active discussed in more detail. drug plus excipients. This core is coated with a solution of 0372 INDAS was developed specifically to improve the insoluble polymers and soluble excipients. Once the tablet is solubility and absorption characteristics of poorly water ingested, the fluid of the gastrointestinal tract may dissolve soluble drugs. Solubility and, in particular, dissolution within the Soluble excipients in the outer coating leaving Substan the fluids of the gastrointestinal tract is a key factor in deter tially the insoluble polymer. What results is a network of tiny, mining the overall oral bioavailability of poorly water soluble narrow channels connecting fluid from the gastrointestinal drug. By enhancing solubility, one can increase the overall tract to the inner drug core of water soluble drug. This fluid bioavailability of a drug with resulting reductions in dosage. passes through these channels, into the core, dissolving the INDAS takes the form of a high energy matrix tablet, produc drug, and the resultant Solution of drug may diffuse out in a tion of which is comprised of two distinct steps: the drug in controlled manner. This may permit both controlled dissolu question is converted to an amorphous form through a com tion and absorption. An advantage of this system is that the bination of energy, excipients, and unique processing proce drug releasing pores of the tablet are distributed over substan dures. tially the entire surface of the tablet. This facilitates uniform 0373) Once converted to the desirable physical form, the drug absorption reduces aggressive unidirectional drug deliv resultant high energy complex may be stabilized by an ery. MODAS represents a very flexible dosage form in that absorption process that utilizes a novel polymer cross-linked both the inner core and the outer semipermeable membrane technology to prevent recrystallization. The combination of may be altered to suit the individual delivery requirements of the change in the physical state of an active agent coupled a drug. In particular, the addition of excipients to the inner with the solubilizing characteristics of the excipients core may help to produce a microenvironment within the employed enhances the solubility of the active agent. The tablet that facilitates more predictable release and absorption resulting absorbed amorphous drug complex granulate may rates. The addition of an immediate release outer coating may be formulated with a gel-forming erodible tablet system to allow for development of combination products. promote substantially smooth and continuous absorption. 0377 Additionally, PRODAS may be used to deliver an 0374 IPDAS is a multi-particulate tablet technology that active agent. PRODAS is a multi particulate drug delivery may enhance the gastrointestinal tolerability of potential irri technology based on the production of controlled release mini tant and ulcerogenic drugs. Intestinal protection is facilitated tablets in the size range of 1.5 to 4 mm in diameter. The by the multi-particulate nature of the IPDAS formulation PRODAS technology is a hybrid of multi particulate and which promotes dispersion of an irritant lipoate throughout hydrophilic matrix tablet approaches, and may incorporate, in the gastrointestinal tract. Controlled release characteristics of one dosage form, the benefits of both these drug delivery the individual beads may avoid high concentration of drug systems. being both released locally and absorbed systemically. The 0378. In its most basic form, PRODAS involves the direct combination of both approaches serves to minimize the compression of an immediate release granulate to produce potential harm of an active agent with resultant benefits to individual mini tablets that contain an active agent. These patients. mini tablets are Subsequently incorporated into hard gels and 0375 IPDAS is composed of numerous high density con capsules that represent the final dosage form. A more benefi trolled release beads. Each bead may be manufactured by a cial use of this technology is in the production of controlled two-step process that involves the initial production of a release formulations. In this case, the incorporation of various micromatrix with embedded active agent and the Subsequent polymer combinations within the granulate may delay the coating of this micromatrix with polymer Solutions that form release rate of drugs from each of the individual mini tablets. a rate-limiting semipermeable membrane in vivo. Once an These mini tablets may subsequently be coated with con IPDAS tablet is ingested, it may disintegrate and liberate the trolled release polymer solutions to provide additional beads in the stomach. These beads may subsequently pass delayed release properties. The additional coating may be into the duodenum and along the gastrointestinal tract, e.g., in necessary in the case of highly water soluble drugs or drugs a controlled and gradual manner, independent of the feeding that are perhaps gastroirritants where release can be delayed state. Release of the active agent occurs by diffusion process until the formulation reaches more distal regions of the gas through the micromatrix and Subsequently through the pores trointestinal tract. One value of PRODAS technology lies in in the rate controlling semipermeable membrane. The release the inherent flexibility to formulation whereby combinations rate from the IPDAS tablet may be customized to deliver a of mini tablets, each with different release rates, are incorpo drug-specific absorption profile associated with optimized rated into one dosage form. As well as potentially permitting clinical benefit. Should a fast onset of activity be necessary, controlled absorption over a specific period, this also may immediate release granulate may be included in the tablet. permit targeted delivery of drug to specific sites of absorption The tablet may be broken prior to administration, without throughout the gastrointestinal tract. Combination products Substantially compromising drug release, if a reduced dose is also may be possible using mini tablets formulated with dif required for individual titration. ferent active ingredients. 0376) MODAS is a drug delivery system that may be used 0379 DUREDAS is a bilayer tableting technology that to control the absorption of water soluble agents. Physically may be used to an active agent. DUREDAS was developed to MODAS is a non-disintegrating table formulation that provide for two different release rates, or dual release of a manipulates drug release by a process of rate limiting diffu drug from one dosage form. The term bilayer refers to two sion by a semipermeable membrane formed in vivo. The separate direct compression events that take place during the diffusion process essentially dictates the rate of presentation tableting process. In an exemplary embodiment, an immedi of drug to the gastrointestinal fluids, such that the uptake into ate release granulate is first compressed, being followed by US 2013/0224259 A1 Aug. 29, 2013 28 the addition of a controlled release element which is then through an outlet tube of the nebulizer. The resulting mist compressed onto this initial tablet. This may give rise to the penetrates into the respiratory tract of the patient. characteristic bilayer seen in the final dosage form. 0387. A powder composition containing an active agent, 0380. The controlled release properties may be provided with or without a lubricant, carrier, or propellant, can be by a combination of hydrophilic polymers. In certain cases, a administered to a mammal in need of therapy. This embodi rapid release of an active agent may be desirable in order to ment of the invention can be carried out with a conventional facilitate a fast onset of therapeutic effect. Hence one layer of device for administering a powder pharmaceutical composi the tablet may be formulated as an immediate release granu tion by inhalation. For example, a powder mixture of the late. By contrast, the second layer of the tablet may release the compound and a suitable powder base such as lactose or drug in a controlled manner, e.g., through the use of hydro starch may be presented in unit dosage form in for example philic polymers. This controlled release may result from a capsular or cartridges, e.g. gelatin, or blister packs, from combination of diffusion and erosion through the hydrophilic which the powder may be administered with the aid of an polymer matrix. inhaler. 0381. A further extension of DUREDAS technology is the 0388. There are several different types of inhalation meth production of controlled release combination dosage forms. odologies which can be employed in connection with the In this instance, two different active agents may be incorpo present invention. An active agent can be formulated in basi rated into the bilayer tablet and the release of drug from each cally three different types of formulations for inhalation. layer controlled to maximize therapeutic effect of the combi First, an active agent can beformulated with low boiling point nation. propellants. Such formulations are generally administered by 0382 An active agent can be incorporated into any one of conventional meter dose inhalers (MDIs). However, conven the aforementioned controlled released dosage forms, or tional MDI’s can be modified so as to increase the ability to other conventional dosage forms. The amount of active agent obtain repeatable dosing by utilizing technology which mea contained in each dose can be adjusted, to meet the needs of Sures the inspiratory Volume and flow rate of the patient as the individual patient, and the indication. One of skill in the discussed within U.S. Pat. Nos. 5,404.871 and 5,542,410. art and reading this disclosure will readily recognize how to 0389. Alternatively, an active agent can be formulated in adjust the level of an active agent and the release rates in a aqueous or ethanolic solutions and delivered by conventional controlled release formulation, in order to optimize delivery nebulizers. Lastly, an active agent can be formulated into dry of the active agent and its bioavailability. powderformulations. Such formulations can be administered Inhalational Formulations by simply inhaling the dry powder formulation after creating 0383 an aerosol mist of the powder. 0384 An active agent will in some embodiments be administered to a patient by means of a pharmaceutical deliv 0390 Dosages ery system for the inhalation route. An active agent may be 0391 Although the dosage used will vary depending on formulated in a form suitable for administration by inhala the clinical goals to be achieved, a Suitable dosage range is tion. The inhalational route of administration provides the one which provides up to about 1 g to about 1,000 ug or advantage that the inhaled drug can directly target the lung. about 10,000 g of an active agent and can be administered in An inhalational pharmaceutical delivery system is one that is a single dose. Alternatively, a target dosage of an active agent suitable for respiratory therapy by delivery of an active agent can be considered to be about in the range of about 0.1-1000 to mucosal linings of the bronchi. This invention can utilize a uM, about 0.5-500 uM, about 1-100 uM, or about 5-50 uM in system that depends on the power of a compressed gas to a sample of host blood drawn within the first 24–48 hours after expel an active agent from a container. An aerosol or pressur administration of the agent. ized package can be employed for this purpose. 0392 Those of skill will readily appreciate that dose levels 0385 As used herein, the term “aerosol is used in its can vary as a function of the specific compound, the severity conventional sense as referring to very fine liquid or Solid of the symptoms and the susceptibility of the subject to side particles carries by a propellant gas under pressure to a site of effects. Preferred dosages for a given compound are readily therapeutic application. When a pharmaceutical aerosol is determinable by those of skill in the art by a variety of means. employed in the context of the present disclosure, the aerosol 0393 Routes of Administration contains an active agent, which can be dissolved, Suspended, 0394 An active agent is administered to an individual or emulsified in a mixture of a fluid carrier and a propellant. using any available method and route Suitable for drug deliv The aerosol can be in the form of a solution, Suspension, ery, including in Vivo and ex vivo methods, as well as sys emulsion, powder, or semi-solid preparation. Aerosols temic and localized routes of administration. employed in the present invention are intended for adminis 0395 Conventional and pharmaceutically acceptable tration as fine, Solid particles or as liquid mists via the respi routes of administration include intranasal, intramuscular, ratory tract of a patient. Various types of propellants known to intratracheal, intracranial, Subcutaneous, intradermal, topical one of skill in the art can be utilized. Suitable propellants application, intravenous, rectal, nasal, oral and other enteral include, but are not limited to, hydrocarbons or other suitable and parenteral routes of administration. Routes of adminis gas. In the case of the pressurized aerosol, the dosage unit tration may be combined, if desired, or adjusted depending may be determined by providing a value to deliver a metered upon the agent and/or the desired effect. The composition can amount. be administered in a single dose or in multiple doses. In some 0386. An active agent can also be formulated for delivery embodiments, the composition is administered orally. In with a nebulizer, which is an instrument that generates very other embodiments, the composition is administered intrave fine liquid particles of Substantially uniform size in a gas. For nously. In other embodiments, the composition is adminis example, a liquid containing an active agent is dispersed as tered via an inhalational route. In other embodiments, the droplets. The small droplets can be carried by a current of air composition is administered intramuscularly. US 2013/0224259 A1 Aug. 29, 2013 29

0396 The agent can be administered to a host using any Fibrosis available conventional methods and routes suitable for deliv ery of conventional drugs, including systemic or localized 0403. The present disclosure provides methods of treating routes. In general, routes of administration contemplated by fibrosis, e.g., fibrosis affecting any tissue including, for the invention include, but are not necessarily limited to, example, fibrosis of an internal organ, a cutaneous or dermal enteral, parenteral, or inhalational routes. fibrotic disorder, fibrotic conditions of the eye, and vascular 0397 Parenteral routes of administration other than inha fibrosis. Fibrosis of internal organs (e.g., liver, lung, kidney, lation administration include, but are not necessarily limited heart blood vessels, gastrointestinal tract) occurs in disorders to, topical, transdermal, Subcutaneous, intramuscular, Such as pulmonary fibrosis, idiopathic fibrosis, autoimmune intraorbital, intracapsular, intraspinal, intrasternal, and intra fibrosis, myelofibrosis, liver fibrosis, liver cirrhosis, veno venous routes, i.e., any route of administration other than occlusive disease, mesangial proliferative glomerulonephri through the alimentary canal. Parenteral administration can tis, crescentic glomerulonephritis, diabetic nephropathy, be carried to effect systemic or local delivery of the agent. renal interstitial fibrosis, renal fibrosis in subjects receiving Where systemic delivery is desired, administration typically cyclosporin, endomyocardial fibrosis, bronchiolitis obliter involves invasive or systemically absorbed topical or mucosal ans (a fibrotic process that can occur after lung transplanta administration of pharmaceutical preparations. tion, after exposure to a toxin, or after an infection), and the like. Liver fibrosis, used interchangeably herein with “hepatic 0398. The agent can also be delivered to the subject by fibrosis.” can occur in the context of a chronic hepatitis infec enteral administration. Enteral routes of administration tion, or in the context of an injury (e.g., exposure, Such as include, but are not necessarily limited to, oral and rectal (e.g., chronic exposure, to a toxin). Dermal fibrotic disorders using a Suppository) delivery. include, for example, Scleroderma, morphea, keloids, hyper 0399. By treatment is meant at least an amelioration of the trophic scars, familial cutaneous collagenoma, and connec symptoms associated with the pathological condition afflict tive tissue nevi of the collagen type. Fibrotic conditions of the ing the host, where amelioration is used in a broad sense to eye include conditions such as diabetic retinopathy, post refer to at least a reduction in the magnitude of a parameter, Surgical scarring (for example, after glaucoma filtering Sur e.g. symptom, associated with the pathological condition gery, or after Strabismus Surgery), and proliferative vitreore being treated, such as a neurological disorder and pain that tinopathy. Fibrosis can be triggered by interventional therapy, may be associated therewith. As such, treatment also includes where such fibrosis includes, e.g. restenosis (e.g., restenosis situations where the pathological condition, or at least symp following balloon angioplasty or following atherectomy). toms associated therewith, are completely inhibited, e.g. pre vented from happening, or stopped, e.g. terminated, such that Vascular fibrosis includes, e.g., atherosclerosis, peripheral the host no longer Suffers from the pathological condition, or arterial disease, and the like. at least the symptoms that characterize the pathological con 0404 In some cases, an effective amount of a compound dition. of one of one of Formulae I-X is an amount that reduces 0400 A variety of hosts (wherein the term “host' is used collagen production by a fibroblast. For example, in some interchangeably herein with the terms “subject' and instances, an effective amount of a compound of one of one of “patient’) are treatable according to the subject methods. Formulae I-X is an amount that reduces collagen production Generally such hosts are “mammals” or “mammalian, where by a fibroblast by at least about 10%, at least about 15%, at these terms are used broadly to describe organisms which are least about 20%, at least about 25%, at least about 30%, at within the class mammalia, including the orders carnivore least about 35%, at least about 40%, at least about 45%, at (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and least about 50%, or more than 50%, compared to the amount rats), non-human primates, and primates (e.g., humans, chim of collagen produced by the fibroblast in the absence of the panzees, and monkeys). In some embodiments, the hosts will compound. be humans. Liver Fibrosis Treatment Methods 0405. In some cases, a subject method can be used to treat 04.01 The present disclosure provides a method of treating liver fibrosis. In some cases, an effective amount of a com a patient Suffering from a disorder characterized by excessive pound of one of one of Formulae I-X is an amount that, when NO production and/or elevated DDAH activity, the method administered as monotherapy or combination therapy to an comprising administering to said patient an effective amount individual having liver fibrosis, is effective to increase liver of a compound of one of Formulae I-X, e.g., where a com function, or stabilize liver function. pound of Formula Ia or a compound of Formula III is admin 0406. As used herein, the term “liver function” refers to a istered as an inhalational formulation. Disorders character normal function of the liver, including, but not limited to, a ized by, or resulting from, excessive NO production and/or synthetic function, including, but not limited to, synthesis of elevated DDAH activity include, e.g., fibrosis; sepsis: proteins such as serum proteins (e.g., albumin, clotting fac migraine headache, inflammation, autoimmune diseases, and tors, alkaline phosphatase, aminotransferases (e.g., alanine certain cancers. In some cases, DDAH is reversibly inhibited. transaminase, aspartate transaminase), 5'-nucleosidase, 04.02 Excessive NO production refers to NO production Y-glutaminyltranspeptidase, etc.), synthesis of bilirubin, Syn such that NO is presentata level that is greater than (e.g., 10% thesis of cholesterol, and synthesis of bile acids; a liver meta greater, 20% greater, 50% greater, or more than 50% greater) bolic function, including, but not limited to, carbohydrate a normal control level of NO. Similarly, elevated DDAH metabolism, amino acid and ammonia metabolism, hormone activity is DDAH activity that is greater than (e.g., 10% metabolism, and lipid metabolism; detoxification of exog greater, 20% greater, 50% greater, or more than 50% greater) enous drugs; a hemodynamic function, including splanchnic a normal control level of DDAH activity. and portal hemodynamics; and the like. US 2013/0224259 A1 Aug. 29, 2013 30

0407. In some cases, an effective amount of a compound of each stage in the METAVIR system are as follows: score: of one of one of Formulae I-X is an amount that, when 0, no fibrosis; score: 1, stellate enlargement of portal tract but administered as monotherapy or combination therapy to an without septa formation; score: 2, enlargement of portal tract individual having liver fibrosis, is effective to increase an with rare septa formation; score: 3, numerous septa without index of liver function by at least about 10%, at least about cirrhosis; and score: 4, cirrhosis. 20%, at least about 25%, at least about 30%, at least about 0412. Knodell's scoring system, also called the Hepatitis 35%, at least about 40%, at least about 45%, at least about Activity Index, classifies specimens based on scores in four 50%, at least about 55%, at least about 60%, at least about categories of histologic features: I. Periportal and/or bridging 65%, at least about 70%, at least about 75%, or at least about necrosis; II. Intralobular degeneration and focal necrosis; III. 80%, or more, compared to the index of liver function in an Portal inflammation; and IV. Fibrosis. In the Knodell staging untreated individual, or to a placebo-treated individual. Those system, scores are as follows: score: 0, no fibrosis; score: 1. skilled in the art can readily measure such indices of liver mild fibrosis (fibrous portal expansion); score: 2, moderate function, using standard assay methods, many of which are fibrosis; score: 3, severe fibrosis (bridging fibrosis); and commercially available, and are used routinely in clinical score: 4, cirrhosis. The higher the score, the more severe the Settings. liver tissue damage. Knodell (1981) Hepatol. 1:431. 0408 Serum markers of liver fibrosis can also be mea 0413. In the Scheuer scoring system scores are as follows: Sured as an indication of the efficacy of a Subject treatment score: 0, no fibrosis; score: 1, enlarged, fibrotic portal tracts; method. Serum markers of liver fibrosis include, but are not score: 2, periportal or portal-portal septa, but intact architec limited to, hyaluronate, N-terminal procollagen III peptide, ture; score: 3, fibrosis with architectural distortion, but no 7S domain of type IV collagen, C-terminal procollagen I obvious cirrhosis; score: 4, probable or definite cirrhosis. peptide, and laminin. Additional biochemical markers of liver Scheuer (1991).J. Hepatol. 13:372. fibrosis include a2-macroglobulin, haptoglobin, gamma 0414. The Ishak scoring system is described in Ishak globulin, apolipoprotein A, and gamma glutamyl transpepti (1995).J. Hepatol. 22:696-699. Stage 0, No fibrosis: Stage 1, dase. Fibrous expansion of some portal areas, with or without short 04.09. In some cases, an effective amount of a compound fibrous septa; stage 2, Fibrous expansion of most portal areas, of one of one of Formulae I-X is an amount that, when with or without short fibrous septa; stage 3, Fibrous expan administered as monotherapy or combination therapy to an sion of most portal areas with occasional portal to portal (P-P) individual having liver fibrosis, is effective to reduce a serum bridging; stage 4, Fibrous expansion of portal areas with levelofa marker of liver fibrosis by at least about 10%, at least marked bridging (P-P) as well as portal-central (P-C); stage 5, about 20%, at least about 25%, at least about 30%, at least Marked bridging (P-P and/or P-C) with occasional nodules about 35%, at least about 40%, at least about 45%, at least (incomplete cirrhosis); stage 6, Cirrhosis, probable or defi about 50%, at least about 55%, at least about 60%, at least nite. about 65%, at least about 70%, at least about 75%, or at least 0415. The benefit of anti-fibrotic therapy can also be mea about 80%, or more, compared to the level of the marker in an Sured and assessed by using the Child-Pugh scoring system untreated individual, or to a placebo-treated individual. Meth which comprises a multicomponent point system based upon ods of measuring serum markers include immunological abnormalities in serum bilirubin level, serum albumin level, based methods, e.g., enzyme-linked immunosorbent assays prothrombin time, the presence and severity of ascites, and (ELISA), radioimmunoassays, and the like, using antibody the presence and severity of encephalopathy. Based upon the specific for a given serum marker. presence and severity of abnormality of these parameters, 0410. Whether a subject treatment method is effective in patients may be placed in one of three categories of increasing reducing liver fibrosis is determined by any of a number of severity of clinical disease: A, B, or C. well-established techniques for measuring liver fibrosis and 0416. In some cases, an effective amount of a compound liver function. Liver fibrosis reduction can be determined by of one of one of Formulae I-X is an amount that, in mono analyzing a liver biopsy sample. An analysis of a liver biopsy therapy or combination therapy, when administered to an comprises assessments of two major components: necroin individual having liver fibrosis, effects a change of one unit or flammation assessed by "grade' as a measure of the severity more in the fibrosis stage based on pre- and post-therapy liver and ongoing disease activity, and the lesions of fibrosis and biopsies. In some cases, liver fibrosis is reduced by at least parenchymal or vascular remodeling as assessed by 'stage' one unit in the METAVIR, the Knodell, the Scheuer, the as being reflective of long-term disease progression. See, e.g., Ludwig, or the Ishak scoring system. Brunt (2000) Hepatol. 31:241-246; and METAVIR (1994) 0417 Secondary, or indirect, indices of liver function can Hepatology 20:15-20. Based on analysis of the liver biopsy, a also be used to evaluate the efficacy of treatment with. Mor score is assigned. A number of standardized scoring systems phometric computerized semi-automated assessment of the exist which provide a quantitative assessment of the degree quantitative degree of liver fibrosis based upon specific stain and severity of fibrosis. These include the METAVIR, ing of collagen and/or serum markers of liver fibrosis can also Knodell, Scheuer, Ludwig, and Ishak scoring systems. be measured as an indication of the efficacy of a subject 0411. The METAVIR scoring system is based on an analy treatment method. Secondary indices of liver function sis of various features of a liver biopsy, including fibrosis include, but are not limited to, serum transaminase levels, (portal fibrosis, centrilobular fibrosis, and cirrhosis); necrosis prothrombin time, bilirubin, platelet count, portal pressure, (piecemeal and lobular necrosis, acidophilic retraction, and albumin level, and assessment of the Child-Pugh score. ballooning degeneration); inflammation (portal tract inflam mation, portal lymphoid aggregates, and distribution of portal Pulmonary Fibrosis inflammation); bile duct changes; and the Knodell index 0418. The present disclosure provides methods of treating (scores of periportal necrosis, lobular necrosis, portal inflam pulmonary fibrosis. Pulmonary fibrosis can becaused by, e.g., mation, fibrosis, and overall disease activity). The definitions chronic inflammatory processes such as sarcoidosis, Wegen US 2013/0224259 A1 Aug. 29, 2013 er's granulomatosis, etc.; infections; environmental agents extensive than reticular opacity), and mediastinal adenopathy (e.g., asbestos, silica, exposure to certain gases); exposure to (or, if present, is not extensive enough to be visible on chest ionizing radiation (Such as radiation therapy to treat a tumor X-ray). A diagnosis of definite IPF is made if characteristics in the chest); chronic conditions (e.g., systemic lupus erythe (1), (2), and (3) are met. A diagnosis of probable IPF is made mato Sus; rheumatoid arthritis; etc.); and certain medications. if characteristics (1) and (3) are met. Pulmonary fibrosis can be caused by, or exacerbated by, the 0424. In some embodiments, an effective amount of a use of tobacco. In some patients, the cause of the pulmonary compound of one of Formulas I-X is an amount that, in fibrosis is not understood; where the cause of pulmonary monotherapy or combination therapy, when administered to fibrosis is not understood, the pulmonary fibrosis is referred an individual having pulmonary fibrosis, is effective to to as "idiopathic pulmonary fibrosis” (IPF). decrease IPF disease progression by at least about 10%, at 0419. In some embodiments, an effective amount of a least about 15%, at least about 20%, at least about 25%, at compound of one of one of Formulae I-X is an amount that, in least about 30%, at least about 35%, at least about 40%, at monotherapy or combination therapy, when administered to least about 45%, at least about 50%, at least about 55%, at an individual having pulmonary fibrosis, is effective to reduce least about 60%, at least about 65%, at least about 70%, or the pulmonary fibrosis or reduce the rate of progression of the more, compared with a placebo control or an untreated con pulmonary fibrosis by at least about 10%, at least about 15%, trol. at least about 20%, at least about 25%, at least about 30%, at 0425 Disease progression is the occurrence of one or least about 35%, at least about 40%, at least about 45%, or at more of the following: (1) a decrease in predicted forced vital least about 50%, or more, compared with the degree of pull capacity (FVC) of 10% or more; (2) an increase in A-a gra monary fibrosis in the individual prior to treatment or com dient of 5 mm Hg or more; (3) a decrease of 15% or more in pared to the rate of progression of pulmonary fibrosis that single breath diffusing capacity (DL). Whether disease would have been experienced by the patient in the absence of progression has occurred is determined by measuring one or the monotherapy or combination therapy. more of these parameters on two consecutive occasions 4 to 0420. In some embodiments, an effective amount of a 14 weeks apart, and comparing the value to baseline. compound of one of Formulas I-X is an amount that, in 0426. Thus, e.g., where an untreated or placebo-treated monotherapy or combination therapy, when administered to individual exhibits a 50% decrease in FVC over a period of an individual having pulmonary fibrosis, is effective to time, an individual administered with an effective amount of increase the function of, or to reduce the rate of deterioration a compound of one of Formulas I-X exhibits a decrease in of a lung of the individual by at least about 10%, at least about FVC of 45%, about 42%, about 40%, about 37%, about 35%, 15%, at least about 20%, at least about 25%, at least about about 32%, about 30%, or less, over the same time period. 30%, at least about 35%, at least about 40%, at least about 0427. In some embodiments, an effective amount of a 45%, or at least about 50%, or more, compared to the basal compound of one of Formulas I-X is an amount that, in level of lung function in the individual prior to the mono monotherapy or combination therapy, when administered to therapy or combination therapy or compared to the rate of an individual having pulmonary fibrosis, is effective to deterioration in lung function that would have been experi increase progression-free Survival time, e.g., the time from enced by the individual in the absence of the monotherapy or baseline (e.g., a time point from 1 day to 28 days before combination therapy. The severity of the disease, and its beginning of treatment) to death or disease progression is response to treatment, may be assessed by pulmonary func increased by at least about 10%, at least about 20%, at least tion testing, e.g., spirometry, to assess lung Volumes, compli about 25%, at least about 30%, at least about 40%, at least ance, and gas diffusion; by imaging methods, such as com about 50%, at least about 60%, at least about 70%, at least puterized tomography, to assess lung Volumes and fibrosis; by about 80%, at least about 90%, at least about 2-fold, at least functional testing Such as treadmill exercise testing; by Sur about 3-fold, at least about 4-fold, at least about 5-fold, or vival free of hospitalization; or by extension of life. more, compared a placebo-treated or an untreated control individual. Thus, e.g., in some embodiments effective Idiopathic Pulmonary Fibrosis amounts are any dosages that is effective to increase the 0421. The present invention disclosure provides methods progression-free Survival time by at least about 1 week, at of treating idiopathic pulmonary fibrosis (IPF). The methods least about 2 weeks, at least about 3 weeks, at least about 4 generally involve administering to an individual in need weeks, at least about 2 months, at least about 3 months, at thereof a compound of one of Formulas I-X. least about 4 months, at least about 5 months, at least about 6 0422. In some embodiments, a diagnosis of IPF is con months, at least about 8 months, at least about 10 months, at firmed by the finding of usual interstitial pneumonia (UIP) on least about 12 months, at least about 18 months, at least about histopathological evaluation of lung tissue obtained by Sur 2 years, at least about 3 years, or longer, compared to a gical biopsy. The criteria for a diagnosis of IPF are known. placebo-treated or untreated control. Ryu et al. (1998) Mayo Clin. Proc. 73:1085-1101. 0428. In some embodiments, an effective amount of a 0423. In other embodiments, a diagnosis of IPF is a defi compound of one of Formulas I-X is an amount that, in nite or probable IPF made by high resolution computer monotherapy or combination therapy, when administered to tomography (HRCT). In a diagnosis by HRCT, the presence an individual having pulmonary fibrosis, is effective to of the following characteristics is noted: (1) presence of increase at least one parameter of lung function, e.g., an reticular abnormality and/or traction bronchiectasis with effective amount of a compound of any one of Formulas I-X basal and peripheral predominance; (2) presence of honey is any dosage that increases at least one parameter of lung combing with basal and peripheral predominance; and (3) function by at least about 10%, at least about 20%, at least absence of atypical features Such as micronodules, peribron about 25%, at least about 30%, at least about 40%, at least chovascular nodules, consolidation, isolated (non-honey about 50%, at least about 60%, at least about 70%, at least comb) cysts, ground glass attenuation (or, if present, is less about 80%, at least about 90%, at least about 2-fold, at least US 2013/0224259 A1 Aug. 29, 2013 32 about 3-fold, at least about 4-fold, at least about 5-fold, or 0435. In some cases, a subject method of treating sepsis more, compared to an untreated individual or a placebo comprises administering to an individual in need thereofan treated control individual. In some of these embodiments, a effective amount of a compound of one of Formulas I-X; and determination of whether a parameter of lung function is administering a second therapeutic agent, e.g., an antibiotic. increased is made by comparing the baseline value with the value at any time point after the beginning of treatment, e.g., Individuals Suitable for Treatment 48 weeks after the beginning of treatment, or between two time points, e.g., about 4 to about 14 weeks apart, after the 0436 Individuals suitable for treatment with a subject beginning of treatment. method include individuals who have been diagnosed as hav 0429. In some embodiments, an effective amount of a ing a disorder characterized by excessive NO production compound of one of Formulas I-X is an amount that, in and/or elevated DDAH activity, where such individuals monotherapy or combination therapy, when administered to include, e.g., individuals who have been diagnosed as having an individual having pulmonary fibrosis, is effective to fibrosis, individuals who have been diagnosed as having sep increase the FVC by at least about 10% at least about 20%, at sis, etc. In some cases, individuals who have gastritis or least about 25%, at least about 30%, at least about 40%, at gastric ulcer are specifically excluded. least about 50%, at least about 60%, at least about 70%, at 0437. In some cases, the individual is a human. In other least about 80%, at least about 90%, at least about 2-fold, at instances, the individual is a non-human mammal, e.g., a least about 3-fold, at least about 4-fold, at least about 5-fold, canine, a feline, a rodent (e.g., a mouse; a rat), a non-human or more compared to baseline on two consecutive occasions 4 primate, an ungulate, etc. As one non-limiting example, in to 14 weeks apart. Some cases, the non-human mammal is a canine, e.g., a dog 0430. In some of these embodiments, an effective amount that has IPF, e.g., a terrier such as a West Highland white of a compound of one of Formulas I-X is an amount that, in terrier. Webb and Armstrong (2002) Can. Vet. 43.703. monotherapy or combination therapy, when administered to an individual having pulmonary fibrosis, results in a decrease Screening Methods in alveolar:arterial (A-a) gradient of at least about 2 mm. Hg, 0438. The present disclosure provides methods for identi at least about 7 mm Hg, at least about 10 mmHg, at least about fying an agent that inhibits enzymatic activity of DDAH. The 12 mm Hg, at least about 15 mm Hg, or more, compared to methods generally involve contacting a DDAH polypeptide baseline. with a DDAH substrate and a test agent; and determining the 0431. In some of these embodiments, an effective amount effect, if any, of the test agent on DDAH activity. Determining of a compound of one of Formulas I-X is an amount that, in the effect of the test agent on DDAH activity involves detect monotherapy or combination therapy, when administered to ing the product of action of DDAH on the DDAH substrate. A an individual having pulmonary fibrosis, increases the single test agent that reduces the amount of product produced, com breath DL by at least about 15%, at least about 20%, at least pared to the amount of product produced in the absence of the about 30%, at least about 40%, at least about 50%, at least test agent, is considered a DDAH inhibitor. Test agents that about 60%, at least about 70%, at least about 80%, at least are DDAH inhibitors are candidate agents for treating a dis about 90%, at least about 2-fold, at least about 3-fold, at least ease associated with excessive NO production and/or about 4-fold, at least about 5-fold, or more, compared to elevated DDAH activity. In some cases, a subject Screening baseline. DL is the lung diffusing capacity for carbon mon method can be used to exclude a test agent from further oxide, and is expressed as mL CO/mm Hg/second. development when DDAH inhibition is not a desired effect. 0432 Parameters of lung function include, but are not 0439. As used herein, the term “determining refers to limited to, forced vital capacity (FVC); forced expiratory both quantitative and qualitative determinations and as such, Volume (FEV); total lung capacity; partial pressure of arte the term “determining is used interchangeably herein with rial oxygen at rest; partial pressure of arterial oxygen at maxi 'assaying.” “measuring and the like. mal exertion. Lung function can be measured using any 0440 The terms “candidate agent,” “test agent,” “agent.” known method, including, but not limited to, spirometry. “substance.” and “compound are used interchangeably herein. Candidate agents encompass numerous chemical Sepsis classes, typically synthetic, semi-synthetic, or naturally-oc 0433. The present disclosure provides methods for treat curring inorganic or organic molecules. Candidate agents ing sepsis. The methods generally involve administering to an include those found in large libraries of synthetic or natural individual in need thereofan effective amount of a compound compounds. For example, synthetic compound libraries are of one of Formulas I-X. commercially available from Maybridge Chemical Co. (Tre 0434. In some embodiments, an effective amount of a villet, Cornwall, UK), ComGenex (South San Francisco, compound of one of Formulas I-X is an amount that is effec Calif.), and MicroSource (New Milford, Conn.). A rare tive to reduce an adverse symptom of sepsis. For example, in chemical library is available from Aldrich (Milwaukee, Wis.). Some embodiments, an effective amount of a compound of Alternatively, libraries of natural compounds in the form of one of Formulas I-X is an amount that is effective to reduce bacterial, fungal, plant and animal extracts are available from the use of pressor agents needed to reverse the hypotension Pan Labs (Bothell, Wash.) or are readily producible. associated with sepsis by at least about 5%, at least about 0441 Candidate agents may be small organic or inorganic 20%, at least about 30%, at least about 40%, at least about compounds having a molecular weight of more than 50 and 50%, at least about 60%, at least about 70%, at least about less than about 10,000 daltons, e.g., a candidate agent may 80%, or more than 80%, compared to the use of pressor agents have a molecular weight of from about 50 daltons to about before treatment with the compound. Pressor agents (vaso 100 daltons, from about 100 daltons to about 150 daltons, pressors) include, e.g., epinephrine, isoproterenol, norepi from about 150 daltons to about 200 daltons, from about 200 nephrine, and the like. daltons to about 500 daltons, from about 500 daltons to about US 2013/0224259 A1 Aug. 29, 2013

1000 daltons, from about 1,000 daltons to about 2500 daltons, Dimethyl-1-phenyl-3-pyrazolin-5-one) and 2,3-butanedione from about 2500 daltons to about 5000 daltons, from about Oxime. For example, DDAH (e.g., recombinant human 5000 daltons to about 7500 daltons, or from about 7500 DDAH1) is mixed with ADMA in the presence of screening daltons to about 10,000 daltons. Candidate agents may com buffer in 384-well plates. Plates are incubated at 37° C. for 4 prise functional groups necessary for structural interaction hours. Subsequently, color developing reagent (containing 2 with proteins, particularly hydrogen bonding, and may volumes of antipyrine and 1 volume of 2,3-Butanedione include at least an amine, carbonyl, hydroxyl or carboxyl oxime reagents) are added and the plates are incubated at 60° group, and may contain at least two of the functional chemical C. for 90 min prior to spinning them at 1,500 rpm for 5 min. groups. The candidate agents may comprise cyclical carbon In this assay, absorbance is proportional to the concentration or heterocyclic structures and/or aromatic or polyaromatic of citrulline generated by DDAH. structures substituted with one or more of the above func 0447. As another example, an assay to determine DDAH tional groups. Candidate agents are also found among bio enzymatic activity is a fluorimetric assay. For example, molecules including peptides, Saccharides, fatty acids, Ste DDAH is mixed with the artificial substrate S-methyl-thio roids, purines, pyrimidines, derivatives, structural analogs or citrulline (SMTC) and a test agent; and the effect of the test combinations thereof. agent on DDAH activity is determined by measuring the 0442 Assays of the invention include controls, where suit amount of methanethiol produced. DDAH metabolizes able controls include a sample (e.g., a sample comprising the SMTC into L-citrulline and methanethiol (CH, SH). The DDAH polypeptide and the DDAH substrate in the absence of thiol released from the reaction can be monitored fluorimetri the test agent). Generally a plurality of assay mixtures is run cally by adding 7-Diethylamino-3-(4-maleimidophenyl)-4- in parallel with different agent concentrations to obtain a methylcoumarin (CPM). The effect of small molecules that differential response to the various concentrations. Typically, directly regulate DDAH activity can be monitored by com one of these concentrations serves as a negative control, i.e. at paring their fluorescence readout with that of no compound Zero concentration or below the level of detection. addition (control; vehicle). 0443) A variety of other reagents may be included in the 0448. A candidate agent can be assessed for any cytotoxic screening assay. These include reagents like salts, neutral activity it may exhibit toward a living cell, using well-known proteins, e.g. albumin, detergents, etc., including agents that assays, such as trypan blue dye exclusion, an MTT (3-(4.5- are used to facilitate optimal enzyme activity and/or reduce dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bro non-specific or background activity. Reagents that improve mide) assay, and the like. Agents that do not exhibit cytotoxic the efficiency of the assay, such as protease inhibitors, anti activity are considered candidate agents. microbial agents, etc. may be used. The components of the 0449 In many embodiments, the screening method is car assay mixture are added in any order that provides for the ried out in vitro, in a cell-free assay. In some embodiments, requisite activity. Incubations are performed at any suitable the in vitro cell-free assay will employ a purified DDAH temperature, typically between 4°C. and 40°C. Incubation polypeptide, where “purified’ refers to free of contaminants periods are selected for optimum activity, but may also be or any other undesired components. Purified DDAH polypep optimized to facilitate rapid high-throughput screening. Typi tide that is suitable for a Subject Screening method is at least cally between 0.1 hour and 1 hour will be sufficient. about 50% pure, at least about 60% pure, at least about 70% 0444. In some embodiments, a test compound of interest pure, at least about 75% pure, at least about 80% pure, at least has an ICs of from about 1 nM to about 1 mM, e.g., from about 85% pure, at least about 90% pure, at least about 95% about 1 nM to about 10 nM, from about 10 nM to about 15 pure, at least about 98% pure, at least about 99% pure, or nM, from about 15 nM to about 25 nM, from about 25 nM to greater than 99% pure. about 50 nM, from about 50 nM to about 75 nM, from about 0450 ADDAH polypeptide suitable for use in a subject 75 nM to about 100 nM, from about 100 nM to about 150 nM, screening method can comprise an amino acid sequence hav from about 150 nM to about 200 nM, from about 200 nM to ing at least about 85%, at least about 90%, at least about 95%, about 250 nM, from about 250 nM to about 300 nM, from at least about 98%, at least about 99%, or 100%, amino acid about 300 nM to about 350 nM, from about 350 nM to about sequence identity to a contiguous stretch of from about 250 400 nM, from about 400 nM to about 450 nM, from about 450 amino acids to 285 amino acids of the amino acid sequence of nM to about 500 nM, from about 500 nM to about 750 nM, a DDAH polypeptide as depicted in FIG. 15. from about 750 nM to about from about 1 uM to about 10 uM, 0451 A DDAH polypeptide is readily prepared in a vari from about 10 uM to about 25uM, from about 25uM to about ety of host cells Such as unicellular microorganisms, or cells 50 uM, from about 50 uM to about 75uM, from about 75uM of multicellular organisms grown in in vitro culture as uni to about 100 uM, from about 100 uM to about 250 uM, from cellular entities. Suitable host cells include bacterial cells about 250 uM to about 500 uM, or from about 500 uM to Such as Escherichia Coli; yeast cells such as Saccharomyces about 1 mM. cerevisiae, Pichia pastoris, Hansenula polymorpha, 0445 Enzymatic activity of DDAH can be determined Kluyveromyces lactis, Yarrowia lipolytica, Candida utilis, using any known assay. Colorimetric assays and fluorimetric Schizosaccharomyces pombe, and the like; insect cells Such assays can be used, depending on the nature of the product as Drosophila melanogaster cells; amphibian cells Such as produced by action of DDAH on the DDAH substrate. The Xenopus cells; mammalian cells, such as CHO cells, 3T3 following are exemplary, non-limiting assays. cells, and the like. 0446. In some cases, an assay to determine DDAH enzy 0452. In some embodiments, the in vitro cell-free assay matic activity is a colorimetric assay that detects product will employ a fusion protein, comprising a DDAH polypep (L-citrulline) formation from the substrate ADMA. DDAH tide fused in-frame to a fusion partner. In some embodiments, can be mixed with ADMA and a test agent; and the effect of the fusion partner is attached to the amino terminus of the the test agent on DDAH activity is determined by measuring DDAH polypeptide. In other embodiments, the fusion partner the amount of L-citrulline produced, using antipyrine (2,3- is attached to the carboxyl terminus of the DDAH polypep US 2013/0224259 A1 Aug. 29, 2013 34 tide. In other embodiments, the fusion partner is fused in Example 1 frame to the DDAH polypeptide at a location internal to the DDAH polypeptide. Suitable fusion partners include immu Compounds of Formula I as Novel Class of nological tags such as epitope tags, including, but not limited Dimethylarginine Dimethylaminohydrolase (DDAH) to, hemagglutinin, FLAG, and the like; proteins that provide Inhibitors for a detectable signal, including, but not limited to, fluores cent proteins, enzymes (e.g., B-galactosidase, luciferase, 0457. A high throughput screen was conducted using the horse radish peroxidase, etc.), and the like; polypeptides that Stanford High Throughput Bioscience Center (HTBC) facilitate purification or isolation of the fusion protein, e.g., chemical library to search for modulators of Dimethylargin metal ion binding polypeptides Such as 6H is tags (e.g., ine dimethylaminohydrolase (DDAH) activity. DDAH/6His), glutathione-S-transferase, and the like: polypeptides that provide for subcellular localization; and Materials and Methods polypeptides that provide for secretion from a cell. Measurement of DDAH Activity: 0453. In some embodiments, the fusion partner is an epitope tag. In some embodiments, the fusion partner is a 0458. The enzymatic activity of DDAH was monitored metal chelating peptide. In some embodiments, the metal using a colorimetric assay that detects product (L-citrulline) chelating peptide is a histidine multimer, e.g., (His). In some formation from the substrate ADMA. In brief, recombinant embodiments, a (His) multimer is fused to the amino termi human DDAH1 (rhDDAH1) was mixed with ADMA in the nus of a DDAH polypeptide; in other embodiments, a (His) presence of screening buffer in 384-well plates. Plates were multimer is fused to the carboxyl terminus of a DDAH incubated at 37°C. for 4 hours. Subsequently, color develop polypeptide. The (His)-DDAH fusion protein is purified ing reagent (containing 2 Volumes of Antipyrine and 1 Vol using any of a variety of available nickel affinity columns (e.g. ume of 2,3-Butanedione oxime reagents) was added and the His-bind resin, Novagen). plates were incubated at 60° C. for 90 min prior to spinning 0454. In some embodiments, a subject screening method them at 1,500 rpm for 5 min. In this assay absorbance is is carried out in vitro in a cell, e.g., a cell grown in cell culture proportional to the concentration of citrulline generated by as a unicellular entity. Suitable cells include, e.g., eukaryotic DDAH, and was measured using an AnalystGT plate reader at cells, e.g., mammalian cells such as human umbilical vein 485 nm using a dichroic beamsplitter. endothelial cells (HUVEC; e.g., AmericanType Culture Col Orthogonal Assay to Measure DDAH Activity: lection (ATCC)CRL-1730), human microvascular endothe lial cells (HMEC-1; ATCC CRL-4025), PC3 cells (ATCC 0459. The activity of human DDAH1 was quantified using CRL1435), MDA-MB-231 cells (ATCC HTB26), MCF-7 fluorimetric assay by incubating the enzyme with an artificial cells (ATCC HTB22), HeLa cells (ATCC No. CCL-2), CHO substrate S-methyl-thiocitrulline (SMTC). DDAH metabo cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 lizes SMTC into L-citrulline and methanethiol (CH, SH). cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells The thiol released from the reaction can be monitored fluo (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., rimetrically by adding 7-Diethylamino-3-(4-maleimidophe ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS nyl)-4-methylcoumarin (CPM). The effect of small mol cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse ecules that directly regulate DDAH activity can be monitored L cells (ATCC No. CCLI.3), human embryonic kidney by comparing their fluorescence (Top Read, Ex 360-35, Em (HEK) cells (ATCC No. CRL1573), and the like. 460-40, Dichroic 425) readout with that of no compound 0455. In some cases, a cell in vitro is contacted with a test addition (vehicle). agent; and the effect of the test agent is determined by assay ing the effect of the test agent on DDAH activity in lysates Results made from the cells. 0460. The high throughput based search for new chemical entities (NCEs) that regulate DDAH activity revealed several EXAMPLES novel small molecules that are potent inhibitors of DDAH in a dose-dependent fashion. Among the DDAH inhibitors, four 0456. The following examples are put forth so as to pro members of the proton pump inhibitors (PPIs) were discov vide those of ordinary skill in the art with a complete disclo ered: Omeprazole (identified in 3 independent screens as Sure and description of how to make and use the present STF-006355 in Biomol FDA (BMF)-6 with ICso-61.4 uM), invention, and are not intended to limit the scope of what the BMF-7 (IC.s 20 uM) and in MicroSource-2 as STF-006515 inventors regard as their invention nor are they intended to (ICso-85 uM); Pantoprazole (identified in BMF-7 as STF represent that the experiments below are all or the only 006577 (ICs-63 uM); Lansoprazole (identified in experiments performed. Efforts have been made to ensure MicroSource-10 as STF-001701 (ICs51 uM) and Tenato accuracy with respect to numbers used (e.g. amounts, tem prazole identified in BMF-8 as STF-008450 (ICs—7.8 uM). perature, etc.) but some experimental errors and deviations The dose-dependent inhibition by each of the PPI inhibitors is should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecu shown in FIG. 1. lar weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, Compounds for Further Validation: e.g., bp, base pair(s): kb, kilobase(s); pl. picoliter(s); S or sec, 0461 Validation studies were performed by obtaining second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s): fresh powders of each of the compounds directly from ven kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intra dors and preparing them in a different location and indepen muscular(ly); i.p., intraperitoneal(ly); s.c., Subcutaneous(ly); dent of the tools used to initially perform the HTS screen. and the like. Stocks of each of the compounds were made using DMSO US 2013/0224259 A1 Aug. 29, 2013

that has never been stored in the HTS facility and the prepared fluency. The cells were then washed with phosphate buffered reagents were stored in freezers other than where the HTS saline (PBS) and plain DMEM (DMEM with no addition of screen compounds are kept. serum or the other Supplements) was mixed with esomepra Zole (20 uM final concentration from 100 mM stock) and Validation: added to the cells. In parallel, cells were also treated with 0462 Compounds were validated for their direct effect on vehicle (equal volume of dimethylsulfoxide (DMSO) or a DDAH activity using the fluorometric assay described above. known DDAH inhibitor (L-257; Leiper J et al; Nature Med The validation study has confirmed the ability of the PPIs to 2007: 13(2): 198-203). After ~4 hrs of incubation, the media directly inhibit DDAH activity (FIG. 2). As shown in FIG. 2, was aspirated from each flask and the cells were cultured in five of six PPIs inhibited DDAH significantly (p<0.05). fully supplemented DMEM as described above and incubated Ebselen was used as a positive assay control. The data shown at 37° C./5% CO, for another 20 hrs. The serum was initially is from triplicate experiments (Mean+/-SEM). In parallel, withdrawn due to the possibility that esomeprazole would the compounds were also cross-validated to rule out the pos interact with CyS containing serum proteins and fail to inter sibility that their apparent activity is caused by non-specific nalize at effective concentrations. reaction quenching. 0469. After 24 hrs, the cells were harvested. Total cellular protein was estimated as described and ADMA was measured Testing of Additional Members of PPI Class: by enzyme-linked immunosorbent assay (ELISA) following the manufacturer's recommendations; DLD Diagnostika, 0463. An additional two compounds (Esomeprazole and Hamburg, Germany). Rabeprazole) were obtained as powders and tested for their 0470 The ADMA study demonstrated that esomeprazole effect on DDAH activity as described above. Both esomepra increased intracellular ADMA (by ~37%) compared to Zole and rabeprazole also significantly inhibited DDAH vehicle control. L-257 (a known DDAH inhibitor; Leiper Jet activity (FIG. 3). al; Nat. Med. 2007 February: 13(2):198-203.) also increased Cross Validation using an Orthogonal Assay: ADMA by 68% (FIG. 5, data are Mean+/-SEM from dupli 0464 Validation study using the CPM assay confirmed cates). In FIG. 5, endothelial cells were treated wtiht eh that all the six members of the PPI class (tenatoprazole, indicated small molecules (20 uM) or vehicle for 24 hours. omeprazole, pantoprazole, lansoprazole, esomeprazole and These data indicate that PPIs (esomeprazole) may inhibit rabeprazole) inhibited DDAH activity significantly (p<0.05). DDAH activity in mammalian cells. To further confirm these data, the above described orthogonal colorimetric assay was used to independently test the effect of Example 2 these PPIs on DDAH activity. Interestingly, all the 6 PPIs significantly inhibited DDAH activity (p<0.0001; see FIG.3) PD 404, 182 as a Novel and Potent Inhibitor of and reduced the production of L-citrulline substantially (p<0. dimethylarginine dimethylaminohydrolase (DDAH) 0001; see FIG. 4). In the results shown in FIG.3, Ebselen was used as a positive assay control. The data presented in FIG. 3 0471. In the high throughput screening of the Stanford are from triplicate experiments (Mean+/-SEM). chemical library, it was discovered that the small molecule 0465. The data in FIG. 4 show that production of L-citrul PD 404, 182 (CAS #72596-74-8) is a very potent inhibitor of line from ADMA was significantly reduced (p<0.05) when human DDAH1 (50% inhibitory concentration=ICso-8.714 DDAH was pre-incubated with each of the six PPIs. In FIG.4, uM). This compound was deposited in the Library of Phar L-citrulline concentration was calculated from a standard macologically Active Compounds (LOPAC) library obtained curve. Data are from triplicate experiments (Mean-SEM). by Stanford High Throughput Bioscience Center (HTBC) 0466. The discovery and validation of four members of the from Sigma and coded as STF-001938. As described below, PPIs as direct inhibitors of DDAH activity in independent PD-404 directly inhibits DDAH activity in a dose-dependent screening efforts and in more than one library indicates that manner (FIG. 6). the class of proton pump inhibitors might be acting on human DDAH1 through similar mechanism possibly by interacting Measurement of DDAH Activity: with the active site cysteine (Cys 273). This finding is 0472. The enzymatic activity of DDAH was monitored strengthened by the discovery of two additional members of using a colorimetric assay that detects product (L-citrulline) the PPI class as significant inhibitors of DDAH activity and formation from the substrate ADMA, as described in by the cross-validation of the findings using an orthogonal Example 1. In brief, recombinant human DDAH1 (rhD assay. DAH1) was mixed with ADMA in the presence of screening buffer in 384-well plates. Plates were incubated at 37° C. for Validation of Proton Pump Inhibitors (PPIs) as DDAH 4 hours. Subsequently, color developing reagent (containing Inhibitors in Cell-based Assay: 2 volumes of antipyrine and 1 volume of 2,3-Butanedione Oxime reagents) was added and the plates were incubated at 0467. After validating the inhibition of DDAH activity by 60° C. for 90 min prior to spinning them at 1,500 rpm for 5 members of the PPI in vitro using a primary and an orthogonal min. In this assay absorbance is proportional to the concen assay, esomeprazole (Nexium) was taken as a prototype to tration of citrulline generated by DDAH, and was measured study its effect on intracellular ADMA. using an AnalystGT plate reader at 485 nm using a dichroic 0468 First, human microvascular endothelial cells (HMEC-1; ATCC) were seeded in 75 cm cell culture flasks beamsplitter. and cultured in fully supplemented DMEM (Invitrogen cat Orthogonal Assay to Measure DDAH Activity: #11995; supplemented with 10% fetal bovine serum (FBS), 4 mM HEPES: GIBCO 15630 and penicillin/streptomycin). 0473. The activity of human DDAH1 was quantified using The cells were incubated at 37° C./5% CO, until -60% con fluorimetric assay by incubating the enzyme with an artificial US 2013/0224259 A1 Aug. 29, 2013 36 substrate S-methyl-thiocitrulline (SMTC), as described in flask and the cells were cultured in fully supplemented Example 1. DDAH metabolizes SMTC into L-citrulline and DMEM as described above and incubated at 37° C./5% CO, methanethiol (CH, SH). The thiol released from the reac for another 20 hrs. The serum was initially withdrawn due to tion can be monitored fluorimetrically by adding 7-Diethy the possibility that PD 404 would interact with Cys contain lamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). ing serum proteins and fail to internalize at effective concen The effect of small molecules that directly regulate DDAH trations. A recent study has demonstrated that PD 404 loses activity can be monitored by comparing their fluorescence Some of its bioactivity upon interaction with human serum (Top Read, Ex 360-35, Em 460-40, Dichroic 425) readout (Chamoun A et al. Antimicrob. Agents Chemotherapy. doi: with that of no compound addition (vehicle). 10.1128/AAC.05722-11). 0480. After 24 hours, the cells were harvested. Total cel Results: lular protein was estimated as described and ADMA was measured by ELISA following the manufacturer's recom 0474 The high throughput-based search for new chemical mendations; DLD Diagnostika, Hamburg, Germany). entities (NCEs) that regulate DDAH activity revealed several 0481 Result: novel small molecules that are potent inhibitors of DDAH in 0482. The ADMA study demonstrated that PD 404 (20 a dose-dependent fashion. Among the DDAH inhibitors, PD uM) significantly increased intracellular ADMA (by -84%) 404 was discovered (Stanford ID #: STF-001938) as shown in compared to vehicle control. L-257 (20 uM) also increased FIG. 6 (ICs-8.714 uM). ADMA by 68% (FIG. 9, data are Mean+/-SEM from dupli cates) compared to vehicle control. Endothelial cells were Compound for Further Validation: treated with the indicated small molecules (20 uM) or vehicle 0475 Validation study was performed by obtaining fresh for 24 hours. These data indicate that PD 404 inhibits DDAH powder of PD 404 directly from Sigma (Cat #P2742 at >98% activity in mammalian cells. high performance liquid chromatography (HPLC) purity)and preparing it in a different location and independent of the Example 3 tools used to initially perform the HTS screen. Stock concen tration was made using DMSO that has never been stored in Development of a Dimethylarginine the HTS facility and the prepared reagent was stored in a Dimethylaminohydrolase (DDAH) Assay for High freezer other than where the HTS screen compounds are kept. Throughput Chemical Screening Validation: Materials and Methods 0476 PD 404 was validated for its direct effect on DDAH 0483 The chemical library of the Stanford High Through activity using the fluorometric assay described above. The put Bioscience Center (HTBC) contains over 130,000 small validation study has confirmed the ability of PD 404 (50 uM) molecules selected from diverse sources including Sigma, to directly inhibit DDAH activity (FIG. 7, data is Mean+/- ChemDiv, MicroSource, ChemBridge, the NIH clinical col SEM from triplicate experiments using CPM fluorometric lection (NIH CC), National Cancer Institute (NCI), natural assay), compared to vehicle control. In parallel, the com products and FDA approved drug libraries using stringent pound was also cross-validated to rule out the possibility that criteria to maximize diversity and medicinal drug-like prop its apparent activity is caused by non-specific reaction erties (http://htbc(dot)Stanford(dot)edu/). The orthogonal quenching. assays, using hits derived from the HTBC, were conducted Cross Validation using an Orthogonal Assay: using chemicals purchased from Sigma-Aldrich (St. Louis, 0477 Validation study using the CPM assay confirmed Mo.) unless indicated otherwise. For generation of DDAH that PD 404 inhibited DDAH activity significantly (p<0.05). protein, E. coli BL21 strain (Invitrogen) was used. The plas To further confirm these data, the above described orthogonal mid construct pGEX-6P-1-DDAH1 was used. Empty vector colorimetric assay was used to independently test the effect of control, enzyme purification and cleavage reagents were from this compound on DDAH activity. Interestingly, PD 404 (50 GE Healthcare (Piscataway, N.J.). Clear and black 384-well uM) significantly inhibited DDAH activity (p<0.0001; FIG. plates were from E&K Scientific (Santa Clara, Calif.). Anti 8, data is Mean+/-SEM from triplicate experiments), com bodies directed against DDAH-1 (Abcam: Cambridge, pared to vehicle control. Mass.) and GST (GE Healthcare) were obtained from com mercial purveyors. Validation of PD 404 as DDAH Inhibitorin Cell-based Assay: Production of Recombinant Human DDAH1: 0478 After validating the inhibition of DDAH activity by PD 404 in vitro using a primary and an orthogonal assay, its 0484 Human DDAH1 was expressed in E. coli BL21 Star effect on intracellular ADMA was studied. (DE3) strain for protein production. In parallel, cells were 0479 First, human microvascular endothelial cells also transformed with empty vector. Positive clones were (HMEC-1; ATCC) were seeded in 75 cm cell culture flasks selected by polymerase chain reaction (PCR) and the clones and cultured in fully supplemented DMEM (Invitrogen cat harboring DDAH were subsequently inoculated into LB #11995; supplemented with 10% FBS, 4 mM HEPES: broth. Bacteria were grown at 37°C. (225 rpm) for 36 hours GIBCO 15630 and penicillin/streptomycin). The cells were and preinduction samples were removed prior to inducing the incubated at 37° C./5% CO, until -60% confluency. The cells remaining culture by adding isopropyl-beta-D-thiogalacto were then washed with PBS and plain DMEM (DMEM with pyranoside (IPTG; 0.1 mM final concentration) at 25°C. for no addition of serum or the other Supplements) was mixed 18 hours. The cells were harvested by centrifugation and the with PD 404 (20 uM final conc from 100 mM stock) and Supernatant was discarded prior to lysing them with cell dis added to the cells. In parallel, cells were also treated with ruption buffer (containing 20 mM Tris-HCl; pH 8.0: 150 mM vehicle (equal volume of DMSO) or a known DDAH inhibi NaCl; 2 mM f-mercaptoethanol; 1 mM phenylmethylsulfo tor (L-257; LeiperJetal; Nature Med 2007: 13(2): 198-203). nyl fluoride (PMSF); 1 mM benzamidine and 10 mM DNAse After ~4hrs of incubation, the media was aspirated from each 1) and with 1% triton X-100 and lysozyme to break the US 2013/0224259 A1 Aug. 29, 2013 37 peptidoglycan layer. The lysate was centrifuged at 20,000 g SMTC into L-citrulline and methanethiol (CH, SH). In for 40 min at 4°C. and the supernatant was transferred into brief, DDAH (30 nM final concentration) was mixed with clean tubes for SDS-PAGE and Western analyses. The protein SMTC (100 uM final concentration), CPM (50 uM final con was purified using Glutathione Sepharose 4B column in a centration) and screening buffer (containing a final concen batch mode according to the manufacturers recommenda tration of 0.01% Triton-X100 and 1 mM EDTA). The reaction tions. The GST-tag was cleaved off the recombinant protein mix was added to black 384-well plates to validate primary using Precision Protease. The purified protein was eluted, hits that modulate DDAH activity. The release of CH, SH SDS-PAGE analyzed, and its identity was confirmed by West was monitored fluorimetrically by adding 7-Diethylamino-3- ern and Mass Spectroscopy. (4-maleimidophenyl)-4-methylcoumarin (CPM) aS described in Linsky (Linsky, T. & Fast, W. A continuous, DDAH Activity Assay: fluorescent, high-throughput assay for human dimethylargi 0485 The L-citrulline assay was based upon an original nine dimethylaminohydrolase-1. J Biomol Screen 16, 1089 test-tube method developed by Prescott and Jones in 1969 1097 (2011)). (Prescott, L. M. & Jones, M. E. Modified methods for the determination of carbamylaspartate. Anal Biochem 32, 408 Application of the L-Citrulline Assay to Cells: 419 (1969)), which was adapted and optimized for a micro 0489. The feasibility of the citrulline assay in cell culture plate format. Subsequently, the activity of DDAH was quan was validated by measuring the levels of L-citrulline in cell tified by detecting its conversion of ADMA to citrulline using lysates applying a protocol similar to that described above. the optimized protocol. The assay was scaled up to a 384-well First, primary microvascular endothelial cells (HMVECs) format for high throughput chemical screening. were seeded in 75 cm cell-culture flasks until confluency. The cells were exposed to 1 mM of L-Arginine for 24 hours in High Throughput Screening of Small Molecules: the presence of vehicle control. The cells were washed with 0486) Over 130,000 small molecules deposited in the PBS and then dissociated with 3 mL of Accutase for 3 minutes Stanford High-throughput Bioscience Center (HTBC) were at 37° C./5% CO. The cells were then pelleted down by screened using the enzymatic assay to identify chemicals that centrifugation and lysed by adding lysis buffer (containing regulate DDAH activity. In brief, recombinant human 100 mMNaHPO4; 1% NP-40; 1x protease and phosphatase DDAH1 (rhDDAH1) was mixed with ADMA in the presence inhibitors). The suspension was kept on ice for 30 minutes of screening buffer in 384-well plates using a Staccato mul prior to centrifugation at 13,000 rpm for 30 minutes at 4°C. tidrop. Small molecules (100 nL each) were then added to the Finally, the cell debris was removed and the supernatant was wells using a robotic arrayer to yield a final compound screen collected for the citruline assay. The citrulline assay was ing concentration of up to 50 uM. Plates were incubated at 37° performed in a microplate assay as described above by trans C. for 4 hours. Subsequently, color developing reagent (con ferring equimolar amounts of cell lysate and adding 0.5 Vol taining 2 volumes of Antipyrine and 1 Volume of 2,3-Butane ume of color-developing reagent. The mix was incubated at dione oxime reagents) was added using Velocity 11 system 60° C. for 90 minutes and absorbance was measured as and the plates were sealed using an automated plate sealer. described above. Known concentrations of commercial cit Finally, color was developed by incubating the plates at 60° C. rulline were used to construct standard curves and to estimate for 90 minutes prior to spinning them at 1,500 rpm for 5 the concentration of citrulline in the samples. minutes. In this assay absorbance is proportional to the con centration of citruline generated by DDAH, and was mea Results Sured using an AnalystGT plate reader at 485 nm using a dichroic beamsplitter. The signal-to-noise ratio of separation 0490. A human DDAH-1 (858 bp)-encoding plasmid was was calculated using an established formula in Zhang. Successfully transformed into an E. coli system and the poly (Zhang, J. H., Chung, T. D. & Oldenburg, K. R. A Simple merase chain reaction (PCR)-positive clones were used for Statistical Parameter for Use in Evaluation and Validation of the production of rhDDAH1. Westernblot analysis using both High Throughput Screening Assays. J Biomol Screen 4. anti-DDAH1 and anti-glutathione-S-transferase (GST) anti 67-73 (1999)). bodies confirmed that rhDDAH1 tagged with GST (56.5kDa) was expressed only in the cells transformed with the vector Identification of Primary Hits: encoding DDAH and induced by IPTG (FIG.10). In FIG. 10, Western blot analysis shows the production of GST-DDAH 0487. Inhibitors were defined as compounds that reduce (56.5 kDa). In FIG. 10, lanes 1.3: pre-induction; lane-2: post absorbance by at least 30% compared to control wells. The induction sample of an empty vector, lane 4: isopropyl B-D- hits were validated using 8-point full dose response study (50 1-thiogalactopyranoside (IPTG) induction of DDAH vector. uM to 0.39 uM in serial dilutions). A total of over 150 com Lane-M is SeeBlue Plus molecular weight marker. pounds, about 0.12% of the total compounds, caused a reduc 0491 Sepharose column purification and subsequent tion in absorbance of at least 30%. To determine which of Sodium dodecyl Sulfate-polyacrylamide gel electrophoresis these hits were true inhibitors of DDAH activity, a modifica (SDS-PAGE) (FIG. 11A) and Western blot analyses (FIG. tion of a validated secondary fluorometric assay was used, as 11B) revealed that the protein can be purified to a single band described below. In parallel, compounds were also cross for use in screening experiments. FIG. 11A shows SDS validated by adding them in reaction mix containing all the PAGE analysis of purified human DDAH1. In FIG. 11A, components described above with the exception of the PI pre-induction; LY=lysate; FT=flow through; W1 wash; enzyme to rule out the possibility that their apparent activity E=eluent and M=1 kb-marker. FIG. 11B shows Western blot is caused by non-specific reaction quenching and not directly showing purified (after GST cleavage) recombinant human inhibiting DDAH. DDAH1 (-37 kDa). In FIG. 11B, GIPurified fractions of DDAH1 (lanes 1-9) were probed using anti-DDAH1 anti Secondary Assay to Validate Potential DDAH Inhibitors: body. Despite earlier reports of difficulties producing soluble 0488 For the secondary assay, a fluorimetric assay that protein in an E. coli (BL 21) system, the protein yield was uses SMTC as a substrate was adapted. DDAH metabolizes consistently above 2 mg/L culture, probably due to optimal US 2013/0224259 A1 Aug. 29, 2013

induction time, RNAse and major protease deficient produc primary human endothelial cell lysate. Cells were treated tion system used in the experiments. with L-Arginine or Vehicle for 24h and assayed for L-Citrul 0492 To find small molecule modulators of DDAH activ line levels. Values are mean+/-SEM. Experiments were per ity, an enzymatic assay that would lend itself to high through formed in triplicates. *p-0.05. put Screening was developed. A microplate-based L-citrulline assay to measure DDAH activity has been previously Validation Assay reported in Knipp (Knipp, M. & Vasak, M. A colorimetric 96-well microtiter plate assay for the determination of enzy 0494. The concentration of SMTC as a substrate in the matically formed citruline. Anal Biochem 286:257-264 colorimetric citrulline assay described above was optimized. (2000)); however, this protocol requires harsh conditions Subsequently, this substrate was used in the fluorimetric including heating the plates to a temperature of ~100°C. In a assay near its k(k~1 min') at 100 uM Substrate concen high throughput format, these conditions could cause sample tration (K-1 LM), detecting the metabolite methanethiol evaporation and deformation of the reaction plates and thus (CH SH) using the CPM reagent. This study, in conjunc this assay is not practical for the purpose of comprehensive tion with the citrulline assay, narrowed down the number of screening. Accordingly, the conditions, including the amount hits by reducing potential false positives, such as pan-assay of enzyme, Substrate, incubation time and temperature, were interference compounds (PAINS). About 70% of the selected optimized for a high throughput assay (FIG. 12). FIG. 12 hits validated in the primary assay (about 35 compounds were shows a citruline assay showing that the conversion of retested in the secondary assay) were also inhibitors in the ADMA to L-citrulline by DDAH1 is proportional to time, secondary fluorimetric assay. In addition, the validation assay temperature and enzyme concentration. Data are averaged also confirmed known DDAH inhibitors such as chloromer from at least duplicate experiments. curibenzoate and ebselen and identified several new, potent 0493. In brief, various amounts of rhDDAH1 (0.1 to 10 inhibitors of human DDAHL Not surprisingly these include uM) were incubated with various concentrations of ADMA (1 other mercury-containing compounds such as phenylmercu uM to 8 mM) for various time points (30 minto 10h) at room ric acetate (ICso-0.78 LM) and quinone-type compounds temperature or 37°C. In the final screening assay, catalytic such as ChemDiv 2548-0707 (ICs5.5 uM) and 2548-0703 activity (k -0.7 min') conditions at 0.3 uM of DDAH, 500 (ICso-9.0 LM). However, it also includes more structurally uM of ADMA (K-180 uM) and 4 hours of incubation time novel compounds such as 4-mercapto-5-methoxy-2-phenyl at 37°C. were used. The reactions were stopped and color was 3(2H)-pyridazinone (4-MMP, ICs-12.7 LM) and developed as described above followed by incubation at 60° SCH-202676 (ICs-13.3 uM) (FIG. 14). FIG. 14 shows curve C. for 90 min. Absorbance, proportional to DDAH activity, fit data showing inhibition of human DDAH-1 activity by was measured at 485 nmit20 nm. In order to screen the entire selected Small molecules using the CPM assay: A) ChemDiv library, the biochemical properties of the assay were opti Compound 2548-0707; B) ChemDiv Compound 2548-0703; mized, as described above. The technique was modified so as C) 4-MMP; and D) SCH-202676. The inhibitory concentra to reduce the number of steps and facilitate robotic handling tion at 50% (ICs) was calculated using Assay Explorer Soft and throughput. Subsequently, HTS was performed using Ware 384-well plates. The Stanford HTBC is equipped with a Cali 0495. In addition, the time-dependence kinetics study per Life Sciences workstation for sample preparation and with two of the compounds (phenylmercuric acetate and analysis, Titertek multidrops, microplate dispenser and auto 4-chloromercuribenzoic acid) indicates progressive inhibi mated liquid handler and laboratory robotics for screening of tion suggesting that their mode of inhibition might be irre chemical libraries. The Z'-score was consistently found to be versible. between 0.7 and 0.8, indicating the robustness of the assay. 0496 Furthermore, the follow-up study on selected Furthermore, the feasibility of the assay was demonstrated in inhibitors using the citruline assay also confirmed the activ cell culture study by quantifying the amount of L-citruline in ity of these inhibitors against human DDAH1. Given their endothelial cells treated with L-arginine or vehicle. As structure, it is possible that many of the compounds inhibit expected, pre-treatment with L-Arginine increased the DDAH by reacting with the active site cysteine. Additional amount of intracellular L-citrulline significantly (FIG. 13; inhibitors of human DDAH1 and their potencies are shown in p-0.05). In FIG. 13, M-measurement of L-citrulline from Table 1. TABLE 1 Inhibitors of human DDAH-1. The following compounds were validated in full dose-response curves.The inhibito concentration at 50% (ICs) was calculated using Assay Explorer Software.

Molecular Name ICs (IM) Hill Slope

13.1 1.8 US 2013/0224259 A1 Aug. 29, 2013 39

TABLE 1-continued

Inhibitors of human DDAH-1. The following compounds were validated in full dose-response curves.The inhibitory concentration at 50% (ICs) was calculated using Assay Explorer software.

Molecular Name ICs (IM) Hill Slope

O 23.3 1.6

HO O

N

O

S OH 9.9 1.7 / A re-ch, N O

HC 21.2 1.1

NH

O NH

O

HN 17.7 1.3

O S

/ \ OH 45.8 1.1

O H3C O

CH O O 13.7 1.7 N N CH3 2 s/ HC N

CH O O 86 1.7

N CH 3

2 N HC N S CH US 2013/0224259 A1 Aug. 29, 2013 40

TABLE 1-continued

Inhibitors of human DDAH-1. The following compounds were validated in full dose-response curves.The inhibitory concentration at 50% (ICso) was calculated using Assay Explorer Software.

Molecular Name ICs (IM) Hill Slope

CH O O 12.8 1.6 N 2 S X-/ \ S HC N

CH3 O O 7.1 2.3 N N CH3 H3C N2Ns/ \ O/-

CH3 O O 14.1 1.6 N N CH3 H3C N2N's/

CH O O 11 1.5 N N O HC N 2N's/ O

CH3 O O 134 1.2 N N 2Ns/

C US 2013/0224259 A1 Aug. 29, 2013 41

TABLE 1-continued Inhibitors of human DDAH-1. The following compounds were validated in full dose-response curves.The inhibitory concentration at 50% (ICso) was calculated using Assay Explorer Software. Molecular Name ICs (IM) Hill Slope

CH3 O O 1O.S 1.5 N N

H3C N2Ns/

o CH

CH O O 9.9 1.5 N

2 N HC N S y H. O O / H3C

CH O O S.1 1.7 N N

HC N2Ns/

H3C

CH O O 11.8 2 N N

HC N2Ns/

O-CH 3.3 1.4 CH O O

N N )-

HC N2Ns/

CH3 39.9 0.4 US 2013/0224259 A1 Aug. 29, 2013 42

TABLE 1-continued Inhibitors of human DDAH-1. The following compounds were validated in full dose-response curves.The inhibitory concentration at 50% (IC was calculated using Assay Explorer Software. Molecular Name ICs (IM) Hill Slope O 13.2 1

NH

Example 4 followed by 22 hours low serum (0.1% fetal bovine serum; FBS) in order to synchronize them. Benzodisothiazol-3(2H)-one as a potent inhibitor 0502. On day-3, the cells were stimulated with fully of Dimethylarginine Dimethylaminohydrolase supplemented media (containing 10% FBS) in the absence or (DDAH) presence of recombinant transforming growth factor-1 (TGF B1) (at 10 ng/mL final conc) in the presence of a DDAH 0497 Experiments were performed using the same proto inhibitor (DDAHi; the proton pump inhibitor (PPI) lansopra cols as in the Examples described above to test inhibition of Zole at a final concentration of 20 uM) or vehicle for 24 hours. DDAH activity by benzodisothiazol-3(2H)-one. FIG. 17 0503. The acid-soluble collagen content in each well was shows curve fit data showing inhibition of DDAH activity by determined in the conditioned media using the Sircol col benzodisothiazol-3(2H)-one. Benzodisothiazol-3(2H)- lagen assay (Accurate Chemical and Scientific Corp; CLRS one had an ICs-13.2 uM and a Hill slope of 1 (see Table I). 1000) following the recommended protocol. 0504 The amount of collagen in each well was estimated Example 5 from a standard curve and the collagen content in each sample was normalized to total cellular protein from the respective Effect of PPIs on Collagen Production by Lung well and was expressed as ug collagen per milligram of pro Fibroblasts tein. Materials and Methods Results 0498 Collagen Production in Quiescent Lung Fibroblasts (0505. The IPF fibroblasts seeded in the presence of TGF-B Isolated from IPF Patients: showed significant elaboration of collagen production (over 2-fold increase) compared to the cells seeded in the absence Characterization of IPF Lung Fibroblasts: of TGF-B. However, treatment with a proton pump inhibitor 0499 Lung fibroblasts from patients with late-stage IPF (PPI) nearly abolished the effect of TGF-?3. were isolated and extensively characterized by immunofluo rescence staining for the expression of pan-mesenchymal Example 6 markers such as Vimentin and collagen IV: fibroblasts marker: Effect of PPIs on Alveolar Epithelial Cell Fibroblast Specific Protein (FSP1; S 100A4); and smooth Proliferation muscle cell markers such as alpha Smooth muscle actin (C-SMA); skeletal-myosin heavy chain (sk-MHC); Caldes Materials and Methods: mon; Calponin and Desmin. In addition, endothelial pheno type was ruled out by negative staining for the endothelial Proliferation Assay in Quiescent Lung Epithelial Cell Line: cell-specific marker CD31. 0506 Human Lung Carcinoma Cell Line with Type II 0500. The sources of the antibodies, their catalog num Alveolar Epithelial Cell Property: bers, and the working dilutions used in this characterization 0507 Human lung carcinoma cell line (A-549) was pur were as follows: Vimentin (Sigma V5255; Mouse at 1:100 chased from the American Type Culture Collection (ATCC: dilution); FSP1 (Rabbit; Abcam ab41532 at 1:150); Collagen Manassas, Va.; Cat #CCL-185). This cell line is widely used IV (Rabbit; Abcam: ab6586 at 1:100); C-SMA (Sigma to study human alveolar epithelial (AT II) cells due to the A2547: Mouse at 1:400 dilution); skeletal-MHC (Mouse: similarities with primary AT II cells (Lieber Metal “A con Abcam ab32330 at 1:300); Caldesmon (Rabbit; Abcam tinuous tumor-cell line from a human lung carcinoma with ab32330 at 1:250); Calponin (Mouse; Abcam ab700 at properties of type II alveolar epithelial cells': Int. J. Cancer 1:300): Desmin (Mouse: Sigma D1033 at 1:100) and CD31 1976; 17(1): 62-70; PMID: 175022). A detailed description (Dako M0823: Mouse; at 1:100 dilution). and characterization (including additional references) of this cell line are available at the ATCC web site www(dot)atcc Collagen Assay: (dot)org). 0501 Cells were seeded at 4x10" cells/well in a 6-well 0508. The cells were cultured in Kaighn's Modification of plate (1.2 mL media per well) and incubated overnight at 37° Ham's F-12K (Invitrogen/Gibco cat #21127) supplemented C./5% CO. The next day, the conditioned media was aspi with FBS (10%), Pen/Step (1%) and HEPES (2 mL in 500 mL rated and the cells were rinsed with phosphate-buffered saline Media). It was also established that the cells could as well be (PBS) prior to (fetal bovine) serum starving them for 2 hours cultured in standard Dulbecco's Modified Eagle Medium US 2013/0224259 A1 Aug. 29, 2013

(DMEM; Gibco cat #11995-065) supplemented with FBS shown to recover (Figure). This mode of enzymatic inhibition (10%), Pen/Step (1%) and HEPES (2 mL in 500 mL Media). by Small molecules is a pharmacologically desirable prop erty. BrdU Cell Proliferation Assay: 0516 FIG. 20. A dilution assay demonstrating reversible 0509 Cells were seeded at 5x10 cells/well in a 96-well inhibition of DDAH activity by PPIs. Omeprazole (IC-60 plate (200 uL media per well) & incubated overnight at 37° uM) showed reversible inhibition of DDAH as indicated by C./5% CO. The next day, the conditioned media was aspi DDAH enzymatic activity recovery upon dilution of the rated and the cells were PBS-rinsed prior to (fetal bovine) inhibitor. serum starving them for 2 hours followed by 22 hours low serum (0.1% FBS) in order to synchronize them. Example 8 0510. On day-3, 2 hours before the end of the 24 hours low serum period, DDAH inhibitors (Proton pump inhibitors; Effect of a PPI (Omeprazole) on Cellular ADMA PPIs) were added in a 100 uL solution of 0.1% FBS DMEM. 0517. To test the effects of a PPI (Omeprazole) on intrac The following DDAH inhibitors were used: Lansoprazole; ellular ADMA production, human lung epithelial cells Esomeprazole; and Rabeprazole. A "vehicle only sample (A-549) were treated with vehicle, PPI (Omeprazole at 10, served as a “no DDAH control. In addition, Media only 20, 50 or 100 uM) or a known DDAH inhibitor (L-257; Leiper (blank control) without cells, and Cells only (background Jetal; Nature Med 2007: 13(2): 198-203) for 24 hours. After control) were included in the assay. Two hours later, 100LL of 24 hours, ADMA was measured by enzyme-linked immun DMEM containing 10% FBS was added to each well in order osorbent assay (ELISA). As shown in FIG. 21, an increase in to serum-stimulate (for 24 hours) the cells and make the total intracellular ADMA was observed when human A-549 lung well content to 200 uL. Four hours prior to the end of the 24 epithelial cells were treated with Omeprazole. Data are hrs, 20 uL of 1:500 diluted Bromodeoxyuridine (BrdU) (di Mean-SEM from duplicate experiments. *p-0.05 compared luted in fully-supplemented DMEM) was added to all wells except the “Cells only” control wells. to vehicle (“Veh'). 0511. The incorporation of BrdU into newly synthesized Example 9 DNA by actively proliferating cells was detected immu nochemically using an Antibody directed against BrdU using the recommended protocol and the BrdU Cell Proliferation Effect of PPI on Nitric Oxide Production Assay kit (Millipore; Cat #2750). 0518. To test the effects of PPI (Omeprazole) on nitric oxide production, human alveolar epithelial cells (A-549) Results were treated with vehicle, PPI (Omeprazole) or a known DDAH inhibitor (L-257; Leiper J et al; Nature Med 2007: 0512. The results are shown in FIG. 19. The cells seeded in 13(2): 198-203) for 24 hours. Total nitrite (NOx) was mea the absence of the DDAH inhibitors showed continued incor sured using Griess reaction. Data are Mean-SEM from dupli poration of BrdU indicating active proliferation. However, cate experiments. *p-0.05. the treatment PPIs significantly reduced the incorporation of BrdU indicating inhibition of active cell proliferation. This 0519. As shown in FIG. 22, Omeprazole reduced the lev finding indicates that inhibition of DDAH using the PPIs can els of nitric oxide in the human alveolar epithelial cell line. regulate the over-proliferation of alveolar epithelial cells as seen in lung fibrosis. Example 10 0513 FIG. 19. Treatment with proton pump inhibitors (PPIs; 20 uM) ameliorated serum-induced lung alveolar epi Effect of PPI on Collagen Production by IPF thelial cell proliferation. Human lung epithelial cell line Fibroblasats (A-549) cells were rendered quiescent, then serum-stimu 0520 Lung fibroblasts were isolated from patients diag lated to induced re-entry into the cell cycle. BrdU (4 h) incor nosed with IPF according to International Standards. Isolated poration was assessed. Data are meantSEM (duplicates). fibroblasts were seeded at 6x10" and synchronized the fol lowing day. On day-3, the cells were stimulated with serum Example 7 stimulation in the absence or presence of recombinant trans forming growth factor-1 (TGF-31) in the presence of the Inhibition of DDAH by PPIs is Reversible proton pump inhibitor Omeprazole (final concentration 50 or 0514. In order to understand the mechanism by which 100 uM)); L-257 (final concentration 50 uM) or TGF-131 PPIs inhibit DDAH activity and to determine whether the inhibitor A83-01 (final concentration 20 uM) controls; or inhibition mechanism is reversible, a dilution study was con vehicle for 24 hours. ducted, in which DDAH (at a 100-fold excess to the final 0521. The acid-soluble collagen content was determined concentration used) was pre-incubated with excess PPI using the Sircol collagen assay (Accurate Chemical and Sci (100x, 10x or 1x the ICs value) and then inhibition of enzy entific Corp; CLRS 1000) following the recommended pro matic activity was determined using a fluorometric assay as tocol. described above. For a reversible inhibitor that binds to a 0522. As shown in FIG. 23, the IPF fibroblasts seeded in single site of an enzyme (1:1 stoichiometry), it is anticipated the presence of TGF-3 showed significant elaboration of col that inhibition can be saturated. lagen production compared to the cells seeded in the absence 0515. The data are shown in FIG. 20. Inhibition of DDAH of TGF-B. However, treatment with Omeprazole, L-257, or activity by PPIs was found to be reversible upon dilution of A83-01 reduced TGF-B induced collagen synthesis. Data are the inhibitors; hence the enzymatic activity of DDAH was Mean+SEM from duplicate experiments. *p-0.05. US 2013/0224259 A1 Aug. 29, 2013 44

Example 11 1. A pharmaceutical formulation comprising: a) a dimethylarginine dimethylaminohydrolase (DDAH) Effect of PPIs on Alveolar Epithelial Cell inhibitor of one of the following formulas: Proliferation

0523 To determine the effects of PPI (Omeprazole) on i) Formula Ia: alveolar epithelial cell proliferation, a BrdU Cell Prolifera tion Assay was carried out. Omeprazole or L-257 control (Ia) were added to either a human or mouse alveolar epithelial cell sample. A “vehicle only’ sample served as a “no DDAH' control. 0524. The results are shown in FIGS. 24 and 25. The cells (R), H \ / seeded in the absence of the DDAH inhibitors showed con tinued incorporation of BrdU indicating active proliferation. R5 R4 However, treatment with Omeprazole and L-257 reduced incorporation of BrdU in human (FIG. 24) and mouse cell wherein lines (FIG. 25). Q is Nor CH: Example 12 R" is selected from alkyl, substituted alkyl, hydroxy, alkoxy, Substituted alkoxy, amino, Substituted amino, carboxyl, carboxyl ester, cyano, halogen, acyl, aminoa Effect of PPI on Apoptosis cyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Sub 0525) To test the effects of PPI (Omeprazole) on human stituted alkynyl: alveolar epithelial cell apoptosis, human alveolar epithelial R. R. R. and Rare independently selected from hydro cells (A-549) were treated with vehicle, PPI (Omeprazole) or gen, alkyl, Substituted alkyl, hydroxy, alkoxy, Substi a known DDAH inhibitor (L-257; Leiper J et al; Nature Med tuted alkoxy, amino, Substituted amino, carboxyl, car 2007: 13(2): 198-203) in the presence of an apoptosis-inducer boxyl ester, cyano, halogen, acyl, aminoacyl, nitro, (1 uM A23 187) for 24 hours. Apoptosis as measured by alkenyl, Substituted alkenyl, alkynyl, and Substituted mono- and oligo-nucleosome enrichment was determined alkynyl; and using Cell Death Detection ELISA''' (Roche). Histone DNA complex (His-DNACPX) was included as a control. As m is an integer from Zero to four, shown in FIG. 26, Omeprazole reduced the level of apoptosis in human alveolar epithelial cells. Data shown are Mean+/- SEM from Duplicate Experiments. * p-0.05.

Example 13 i) Formula Ib: (Ib) 0526 Cytotoxicity of PPIs...To determine whether PPIs are O cytotoxic to human alveolar epithelial cells (A-549) were treated with vehicle, or varying concentrations of PPI (Ome 1y prazole) or a known DDAH inhibitor (L-257; Leiper J et al; A4N Nature Med 2007: 13(2): 198-203) and cytotoxicity was (R), assessed by the release of lactate dehydrogenase (LDH) into conditioned media. As shown in FIG. 27, PPI (Omeprazole) is not cytotoxic to human alveolar epithelial cells at least up to 300 LM, a concentration 3-6 fold higher than the concentra tion of PPIs at which anti-fibrotic, anti-proliferative, and anti apoptotic effects were observed as described herein. Cytotox icity was also not observed at least up to 300 uM in mouse wherein alveolar epithelial (MLE-12) cells (FIG. 28). Mean+/-SEM from Duplicate Experiments. * p-0.05. R" is selected from alkyl, substituted alkyl, hydroxy, alkoxy, Substituted alkoxy, amino, Substituted amino, 0527. While the present invention has been described with carboxyl, carboxyl ester, cyano, halogen, acyl, aminoa reference to the specific embodiments thereof, it should be cyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Sub understood by those skilled in the art that various changes stituted alkynyl: may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In R. R. R. and R are independently selected from is addition, many modifications may be made to adapt a par selected from alkyl, substituted alkyl, hydroxy, alkoxy, ticular situation, material, composition of matter, process, Substituted alkoxy, amino, Substituted amino, carboxyl, process step or steps, to the objective, spirit and scope of the carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, present invention. All such modifications are intended to be alkenyl, Substituted alkenyl, alkynyl, and Substituted within the scope of the claims appended hereto. alkynyl; and US 2013/0224259 A1 Aug. 29, 2013 45

m is an integer from Zero to four, -continued OCH i) Formula Ic: HCO N N (Ic) )—s NF N N 2 21 N \, H CCA-4NN )-( = R3, (Tenatoprazole) (R). H \ / OCH R5 R4 HCO N \ ; and wherein Xu ( N / R" is selected from alkyl, substituted alkyl, hydroxy, N O alkoxy, Substituted alkoxy, amino, Substituted amino, (Esomeprazole) carboxyl, carboxyl ester, cyano, halogen, acyl, aminoa Na cyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Sub stituted alkynyl: N N-l R. R. R', and R are independently selected from is )—sV O selected from alkyl, substituted alkyl, hydroxy, alkoxy, N O \- Substituted alkoxy, amino, Substituted amino, carboxyl, O- . carboxyl ester, cyano, halogen, acyl, aminoacyl, nitro, (Rabeprazole) alkenyl, Substituted alkenyl, alkynyl, and Substituted alkynyl; and 5. The formulation of claim 1, wherein the DDAH inhibitor m is an integer from Zero to four, is formulated with a fluid carrier and a propellant. and 6. The formulation of claim 1, wherein the DDAH inhibitor is in a dry powder formulation. b) a flowable formulation suitable for delivery by inhala 7. A package for use in treating a disorder associated with tion. excessive NO production and/or elevated DDAH activity, the 2-3. (canceled) package comprising a container having therein the formula tion of claim 1. 4. The pharmaceutical formulation of claim 1, wherein the 8. The package of claim 7, wherein the package: DDAH inhibitor is selected from: i) is a metered dose inhaler, and the DDAH inhibitor is formulated with a propellant; OCH ii) is a dry powder inhaler, and the DDAH inhibitor is HC formulated in a dry powder formulation; or iii) is a nebulizer, and the DDAH inhibitor is in an aqueous e CH3: or ethanolic Solution. H3CO N N h 9-10. (canceled) 11. A metered dose inhaler having therein the formulation X- N of claim 1, wherein the DDAH inhibitor is formulated with a NH O propellant. (Omeprazole) 12. A dry powder inhaler having therein the formulation of H3CO OCH3: claim 1, wherein the DDAH inhibitor is formulated in a dry powder formulation. 13. A nebulizer having therein the formulation of claim 1, F. O N / \ wherein the DDAH inhibitor is in an aqueous or ethanolic N o Solution. X- N 14. An aerosol comprising a pharmaceutically active H O DDAH inhibitor of Formula Ia, Formula Ib, or Formula Ic. 15. The aerosol of claim 14, wherein the DDAH inhibitoris (Pantoprazole) contained within aerosolized particles having a diameter in a Nie range of from about 0.25um to about 12 um. 16. The aerosol of claim 14, wherein the aerosol is in the N h N N form of a solution, a Suspension, a powder, or a semi-solid O preparation. NX O 17-19. (canceled) CF3: 20. A pharmaceutical formulation comprising a dimethy (Lansoprazole) larginine dimethylaminohydrolase (DDAH) inhibitor of the formula: US 2013/0224259 A1 Aug. 29, 2013 46

wherein (III) R" is selected from hydrogen, alkyl, substituted alkyl, alkoxy, Substituted alkoxy, cycloalkyl, Substituted -- (R), cycloalkyl, aryl, Substituted aryl, heterocyclyl, and Sub J stituted heterocyclyl: rry R’ is selected from hydrogen, alkyl, and substituted alkyl: ? 2 1s RandR are independently selected from is selected from hydrogen, alkyl, Substituted alkyl, hydroxy, alkoxy, Sub stituted alkoxy, amino, Substituted amino, carboxyl, car wherein boxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alk Q is S. O. NH or CH: enyl, Substituted alkenyl, alkynyl, and Substituted Q is N or CH: alkynyl: Q is N or CH: R" is selected from alkyl, substituted alkyl, hydroxy, alkoxy, Substituted alkoxy, amino, Substituted amino, ii) Formula IIb: carboxyl, carboxyl ester, cyano, halogen, acyl, aminoa cyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Sub (IIb) stituted alkynyl: CH3 O O R R is selected from alkyl, substituted alkyl, hydroxy, \ alkoxy, Substituted alkoxy, amino, Substituted amino, N V carboxyl, carboxyl ester, cyano, halogen, acyl, aminoa N R2 cyl, nitro, alkenyl, Substituted alkenyl, alkynyl, and Sub 2 / stituted alkynyl: H3C N S m is an integer from Zero to four, and n is an integer from Zero to three; and a flowable formulation suitable for delivery by inhalation. wherein 21. The pharmaceutical composition of claim 20, wherein R" is selected from hydrogen, alkyl, substituted alkyl, the DDAH inhibitor is: alkoxy, Substituted alkoxy, cycloalkyl, Substituted cycloalkyl, aryl, substituted aryl, heterocyclyl, and sub stituted heterocyclyl: R’ is selected from hydrogen, alkyl, and substituted alkyl: and b) a pharmaceutically acceptable excipient. ls 26. (canceled) S NH. 27. The pharmaceutical composition of claim 25, wherein the DDAH inhibitor is selected from: 22. The formulation of claim 20, wherein the DDAH inhibitor is formulated with a fluid carrier and a propellant, or wherein the DDAH inhibitor is in a dry powder formulation. 23. A package for use in treating a disorder associated with CH3 O O excessive NO production and/or elevated DDAH activity, the H package comprising a container having therein the formula N N )- CH3: tion of claim 20. 2 M 24. The package of claim 23, wherein the package is a H3C N S metered dose inhaler, and the DDAH inhibitor is formulated with a propellant; wherein the package is a dry powder inhaler, and the DDAH inhibitor is formulated in a dry pow der formulation; or wherein the package is a nebulizer, and CH3 O O the DDAH inhibitor is in an aqueous or ethanolic solution. 25. A pharmaceutical formulation comprising: N CH3 a) a dimethylarginine dimethylaminohydrolase (DDAH) 2 N inhibitor of one of the following formulas: H3C N S CH3:

CH O O i) Formula IIa: (IIa) N R3 O O R1 2 S N S N /V HC N N R2 2Ns/ US 2013/0224259 A1 Aug. 29, 2013 47

-continued -continued CH O O CH3 O O H N N CH3: N N )- 2 M / 2 M HC N S O H3C N S CH O O s N O -CH3; and N CH3: CH O O / HC N 2N's/ N

HC N 2N's/ CH O O

CH3 O O N N N HC N2N's/ N O; C H3C N2Ns/ O 28. The formulation of claim 25, wherein the DDAH CH O O inhibitor is formulated with a fluid carrier and a propellant, or wherein the DDAH inhibitor is in a dry powder formulation. N 29. A package for use in treating a disorder associated with N excessive NO production and/or elevated DDAH activity, the HC N2Ns/ package comprising a container having therein the formula tion of claim 25. 30. The package of claim 29, wherein the package is a metered dose inhaler, and the DDAH inhibitor is formulated Cl; with a propellant; wherein the package is a dry powder CH O O inhaler, and the DDAH inhibitor is formulated in a dry pow der formulation; or wherein the package is a nebulizer, and N the DDAH inhibitor is in an aqueous or ethanolic solution. N 31. A method of treating an individual suffering from a disorder characterized by excessive NO production and/or HC N 2Ns/ elevated DDAH activity, the method comprising administer ing to the individual an effective amount of a pharmaceutical formulation of claim 1. 32. The method of claim 72, wherein the formulation is administered by injection, or wherein the formulation is O administered with a carrier in the form of normal saline solu tion. CH3: 33. (canceled) CH3 O O 34. The method of claim 31, wherein the formulation is administered locally to the airways of the patient. N 35. The method of claim 34, wherein the formulation is 2 N administered by inhalation, by insufflating an aerosol com H3C N S H. prising the DDAH inhibitor, in a dry powder formulation, O O using a nebulizer. 36.-38. (canceled) / 39. The method of claim 31, wherein the DDAH inhibitor H3C is in an aqueous or ethanolic solution. CH3 O O 40. The method of claim 31, wherein the individual is a human, or is a non-human mammal. N 41. The method of claim 31, wherein the disorder is fibro N S1S. 2Ns/ 42. An in vitro method of identifying an agent that inhibits H3C N enzymatic activity of a dimethylarginine dimethylaminohy drolase (DDAH) polypeptide, the method comprising: a) contacting the DDAH polypeptide and a DDAH sub strate with a test agent; and US 2013/0224259 A1 Aug. 29, 2013 48

b) determining the effect, if any, of the test agent on DDAH 46. The pharmaceutical formulation of claim 44, wherein enzymatic activity, wherein the Substrate is asymmetric the DDAH inhibitor is dimethylarginine, and the determining step is a colori metric assay for L-citrulline, wherein a test agent that decreases DDAH activity, compare to a control, is an agent that inhibits DDAH activity. 43. The method of claim 42, wherein the substrate is ADMA, and said determining step comprises reacting L-cit NH. rulline with 2,3-Dimethyl-1-phenyl-3-pyrazolin-5-one and 2,3-butanedione oxime. 44. A pharmaceutical formulation comprising: 47. The formulation of claim 44, wherein the DDAH a) a dimethylarginine dimethylaminohydrolase (DDAH) inhibitor is formulated with a fluid carrier and a propellant, or inhibitor of one of the following formulas: wherein the DDAH inhibitor is in a dry powder formulation. 48. (canceled) 49. A package for use in treating a disorder associated with i) Formula Xa excessive NO production and/or elevated DDAH activity, the (Xa) package comprising a container having therein the formula R O tion of claim 44. R2 50. The package of claim 49, wherein the package: N a) is a metered dose inhaler, and the DDAH inhibitor is NR', formulated with a propellant; R3 % S b) is a dry powder inhaler, and the DDAH inhibitor is formulated in a dry powder formulation; or c) is a nebulizer, and the DDAH inhibitor is in an aqueous wherein or ethanolic Solution. R", Rand Rare independently selected from hydrogen, 51-52. (canceled) alkyl, substituted alkyl, hydroxy, alkoxy, substituted 53. A metered dose inhaler having therein the formulation alkoxy, amino, Substituted amino, carboxyl, carboxyl of claim 44, wherein the DDAH inhibitor is formulated with ester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, a propellant. Substituted alkenyl, alkynyl, and Substituted alkynyl; 54. A dry powder inhaler having therein the formulation of R" is selected from hydrogen, alkyl, substituted alkyl, claim 44, wherein the DDAH inhibitor is formulated in a dry alkoxy, Substituted alkoxy, cycloalkyl, Substituted powder formulation. cycloalkyl, aryl, Substituted aryl, heterocyclyl, and Sub 55. A nebulizer having therein the formulation of claim 44, stituted heterocyclyl; and wherein the DDAH inhibitor is in an aqueous or ethanolic Q is N or CR, where R, if present, is selected from Solution. hydrogen, alkyl, Substituted alkyl, hydroxy, alkoxy, Sub 56. An aerosol comprising a pharmaceutically active stituted alkoxy, amino, Substituted amino, carboxyl, car boxyl ester, cyano, halogen, acyl, aminoacyl, nitro, alk DDAH inhibitor of claim 44. enyl, Substituted alkenyl, alkynyl, and Substituted 57. The aerosol of claim 56, wherein the DDAH inhibitoris alkynyl: contained within aerosolized particles having a diameter in a range of from about 0.25um to about 12 um. 58. The aerosol of claim 56, wherein the aerosol is in the form of a solution, a Suspension, a powder, or a semi-solid ii) Formula Xb: preparation. (Xb) RI 59-61. (canceled) O 62. A method of treating an individual suffering from a R2 disorder characterized by excessive NO production and/or elevated DDAH activity, the method comprising administer NH, M ing to the individual an effective amount of a pharmaceutical R4 S formulation of claim 44. 63. The method of claim 62, wherein the formulation is a R5 formulation of claim 44, and is administered with a carrier in the form of normal saline solution. wherein 64. The method of claim 63, wherein the formulation is R. R. Rand Rare independently selected from hydro administered locally to the airways of the patient. gen, alkyl, Substituted alkyl, hydroxy, alkoxy, Substi 65. The method of claim 63, wherein the formulation is tuted alkoxy, amino, Substituted amino, carboxyl, car administered i) by inhalation; ii) by insufflating an aerosol boxyl ester, cyano, halogen, acyl, aminoacyl, nitro, comprising the DDAH inhibitor; iii) in a dry powder formu alkenyl, Substituted alkenyl, alkynyl, and Substituted lation; iv) using a nebulizer. alkynyl: 66-68. (canceled) and 69. The method of claim 62, wherein the DDAH inhibitor b) a flowable formulation suitable for delivery by inhala is in an aqueous or ethanolic solution. tion. 70. The method of claim 62, wherein the individual is a 45. (canceled) human or is a non-human mammal. US 2013/0224259 A1 Aug. 29, 2013 49

71. The method of claim 62, wherein the disorder is fibro S1S. 72. A method of treating an individual suffering from a disorder characterized by excessive NO production and/or elevated DDAH activity, the method comprising administer ing to the individual an effective amount of a pharmaceutical formulation of claim 25.

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