US 2005.0085486A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0085486A1 Gonzalez-Cadavid et al. (43) Pub. Date: Apr. 21, 2005
(54) METHODS OF USE OF INHIBITORS OF Publication Classification PHOSPHODESTERASES AND MODULATORS OF NITRICOXIDE, (51) Int. Cl." ...... A61K 31/519 REACTIVE OXYGEN SPECIES, AND (52) U.S. Cl...... 514/252.16; 514/262.1 METALLOPROTEINASES IN THE TREATMENT OF PEYRONIES DISEASE, (57) ABSTRACT ARTERIOSCLEROSIS AND OTHER The present methods and compositions are of use for treat FIBROTC DISEASES ment of conditions involving fibrosis, Such as Peyronies disease plaque, penile corporal fibrosis, penile veno-Occlu (76) Inventors: Nestor F. Gonzalez-Cadavid, Sive dysfunction, Dupuytren's disease nodules, vaginal Pasadena, CA (US); Jacob Rajfer, fibrosis, clitoral fibrosis, female Sexual arousal disorder, Rolling Hills Estates, CA (US) abnormal wound healing, keloid formation, general fibrosis of the kidney, bladder, prostate, skin, liver, lung, heart, Correspondence Address: intestines or any other localized or generalized fibrotic BLAKELY SOKOLOFFTAYLOR & ZAFMAN condition, Vascular fibrosis, arterial intima hyperplasia, ath 12400 WILSHIRE BOULEVARD erosclerosis, arteriosclerosis, restenosis, cardiac hypertro SEVENTH FLOOR phy, hypertension or any condition characterized by exces LOS ANGELES, CA 90025-1030 (US) sive fibroblast or smooth muscle cell proliferation or deposition of collagen and extracellular matrix in the blood (21) Appl. No.: 10/779,069 vessels and/or heart. In certain embodiments, the composi (22) Filed: Feb. 13, 2004 tions may comprise a PDE-4 inhibitor, a PDE-5 inhibitor, a compound that elevates cGMP and/or PKG, a stimulator of (30) Foreign Application Priority Data guanylyl cyclase and/or PKG, a combination of a compound that elevates cGMP, PKG or NO with an antioxidant that Oct. 21, 2003 (WO)...... PCT/USO3/33400 decreases ROS, or a compound that increases MMP activity.
Patent Application Publication Apr. 21, 2005 Sheet 2 of 21 US 2005/0085486A1
L-ARG
(A)
SL PXF
12 b
RATO COLLAGEN g (B) LACUNARSPACESFIBERS/CELLS 6 2 O p s L-ARG SIL PXF TGFB1
FIG. 2 Patent Application Publication Apr. 21, 2005 Sheet 3 of 21 US 2005/0085486A1
LARG (A)
Sl PXF
a vs. bNS;a,b vs. C, NS, d vs a p<0.05; d VS. b,CNS; e VS. a, b, c, d p<0.001 50 40 APOPTOTIC 30 NDEX 20 (B) 10
h L-ARG SL PXF SAL TGFB1
FIG. 3 Patent Application Publication Apr. 21, 2005 Sheet 4 of 21 US 2005/0085486A1
N\/WITH (O)
Patent Application Publication Apr. 21, 2005 Sheet 5 of 21 US 2005/0085486A1
(A) CO 120 LL C y 80 H co s' SP 40 ::::: DC *SS : z:W 2 250SL H 200SL o - C 20OPXF O + 33(S. 200 100 % O 2 YY-2 ZYear 2 COLLAGEN ASMA
(B) 2 C - - O O
Skss TGFB1 TREATED
FIG. 5 Patent Application Publication Apr. 21, 2005 Sheet 6 of 21 US 2005/0085486A1
COLLAGEN 1 ASMA (%)STTEOTWIOL
HE|dNOISSERHc|XE-IOÅ1||SNBLN|| (TTHO/OOI)TEOBAILISOd
10 400M 10M 400M
F.G. 6 Patent Application Publication Apr. 21, 2005 Sheet 7 of 21 US 2005/0085486A1
(A) HUMAN RAT (B) HUMAN RAT TA PD TA 861 bp
(C)
Patent Application Publication Apr. 21, 2005 Sheet 8 of 21 US 2005/0085486A1
FIG 8 Patent Application Publication Apr. 21, 2005 Sheet 9 of 21 US 2005/0085486A1
C vs D p<0.05 C vs B p<0.01 (B) 45 A vs C p<0.01 C (n=4) A vs D p<0.05 40 A vs Bp-0.05 35 30 D (n=4) 25 20 15 A (n= 2)
Saline TGF- Bt Fibrin TGF- B1 Fibrin FIG. 9 Patent Application Publication Apr. 21, 2005 Sheet 10 of 21 US 2005/0085486A1
Tunica Plaque 16 17 18 26 30 24 25 29 33 34 35 GAPDH MMP2 GAPDH TIMP1 GAPDH DCN GAPDH TMSB-10 GAPDH PTN
(B) DTunica Plaque
MMP2 TIMP1 DCN TMSB-10 PTN
FIG. 10 Patent Application Publication Apr. 21, 2005 Sheet 11 of 21 US 2005/0085486A1
Saline TGF.IGF-6 Injection n ection
FIG 11 Patent Application Publication Apr. 21, 2005 Sheet 12 of 21 US 2005/0085486A1
HUMAN TISSUE (A) PDE4A 882 bp GAPDH(1) 496 bp
GAPDH(2) 1.1 kb
PDE4B 406 bp
(B) PDE4A 882 bp PDE4B 406 bp
(C)
FIG. 12 Patent Application Publication Apr. 21, 2005 Sheet 13 of 21 US 2005/0085486A1
Patent Application Publication Apr. 21, 2005 Sheet 14 of 21 US 2005/0085486A1
SLDENAFIL
Ne §3 N• Ng PENTOXFYLLINE
§?· §3 Nto N?
PENTOXFYLLINE
%d'IWW79
Concentration (M)
F.G. 14 Patent Application Publication Apr. 21, 2005 Sheet 15 of 21 US 2005/0085486A1
YOUNG OLD OLD+L-NL
(A)
20 A (B)
5
AORTA FEMORAL DORSAL BULBO URETHRAL O YOUNG OLD2OLD+L-N.
FIG. 15 Patent Application Publication Apr. 21, 2005 Sheet 16 of 21 US 2005/0085486A1
PENILE DORSAL ARTERY YOUNG OLD OLDL-NIL (A) Ei. fall, was 'r 3.
iNOS
3-Nitro tyrosine
(B) 12 iNOS 3-Nitrotyrosine
B i 4. s CS
Dorsal Bulbo-urethral Dorsal Bulbourethral
YOUNG OLDZOLD+L-NIL
FIG. 16 Patent Application Publication Apr. 21, 2005 Sheet 17 of 21 US 2005/0085486A1
(A) s 1 O CC
- C D 1. O
(B)
O YOUNG a OLD O 2 OLD+L-N.
AOrta Dorsal Artery Bulbo-urethral Artery
FIG. 17 Patent Application Publication Apr. 21, 2005 Sheet 18 of 21 US 2005/0085486A1
(A) YOUNG OLD OLD+L-NL
(B) 0.25 0. 2
0.15
0.05
YOUNG OLD OLD+L-NL Penile Dorsal Artery
FIG. 18 Patent Application Publication Apr. 21, 2005 Sheet 19 of 21 US 2005/0085486A1
(A) PENILE DORSAL ARTERY
YOUNG - - - OLD - OLDL-NIL
(B) i 2 s O YOUNG P DOLD O b 2OLD+L-NFL O O a. Dorsal Artery Bulbo-urethral Artery
FIG. 19 Patent Application Publication Apr. 21, 2005 Sheet 20 of 21 US 2005/0085486A1
H to 2 O O. t H 9. 0. O C O
D 1. 2 O L c CN 1. 9 CC - ld Co Ltd Co Lt Cd Ud Cd 9 C o cro Cyd CN on - D SPAI-1 Number of Positive Cells/Field 4. 2 U d St - O. t U OL
Os g O C
>a
g c X
ld d led Cd Co Ltd cd Lo go rt N. c. c. C. C. R. v c c O C C C C C C C C Co PAI-1 Mean Optical Density 3. o Patent Application Publication Apr. 21, 2005 Sheet 21 of 21 US 2005/0085486A1
V GN
9
e c)
an
S s V S. n. tr is 9 S CO 3 2. t CD d ce
s CN v- cd go Apoptotic Index (%)
3. o US 2005/0085486A1 Apr. 21, 2005
METHODS OF USE OF INHIBITORS OF increase in TGF-B1 Synthesis. This represents impairment in PHOSPHODESTERASES AND MODULATORS OF the repair process that leads to persistent fibrosis and a loSS NITRICOXIDE, REACTIVE OXYGEN SPECIES, of elasticity of the TA. AND METALLOPROTEINASES IN THE 0008 PD can rarely be alleviated by medical treatment TREATMENT OF PEYRONIES DISEASE, with anti-inflammatory agents (corticosteroids, antihista ARTERIOSCLEROSIS AND OTHER FIBROTC mine), antioxidants (vitamin E, Superoxide dismutase), col DISEASES lagen breakdown (collagenase), Ca channel blockers (Vera pamil), and other antifibrotic compounds (colchicine, RELATED APPLICATIONS Potaba: K aminobenzoate) (Hellstrom and Bivalacqua, 0001. The present application claims the benefit under 35 2000). In most cases, Surgery is the only available option to U.S.C. S 119(e) of U.S. Provisional Application Ser. No. correct the deformity and alleviate the pain So that normal 60/420,281, filed on Oct. 22, 2002, and claims the benefit of Sexual activity can be resumed. A need exists for non PCT Serial No. USO3/33400, filed on Oct. 23, 2003. Surgical methods of treatment of Peyronie's disease and other medical conditions in which fibrosis is important. BACKGROUND OF THE INVENTION 0009 Fibrotic disease is not limited to the reproductive organs, but can be found in other tissues, Such as cardio 0002) 1. Field vascular tissues. Both erectile dysfunction (ED) and cardio 0003. The present methods and compositions relate to the vascular disease, particularly hypertension, are prevalent in field of Peyronie's disease, arteriosclerosis and other fibrotic the aging male (Kloner et al., 2002; Sullivan et al., 2001; conditions. More particularly, the method and compositions Melman et al., 1999). One of the underlying causes of concern use of phosphodiesterase (PDE) inhibitors and hypertension is arteriosclerosis, or arterial Stiffness, due to modulators of nitric oxide, reactive oxygen Species and an acquired fibrosis of the media of the arterial wall (Brei metalloproteinases in the treatment of Such conditions. In thaupt-Grogler and Belz, 1999; Robert, 1999; Intengan and particular embodiments, the inhibitors inhibit type 4 and/or Schiffrin, 2000, 2001; Formieri et al., 1992). Arteriosclerosis type 5 PDEs. is significantly associated with aging, and is recognized by an increase in collagen, and in Some cases by a loSS of 0004 2. Description of Related Art smooth muscle cells (SMC) within the arterial media, which 0005 Peyronie's disease (PD) is a fibromatosis (Hell results in a decrease in the SMC/collagen ratio, often accom strom and Bivalacqua, 2000; Schwarzer et al., 2001; Jarow panied by endothelial dysfunction (Cai and Harrison, 2000). et al., 1997; Devine et al., 1997) of the tunica albuginea 0010. The pathogenesis of aging associated ED, both in (TA), the specialized lining of the corpora cavernosa of the the human and rat, is mostly related to the loss of SMC in penis. Clinically, this usually leads to penile deformation the penile corpora cavernosa by apoptosis, with a corre (curved penis during erection), pain, and quite frequently sponding increase in collagen fibers (Melman and Gingell, erectile dysfunction. The initiating event is believed to be an 1999; Cai and Harrison, 2000; Melman, 2001; Garban et al., external force to the erect penis that results in an injury to the 1995; Ferrini et al., 2001a). The clinical result of this aging TA of the corpora and the TA fails to heal normally (Jarow process in the penis is defective cavernoSal SMC relaxation et al., 1997; Devine et al., 1997; Diegelmann, 1997; Sherratt leading to Veno-occlusive dysfunction (Breithaupt-Grogler and Dallon, 2002). In the detumesced state, the only indi and Belz, 1999; Rogers et al., 2003), the most common cation of the disease is the palpation of a knot or Scar within cause of ED. the TA, which in its most Severe State presents as a calcified plaque. 0011. In the arterial tree, excessive collagen deposition in the media, with or without loss of SMC, leads to defective 0006 PD affects about 5% of men in the USA, and VaSO-relaxation and clinically may present as hypertension translating into about 3-4 million affected American males. (Breithaupt-Grogler and Belz, 1999; Robert, 1999; Intengan Although the condition is not always associated with erectile and Schiffrin, 2000, 2001). Because the penis may be dysfunction, patients usually present to the physician with considered a specialized extension of the vascular tree, the either recognition of a palpable plaque on the penile Shaft, common alterations observed in the SMC of both the penis pain with tumescence, impotence and/or difficulty with and peripheral vascular System in the aging male, leading to intromission that is due to curvature of the erect penis. Since ED and hypertension, respectively, Suggest that both condi the disorder was first described in 1743, no medical treat tions may share a common etiology. ment has ever proven to be beneficial in combating the condition, thereby highlighting the need to develop novel 0012. A need exists for effective methods to treat and/or approaches to combat this disorder. ameliorate the Symptoms of a variety of fibrotic disease, Such as PD, ED and arteriosclerosis. No effective method of 0007. There may also be a genetic predisposition to treatment currently exists that is directed towards the developing PD, Since it is associated with other contractures molecular pathways underlying excessive collagen deposi Such as Dupuytren’s disease (palmar fascia; 10-20% inci tion. dence or more in PD) (Connelly, 1999) and Ledeshore's disease (plantar fascia). The pathophysiology is character SUMMARY OF THE INVENTION ized by localized disruption of the TA, increased microvas cular permeability, persistent fibrin (deficient fibrinolysis) 0013 Certain embodiments of the present invention full and collagen deposition, perivascular inflammation, disor fill an unresolved need in the art, by providing novel ganization and loss of elastic fibers (release of elastase by methods for therapeutic treatment of Peyronie's disease, macrophages), disorganized collagen bundles, and an erectile dysfunction, arteriosclerosis and other fibroses. In US 2005/0085486A1 Apr. 21, 2005
Some embodiments, PD plaques and/or other fibrotic con injection and sildenafil in water. PXF received a TGF-31 ditions can be pharmacologically arrested or reduced in Size, injection and pentoxifylline in water. (B) QIA (n=5 per by decreasing collagen Synthesis and inducing myofibro group) as in FIG. 2. blast apoptosis by increasing the NO/ROS ratio, the levels of cGMP, or the activation of its effector, PKG in the TA and/or 0020 FIG. 4. Expression of PDE-5 mRNA and protein in Stimulating collagen degradation by activating the MMPS the human PD plaque and normal tunica albuginea, and their and/or down-regulating the expression of the MMP inhibi homologous tissues in the TGF-1 rat model of PD. (A) tors (TIMP), by increasing NO/cGMP levels and/or the Ethidium bromide-stained DNA bands obtained by RT/PCR thymosins in the TA. from RNAS isolated from the respective tissues, and frac 0.014 Particular embodiments of the invention may be tionated on agarose gels. (B) Luminol-stained protein bands directed towards increasing levels of c(GMP and/or cAMP by obtained by western blot on polyacrylamide gels. PS.: penile selective inhibition of phosphodiesterase (PDE) isoforms. shaft; TA: tunica albuginea; PD: Peyronie's disease; CC: PDE isoforms of interest in the TA and in PD plaque tissues corpora cavernosa; CER: cerebellum; CRU: penile crura. include PDE5A-3, PDE4A, PDE4B and PDE4D. As non (C) Microphotgraphs (200x) of sections from human and limiting examples, pentoxifylline and Similar compounds act untreated rat tissues. D.ART: dorsal artery; ART: artery. as a non-specific cAMP-PDE inhibitor and increase cAMP Arrows show positive cells for PDE-5. levels, while Sildenafil and Similar compounds Selectively 0021 FIG. 5. Inhibition of collagen I synthesis and inhibit PDE5A and increase coMP levels. myofibroblast differentiation by PDE inhibitors in fibroblast 0.015. Other embodiments may involve increasing NO cultures from a human PD plaque. (A) QIA evaluation of levels, for example by administering L-arginine, a Stimulator collagen I and ASMA expression. Control (C) no addition; of NOS activity. As shown in the following examples, 50SIL: sildenafil (50 nM); 200SIL: sildenafil (200 nM); pentoxifylline, Sildenafil and L-arginine all act to reduce the 200PXF:pentoxifylline (200 nM). (B) DAB-stained immu expression of collagen I and C-Smooth muscle actin. Long nocytochemical detection of collagen III in PD cells incu term administration of nitrergic agents, Such as pentoxifyl bated for 3 days in DME-serum free medium in the presence line, Sildenafil and L-arginine may be of use to reduce PD or absence of 5 ng/ml of TGF-B1 (200x). plaque size and collagen/fibroblast ratio and may reverse or 0022 FIG. 6. Effects on collagen I synthesis and myo prevent the further development of the fibrosis observed in fibroblast differentiation in fibroblast cultures from the PD, ED, arteriosclerosis and other fibrotic conditions. human PD plaque by a cQMP analog (8-Br cGMP), esti mated by immunocytochemistry. Cells were incubated for 3 BRIEF DESCRIPTION OF THE DRAWINGS days with the indicated concentration and collagen I and 0016. The following drawings form part of the present ASMA were immuno-cytochenmically detected. Values are Specification and are included to further demonstrate certain means--/-SEM for three separate incubations. p.<0.05 were embodiments of the claimed subject matter. The embodi as follows: panel A: a VS b.c., panel D: a VS c, all others were ments may be better understood by reference to one or more non-significant. of these drawings in combination with the detailed descrip 0023 FIG. 7. Expression of PDE-5 mRNA and protein in tion presented herein. fibroblast cultures from human PD plaque and human and 0017 FIG.1. The effect of NO, TGF-B1, ROS and coMP rat normal tunica albuginea. (A) Ethidium bromide staining on fibroblast/myoblast differentiation and collagen deposi of PDE-5A cDNA bands generated from cell RNA by tion. RT-PCR and fractionated on agarose gels. (B) Luminol detection of PDE-5 protein bands obtained by western blot 0.018 FIG. 2. Inhibition of collagen deposition in the of cell extracts on PAGE. Arrows indicate PDE-5A variants. fibrotic plaque induced by TGF-B1 in the rat TA, by long DUP. cells from Dupuytren's nodules. (C) Microphoto term oral treatment with L-arginine and PDE inhibitors, graphs (200x) of cell cultures stained with the indicated estimated by Masson staining. (A) Microphotographs (40x) antibodies and counter-Stained with Meyer haematoxylin of croSS Sections of half of the rat penis. Dark arrows indicate the outer extent of plaque development and of 0024 FIG. 8. Gene transfer to the tunica albuginea of tunical thickening. Light arrowheads (lower right-hand cor plasmid and adenoviral cDNA constructs facilitated by ner of each panel) indicate the site of TGF-B1 injection. The electroporation. Both the pCMV-Bgal and the AdW-CMV control (-) was injected with TGF-B1 injection, no treatment Bgal constructs were injected into the tunica albuginea of the was given. L-ARG received a TGF-31 injection and L-argi rat, followed by electroporation, and 10 days later rats were nine in water. SIL received a TGF-B1 injection and sildenafil Sacrificed and frozen fixed tissue Sections were stained with in water. PXF received a TGF-31 injection and pentoxifyl X-gal, and counterStained with neutral red. TA: tunica line in water. (B) QIA evaluation (n=5 per group). Five albuginea. (200x magnification). Sections/animal, three fields/Section. 0025 FIG. 9. Induction of TGF-B1 expression in the 0.019 FIG. 3. Stimulation of apoptosis, as estimated by Peyronie-like plaque induced by fibrin in the tunica albug TUNEL, in the fibrotic plaque induced by TGF-31 injection inea of the rat. (A) Sections adjacent to the ones for plaques into the rat TA, following oral treatment with L-arginine, shown on FIG. 11 were immunostained with an antibody for sildenafil or pentoxiphylline. (A) Microphotographs (400x) TGF-B1. Arrows point to cells with intense staining. (220x of tissue Sections. Arrows indicate apoptotic cells in the Site magnification)(B) Image quantitation of positive cells in the of the plaque. The control (-) was injected with TGF-B1 average field of the fibrotic plaque (n=5). Values are injection, no treatment was given. L-ARG received a TGF means--/-SEM, and p values are as indicated. (200x mag B1 injection and L-arginine in water. SIL received a TGF-31 nification) US 2005/0085486A1 Apr. 21, 2005
0026 FIG. 10. Confirmation by RT/PCR of alterations in as indicated. Nitrotyrosine is a marker for peroxynitrite. (A) the expression of certain genes in the human Peyronies Micrographs from Selected arteries and tissue Sections, as plaque as compared to the normal tunica albuginea. indicated. Bar=50 um. (B) QIA as on FIG. 15, expressed as 0027 FIG. 11. PD-like plaque similar to the one induced intensity of immunostaining per area, as means--/-SEM. For in the rat can be elicited in the tunica albuginea of the mouse iNOS: Dorsal: A vs. Bp-0.05; Avs. C. N.S.; B vs C: p<0.05; by TGF-f1 injection, detected by Masson staining. Low (4x, Bulbo-urethral: A vs. B: p<0.05; A vs. C. N.S; B vs. C: top); and high (100x, bottom) magnifications of mouse penis p<0.05. For 3-nitrotyrosine: Dorsal: A vs. B: p<0.001; A vs. injected with saline and TGF-31. Light arrows show the site C: p<0.05; B vs. C. p-0.05; Bulbourethral: A vs. Bp-0.01; of TGF-31 injection. Boxes represent the area of high A vs. C. N.S; B vs. C. p-0.01. magnification. CC corpora cavernosa; UR: urethra; TA: tunica albuginea; Pl: plaque; NB: nerve bundle. 0033 FIG. 17. Intensification by iNOS blockade of aging-related oxidative StreSS in the arterial media. Tissue 0028 FIG. 12. Expression of PDE-4 mRNA and protein sections were immunostained for Cu, Zn" SOD and for in the human PD plaque and normal tunica albuginea, and Mn" SOD. (A) Micrographs from selected arteries and their homologous tissues in the TGF-31 rat model of PD, tissue sections only for Cui Zn" SOD, as indicated. and in fibroblasts cultured from these tissues. (A) Ethidium Bar=50 um. (B) QIA as on FIG. 16, expressed as intensity bromide staining of DNA generated from PD and normal of immunostaining per area, as means-/-SEM. Dorsal: A VS human TA tissue by RT/PCR with primers for PDE4A, PDE4B, and GAPDH (reference gene), separated by agarose C p<0.05; A vs. NS; B vs. NS; Bulbourethral: A vs. C gel electrophoresis. (B) PDE4 mRNA in rat penile shaft P-0.001; A vs. BP-0.05; B vs. P-0.01A vs B, C p-0.001; (PS), rat TA cells, or human TA or PD cells. TA: tunica B vs. C. NS. Mn'" SOD gave essentially similar results (not albuginea, PD: Peyronie's disease, CC: corpora cavernosa shown). smooth muscle; PS: penile shaft. (C) Luminol-stained pro 0034 FIG. 18. Differential expression of another marker tein bands on western blots of human tissue and cell extracts of oxidative StreSS, heme oxygenase 1, in the adventitia of with the antibody against PDE4A. the arterial wall. Sections were immunostained with an 0029 FIG. 13. Immunodetection of PDE-4 protein in the antibody against heme oxygenase I and counterstained with rat penis and cultures of rat and human tunica albuginea hematoxylin. (A) Micrographs from the dorsal artery. Bar= fibroblasts. Microphotographies of tissue sections (top pan 50 um. (B) QIA as on previous figures. Values expressed as els) or cell cultures (middle and botom panels), as indicated, means+/-SEM. *p-0.05: young vs old (t test). Submitted to immunodetection with antibodies against PDE4A or PDE4D, and counterstained with Meyer haema 0035 FIG. 19. Reduction by iNOS blockade of the toxylin. aging-related Stimulation of apoptosis in the media of the penile arteries. Sections were immunostained with the 0030 FIG. 14. Effect of pentoxifylline and sildenafil on TUNEL procedure and counterstained with methyl green. cAMP and c(GMP levels in fibroblast cultures from human (A) Micrographs from Selected arteries and tissue sections, PD plaque, estimated by enzyme immunoassayS. Cells were as indicated. Bar=50 um (B) QIA as on previous figures, incubated for 3 days in fibroblast growth medium (FGM)/ expressed as apoptotic index (percent number of apoptotic 10% fetal bovine serum, in the presence of SNAP (100 uM; cells/total number of cells), as means+/-SEM. Dorsal: A vs. medium changed daily) added 4 hs prior to the PDE inhibi B, C p<0.001; B vs. C p-0.05; Bulbourethral: A vs. C tors, and increasing concentrations of Sildenafil or pentoxi p-0.001; A vs. p-0.05; B vs C p-0.01 fylline. c6MP and cAMP levels were measured in cell homogenates. Results are the means of 2 Separate experi 0036 FIG. 20. Intensification by iNOS blockade of the ments conducted in triplicate. (A) coMP levels in the aging-related Stimulation of PAI expression in the media of presence of sildenafil. (B) coMP levels in the presence of the penile arteries. Sections were immunostained with an pentoxifylline. (C) cAMP levels in the presence of pentoxi antibody against PAI and counterstained with hematoxylin. fylline. (A) Micrographs from the dorsal artery. (B) QIA for both the 0031 FIG. 15. Intensification by iNOS blockade of dorsal penile and bulbourethral arteries, as on previous aging-related fibrosis in the arterial media. Old male rats figures. Bar=50 Itm Values expressed as means-/-SEM. a were given L-NIL for 3 weeks, or left untreated. Young vs. b.c. p<0.001. untreated rats Served as controls. Tissue Sections were 0037 FIG. 21. Effect of 8 Br-cGMP on apoptosis in obtained from penis (to visualize the dorsal and bulbo fibroblasts from human cultured PD plaque, estimated by urethral penile arteries) and from the aorta and femoral TUNEL. (A) Microphotographs (200x) of apoptotic cells in artery, and stained with Masson (SMC: red; collagen: blue). incubations receiving no addition and 400 uM 8 Br-cGMP (A) Micrographs from Selected arteries and tissue sections, for 3 days. (B) Apoptotic index, as means+/-SEM for three as indicated. Bar=50 um. (B) Quantitative image analysis Separate incubations. a VS b,c p>0.05. (QIA) expressed as ratios of areas occupied by SMC and collagen, as means-/-SEM. Aorta: A vs. B, C p<0.001; B vs. 0038 Table 1. Differential profiles of selected gene C p<0.01; Femoral: A Vs B, C p-0.001; B vs. C p-0.01; expression in human Peyronies plaques and Dupuytren’s Dorsal: A Vs B, C P-0.001 B vs. P-0.05; Bulbourethral: A nodules against their respective control tissues determined vs B, C p-0.001; B vs. C: NS. with a DNA microarray assay. 0032 FIG. 16. Reduction by iNOS blockade of the 0039 Table 2. Effect of aging on arterial wall thickness aging-related Stimulation of the nitrosative pathway in the and lumen diameter in large and Small arteries in the rat n=5; media of the penile arteries. Sections were immunostained **: p-0.01. US 2005/0085486A1 Apr. 21, 2005
DETAILED DESCRIPTION OF ILLUSTRATIVE 0066. As used herein, an “inhibitor” of PDE5 means any EMBODIMENTS compound or combination of compounds that acts to decrease the activity of PDE5, either directly or indirectly. 0040 Abbreviations and Definitions An inhibitor can be a molecule, an atom, or a combination 0041. The following abbreviations are used herein. Other of molecules or atoms without limitation. The term “antago abbreviations not listed below have their plain and ordinary nist” of PDE5 is generally synonymous with an “inhibitor” meaning. of PDE5. Inhibitors may act directly on PDE5 by, for example, binding to and blocking the catalytic Site or Some 0042 ASMA: C-Smooth muscle actin; other functional domain of PDE5 that is required for activity. 0043 ED: erectile dysfunction; An inhibitor may also act indirectly, for example, by facili tating or interfering with the binding of PDE5 to another 0044) iNOS: inducible NOS (also NOS II); protein or peptide. 0045 L-NAME: L-N-Nitro-L-arginine methyl 0067. This application also concerns, at least in part, esther; isolated proteins and nucleic acids for type 4 phosphodi 0046 L-NIL: L-iminoethyl-L-lysine; esterase (PDE4, e.g., GenBank Accession Nos. NM006203, NM002600, NM006202, NPO06194, NPO02591, 0047 MMP: matrix metalloproteinase; NP006193), as well as methods of therapeutic treatment of fibrotic diseases directed towards Such proteins. In the 0.048 nNOS: neuronal NOS; present disclosure, reference to “PDE4' or “type 4 phos 0049) NO: nitric oxide; phodiesterase' without further qualification or limitation means any or all isoforms of PDE4. The terms “PDE4 0050 NOS: nitric oxide synthase; isoform” and PDE4 “inhibitor” or “antagonist” are used 0051) PAI: plasminogen activator inhibitor; consistently with the corresponding terms defined above for 0052 PD: Peyronie's disease; PDE5. 0068 Peyronie's Disease and Other Fibrotic Conditions 0053) PDE: phosphodiesterase; 0069 Peyronie's Disease 0054) PKG: protein kinase G; 0070 Peyronie's disease (PD) is a localized fibrosis of 0055) PPC: pluripotent cells; the tunica albuginea (TA) of the penis (Hellstrom and QIA: quantitative image analysis, Bivalacqtia, 2000; Gonzalez-Cadavid et al., 2002; Gholami 0056) et al., 2002) affecting close to 5% of the male population 0057) ROS: reactive oxygen Species, (Schwarzer et al., 2001). The leading theory of the etiology of PD is that it results from an abnormal wound healing 0058 SMC: Smooth muscle cell; process of the TA Subsequent to an injury, usually during 0059) SNAP: S-Nitroso-N-acetyl penicillamine; coitus (Hellstrom and Bivalacqua, 2000; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002, Jarow and Lowe, 1997; 0060) TA: tunica albuginea; Devine et al., 1997). It is assumed that at the time of the 0061 TGF-B 1: transforming growth factor-f1; injury, extravasation of blood borne proteins, mainly fibrin, into the TA occurs (Somers and Dawson, 1997; Van de 0062) TIMP: tissue inhibitor of MMP Water, 1997; Herrick et al., 1999). Some of these foreign proteins may induce a Severe but local inflammatory 0.063 AS used herein, “a” or “an” may mean one or more response in the TA resulting in the local release of pro than one of an item. fibrotic factors, mainly transforming growth factor B1 (TGF 0064. This application concerns, at least in part, isolated f31) and reactive oxygen species (ROS) (Hellstrom and proteins and nucleic acids for type 5 phosphodiesterase Bivalacqua, 2000; Gonzalez-Cadavid et al., 2002; Gholami (PDE5, e.g., GenBank Accession Nos. NM033437, et al., 2002), which then trigger excessive deposition and NM033431, NM033430, NM001083, NP246273, disorganization of the collagen fibers. NP237223, NP236914, NP001074), as well as methods of 0071. Within the injured TA of some individuals, the therapeutic treatment of fibrotic diseases directed towards above process may result in: a) an increase in the differen such proteins. In the present disclosure, reference to “PDE5” tiation of TA fibroblasts into myofibroblasts (Vernet et al., or “type 5 phosphodiesterase” without further qualification 2002); b) an increased deposition of collagen fibers by both or limitation means any or all isoforms of PDE5. the TA fibroblasts and myofibroblasts (Vernet et al., 2002; 0065. A “PDE5 isoform” is a variant of type 5 phos Ferrini et al., 2002); c) a decrease in apoptosis of the TA phodiesterase that differs in its primary structure (i.e., amino fibroblasts/myofibroblasts, and d) a decrease in the natural acid sequence) from other isoforms of PDE5. The term breakdown and reorganization of newly deposited collagen encompasses, but is not limited to, isoforms that are pro fibers that is normally performed by the matrix metallopro duced by truncation, amino acid Substitution (mutation) or teinases (MMP) (Mignatti et al., 1996; Arthur, 2000). The by alternative mRNA splicing, So long as Some difference in MMPs are the collagenolytic enzymes that are involved in amino acid Sequence results. For the purposes of the present the natural turnover of collagen in the wound healing invention, other types of covalent modification would be process. At its extreme, this process may become excessive, considered to fall within the Scope of a single isoform. For with newly deposited collagen and extracellular matrix in a example, both phosphorylated and unphosphorylated forms tissue that fails to “heal and reorganize normally” (Mignatti of PDE5 would be considered to represent the same isoform. et al., 1996) eventually becoming calcified (Gelbard, 1988; US 2005/0085486A1 Apr. 21, 2005
Muralidhar et al., 1996). Calcification may occur by osteo regulation of fibroblast replication and myofibroblast differ blasts that are transformed from pluripotent cells (PPC) entiation; and c) the consequent or independent reduction in among the fibroblasts and/or myofibroblasts within the TA the transcriptional expression of collagen I. by either autocrine and/or paracrine factor(s). 0076 An additional mechanism for NO to counteract 0.072 The primary cell that is involved in collagen syn fibrosis may involve stimulation of myofibroblast and/or thesis in the wound healing process is the fibroblast (Singer fibroblast programmed cell death. The induction of apopto and Clark, 1999). For wound closure, some of the fibroblasts sis by NO is well documented, either in vitro by NO donors, must differentiate into myofibroblasts (Vernet et al., 2002; such as S-Nitroso-N-acetyl penicillamine (SNAP) (Sikka et Gonzalez-Cadavid et al., 2002; Gholami et al., 2002; al., 2002; Nishio et al., 1996) or inducible nitric oxide Muralidhar et al., 1996; Singer and Clark, 1999, Powel et al., synthase (iNOS) expression (Nishio et al., 1996; Tain et al., 1999), cells that are intimately involved in the terminal 2002), or in vivo by neuronal NOS (nNOS) activation Stages of the wound healing process. Once this proceSS is (Ferrini et al., 2001b), iNOS induction (Ferrini et al., 2001b; completed, the myofibroblast is normally eliminated from Watanabe et al., 2002), or administration of the NOS sub the wound by apoptosis (Gabbiani, 1996). If myofibroblasts strate L-arginine (Wang et al., 1999; Holm et al., 2000). The persist and do not undergo pre-programmed cell death, they proposed antifibrotic role of iNOS is in agreement with may continue to Synthesize additional collagen and extra indirect results obtained in animal models of kidney and cellular matrix, leading to an increase in fibrosis. Within the cardiac fibrosis, where general NOS inhibitors (not isoform TA, this increase in fibrosis may lead to the clinical recog Specific), Such as L-N-Nitro-L-arginine methyl esther nition of a palpable Peyronie's plaque. (L-NAME), cause or exacerbate fibrosis (Chatziantoniou et al., 1998; Boffa et al., 1999; Pechanova et al., 1999). 0073. In addition to being a stimulator of the differentia L-arginine Supplementation has been shown to be anti tion of fibroblasts into myofibroblasts, TGF-31 can be fibrotic in vascular and renal disease (Peters et al., 2000), but secreted by both fibroblasts and myofibroblasts (Powel et al., has not been tested on the PD plaque. 1999; Tomasek et al., 1999; Walker et al., 2001). In other fibrotic conditions, like cardiac and renal fibrosis, TGF-31 0077 Thus, pharmacologic inhibition of the pro-fibrotic has been shown to not only increase the replication and process and/or Stimulation of the anti-fibrotic processes, differentiation of fibroblasts into myofibroblasts, but also to may halt the progression and/or reverse the process of PD. inhibit apoptosis of the myofibroblasts (Desmouliere, 1995; More globally, Such results may be extrapolated to more Chipev et al., 2000). This self-perpetuating cycle of cellular life-threatening fibrotic conditions Such as renal, lung, liver, differentiation of fibroblasts into myofibroblasts and contin and cardiac fibrosis (Nagase and Brew, 2002; Martinez ued TGF-B1 Secretion by these same two cell types, as a Hernandez, 1994; Schuppan et al., 2000). The results dis result of the exposure of these cells to TGF-31 itself, may closed herein provide novel avenues of therapy for not only ultimately lead to excessive collagen Synthesis. Coupled PD but also for fibrosis in general. with a disorganization of collagen fibers and a decrease in their degradation by at least a partial inhibition of the MMPs 0078. The interaction within the TA between the pro (Iredale, 1997), this may explain the continued growth of the fibrotic and anti-fibrotic factors acting on fibroblasts and PD plaque. myofibroblasts and their respective differentiation and apo ptotic processes is outlined in FIG. 1. One of the major 0074) In response to the pro-fibrotic effects of TGF-B1 functions of NO produced by iNOS in the PD tissue is to and ROS in the TA, the injured TA tissue attempts to react with the pro-fibrotic compound ROS to form perox counteract these pro-fibrotic processes by releasing the ynitrite (Beckmann and Koppenol, 1996; Ferrini et al., anti-fibrotic compound, nitric oxide (NO). NO in the TA is 2001a; Vernet et al., 1998). Peroxynitrite is known to induce Synthesized by the inducible nitric oxide Synthase enzyme apoptosis in most cell types, and Specifically of the collagen (iNOS) (Vernet et al., 2002; Gonzalez-Cadavid et al., 2002; producing cells such as the fibroblast and myofibroblast Gholami et al., 2002; Ferrini et al., 2002). Therefore, in the (Heigold et al., 2002; Duffield et al., 2000; Zhang and Phan, development of PD, there may be a constant battle between 1999). In addition, NO stimulates guanylyl cyclase to pro pro-fibrotic and anti-fibrotic processes within the TA, as is duce coMP Gonzalez-Cadavid et al., 1999), which in turn evident from the results from DNA microarrays discussed in stimulates PKG (protein kinase G) (Sinnaeve et al., 2002; the Examples below, with the winner determining whether Wollert et al., 2002). Like NO, both cgMP and PKG inhibit normal healing or a fibrotic condition ensues. collagen Synthesis and are anti-fibrotic (Sinnaeve et al., 2002; Wollert et al., 2002; Hofmann et al., 2000; Chen et al., 0075 AS discussed in the Examples below, studies on the 1999a, 1999b; Redondo et al., 1998). c6MP is normally corresponding human tissues and cells combined with data degraded to inactive GMP by the phosphodiesterase (PDE) from rat models have shown that the development of the PD enzymes (Corbin and Francis, 1999; Uckert et al., 2001). plaque is associated with expression of inducible nitric oxide Some of the inhibitors of these enzymes, like pentoxifylline, synthase (INOS) and stimulation of nitric oxide (NO) syn have also been shown to be anti-fibrotic in animal models thesis, in conjunction with an increase in Oxidative StreSS and ROS levels (Vernet et al., 2002; Gholami et al., 2002; and humans (Fischer et al., 2001; Desmouliere et al., 1999; Hellstrom and Bivalacqua, 2000; Sikka et al., 2002). The Kremer et al., 1999). specific inhibition of iNOS activity with L-iminoethyl-L- 0079 The accumulation of collagen, which is one of the lysine (L-NIL) exacerbates fibrosis in the TGF-B1 rat model, histological hallmarks of tissue fibrosis, may in part be also consistent with a model wherein NO produced by iNOS due to the inactivation of the MMP enzymes that degrade the plays an antifibrotic role in PD by at least three mechanisms: already laid-down collagen fibers during its natural turnover a) the quenching of the pro-fibrotic ROS by a reaction cycle (Mignatti et al., 1996; Arthur, 2000). MMPs can be leading to the formation of peroxynitrite; b) the down inactivated by TIMPs, the tissue inhibitors of MMP, that US 2005/0085486A1 Apr. 21, 2005 have been shown to increase in fibrotic conditions (Iredale, erosclerosis, arteriosclerosis, restenosis, cardiac hypertro 1997; McCrudden and Iredale, 2000; Arthur, 2000). Another phy or any other condition characterized by excessive fibro anti-fibrotic effect of NO is that it stimulates MMP activity blast or Smooth muscle cell proliferation or deposition of (Sasaki et al., 1998; Okamoto et al., 1997) and inhibits the collagen and extracellular matrix in the blood vessels and/or expression of TIMP (Darby et al., 2002; Bugno et al., 1999). heart. Both the vagina and clitoris are known to undergo 0080. The results disclosed below provide a series of fibrosis and hardening with aging, menopause and estrogen/ approaches (Magee et al., 2002b; Ferrini et al., 2002) testosterone deficiency. Together with poor lubrication, the focused on the role of the myofibroblast and the interaction vaginal/clitoral fibrosis contribute to the development of between NO and ROS (the NO/ROS balance) in the patho female sexual arousal disorder, affecting about 30 to 40% of genesis of the PD plaque and in arteriosclerosis, particularly women. (Traish et al., Arch. Sex Behav. 31:393-400, 2002; of penile arteries. These include the use of the established Park et al., Intl. J. Impot. Res. 13:116-124, 2001; Berman et TGF-31 rat model of PD (Ferrini et al., 2002; Vernet et al., al., J. Sex Marital Ther. 27:411-420, 2001; Berman et al., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002), Urology 54:385-391, 1999; Berman et al., Fertil. Steril., the establishment and study of cell cultures from the human 79:925-931, 2003.) The mechanisms of fibrosis are similar PD and normal TA tissues (Vernet et al., 2002; Gonzalez for a number of different organs and disease States. Cadavid et al., 2002; Gholami et al., 2002), the application 0083) A distinction exists between long-term (weeks, of quantitative image analysis (OIA) of tissue Sections and months, years) continuous treatment with, for example, a cells Subjected to histochemistry and immunohistochemistry PDE5 inhibitor Such as Sildenafil to maintain a constant level (Ferrini et al., 2002; Vernet et al., 2002; Gonzalez-Cadavid of these agents in order to arrest or regreSS a fibrotic et al., 2002; Gholami et al., 2002), the use of selective condition, Versus on demand (prior to the Sexual act) single inhibitors of some of the biochemical pathways shown in pill, short-term treatment with sildenafil or other PDE5 FIG. 1 (Ferrini et al., 2002; Vernet et al., 2002; Gonzalez inhibitors to obtain Smooth muscle vasodilation in the penis Cadavid et al., 2002; Gholami et al., 2002), DNA microar (male penile erection) or vagina/clitoris (female Sexual rays for multiple gene expression (Magee et al., 2002b), the arousal) upon Sexual stimulation. Current studies with discovery that the same processes that occur in the PD Sildenafil are Symptomatic to treat defects in vaginal/clitoral fibrotic plaque are also involved in aging-related fibrosis of or penile vasodilation exclusively during a Sexual act and are the arterial wall media leading to arteriosclerosis, which by not addressed to the long-term cure of underlying tissue affecting the penile arteries would cause ED, and other fibrosis. Additional details relevant to the treatment of molecular biology assays that provide an integrated analysis fibrotic conditions are disclosed in the Examples Section of the molecular pathophysiology of this condition, with below as well as in the references of Vernet et al. (2002), corresponding novel approaches to therapeutic intervention Gonzalez-Cadavid et al. (2002) and Gholami et al. (2002), of fibrotic disease directed towards the underlying molecular the entire texts of which are Specifically incorporated herein pathways. by reference. 0081. The combination of 1) an agent that increases NO, 0084 Peripheral Vascular Disease, Erectile Disfunction cGMP or cAMP levels, with 2) a compound that reduces and Hypertension oxidative stress and ROS levels, such as an antioxidant, will preserve the antifibrotic effects of agents in the first category, 0085 One of the prevalent views of peripheral vascular without an undesirable excessive level of apoptosis that may disease is that it is caused by oxidative damage to the arterial lead to cytotoxicity in cells other than myofibroblasts (e.g., wall by reactive oxygen species (ROS), that cause lipid Smooth muscle cells, neurons, endothelial cells, etc.) By peroxidation and other alterations (Cai and Harrison, 2000; reducing ROS with the antioxidant, the formation of del Berry et al., 2001; Zalba et al., 2000). These compounds are eterious levels of peroxynitrite (the product of ROS quench mainly produced by Xanthine oxidase, NADPH oxidase, as ing by NO) would be reduced to the minimum required for well as mitochondrial enzymes, and are counteracted by effective antifibrotic effects on myofibroblasts and fibro heme-oxygenase I and Superoxide dismutase (SOD), which blasts involved in excessive collagen and extracellular can reduce ROS by acting as endogenous antioxidants. In matrix Synthesis, without damage to other tissues. In the addition to causing endothelial damage, ROS are known case of the combination of an antioxidant with agents in the stimulators of collagen deposition and SMC proliferation first category raising c(GMP or cAMP levels, the reduction in (Berry et al., 2001; Zalba et al., 2000) in the vascular wall. ROS would allow more endogenous NO levels to be pre Xanthine oxidase and SOD are also present in the penile Served. Therefore, the combination of agents may be more corpora cavernosa (Jones et al., 2002), and oxidative stress effective and Safe than a Single agent in either category due to ROS has been postulated to be central to impaired alone. cavernosal function in aging-related ED (Jones et al., 2002; Khan et al., 2001; Bivalacqua et al., 2003). 0082 The skilled artisan will realize that the methods and compositions disclosed herein are of use not only for treat 0.086 Besides antioxidants, nitric oxide (NO) also ment of Peyronie's disease and ED due to loss of cavernosal quenches ROS in the vasculature, as shown by the increase Smooth muscle in the trabecular spaces and penile arteries, in ROS levels and the development of cardiac and renal but also for other conditions involving fibrosis, Such as fibrosis and vascular Stiffness when there is long-term sys penile corporal fibrosis, Dupuytren's disease nodules, vagi temic blockade of NOS activity with NOS inhibitors (Kita nal fibrosis, clitoral fibrosis, female Sexual arousal disorder, moto et al., 2000; Gonzalez et al., 2000; Usui et al., 1999). abnormal wound healing, keloid formation, general fibrosis The ROS-quenching and anti-fibrotic effects of NO are not of the kidney, bladder, prostate, skin, liver, lung, heart, limited to the SMC and can be demonstrated in other intestines or any other localized or generalized fibrotic non-vascular conditions (Ferrini et al., 2002; Vernet et al., condition, Vascular fibrosis, arterial intima hyperplasia, ath 2002). In this process, NO reduces ROS levels through the US 2005/0085486A1 Apr. 21, 2005 formation of peroxynitrite (Cai and Harrison, 2000; Jones et hyperplasia in atherOSclerosis and restenosis and ameliorate al., 2002; Ferrini et al., 2002; Vernet et al., 2002; Gewaltig fibrosis in other systems (Ferrini et al., 2002; Vernet et al., and Kojda, 2002), thereby increasing the NO/ROS ratio. NO 2002; Gewaltig et al., 2002; Behr-Roussel et al., 2000). But is also postulated to not only inhibit collagen Synthesis excessive peroxynitrite may also be noxious, if it leads to a directly, but to favor collagen degradation by Stimulating loss of SMC and the subsequent impairment of tissue metalloproteinases and down-regulating expression of their relaxation. We propose that during aging, iNOS induction in inhibitors, such as the plasminogen activator inhibitor (PAI) the vasculature is not restricted to the cavernosal SMC (Li et al., 2000; Kaikita et al., 2002). The predominance of (Ferrini et al., 2001a) and large arteries (Goettsch et al., nitrosative pathways over oxidative StreSS is proposed to be 2001; Chou et al., 1998; Cernadas et al., 1998), but is protective against fibrosis (Ferrini et al., 2002; Vernet et al., generalized to the wall of the entire peripheral vascular tree. 2002), ED (Jones et al., 2002), atherosclerosis, and hyper This proceSS would aim to counteract oxidative StreSS and tension (Gewaltig and Kojda, 2002; Cheng et al., 2001). metalloproteinase inhibition, and the Subsequent decrease in 0087. The NO/ROS balance also directly modulates the the SMC/collagen ratio that causes loSS of compliance and relaxation of the vascular and penile Smooth muscle. The NO-induced vaso-relaxation. As disclosed in the following NO produced by the endothelial NOS in the vascular endot Examples, we have examined large and Small (resistance) helium controls blood pressure by relaxing the arterial SMC arteries in both young and aged rats for SMC/collagen ratio, (González-Cadavid et al., 1999). In the penile corpora iNOS, peroxynitrite, heme oxygenase I, SOD, PAI, and cavernosa, NO as a mediator of penile erection is produced SMC apoptosis, and determined how these parameters were by the neuronal NOS, specifically the PnNOS variant (Berry affected in aged rats when iNOS activity was specifically et al., 2001), localized in the nerve terminals, and to a lesser inhibited with L-NML. extent by endothelial NOS in the lacunar and sinusoidal 0090 NO/cGMP Inhibition of Fibrogenic Pathways endothelium of the penis (González-Cadavid et al., 1999). In experimental animals, reduction in NOS levels in the vas 0091. In molecular terms, the fibrotic process occurring culature and penile corpora is associated with hypertension during abnormal wound healing, e.g., in dermal wounds, is (Gewaltiget al., 2002) and with ED, respectively (González essentially an increased and disorganized collagen deposi Cadavid et al., 1999; Garban et al., 1995; Berry et al., 2001). tion impairing granulation tissue formation. This is accom If oxidative stress becomes excessive, the reaction of ROS panied by an increase in the local production and Secretion with NO to form peroxynitrite reduces NO concentration in of TGF-1 (Klar and Morrisey, 1998; Badalamente et al., the tissues, which may lead to hypertension and ED by 1996; Wahl, 1997), a factor which: a) stimulates collagen impairing NO dependent SMC relaxation. synthesis (Tiggelman et al., 1997; Faouzi et al., 1999), b) inhibits collagenolysis (van der Zee et al., 1997) and fibrin 0088. It is still unknown to what extent these neuronal olysis (Holmdahl et al., 2001), c) enhances the release of and endothelial NOS isoforms participate in producing NO ROS (Casini et al., 1997; Muriel, 1998a), and d) transcrip as an antifibrotic mechanism. In contrast, more direct evi tionally represses iNOS (Hung et al., 1995). dence has emerged recently on the role of the inducible isoform of NOS (iNOS) (Kibbe et al., 1999) in reducing 0092 ROS are hydroxyl radicals and Superoxide anions ROS and modulating the SMC/collagen ratio in different that are quenched by NO to primarily form peroxynitrite tissues. iNOS is spontaneously induced in the corpora cav (Poli, 2000; Curtin et al., 2002; Cattell, 2002; Kim et al., ernosa (Ferrini et al., 2001a) and brain (Vernet et al., 1998; 2001; Fanet al., 2000; Ito et al., 1992). The balance between Ferrini et al., 2001b) during aging, and in certain fibrotic NO and ROS levels is known to be considerably altered in conditions (Ferrini et al., 2002; Vernet et al., 2002). In the other fibrotic conditions affecting liver (cirrhosis), lung vasculature, iNOS is also induced in the media in aging (pulmonary fibrosis), kidney (renal fibrosis), heart (cardiac related arterial stiffness (Goettsch et al., 2001; Chou et al., hypertrophy), and the vascular tree (arterial medial hyper 1998; Cernadas et al., 1998), transplant arteriosclerosis (Lee plasia). This abnormal ratio between NO and ROS is et al., 1999), and atherosclerosis (Ihrig et al., 2001; Niu et believed to be due to both a decrease in local NO synthesis al., 2001; Behr-Roussel et al., 2000), and it is assumed to (presumably via iNOS) and an increase in ROS (Casini et inhibit collagen deposition and prevent medial hyperplasia al., 1997; Muriel, 1998a; Curtin et al., 2002; Cattell, 2002; via induction of SMC apoptosis and/or inhibition of SMC Kim et al., 2001; Fan et al., 2000). ROS, produced by the replication (Gewaltig and Kojda, 2002; Kibbe et al., 1999; macrophages and neutrophils, have been shown to induce Niu et al., 2001). The specific inhibition of iNOS activity by lipid peroxidation in cell membranes and increase vascular L-N-(iminoethyl)-lysine acetate (L-NIL) (Ferrini et al., permeability and leakage of fibrinogen and other clotting 2002; Vernet et al., 2002; Behr-Roussel et al., 2000), or the factors into tissue (Cattell, 2002; Kim et al., 2001). ROS blockade of iNOS expression in the iNOS knockout mouse generation during oxidative StreSS is accompanied by a (Ihrig et al., 2001; Niu et al., 2001; Hochberg et al., 2000), considerable induction of heme-oxygenase-1 (HO-1) causes fibrosis in non-vascular tissues, a decrease in NO/per (Foresti et al., 1999; Nathan, 1997), the enzyme that protects Oxynitrite levels, an increase in ROS, and a reduction in the against oxidative StreSS and acts as an anti-apoptotic and SMC/collagen ratio. anti-inflammatory (Ryter and Choi, 2002) response. HO-1 can also be elicited by peroxynitrite. 0089. Despite the fact that a certain predominance of the nitrosative over the oxidative pathways may preserve the 0093. Among the several regulators of collagen deposi normal integrity and function of blood vessels and corpora tion and wound healing, NO is particularly interesting as an cavernosa, an excessive production of NO and peroxynitrite, inhibitor of fibrosis. In the case of PD, NO appears to be may also induce apoptosis and cell loss (Ferrini et al., 2001a, produced by the induction of iNOS in the TA (Ferrini et al., 2002; Vernet et al., 2002; Kibbe et al., 1999). Depending on 2002; Vernet et al., 2002; Gonzalez-Cadavid et al., 2002; the context, this may be beneficial by preventing media Gholami et al., 2002). This iNOS isoform is involved in US 2005/0085486A1 Apr. 21, 2005 producing persistent high levels of NO by transcriptional promote apoptosis (Loweth et al., 1997; Sirotkin et al., 2000; induction, essentially as a defensive mechanism during Taimor et al., 2000). PDE inhibitors, by elevating c(3MP, inflammation (Nathan, 1997a, 1997b). iNOS is physiologi also cause similar effects in vitro (Schade et al., 2002; Horio cally expressed in the adult at very low basal levels, if at all. et al., 1999; Thompson et al., 2000), and in particular induce Only upon induction by cytokines, Such as tumor necrosis apoptosis in vivo (Chan et al., 2002; Takuma et al., 2001). factor C. (TNFC), interleukin 1B (IL-1B), interferon-Y Some of these PDE inhibitors, like pentoxifylline, are active (INFY), and related factors, does iNOS induction take place. in preventing experimental fibrosis in the lung, liver, and It can under certain chronic conditions lead to a high, and heart (Fischer et al., 2001; Desmouliere et al., 1999; Kremer Some times excessive production of NO that acts as either a et al., 1999), and are currently being used for the treatment cytotoxic agent, or, in the Specific case of collagen, inhibits of human liver fibrosis (Windmeier and Gressner, 1997) and fiber deposition. These conditions include inflammation, Crohn's disease (Reimund et al., 1997). infections, cancer, degenerative diseases and aging, where the factors triggering this increased iNOS response are 0097 Role of Myofibroblast in Fibrosis unknown (Kibbe et al., 1999; Wang et al., 2002a; Miller and 0098. One of the major pathological findings in tissue Sandoval, 1999). Additionally, many NO metabolites, par fibrosis is the presence of activated and proliferating myo ticularly peroxynitrite, trigger localized apoptosis and tissue fibroblasts. These cells not only play an important role in the toxicity (Nathan, 1997a). contraction phase of normal wound healing but they also are 0094. The specific role of NO as a regulator of wound responsible for the development of tissue fibrosis or of a scar healing is well established in vivo and in vitro (Curtin et al., (Powel et al., 1999; Tomasek et al., 1999; Walker et al., 2002; Cattell, 2002; kim et al., 2001; Hogaboam et al., 1998; 2001). The myofibroblast (FIG. 1) is the cell widely Rizvi and Myers, 1997; Cao et al., 1997; Chatziantoniou et believed to generate the contracture in Dupuytren's disease, al., 1998; Kolpakov et al., 1995). NO donors and the NOS the condition present in 10-20% of PD cases (Connelly, Substrate, L-arginine, have been shown to inhibit collagen 1999). It is believed that after fulfilling its role in wound fiber ((Curtin et al., 2002; Cattell, 2002; kim et al., 2001; contraction and in Secreting collagen during healing, the Hogaboam et al., 1998; Rizvi and Myers, 1997; Cao et al., myofibroblast disappears by programmed cell death (apop 1997; Chatziantoniou et al., 1998; Kolpakov et al., 1995) tosis) (Gabbiani, 1996). However, in Dupuytren’s disease and fibrin deposition (Westenfeld et al., 2002; Dambisya and and other fibrotic conditions the myofibroblast persists, Lee, 1996; Catani et al., 1998; Dambisya et al., 1996), and resulting in a persistent fibrosis and contracture (Kloen, TGF-B1 synthesis (Craven et al., 1997). The experimental 1999; Badalamente and Hurst, 1999). Morphologically, decrease of NO synthesis by NOS inhibition, or the reduc myofibroblasts are intermediate between the fibroblast and tion of iNOS induction, leads to impaired wound healing the Smooth muscle cell. Phenotypically, they express large (Schaffer et al., 1997a), and also to fibrosis, as documented bundles of actin filaments (actin, myosin, and associated in myocardial hypertrophy, coronary vascular remodeling proteins: “stress fibers”), with a fibrillar space material following an infarct, cystic fibrosis, obstructive nephropa named the “fibronexus', composed of fibronectin. Myofi thy, and pulmonary fibrosis (Takemoto et al., 1997; babal et broblasts can be identified by the detection of both C. Smooth al., 1997; Numaguchi et al., 1995; Ikeda et al., 1997; Moreno muscle actin (ASMA) (absent in fibroblasts), and vimentin et al., 1996; Morrissey et al., 1996; Kelley and Drumm, (absent in Smooth muscle cells). 1998). Physiologically, the reduction of NO synthesis may 0099] It is believed that myofibroblasts can originate occur by either transcriptional blockade of iNOS induction from either fibroblasts, Smooth muscle cells, or from an as (Geller and Billiar, 1998; Forstermann et al., 1998), and in yet uncharacterized stem cell (Powel et al., 1999; Tomasek certain cases, down-regulation of eNOS (Forstermann et al., et al., 1999; Walker et al., 2001). Upon the appropriate 1998), or by inhibition of NOS activity by advanced glyca stimulation, the myofibroblast may revert back to a fibro tion-end products (AGE) (Jiaan et al., 1995) or a natural blast or smooth muscle cell. Myofibroblasts express recep NOS competitive inhibitor such as asymmetric dimethyl tors for TGF-B1, PDGF, bFGF, endothelin, and prostaglan arginine (ADMA) (Boger et al., 1998). dins. All these factors generate in culture, an “activated” 0095. In contrast to the anti-fibrotic effect of NO that myofibroblast that is able to proliferate. In liver fibrosis, this would occur as a defense against fibrosis, in the early Stages activated form of the myofibroblast can be transformed into of normal wound healing NO actually Stimulates collagen the non-proliferating “stellate” form by either cAMP or synthesis (Schaffer et al., 1997b; Yamasaki et al., 1998; PGE2 (Powel et al., 1999; Tomasek et al., 1999; Walker et Thornton et al., 1998; Sherratt and Dallon, 2002; Diegel al., 2001; Wu and Zern, 2000). The activated myofibroblast mann, 1997). Therefore, the anti-fibrotic effects of NO may is then able to secrete cytokines, TGF-B1 and other growth be the result of a continuous and high level of local NO factors, and inflammatory mediators. Within the latter cat synthesis, like the one produced upon iNOS induction egory, the cell has been shown to release NO and ROS, and (Ferrini et al., 2002; Vernet et al., 2002). This shows the matrix proteins involved in wound repair and fibrosis, Such importance of the local levels of NO for either facilitating as collagens I, III, IV, VI, and XVIII, laminins, proteogly normal collagen deposition (wound healing) or preventing cans, adhesion molecules and MMPs (Powel et al., 1999; its accumulation (fibrosis). Tomasek et al., 1999; Walker et al., 2001). 0096) Anti-fibrotic effects of NO may also be mediated 0100. The myofibroblast is involved in functions such as by c0MP through guanylyl cyclase activation (Gonzalez wound repair in Skin and repair of the myocardium after Cadavid et al., 1999).. c6MP analogs inhibit collagen syn myocardial infarction. This cell has been implicated in the thesis (Chen et al., 1999a, 1999b; Redondo et al., 1998), pathophysiology of the Dupuytren contracture, keloids, fibroblast replication (Chiche et al., 1998; Pandey et al., myocardial fibrosis, ischemia reperfusion injury, coronary 2000), myofibroblast differentiation (Tao et al., 1999), and artery restenosis, glomerulonephritis, liver cirrhosis, pulmo US 2005/0085486A1 Apr. 21, 2005
nary interstitial fibrosis, and many other fibrotic conditions NOS, MMP or another protein may be incorporated into (Powel et al., 1999; Tomasek et al., 1999; Walker et al., expression vectors for therapeutic use in fibrosis. AS dis 2001). However, in PD, there are few reports on the role of cussed below, a gene encoding a given protein may contain the myofibroblasts, other than the description of the original a variety of different bases and yet still produce a corre culture of fibroblasts from the PD plaque (Somers et al., sponding polypeptide that is indistinguishable functionally, 1982) and a few more recent studies (Anderson et al., 2000a, and in Some cases Structurally, from the known Sequences of 2000b, Mulhall et al., 2001a, 2001b) and our own work Such genes. It is a matter of routine for the skilled artisan to (Vemet et al., 2002; Gonzalez-Cadavid et al., 2002; Gholami obtain known genomic and/or cDNA sequences encoding et al., 2002). In cell culture, PD fibroblasts demonstrate a various proteins from publicly available Sources, Such as faster replication rate as compared to those from the normal GenBank. TA, a higher production of a pro-fibrotic agent (basic fibroblast growth factor), and a potential alteration of the 0107 Any reference to a nucleic acid should be read as p53 pathway that normally represses cell replication and encompassing a host cell containing that nucleic acid and, in favors apoptosis, which would indicate a Sort of “immor Some cases, capable of expressing the product of that nucleic talization” in culture (Anderson et al., 2000a, 2000b, Mul acid. Cells expressing nucleic acids of the present invention hall et al., 2001a, 2001b). Other groups have studied fibro may prove useful in the context of Screening for agents that blast cultures from the Peyronie's plaque but did not focus induce, repress, inhibit, augment, interfere with, block, on their myofibroblast content (Duncan et al., 1991; abrogate, Stimulate, or enhance the catalytic activity and/or E1-Sakka et al., 1997a). regulatory properties of PDE4 and/or PDE5. 0101 Animal Models of PD 0.108 Nucleic acids according to the present invention may contain an entire gene, a cDNA, or a domain of a 0102) An animal model of PD has recently been devel protein that expresses catalytic activity. The nucleic acid oped (E1-Sakka et al., 1997b; El-Sakka et al., 1998), based may be derived from genomic DNA, i.e., cloned directly on the administration of a Synthetic heptapeptide of human from the genome of a particular organism. In preferred TGF-31 directly into the TA of the rat. After 45 days, the animal develops histological alterations and collagen depo embodiments, however, the nucleic acid would comprise sition resembling those observed in the human PD plaque complementary DNA (cDNA). (El-Sakka et al., 1997a, 1997b, 1998). The administration of 0109 The DNA segments of the present invention the full human TGF-B1 protein to the TA leads to a similar include those encoding biologically functional equivalent process in the rat model (Vernet et al., 2002; Gonzalez proteins and peptides. Such Sequences may arise as a con Cadavid et al., 2002; Gholami et al., 2002; El-Sakka et al., Sequence of codon redundancy and amino acid functional 1999, 1998). equivalency that are known to occur naturally within nucleic 0103) Another potentially useful animal model for the acid Sequences and the proteins thus encoded. Alternatively, study of the pathophysiology of the PD plaque is the functionally equivalent proteins or peptides may be created collagen I promoter transgenic mouse (Fakhouri et al., 2001; via the application of recombinant DNA technology, in Tharaux et al., 2000). This mouse carries the regulatory which changes in the protein Structure may be engineered, region of the collagen I-C2 gene linked to two reporter based on considerations of the properties of the amino acids genes, luciferase and B-galactosidase, So that whenever being eXchanged. Changes designed by man may be intro collagen mRNA Synthesis is stimulated luciferase and C-ga duced through the application of Site-directed mutagenesis lactosidase will be expressed. Both proteins can be esti techniqueS or may be introduced randomly and Screened mated by a chemiluminescence reaction in tissue homoge later for the desired function, as described below. nates, and B-galactosidase Specifically by the development 0110 Expression Vectors of a blue color in tissue Sections. 0.111) Nucleic acids encoding proteins or peptides may be 0104. This collagen 1 promoter mouse model has incorporated into expression vectors for production of the recently been used (Dussaule et al., 2000) to demonstrate the encoded proteins or peptides. Non-limiting examples of link between NO, endothelin, and collagen Synthesis in expression Systems known in the art include bacteria Such as kidney fibrosis, which is characterized by collagen I accu E. coli, yeast Such as Pichia pastoris, baculovirus, and mulation. In essence, by giving the NOS inhibitor L-NAME mammalian expression systems such as in COS or CHO to these transgenic mice for up to 14 weeks, it was possible cells. A complete gene can be expressed or, alternatively, to induce nephroangio- and glomerulo-fibrosis, accompa fragments of the gene encoding portions of polypeptide can nied by an increase in luciferase levels and an increased be produced. urinary excretion rate of endothelin. The blockade of endot helin receptors with the Selective ET antagonist boSentan 0112 The gene or gene fragment encoding a polypeptide reduced collagen deposition in the L-NAME animals, and may be inserted into an expression vector by Standard abolished collagen I promoter activation, as quantitated by Subcloning techniques. An E. coli expression vector may be luciferase activity. This animal model demonstrated that NO used which produces the recombinant polypeptide as a inhibition induces an early activation of the collagen I gene fusion protein, allowing rapid affinity purification of the in the kidney arterioles and glomeruli, Suggesting that NO protein. Examples of Such fusion protein expression Systems inhibits collagen deposition and the endothelin-mediated are the glutathione S-transferase System (Pharmacia, Piscat fibrogenic effect, as confirmed by other studies (Boffa et al., away, N.J.), the maltose binding protein system (NEB, 1999; Tharaux et al., 1999). Beverley, Mass.), the FLAG system (IBI, New Haven, 0105 Nucleic Acids Conn.), and the 6xHis system (Qiagen, Chatsworth, Calif.). 0106. In certain embodiments of the present invention, 0113 Some of these systems produce recombinant genes encoding one or more isoforms of PDE4, PDE5, PKG, polypeptides bearing only a Small number of additional US 2005/0085486A1 Apr. 21, 2005
amino acids, which are unlikely to affect the antigenic ability tide. These typically are Smaller insertions than the fusion of the recombinant polypeptide. For example, both the proteins described above and are introduced, for example, to FLAG system and the 6xHis system add only short disrupt a protease cleavage Site. Sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of the polypeptide 0118. The engineering of DNA segment(s) for expression to its native conformation. Other fusion Systems are in a prokaryotic or eukaryotic System may be performed by designed to produce fusions wherein the fusion partner is techniques generally known to those of skill in recombinant easily excised from the desired polypeptide. In one embodi expression. It is believed that Virtually any expression Sys ment, the fusion partner is linked to the recombinant tem may be employed in the expression of the claimed polypeptide by a peptide Sequence containing a specific nucleic acid Sequences. recognition Sequence for a protease. Examples of Suitable 0119) As used herein, the terms “engineered” and Sequences are those recognized by the Tobacco Etch Virus “recombinant cells are intended to refer to a cell into which protease (Life Technologies, Gaithersburg, Md.) or Factor an exogenous DNA segment or gene, Such as a cDNA or Xa (New England Biolabs, Beverley, Mass.). gene has been introduced through the hand of man. There fore, engineered cells are distinguishable from naturally 0114. The expression system used may also be one driven occurring cells that do not contain a recombinantly intro by the baculovirus polyhedron promoter. The gene encoding duced exogenous DNA segment or gene. Recombinant cells the polypeptide may be manipulated by Standard techniques include those having an introduced cDNA or genomic gene, in order to facilitate cloning into the baculovirus vector. One and also include genes positioned adjacent to a heterologous baculovirus vector is the pBlueBac vector (Invitrogen, Sor promoter not naturally associated with the particular intro rento, Calif.). The vector carrying the gene for the polypep tide is transfected into Spodoptera frugiperda (Sf9) cells by duced gene. Standard protocols, and the cells are cultured and processed 0120) To express a recombinant encoded protein or pep to produce the recombinant antigen. See U.S. Pat. No. tide, whether mutant or wild-type, in accordance with the 4,215,051 (incorporated by reference). present invention one would prepare an expression vector that comprises one of the claimed isolated nucleic acids 0115 Amino acid sequence variants of the polypeptide under the control of, or operatively linked to, one or more may also be prepared. These may, for instance, be minor promoters. To bring a coding Sequence “under the control Sequence variants of the polypeptide which arise due to of a promoter, one positions the 5' end of the transcription natural variation within the population or they may be initiation site of the transcriptional reading frame generally homologues found in other species. They also may be between about 1 and about 50 nucleotides “downstream” Sequences which do not occur naturally but which are (i.e., 3) of the chosen promoter. The “upstream” promoter sufficiently similar that they function similarly and/or elicit Stimulates transcription of the DNA and promotes expres an immune response that croSS-reacts with natural forms of Sion of the encoded recombinant protein. This is the mean the polypeptide. Sequence variants may be prepared by ing of “recombinant expression' in this context. Standard methods of Site-directed mutagenesis Such as those described herein. 0121 Many standard techniques are available to con Struct expression vectors containing the appropriate nucleic 0.116) Substitutional variants typically contain an alterna acids and transcriptional/translational control Sequences in tive amino acid at one or more sites within the protein, and order to achieve protein or peptide expression in a variety of may be designed to modulate one or more properties of the host-expression Systems. Cell types available for expression polypeptide Such as Stability against proteolytic cleavage. include, but are not limited to, bacteria, Such as E. coli and Substitutions preferably are conservative, that is, one amino B. Subtilis transformed with recombinant bacteriophage acid is replaced with one of Similar size and charge. Con DNA, plasmid DNA or cosmid DNA expression vectors. Servative Substitutions are well known in the art and include, for example, the changes of arginine to lysine; asparagine to 0122) Promoters that are most commonly used in recom glutamine or histidine, aspartate to glutamate; cysteine to binant DNA construction include the f-lactamase (penicil Serine, glutamine to asparagine; glutamate to aspartate; linase), lactose and tryptophan (trp) promoter Systems. histidine to asparagine or glutamine, isoleucine to leucine or While these are the most commonly used, other microbial Valine; leucine to Valine or isoleucine; lysine to arginine or promoters have been discovered and utilized, and details glutamine; methionine to leucine or isoleucine, phenylala concerning their nucleotide Sequences have been published, nine to tyrosine, leucine or methionine, Serine to threonine; enabling those of skill in the art to ligate them functionally threonine to Serine; tryptophan to tyrosine; tyrosine to with plasmid vectors. tryptophan or phenylalanine; and Valine to isoleucine or 0123 For expression in Saccharomyces, the plasmid leucine. YRp7, for example, is commonly used (Stinchcomb et al., 0117 Insertional variants include fusion proteins such as Nature, 282: 39, 1979; Tschemper et al., Gene, 10:157, those used to allow rapid purification of the polypeptide and 1980). This plasmid contains the trpl gene which provides a also may include hybrid proteins containing Sequences from Selection marker for a mutant Strain of yeast lacking the other proteins and polypeptides which are homologues of ability to grow in tryptophan, for example ATCC No. 44076 the polypeptide. For example, an insertional variant may or PEP4-1. The presence of the trpl lesion as a characteristic include portions of the amino acid Sequence of the polypep of the yeast host cell genome then provides an effective tide from one Species, together with portions of the homolo environment for detecting transformation by growth in the gous polypeptide from another species. Other insertional absence of tryptophan. variants may include those in which additional amino acids 0.124 Suitable promoting Sequences in yeast vectors are introduced within the coding Sequence of the polypep include the promoters for 3-phosphoglycerate kinase (Hitze US 2005/0085486A1 Apr. 21, 2005 man et al., J. Biol. Chem., 255:2073, 1980) or other glyco vaccinia virus 7.5K promoter). Further, it is also possible, lytic enzymes (Hess et al., J. Adv. Enzyme Reg., 7:149, 1968; and may be desirable, to utilize promoter or control Holland et al., Biochemistry, 17:4900, 1978), such as eno Sequences normally associated with the desired gene lase, glyceraldehyde-3-phosphate dehydrogenase, hexoki Sequence, provided Such control Sequences are compatible nase, pyruvate decarboxylase, phosphofructokinase, glu with the host cell systems. cose-6-phosphate isomerase, 3-phosphoglycerate mutase, 0.130. A number of viral based expression systems may pyruvate kinase, triosephosphate isomerase, phosphoglu be utilized, for example, commonly used promoters are cose isomerase, and glucokinase. In constructing Suitable derived from polyoma, Adenovirus 2, and most frequently expression plasmids, the termination Sequences associated Simian Virus 40 (SV40). The early and late promoters of with these genes are also ligated into the expression vector SV40 virus are particularly useful because both are obtained 3' of the Sequence desired to be expressed to provide easily from the virus as a fragment that also contains the polyadenylation of the mRNA and termination. SV40 viral origin of replication. Smaller or larger SV40 0.125 Other suitable promoters, which have the addi fragments may also be used, provided there is included the tional advantage of transcription controlled by growth con approximately 250 bp Sequence extending from the Hind ditions, include the promoter region for alcohol dehydroge site toward the Bgl I site located in the viral origin of nase 2, isocytochrome C, acid phosphatase, degradative replication. enzymes associated with nitrogen metabolism, and the 0131). In cases where an adenovirus is used as an expres aforementioned glyceraldehyde-3-phosphate dehydroge Sion vector, the coding Sequences may be ligated to an nase, and enzymes responsible for maltose and galactose adenovirus transcription/translation control complex, e.g., utilization. the late promoter and tripartite leader Sequence. This chi 0126. In addition to micro-organisms, cultures of cells meric gene may then be inserted in the adenovirus genome derived from multicellular organisms may also be used as by in vitro or in Vivo recombination. Insertion in a non hosts. In principle, any Such cell culture is workable, essential region of the viral genome (e.g., region E1 or E3) whether from vertebrate or invertebrate culture. In addition will result in a recombinant virus that is viable and capable to mammalian cells, these include insect cell Systems of expressing proteins in infected hosts. infected with recombinant virus expression vectors (e.g., 0132) Specific initiation signals may also be required for baculovirus); and plant cell Systems infected with recombi efficient translation of the claimed isolated nucleic acid nant virus expression vectors (e.g., cauliflower mosaic virus, coding sequences. These signals include the ATG initiation CaMV; tobacco mosaic virus, TMV) or transformed with codon and adjacent Sequences. Exogenous translational con recombinant plasmid expression vectors (e.g., Ti plasmid) trol Signals, including the ATG initiation codon, may addi containing one or more coding Sequences. tionally need to be provided. One of ordinary skill in the art 0127 Examples of useful mammalian host cell lines are would readily be capable of determining this and providing VERO and HeLa cells, Chinese hamster ovary (CHO) cell the necessary Signals. It is well known that the initiation lines, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and codon must be in-frame (or in-phase) with the reading frame MDCK cell lines. In addition, a host cell strain may be of the desired coding Sequence to ensure translation of the chosen that modulates the expression of the inserted entire insert. These exogenous translational control signals Sequences, or modifies and processes the gene product in the and initiation codons may be of a variety of origins, both Specific fashion desired. Such modifications (e.g., glycosy natural and Synthetic. The efficiency of expression may be lation) and processing (e.g., cleavage) of protein products enhanced by the inclusion of appropriate transcription may be important for the function of the encoded protein. In enhancer elements or transcription terminators (Bittner et preferred embodiments of the invention, the host cells are al., Methods in Enzymol, 153: 516-544, 1987). human cells inside a Subject with a fibrotic condition. 0133. In eukaryotic expression, one will also typically 0128. Different host cells have characteristic and specific desire to incorporate into the transcriptional unit an appro mechanisms for the post-translational processing and modi priate polyadenylation site (e.g., 5'-AATAAA-3') if one was fication of proteins. Appropriate cells lines or host Systems not contained within the original cloned Segment. Typically, may be chosen to ensure the correct modification and the poly A addition site is placed about 30 to 2000 nucle processing of the foreign protein expressed. Expression otides “downstream” of the termination site of the protein at vectors for use in mammalian cells ordinarily include an a position prior to transcription termination. origin of replication (as necessary), a promoter located in front of the gene to be expressed, along with any necessary 0134) Nucleic Acid Delivery ribosome binding sites, RNA splice Sites, polyadenylation 0.135 Liposomal Formulations Site, and transcriptional terminator Sequences. The origin of 0.136. In certain broad embodiments of the invention, the replication may be provided either by construction of the oligo- or polynucleotides and/or expression vectors may be vector to include an exogenous origin, Such as may be entrapped in a liposome. Liposomes are vesicular Structures derived from SV40 or other viral (e.g., Polyoma, Adeno, characterized by a phospholipid bilayer membrane and an VSV, BPV) source, or may be provided by the host cell inner aqueous medium. Multilamellar liposomes have mul chromosomal replication mechanism. If the vector is inte tiple lipid layerS Separated by aqueous medium. They form grated into the host cell chromosome, the latter is often Spontaneously when phospholipids are Suspended in an Sufficient. excess of aqueous Solution. The lipid components undergo 0129. The promoters may be derived from the genome of Self-rearrangement before the formation of closed Structures mammalian cells (e.g., metallothionein promoter) or from and entrap water and dissolved Solutes between the lipid mammalian viruses (e.g., the adenovirus late promoter; the bilayers (Ghosh and Bachhawat, In: Liver Diseases, Tar US 2005/0085486A1 Apr. 21, 2005 geted Diagnosis and Therapy. Using Specific Receptors and T cells and tumor necrosis factor and appear to be important Ligands, Wu et al. (Eds.), Marcel Dekker, New York, pp for viral propagation. Functions associated with the E4 87-104, 1991). Also contemplated are cationic lipid-nucleic proteins include DNA replication, late gene expression, and acid complexes, Such as lipofectamine-nucleic acid com host cell shutoff. The late gene products include most of the plexes. Virion capsid proteins, and these are expressed only after 0.137 In certain embodiments of the invention, the lipo most of the processing of a Single primary transcript from Some may be complexed with a hemagglutinating virus the major late promoter has occurred. The major late pro (HVJ). This has been shown to facilitate fusion with the cell moter (MLP) exhibits high efficiency during the late phase membrane and promote cell entry of liposome-encapsulated of the infection (Stratford-Perricaudet and Perricaudet, In: DNA (Kaneda et al., Science, 243:375-378, 1989). In other Human Gene Transfer, O. Cohen-Haguenauer et al., eds., embodiments, the liposome may be complexed or employed John Libbey Eurotext, France, pp. 51-61, 1991). in conjunction with nuclear non-histone chromosomal pro 0.142 AS only a Small portion of the viral genome appears teins (HMG-1). In that such expression vectors have been to be required in cis (Tooze, Molecular Biology of DNA employed in transfer and expression of a polynucleotide in Tumor Viruses, 2nd ed., Cold Spring Harbor Laboratory, Vitro and in Vivo, they may be applicable for the present Cold Spring Harbor, N.Y., 1991), adenovirus-derived vec invention. Liposomes within the Scope of the present inven tors offer excellent potential for the substitution of large tion can be prepared in accordance with known laboratory DNA fragments when used in connection with cell lines techniques. In one embodiment, liposomes are prepared by such as 293 cells. Ad5-transformed human embryonic kid mixing liposomal lipids, in a Solvent in a container, e.g., a ney cell lines (Graham et al., J. Gen. Virol, 36:59-72, 1977) glass, pear-shaped flask. The container should have a Vol have been developed to provide the essential viral proteins ume ten-times greater than the Volume of the expected in trans. Suspension of liposomes. Using a rotary evaporator, the 0.143 Advantages of adenovirus vectors over retroviruses Solvent is removed at approximately 40DC under negative include the higher levels of gene expression. Adenovirus pressure. The solvent normally is removed within about 5 replication is independent of host gene replication, unlike min to 2 hours, depending on the desired Volume of the retroviral Sequences. Because adenovirus transforming liposomes. The composition can be dried further in a des genes in the E1 region can be readily deleted and Still iccator under Vacuum. The dried lipids generally are dis provide efficient expression vectors, oncogenic risk from carded after about 1 week because of a tendency to dete adenovirus vectors is thought to be low (Grunhaus and riorate with time. Horwitz, Seminar in Virology, 3:237-252, 1992). 0.138. The dried lipids or lyophilized liposomes prepared as described above may be reconstituted in a Solution of 0144. In general, adenovirus gene transfer Systems are nucleic acid and diluted to an appropriate concentration with based upon recombinant, engineered adenovirus which is an Suitable Solvent. The mixture is then Vigorously shaken in rendered replication-incompetent by deletion of a portion of a vortex mixer. Unencapsulated nucleic acid is removed by its genome, Such as E1, and yet Still retains its competency centrifugation at 29,000 g and the liposomal pellets for infection. Sequences encoding relatively large foreign washed. The washed liposomes are resuspended at an appro proteins can be expressed when additional deletions are priate total phospholipid concentration, e.g., about 50-200 made in the adenovirus genome. For example, adenoviruses mM. The amount of nucleic acid encapsulated can be deleted in both E1 and E3 regions are capable of carrying up determined in accordance with Standard methods. After to 10 kB of foreign DNA and can be grown to high titers in determination of the amount of nucleic acid encapsulated in 293 cells (Stratford-Perricaudet and Perricaudet, 1991). Per the liposome preparation, the liposomes may be diluted to Sistent expression of transgenes following adenoviral infec appropriate concentration and Stored at 4DC until use. tion has also been reported. 0.145) Other Viral Vectors as Expression Constructs. 0139 Alternative Delivery Systems Other viral vectors may be employed as expression con 0140 Adenoviruses: Human adenoviruses are double Structs in the present invention. Vectors derived from Viruses Stranded DNA tumor viruses with genome sizes of approxi Such as vaccinia virus (Baichwal and Sugden, In: Gene mate 36 kB. As a model System for eukaryotic gene expres Transfer, Kucherlapati R, ed., New York, Plenum Press, pp. Sion, adenoviruses have been widely Studied and well 117-148, 1986) adeno-associated virus (AAV) (Baichwal characterized, which makes them an attractive System for and Sugden, 1986) and herpes viruses may be employed. development of adenovirus as a gene transfer System. This They offer Several attractive features for various mammalian group of viruses is easy to grow and manipulate, and they cells (Horwich, et al., J. Virol, 64.642-650, 1990). exhibit a broad host range in vitro and in vivo. In lytically infected cells, adenoviruses are capable of Shutting off host 0146) With the recent recognition of defective hepatitis B protein Synthesis, directing cellular machineries to Synthe Viruses, new insight was gained into the Structure-function Size large quantities of viral proteins, and producing copious relationship of different viral Sequences. In vitro Studies showed that the virus could retain the ability for helper amounts of Virus. dependent packaging and reverse transcription despite the 0.141. The E1 region of the genome includes E1A and deletion of up to 80% of its genome (Horwich et al., 1990). E1B, which encode proteins responsible for transcription This Suggested that large portions of the genome could be regulation of the viral genome, as well as a few cellular replaced with foreign genetic material. The hepatotropism genes. E2 expression, including E2A and E2B, allows Syn and persistence (integration) were particularly attractive thesis of viral replicative functions, e.g. DNA-binding pro properties for liver-directed gene transfer. Chang et al. tein, DNA polymerase, and a terminal protein that primes recently introduced the chloramphenicol acetyltransferase replication. E3 gene products prevent cytolysis by cytotoxic (CAT) gene into duck hepatitis B virus genome in the place US 2005/0085486A1 Apr. 21, 2005 of the polymerase, Surface, and pre-Surface coding complementary to the mRNA start site. One can readily test Sequences. It was cotransfected with wild-type virus into an Such constructs simply by testing the constructs in Vitro to avian hepatoma cell line. Culture media containing high determine whether levels of the target protein are affected. titers of the recombinant Virus were used to infect primary Similarly, detrimental non-specific inhibition of protein Syn duckling hepatocytes. Stable CAT gene expression was thesis also can be measured by determining target cell detected for at least 24 days after transfection (Chang et al., viability in vitro. Hepatology, 14:124A, 1991). 0154 As used herein, the terms “complementary” or 0147 Non-viral Methods. Several non-viral methods for “antisense” mean polynucleotides that are Substantially the transfer of expression vectors into cultured mammalian complementary to the target Sequence over their entire cells also are contemplated by the present invention. These length and have very few base mismatches. For example, include calcium phosphate precipitation (Graham and Van Sequences of fifteen bases in length may be termed comple der Eb, Virology, 52:456-467, 1973) DEAE-dextran (Gopal, mentary when they have a complementary nucleotide at Mol. Cell Biol. 5:1188–1190, 1985), lipofectamine-DNA thirteen or fourteen nucleotides out of fifteen. Sequences complexes, and receptor-mediated transfection (Wu and Wu, that are “completely complementary' will be sequences Biochemistry, 27: 887-892, 1988; Wu and Wu, J. Biol. which are entirely complementary throughout their entire Chem., 262: 4429-4432, 1987). Some of these techniques length and have no base mismatches. may be Successfully adapted for in Vivo or eX Vivo use. 0.155) Other sequences with lower degrees of homology 0.148. In one embodiment of the invention, the expression also are contemplated. For example, an antisense construct construct may simply consist of naked recombinant vector. that has limited regions of high homology, but also contains Transfer of the construct may be performed by any of the a non-homologous region (e.g., a ribozyme) could be methods mentioned above which physically or chemically designed. These molecules, though having less than 50% permeabilize the cell membrane. For example, Dubensky et homology, would bind to target Sequences under appropriate al. (Proc. Nat. Acad. Sci. USA, 81:7529-7533, 1984) injected conditions. polyomavirus DNA in the form of CaPO precipitates into liver and Spleen of adult and newborn mice demonstrating 0156 Although the antisense sequences may be full active viral replication and acute infection. length cDNA copies, or large fragments thereof, they also may be shorter fragments, or "oligonucleotides, defined 0149 Anti-Sense herein as polynucleotides of 50 or leSS bases. Although 0150. The term “antisense” is intended to refer to poly shorter oligomers (8-20) are easier to make and increase in nucleotide molecules complementary to a portion of a Vivo accessibility, numerous other factors are involved in targeted gene or mRNA species. “Complementary' poly determining the Specificity of base-pairing. For example, nucleotides are those that are capable of base-pairing both binding affinity and Sequence Specificity of an oligo according to the Standard Watson-Crick complementarity nucleotide to its complementary target increase with increas rules. That is, the larger purines will base pair with the ing length. It is contemplated that oligonucleotides of 8, 9, Smaller pyrimidines to form combinations of guanine paired 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, with cytosine (G:C) and adenine paired with either thymine 50 or 100 base pairs will be used. While all or part of the (A:T) in the case of DNA, or adenine paired with uracil gene Sequence may be employed in the context of antisense (A:U) in the case of RNA. Inclusion of less common bases construction, Statistically, any Sequence of 14 bases long Such as inosine, 5-methylcytosine, 6-methyladenine, hypox should occur only once in the human genome and, therefore, anthine and others in hybridizing Sequences does not inter Suffice to specify a unique target Sequence. fere with pairing. O157. In certain embodiments, one may wish to employ 0151. Antisense polynucleotides, when introduced into a antisense constructs which include other elements, for target cell, Specifically bind to their target polynucleotide example, those which include C-5 propyne pyrimidines. and interfere with transcription, RNA processing, transport, Oligonucleotides which contain C-5 propyne analogues of translation and/or Stability. AntiSense RNA constructs, or uridine and cytidine have been shown to bind RNA with DNA encoding such antisense RNAs, may be employed to high affinity and to be potent antisense inhibitors of gene inhibit gene transcription or translation or both within a host expression (Wagner et al., Science, 260:1510-1513, 1993). cell, either in vitro or in Vivo, Such as within a host animal, 0158 Alternatively, the antisense oligo- and polynucle including a human Subject. otides according to the present invention may be provided as 0152 The intracellular concentration of monovalent cat RNA via transcription from expression constructs that carry ion is approximately 160 mM (10 mM Na"; 150 mM K"). nucleic acids encoding the oligo- or polynucleotides. The intracellular concentration of divalent cation is approxi Throughout this application, the term "expression construct” mately 20 mM (18 mM Mg"; 2 mM Ca"). The intracellular is meant to include any type of genetic construct containing protein concentration, which would Serve to decrease the a nucleic acid encoding a product in which part or all of the Volume of hybridization and, therefore, increase the effec nucleic acid Sequence is capable of being transcribed. Typi tive concentration of nucleic acid species, is 150 mg/ml. cal expression vectors include bacterial plasmids or phage, Constructs can be tested in Vitro under conditions that mimic such as any of the puC or BluescriptTM plasmid series or these in Vivo conditions. Viral vectors adapted for use in eukaryotic cells. 0153 Antisense constructs may be designed to bind to 0159. In preferred embodiments, the nucleic acid encodes the promoter and other control regions, exons, introns or an antisense oligo- or polynucleotide under transcriptional even exon-intron boundaries of a gene. It is contemplated control of a promoter. The term promoter will be used here that effective antisense constructs may include regions to refer to a group of transcriptional control modules that are US 2005/0085486A1 Apr. 21, 2005
clustered around the initiation site for RNA polymerase II. Sequence. Synthetic siRNAS may also be introduced into Promoters are composed of discrete functional modules, cells to inhibit expression of one or more Selected genes. each consisting of approximately 7-20 bp of DNA, and SiRNAS may be generated by Standard Solid-phase oligo containing one or more recognition Sites for transcriptional nucleotide Synthesis, by RNA-Specific endonuclease cleav activator or repressor proteins. age of double-stranded RNA, or by expression of transfected 0160. At least one module in each promoter functions to DNA templates incorporating promoter Sequences for RNA position the start site for RNA synthesis. The best known polymerase III. Introduction of siRNA into a mammalian example of this is the TATA box, but in some promoters cell results in the targeted destruction of messenger RNAS of lacking a TATA box, Such as the promoter for the mamma the same Sequence. Commercial products for siRNAS are lian terminal deoxynucleotidyl transferase gene and the available from a number of Sources, Such as Gene Therapy promoter for the SV40 late genes, a discrete element over Systems, Inc. (San Diego, Calif.), Promega (Madison, Wis.) lying the Start Site itself helps to fix the place of initiation. and Sirna Therapeutics (Boulder, Colo.). 0.161 Additional promoter elements regulate the fre 0.167 Methods for design of siRNA sequences are pub quency of transcriptional initiation. Typically, these are licly available. For example, the siRNA Target Finder may located in the region 30-110 bp upstream of the start site, be used online at the Ambion website. Target mRNA although a number of promoters have recently been shown Sequences are input into the program, which then Scans for to contain functional elements downstream of the Start Site 21 nucleotide Sequences that begin with an AA dinucleotide. as well. A variety of Specific eukaryotic promoter elements The program selects for siRNAs with about a 30 to 50% GC are known in the art and any Such known element may be content, avoiding Sequences with 4-6 poly T Stretches that used in the practice of the claimed invention. Depending on would function as terminators for RNA Polymerase III the promoter, it appears that individual elements can func transcription. After selection of two to four siRNA candi tion either co-operatively or independently to activate tran dates, the generated Sequences may be searched for homol Scription. The particular promoter that is employed to con ogy (for example, using the BLAST Search engine on the trol the expression of a nucleic acid encoding the inhibitory NCBI server) to other untargeted mRNA sequences. SiR peptide is not believed to be important, So long as it is NAS with homology to non-targeted Sequences are elimi capable of expressing the peptide in the targeted cell. nated from consideration. SiRNA expression cassettes may also be obtained from Ambion (Austin,Tex.). SiRNAS may 0162 Enhancers are genetic elements that increase tran be purchased and used according to the manufacturer's Scription from a promoter located at a distant position on the instructions to provide targeted inhibition of the expression Same molecule of DNA. Enhancers are organized much like of specific genes, such as PDE-4 and/or PDE-5. promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional 0168 Ribozymes proteins. Any promoter/enhancer combination known in the 0169. Another method for inhibiting the expression of art (e.g., the Eukaryotic Promoter Data Base) also could be Specific genes within the Scope of the present invention is via used to drive expression of the gene. ribozymes. Ribozymes are RNA-protein complexes that 0163 Where a cDNA insert is employed, typically one cleave nucleic acids in a site-specific fashion. Ribozymes will typically include a polyadenylation signal to effect have specific catalytic domains that possess endonuclease proper polyadenylation of the gene transcript. The nature of activity (Kim and Cech, 1987; Gerlach et al., 1987; Forster the polyadenylation Signal is not believed to be crucial to the and Symons, 1987). For example, a large number of Successful practice of the invention, and any Such Sequence ribozymes accelerate phosphoester transfer reactions with a may be employed, Such as human growth hormone and high degree of Specificity, often cleaving only one of Several SV40 polyadenylation signals. Also contemplated as an phosphoesters in an oligonucleotide Substrate (Cech et al., element of the expression construct is a terminator. These 1981; Michel and Westhof, 1990; Reinhold-Hurek and elements can Serve to enhance message levels and to mini Shub, 1992). This specificity has been attributed to the mize read through from the construct into other Sequences. requirement that the Substrate bind via Specific base-pairing 0164. In certain embodiments of the invention, the deliv interactions to the internal guide sequence (“IGS") of the ery of a nucleic acid in a cell may be identified in vitro or ribozyme prior to chemical reaction. in Vivo by including a marker in the expression construct. 0170 Ribozyme catalysis has primarily been observed as The marker would result in an identifiable change to the part of Sequence-specific cleavage/ligation reactions involv transfected cell permitting identification of expression. ing nucleic acids (Joyce, 1989; Cech et al., 1981). For Enzymes Such as herpes simplex virus thymidine kinase (tk) example, U.S. Pat. No. 5,354,855 reports that certain (eukaryotic) or chloramphenicol acetyltransferase (CAT) ribozymes can act as endonucleases with a Sequence Speci (prokaryotic) may be employed. ficity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, 0.165 siRNA Sequence-specific ribozyme-mediated inhibition of gene 0166 Small interfering RNAs (siRNAs) are short RNA expression may be particularly Suited to therapeutic appli molecules (typically from 21 to 23 nucleotides in length) cations (Scanlon et al., 1991; Sarver et al., 1990; Sioud et al., that may be used to induce targeted gene Silencing by RNA 1992). It was reported that ribozymes elicited genetic interference (Myers et al., Nature Biotechnology 21:324 changes in Some cells lines to which they were applied. The 328, 2003; Elbashir, Nature 411:494-498, 2001; Caplen et altered genes included the oncogenes H-ras, c-foS and genes al., Proc. Natl. Acad. Sci. USA 98: 974247, 2001). SiRNAs of HIV. Most of this work involved the modification of a occur naturally in vivo when double-stranded RNA is target mRNA, based on a specific mutant codon that is cleaved by ribonuclease III to produce a short siRNA cleaved by a specific ribozyme. US 2005/0085486A1 Apr. 21, 2005
0171) Several different ribozyme motifs have been necessary to prepare pharmaceutical compositions in a form described with RNA cleavage activity (Symons, 1992). appropriate for the intended application. Generally, this will Examples that are expected to function equivalently include entail preparing compositions that are essentially free of Sequences from the Group I Self Splicing introns including pyrogens, as well as other impurities that could be harmful Tobacco Ringspot Virus (Prody et al., 1986), Avocado to humans or animals. Sunblotch Viroid (Palukaitis et al., 1979; Symons, 1981), 0177 Aqueous compositions of the present invention and Lucerne Transient Streak Virus (Forster and Symons, comprise an effective amount of inhibitor or activator, 1987). Sequences from these and related viruses are referred dissolved or dispersed in a pharmaceutically acceptable to as hammerhead ribozyme based on a predicted folded carrier or aqueous medium. Such compositions also are Secondary Structure. referred to as innocula. The phrase “pharmaceutically or 0172 Other suitable ribozymes include sequences from pharmacologically acceptable” refers to molecular entities RNase P with RNA cleavage activity (Yuan et al., 1992, and compositions that do not produce adverse, allergic, or Yuan and Altman, 1994, U.S. Pat. Nos. 5,168,053 and other untoward reactions when administered to an animal or 5,624,824), hairpin ribozyme structures (Berzal-Herranz et a human. AS used herein, "pharmaceutically acceptable al., 1992; Chowrira et al., 1993) and Hepatitis Delta virus carrier' includes any and all Solvents, dispersion media, based ribozymes (U.S. Pat. No. 5,625,047). The general coatings, antibacterial and antifungal agents, isotonic and design and optimization of ribozyme directed RNA cleavage absorption delaying agents and the like. The use of Such activity has been discussed in detail (Haseloff and Gerlach, media and agents for pharmaceutically active Substances is 1988, Symons, 1992, Chowrira et al., 1994; Thompson et al., well known in the art. Except insofar as any conventional 1995). media or agent is incompatible with the inhibitors or acti 0173 The other variable on ribozyme design is the selec Vators of the present invention, its use in therapeutic com tion of a cleavage site on a given target RNA. Ribozymes are positions is contemplated. Supplementary active ingredients targeted to a given Sequence by Virtue of annealing to a site also can be incorporated into the compositions. by complimentary base pair interactions. Two Stretches of 0.178 The active compositions of the present invention homology are required for this targeting. These Stretches of may include classic pharmaceutical preparations. Adminis homologous Sequences flank the catalytic ribozyme struc tration of these compositions according to the present inven ture defined above. Each Stretch of homologous Sequence tion will be via any common route So long as the target tissue can vary in length from 7 to 15 nucleotides. The only is available via that route. This includes oral, nasal, buccal, requirement for defining the homologous Sequences is that, rectal, vaginal or topical. Alternatively, administration may on the target RNA, they are separated by a specific Sequence be by orthotopic, intradermal, Subcutaneous, intramuscular, that is the cleavage Site. For hammerhead ribozymes, the intraperitoneal or intravenous injection. Such compositions cleavage Site is a dinucleotide Sequence on the target normally would be administered as pharmaceutically RNA-a uracil (U) followed by either an adenine, cytosine acceptable compositions. or uracil (A.C or U) (Perriman et al., 1992; Thompson et al., 0179 The active compounds also may be administered 1995). The frequency of this dinucleotide occurring in any parenterally or intraperitoneally. Solutions of the active given RNA is statistically 3 out of 16. Therefore, for a given compounds as free base or pharmacologically acceptable target messenger RNA of 1000 bases, 187 dinucleotide Salts can be prepared in water Suitably mixed with a Surfac cleavage Sites are Statistically possible. tant, Such as hydroxypropylcellulose. Dispersions also can 0.174. The large number of possible cleavage sites in be prepared in glycerol, liquid polyethylene glycols, and genes of moderate size, coupled with the growing number of mixtures thereof and in oils. Under ordinary conditions of Sequences with demonstrated catalytic RNA cleavage activ Storage and use, these preparations contain a preservative to ity indicates that a large number of ribozymes that have the prevent the growth of microorganisms. potential to downregulate gene expression are available. Additionally, due to the Sequence variation among different 0180. The pharmaceutical forms suitable for injectable genes, ribozymes could be designed to Specifically cleave use include Sterile aqueous Solutions or dispersions and individual genes or gene products. Designing and testing Sterile powders for the extemporaneous preparation of Sterile ribozymes for efficient cleavage of a target RNA is a proceSS injectable Solutions or dispersions. In all cases the form must well known to those skilled in the art. Examples of Scientific be Sterile and must be fluid to the extent that easy Syring methods for designing and testing ribozymes are described ability exists. It must be stable under the conditions of by Chowrira et al., (1994) and Lieber and Strauss (1995), manufacture and Storage and must be preserved against the each incorporated by reference. The identification of opera contaminating action of microorganisms, Such as bacteria tive and preferred Sequences for use in ribozymes targeted to and fungi. The carrier can be a Solvent or dispersion medium Specific genes is simply a matter of preparing and testing a containing, for example, water, ethanol, polyol (for example, given Sequence, and is a routinely practiced “Screening” glycerol, propylene glycol, and liquid polyethylene glycol, and the like), Suitable mixtures thereof, and vegetable oils. method known to those of skill in the art. The proper fluidity can be maintained, for example, by the 0175 Formulations and Routes for Administration to use of a coating, Such as lecithin, by the maintenance of the Patients required particle size in the case of dispersion and by the use 0176). In certain embodiments, the inhibitors or activators of Surfactants. of PDE5, PKG, NOS, MMP or another protein and/or 0181. The prevention of the action of microorganisms stimulators or agonists of c(GMP may be used for therapeutic can be brought about by various antibacterial and antifungal treatment of medical conditions, Such as Peyronie's disease. agents, for example, parabens, chlorobutanol, phenol, Sorbic Where clinical applications are contemplated, it will be acid, thimerosal, and the like. In many cases, it will be US 2005/0085486A1 Apr. 21, 2005 preferable to include isotonic agents, for example, SugarS or preferred modes for its practice. However, those of skill in Sodium chloride. Prolonged absorption of the injectable the art should, in light of the present disclosure, appreciate compositions can be brought about by the use in the com that many changes can be made in the Specific embodiments positions of agents delaying absorption, for example, alu which are disclosed and still obtain a like or similar result minum monoStearate and gelatin. without departing from the Spirit and Scope of the invention. 0182 Sterile injectable solutions are prepared by incor The results disclosed below are generally addressed to the porating the active compounds in the required amount in the following areas. appropriate Solvent with various other ingredients enumer 0187 NO/cGMP ated above, as required, followed by filtered Sterilization. Generally, dispersions are prepared by incorporating the 0188 The antifibrotic effects of agents that, either orally various Sterilized active ingredients into a Sterile vehicle or via gene transfer, stimulate the NO/cGMP pathways and which contains the basic dispersion medium and the increase NO levels and coMP levels or PKG activity or required other ingredients from those enumerated above. In agents that inhibit oxidative stress by decreasing ROS the case of Sterile powders for the preparation of Sterile levels. For example, by a) gene transfer of the sense cDNA injectable Solutions, the preferred methods of preparation for iNOS or nNOS in a single transfection; b) long-term are vacuum-drying and freeze-drying techniques which administration of an oral NO donor (e.g. molsidomine), or yield a powder of the active ingredient plus any additional the NOS Substrate (L-arginine) that produces a continuously desired ingredient from a previously Sterile-filtered Solution elevated level of NO. Alternatively, by increasing c(GMP thereof. and/or stimulating PKG by a) identifying PDE isoforms present in the affected tissue and using oral PDE inhibitors 0183 The compositions of the present invention may be Such as Sildenafil, Zaprinast, and pentoxifylline; b) by gene formulated in a neutral or salt form. Pharmaceutically transfer of PKG1 cDNA, or its mutated version, to increase acceptable Salts include the acid addition Salts which are the level of PKG activation. In other alternatives, by reduc formed by reaction of basic groups with inorganic acids Such ing the concentration of ROS by a) early (to arrest the as, for example, hydrochloric or phosphoric acids, or Such development of the PD-like plaque, arteriosclerotic plaque organic acids as acetic, oxalic, tartaric, mandelic, and the or other fibrotic lesion) or late (to induce regression of an like. Salts formed with free acidic groups can also be derived already formed plaque) administration of oral antioxidants from inorganic baseS Such as, for example, Sodium, potas such as vitamin E or S-adenosyl methionine (SAME); b) sium, ammonium, calcium, or ferric hydroxides, and Such combination therapy with antioxidant (Vitamin E or SAME) organic bases as isopropylamine, trimethylamine, histidine, combined with one or more NO donors (molsidomine or procaine and the like. L-arginine) or cGMP/PKG therapy (sildenafil, Zaprinast, 0184. Upon formulation, solutions will be administered pentoxifylline, or PKG1 cDNA), to induce regression of the in a manner compatible with the dosage formulation and in plaque (late treatment). Such amount as is therapeutically effective. The formulations 0189 MMP are easily administered in a variety of dosage forms Such as injectable Solutions, drug release capsules and the like. For 0190. Stimulation of MMP (collagenolysis) induced by parenteral administration in an aqueous Solution, for thymosin peptides or other MMP activators. Correlating example, the solution should be suitably buffered if neces MMP inhibition in fibrosis with the levels of TIMP1, an Sary and the liquid diluent first rendered isotonic with inhibitor of MMP. Use of the MMP inducers thymosin B-4 Sufficient Saline or glucose. These particular aqueous Solu and 10 are to stimulate MMP activity. tions are especially Suitable for intravenous, intramuscular, 0191) Materials and Methods Subcutaneous and intraperitoneal administration. 0185. In this connection, sterile acqueous media which can 0192 Human Tissues and Cell Cultures be employed will be known to those of skill in the art in light 0193 Human tissue: Human TA was obtained from non of the present disclosure. For example, one dosage could be PD patients (n=4), two undergoing partial penectomy due to dissolved in 1 ml of isotonic NaCl Solution and either added penile cancer and two undergoing penile prosthesis Surgery. to 1000 ml of hypodermoclysis fluid or injected at the Plaque tissue was isolated from PD patients (n=8) who proposed Site of infusion. Some variation in dosage will underwent a Surgical procedure to treat this condition (Ver necessarily occur depending on the condition of the Subject net et al., 2002; Ferrini et al., 2002; Magee et al., 2002b: being treated. The perSon responsible for administration Davila et al., 2003b). Fragments of newly obtained tissue are will, in any event, determine the appropriate dose for the stored for 24h in “RNA-later” (Ambion, Inc., Austin,Tex.), individual Subject. Moreover, for human administration, for RNA analysis, in 4% formalin for histochemistry and preparations should meet Sterility, pyrogenicity, general immunohistochemistry, or in culture medium (DMEM/10% safety and purity standards as required by FDA Office of fetal calf serum) or fibroblast growth medium (FGM-2) BiologicS Standards. (Clonetics, Walkersville, Md.) with 20% fetal bovine serum, for protein analysis or cell culture. Tissues were then frozen EXAMPLES at -80 C. until further use, except for fixed portions that were stored at 4 C. in PBS until paraffin embedding or 0186 The following examples are included to demon cryoSectioning, and pieces used for cell cultures. strate preferred embodiments of the invention. It should be appreciated by those of Skill in the art that the techniques 0194 Primary human cell cultures: Human fibroblast disclosed in the examples which follow represent techniques primary cultures were obtained from fragments of PD discovered by the inventors to function well in the practice plaque or TA essentially according to Smith and Liu (2002), of the invention, and thus can be considered to constitute and their purity was established by immunohistochemistry, US 2005/0085486A1 Apr. 21, 2005
as detailed below. New primary cultures were obtained from 0199 TGF-B1-collagen I promoter mouse model. The fragments of PD plaque or TA that were washed in Hanks transgenic line pGB 19.5/13.5 was obtained from George solution, minced in a fibroblast growth medium (FGM) Bou-Gharios (London, England). These animals harbor the (BioWhittaker Inc., Walkersville, Md.) and 20% fetal bovine promoter of the C2 chain of the mouse collagen type I gene serum (FBS), and plated onto a 25 cm culture flask per linked to the E. coli 3-galactosidase, that is expressed in specimen (Vernet et al., 2002). Fragments were left undis cells and tissueS where collagen I is normally expressed turbed until attachment for about 1 week. Once the mono (Fakhouri et al., 2001; Tharaux et al., 2000; Dussaule et al., layer Started to develop, the fragment was removed. Medium 2000). Animals were injected into the TA as above with with 10% FBS was changed once a week and when cells TGF-31, and sacrificed. achieved approximately 80% confluence (3-4 weeks) they were trypsinized and Split onto 10 cm plates. Cells were 0200 Arterial Tree Rodent Model. Young (3-month) and allowed to grow again to 80% confluence, with medium aged (22-24 month) male Brown Norway rats were obtained changed twice a week. The cells collected from this passage from the NIH/NIA colony (Harlan Sprague-Dawley, Inc., were considered as passage 1. Successive passages were San Diego, Calif.), and maintained under controlled tem performed at 1/3 split ratio, and Studies were carried out on perature and lighting. One half of the aged animals were cells from passages 3 onwards. Studies were performed with treated for 3 weeks with L-NIL at 0.1 g/l in the drinking PD cells from the 4" to the 10" passages. Cells were water, while the rest of the animals received plain drinking incubated on: a) 75 cm' flasks for RNA isolation; b) 6-well water. Animals were anesthetized, pretreated with heparin, plates for protein isolation; and c) 8-well removable cham and perfused through the left ventricle with saline followed bers for cytochemistry and immunocytochemistry. Treat by 4% formalin. The abdominal aorta, brachial and femoral ments with different additions were initiated 24 hours after neurovascular bundles as well as the penis, denuded of its plating and continued for different periods. skin, were removed and post-fixed overnight in 4% forma lin, and washed and stored in PBS at 4 C until paraffin 0195 Cells incubated in 8-well chamber slides were embedding. allowed to grow to 50-60% confluence. At this point, cells received in duplicate sildenafil, pentoxifylline, or 8-Br 0201 General Procedures cGMP at the concentrations indicated, and were allowed to 0202 Injection-electroporation. Injection into the TA was propagate for 3 days without changing medium. In certain performed with the appropriate AdW or plasmid cDNA cases SNAP was added and replaced daily after changing the constructs at doses described below, and electroporation was medium (Vernet et al., 2002). All studies were done in applied at 100 volts, 8 pulses/sec, 40 ms (Magee et al., duplicate or triplicate. For the isolation of rat TA fibroblasts, 2002a). the TA was carefully dissected from rat corpora cavernosa tissue, and cultures were developed and their purity tested as 0203 Minipump implantation. Alza osmotic minipumps in the case of the human tissues. (Alza Corp, Palo Alto, Calif.), #2001D, delivering 8 ul/hr of a Saline Solution (100 ul) containing the Selected compound 0.196 Rodent Models and Tissue Processing during a period of 24 hs for “short-term' treatment, or 0.25 0197) TGF-B1 rat model. Male Fisher 344 rats, 9-11 ul/hour, for 2 weeks (Alzait 1002) for “long-term” treatment, month old purchased from the NIH/NIA colony (Harlan were implanted in a Subcutaneous tunnel over the inguinal Sprague-Dawley, Inc., San Diego, Calif.) and maintained canal, and attached to the abdominal muscles with a non under controlled temperature and lighting, were anesthetized absorbable suture. A delivery catheter from the minipump and injected in the penile TA at the middle of the penis with was placed through the tissues to the penile crura and either vehicle only (Saline, group 1) or 0.5 lig recombinant Sutured to the perineal muscles, as previously described human TGF-B1 (Biotech Diagnostic, Laguna Niguel, Calif., (Garban et al., 1997; Gelman et al., 1998). groups 2-5) as disclosed (Ferrini et al., 2002; Vernet et al., 0204 Detection of PDE mRNA Expression in Tissues 2002). After the injection, groups 1 and 2 were given and Cells drinking water while the other groups received water with L-arginine (2.25 g/kg/day, group 3) (Moody et al., 1997), or 0205 Total RNA was isolated from the human TA and Sildenafil (10 mg/kg/day, group 4) or pentoxifylline (10 PD tissues, from their respective fibroblast cultures, and mg/kg/day, group 5). Forty-five days later, or as indicated, from rat TA and penile shaft tissues, and their respective animals were Sacrificed and perfused through the left ven fibroblast and smooth muscle cell cultures, by the Trizol tricle with saline followed by 4% formalin (Ferrini et al., procedure (Gibco BRL, Gaithesburg, Md.). RNA was then 2002; Vernet et al., 2002). After the penises were excised, submitted (1 ug) to reverse transcription (Vernet et al., 2002; the penile skin was denuded by removing the glans and Magee et al., 2002b; Ferrini et al., 2001 b) using Superscript adhering non-crural tissue. The penile shaft was separated II RNase H reverse transcriptase (Gibco BRL) and random from the crura and 2-3 mm transverse slices were cut around primers (0.25 ug), followed by PCR with the respective gene the site of the saline or TGF-31 injection. All tissues were specific primers (Kuthe et al., 2001): a) for human PDE5A, post-fixed overnight in 4% formalin, washed in PBS and on nt 1027-1049 (forward) and nt 1788-1764 (reverse) of the stored at 4 C. respective cDNA (Genbank #158526); encompassing a 762 bp band common to the three variants 1-3; b) for rat PDE5A, 0198 TGF-B1-iNOS knock-out mouse model. The iNOS the primers on nt 1905-1924 (forward) and nt 2479-2460 knockout strain (B6;129PNOS2 tively); as the source of the expected 883 bp (A) and 406 bp Serum and incubated with monoclonal primary antibodies (B) bands; e) for rat PDE4, the primers on nt 241-260 for ASMA and vimentin (Sigma Immunohistology Kits, (forward) and nt 656-637 (reverse) of the respective cDNA Sigma Chemical Co., St. Louis, Mo.), collagen I, and col (Genbank #M25350), generating a band of 416 bp. PCR lagen III (1:40) (Chemicon International, Temecula, Calif.), products were separated by electrophoresis on 1% agarose overnight at 4 C. (Vernet et al., 2002; Ferrini et al., 2002). gels and Stained with ethidium bromide. For densitometry, Processing was according with the manufacturer's instruc normalization was performed against the GAPDH house tions for ASMA, Vimentin and collagen, consisting in the keeping gene fragment generated in the Same PCR reaction. respective monoclonal antibodies and an anti-mouse bioti nylated secondary antibody, followed by avidin-biotinylated 0206) Detection of PDE5 and 4 Protein Expression in HRP and the 3-amino-9-ethylcarbazol (AEC) chromogen. Tissue and Cell Extracts For PDE5A, the antibodies were as described above. Nega 0207 Tissue extracts were obtained by homogenizing in tive controls omitted the first antibodies or were replaced by a 1:6 wit/vol ratio in a buffer containing 0.32 M sucrose, 20 IgG isotype at the same concentration of the first antibodies. mM HEPES (pH 7.2), 0.5 mM EDTA, 1 mM dithithreitol Counterstaining was done with Mayer's hematoxylin. All and protease inhibitors (3 uM leupeptin, 1 uM pepstatin A, the slides were mounted with Aqua Mount (Lerner, Pitts 1 mM phenylmethyl sulfonyl fluoride). In the case of cell burgh, Pa.). For PDE4, the anti PDE4A and PDE4B affinity extracts 0.5 ml of this solution per 10 cm Petri dish was used. purified IgGs used for western blot was employed, and in The particulate and cytosolic fractions were obtained by addition the anti-PDE4A4 and anti-PDE4D (detecting vari homogenization of the cells in a Polytron Homogenizer, ants 1-5) from the same source (FabGennix Inc.) were used. (Brinkmann, Switzerland), and centrifugation at 12,000xg 0213. In the case of tissue sections, the determinations of for 60 min. the collagen/Smooth muscle ratio were carried out with 0208 Equal amounts of protein (30 ug) were run on 7.5% Masson trichrome (Ferrini et al., 2002; Davila et al., 2003b) polyacrylamide gels, and Submitted to western blot immu on adjacent 5 um paraffin-embedded croSS-Sections from the nodetection with polyclonal anti-mouse PDE5 (against human normal tunical or plaque tissues, or from a 2 mm area cGMP binding region) IgG (1:1000) (Calbiochem, La Jolla, around the site of injection in the rat saline- and TGF-B1 Calif.), and a Secondary donkey anti-mouse IgG linked to injected Shaft tissues. Other distal Sections were obtained horse radish-peroxidase (Amersham Pharmacia, Piscataway, along the rat penile shaft. N.J.), followed by a luminol reaction (Simko and Simko, 0214 SMC and collagen fibers within the corporal tissue 2000; Magee et al., 2002b; Ferrini et al., 2001b). Human and vascular tree were estimated by Masson trichromic PDE5 does not cross-react with other PDE5 isoforms. Nega Staining (Sigma Diagnostic, St. Louis, Mo.) (Ferrini et al., tive controls were performed without primary antibody. 2002; Vernet et al., 2002; Davila et al., 2203b) in sections 0209 For PDE4 immunodetection, the following affinity adjacent to those used for immunohistochemical Staining, purified IgGs were used (FabCennix Inc., Shreveport, La.): followed by image analysis to measure the ratio between a) anti-PDE4A selective antibody (detecting variants iden SMC (red) and collagen fibers (blue). The results were tified by 1, 5, 8, X, and unassigned); b) anti-PDE4B (detect expressed as red/blue ratios per area (see below). In the ing variants 1-4), and anti-PDE4D (detecting variants 1-5) arterial tree, the intima/media thickness (IMT), and the (Salanova et al., 1999). diameter of the lumen were also measured. 0210. The presence of PDEs in the PD fibroblasts in 0215. The determinations of iNOS, nitrotyrosine, heme culture was confirmed by the ability of increasing concen oxygenase I, PAI-I (Davila et al., 2003b), manganese Super trations of Sildenafil and pentoxifylline to raise the basal oxide dismutase (MnSOD), and CuZn SOD (Cu/Zn SOD) cGMP and cAMP levels in triplicate wells, either in the (Martin et al., 1994) were carried out on 5 um paraffin absence or the presence of the NO donor, SNAP (S-nitroso embedded adjacent tissue Sections, that. were quenched for N-acetyl penicillamine) (Alexis Biochemicals, San Diego, endogenous peroxidase activity after deparaffinization and Calif.) added daily at 100 uM, as measured by c0MP and rehydration. Sections were blocked with normal goat Serum, cAMP EIA (enzyme immuno absorption) kits (Cayman and incubated with polyclonal IgG antibodies against mouse Chemical, Ann Arbor, Mich.). Experiments were performed iNOS (1:500) (Transduction Laboratories, Lexington, KT), in duplicate. Values were expressed as pmoles cGMP or nitrotyrosine (1:100) (Upstate, Lake Placid, N.Y.), Mn SOD cAMP/mg protein. To normalize for differences between and Cu/Zn SOD (Oxygen, Portland, Oreg.) (1:800 and 1:500 experiments, the changes in coMP and cAMP levels exerted respectively), heme oxygenase I (Stressgen, San Diego, by Sildenafil and pentoxifylline were expressed as % of their Calif.), or PAI-1 (Abcam Ltd, Cambridge, UK). For negative respective control values in the absence of the PDE inhibi controls the first antibodies were replaced by IgG isotype. torS. The detection was based on a secondary anti-rabbit bioti nylated antibody (1:200) for iNOS and nytrotyrosine (Cal 0211. Histochemical and Immunohistochemical Determi biochem, La Jolla, Calif.), or anti-sheep biotinylated anti nations body (1:200) for Cu/Zn and Mn SOD, followed by the ABC 0212. In the case of cell cultures, at completion of incu complex (1:100) (Calbiochem) and 3,3' diaminobenzidine bations, slides were removed from the chambers and cells (spelling) (DAB) (Sigma, St Louis Mo.). Sections were were fixed for immunodetection for 20 min in 4% buffered counterstained with hematoxylin. formalin at room temperature for C-Smooth muscle actin (ASMA) (as a myofibroblast marker), Vimentin (as a general 0216) TUNEL Assay for Apoptosis fibroblast marker), and in certain cases for PDE5 and PDE4, 0217. The TUNEL assay (Ferrini et al., 2001a, 2001b) or in ethanol at -20C for collagen I and III (Vernet et al., was performed in the adjacent matched tissue Sections used 2002). The cells were quenched, blocked with normal goat for collagen, iNOS or nitrotyrosine Staining, applying the US 2005/0085486A1 Apr. 21, 2005 Apoptag Oncor kit (Oncor, Gaithersburg, Md.). In brief, data was established using the Wilk-Shapiro test, and the after deparaffinization and rehydration, Sections were incu outcome measures between two groups were compared by bated with proteinase K (20 ug/ml) and endogenous peroxi the t test. Multiple comparisons among the different groups dase activity was quenched with 2% H.O. Sections were were analyzed by a single factor analysis of variance incubated with digoxigenin-conjugated nucleotides and (ANOVA), followed by post-hoc comparisons with the TdT, and Subsequently treated with antidigoxigenin-peroxi Student-Neuman Keuls test, according to the Graph Pad dase. To detect immunoreactive cells, Sections were Stained prism V. 30. Differences among groups were considered with 0.5% DAB/0.01% HO, and counterstained with 0.5% significant at P-0.05. methyl green. As a negative control, buffer was Substituted for the TdT enzyme. Testicular sections from old animals Example 1 were used as positive control. For cell cultures, the cells were fixed in 4% formaldehyde for 30 min on ice, and Spontaneous iNOS Induction. In Vivo in the PD post-fixed with ethanol-acetic acid 2/1 for 5 min at -20 C. Plaque Leads to Increased NO Synthesis, Then the above procedure was applied, except that the Peroxynitrite Formation, and Fibroblast Apoptosis proteinase K was omitted. 0224. It has been reported that aging perse results in the 0218 Quantitative Image Analysis (QIA) spontaneous induction of iNOS and the formation of the NO 0219. The quantitation of the staining obtained by either metabolite, peroxynitrite, in both the rat hypothalamus (Fer histochemical or immunohisto/cytochemical techniques was rini et al., 2001b; Vernet et al., 1998) and corpora cavernosa performed by computerized densitometry using the (Ferrini et al., 2001a). This was accompanied by apoptosis ImagePro 4.01 program (Media Cybernetics, Silver Spring, of both the neurons and the cavernoSal Smooth muscle. In Md.), coupled to an Olympus BHS microscope equipped the TGF-B1 induced rat model of PD, a similar iNOS with a Spot RT digital camera or VCC video camera (Wang induction in the TA (Bivalacqua et al., 2000; Hellstrom, et al., 2001, Ferrini et al., 2001a, 2001b; Davila et al., 2001) has been reported. Initially, it was assumed that this 2003b). process of iNOS induction was deleterious to the TA. 0220. The number of positive cells was counted in a computerized grid against the total number of cells deter 0225. As proposed herein, it is believed that iNOS induc mined by counterstaining, and results were expressed as a tion in the TA, and perhaps in fibrosis in general, is a percentage of positive cells over total cells. In addition, the beneficial, anti-fibrotic, cellular defense mechanism. The integrated optical density (IOD) was obtained by measuring locally produced NO from elevated iNOS would inhibit the density per object and multiplying it by the respective collagen deposition, oppose pro-fibrotic agents, and induce area. The Sum of all the individual values in the field was apoptosis of myofibroblasts, which pathologically persist in then divided by the number of positive cells, to obtain the the PD plaque. This model has been examined in the TA mean IOD/positive cell, as a measure of average immunore (Ferrini et al., 2002; Vernet et al., 2002; Gonzalez-Cadavid activity/cell. In certain cases, results were expressed as the et al., 2002; Gholami et al., 2002) by quantitative image unweighted average optical density per area (O.D/AREA), analysis (OIA) of immunohistochemical and histochemical to determine the relative concentration of immunoreactive Stained tissue Sections, and measurement of RNA and pro antigen. For collagen/smooth muscle Staining, the ratio tein expression by quantitative RT/PCR, northern blots, between the width of the area Stained positive for collagen western blots, DNA microarrays, and other procedures (n=5 (blue) divided by the total area of the lacunar spaces plus to 9 per experimental group). All results discussed below are Smooth muscle (white+red) was employed. The apoptotic Significant (p<0.05), unless Stated otherwise. index (rate of programmed cell death) was calculated as the 0226. It was first observed that in the human PD plaque, percentage of apoptotic cells within the total number of cells iNOS induction as seen by immunohistochemistry occurs in a given area (non-apoptotic nuclei plus apoptotic cells). In spontaneously in discrete cells (Ferrini et al., 2002). These all cases, five non-overlapping fields were Screened per cells were identified as fibroblasts and myofibroblasts based tissue Section or per well. Three Sections per tissue specimen on Vimentin as markers for both cell types and alpha Smooth from groups of five animals, or two wells per experimental muscle actin (ASMA) for myofibroblasts (Vernet et al., point in cell incubations, were then used to calculate the 2002). iNOS expression as measured by immunohistochem means-/-SEM. istry was also detected in the rat PD-like plaque 45 days after 0221 For iNOS, nitroyrosine, heme-oxygenase, PAI-1, TGF-31 injection in the TA, in comparison to control tissue MnSOD and Cu/Zn SOD determination, at least 6 sections obtained from rats injected with saline (Ferrini et al., 2002; per specimen were analyzed. Each Slide assayed had its Vernet et al., 2002). In addition, in plaque tissue from both corresponding negative control. In certain cases, the number rat and human, iNOS induction was accompanied by of immuno-positive cells was determined as a percentage of increased peroxynitrite, a product formed by the reaction of the total counterstained nuclei in a computerized grid. In the NO with ROS (Ferrini et al., 2002). In contrast to its Masson staining, the ratio between SMC (red) and collagen inductive effects on cell apoptosis, peroxynitrite does not fibers (blue) was obtained and expressed per area. The rate induce collagen deposition or fibrosis, which means it is not of programmed cell death (apoptotic index) was expressed pro-fibrotic per se (Okamoto et al., 1997), and therefore, as the percentage of apoptotic cells within the total number differs considerably from one of the compounds from which of cells in a given area (non-apoptotic nuclei plus apoptotic it originates, ROS, that is highly pro-fibrotic (Casini et al., cells). 1997; Muriel et al., 1998a; Hung et al., 1995; Poli, 2000; 0222 Statistical Analysis Curtin et al., 2002; Cattell, 2002; Kim et al., 2001). 0223 Values were expressed as mean (M)+/-standard 0227. The fibrotic plaque was visualized in the rat model error of the mean (SEM). The normality distribution of the by Masson Staining showing disorganization of collagen US 2005/0085486A1 Apr. 21, 2005 fibers and intensification of collagen deposition and thick inhibitor (Ferrini et al., 2002, Vernet et al., 2002). In the ening of the TA (Ferrini et al., 2002). In the case of the TGF-31 injected rat model, treatment with L-NIL, which human plaque, Similar changes were revealed with Masson lowers NO derived from iNOS, induced a remarkable expan Staining. We further observed an increase in collagen I Sion and thickening of the TA that was due to excessive mRNA levels and protein-bound hydroxy-proline, which are collagen fiber deposition (Ferrini et al., 2002). We also additional direct measurements of tissue collagen content observed a considerable increase in peroxynitrite as indi (Ferrini et al., 2002). These initial results demonstrated that cated by nitrotyrosine formation in the TA (Ferrini et al., iNOS was strongly expressed in PD tissue and may play an 2002). These observations further support the role of NO important role in the plaque. from iNOS in reducing the growth of the plaque in the rat 0228 To examine the specific role of NO on PD plaque TA. formation, overall NOS activity was increased in the TA by 0231. The effect of L-NIL in increasing collagen in the treating the animals with the oral NOS Substrate, L-arginine. TA of the TGF-B1 rat model may be due to an increase in The long-term oral administration of the NOS substrate, collagen Synthesis, a decrease in its normal breakdown, or L-arginine, in the drinking water (2.25%), leads to a stimu both. To determine whether the larger PD plaque in the lation of NOS activity and NO synthesis in the whole penis L-NIL treated rat is due, at least in part, to an increase in (Moody et al., 1997) and should also be increased in the TA. collagen I Synthesis (the most prevalent collagen protein in In addition, NO has been shown to be a down-regulator of the TA), and not simply to the inhibition of collagenolysis by collagen synthesis (Haig et al., 1994). If NO has a down the MMPs, we injected a cDNA plasmid construct of the regulatory effect on collagen Synthesis in the plaque, then collagen I promoter driving the expression of a reporter gene overproduction of NO should inhibit this collagen synthesis (Magee et al., 2002a) into the site of the original TGF-B1 or and prevent development or halt progression of the plaque. Saline injection, 10 days prior to Sacrifice and 35 days after It was observed that increasing NO by oral L-arginine the TGF-31 injection. This plasmid is an indicator of col treatment for 45 days led to a considerable decrease in the lagen I transcriptional activity within the rat PD plaque. size of the TGF-B1-induced plaque (FIG. 2A, top panels). Expression of the reporter B-galactosidase, measured by The plaque in the rat model consists of disorganized col luminometry in tissue extracts from areas at and around the lagen fibers and thickening of the TA that Seems to spread plaque, was considerably intensified in comparison to the extensively from the site of the TGF-?31 injection. This control TA (Vernet et al., 2002). This suggests that the observed decrease in plaque development by oral L-arginine reduction in NO by L-NIL inhibition of iNOS, directly or treatment was also verified by quantitating the ratio of the indirectly, activates pro-fibrotic factors such as ROS to areas in the TA occupied by collagen fibers to the areas further activate the collagen I promoter. occupied by the cells and lacunar spaces (FIG. 2B). Changes in the width of the tunica as measured by QIA also Example 3 confirmed these results (not shown). These observations demonstrate that NO, from oral NO donors, seems to play an The Inhibition of PDE Activity. In Vivo Reduces anti-fibrotic role in the TA of the TGF-31 injected rat model Collagen Deposition and Intensifies Fibroblast of PD. Apoptosis in the PD-Like Plaque in the Animal 0229. Another major effect of high levels of NO in any Model tissue is its conversion, by its interaction with ROS, into 0232) Numerous studies have documented that increasing peroxynitrite, which is a known inducer of apoptosis (Beck the levels of coMP by inhibition of PDE enzymes, either mann et al., 1996; Ferrini et al., 2001a; Vernet et al., 1998; with non-specific PDE inhibitors, such as pentoxifylline Heigold et al., 2002; Duffield et al., 2000; Zhang et al., (Corbin and Francis, 1999; Uckert et al., 2001; Fischer et al., 1999). NO in the TA may also act to increase apoptosis of 2001; Desmouliere et al., 1999; Kremer et al., 1999), or those cells within the PD plaque that are responsible for specific isoform inhibitors for PDE-5 such as exisulind promoting collagen Synthesis. In the PD animal model given (Chan et al., 2002; Takuma et al., 2001), can inhibit collagen oral L-arginine (2.25%), there appeared to be an increase in Synthesis and fibrosis and can induce apoptosis in Vivo and the number of apoptotic cells per field in the PD-like plaque in cultured cells (Chiche et al., 1998; Pandey et al., 2000; as compared to the control animals (FIG. 3A, top panels). Tao et al., 1999; Loweth et al., 1997; Sirotkin et al., 2000; But when an apoptotic index (apoptotic cells/total number of Taimor et al., 2000; Schade et al., 2002; Horio et al., 1999; cells) was used to quantify apoptosis, no significant differ Thompson et al., 2000). Thus, elevating c(GMP levels may ence was observed (FIG. 3B), because of the parallel be able to inhibit tunical plaque formation. A Study was increase in cell number. performed to determine whether the antifibrotic effects of NO in human and rat PD may be at least partially mediated Example 2 by the elevation of its downstream product, c0MP. 0233 Pentoxifylline (a non-specific, generalized PDE Inhibition of iNOS Activity In Vivo Stimulates inhibitor), and sildenafil (specific PDE-5 inhibitor) were Both Collagen Synthesis and Collagen Fiber given orally to the rat in their drinking water (100 mg/l for Deposition in the Rat PD-Like Plaque each PDE inhibitor) for 45 days following TGF-B1 injection 0230. The studies with L-arginine detailed in Example 1 to initiate the plaque in the rat model. FIG. 2A, bottom show modulation of the size of the PD plaque by NO. To panels shows that, as assessed by Masson Staining in terms determine whether the NO involved in this anti-fibrotic of collagen fiber/cell-lacunae area ratio, and width of the process emanated from iNOS, we studied in the TGF-31 rat tunica (not shown), there was considerable reduction in model the effects of specifically blocking iNOS activity by plaque size induced by the PDE inhibitors. This was accom the long-term oral administration of L-NIL, a specific iNOS panied by an intensification of the apoptotic index, espe US 2005/0085486A1 Apr. 21, 2005 cially for pentoxifylline (FIG. 3A, bottom panels). The prised of both TA and corpora cavernoSa was assayed. finding that pentoxifylline is more effective than sildenafil in Immunocytochemical detection with an antibody detecting inducing apoptosis may be due to a number of possible all three variants of PDE5A revealed that it was expressed in mechanisms. Since pentoxifylline inhibits multiple PDE discrete cells interspersed among collagen fibers in the isoforms, it may suggest that other PDEs besides PDE5 may normal human TA and the PD plaque (FIG. 4C, upper play a role in elevating c(GMP levels within the TA that will panels). PDE5A was also detected in the media of the of the lead to apoptosis of cells within the plaque. Additionally, dorsal artery and those within the corpora cavernosa, and in PDE inhibitors may act differentially on the three processes both the corporal smooth muscle and TA of the penis (FIG. that ultimately would inhibit fibrosis development, namely 4C, lower panels). myofibroblast apoptosis, collagen Synthesis, and collagen 0237) PDE5 mRNA was also identified by RT/PCR in the degradation. Although we have not identified the cells fibroblasts cultured from the human normal TA and PD undergoing apoptosis, it is likely that they are fibroblasts/ plaque, and from the rat TA (FIG. 7A), and the respective myofibroblasts, because those are the predominant cellular protein was detected by Western blot in the human cells as component of the rat TA, and because of the direct demon a single PDE5A-3 variant, which agrees with what was stration of the effects of these treatments on cultures of observed in vivo in the TA and PD plaque, (FIG. 7B). The human fibroblasts and myofibroblasts (see below). rat TA fibroblasts also express the 3 variant, accompanied by equal amounts of the 1 variant, despite the latter larger Example 4 variant was not detected in the rat penile shaft. Immunocy tochemical detection (FIG. 4C, upper panels) confirmed the Presence of PDE5 in the Human Penile Tunica expression of PDE5A in the three types of cells, namely Albuginea and PD Plaque, in the Rat Tunica fibroblasts from the human normal TA and PD plaque, and Albuginea, and in Fibroblasts Cultured From These from the rat normal TA. However, in the latter case, as Tissues opposed to the human cell cultures derived from tissues 0234. As an initial assessment of PDE isoforms reasonably free from contamination by cavernoSal Smooth expressed in the TA, RT-PCR was performed on splice muscle, the rat fibroblasts were obtained from whole corpora products of PDE5A (Kim et al., 2000). The observed effects cavernosa including the Smooth muscle. By Successive of sildenafil (specific PDE5 inhibitor) and to a partial extent passages in fibroblast culture medium (Smith et al., 2002), pentoxifylline (has some PDE5 inhibitor activity) are prob rather pure fibroblast cultures were Selected, as evidenced ably mediated by the PDE5A isoform. RT/PCR with primers from vimentin staining, and Some were myofibroblasts, as common to the 3 PDE5A variants (Uckert et al., 2001; Lin seen with ASMA staining (FIG. 7A, bottom panels). et al., 2000a, 2002a) have shown that this enzyme is expressed in both human TA and PD tissues, as well as Example 5 control tissue, the corpora cavernosa (FIG. 4A). FIG. 4A shows the ethidium bromide staining of PCR DNA frag Presence of PDE4 Variants in the Human Penile ments from reactions carried out in duplicate and fraction Tunica Albuginea and PD Plaque, in the Rat Tunica ated by agarose gel electrophoresis. The 575 bp PDE5A Albuginea, and in Fibroblasts Cultured From These DNA band was generated as expected from the rat penile Tissues shaft (PS) and the 762 bp from the human corpora cavernosa (CC) RNAS, and was amplified to a similar level in total 0238 PDE4 mRNA Studies RNA from the human TA and PD. No RNA was extracted 0239). Since the cAMP-dependent PDE inhibitor, pen from the normal TA and the TGF-B1-induced PD-like plaque toxifylline, has been used previously as an antifibrotic in the rat, due to the difficulty in dissecting large amounts of compound (Lee et al., 1997; Becker et al., 2001; Raetsch et tissue to avoid contamination by cavernoSal Smooth muscle. al., 2002), we investigated by RT/PCR whether PDE4 is 0235 PDE5A expression was confirmed at the protein expressed in the TA and PD tissues and the respective cell level by Western blot assays of tissue extracts, as shown by cultures, utilizing primers for two (A and B) of the three the luminol-stained protein bands (FIG. 4B), that can dis variants. FIG. 12A shows that PDE4A and B mRNAS are criminate the three splicing variant proteins of PDE5A expressed in the human normal TA and in the PD tissue. designated as 1, 2, and 3 with respective apparent sizes of Both variants were also detected in human corpora cavern 100, 92, and 83 kDa, respectively (Lin et al., 2000a, 2002a). osa tissue containing mainly Smooth muscle (not shown). The three variants were detected as expected in the rat Confirming these results, PDE4A and B mRNAs were also cerebellum (CER), our control tissue, whereas in the rat found in the fibroblasts cultured from human TA and PD penile crura (CRU) and shaft (PS), the predominant forms (FIG. 12B). In the case of the rat, PDE4 mRNA (without were the 1 and 3, respectively, with only traces of variant 2 variant discrimination) was detected in the TA cells, and to in the crura, and a band Smaller than the 3 variant in the a lesser degree in the penile shaft tissue, thus Suggesting that penile shaft. This PDE5A-3 variant, accompanied by smaller PDE4 in the rat TA fibroblast cultures does not arise from amounts of the 2 variant, was also expressed in the human contamination with Smooth muscle, which in any case had corpora cavernosa (CC), and in the TA and PD plaque. Some been reasonably excluded above by immunocytochemistry. PDE5A-1 variant was also detected in the human CC. 0240 PDE4 Western Blot. 0236 Immunohistochemistry in human PD and TA sec 0241 Confirmation of the expression of PDE4A at the tions confirmed the expression of PDE5 in both tissues, at a protein level was obtained by western blot with an antibody higher level in PD. In the rat, it is extremely difficult to for the different variants, in extracts from the human cul isolate pure TA and PD-like tissue totally free from corpora tured cells used for the identification of PDE4A in TA and cavernosa cells. Therefore, the whole rat penile shaft, com PD plaque. FIG. 12C shows an intense 76 kDa band that US 2005/0085486A1 Apr. 21, 2005 22 would correspond to a variant identified in testis (Salanova 0246. In order to determine whether the PDE inhibitors et al., 1999), as well as a minor 102 kDa band for the may reduce collagen Synthesis, the PD cells were incubated So-called PDE4AX, also seen in the testis. The 76 kDa with or without the drugs at lower concentrations: pentoxi protein is very intense in the three human tissues: TA, PD fylline at 200 nM, and sildenafil, at 50 and 200 nM. After 3 plaque, and corpora cavernosa, but the 102 kDa band was days, cells were fixed and the intracellular deposition of virtually not detected. No PDE4B could be visualized when collagen I and III was determined by immunocytochemistry the western blot membranes were Stripped and reacted with with Specific antibodies against the two isoforms. The anti an antibody specific for this isoform (not shown). body against collagen I elicited an intense granular and perinuclear staining (not shown). In contrast, collagen III 0242 PDE4 Immunohistochemistry was detected in only in about 30% of the cells, and stained 0243 Immunodetection with the PDE4A antibody iden more diffusely and rather lightly, even when cells where tified cells all along the internal side of the TA, as well as in treated with TGF-B1 (10 ng/ml), a known stimulator of the corpora cavernoSa Smooth muscle, expressing PDE4A collagen III synthesis (FIG. 5B). (FIG. 13, top). An antibody specific for PDE4D also showed 0247 Quantitation by image analysis (FIG. 5A) in the cells reactive for this isoform, evidencing that both PDE4 cultured human PD fibroblasts indicated that in the absence genes are expressed in the TA and corpora cavernosa (FIG. of additions, most of the cells (100%) expressed collagen I, 13, top). A similar situation is seen in the rat TA fibroblasts and that both pentoxifylline and sildenafil at 200 nM com in culture, with considerable expression of PDE4A in most pletely inhibited collagen Synthesis in a Small number of cells, whereas only a fraction of the cells express PDE4D cells (5-15% of the total, FIG. 5A top), and significantly (FIG. 13, middle). In contrast, most of the human TA reduced (30-40% decrease) the average intensity of expres fibroblasts were intensively stained with antibodies against sion per cell (FIG. 5A, bottom). In contrast, the PDE the A and D isoforms (FIG. 13, bottom). Despite the fact that inhibitors did not decrease, but even increased, the Synthesis the PDE4B mRNA was identified by RT/PCR (see FIG. 12), of collagen III (not shown). More drastically than in the case virtually no protein reactivity for this isoform was observed of collagen I, both of the PDE inhibitors (pentoxifylline and by imunodetection in tissue sections or cell cultures (not sildenafil) significantly reduced the number of ASMA posi shown). A similar situation occurred with one of the variants tive cells (myofibroblasts) from 37% in the control to about of PDE4A (PDE4A4), utilizing a specific antibody, different 24%. The average ASMA expression per cell was signifi from the general used above for PDE4A that according to the cantly reduced by the PDE inhibitors by more than 90% in Supplier does not detect variant 4. all cases. Example 6 0248. To show that in the case of sildenafil Some of the effects are mediated by the elevation of c(GMP, the PD Incubation of PD Fibroblast Cultures With PDE fibroblasts were then incubated for 3 days with 8-Br-cGMP, Inhibitors or a coMP Analog Reduces Collagen I and a significant (30%) reduction in the number of cells Synthesis and Myofibroblast Differentiation, and expressing collagen I was obtained at 10 uM 8 Br-cGMP Increases Apoptosis (FIG. 6), although a higher concentration (400 uM) did not induce further decrease (FIG. 6). In contrast to the effects of 0244 Verification that the PDE5A and PDE4 proteins the PDE inhibitors observed in the previous experiments, detected in the TA and PD cells and tissues are enzymatically collagen I expression per cell was reduced only moderately active was obtained by measuring the levels of c(GMP in cell and non-significantly by the coMP analog. The differentia extracts of the PD fibroblast cultures, with a basal mean tion of fibroblasts into myofibroblasts measured by the level value--/-SEM of 5.0+/-0.4 pmol/mg protein (n=5) in the of ASMA expression per cell was decreased significantly by absence of additions. Levels of c(GMP increased 5.0-fold 400 uM8 Br-cGMP, as in the case of the PDE inhibitors, but with 100 uM SNAP (an NO donor) for 3 days, with fresh there was no effect on the relative number of positive cells daily replacement of medium with SNAP. AccMP analog (FIG. 6). able to enter the cell, 8 Br-cGMP, at 10 uM, was also able to increase coMP levels by 6.4-fold, and with 100 uM 8 0249. The increase of coMP in the PD cells incubated Br-cGMP, the levels of c(GMP were dramatically elevated by with 8 Br-cGMP leads to a stimulation of apoptosis, as 38.7-fold. The basal levels of cAMP were 42.6+/-12.7 shown by an increase in apoptotic bodies detected with the pmol/mg protein in the absence of SNAP and did not vary TUNEL technique (FIG. 21A). However, because of vari significantly when measured after 3 days with SNAP. ability between experiments, the considerable 2.3 fold increase measured by image analysis did not achieve Statis 0245. The coMP-dependent PDE5 inhibitor sildenafil tical significance (FIG. 21B). did not significantly stimulate coMP levels in the absence of SNAP (not shown). However, in the presence of the NO Example 7 donor, the coMP levels expressed as % of the basal control levels in the absence of Sildenafil, were increased dose Increase in NO Levels in Fibroblast Cultures From dependently by Sildenafil after the 3-day incubation, as Human Normal TA and PD Leads to Peroxynitrite expected (FIG. 14A). When cells were incubated with this Formation, Fibroblast Apoptosis, and Reduction of NO donor, increasing concentrations of pentoxifylline, also Intracellular Collagen as expected, did not increase significantly c0MP levels expressed as % of control levels (FIG. 14B), but were very 0250 We have developed primary cell cultures from effective in increasing cAMP levels (FIG. 14C), thus con human normal TA and PD plaque, obtained from different firming its role as a cAMP-dependent PDE inhibitor with patients, that were dissected to avoid contamination with little or no effect on coMP-dependent PDE. corporal Smooth muscle. These cultures contain fibroblasts US 2005/0085486A1 Apr. 21, 2005 23 and some myofibroblasts, as shown by a 100% immuno positive by immunocytochemistry (FIG. 7C, top panels). reactivity with a vimentin antibody (Vernet et al., 2002) and Cells derived from human TA tissue were clearly fibroblasts represent the main cellular component of the original tis with some myofibroblast differentiation, as assayed with SCS. vimentin and ASMA markers (FIG. 7C, bottom panels). 0251 The main features of these cultures, demonstrated Example 9 by QIA immunocytochemistry, are: 1) a Substantial morpho logical difference between the TA and PD plaque cells ROS Levels are Increased in the Human and Rat (Vernet et al., 2002), that corresponds to the observations in PD-Like Plaque Tissues and are Reduced by NO Vivo. PD cells change from Small, more Spindle shaped cells to much bigger, polygonal cells with bigger nuclei and 0255 ROS plays an important role in the development expansions, and in certain cases typical "stellate' appear and maintenance of many fibrotic disorders including PD, ance whereas TA cells do not Substantially change in mor by stimulating collagen synthesis (Poli, 2000; Curtin et al., phology. 2) These fibroblasts, particularly those of PD 2002; Cattell, 2002; Kim et al., 2001; Fan et al., 2000; origin, are able to differentiate into vimentin--/ASMA+ Higuchi et al., 1999). Therefore, the interplay and reactivity myofibroblasts comprising about 30% of the cells in culture of ROS with NO may be an important therapeutic target. We (Vemet et al., 2002), and this percentage of myofibroblasts have previously shown that heme-oxygenase I immunore is also seen in vivo in the plaque (Vemet et al., 2002). 3) The activity, a marker for the strong pro-fibrotic factor ROS, is cultures can be induced to express iNOS, Synthesize col increased in the PD plaque in comparison to normal TA in lagen I, and undergo apoptosis both in vitro and in vivo the human and the TGF-B1 rat model of PD (Ferrini et al., (Vernet et al., 2002). 4) Their responses to different agents, 2002). Additionally, when iNOS activity was blocked with specifically the inhibitory action of an NO donor, SNAP, on L-NIL in the TGF-B1 rat model, there was a considerable a) collagen I Synthesis in all the cells, and b) myofibroblast elevation of ROS levels (Ferrini et al., 2002). The same differentiation (Vernet et al., 2002), and their response to inverse correlation between NO and ROS was observed PDE inhibitors in cell culture (FIG. 5) resemble the utilizing Superoxide dismutase immunodetection, which responses observed in vivo in the animal model of PD measures the antioxidative response in both human and PD treated with NO donors or PDE inhibitors (FIG. 3). 5) tissue and in human fibroblast cultures (not shown). This Collagen III, is also synthesized but to a lower eXtent than reduction in the NO/ROS ratio is associated with a consid collagen I and is not significantly affected by NO or PDE erable Stimulation of collagen deposition and collagen Syn inhibitors (FIG. 5). thesis (Ferrini et al., 2002). 0252) The use of an NO donor SNAP or iNOS induction Example 10 with a cytokine cocktail increased intracellular NO reduced fibroblast numbers in both normal human TA and PD Use of Gene Transfer and Reporter Gene cultures (Vernet et al., 2002). This process is associated with Expression for Analyzing PD the production of peroxynitrite, as evidenced by nitroty 0256 Several of the therapeutic approaches disclosed rosine formation (not shown) and resembles the increase in herein are based on gene transfer of cDNA constructs to the apoptosis Seen in Vivo after Sildenafil and pentoxifylline penile TA (e.g., iNOS, PKG). Therapeutic administration of treatmentS. recombinant cDNA may lead to an elevated expression of the corresponding anti-fibrotic protein. In the case of the Example 8 penis, we have utilized plasmid and adenoviral constructs of iNOS and penile nNOS (PnNOS) (Magee et al., 2002a), The Increase in c(GMP Levels in Fibroblast including the use of plasmid and adenoviral constructs Cultures Leads to Fibroblast Apoptosis and the expressing f-galactosidase as a reporter gene, and electropo Reduction in Intracellular Collagen I ration to enhance viral and plasmid uptake during transfec 0253) To verify that the effects of pentoxifylline and tion (Magee et al., 2002a). sildenafil described above were due to an elevation in c(GMP 0257 Such constructs can penetrate and spread into the levels, we incubated human PD cells with a stable c(GMP TA, as shown by X-gal staining (FIG. 8) suggesting that the analog able to traverse the cell membrane, 8-BrcGMP. This direct injection to the TA with and without electroporation is compound (with levels as low as 10 uM) inhibited collagen feasible for targeting genes to the TA for arresting or I production by the cells (FIG. 6), and increased apoptosis, reversing the growth and development of the PD plaque. as determined by TUNEL (Ferrini et al., 2001a; Vernet et al., 1998), from 7.5+/-0.4 (control) to 20.5+/-1.1 (100 uM Example 11 8-BrcGMP) cells per field. The intracellular coMP levels increased from 0.02 to 1.83 nmoles/10 cells with 100 uM Confirmation of the Pro-Fibrotic Role of TGF-B1 8-BrcGMP in control vs. treated cells, respectively. Expression in Another Model of PD 0254 The effects of the PDE inhibitors, as seen in in vivo 0258. The TGF-B1 rat model for PD is a very valuable experiments, are most likely mediated in part by PDE-5, as tool, and since its introduction in 1997 (E1-Sakka et al., shown by the presence of PDE5A mRNA (FIG. 7A), and 1997b, 1998, 1999), we have been able to study various specifically, the PDE5A-3 protein variant as in the in vivo aspects of the pathophysiology of PD, Some of which are derived tissues (FIG. 7B), in both the human TA and human presented above. However, in a different experimental PD derived cells. The fibroblast cultures obtained from the design based on the ubiquitous finding of fibrin in histo rat TA express all three PDE-5 variants. All human and/or rat logical Samples of human PD tissue (not shown), we have cell cultures of normal TA and PD tissues were PDE5 recently re-confirmed the importance of TGF-B1 as the main US 2005/0085486A1 Apr. 21, 2005 24 profibrotic factor in eliciting the PD-like plaque in the TA of thymosin-fi family (Table 1). These proteins stimulate MMP the rat. We observed considerable expression of this factor activity, cross-link to fibrin and collagen, and promote in a tunical lesion induced by the injection of a preparation wound healing (Huffet al., 2002; Malinda et al., 1999; Sosne of human fibrin (fibrinogen/thrombin/aprotinin) in the rat TA et al., 2002). Their increased synthesis in the PD plaque may (FIG. 9). This tunical lesion, caused by fibrin and mediated be another manifestation of a defense mechanism that is by TGF-31, is fully developed at 3 weeks, as visualized by unable to control or arrest the progression of fibrosis. The Masson Staining, and is indistinguishable from the one administration of thymosin-B4 has been proposed for wound caused by the injection of TGF-B1 alone. However, the healing (Huffet al., 2002; Malinda et al., 1999; Sosne et al., TGF-31 model requires 6 weeks to develop, whereas this 2002), and, as stated above, PD is likely the result of an fibrin induced model only requires 3 weeks to fully develop injury that does not heal properly. It should be possible to (not shown). The fibrin induced plaque is accompanied by further up-regulate this endogenous defense mechanism by detection of fibrin in the lesion (similar to what is seen in the pharmacologically increasing thymosin levels in the TGF human, but absent from the TGF-B1 injected rat model), B1-induced lesions in the rat model of PD. disorganization of elastin fibers, expression of iNOS and heme-oxygenase I, and an increased level of apoptosis. Save Example 14 for the presence of fibrin in the TA, all findings are similar to the ones observed in the TGF-31 injected model (not Investigating the Role of NO and ROS in PD shown). Using the iNOS Knockout Mouse Model Example 12 0262 The blockade of iNOS activity in the rat by long term oral L-NIL administration (Ferrini et al., 2002) is only partially effective in inhibiting iNOS. Therefore, the iNOS The TA and the PD Plaque are Tissues in Constant knockout mouse (Hochberg et al., 2000) is of use for studies Turnover, and Collagenase Inhibition May Play a where iNOS expression is completely absent. NO in this Role in Collagen Accumulation animal model can be synthesized only by the other NOS 0259. As disclosed above, collagen synthesis is stimu isoforms, namely eNOS and nNOS. These isoforms are in lated in the TGF-B1 animal model of PD, particularly when general constitutive and as Such are difficult to induce. It NO synthesis is partially inhibited by L-NIL. Data obtained therefore Seems unlikely that they would play a significant by DNA microarray analysis (Clontech) has allowed us to anti-fibrotic role. The iNOS knockout mouse has previously define and compare changes in the profile of multiple gene been used to show that experimental urethral fibrosis is expressions at the mRNA level in the PD plaque versus intensified as a consequence of the iNOS knockout (Tanaka normal TA. Data (Table 1), obtained in 9 patients and 9 et al., 2002). We have tested whether it is possible to develop control subjects indicated that: a) PD, like its related disor a PD-like plaque by injection of TGF-31 into the TA, der Dupuytren's contracture, is a condition that Seems to be injecting 0.2 ug of TGF-31 into the TA of a wild type mouse. in a State of dynamic flux, with alterations in the expression FIG. 11 shows plaque formation within the TA at 6 weeks of mRNAS for genes related to collagen turnover and tissue as shown with Masson Staining. remodeling, extracellular matrix Synthesis and degradation, and cell replication and apoptosis. This Suggests that the Example 15 fibrosis of PD is not a terminal event but a dynamic one, and that it is possible to pharmacologically affect its Steady State Therapeutic Intervention in PD and alter its direction by: a) inhibiting collagen synthesis and/or fibroblast differentiation and replication, Since a 0263. The results above demonstrate that PDE5 and 4 are Subset of differentially expressed genes are related to these both expressed in the human and rat normal tunica albug processes; and/or b) by Stimulating collagen breakdown, inea, and the respective PD and PD-like fibrotic plaques, as Since there is a considerable increase in the expression of well as in the cell cultures obtained from these tissues. The results also demonstrate the inhibition of a TGF-B1-induced different types of MMPs (e.g. MMP2 and MMP9). The fibrotic plaque in the rat model of PD, through the reduction increased expression of MMPs was confirmed in human PD of collagen deposition and possibly an increase in apoptosis by RT/PCR (FIG. 10). of the resident fibroblasts and myofibroblasts, by long-term 0260 MMPS may play an important role in extracellular oral administration of the respective PDE5 and cAMP matrix remodeling in the PD plaque. The inhibition of MMP dependent PDE inhibitors, sildenafil and pentoxifylline, and may occur by increased activity of the MMP inhibitors the NOS substrate, L-arginine. (TIMP). We have verified the increased expression of TIMP in PD tissue using the more sensitive RT/PCR procedure, 0264. The in vitro effects of both PDE inhibitors and a which showed a 2-fold stimulation of TIMP1. The increased cGMP analog, 8 Br-cGMP, on fibroblast cultures obtained from the human PD plaque, indicate that these agents may expression of TIMP1 should lead to an increased inhibition be effective against fibrosis by reducing the relative number of MMP. of fibroblasts/myofibroblasts through the induction of apo Example 13 ptosis of these cells. We also found that these compounds a) interfere with fibroblast differentiation into myofibroblasts, The Expression of a Family of Wound the cells that are key players in tissue fibrosis, and b) Healing-Related Peptides, the Thymosins, is down-regulate the Synthesis of collagen I but not collagen m. The effects of sildenafil may be exerted through the inhibi Increased in Human PD tion of PDE-5, and in the case of pentoxifylline through a 0261. In the DNA microarray study mentioned above, we cAMP-dependent PDE, potentially PDE4. The results open found increased expression of peptides belonging to the a new approach for the treatment of PD and, by extension, US 2005/0085486A1 Apr. 21, 2005 25 tissue fibrosis, based on the use of PDE inhibitors and other with toxicity. In addition, it is possible that local adminis enhancers of PDK activity, and possibly of compounds and tration of either L-arginine or the PDE inhibitors, e.g. by biologicals that enhance NO Synthesis. injection into the plaque or in vehicles able to traverse the 0265). The reduction of the fibrotic plaque observed in skin and TA may considerably reduce the effective dosage. vivo in animals receiving L-arginine, coincides with its 0268. It is unknown why administration of L-arginine, effects in preventing experimental ethanol-induced inflam which should increase NO synthesis and hence coMP levels matory and fibrotic changes in liver, kidney, lung, and and has been shown to be effective in arresting the growth cardiovascular system (Nanjiet al., 2001; Peters et al., 2000; of the TGF-B1 induced plaque in the rat model of PD, failed Simko and Simko, 2000; Susic et al., 1999; Song et al., to stimulate apoptosis, as could be expected from its effects 1998; Bing et al., 2002; Alves et al., 2002). The action of increasing it in vivo in the Smooth muscle of the pulmonary L-arginine may be mediated by the stimulation of NOS arteries (Wang et al., 1999; Holm et al., 2000). However, the activity. This was previously shown by the increase of absence of a stimulation of the apoptotic index in the PD L-arginine levels in the penis and the improvement of plaque by L-arginine may agree with the decrease in apop erectile dysfunction in the aging rat by NOS stimulation tosis observed in liver transplants which is in line with the achieved after a regimen of L-arginine administration of 2.2 anti-apoptotic effects of NO in certain conditions and tissues g/kg/day (Moody et al., 1997). This dose is within the range (Wang et al., 2002b). In any case, not only c0MP but its normally employed as vasculoprotective for long-term Stud down-stream compound in the NO-cGMP cascade, PKG, is ies in the rat (Bing et al., 2002; Alves et al., 2002). also effective in preventing fibrosis and remodeling in 0266 The in vivo and in vitro results showing an inhi balloon-injury and arterial restenosis (Wollert et al., 2002; bition of collagen synthesis and stimulation of apoptosis in Chiche et al., 1998), as shown by gene transfer of the PKG the PD-like plaque and in PD cells by both sildenafil and cDNA in rats. pentoxifylline, are in good agreement with the extensive use 0269. The results demonstrating the presence of PDE5A pentoxifylline as an antifibrotic agent in liver and vascular and PDE4 in the TA and PD plaque in the human and rat, and fibrosis (Becker et al., 2001; Raetsch et al., 2002; Chen et al., in their respective fibroblast cultures, provide a rationale for 1999; Tarcin et al., 2003). The fact that the coMP analog the anti-fibrotic effects of PDE inhibitors on the PD animal 8-Br-cGMP inhibited collagen I synthesis and induced apo model and on the PD cell cultures. The PDE5A1 and ptosis in PD cells suggests that in the case of Sildenafil the PDE5A2 proteins have been previously localized in human in vivo effects on the function of the fibroblasts/myofibro penile corpora cavernosa (Lin et al., 2000a). The PDE5A3 blasts in the TA may be mediated by the elevation of c(GMP variant was also found in corpora cavernosa and confined to levels. In addition, c0 MP analogs, PKG activators, and PDE tissues with a smooth muscle or cardiac muscle component, inhibitors have been shown to inhibit collagen synthesis and is twice as sensitive as PDE5A1 to sildenafil, but, as (Redondo et al., 1998; Wollert et al., 2002), and induce with PDE5A1 and 2, is subject to transcriptional up-regu apoptosis (Sirotkin et al., 2000), and some of the PDE lation by both cAMP and coMP (Lin et al., 2002a; Turko et inhibitors like Sulindac sulfone (Exisulind) are effective as al., 1999). As to PDE4, cAMP can activate PKG nearly as anticancer agents because of their intense pro-apoptotic effectively as cCMP, so that eventually, the inhibition of action (Piazza et al., 2001; Thompson et al., 2000). How PDE4 may cause PKG effects (e.g., counteracting fibrosis) ever, since pentoxifylline did not affect cOMP levels in the similar to those exerted by as the inhibition of PDE5A. human PD fibroblasts, and the drug is considered to be a non-specific inhibitor of cAMP-PDE (Lin et al., 2002c; 0270. The results reported above indicate that pharma Liang et al., 1998), and at least in Some cell types does not cological interventions aimed at elevating NO, c0MP, or affect cOMP levels (Chen et al., 1999), the increase in cAMP PKG levels, and possibly cAMP, in the penis are of use for may also have played a role in the antifibrotic effects the treatment of PD, and potentially, for other fibrotic observed with pentoxifylline. Whether this occurs via the conditions. This work has not addressed the question on inhibition of PDE4 present in TA and PD remains to be whether intervention would induce regression of an already established. Pentoxifylline may also act through its blockade well-formed plaque, but comparison of multiple gene expression profiles in human PD and the related of PDGF-induced activation of the mitogen activated protein Dupuytren's disease suggest that both conditions are in a kinase system (Souness et al., 2000) and of other cytokine dynamic cell and protein turnover involving replication, mediated fibrogenic mechanisms (Raetsch et al., 2002). differentiation, apoptosis, and collagen and extracellular 0267. The daily dose of pentoxifylline used was /s of the matrix synthesis and breakdown (Magee et al., 2002b: oral dose normally employed in rats for the long-term Gonzalez-Cadavid et al., 2002; Gholamiet al., 2002). There treatment of fibrosis (Chen et al., 1999; Tarcin et al., 2003), fore, modulation of any of these processes may involute the and in the case of sildenafil, it is % to /7 of the chronic plaque, as has been observed in generalized fibrotic condi dosage used in recent studies in rats (Sebkhi et al., 2003). tions (Lee et al., 2001; Lai et al., 2000). When the 10 mg/kg/day dose is translated into the equiva lent dose in humans by correcting for differences in the body Example 16 weight/skin area (Freireich et al., 1966), it is roughly 1.5 mg/kg which is about the dose ingested by men with an on Intensification of Aging-Related Fibrosis in the demand single 100 mg tablet. The Selected dose was dis Arterial Media by iNOS Inhibition pensed in 24 hours and not as a bolus administration, So that concentrations at a given time should be much lower, 0271 In order to determine whether aging per se is considering the short half-life (about 4-6 hours) of sildenafil. associated with an intensification of collagen deposition and Therefore, the daily doses of the PDE inhibitors tested in the a relative loss of SMC in the media from the aorta to the current work are not supra-pharmacological or associated peripheral resistant arteries (Breithaupt-Grogler and Belz, US 2005/0085486A1 Apr. 21, 2005 26 1999; Robert, 1999; Integan and Schiffrin, 2000), staining aging. The iNOS staining in these vessels was mainly was performed on Sections from the abdominal aorta, femo confined to the media and intima. A similar finding was seen ral, and brachial arteries as well as from the penile shaft in the aorta, brachial, and femoral arteries (not shown). focusing on two peripheral putative resistance arteries: the When L-NIL, the inhibitor of iNOS activity, was given, bulbourethral and dorsal arteries of the penis. FIG. 15A there was a reduction in iNOS expression, which combined shows that in the media of the dorsal penile artery, few with the direct decrease of iNOS activity, led to a reduction collagen fibers were present in the young rats but were in peroxynitrite formation. Quantitation by image analysis considerably increased in the aged animals, resembling the confirmed these changes in the resistant dorsal and bul Situation Seen in the aorta. Consistent with the model that bourethral arteries of the penis (FIG. 16B), as well as in the iNOS may act as antifibrotic agent within the vascular tree, aorta and femoral arteries (not shown). The brachial artery the administration of L-NIL, a specific inhibitor of iNOS was not Subjected to image analysis. activity, for 3 weeks to the aged rats led to a further increase in the collagen fibers within the media of the aorta and the Example 18 dorsal penile artery. 0272 Image analysis was performed in all arteries with Effects of iNOS Inhibition on ROS Production, the exception of the brachial (FIG. 15B), on 5 animals per Apoptosis, and PAI in the Arterial Media experimental group, and 6 Sections per animal (3-4 fields per 0275 Utilizing the Cu/Zn SOD as an indirect marker of Section). In all vessels studied, there was a marked reduction ROS, the production of ROS in the arterial media was found in the SMC/collagen ratio with aging. Following iNOS to be considerably increased with aging in the femoral, blockade by L-NIL, there was a further exacerbation in the brachial and resistant arteries but not in the aorta, and this amount of collagen within the media (with the exception of process was further increased with iNOS inhibition by the bulbourethral artery), Suggesting that the decrease in NO L-NIL (FIG. 17A). This was additionally confirmed by production by the inhibition of iNOS leads to an intensifi image analysis (FIG. 17B), that indicated that L-NIL block cation of the aging-related fibrosis. These alterations were ade of iNOS activity raised Cu/Zn SOD by 40 to 50%. The not accompanied in the resistant arteries by a significant Mn SOD gave similar results from the aorta to the resistant increase in the intima/media thickness (IMT), whereas in the arteries (not shown). Another antioxidant enzyme, heme aorta and femoral the IMT was higher (Table 2). The oxygenase I, demonstrated the same aging related changes measurements of the luminal diameter (Table 2) confirmed in the penile dorsal artery, as observed for both SOD the clinical observation that the dorsal and bulbo-urethral enzymes, but remarkably, L-NIL did not induce a further arteries, with a luminal diameter well below 350 um, fall Significant change in the expression of this enzyme (FIG. within the definition of resistance arteries (Intengan and 18). The localization of virtually all the expression of heme Schriffin, 2000; Moore and Schiffrin, 2001). oxygenase-1 was in the arterial adventitia, rather than in the Example 17 media as seen for the SOD enzymes. 0276. The NO/ROS balance was significantly altered iNOS Induction and Peroxynitrite Deposition in the throughout the entire arterial media by iNOS inhibition with Arterial Media With Aging L-NIL via a reduction in NO synthesis (denoted by perox ynitrite) and a stimulation of ROS formation (denoted by the 0273 All the antibodies used in this work have been antioxidant enzymes). Apoptosis of the SMC within the validated (Ferrini et al., 2001a, 2002; Vernet et al., 2002; media of the penile resistance arteries increased with aging, Goettsch et al., 2001), and in the case of the iNOS antibody and decreased Subsequently in the old animals receiving it was additionally tested in the current work by immuno L-NIL treatment (FIG. 19A). The apoptotic index was cytochemistry against rat fibroblast cultures (penile tunica calculated for both the dorsal and bulbourethral penile albuginea, see Ferrini et al., 2002; Vernet et al. 2002) arteries by image analysis, and was higher in aged compared induced to express iNOS with a cytokine cocktail, producing 130 uM nitrites (Hung et al., 1995). These cells were to young rats but L-NIL treatment resulted in a reduction in intensively stained, in comparison to uninduced cells (<10 this index (FIG. 19B). uM nitrites) that were negative (not shown). Western blots 0277 Aging alone or in combination with iNOS inhibi revealed the expected single 130 kDa band in extracts of the tion affected the expression of PAI-1, a well characterized induced cells, also detected by the corresponding mono inhibitor of metalloproteinases (Liet al., 2000; Kaikita et al., clonal antibody, and this band was absent in aorta extracts 2002). Inhibition of PAI is associated with an increase in from a young iNOS knockout mouse that had received LPS collagen fibers due to its interference with metalloprotein (4 mg/kg) to induce iNOS, whereas the band was visible in ases that are involved with the breakdown of collagen. the respective extract from the Similarly treated wild type Compared to young animals, PAI expression was consider animal (not shown). ably increased in the arterial media with aging, and was even further stimulated by iNOS inhibition, as seen in the resis 0274) This antibody showed that iNOS is increased with tant artery (FIG.20A). Quantitative image analysis for both aging in parallel with collagen deposition in the arterial the mean intensity of expression (FIG.20B) and the number media throughout the vascular tree, confirmed by detection of PAI positive cells (FIG. 20B) indicated that the increase of nitrotyrosinylated proteins. The latter arise from perOX in PAI by aging alone was between 2- and 5-fold, respec ynitrite produced by the reaction between NO and ROS, and tively. However, the effect of L-NIL on PAI expression in the therefore are an indirect measure of NOS activity. FIG.16A shows negligible iNOS expression and nitrotyrosine forma aged media was negligible (FIG. 20B). tion in the dorsal artery of the penis of the young animals, 0278. These results indicate that the arterial media from and a remarkable intensification of both processes with the aorta to the Small resistant arteries undergoes many of US 2005/0085486A1 Apr. 21, 2005 27 the changes that occur within the corporal tissue with aging, apoptosis (Connat et al., 2001; Asai et al., 2000), would namely: a) a reduction in the SMC/collagen ratio; b) an explain the clinical observation in humans of diminished increase in markers of oxidative StreSS, and of inhibitors of arterial elasticity associated with aging, which in Some collagen degradation, Such as PAI, which are known pro instances is compounded by a reduction of the arterial lumen fibrotic factors; and c) the spontaneous induction of iNOS, due to media/intimal thickening (Moore and Schiffrin, which is believed to act as an anti-fibrotic agent (Vernet et 2001). The fact that different vessels in the arterial tree, al., 2002; Ferrini et al., 2002; Hochberg et al., 2000). How regardless of size or location, seem to experience fibrosis of ever, the increase in SMC apoptosis in the media of the the media may explain dysfunctional vasorelaxation or resistant arteries of the penis, presumably leading to a impaired perfusion of many organs that occurs with aging reduction in the absolute SMC content, contrasts with what (Breithaupt-Grogler and Belz, 1999; Robert, 1999; Integan has been reported for large vessels Such as the aorta and the and Schiffrin, 2000, Formieri et al., 1992; Garban et al., femoral artery (Connat et al., 2001; Asai et al., 2000), but 1995; Ferrini et al., 2001a; Rogers et al., 2003; Berry et al., does agree with the process described in the corporal SMC 2001). The ability of the resistant arteries to relax normally (Garban et al., 1995; Ferrini et al., 2001a). is fundamental for the control of the systemic blood pres 0279. Our results also confirm the role for NO derived Sure, and as exemplified by the dorsal penile and bulboure from iNOS produced by the SMCs of the media in combat thral arteries in this study, they showed an intensification of ing aging-related fibrosis within the media, as evidenced SMC loss due to apoptosis without a change in IMT, which both by the increase in ROS and an intensification of fibrosis agrees with has been previously reported for the mesenteric within the media of the arterial wall when iNOS activity is Small resistant arteries in hypertension (Rizzoniet al., 2000). inhibited. The excessive deposition of collagen fibers 0282 Although NO has been shown in animal models to observed in the arterial media of the aged rats is thought to be protective against atherosclerosis and restenosis in the lead to arterial StiffneSS or arteriosclerosis in the vascular vascular System (Gewaltig and Kojda, 2002, Cheng et al., System. Because of the apoptosis occurring in the SMC, the 2001), and fibrosis throughout the vascular tree and other relative reduction in the SMC/collagen ratio is intensified in organs (Ferrini et al., 2002; Vernet et al., 2002; Gewaltig and the resistant arteries of the penis, in comparison to the larger Kojda, 2002), the concept that NO may prevent aging arteries, e.g. the aorta. This process may be a primary factor related arteriosclerosis is novel. In fact, the pro-apoptotic involved in the development of essential hypertension, action of NO (Gewaltig and Kojda, 2002; Kibbe et al., 1999) which is very prevalent with aging. would Suggest that it decreases the SMC/collagen balance through increased cell death. We have found in the aged 0280. In the case of the penile arteries, the data also animals treated with L-NIL an association between NOS indicate that a reduction in the ability of penile vessels to inhibition and Subsequent reduction of nitrotyrosine forma relax normally during cavernoSal nerve Stimulation leading tion, with a decrease of apoptosis, which would Suggest that to an erection, may contribute in part to the high prevalence NO does cause some SMC loss in the penile resistant arteries of ED associated with aging. In addition to this aging-related Similar to what has been previously assumed to occur in the fibrosis of the arterial media (Breithaupt-Grogler and Belz, corpora cavernosa (Ferrini et al., 2001a). However, an 1999; Robert, 1999; Integan and Schiffrin, 2000; Formieriet increased apoptotic indeX may be balanced by a Stimulation al., 1992), it is well documented (Grein and Schubert, 2002) of cell replication or tissue remodeling (Ingengan and Schif that Similar fibrotic changes occur within the penile corporal frin, 2001), and what really matters physiologically is the net Sinusoids. The corporal tissue comprises primarily of a balance between both processes. In the data above, the syncytium of vascular SMC with an endothelium lining relative number of SMC in the arterial media (represented which is biologically and physiologically indistinguishable by the Smooth muscle/collagen ratio), was severely reduced from the one present in the media and intima of the vascular when NO synthesis was diminished by L-NIL. This, tree (Krall et al., 1988) and may be considered a highly together with the well known effects of NO in scavenging evolved extension of these arterial tissues. Therefore, insults the profibrotic compound, ROS, thereby decreasing collagen that afflict the arterial media may also afflict the corporal SMCs, resulting in defective vaso-relaxation in both the Synthesis and down-regulating its breakdown (see Ferrini et corporal tissue (ED) and the arterial tree (hypertension). al., 2002; Vernet et al., 2002), would support the view of an Indeed, the prevalence of ED and hypertension in man overall beneficial role of NO in preventing arterial stiffness seems to parallel each other as a function of age (Sullivan et and loSS of compliance of the corpora cavernosa. al., 2001; Melman and Gingell, 1999), and many disorders 0283. A final question is whether collagen accumulation that damage one of these vascular tissues also seem to with aging is at least partially mediated via the regulation of impact the other e.g. diabetes, chronic renal failure, etc. In PAI-1, TIMP1 and other metalloproteinase inhibitors (Liet all these disorders, vascular oxidative StreSS and fibrosis, al., 2000; Kaikita et al., 2002), that increase in different leading to arteriosclerosis, are common denominators at the types of fibrosis. The current results with PAI, combined histological and molecular and levels. with previous data where we observed considerable metal loproteinase and PAI mRNA expression in the fibrotic 0281. The results on the abdominal aorta and the rest of plaque of Peyronie's disease in the human and rat (Magee et the Smaller arteries and arterioles are in agreement with al., 2002a), would Suggest that although the increase in the previous Studies from other groups showing in the aging rat pro-fibrotic PAI may induce a compensatory elevation of both an intensification of oxidative stress (van der Loo et al., 2000; Demaree et al., 1999) and collagen deposition metalloproteinase levels, the enzyme would remain inhib (Goettsch et al., 2001; Chou et al., 1998; Csiszar et al., ited and the net result would be an impaired collagen 2002), that is the likely cause of the reduction of the breakdown. SMC/collagen ratio within the media. This alteration, that in 0284. In conclusion, the results indicate that within the the large vessels does not appear to be caused by SMC arterial System and the cavernosal tissue it may be possible US 2005/0085486A1 Apr. 21, 2005 28 to pharmacologically modulate a) the NO/ROS balance with group. The dose of molsidomine used is based on the report NO donors or other NO generators together with antioxi in which it was utilized for 22 days to protect against dants, and b) the PAI/MMP balance with agents modifying tubulo-interstitial injury in a rat model of chronic glomerular their relative expression. Such novel therapies may consti disease (Uckert et al., 2001), and is calculated to be equiva tute viable approaches for the prevention and/or therapy of lent to approximately 15 mg/kg/day in the rat. In the case of vascular disorders that involve the arterial media and the L-arginine, it is given only as a late treatment, but at 2 doses: corpora. 22.5 and 10.0 g/l (in drinking water) (2 groups). Previously, the 22.5 g/l dose of L-arginine was used for 45 days to Example 19 elevate NOS activity in the rat penis (Moody et al., 1997), and to inhibit the plaque with the early treatment is equiva Gene Therapy With iNOS cDNA lent to roughly 2.8 g/kg/day. 0285 All studies throughout this section, unless specifi cally indicated, are performed in the rat model where the Example 21 PD-like plaque is initiated by the injection of TGF-1 (0.5 ug) into the TA. TGF-B1 transcriptionally amplifies its own Oral PDE Inhibitors to Regress the PD Plaque Synthesis, allowing for a Single injection. Saline-injected TA 0289 Sildenafil (specific PDE5 inhibitor), and pentoxi are used as controls. The AdW-CMV-iNOS construct has fylline (non-specific PDE inhibitor), are given at doses of been prepared by subcloning the iNOS cDNA driven by the 100 mg/l in the drinking water, as well as a control (drinking strong CMV promoter (Garban et al., 1997), from a plasmid water only), beginning on day 45 (3 groups). The study may construct into an AdW plasmid vector, and purifying the AdW be repeated at double or possibly quadruple the dosage construct, as previously described for PnNOS (Magee et al., above (2 groups). Reduction in plaque size was observed 2002a). This AdW vector is replication-defective and helper with both sildenafil and pentoxifylline during the entire time dependent, and therefore is non-infectious and totally of plaque development (early treatment), and this type of innocuous. In addition, it lackS virtually all the original viral treatment may be as effective in regressing the plaque (late Sequences that may be immunogenic. This AdW construct treatment). can be transfected into the TA, and it has been cloned and utilized for other therapeutic purposes in the penis (Magee 0290 Eleven PDE mRNAs for the respective isoforms et al., 2002a). have so far been identified by RT/PCR in the human (Kim et al., 2000). Due to the fact that pentoxifylline is more 0286 Rats are injected in the TA at the same site as the effective than sildenafil at inhibiting apoptosis in the PD TGF-B1 was injected 5 days earlier (as evidenced by a plaque, it is likely that more than one PDE gene product may non-absorbable suture) with 10 and 10 vp of either Adv be involved in plaque development, and the pure PDE4 CMV-iNOS in 50 ul saline, or with vehicle (saline) only. inhibitor, rolipram (Uckert et al., 2001) may identify a This 5-day waiting period between the TGF-B1 injection and related, relevant PDE. The increase in cAMP may act the cDNA construct avoids any interference of the viral directly, or through stimulating the synthesis of c(GMP (Kim preparation with the injected TGF-31, and/or its dispersion et al., 2000). Rolipram, is given to early and late treatment by the electroporation applied to enhance transfection of the groups (2 groups), at the same dose, in order to determine iNOS construct. The resulting 4 groups of rats are allowed whether increasing cAMP levels is as, or more, effective to develop the plaque for 40 more days, Sacrificed, and the than c(GMP area around the plaque is excised, fixed, paraffin-embedded, and sectioned (Ferrini et al., 2002, Vernet et al., 2002). In Example 22 another 2 groups of rats injected with TGF-B1 to induce a plaque, the cDNA construct and Saline are injected 45 days after the TGF-B1 injection (when the plaque has already Gene Therapy with PKG formed) and 30 days later the animals are sacrificed. 0291. The AdW-PKG (wild type) and AdW-PKGcat 0287. In the “early” treatment groups (iNOS given 5 days (mutated) are injected into the TA (2 groups). The consti after TGF-B1 injection), the TA of the iNOS-treated animals tutively active PKG1 mutant consists of the carboxy-termi shows, in comparison with controls: 1) a decrease in the size nal catalytic domain without the amino-terminal regulatory of the plaque as evidenced by Masson, collagen I/III Stain domain where cQMP binds (Wollert et al., 2002). Both the ing, and hydroxyproline content; 2) a higher expression of wild type and mutated constructs are obtained from Dr. iNOS and nitrotyrosine; 3) decrease of ROS; and 4) increase Stefan Janssens (Center for Transgene Technology and Gene in the apoptotic index of the fibroblasts/myofibroblasts. In Therapy, University of Leuven, Belgium). the “late” treatment group, where the iNOS is injected 45 days after TGF-31 injection, at least Some regression of the Example 23 plaque is obtained. Screening of Other cGMP-Dependent PDE Example 20 Isoforms 0292 Tissues from human normal TA and PD plaque Oral NO Donors or NOS Substrate preserved in RNA later (Magee et al., 2002b; Ferrini et al., 0288 Plaques are induced in the TA of rats by TGF-B1 2002) or the previously isolated RNAS from these tissues, injection (Ferrini et al., 2002). Drinking water containing conserved at -80 C., are used. In the case of the rat model, molsidomine (N-ethoxycarbonyl-3-morpho-linosydnom a 2-3 mm transverse Section is obtained at the Site of Saline ine), at 0.12 g/l (Benigniet al., 1999) (freshly prepared each or TGF-31 injection 45 days after plaque initiation. RNA is day) is given to 2 groups of rats, an early and a late treatment isolated from 2 groups of human and 2 groups of rat tissues. US 2005/0085486A1 Apr. 21, 2005 29 Example 24 are deemed to be within the Spirit, Scope and concept of the invention as defined by the appended claims. Oral Antioxidant REFERENCES 0293 The antioxidant vitamin E (C-tocopherol) is given in a specially prepared oral diet, So that the animal receives 0297. The following references, to the extent that they in an early treatment phase approximately 200 IU/kg/bw/ provide exemplary procedural or other details Supplemen day, whereas controls receive the normal diet containing leSS tary to those Set forth herein, are specifically incorporated than 20 IU/kg (Gonca et al., 2000). The third group (3 herein by reference. groups) receive twice a day an intramuscular injection (10 mg/kg) of another antioxidant compound that ameliorates 0298 Alves, M. A. Barao, L. N. Odo, G. Nascimento oxidative StreSS and lipid peroxidation, the glutathione pre Gomes, C. Franco Md Mdo, D. Nigro, S. R. Lucas, F. cursor S-adenosyl-L-methionine (SAM) (Muriel et al., R. Laurindo, L.I. Brandizzi, F. Zaladek Gil. L-Arginine 1998b). 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Plasmid preparations of a cDNA encoding both peptides (200 ug/rat) 0306 Behr-Roussel D, Rupin A, Simonet S, et al. are also given by injection/electroporation to the TA (2 Effect of chronic treatment with the inducible nitric groups), as a late treatment (i.e. 45 days after the TGF-31 oxide synthase inhibitor N-iminoethyl-L-lysine or with injection, to induce the PD like plaque). L-arginine on progression of coronary and aortic ath erosclerosis in hypercholesterolemic rabbits. Circula 0296 All of the COMPOSITIONS, METHODS and tion. 2000; 102:1033-1038. APPARATUS disclosed and claimed herein can be made and executed without undue experimentation in light of the 0307 Benigni A, Zoa C, Noris M, Coma D, Benedetti present disclosure. 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Vascular oxidant stress: molecu US 2005/0085486A1 Apr. 21, 2005 39 lar mechanisms and pathophysiological implications. J 3. The method of claim 2, wherein the administration of Physiol Biochem. 2000; 56:57-64 a PDE inhibitor comprises long term oral prevention or 0556 Zhang H Y, Phan S H (1999) Inhibition of treatment with Sildenafil, tadalafil, Vardenafil, pentoxifyllin, myofibroblast apoptosis by transforming growth factor rolipram or another inhibitor of PDE5A and/or PDE-4. 4. The method of claim 3, wherein the PDE inhibitor is beta (1). Am J Respir Cell Mol Biol 21(6):658-65. administered at the same dosage per unit of body weight as 0557. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell J the dosages administered to rodents. W. Katz AJ, Benhaim P. Lorenz HP, Hedrick M H 5. The method of claim 3, wherein the PDE inhibitor is (2001) Multilineage cells from human adipose tissue: administered at a different dosage than the dosages admin implications for cell-based therapies. Tissue Engineer istered to rodents. ing 7(2): 211-228. 6. The method of claim 1, wherein the condition is Peyronie's disease plaque, penile corporal fibrosis, penile TABLE 1. Veno-occlussive dysfunction, Dupuytren’s disease nodules, Dupuytren Peyronie vaginal fibrosis and/or clitoral fibrosis in female Sexual Protein/gene n: Mean SE n* * Mean SE arousal disorder, abnormal wound healing, keloid formation, matrix 9 29 - 10 2 4.7 2.6 general fibrosis of the kidney, bladder, prostate, Skin, liver, metalloproteinase 2 lung, heart, intestines or any other localized or generalized matrix 2 50.8 - 0.8 fibrotic condition. metalloproteinase 9 7. The method of claim 6, wherein the condition is thymosin beta-10 9 5.9 + 2.6 5 5.5 + 1.3 (TMSB10) Peyronie's disease plaque or Dupytren’s disease nodules. thymosin beta 4 8 5.9 1.5 5 2.5 + 0.9 8. The method of claim 1, wherein the condition is prothymosin alpha 2 2.6 O.O 2 6.2 - 3.8 vascular fibrosis, arterial intima hyperplasia, atherosclerosis, Osteoblast specific 5 5.6+ 1.4 3 4.3 - 0.5 factor 1 (OSF-1) arteriosclerosis, restenosis, hypertension, cardiac hypertro Osteoblast specific 4 26.7 - 12.7 phy or any other condition characterized by excessive fibro factor 2 (OSF2) blast or Smooth muscle cell proliferation or deposition of rho. GDP dissociation 6 3.5 - 1.4 2 18.3 2.4 collagen and extracellular matrix in the blood vessels and/or inihibitor 1 (RHO-GDI 1) heart. n* = 9 patients 9. The method of claim 8, wherein the condition is n** = 10 patients arteriosclerosis or hypertension. 10. The method of claim 2, wherein the PDE-4 or PDE-5 0558) inhibitor is administered orally, by injection, by local admin istration to the penis or other target tissue. TABLE 2 11. The method of claim 2, wherein the PDE inhibitor INTIMAMEDIA comprises an antisense or siRNA vector for PDE-4 and/or THICKNESS PDE-5. 12. The method of claim 2, wherein the PDE-5 inhibitor YOUNG OLD OLD + L-NIL is specific for PDE-5 variant 3. (um) (um) (um) 13. A method of preventing or treating a condition involv AORTA 73.4 8.6** 93.0 - 6.6** 85.2 6.6 ing fibrosis comprising: FEMORAL 49.77.1 63.5 - 2.4 51.8 - 4.6 PENILE DORSAL 17.O. 23 18.6+ 1.4 23.6 3.3 a) administering a compound that elevates cGMP or PKG BULBO URETHRAL 10.5 - 1.3 10.1 - 1.6 9.9 O.7 to an individual with a condition involving fibrosis, and b) preventing or treating the fibrosis. 0559) 14. The method of claim 13, wherein the compound is a Stimulator of guanylyl cyclase and/or PKG. 15. The method of claim 13, wherein the compound is a LUMEN DIAMETER gene therapy vector comprising a PKG cDNA. 16. The method of claim 13, wherein the condition is YOUNG OLD OLD + L-NIL Peyronie's disease plaque, penile corporal fibrosis, penile (um) (um) (um) Veno-occlussive dysfunction, Dupuytren’s disease nodules, PENILE DORSAL 100.8 - 13.7 118.7 6.6 105.5 - 16. vaginal fibrosis, clitoral fibrosis, female Sexual arousal dis BULBO URETHRAL 40.1 - 5.6 42.6 6.8 47.3 - 6.6 order, abnormal wound healing, keloid formation, general fibrosis of the kidney, bladder, proState, Skin, liver, lung, heart, intestines or any other localized or generalized fibrotic 1. A method of preventing or treating a condition involv condition, Vascular fibrosis, arterial intima hyperplasia, ath ing fibrosis comprising: erosclerosis, arteriosclerosis, restenosis, cardiac hypertro phy, hypertension or any condition characterized by exces a) administering a PDE inhibitor to an individual with a sive fibroblast or smooth muscle cell proliferation or condition involving fibrosis, and deposition of collagen and extracellular matrix in the blood b) preventing or treating the fibrosis. vessels and/or heart. 2. The method of claim 1, wherein the PDE inhibitor 17. A method of preventing or treating a condition involv inhibits PDE-4 and/or PDE-5. ing fibrosis comprising: US 2005/0085486A1 Apr. 21, 2005 40 a) administering a compound that elevates NO(NO donor a) administering a compound that increases MMP activity or generator) and/or decreases ROS to an individual to an individual with a condition involving fibrosis, and with a condition involving fibrosis, and b) preventing or treating the fibrosis. b) preventing or treating the fibrosis. 23. The method of claim 22, wherein the compound is an 18. The method of claim 17, wherein the compound is an MMP activator, an antagonist of an MMP inhibitor, or a gene NO donor, an NO generator, a gene therapy vector com therapy vector comprising an MMP cDNA. prising an NOS cDNA, or a PDE inhibitor. 24. The method of claim 23, wherein the compound is a 19. The method of claim 18, further comprising admin Beta thymosin or another peptide in the thymosin family. istering an antioxidant in combination with the NO donor, NO generator, gene therapy vector comprising an NOS 25. The method of claim 1, wherein said administering cDNA, or PDE inhibitor. comprises long term continuous treatment for weeks, 20. The method of claim 19, wherein the combination is months or years. effective to provide an antifibrotic effect without damaging 26. The method of claim 13, wherein said administering non-fibrotic tissues by excessive peroxynitrite formation. comprises long term continuous treatment for weeks, 21. The method of claim 17, wherein the condition is months or years. Peyronies disease plaque, penile corporal fibrosis, penile 27. The method of claim 17, wherein said administering Veno-occlussive dysfunction, Dupuytren’s disease nodules, comprises long term continuous treatment for weeks, vaginal fibrosis, clitoral fibrosis, female Sexual arousal dis months or years. order, abnormal wound healing, keloid formation, general 28. The method of claim 22, wherein said administering fibrosis of the kidney, bladder, proState, Skin, liver, lung, comprises long term continuous treatment for weeks, heart, intestines or any other localized or generalized fibrotic months or years. condition, Vascular fibrosis, arterial intima hyperplasia, ath 29. A method comprising: erosclerosis, arteriosclerosis, restenosis, cardiac hypertro phy, hypertension or any condition characterized by exces administering an amount of a cyclic guanosine 3',5'- sive fibroblast or smooth muscle cell proliferation or monophosphate (CGMP) type 5 phosphodiesterase deposition of collagen and extracellular matrix in the blood (PDE 5) inhibitor to effect a condition comprising vessels and/or heart. fibrosis. 22. A method of preventing or treating a condition involv ing fibrosis comprising: