WO2004037183A2 - Procedes d'utilisation d'inhibiteurs de phospodiesterases et modulateurs d'acide nitrique, especes d'oxygene reactives, et metalloproteinases dans le traitement de la maladie de peyronie, de l'arteriosclerose et d'autres maladies fibrotiques - Google Patents

Procedes d'utilisation d'inhibiteurs de phospodiesterases et modulateurs d'acide nitrique, especes d'oxygene reactives, et metalloproteinases dans le traitement de la maladie de peyronie, de l'arteriosclerose et d'autres maladies fibrotiques Download PDF

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WO2004037183A2
WO2004037183A2 PCT/US2003/033400 US0333400W WO2004037183A2 WO 2004037183 A2 WO2004037183 A2 WO 2004037183A2 US 0333400 W US0333400 W US 0333400W WO 2004037183 A2 WO2004037183 A2 WO 2004037183A2
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fibrosis
collagen
pde
cells
plaque
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PCT/US2003/033400
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WO2004037183A3 (fr
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Nestor F. Gonzalez-Cadavid
Jacob Rajfer
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Harbor-Ucla Research And Education Institute
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Priority to AU2003286555A priority Critical patent/AU2003286555A1/en
Priority to US10/779,069 priority patent/US8133903B2/en
Publication of WO2004037183A2 publication Critical patent/WO2004037183A2/fr
Publication of WO2004037183A3 publication Critical patent/WO2004037183A3/fr
Priority to US13/372,094 priority patent/US20120141454A1/en
Priority to US14/215,444 priority patent/US20140200219A1/en
Priority to US15/277,550 priority patent/US20170239257A1/en
Priority to US15/832,918 priority patent/US20180318307A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings

Definitions

  • the present methods and compositions relate to the field of Peyronie's disease, arteriosclerosis and other fibrotic conditions. More particularly, the method and compositions concern use of phosphodiesterase (PDE) inhibitors and modulators of nitric oxide, reactive oxygen species and metalloproteinases in the treatment of such conditions, particular embodiments, the inhibitors inhibit type 4 and/or type 5 PDEs.
  • PDE phosphodiesterase
  • Peyronie's disease is a fibromatosis (Hellstrom and Bivalacqua, 2000; Schwarzer et al., 2001; Jarow et al., 1997; Devine et al., 1997) of the tunica albuginea (TA), the specialized lining of the corpora cavernosa of the penis. Clinically, this usually leads to penile deformation (curved penis during erection), pain, and quite frequently erectile dysfunction.
  • the initiating event is believed to be an external force to the erect penis that results in an injury to the TA of the corpora and the TA fails to heal normally (Jarow et al., 1997; Devine et al., 1997; Diegelmann, 1997; Sherratt and Dallon, 2002).
  • the only indication of the disease is the palpation of a knot or scar within the TA, which in its most severe state presents as a calcified plaque.
  • PD affects about 5% of men in the USA, and translating into about 3-4 million affected American males.
  • PD can rarely be alleviated by medical treatment with anti-inflammatory agents (corticosteroids, antihistamine), antioxidants (vitamin E, superoxide dismutase), collagen breakdown (coUagenase), Ca channel blockers (verapamil), and other antifibrotic compounds (colchicine, Potaba: K aminobenzoate) (Hellstrom and Bivalacqua, 2000).
  • anti-inflammatory agents corticosteroids, antihistamine
  • vitamin E superoxide dismutase
  • collagen breakdown coUagenase
  • Ca channel blockers verapamil
  • other antifibrotic compounds colchicine, Potaba: K aminobenzoate
  • Fibrotic disease is not limited to the reproductive organs, but can be found in other tissues, such as cardiovascular tissues. Both erectile dysfunction (ED) and cardiovascular disease, particularly hypertension, are prevalent in the aging male (Kloner et al., 2002; Sullivan et al., 2001; Melman et al., 1999).
  • ED erectile dysfunction
  • hypertension is prevalent in the aging male (Kloner et al., 2002; Sullivan et al., 2001; Melman et al., 1999).
  • arteriosclerosis or arterial stiffness, due to an acquired fibrosis of the media of the arterial wall (Breitmaschine-Grogler and Belz, 1999; Robert, 1999; Intengan and Schiffrin, 2000, 2001; Fornieri et al., 1992).
  • Arteriosclerosis is significantly associated with aging, and is recognized by an increase in collagen, and in some cases by a loss of smooth muscle cells (SMC) within the arterial media, which results in a decrease in the SMC/collagen ratio, often accompanied by endothelial dysfunction (Cai and Harrison, 2000).
  • SMC smooth muscle cells
  • the pathogenesis of aging associated ED is mostly related to the loss of SMC in the penile corpora cavernosa by apoptosis, with a corresponding increase in collagen fibers (Melman and Gingell, 1999; Cai and Harrison, 2000; Melman, 2001; Garban et al., 1995; Ferrini et al., 2001a).
  • the clinical result of this aging process in the penis is defective cavernosal SMC relaxation leading to veno- occlusive dysfunction (Breithaupt-Grogler and Belz, 1999; Rogers et al., 2003), the most common cause of ED.
  • Certain embodiments of the present invention fulfill an unresolved need in the art, by providing novel methods for therapeutic treatment of Peyronie's disease, erectile dysfunction, arteriosclerosis and other fibroses.
  • PD plaques and/or other fibrotic conditions can be pharmacologically arrested or reduced in size, by decreasing collagen synthesis and inducing myofibroblast apoptosis by increasing the NO/ROS ratio, the levels of cGMP, or the activation of its effector, PKG in the TA and/or stimulating collagen degradation by activating the MMPs and/or down-regulating the expression of the MMP inhibitors (TIMP), by increasing NO/cGMP levels and/or the thymosins in the TA.
  • TIMP MMP inhibitors
  • Particular embodiments of the invention may be directed towards increasing levels of cGMP and/or cAMP by selective inhibition of phosphodiesterase (PDE) isoforms.
  • PDE isoforms of interest in the TA and in PD plaque tissues include PDE5 A- 3, PDE4A, PDE4B and PDE4D.
  • pentoxifylline and similar compounds act as a non-specific cAMP-PDE inhibitor and increase cAMP levels
  • sildenafil and similar compounds selectively inhibit PDE5A and increase cGMP levels.
  • Other embodiments may involve increasing NO levels, for example by administering L-arginine, a stimulator of NOS activity.
  • pentoxifylline, sildenafil and L-arginine all act to reduce the expression of collagen I and ⁇ -smooth muscle actin.
  • Long-term administration of nitrergic agents, such as pentoxifylline, sildenafil and L-arginine may be of use to reduce PD plaque size and collagen/fibroblast ratio and may reverse or prevent the further development of the fibrosis observed in PD, ED, arteriosclerosis and other fibrotic conditions.
  • FIG. 1 The effect of NO, TGF- ⁇ l, ROS and cGMP on fibroblast/myoblast differentiation and collagen deposition.
  • FIG. 2. Inhibition of collagen deposition in the fibrotic plaque induced by TGF- ⁇ l in the rat TA, by long-term oral treatment with L-arginine and PDE inhibitors, estimated by Masson staining.
  • A Microphotographs (40X) of cross sections of half of the rat penis. Dark arrows indicate the outer extent of plaque development and of tunical thickening. Light arrowheads (lower right-hand corner of each panel) indicate the site of TGF- ⁇ l injection. The control (-) was injected with TGF- ⁇ l injection, no treatment was given.
  • L-ARG received a TGF- ⁇ l injection and L-arginine in water.
  • SIL received a TGF- ⁇ l injection and sildenafil in water.
  • PXF received a TGF- ⁇ l injection and pentoxifylline in water.
  • FIG. 3 Stimulation of apoptosis, as estimated by TUNEL, in the fibrotic plaque induced by TGF- ⁇ l injection into the rat TA, following oral treatment with L-arginine, sildenafil or pentoxiphylline.
  • A Microphotographs (400X) of tissue sections. Arrows indicate apoptotic cells in the site of the plaque. The control (-) was injected with TGF- ⁇ l injection, no treatment was given.
  • L-ARG received a TGF- ⁇ l injection and L- arginine in water.
  • SIL received a TGF- ⁇ l injection and sildenafil in water.
  • PXF received a TGF- ⁇ l injection and pentoxifylline in water.
  • FIG. 4 Expression of PDE-5 mRNA and protein in the human PD plaque and normal tunica albuginea, and their homologous tissues in the TGF- ⁇ l rat model of PD.
  • A Ethidium bromide-stained DNA bands obtained by RT/PCR from RNAs isolated from the respective tissues, and fractionated on agarose gels.
  • B Luminol-stained protein bands obtained by western blot on polyacrylamide gels.
  • PS penile shaft
  • TA tunica albuginea
  • PD Peyronie's disease
  • CC corpora cavernosa
  • CER cerebellum
  • CRU penile crura.
  • C Microphotgraphs (200X) of sections from human and untreated rat tissues.
  • D.ART dorsal artery; ART: artery. Arrows show positive cells for PDE-5.
  • FIG. 5 Inhibition of collagen I synthesis and myofibroblast differentiation by PDE inhibitors in fibroblast cultures from a human PD plaque.
  • A QIA evaluation of collagen I and ASMA expression. Control
  • C no addition; 50SE : sildenafil (50 nM); 200SIL: sildenafil (200 nM); 200PXF:pentoxifylline (200 nM).
  • B DAB-stained immunocytochemical detection of collagen III in PD cells incubated for 3 days in DME- serum free medium in the presence or absence of 5 ng/ml of TGF- ⁇ 1(200X).
  • FIG. 6 Effects on collagen I synthesis and myofibroblast differentiation in fibroblast cultures from the human PD plaque by a cGMP analog (8-Br cGMP), estimated by immunocytochemistry. Cells were incubated for 3 days with the indicated concentration and collagen I and ASMA were immuno-cytochenmically detected. Values are means+/-SEM for three separate incubations. p ⁇ 0.05 were as follows: panel A: a vs b,c; panel D: a vs c; all others were non-significant.
  • FIG. 7 Expression of PDE-5 mRNA and protein in fibroblast cultures from human PD plaque and human and rat normal tumca albuginea.
  • A Ethidium bromide staining of PDE-5 A cDNA bands generated from cell RNA by RT-PCR and fractionated on agarose gels.
  • B Luminol detection of PDE-5 protein bands obtained by western blot of cell extracts on PAGE. Arrows indicate PDE-5 A variants.
  • DUP cells from Dupuytren's nodules.
  • C Microphotographs (200x) of cell cultures stained with the indicated antibodies and counter-stained with Meyer haematoxylin
  • FIG. 8 Gene transfer to the tunica albuginea of plasmid and adenoviral cDNA constructs facilitated by electroporation. Both the pCMN- ⁇ gal and the AdN-CMN- ⁇ gal constructs were injected into the tunica albuginea of the rat, followed by electroporation, and 10 days later rats were sacrificed and frozen fixed tissue sections were stained with X-gal, and counterstained with neutral red. TA: tunica albuginea. (200X magnification).
  • FIG. 9 Induction of TGF- ⁇ l expression in the Peyronie-like plaque induced by fibrin in the tunica albuginea of the rat.
  • A Sections adjacent to the ones for plaques shown on FIG. 11 were immunostained with an antibody for TGF- ⁇ l. Arrows point to cells with intense staining.
  • FIG. 10 Confirmation by RT/PCR of alterations in the expression of certain genes in the human Peyronie's plaque as compared to the normal tunica albuginea.
  • FIG. 11 PD-like plaque similar to the one induced in the rat can be elicited in the tunica albuginea of the mouse by TGF- ⁇ l injection, detected by Masson staining. Low (4X, top); and high (100X, bottom) magnifications of mouse penis injected with saline and TGF- ⁇ l. Light arrows show the site of TGF- ⁇ l injection. Boxes represent the area of high magnification.
  • FIG. 12 Expression of PDE-4 mRNA and protein in the human PD plaque and normal tunica albuginea, and their homologous tissues in the TGF- ⁇ l rat model of PD, and in fibroblasts cultured from these tissues.
  • A Ethidium bromide staining of DNA generated from PD and normal human TA tissue by RT/PCR with primers for PDE4A, PDE4B, and GAPDH (reference gene), separated by agarose gel electrophoresis.
  • B PDE4 mRNA in rat penile shaft (PS), rat TA cells, or human TA or PD cells.
  • TA tunica albuginea
  • PD Peyronie's disease
  • CC corpora cavernosa smooth muscle
  • PS penile shaft.
  • C Luminol-stained protein bands on western blots of human tissue and cell extracts with the antibody against PDE4A.
  • FIG. 13 hnmunodetection of PDE-4 protein in the rat penis and cultures of rat and human tunica albuginea fibroblasts. Microphotographies of tissue sections (top panels) or cell cultures (middle and botom panels), as indicated, submitted to immunodetection with antibodies against PDE4A or PDE4D, and counterstained with Meyer haematoxylin.
  • FIG. 14 Effect of pentoxifylline and sildenafil on cAMP and cGMP levels in fibroblast cultures from human PD plaque, estimated by enzyme immunoassays.
  • Cells were incubated for 3 days in fibroblast growth medium (FGM)/10% fetal bovine serum, in the presence of SNAP (100 uM; medium changed daily) added 4 hs prior to the PDE inhibitors, and increasing concentrations of sildenafil or pentoxifylline.
  • FGM fibroblast growth medium
  • SNAP 100 uM; medium changed daily
  • FIG. 16 Quantitative image analysis (QIA) expressed as ratios of areas occupied by SMC and collagen, as means+/-SEM.
  • Aorta A vs. B, C pO.OOl; B vs. C p ⁇ 0.01; Femoral: A vs B, C pO.OOl; B vs. C pO.01; Dorsal: A vs B, C PO.OOl B vs. PO.05; Bulbourethral: A vs B, C pO.OOl; B vs. C: NS.
  • FIG. 16 Reduction by iNOS blockade of the aging-related stimulation of the nitrosative pathway in the media of the penile arteries.
  • Nitrotyrosine is a marker for peroxynitrite.
  • B QIA as on FIG. 15, expressed as intensity of immunostaining per area, as means+/-SEM.
  • Dorsal A vs. B pO.05; A vs. C: N.S.; B vs C: pO.05; Bulbo-urethral: A vs. B: p .05; A vs. C: N.S; B vs. C: p .05.
  • FIG. 17 Intensification by iNOS blockade of aging-related oxidative stress in the arterial media. Tissue sections were immunostained for Cu 2+ Zn 2+ SOD and for Mn 2+ SOD.
  • B QIA as on FIG.
  • FIG. 18 Differential expression of another marker of oxidative stress, heme oxygenase 1, in the adventitia of the arterial wall. Sections were immunostained with an antibody against heme oxygenase I and counterstained with hematoxylin.
  • B QIA as on previous figures. Values expressed as means+/-SEM. *pO.05: young vs old (t test).
  • FIG. 19 Reduction by iNOS blockade of the aging-related stimulation of apoptosis in the media of the penile arteries. Sections were immunostained with the TUNEL procedure and counterstained with methyl green.
  • B QIA as on previous figures, expressed as apoptotic index (percent number of apoptotic cells/total number of cells), as means+/-SEM.
  • FIG. 20 Intensification by iNOS blockade of the aging-related stimulation of PAI expression in the media of the penile arteries. Sections were immunostained with an antibody against PAI and counterstained with hematoxylin.
  • A Micrographs from the dorsal artery.
  • FIG. 21 Effect of 8 Br-cGMP on apoptosis in fibroblasts from human cultured PD plaque, estimated by TUNEL.
  • Table 1 Differential profiles of selected gene expression in human Peyronie's plaques and Dupuytren's nodules against their respective control tissues determined with a DNA microarray assay.
  • ASMA -smooth muscle actin
  • ED erectile dysfunction
  • iNOS inducible NOS (also NOS II);
  • L-NAME L-N ⁇ -Nitro-L-arginine methyl esther
  • L-NIL L-iminoethyl-L-lysine
  • MMP matrix metalloproteinase
  • nNOS neuronal NOS
  • NOS nitric oxide synthase
  • PAI plasminogen activator inhibitor
  • PD Peyronie's disease
  • PDE phosphodiesterase
  • PKG protein kinase G
  • PPC pluripotent cells
  • ROS reactive oxygen species
  • SMC smooth muscle cell
  • SNAP S-Nitroso-N-acetyl penicillamine
  • TA tunica albuginea
  • TGF- ⁇ l transforming growth factor- ⁇ l
  • TIMP tissue inhibitor of MMP
  • This application concerns, at least in part, isolated proteins and nucleic acids for type 5 phosphodiesterase (PDE5, e.g., GenBank Accession Nos. NM033437, NM033431, NM033430, NM001083, NP246273, NP237223, NP236914, NP001074), as well as methods of therapeutic treatment of fibrotic diseases directed towards such proteins.
  • PDE5 type 5 phosphodiesterase
  • a "PDE5 isoform” is a variant of type 5 phosphodiesterase that differs in its primary structure (i.e., amino acid sequence) from other isoforms of PDE5.
  • the term encompasses, but is not limited to, isoforms that are produced by truncation, amino acid substitution (mutation) or by alternative mRNA splicing, so long as some difference in amino acid sequence results.
  • other types of covalent modification would be considered to fall within the scope of a single isoform.
  • both phosphorylated and unphosphorylated forms of PDE5 would be considered to represent the same isoform.
  • an "inhibitor” of PDE5 means any compound or combination of compounds that acts to decrease the activity of PDE5, either directly or indirectly.
  • An inhibitor can be a molecule, an atom, or a combination of molecules or atoms without limitation.
  • the term "antagonist" of PDE5 is generally synonymous with an “inhibitor” of PDE5.
  • Inhibitors may act directly on PDE5 by, for example, binding to and blocking the catalytic site or some other functional domain of PDE5 that is required for activity.
  • An inhibitor may also act indirectly, for example, by facilitating or interfering with the binding of PDE5 to another protein or peptide.
  • PDE4 type 4 phosphodiesterase
  • PDE4 e.g., GenBank Accession Nos. NM006203, NM002600, NM006202, NP006194, NP002591, NP006193
  • PDE4 isoform and PDE4 "inhibitor” or "antagonist” are used consistently with the corresponding terms defined above for PDE5.
  • Peyronie's disease is a localized fibrosis of the tunica albuginea (TA) of the penis (Hellstrom and Bivalacqua, 2000; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002) affecting close to 5% of the male population (Schwarzer et al., 2001).
  • the leading theory of the etiology of PD is that it results from an abnormal wound healing process of the TA subsequent to an injury, usually during coitus (Hellstrom and Bivalacqua, 2000; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002; Jarow and Lowe, 1997; Devine et al., 1997).
  • TGF- ⁇ l transforming growth factor ⁇ l
  • ROS reactive oxygen species
  • the above process may result in: a) an increase in the differentiation of TA fibroblasts into myofibroblasts (Vernet et al., 2002); b) an increased deposition of collagen fibers by both the TA fibroblasts and myofibroblasts (Vernet et al., 2002; Ferrini et al., 2002); c) a decrease in apoptosis of the TA fibroblasts/myofibroblasts, and d) a decrease in the natural breakdown and reorganization of newly deposited collagen fibers that is normally performed by the matrix metalloproteinases (MMP) (Mignatti et al., 1996; Arthur, 2000).
  • MMP matrix metalloproteinases
  • the MMPs are the collagenolytic enzymes that are involved in the natural turnover of collagen in the wound healing process. At its extreme, this process may become excessive, with newly deposited collagen and extracellular matrix in a tissue that fails to "heal and reorganize normally" (Mignatti et al, 1996) eventually becoming calcified (Gelbard, 1988; Muralidhar et al., 1996). Calcification may occur by osteoblasts that are transformed from pluripotent cells (PPC) among the fibroblasts and/or myofibroblasts within the TA by either autocrine and/or paracrine factor(s).
  • PPC pluripotent cells
  • the primary cell that is involved in collagen synthesis in the wound healing process is the fibroblast (Singer and Clark, 1999).
  • some of the fibroblasts must differentiate into myofibroblasts (Vernet et al., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002; Muralidhar et al., 1996; Singer and Clark, 1999, Powel et al, 1999), cells that are intimately involved in the terminal stages of the wound healing process.
  • myofibroblast is normally eliminated from the wound by apoptosis (Gabbiani, 1996).
  • myofibroblasts persist and do not undergo pre-programmed cell death, they may continue to synthesize additional collagen and extracellular matrix, leading to an increase in fibrosis. Within the TA, this increase in fibrosis may lead to the clinical recognition of a palpable Peyronie's plaque.
  • TGF- ⁇ l can be secreted by both fibroblasts and myofibroblasts (Powel et al., 1999; Tomasek et al., 1999; Walker et al., 2001). In other fibrotic conditions, like cardiac and renal fibrosis, TGF- ⁇ l has been shown to not only increase the replication and differentiation of fibroblasts into myofibroblasts, but also to inhibit apoptosis of the myofibroblasts (Desmouhere, 1995; Chipev et al., 2000).
  • NO in the TA is synthesized by the inducible nitric oxide synthase enzyme (iNOS) (Vernet et al., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002; Ferrini et al., 2002).
  • iNOS inducible nitric oxide synthase enzyme
  • L- iminoethyl-L-lysine L- iminoethyl-L-lysine
  • iNOS plays an antifibrotic role in PD by at least three mechanisms: a) the quenching of the pro-fibrotic ROS by a reaction leading to the formation of peroxynitrite; b) the down-regulation of fibroblast replication and myofibroblast differentiation; and c) the consequent or independent reduction in the transcriptional expression of collagen I.
  • An additional mechanism for NO to counteract fibrosis may involve stimulation of myofibroblast and/or fibroblast programmed cell death.
  • NO donors such as S-Nitroso-N-acetyl penicillamine (SNAP) (Sikka et al, 2002; Nishio et al, 1996) or inducible nitric oxide synthase (iNOS) expression (Nishio et al., 1996; Tain et al., 2002), or in vivo by neuronal NOS (nNOS) activation (Ferrini et al., 2001b), iNOS induction (Ferrini et al., 2001b; Watanabe et al., 2002), or administration of the NOS substrate L-arginine (Wang et al., 1999; Holm et al., 2000).
  • SNAP S-Nitroso-N-acetyl penicillamine
  • iNOS inducible ni
  • pharmacologic inhibition of the pro-fibrotic process and/or stimulation of the anti-fibrotic processes may halt the progression and/or reverse the process of PD. More globally, such results may be extrapolated to more life-threatening fibrotic conditions such as renal, lung, liver, and cardiac fibrosis (Nagase and Brew, 2002; Martinez-Hernandez, 1994; Schuppan et al., 2000). The results disclosed herein provide novel avenues of therapy for not only PD but also for fibrosis in general. [0073] The interaction within the TA between the pro-fibrotic and anti-fibrotic factors acting on fibroblasts and myofibroblasts and their respective differentiation and apoptotic processes is outlined in FIG. 1.
  • Peroxynitrite is known to induce apoptosis in most cell types, and specifically of the collagen-producing cells such as the fibroblast and myofibroblast (Heigold et al., 2002; Duffield et al., 2000; Zhang and Phan, 1999).
  • NO stimulates guanylyl cyclase to produce cGMP Gonzalez-Cadavid et al., 1999), which in turn stimulates PKG (protein kinase G) (Sinnaeve et al., 2002; Wollert et al, 2002).
  • PKG protein kinase G
  • both cGMP and PKG inhibit collagen synthesis and are anti-fibrotic (Sinnaeve et al., 2002; Wollert et al., 2002; Hofinann et al., 2000; Chen et al., 1999a, 1999b; Redondo et al., 1998).
  • cGMP is normally degraded to inactive GMP by the phosphodiesterase (PDE) enzymes (Corbin and Francis, 1999; Uckert et al., 2001).
  • PDE phosphodiesterase
  • the accumulation of collagen which is one of the histological hallmarks of tissue fibrosis, may in part be also due to the inactivation of the MMP enzymes that degrade the already laid-down collagen fibers during its natural turnover cycle (Mignatti et al., 1996; Arthur, 2000). MMPs can be inactivated by TIMPs, the tissue inhibitors of MMP, that have been shown to increase in fibrotic conditions (Iredale, 1997; McCrudden and Iredale, 2000; Arthur, 2000).
  • Another anti-fibrotic effect of NO is that it stimulates MMP activity (Sasaki et al., 1998; Okamoto et al., 1997) and inhibits the expression of TIMP (Darby et al., 2002; Bugno et al., 1999).
  • TGF- ⁇ l rat model of PD include the use of the established TGF- ⁇ l rat model of PD (Ferrini et al., 2002; Vernet et al., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002), the establishment and study of cell cultures from the human PD and normal TA tissues (Vernet et al., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002), the application of quantitative image analysis (QIA) of tissue sections and cells subjected to histochemistry and immunohistochemistry (Ferrini et al., 2002; Vernet et al., 2002; Gonzalez-Cadavid et al, 2002; Gholami et al, 2002), the use of selective inhibitors of some of the biochemical pathways shown in FIG.
  • QIA quantitative image analysis
  • the combination of agents may be more effective and safe than a single agent in either category alone.
  • compositions disclosed herein are of use not only for treatment of Peyronie's disease and ED due to loss of cavernosal smooth muscle in the trabecular spaces and penile arteries, but also for other conditions involving fibrosis, such as penile corporal fibrosis, Dupuytren's disease nodules, vaginal fibrosis, clitoral fibrosis, female sexual arousal disorder, abnormal wound healing, keloid formation, general fibrosis of the kidney, bladder, prostate, skin, liver, lung, heart, intestines or any other localized or generalized fibrotic condition, vascular fibrosis, arterial intima hyperplasia, atherosclerosis, arteriosclerosis, restenosis, cardiac hypertrophy or any other condition characterized by excessive fibroblast or smooth muscle cell proliferation or deposition of collagen and extracellular matrix in the blood vessels and/or heart.
  • fibrosis such as penile corporal fibrosis, Dupuytren's disease nodules, vaginal fibros
  • vaginal/clitoral fibrosis contribute to the development of female sexual arousal disorder, affecting about 30 to 40% of women.
  • the mechanisms of fibrosis are similar for a number of different organs and disease states.
  • a PDE5 inhibitor such as sildenafil
  • ROS reactive oxygen species
  • ROS are known stimulators of collagen deposition and SMC proliferation (Berry et al., 2001; Zalba et al, 2000) in the vascular wall.
  • Xanthine oxidase and SOD are also present in the penile corpora cavernosa (Jones et al., 2002), and oxidative stress due to ROS has been postulated to be central to impaired cavernosal function in aging-related ED (Jones et al., 2002; Khan et al., 2001; Bivalacqua et al., 2003).
  • nitric oxide also quenches ROS in the vasculature, as shown by the increase in ROS levels and the development of cardiac and renal fibrosis and vascular stiffness when there is long-term systemic blockade of NOS activity with NOS inhibitors (Kitamoto et al, 2000; Gonzalez et al, 2000; Usui et al., 1999).
  • the ROS-quenching and anti-fibrotic effects of NO are not limited to the SMC and can be demonstrated in other non-vascular conditions (Ferrini et al., 2002; Vernet et al., 2002).
  • NO reduces ROS levels through the formation of peroxynitrite (Cai and Harrison, 2000; Jones et al., 2002; Ferrini et al., 2002; Vernet et al., 2002; Saltig and Kojda, 2002), thereby increasing the NO/ROS ratio.
  • NO is also postulated to not only inhibit collagen synthesis directly, but to favor collagen degradation by stimulating metalloproteinases and down-regulating expression of their inhibitors, such as the plasminogen activator inhibitor (PAI) (Li et al., 2000; Kaikita et al., 2002).
  • PAI plasminogen activator inhibitor
  • nitrosative pathways over oxidative stress is proposed to be protective against fibrosis (Ferrini et al., 2002; Vernet et al, 2002), ED (Jones et al., 2002), atherosclerosis, and hypertension (Gewaltig and Kojda, 2002; Cheng et al., 2001).
  • the NO/ROS balance also directly modulates the relaxation of the vascular and penile smooth muscle.
  • the NO produced by the endothelial NOS in the vascular endothelium controls blood pressure by relaxing the arterial SMC (Gonzalez-Cadavid et al., 1999).
  • NO as a mediator of penile erection is produced by the neuronal NOS, specifically the PnNOS variant (Berry et al., 2001), localized in the nerve terminals, and to a lesser extent by endothelial NOS in the lacunar and sinusoidal endothelium of the penis (Gonzalez-Cadavid et al., 1999).
  • iNOS inducible isoform of NOS
  • iNOS is also induced in the media in aging-related arterial stiffness (Goettsch et al., 2001; Chou et al., 1998; Cernadas et al., 1998), transplant arteriosclerosis (Lee et al., 1999), and atherosclerosis (Dirig et al., 2001; Niu et al., 2001; Behr-Roussel et al., 2000), and it is assumed to inhibit collagen deposition and prevent medial hyperplasia via induction of SMC apoptosis and/or inhibition of SMC replication (Gewaltig and Kojda, 2002; Kibbe et al., 1999; Niu et al., 2001).
  • L-N- (iminoethyl)-lysine acetate (L-NIL)
  • L-NIL L-N- (iminoethyl)-lysine acetate
  • iNOS induction in the vasculature is not restricted to the cavernosal SMC (Ferrini et al., 2001a) and large arteries (Goettsch et al., 2001; Chou et al., 1998; Cernadas et al., 1998), but is generalized to the wall of the entire peripheral vascular tree. This process would aim to counteract oxidative stress and metalloproteinase inhibition, and the subsequent decrease in the SMC/collagen ratio that causes loss of compliance and NO-induced vaso- relaxation.
  • the fibrotic process occurring during abnormal wound healing is essentially an increased and disorganized collagen deposition impairing granulation tissue formation. This is accompanied by an increase in the local production and secretion of TGF- ⁇ l (Klar and Morrisey, 1998; Badalêt et al., 1996; Wahl, 1997), a factor which: a) stimulates collagen synthesis (Tiggelman et al., 1997; Faouzi et al., 1999), b) inhibits collagenolysis (van der Zee et al., 1997) and fibrinolysis (Holmdahl et al., 2001), c) enhances the release of ROS (Casini et al., 1997; Muriel, 1998a), and d) transcriptionally represses iNOS (Hung et al., 1995).
  • ROS are hydroxyl radicals and superoxide anions that are quenched by NO to primarily form peroxynitrite (Poli, 2000; Curtin et al., 2002; Cattell, 2002; Kim et al., 2001; Fan et al., 2000; Ito et al., 1992).
  • NO and ROS levels are known to be considerably altered in other fibrotic conditions affecting liver (cirrhosis), lung (pulmonary fibrosis), kidney (renal fibrosis), heart (cardiac hypertrophy), and the vascular tree (arterial medial hyperplasia).
  • ROS generation during oxidative stress is accompanied by a considerable induction of heme-oxygenase-1 (HO-1) (Foresti et al., 1999; Nathan, 1997), the enzyme that protects against oxidative stress and acts as an anti-apoptotic and anti-inflammatory (Ryter and Choi, 2002) response.
  • HO-1 can also be elicited by peroxynitrite.
  • NO is particularly interesting as an inhibitor of fibrosis.
  • NO appears to be produced by the induction of iNOS in the TA (Ferrini et al., 2002; Vernet et al., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002).
  • This iNOS isoform is involved in producing persistent high levels of NO by transcriptional induction, essentially as a defensive mechanism during inflammation (Nathan, 1997a, 1997b).
  • iNOS is physiologically expressed in the adult at very low basal levels, if at all.
  • cytokines such as tumor necrosis factor ⁇ (TNF ⁇ ), interleukin l ⁇ (IL-l ⁇ ), interferon- ⁇ (INF ⁇ ), and related factors. It can under certain chronic conditions lead to a high, and some times excessive production of NO that acts as either a cytotoxic agent, or, in the specific case of collagen, inhibits fiber deposition. These conditions include inflammation, infections, cancer, degenerative diseases and aging, where the factors triggering this increased iNOS response are unknown (Kibbe et al, 1999; Wang et al., 2002a; Miller and Sandoval, 1999). Additionally, many NO metabolites, particularly peroxynitrite, trigger localized apoptosis and " tissue toxicity (Nathan, 1997a).
  • NO As a regulator of wound healing is well established in vivo and in vitro (Curtin et al., 2002; Cattell, 2002; kirn et al., 2001; Hogaboam et al, 1998; Rizvi and Myers, 1997; Cao et al, 1997; Chatziantoniou et al, 1998; Kolpakov et al., 1995).
  • NO donors and the NOS substrate, L-arginine have been shown to inhibit collagen fiber ((Curtin et al., 2002; Cattell, 2002; kirn et al., 2001; Hogaboam et al., 1998; Rizvi and Myers, 1997; Cao et al., 1997; Chatziantoniou et al., 1998; Kolpakov et al., 1995) and fibrin deposition (Westenfeld et al., 2002; Dambisya and Lee, 1996; Catani et al., 1998; Dambisya et al., 1996), and TGF- ⁇ l synthesis (Craven et al, 1997).
  • the reduction of NO synthesis may occur by either transcriptional blockade of iNOS induction (Geller and Billiar, 1998; Forstermann et al., 1998), and in certain cases, down-regulation of eNOS (Forstermann et al., 1998), or by inhibition of NOS activity by advanced glycation-end products (AGE) (Jiaan et al., 1995) or a natural NOS competitive inhibitor such as asymmetric dimethyl arginine (ADMA) (Boger et al., 1998).
  • AGE advanced glycation-end products
  • ADMA asymmetric dimethyl arginine
  • the anti-fibrotic effects of NO may be the result of a continuous and high level of local NO synthesis, like the one produced upon iNOS induction (Ferrini et al., 2002; Vernet et al., 2002). This shows the importance of the local levels of NO for either facilitating normal collagen deposition (wound healing) or preventing its accumulation (fibrosis).
  • Anti-fibrotic effects of NO may also be mediated by cGMP through guanylyl cyclase activation (Gonzalez-Cadavid et al, 1999).
  • cGMP analogs inhibit collagen synthesis (Chen et al, 1999a, 1999b; Redondo et al., 1998), fibroblast replication (Chiche et al., 1998; Pandey et al., 2000), myofibroblast differentiation (Tao et al., 1999), and promote apoptosis (Loweth et al., 1997; Sirotkin et al., 2000; Taimor et al, 2000).
  • PDE inhibitors by elevating cGMP, also cause similar effects in vitro (Schade et al., 2002; Horio et al., 1999; Thompson et al., 2000), and in particular induce apoptosis in vivo (Chan et al., 2002; Takuma et al., 2001).
  • PDE inhibitors like pentoxifylline, are active in preventing experimental fibrosis in the lung, liver, and heart (Fischer et al., 2001; Desmouhere et al., 1999; Kremer et al., 1999), and are currently being used for the treatment of human liver fibrosis (Windmeier and Gressner, 1997) and Crohn's disease (Reimund et al., 1997).
  • tissue fibrosis One of the major pathological findings in tissue fibrosis is the presence of activated and proliferating myofibroblasts. These cells not only play an important role in the contraction phase of normal wound healing but they also are responsible for the development of tissue fibrosis or of a scar (Powel et al, 1999; Tomasek et al., 1999; Walker et al., 2001).
  • the myofibroblast (FIG. 1) is the cell widely believed to generate the contracture in Dupuytren's disease, the condition present in 10-20% of PD cases (Connelly, 1999).
  • myofibroblasts are intermediate between the fibroblast and the smooth muscle cell. Phenotypically, they express large bundles of actin filaments (actin, myosin, and associated proteins: "stress fibers"), with a fibrillar space material named the "fibronexus", composed of fibronectin.
  • ASMA ⁇ smooth muscle actin
  • vimentin abent in smooth muscle cells
  • myofibroblasts can originate from either fibroblasts, smooth muscle cells, or from an as yet uncharacterized stem cell (Powel et al., 1999; Tomasek et al., 1999; Walker et al., 2001). Upon the appropriate stimulation, the myofibroblast may revert back to a fibroblast or smooth muscle cell.
  • Myofibroblasts express receptors for TGF- ⁇ l, PDGF, bFGF, endothelin, and prostaglandins. All these factors generate in culture, an "activated" myofibroblast that is able to proliferate.
  • this activated form of the myofibroblast can be transformed into the non-proliferating "stellate" form by either cAMP or PGE2 (Powel et al., 1999; Tomasek et al., 1999; Walker et al., 2001; Wu and Zern, 2000).
  • the activated myofibroblast is then able to secrete cytokines, TGF- ⁇ l and other growth factors, and inflammatory mediators.
  • the cell has been shown to release NO and ROS, and matrix proteins involved in wound repair and fibrosis, such as collagens I, III, IV, VI, and XVIII, laminins, proteoglycans, adhesion molecules and MMPs (Powel et al., 1999; Tomasek et al., 1999; Walker et al, 2001).
  • matrix proteins involved in wound repair and fibrosis such as collagens I, III, IV, VI, and XVIII, laminins, proteoglycans, adhesion molecules and MMPs
  • the myofibroblast is involved in functions such as wound repair in skin and repair of the myocardium after myocardial infarction.
  • This cell has been implicated in the pathophysiology of the Dupuytren contracture, keloids, myocardial fibrosis, ischemia reperfusion injury, coronary artery restenosis, glomerulonephritis, liver cirrhosis, pulmonary interstitial fibrosis, and many other fibrotic conditions (Powel et al., 1999; Tomasek et al., 1999; Walker et al., 2001).
  • PD fibroblasts demonstrate a faster replication rate as compared to those from the normal TA, a higher production of a pro-fibrotic agent (basic fibroblast growth factor), and a potential alteration of the p53 pathway that normally represses cell replication and favors apoptosis, which would indicate a sort of "immortalization” in culture (Anderson et al., 2000a, 2000b; Mulhall et al., 2001a, 2001b). Other groups have studied fibroblast cultures from the Peyronie's plaque but did not focus on their myofibroblast content (Duncan et al., 1991; El-Sakka et al., 1997a).
  • Another potentially useful animal model for the study of the pathophysiology of the PD plaque is the collagen I promoter transgenic mouse (Fakhouri et al., 2001; Tharaux et al., 2000).
  • This mouse carries the regulatory region of the collagen I- ⁇ 2 gene linked to two reporter genes, luciferase and ⁇ -galactosidase, so that whenever collagen mRNA synthesis is stimulated luciferase and ⁇ -galactosidase will be expressed.
  • Both proteins can be estimated by a chemiluminescence reaction in tissue homogenates, and ⁇ -galactosidase specifically by the development of a blue color in tissue sections.
  • This collagen 1 promoter mouse model has recently been used (Dussaule et al., 2000) to demonstrate the link between NO, endothelin, and collagen synthesis in kidney - fibrosis, which is characterized by collagen I accumulation, hi essence, by giving the NOS inhibitor L-NAME to these transgenic mice for up to 14 weeks, it was possible to induce nephroangio- and glomerulo-fibrosis, accompanied by an increase in luciferase levels and an increased urinary excretion rate of endothelin.
  • the blockade of endothelin receptors with the selective ET antagonist bosentan reduced collagen deposition in the L- NAME animals, and abolished collagen I promoter activation, as quantitated by luciferase activity.
  • This animal model demonstrated that NO inhibition induces an early activation of the collagen I gene in the kidney arterioles and glomeruli, suggesting that NO inhibits collagen deposition and the endothelin-mediated fibrogenic effect, as confirmed by other studies (Boffa et al., 1999; Tharaux et al., 1999).
  • genes encoding one or more isoforms of PDE4, PDE5, PKG, NOS, MMP or another protein may be incorporated into expression vectors for therapeutic use in fibrosis.
  • a gene encoding a given protein may contain a variety of different bases and yet still produce a corresponding polypeptide that is indistinguishable functionally, and in some cases structurally, from the known sequences of such genes. It is a matter of routine for the skilled artisan to obtain known genomic and/or cDNA sequences encoding various proteins from publicly available sources, such as GenBank.
  • nucleic acid Any reference to a nucleic acid should be read as encompassing a host cell containing that nucleic acid and, in some cases, capable of expressing the product of that nucleic acid.
  • Cells expressing nucleic acids of the present invention may prove useful in the context of screening for agents that induce, repress, inhibit, augment, interfere with, block, abrogate, stimulate, or enhance the catalytic activity and/or regulatory properties of PDE4 and/or PDE5.
  • Nucleic acids according to the present invention may contain an entire gene, a cDNA, or a domain of a protein that expresses catalytic activity.
  • the nucleic acid may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the nucleic acid would comprise complementary DNA (cDNA).
  • the DNA segments of the present invention include those encoding biologically functional equivalent proteins and peptides. Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.
  • Nucleic acids encoding proteins or peptides may be incorporated into expression vectors for production of the encoded proteins or peptides.
  • Non-limiting examples of expression systems known in the art include bacteria such as E. coli, yeast such as Pichia pastoris, baculovirus, and mammalian expression systems such as in COS or CHO cells.
  • a complete gene can be expressed or, alternatively, fragments of the gene encoding portions of polypeptide can be produced.
  • the gene or gene fragment encoding a polypeptide may be inserted into an expression vector by standard subcloning techniques.
  • An E. coli expression vector may be used which produces the recombinant polypeptide as a fusion protein, allowing rapid affinity purification of the protein.
  • Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, NJ), the maltose binding protein system (NEB, Beverley, MA), the FLAG system (IBI, New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA).
  • fusion systems are designed to produce fusions wherein the fusion partner is easily excised from the desired polypeptide.
  • the fusion partner is linked to the recombinant polypeptide by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Beverley, MA).
  • the expression system used may also be one driven by the baculovirus polyhedron promoter.
  • the gene encoding the polypeptide may be manipulated by standard techniques in order to facilitate cloning into the baculovirus vector.
  • One baculovirus vector is the pBlueBac vector (Invitrogen, Sorrento, CA).
  • the vector carrying the gene for the polypeptide is transfected into Spodoptera frugiperda (Sf9) cells by standard protocols, and the cells are cultured and processed to produce the recombinant antigen. See U.S. Patent No. 4,215,051 (incorporated by reference).
  • Amino acid sequence variants of the polypeptide may also be prepared.
  • sequence variants may, for instance, be minor sequence variants of the polypeptide which arise due to natural variation within the population or they may be homologues found in other species. They also may be sequences which do not occur naturally but which are sufficiently similar that they function similarly and/or elicit an immune response that cross-reacts with natural forms of the polypeptide. Sequence variants may be prepared by standard methods of site-directed mutagenesis such as those described herein.
  • Substitutional variants typically contain an alternative amino acid at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage. Substitutions preferably are conservative, that is, one amino acid is replaced with one of similar size and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine or glutamine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • Insertional variants include fusion proteins such as those used to allow rapid purification of the polypeptide and also may include hybrid proteins containing sequences from other proteins and polypeptides which are homologues of the polypeptide.
  • an insertional variant may include portions of the amino acid sequence of the polypeptide from one species, together with portions of the homologous polypeptide from another species.
  • Other insertional variants may include those in which additional amino acids are introduced within the coding sequence of the polypeptide. These typically are smaller insertions than the fusion proteins described above and are introduced, for example, to disrupt a protease cleavage site.
  • DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of the claimed nucleic acid sequences.
  • engineered and recombinant cells are intended to refer to a cell into which an exogenous DNA segment or gene, such as a cDNA or gene has been introduced through the hand of man. Therefore, engineered cells are distinguishable from naturally occurring cells that do not contain a recombinantly introduced exogenous DNA segment or gene.
  • Recombinant cells include those having an introduced cDNA or genomic gene, and also include genes positioned adjacent to a heterologous promoter not naturally associated with the particular introduced gene.
  • Cell types available for expression include, but are not limited to, bacteria, such as E. coli and B. subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors.
  • Promoters that are most commonly used in recombinant DNA construction include the ⁇ -lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling those of skill in the art to ligate them functionally with plasmid vectors.
  • ⁇ -lactamase penicillinase
  • lactose lactose
  • trp tryptophan
  • the plasmid YR ⁇ 7 for example, is commonly used (Stinchcomb et al, Nature, 282: 39, 1979; Tschemper et al, Gene, 10:157, 1980).
  • This plasmid contains the trp ⁇ gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1.
  • the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Suitable promoting sequences in yeast vectors include the promoters for 3- phosphoglycerate kinase (Hitzeman et al, J. Biol. Chem., 255:2073, 1980) or other glycolytic enzymes (Hess et al, J. Adv.
  • Enzyme Reg 7:149, 1968; Holland et al, Biochemistry, 17:4900, 1978 ), such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
  • promoters which have the additional advantage of transcription controlled by growth conditions, include the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • cultures of cells derived from multicellular organisms may also be used as hosts.
  • any such cell culture is workable, whether from vertebrate or invertebrate culture.
  • mammalian cells these include insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic vims, CaMV; tobacco mosaic vims, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing one or more coding sequences.
  • recombinant virus expression vectors e.g., baculovirus
  • plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic vims, CaMV; tobacco mosaic vims, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing one or more coding sequence
  • Examples of useful mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines.
  • a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the encoded protein.
  • the host cells are human cells inside a subject with a fibrotic condition.
  • Expression vectors for use in mammalian cells ordinarily include an origin of replication (as necessary), a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
  • the origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • an exogenous origin such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • the promoters may be derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian vimses (e.g., the adenovirus late promoter; the vaccinia vims 7.5K promoter). Further, it is also possible, and may be desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.
  • a number of viral based expression systems may be utilized, for example, commonly used promoters are derived from polyoma, Adenovims 2, and most frequently Simian Vims 40 (SV40).
  • the early and late promoters of SV40 vims are particularly useful because both are obtained easily from the vims as a fragment that also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hind site toward the Bgl I site located in the viral origin of replication.
  • the coding sequences may be ligated to an adenovims transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovims genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant vims that is viable and capable of expressing proteins in infected hosts.
  • Specific initiation signals may also be required for efficient translation of the claimed isolated nucleic acid coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be in-frame (or in- phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons may be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements or transcription terminators (Bittner et al, Methods in Enzymol, 153: 516-544, 1987).
  • polyadenylation site e.g., 5'-AATAAA-3'
  • the poly A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.
  • the oligo- or polynucleotides and/or expression vectors may be entrapped in a liposome.
  • Liposomes are vesicular stmctures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • the Hpid components undergo self-rearrangement before the formation of closed stmctures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, Wu et al. (Eds.), Marcel Dekker, New York, pp 87-104, 1991).
  • cationic lipid-nucleic acid complexes such as lipofectamine-nucleic acid complexes.
  • the liposome may be complexed with a hemagglutinating vims (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, Science, 243:375-378, 1989).
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In that such expression vectors have been employed in transfer and expression of a polynucleotide in vitro and in vivo, they may be applicable for the present invention.
  • liposomes within the scope of the present invention can be prepared fn accordance with known laboratory techniques, hi one embodiment, liposomes are prepared by mixing liposomal lipids, in a solvent in a container, e.g., a glass, pear-shaped flask.
  • a container e.g., a glass, pear-shaped flask.
  • the container should have a volume ten-times greater than the volume of the expected suspension of liposomes.
  • the solvent is removed at approximately 40 C under negative pressure.
  • the solvent normally is removed within about 5 min to 2 hours, depending on the desired volume of the liposomes.
  • the composition can be dried further in a desiccator under vacuum.
  • the dried lipids generally are discarded after about 1 week because of a tendency to deteriorate with time.
  • the dried lipids or lyophilized liposomes prepared as described above may be reconstituted in a solution of nucleic acid and diluted to an appropriate concentration with an suitable solvent. The mixture is then vigorously shaken in a vortex mixer. Unencapsulated nucleic acid is removed by centrifugation at 29,000 g and the liposomal pellets washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., about 50-200 mM. The amount of nucleic acid encapsulated can be determined in accordance with standard methods. After determination of the amount of nucleic acid encapsulated in the liposome preparation, the liposomes may be diluted to appropriate concentration and stored at 4 C until use.
  • Adenoviruses Human adenovimses are double-stranded DNA tumor viruses with genome sizes of approximate 36 kB. As a model system for eukaryotic gene expression, adenovimses have been widely studied and well characterized, which makes them an attractive system for development of adenovims as a gene transfer system. This group of viruses is easy to grow and manipulate, and they exhibit a broad host range in vitro and in vivo, h lytically infected cells, adenovimses are capable of shutting off host protein synthesis, directing cellular machineries to synthesize large quantities of viral proteins, and producing copious amounts of vims.
  • the El region of the genome includes E1A and E1B, which encode proteins responsible for transcription regulation of the viral genome, as well as a few cellular genes.
  • E2 expression including E2A and E2B, allows synthesis of viral replicative functions, e.g. DNA-binding protein, DNA polymerase, and a terminal protein that primes replication.
  • E3 gene products prevent cytolysis by cytotoxic T cells and tumor necrosis factor and appear to be important for viral propagation.
  • Functions associated with the E4 proteins include DNA replication, late gene expression, and host cell shutoff
  • the late gene products include most of the virion capsid proteins, and these are expressed only after most of the processing of a single primary transcript from the major late promoter has occurred.
  • MLP major late promoter
  • adeno vims-derived vectors offer excellent potential for the substitution of large DNA fragments when used in connection with cell lines such as 293 cells.
  • Ad5-transformed human embryonic kidney cell lines (Graham et al, J. Gen. Virol, 36:59-72, 1977) have been developed to provide the essential viral proteins in trans.
  • adenovims vectors over retro viruses include the higher levels of gene expression.
  • Adenovims replication is independent of host gene replication, unlike retroviral sequences. Because adenovims transforming genes in the El region can be readily deleted and still provide efficient expression vectors, oncogenic risk from adenovims vectors is thought to be low (Grunhaus and Horwitz, Seminar in Virology, 3:237-252, 1992).
  • adenovims gene transfer systems are based upon recombinant, engineered adenovims which is rendered replication-incompetent by deletion of a portion of its genome, such as El, and yet still retains its competency for infection. Sequences encoding relatively large foreign proteins can be expressed when additional deletions are made in the adenovims genome. For example, adenovimses deleted in both El and E3 regions are capable of carrying up to 10 kB of foreign DNA and can be grown to high titers in 293 cells (Stratford-Perricaudet and Perricaudet, 1991). Persistent expression of transgenes following adenoviral infection has also been reported.
  • Viral Vectors as Expression Constructs.
  • Other viral vectors may be employed as expression constructs in the present invention.
  • Vectors derived from vimses such as vaccinia vims (Baichwal and Sugden, In: Gene Transfer, Kucherlapati R, ed., New York, Plenum Press, pp. 117-148, 1986) adeno-associated vims (AAV) (Baichwal and Sugden, 1986) and herpes vimses may be employed. They offer several attractive features for various mammalian cells (Horwich, et al, J. Virol, 64:642-650, 1990).
  • Non-viral Methods Several non-viral methods for the transfer of expression vectors into cultured mammalian cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and van der Eb, Virology, 52:456- 467, 1973) DEAE-dextran (Gopal, Mol. Cell Biol, 5:1188-1190, 1985), lipofectamine-DNA complexes, and receptor-mediated transfection (Wu and Wu, Biochemistry, 27: 887-892, 1988; Wu and Wu, J Biol Chem., 262: 4429-4432, 1987). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
  • the expression construct may simply consist of naked recombinant vector. Transfer of the constmct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane.
  • Dubensky et al. Proc. Nat. Acad. Sci. USA, 81:7529-7533, 1984
  • injected polyomaviras DNA in the form of CaPO 4 precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection.
  • antisense is intended to refer to polynucleotide molecules complementary to a portion of a targeted gene or mRNA species.
  • “Complementary" polynucleotides are those that are capable of base-pairing according to the standard Watson-Crick complementarity mles. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constmcts, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • the intracellular concentration of monovalent cation is approximately 160 mM (10 mM Na + ; 150 mM K + ).
  • the intracellular concentration of divalent cation is approximately 20 mM (18 mM Mg ; 2 mM Ca ' " " ).
  • the intracellular protein concentration which would serve to decrease the volume of hybridization and, therefore, increase the effective concentration of nucleic acid species, is 150 mg/ml. Constmcts can be tested in vitro under conditions that mimic these in vivo conditions.
  • Antisense constmcts may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that effective antisense constmcts may include regions complementary to the mRNA start site. One can readily test such constmcts simply by testing the constmcts in vitro to determine whether levels of the target protein are affected. Similarly, detrimental nonspecific inhibition of protein synthesis also can be measured by determining target cell viability in vitro. [0139] As used herein, the terms "complementary" or "antisense” mean polynucleotides that are substantially complementary to the target sequence over their entire length and have very few base mismatches.
  • sequences of fifteen bases in length may be termed complementary when they have a complementary nucleotide at thirteen or fourteen nucleotides out of fifteen. Sequences that are "completely complementary” will be sequences which are entirely complementary throughout their entire length and have no base mismatches.
  • sequences with lower degrees of homology are contemplated.
  • an antisense constmct that has limited regions of high homology, but also contains a non-homologous region (e.g., a ribozyme) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • the antisense sequences may be full length cDNA copies, or large fragments thereof, they also may be shorter fragments, or "ohgonucleotides,” defined herein as polynucleotides of 50 or less bases.
  • shorter oligomers (8-20) are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of base-pairing. For example, both binding affinity and sequence specificity of an oligonucleotide to its complementary target increase with increasing length. It is contemplated that ohgonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or 100 base pairs will be used. While all or part of the gene sequence may be employed in the context of antisense construction, statistically, any sequence of 14 bases long should occur only once in the human genome , and, therefore, suffice to specify a unique target sequence.
  • antisense constmcts which include other elements, for example, those which include C-5 propyne pyrimidines.
  • Ohgonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression (Wagner et al, Science, 260:1510-1513, 1993).
  • the antisense oligo- and polynucleotides according to the present invention may be provided as RNA via transcription from expression constructs that carry nucleic acids encoding the oligo- or polynucleotides.
  • expression constmct is meant to include any type of genetic construct containing a nucleic acid encoding a product in which part or all of the nucleic acid sequence is capable of being transcribed.
  • Typical expression vectors include bacterial plasmids or phage, such as any of the pUC or BluescriptTM plasmid series or viral vectors adapted for use in eukaryotic cells.
  • the nucleic acid encodes an antisense oligo- or polynucleotide under transcriptional control of a promoter.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • a variety of specific eukaryotic promoter elements are known in the art and any such known element may be used in the practice of the claimed invention. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
  • the particular promoter that is employed to control the expression of a nucleic acid encoding the inhibitory peptide is not believed to be important, so long as it is capable of expressing the peptide in the targeted cell.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. Any promoter/enhancer combination known in the art (e.g., the Eukaryotic Promoter Data Base) also could be used to drive expression of the gene.
  • a cDNA insert typically one will typically include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed, such as human growth hormone and SV40 polyadenylation signals.
  • a terminator also contemplated as an element of the expression construct. These elements can serve to enhance message levels and to minimize read through from the construct into other sequences.
  • the delivery of a nucleic acid in a cell may be identified in vitro or in vivo by including a marker in the expression constmct.
  • the marker would result in an identifiable change to the transfected cell permitting identification of expression.
  • Enzymes such as herpes simplex vims thymidine kinase (tk) (eukaryotic) or chloramphenicol acetyltransferase (CAT) (prokaryotic) may be employed.
  • siRNAs Small interfering RNAs
  • siRNAs are short RNA molecules (typically from 21 to 23 nucleotides in length) that may be used to induce targeted gene silencing by RNA interference (Myers et al., Nature Biotechnology 21:324-328, 2003; Elbashir, Nature 411:494-498, 2001; Caplen et al., Proc. Natl Acad. Sci. USA 98:9742-47, 2001).
  • SiRNAs occur naturally in vivo when double-stranded RNA is cleaved by ribonuclease III to produce a short siRNA sequence.
  • Synthetic siRNAs may also be introduced into cells to inhibit expression of one or more selected genes.
  • SiRNAs may be generated by standard solid-phase oligonucleotide synthesis, by RNA-specific endonuclease cleavage of double-stranded RNA, or by expression of transfected DNA templates incorporating promoter sequences for RNA polymerase III. Introduction of siRNA into a mammalian cell results in the targeted destruction of messenger RNAs of the same sequence.
  • Commercial products for siRNAs are available from a number of sources, such as Gene Therapy Systems, Inc. (San Diego, CA), Promega (Madison, WI) and Sirna Therapeutics (Boulder, CO).
  • siRNA Target Finder may be used online at the Ambion website.
  • Target mRNA sequences are input into the program, which then scans for 21 nucleotide sequences, that begin with an AA dinucleotide.
  • the program selects for siRNAs with about a 30 to 50% GC content, avoiding sequences with 4-6 polyT stretches that would function as terminators for RNA Polymerase III transcription.
  • the generated sequences may be searched for homology (for example, using the BLAST search engine on the NCBI server) to other untargeted mRNA sequences.
  • SiRNAs with homology to non-targeted sequences are eliminated from consideration.
  • SiRNA expression cassettes may also be obtained from Ambion (Austin, TX). SiRNAs may be purchased and used according to the manufacturer's instmctions to provide targeted inhibition of the expression of specific genes, such as PDE-4 and/or PDE-5.
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987; Gerlach et al, 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al, 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al, 1981).
  • U.S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyme- mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990; Sioud et al, 1992). It was reported that ribozymes elicited genetic changes in some cells lines to which they were applied. The altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.
  • RNA cleavage activity examples include sequences from the Group I self splicing introns including Tobacco Ringspot Vims (Prody et al, 1986), Avocado Sunblotch Viroid (Palukaitis et al, 1979; Symons, 1981), and Lucerne Transient Streak Vims (Forster and Symons, 1987). Sequences from these and related vimses are referred to as hammerhead ribozyme based on a predicted folded secondary stmcture.
  • ribozymes include sequences from RNase P with RNA cleavage activity (Yuan et al, 1992, Yuan and Altinan, 1994, U.S. Patent Nos. 5,168,053 and 5,624,824), hairpin ribozyme structures (Berzal-Herranz et al, 1992; Chowrira et al, 1993) and Hepatitis Delta vims based ribozymes (U.S. Patent No. 5,625,047).
  • Ribozymes are targeted to a given sequence by virtue of annealing to a site by complimentary base pair interactions. Two stretches of homology are required for this targeting. These stretches of homologous sequences flank the catalytic ribozyme structure defined above. Each stretch of homologous sequence can vary in length from 7 to 15 nucleotides. The only requirement for defining the homologous sequences is that, on the target RNA, they are separated by a specific sequence that is the cleavage site.
  • the cleavage site is a dinucleotide sequence on the target RNA - a uracil (U) followed by either an adenine, cytosine or uracil (A,C or U) (Perriman et al, 1992; Thompson et al, 1995).
  • the frequency of this dinucleotide occurring in any given RNA is statistically 3 out of 16. Therefore, for a given target messenger RNA of 1000 bases, 187 dinucleotide cleavage sites are statistically possible.
  • the inhibitors or activators of PDE5, PKG, NOS, MMP or another protein and/or stimulators or agonists of cGMP may be used for therapeutic treatment of medical conditions, such as Peyronie's disease.
  • medical conditions such as Peyronie's disease.
  • Aqueous compositions of the present invention comprise an effective amount of inhibitor or activator, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as innocula.
  • phrases "pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the inhibitors or activators of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, mtraperitoneal or intravenous injection. Such compositions normally would be administered as pharmaceutically acceptable compositions.
  • the active compounds also may be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the prefened methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts which are formed by reaction of basic groups with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free acidic groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. [0166] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drag release capsules and the like.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and mtraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion.
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • NO/cGMP The antifibrotic effects of agents that, either orally or via gene transfer, stimulate the NO/cGMP pathways and increase NO levels and cGMP levels or PKG activity or agents that inhibit oxidative stress by decreasing ROS levels.
  • agents that, either orally or via gene transfer, stimulate the NO/cGMP pathways and increase NO levels and cGMP levels or PKG activity or agents that inhibit oxidative stress by decreasing ROS levels.
  • gene transfer of the sense cDNA for iNOS or nNOS in a single transfection b) long-term administration of an oral NO donor (e.g. molsidomine), or the NOS substrate (L- arginine) that produces a continuously elevated level of NO.
  • an oral NO donor e.g. molsidomine
  • the NOS substrate L- arginine
  • cGMP and/or stimulating PKG by a) identifying PDE isoforms present in the affected tissue and using oral PDE inhibitors such as sildenafil, zaprinast, and pentoxifylline; b) by gene transfer of PKGl cDNA, or its mutated version, to increase the level of PKG activation.
  • oral PDE inhibitors such as sildenafil, zaprinast, and pentoxifylline
  • antioxidants such as vitamin E or S-adenosyl methionine (SAME); b) combination therapy with antioxidant (Vitamin E or SAME) combined with one or more NO donors (molsidomine or L-arginine) or cGMP/PKG therapy (sildenafil, zaprinast, pentoxifylline, or PKGl cDNA), to induce regression of the plaque (late treatment).
  • MMP collagenolysis
  • thymosin peptides or other MMP activators Conelating MMP inhibition in fibrosis with the levels of TEVIPI, an inhibitor of MMP.
  • MMP inducers thymosin ⁇ -4 and 10 are to stimulate MMP activity.
  • RNA-later (Ambion, Inc., Austin, TX)
  • RNA analysis in 4% formalin for histochemistry and immunohistochemistry, or in culture medium (DMEM/10% fetal calf serum) or fibroblast growth medium (FGM-2) (Clonetics, Walkersville, MD) with 20% fetal bovine serum, for protein analysis or cell culture.
  • Tissues were then frozen at -80 °C until further use, except for fixed portions that were stored at 4 °C in PBS until paraffin embedding or cryosectioning, and pieces used for cell cultures.
  • Human fibroblast primary cultures were obtained from fragments of PD plaque or TA essentially according to Smith and Liu (2002), and their purity was established by immunohistochemistry, as detailed below. New primary cultures were obtained from fragments of PD plaque or TA that were washed in Hanks solution, minced in a fibroblast growth medium (FGM) (BioWhittaker Inc., Walkersville, MD) and 20% fetal bovine semm (FBS), and plated onto a 25 cm 2 culture flask per specimen (Vemet et al., 2002). Fragments were left undisturbed until attachment for about 1 week. Once the monolayer started to develop, the fragment was removed.
  • FGM fibroblast growth medium
  • FBS fetal bovine semm
  • TGF- ⁇ l rat model Male Fisher 344 rats, 9-11 month old purchased from the NIH/NIA colony (Harlan Sprague-Dawley, Inc., San Diego, CA) and maintained under controlled temperature and lighting, were anesthetized and injected in the penile TA at the middle of the penis with either vehicle only (saline, group 1) or 0.5 ⁇ g recombinant human TGF- ⁇ l (Biotech Diagnostic, Website, groups 2-5) as disclosed (Ferrini et al., 2002; Vemet et al., 2002).
  • groups 1 and 2 were given drinking water while the other groups received water with L-arginine (2.25 g/kg/day, group 3) (Moody et al., 1997), or sildenafil (10 mg/kg/day, group 4) or pentoxifylline (10 mg/kg/day, group 5). Forty-five days later, or as indicated, animals were sacrificed and perfused through the left ventricle with saline followed by 4% formalin ((Ferrini et al., 2002; Vemet et al., 2002). After the penises were excised, the penile skin was denuded by removing the glans and adhering non-crural tissue.
  • the penile shaft was separated from the crura and 2-3 mm transverse slices were cut around the site of the saline or TGF- ⁇ l injection. . All tissues were post-fixed overnight in 4% formalin, washed in PBS and stored at 4 °C.
  • TGF- ⁇ l -iNOS knock-out mouse model The iNOS knockout strain (B6;129PNOS2 ⁇ TmlLeu>), where iNOS expression was genetically blocked, and the conesponding wild type control (B6;129PFl/y) (Hochberg et al., 2000), were injected (2-3 months old) in the TA with TGF- ⁇ l as in the rat and sacrificed 45 days later.
  • TGF- ⁇ l -collagen I promoter mouse model The transgenic line pGB 19.5/13.5 was obtained from George Bou-Gharios (London, England). These animals harbor the promoter of the ⁇ 2 chain of the mouse collagen type I gene linked to the E. coli ⁇ - galactosidase, that is expressed in cells and tissues where collagen I is normally expressed (Fakhouri et al., 2001; Tharaux et al., 2000; Dussaule et al., 2000). Animals were injected into the TA as above with TGF- ⁇ l, and sacrificed. [0177] Arterial Tree Rodent Model.
  • Minipump implantation Alza osmotic minipumps (Alza Corp, Palo Alto, CA), #2001D, delivering 8 ul/hr of a saline solution (100 ul) containing the selected compound during a period of 24 hs for "short-term” treatment, or 0.25 ul/hour, for 2 weeks (Alza#1002) for "long-term” treatment, were implanted in a subcutaneous tunnel over the inguinal canal, and attached to the abdominal muscles with a non-absorbable suture. A delivery catheter from the minipump was placed through the tissues to the penile crara and sutured to the perineal muscles, as previously described (Garban et al., 1997; Gelman et aL, 1998).
  • Total RNA was isolated from the human TA and PD tissues, from their respective fibroblast cultures, and from rat TA and penile shaft tissues, and their respective fibroblast and smooth muscle cell cultures, by the Trizol procedure (Gibco BRL, Gaithesburg, MD).
  • RNA was then submitted (1 ug) to reverse transcription (Vemet et al., 2002; Magee et al, 2002b; Ferrini et al., 2001b) using Superscript II RNase H " reverse transcriptase (Gibco BRL) and random primers (0.25 ug), followed by PCR with the respective gene specific primers (Kuthe et al., 2001): a) for human PDE5A, on nt 1027-1049 (forward) and nt 1788-1764 (reverse) of the respective cDNA (Genbank #158526); encompassing a 762 bp band common to the three variants 1-3; b) for rat PDE5A, the primers on nt 1905-1924 (forward) and nt 2479-2460 (reverse) of the respective cDNA (Genbank #NM 133584), generating a 575 bp band; c, d) for human PDE4A and B, on nt 942
  • Tissue extracts were obtained by homogenizing in a 1:6 wt/vol ratio in a buffer containing 0.32 M sucrose, 20 mM HEPES (pH 7.2), 0.5 mM EDTA, 1 mM dithithreitol and protease inhibitors (3 ⁇ M leupeptin, 1 ⁇ M pepstatin A, ImM phenylmethyl sulfonyl fluoride).
  • protease inhibitors 3 ⁇ M leupeptin, 1 ⁇ M pepstatin A, ImM phenylmethyl sulfonyl fluoride.
  • the particulate and cytosolic fractions were obtained by homogenization of the cells in a Polytron Homogenizer, (Brinkmann, Switzerland), and centrifugation at 12,000x g for 60 min.
  • Equal amounts of protein (30 ug) were run on 7.5% polyacrylamide gels, and submitted to western blot immunodetection with polyclonal anti-mouse PDE5 (against cGMP binding region) IgG (1:1000) (Calbiochem, La Jolla, CA), and a secondary donkey anti-mouse IgG linked to horse radish-peroxidase (Amersham Pharmacia, Piscataway, NJ), followed by a luminol reaction (Simko and Simko, 2000; Magee et al., 2002b; Ferrini et al., 2001b). Human PDE5 does not cross-react with other PDE5 isoforms.
  • Negative controls were performed without primary antibody.
  • PDE4 immunodetection the following affinity purified IgGs were used (FabGennix Inc., Shreveport, LA): a) anti-PDE4A selective antibody (detecting variants identified by 1, 5, 8, x, and unassigned); b) anti-PDE4B (detecting variants 1-4), and anti-PDE4D (detecting variants 1-5) (Salanova et al., 1999).
  • SMC and collagen fibers within the corporal tissue and vascular tree were estimated by Masson trichromic staining (Sigma Diagnostic, St.Louis, MO) (Ferrini et al., 2002; Vernet et al., 2002; Davila et al, 2203b) in sections adjacent to those used for immunohistochemical staining, followed by image analysis to measure the ratio between SMC (red) and collagen fibers (blue). The results were expressed as red/blue ratios per area (see below). In the arterial tree, the intimamedia thickness (IMT), and the diameter of the lumen were also measured.
  • IMT intimamedia thickness
  • the TUNEL assay (Ferrini et al., 2001a, 2001b) was performed in the adjacent matched tissue sections used for collagen, iNOS or nitrotyrosine staining, applying the Apoptag Oncor kit (Oncor, Gaithersburg, MD). hi brief, after deparaffmization and rehydration, sections were incubated with proteinase K (20 ug/ml) and endogenous peroxidase activity was quenched with 2% H 2 O 2 . Sections were incubated with digoxigenin-conjugated nucleotides and TdT, and subsequently treated with antidigoxigenin-peroxidase.
  • QIA Quantitative image analysis
  • the number of positive cells was counted in a computerized grid against the total number of cells determined by counterstaining, and results were expressed as a percentage of positive cells over total cells.
  • the integrated optical density (IOD) was obtained by measuring the density per object and multiplying it by the respective area. The sum of all the individual values in the field was then divided by the number of positive cells, to obtain the mean IOD/positive cell, as a measure of average immunoreactivity/cell.
  • results were expressed as the unweighted average optical density per area (O.D/AREA), to determine the relative concentration of immunoreactive antigen.
  • apoptotic index rate of programmed cell death
  • iNOS nitroyrosine, heme-oxygenase, PAI-1, MnSOD and Cu/Zn SOD determination
  • nitroyrosine, heme-oxygenase, PAI-1, MnSOD and Cu/Zn SOD determination at least 6 sections per specimen were analyzed. Each slide assayed had its conesponding negative control. In certain cases, the number of immuno-positive cells was determined as a percentage of the total counterstained nuclei in a computerized grid. In the Masson staining, the ratio between SMC (red) and collagen fibers, (blue) was obtained and expressed per area. The rate of programmed cell death (apoptotic index) was expressed as the percentage of apoptotic cells within the total number of cells in a given area (non-apoptotic nuclei plus apoptotic cells).
  • Example 1 Spontaneous iNOS induction in vivo in the PD plaque leads to increased NO synthesis, peroxynitrite formation, and fibroblast apoptosis.
  • aging per se results in the spontaneous induction of iNOS and the formation of the NO metabolite, peroxynitrite, in both the rat hypothalamus (Ferrini et al., 2001b; Vemet et al., 1998) and corpora cavernosa (Ferrini et al., 2001a). This was accompanied by apoptosis of both the neurons and the cavemosal smooth muscle.
  • iNOS induction in the TA is a beneficial, anti-fibrotic, cellular defense mechanism.
  • the locally produced NO from elevated iNOS would inhibit collagen deposition, oppose pro-fibrotic agents, and induce apoptosis of myofibroblasts, which pathologically persist in the PD plaque.
  • This model has been examined in the TA (Ferrini et al, 2002; Vemet et al., 2002; Gonzalez-Cadavid et al., 2002; Gholami et al., 2002) by quantitative image analysis (QIA) of immunohistochemical and histochemica).
  • iNOS expression as measured by immunohistochemistry was also detected in the rat PD-like plaque 45 days after TGF- ⁇ l injection in the TA, in comparison to control tissue obtained from rats injected with saline (Fenini et al., 2002; Vemet et al., 2002).
  • iNOS induction was accompanied by increased peroxynitrite, a product formed by the reaction of NO with ROS (Fenini et al,
  • peroxynitrite does not induce collagen deposition or fibrosis, which means it is not pro-fibrotic per se (Okamoto et al.,
  • Example 2 Inhibition of iNOS activity in vivo stimulates both collagen synthesis and collagen fiber deposition in the rat PD-like plaque.
  • the effect of L-NIL in increasing collagen in the TA of the TGF- ⁇ l rat model may be due to an increase in collagen synthesis, a decrease in its normal breakdown, or both.
  • This plasmid is an indicator of collagen I transcriptional activity within the rat PD plaque.
  • Expression of the reporter ⁇ -galactosidase measured by lummometry in tissue exfracts from areas at and around the plaque, was considerably intensified in comparison to the control TA (Vemet et al, 2002). This suggests that the reduction in NO by L-NIL inhibition of iNOS, directly or indirectly, activates pro-fibrotic factors such as ROS to further activate the collagen I promoter.
  • Example 3 The inhibition of PDE activity in vivo reduces collagen deposition and intensifies fibroblast apoptosis in the PD-like plaque in the animal model.
  • Pentoxifylline (a non-specific, generalized PDE inhibitor), and sildenafil (specific PDE-5 inhibitor) were given orally to the rat in their drinking water (100 mg/1 for each PDE inhibitor) for 45 days following TGF- ⁇ l injection to initiate the plaque in the rat model.
  • FIG. 2A, bottom panels shows that, as assessed by Masson staining in terms of collagen fiber/cell-lacunae area ratio, and width of the tunica (not shown), there was considerable reduction in plaque size induced by the PDE inhibitors. This was accompanied by an intensification of the apoptotic index, especially for pentoxifylline (FIG. 3A, bottom panels).
  • pentoxifylline is more effective than sildenafil in inducing apoptosis may be due to a number of possible mechanisms. Since pentoxifylline inhibits multiple PDE isoforms, it may suggest that other PDEs besides PDE5 may play a role in elevating cGMP levels within the TA that will lead to apoptosis of cells within the plaque. Additionally, PDE inhibitors may act differentially on the three processes that ultimately would inhibit fibrosis development, namely myofibroblast apoptosis, collagen synthesis, and collagen degradation.
  • fibroblasts/myofibroblasts because those are the predominant cellular component of the rat TA, and because of the direct demonstration of the effects of these treatments on cultures of human fibroblasts and myofibroblasts (see below).
  • Example 4 Presence of PDE5 in the human penile tunica albuginea and PD plaque, in the rat tunica albuginea, and in fibroblasts cultured from these tissues
  • FIG. 4A shows the ethidium bromide staining of PCR DNA fragments from reactions carried out in duplicate and fractionated by agarose gel electrophoresis.
  • the 575 bp PDE5A DNA band was generated as expected from the rat penile shaft (PS) and the 762 bp from the human corpora cavernosa (CC) RNAs, and was amplified to a similar level in total RNA from the human TA and PD. No RNA was extracted from the normal TA and the TGF- ⁇ l -induced PD-like plaque in the rat, due to the difficulty in dissecting large amounts of tissue to avoid contamination by cavemosal smooth muscle.
  • PDE5A expression was confirmed at the protein level by western blot assays of tissue extracts, as shown by the luminol-stained protein bands (FIG. 4B), that can discriminate the three splicing variant proteins of PDE5A designated as 1, 2, and 3 with respective apparent sizes of 100, 92, and 83 kDa, respectively (Lin et al., 2000a, 2002a).
  • the three variants were detected as expected in the rat cerebellum (CER), our control tissue, whereas in the rat penile cmra (CRU) and shaft (PS), the predominant forms were the 1 and 3, respectively, with only traces of variant 2 in the cmra, and a band smaller than the 3 variant in the penile shaft.
  • This PDE5A-3 variant accompanied by smaller amounts of the 2 variant, was also expressed in the human corpora cavernosa (CC), and in the TA and PD plaque.
  • Some PDE5A-1 variant was also detected in the human CC.
  • PDE5 mRNA was also identified by RT/PCR in the fibroblasts cultured from the human normal TA and PD plaque, and from the rat TA (FIG. 7A), and the respective protein was detected by western blot in the human cells as a single PDE5A-3 variant, which agrees with what was observed in vivo in the TA and PD plaque, (FIG. 7B).
  • the rat TA fibroblasts also express the 3 variant, accompanied by equal amounts of the 1 variant, despite the latter larger variant was not detected in the rat penile shaft. Immunocytochemical detection (FIG.
  • Example 5 Presence of PDE4 variants in the human penile tunica albuginea and PD plaque, in the rat tunica albuginea, and in fibroblasts cultured from these tissues
  • FIG. 12A shows that PDE4A and B mRNAs are expressed in the human normal TA and in the PD tissue. Both variants were also detected in human corpora cavernosa tissue containing mainly smooth muscle (not shown).
  • PDE4A and B mRNAs were also found in the fibroblasts cultured from human TA and PD (FIG. 12B).
  • PDE4 mRNA (without variant discrimination) was detected in the TA cells, and to a lesser degree in the penile shaft tissue, thus suggesting that PDE4 in the rat TA fibroblast cultures does not arise from contamination with smooth muscle, which in any case had been reasonably excluded above by immunocytochemistry.
  • FIG. 12C shows an intense 76 kDa band that would conespond to a variant identified in testis (Salanova et al., 1999), as well as a minor 102 kDa band for the so-called PDE4Ax, also seen in the testis.
  • the 76 kDa protein is very intense in the three human tissues: TA, PD plaque, and corpora cavernosa, but the 102 kDa band was virtually not detected. No PDE4B could be visualized when the western blot membranes were stripped and reacted with an antibody specific for this isoform (not shown).
  • Example 6 Incubation of PD fibroblast cultures with PDE inhibitors or a cGMP analog reduces collagen I synthesis and myofibroblast differentiation, and increases apoptosis
  • Levels of cGMP increased 5.0-fold with 100 uM SNAP (an NO donor) for 3 days, with fresh daily replacement of medium with SNAP.
  • the basal levels of cAMP were 42.6+/-12.7 pmol/mg protein in the absence of SNAP and did not vary significantly when measured after 3 days with SNAP.
  • the cGMP-dependent PDE5 inhibitor sildenafil did not significantly stimulate cGMP levels in the absence of SNAP (not shown). However, in the presence of the NO donor, the cGMP levels expressed as % of the basal confrol levels in the absence of sildenafil, were increased dose-dependently by sildenafil after the 3-day incubation, as expected (FIG. 14A). When cells were incubated with this NO donor, increasing concentrations of pentoxifylline, also as expected, did not increase significantly cGMP levels expressed as % of confrol levels (FIG. 14B), but were very effective in increasing cAMP levels (FIG. 14C), thus confirming its role as a cAMP-dependent PDE inhibitor with little or no effect on cGMP-dependent PDE.
  • the PD cells were incubated with or without the drags at lower concentrations: pentoxifylline at 200 nM, and sildenafil, at 50 and 200 nM. After 3 days, cells were fixed and the intracellular deposition of collagen I and III was determined by immunocytochemistry with specific antibodies against the two isoforms. The antibody against collagen I elicited an intense granular and perinuclear staining (not shown), h contrast, collagen III was detected in only in about 30% of the cells, and stained more diffusely and rather lightly, even when cells where treated with TGF- ⁇ l (10 ng/ml), a known stimulator of collagen III synthesis (Fig. 5B).
  • FIG. 5A Quantitation by image analysis (FIG. 5A) in the cultured human PD fibroblasts indicated that in the absence of additions, most of the cells (100%) expressed collagen I, and that both pentoxifylline and sildenafil at 200 nM completely inhibited collagen synthesis in a small number of cells (5-15% of the total, FIG. 5A top), and significantly reduced (30-40% decrease) the average intensity of expression per cell (FIG. 5A, bottom). In contrast, the PDE inhibitors did not decrease, but even increased, the synthesis of collagen III (not shown).
  • both of the PDE inhibitors pentoxifylline and sildenafil significantly reduced the number of ASMA positive cells (myofibroblasts) from 37% in the control to about 24%.
  • the average ASMA expression per cell was significantly reduced by the PDE inhibitors by more than 90% in all cases.
  • fibroblasts particularly those of PD origin, are able to differentiate into vimentin+/ASMA+ myofibroblasts comprising about 30%) of the cells in culture (Vemet et al., 2002), and this percentage of myofibroblasts is also seen in vivo in the plaque (Vemet et al., 2002).
  • the cultures can be induced to express iNOS, synthesize collagen I, and undergo apoptosis both in vitro and in vivo (Vemet et al., 2002).
  • Example 8 The increase in cGMP levels in fibroblast cultures leads to fibroblast apoptosis and the reduction in intracellular collagen I.
  • PDE-5 The effects of the PDE inhibitors, as seen in in vivo experiments, are most likely mediated in part by PDE-5, as shown by the presence of PDE5A mRNA (FIG. 7A), and specifically, the PDE5A-3 protein variant as in the in vivo derived tissues (FIG. 7B), in both the human TA and human PD derived cells.
  • the fibroblast cultures obtained from the rat TA express all three PDE-5 variants. All human and/or rat cell cultures of normal TA and PD tissues were PDE5 positive by immunocytochemistry (FIG. 7C, top panels). Cells derived from human TA tissue were clearly fibroblasts with some myofibroblast differentiation, as assayed with vimentin and ASMA markers (FIG. 7C, bottom panels).
  • Example 9 ROS levels are increased in the human and rat PD-like plaque tissues and are reduced by NO.
  • ROS plays an important role in the development and maintenance of many fibrotic disorders including PD, by stimulating collagen synthesis (Poli, 2000; Curtin et al, 2002; Cattell, 2002; Kim et al., 2001; Fan et al., 2000; Higuchi et al, 1999). Therefore, the interplay and reactivity of ROS with NO may be an important therapeutic target.
  • heme-oxygenase I immunoreactivity a marker for the strong pro-fibrotic factor ROS, is increased in the PD plaque in comparison to normal TA in the human and the TGF- ⁇ l rat model of PD (Ferrini et al., 2002).
  • Example 10 Use of gene transfer and reporter gene expression for analyzing PD.
  • cDNA constmcts to the penile TA (e.g., iNOS, PKG).
  • Therapeutic adminisfration of recombinant cDNA may lead to an elevated expression of the conesponding anti-fibrotic protein.
  • plasmid and adenoviral constructs of iNOS and penile nNOS (Magee et al., 2002a), including the use of plasmid and adenoviral constructs expressing ⁇ -galactosidase as a reporter gene, and electroporation to enhance viral and plasmid uptake during transfection (Magee et al., 2002a).
  • constmcts can penetrate and spread into the TA, as shown by X-gal staining (FIG. 8) suggesting that the direct injection to the TA with and without electroporation is feasible for targeting genes to the TA for anesting or reversing the growth and development of the PD plaque.
  • TGF- ⁇ l rat model for PD is a very valuable tool, and since its introduction in 1997 (El-Sakka et al., 1997b, 1998, 1999), we have been able to study various aspects of the pathophysiology of PD, some of which are presented above. However, in a different experimental design based on the ubiquitous finding of fibrin in histological samples of human PD tissue (not shown), we have recently re-confinned the importance of TGF- ⁇ 1 as the main profibrotic factor in eliciting the PD-like plaque in the TA of the rat.
  • the fibrin induced plaque is accompanied by detection of fibrin in the lesion (similar to what is seen in the human, but absent from the TGF- ⁇ l injected rat model), disorganization of elastin fibers, expression of iNOS and heme-oxygenase I, and an increased level of apoptosis. Save for the presence of fibrin in the TA, all findings are similar to the ones observed in the TGF- ⁇ 1 injected model (not shown).
  • Example 12 The TA and the PD plaque are tissues in constant turnover, and coUagenase inhibition may play a role in collagen accumulation.
  • fibrosis of PD is not a terminal event but a dynamic one, and that it is possible to pharmacologically affect its steady state and alter its direction by: a) inhibiting collagen synthesis and/or fibroblast differentiation and replication, since a subset of differentially expressed genes are related to these processes; and/or b) by stimulating collagen breakdown, since there is a considerable increase in the expression of different types of MMPs (e.g. MMP2 and MMP9).
  • MMPs different types of MMPs
  • MMPs may play an important role in extracellular matrix remodeling in the PD plaque.
  • the inhibition of MMP may occur by increased activity of the MMP inhibitors (TIMP).
  • TIMP MMP inhibitors
  • the increased expression of TIMP 1 should lead to an increased inhibition of MMP.
  • Example 13 The expression of a family of wound healing-related peptides, the thymosins, is increased in human PD.
  • thymosin- ⁇ 4 has been proposed for wound healing (Huff et al., 2002; Malinda et al, 1999; Sosne et al, 2002), and, as stated above, PD is likely the result of an injury that does not heal properly. It should be possible to further up-regulate this endogenous defense mechanism by pharmacologically increasing thymosin levels in the TGF- ⁇ l -induced lesions in the rat model of PD.
  • Example 14 Investigating the role of NO and ROS in PD using the iNOS knockout mouse model
  • the iNOS knockout mouse has previously been used to show that experimental urethral fibrosis is intensified as a consequence of the iNOS knockout (Tanaka et al., 2002).
  • FIG. 11 shows plaque formation within the TA at 6 weeks as shown with Masson staining.
  • results above demonstrate that PDE5 and 4 are both expressed in the human and rat normal tunica albuginea, and the respective PD and PD-like fibrotic plaques, as well as in the cell cultures obtained from these tissues.
  • the results also demonstrate the inhibition of a TGF- ⁇ l -induced fibrotic plaque in the rat model of PD, through the reduction of collagen deposition and possibly an increase in apoptosis of the resident fibroblasts and myofibroblasts, by long-term oral adminisfration of the respective PDE5 and cAMP-dependent PDE inhibitors, sildenafil and pentoxifylline, and the NOS substrate, L-arginine.
  • cGMP analogs, PKG activators, and PDE inhibitors have been shown to inhibit collagen synthesis (Redondo et al., 1998; Wollert et al., 2002), and induce apoptosis (Sirotkin et al., 2000), and some of the PDE inhibitors like sulindac sulfone (Exisulind) are effective as anticancer agents because of their intense pro-apoptotic action (Piazza et al., 2001; Thompson et al., 2000).
  • pentoxifylline did not affect cGMP levels in the human PD fibroblasts, and the drag is considered to be a non-specific inhibitor of cAMP-PDE (Lin et al., 2002c; Liang et al., 1998), and at least in some cell types does not affect cGMP levels (Chen et al., 1999), the increase in cAMP may also have played a role in the antifibrotic effects observed with pentoxifylline. Whether this occurs via the inhibition of PDE4 present in TA and PD remains to be established.
  • Pentoxifylline may also act through its blockade of PDGF-induced activation of the mitogen activated protein kinase system (Souness et al., 2000) and of other cytokine-mediated fibrogenic mechanisms (Raetsch et al., 2002).
  • the daily dose of pentoxifylline used was 1/5 of the oral dose normally employed in rats for the long-term treatment of fibrosis (Chen et al., 1999; Tarcin et al., 2003), and in the case of sildenafil, it is 1/2 to 1/7 of the chronic dosage used in recent studies in rats (Sebklii et al., 2003).
  • the 10 mg/kg/day dose is translated into the equivalent dose in humans by conecting for differences in the body 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 demand single 100 mg tablet.
  • the selected dose was dispensed in 24 hours and not as a bolus administration, so that concentrations at a given time should be much lower, considering the short half-life (about 4-6 hours) of sildenafil. Therefore, the daily doses of the PDE inhibitors tested in the current work are not supra-pharmacological or associated with toxicity.
  • local administration of either L- arginine or the PDE inhibitors e.g. by injection into the plaque or in vehicles able to traverse the skin and TA may considerably reduce the effective dosage.
  • the PDE5A3 variant was also found in corpora cavernosa and confined to tissues with a smooth muscle or cardiac muscle component, and is twice as sensitive as PDE5A1 to sildenafil, but, as with PDE5A1 and 2, is subject to transcriptional up-regulation by both cAMP and cGMP (Lin et al., 2002a; Turko et al., 1999).
  • cAMP can activate PKG nearly as effectively as cGMP, so that eventually, the inhibition of PDE4 may cause PKG effects (e.g., counteracting fibrosis) similar to those exerted by as the inhibition of PDE5A.
  • modulation of any of these processes may involute the plaque, as has been observed in generalized fibrotic conditions (Lee et al., 2001; Lai et al., 2000).
  • Example 16 Intensification of aging-related fibrosis in the arterial media by iNOS inhibition
  • 15A shows that in the media of the dorsal penile artery, few collagen fibers were present in the young rats but were considerably increased in the aged animals, resembling the situation seen in the aorta. Consistent with the model that iNOS may act as antifibrotic agent within the vascular tree, the administration of L-NIL, a specific inhibitor of iNOS 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 dorsal penile artery.
  • Example 17 iNOS induction and peroxynitrite deposition in the arterial media with aging
  • FIG. 16A shows negligible ⁇ NOS expression and nitrotyrosine formation in the dorsal artery of the penis of the young animals, and a remarkable intensification of both processes with aging.
  • the iNOS staining in these vessels was mainly confined to the media and intima. A similar finding was seen in the aorta, brachial, and femoral arteries (not shown).
  • Example 18 Effects of iNOS inhibition on ROS production, apoptosis, and PAI in the arterial media
  • heme oxygenase I Another antioxidant enzyme, heme oxygenase I, demonstrated the same aging related changes in the penile dorsal artery, as observed for both SOD enzymes, but remarkably, L-NIL did not induce a further significant change in the expression of this enzyme (FIG. 18).
  • the NO/ROS balance was significantly altered throughout the entire arterial media by iNOS inhibition with L-NIL via a reduction in NO synthesis (denoted by peroxynitrite) and a stimulation of ROS formation (denoted by the antioxidant enzymes).
  • Apoptosis of the SMC within the media of the penile resistance arteries increased with aging, and decreased subsequently in the old animals receiving L-NIL treatment (FIG. 19 A).
  • the apoptotic index was calculated for both the dorsal and bulbourethral penile arteries by image analysis, and was higher in aged compared to young rats but L-NIL treatment resulted in a reduction in this index (FIG. 19B).
  • the corporal tissue comprises primarily of a syncytium of vascular SMC with an endothelium lining which is biologically and physiologically indistinguishable from the one present in the media and intima of the vascular tree (Krall et al., 1988) and may be considered a highly evolved extension of these arterial tissues. Therefore, insults that afflict the arterial media may also afflict the corporal SMCs, resulting in defective vaso-relaxation in both the corporal tissue (ED) and the arterial tree (hypertension).
  • ED corporal tissue
  • hypertension hypertension
  • NO has been shown in animal models to be protective against atherosclerosis and restenosis in the vascular system (Gewaltig and Kojda, 2002; Cheng et al., 2001), and fibrosis throughout the vascular tree and other organs (Ferrini et al., 2002; Vemet et al., 2002;.05.ig and Kojda, 2002), the concept that NO may prevent aging-related arteriosclerosis is novel.
  • the pro-apoptotic action of NO would suggest that it decreases the SMC/collagen balance through increased cell death.
  • a final question is whether collagen accumulation with aging is at least partially mediated via the regulation of PAI-1, TIMPl and other metalloproteinase inhibitors (Li et al., 2000; Kaikita et al., 2002), that increase in different types of fibrosis.
  • PAI-1, TIMPl and other metalloproteinase inhibitors Li et al., 2000; Kaikita et al., 2002
  • the cunent results with PAI combined with previous data where we observed considerable metalloproteinase and PAI mRNA expression in the fibrotic plaque of Peyronie's disease in the human and rat (Magee et al., 2002a), would suggest that although the increase in the pro-fibrotic PAI may induce a compensatory elevation of metalloproteinase levels, the enzyme would remain inhibited and the net result would be an impaired collagen breakdown.
  • the results indicate that within the arterial system and the cavemosal tissue it may be possible to pharmacologically modulate a) the NO/ROS balance with NO donors or other NO generators together with antioxidants, and b) the PAI/MMP balance with agents modifying their relative expression.
  • Such novel therapies may constitute viable approaches for the prevention and/or therapy of vascular disorders that involve the arterial media and the corpora.
  • Example 19 Gene therapy with iNOS cDNA
  • the AdV-CMV-iNOS constmct has been prepared by subcloning the iNOS cDNA driven by the sfrong CMV promoter (Garban et al., 1997), from a plasmid constmct into an AdV plasmid vector, and purifying the AdV constmct, as previously described for PnNOS (Magee et al., 2002a).
  • This AdV vector is replication-defective and helper-dependent, and therefore is non- infectious and totally innocuous. In addition, it lacks virtually all the original viral sequences that may be immunogenic.
  • This AdV constmct can be transfected into the TA, and it has been cloned and utilized for other therapeutic purposes in the penis (Magee et al., 2002a).
  • Rats are injected in the TA at the same site as the TGF- ⁇ l was injected 5 days earlier (as evidenced by a non-absorbable suture) with 10 8 and 10 9 vp of either AdV- CMV-iNOS in 50 ul saline, or with vehicle (saline) only.
  • This 5-day waiting period between the TGF- ⁇ l injection and the cDNA constmct avoids any interference of the viral preparation with the injected TGF- ⁇ l, and/or its dispersion by the electroporation applied to enhance transfection of the iNOS construct.
  • the resulting 4 groups of rats are allowed to develop the plaque for 40 more days, sacrificed, and the area around the plaque is excised, fixed, paraffin-embedded, and sectioned (Ferrini et al., 2002, Vemet et al., 2002).
  • the cDNA constmct and saline are injected 45 days after the TGF- ⁇ l injection (when the plaque has already formed) and 30 days later the animals are sacrificed.
  • the TA of the iNOS-treated animals shows, in comparison with controls: 1) a decrease in the size of the plaque as evidenced by Masson, collagen I/III staining, and hydroxyproline content; 2) a higher expression of iNOS and nitrotyrosine; 3) decrease of ROS; and 4) increase in the apoptotic index of the fibroblasts/myofibroblasts.
  • the iNOS is injected 45 days after TGF- ⁇ l injection, at least some regression of the plaque is obtained.
  • Example 20 Oral NO donors or NOS substrate
  • Plaques are induced in the TA of rats by TGF- ⁇ l injection (Ferrini et al, 2002).
  • Drinking water containing molsidomine N-ethoxycarbonyl-3-morpho-linosydnomine
  • 0.12 g/1 (Benigni et al., 1999) (freshly prepared each day) is given to 2 groups of rats, an early and a late treatment group.
  • the dose of molsidomine used is based on the report in which it was utilized for 22 days to protect against tubulo-interstitial injury in a rat model of chronic glomeralar disease (Uckert et al., 2001), and is calculated to be equivalent to approximately 15 mg/kg/day in the rat.
  • L-arginine it is given only as a late treatment, but at 2 doses: 22.5 and 10.0 g/1 (in drinking water) (2 groups).
  • 22.5 g/1 dose of L-arginine was used for 45 days to elevate NOS activity in the rat penis (Moody et al, 1997), and to inhibit the plaque with the early treatment is equivalent to roughly 2.8 g/kg/day.
  • Example 21 Oral PDE inhibitors to regress the PD plaque
  • Sildenafil specific PDE5 inhibitor
  • pentoxifylline non-specific PDE inhibitor
  • Sildenafil specific PDE5 inhibitor
  • pentoxifylline non-specific PDE inhibitor
  • the study may be repeated at double or possibly quadruple the dosage above (2 groups).
  • Reduction in plaque size was observed with both sildenafil and pentoxifylline during the entire time of plaque development ('early treatment'), and this type of treatment may be as effective in regressing the plaque ('late treatment').
  • AdV-PKG wild type
  • AdV-PKGcat mutated
  • the constitutively active PKGl mutant consists of the carboxy-terminal catalytic domain without the amino-terminal regulatory domain where cGMP binds (Wollert et al., 2002). Both the wild type and mutated constmcts are obtained from Dr. Stefan Janssens (Center for Transgene Technology and Gene Therapy, University of Leuven, Belgium).
  • RNA is isolated from 2 groups of human and 2 groups of rat tissues.
  • the antioxidant vitamin E ( -tocopherol) is given in a specially prepared oral diet, so that the animal receives in an early treatment phase approximately 200 IU/kg/bw/day, whereas controls receive the normal diet containing less than 20 IU/kg (Gonca et al., 2000).
  • the third group (3 groups) receive twice a day an intramuscular injection (10 mg/kg) of another antioxidant compound that ameliorates oxidative stress and lipid peroxidation, the glutathione precursor S-adenosyl-L-methionine (SAM) (Muriel et al., 1998b).
  • SAM glutathione precursor S-adenosyl-L-methionine
  • Example 25 Effect of obliterating iNOS on collagen breakdown. Plaque in the iNOS knockout
  • Example 26 Modulation of MMP through thymosin peptides. Treatment with thymosin ⁇
  • Thymosin ⁇ 4 and 10 are given daily intraperitoneally as a late treatment at 60 ug/day, every other day (2 groups) (Sosne et al., 2002). Such treatment with thymosin- ⁇ 4 (the most abundant thymosin) has been used for promoting healing of dermal wounds (Sosne et al., 2002)). Plasmid preparations of a cDNA encoding both peptides (200 ug/rat) are also given by injection/electroporation to the TA (2 groups), as a late treatment (i.e. 45 days after the TGF- ⁇ l injection, to induce the PD like plaque).
  • Nitric oxide inhibits the synthesis of type-II collagen without altering Col2Al mRNA abundance: prolyl hydroxylase as a possible target. Biochem J 324:305-10.
  • Chipev CC Simman R, Hatch G, Katz AE, Siegel DM, Simon M (2000) Myofibroblast phenotype and apoptosis in keloid and palmar fibroblasts in vitro. Cell Death Differ 7(2):166-76.
  • Davila HH, Magee TR, Rajfer J and Gonzalez-Cadavid NF Gene transfer of antisense PIN (protein inhibitor of NOS) cDNA on erectile dysfunction in aged rats. American Urological Association Meeting, Chicago, II, 2003 a.
  • Fakhouri F Placier S, Ardaillou R, Dussaule JC, Chatziantoniou C (2001) Angiotensin II activates collagen type I gene in the renal cortex and aorta of transgenic mice through interaction with endothelin and TGF-beta. J Am Soc Nephrol 12(12):2701-10.
  • Ferrini M Wang C, Swerdloff R, Sinha Hikim AP, Gonzalez-Cadavid NF (2001b) Aging-related expression of inducible nitric oxide synthase (iNOS) and cytotoxicity markers in rat hypothalamic regions associated with male reproductive function. Neuroendocr 74:1-11.
  • Haig DM Thomson J, Percival A. (1994) The in- vitro detection and quantitation of ovine bone anow precursors of multipotential colony-forming cells. J Comp Pathol 111(1): 73-85.
  • Thymosin beta4 is released from human blood platelets and attached by factor Xllla (transglutaminase) to fibrin and collagen. FASEB J. 16(7):691-6.
  • Intengan HD Schiffrin EL. Vascular remodeling in hypertension. Roles of apoptosis, inflammation, and fibrosis. Hypertension. 2001; 38:581-587.
  • Kim NN Huang Y, Moreland RB, Kwak SS, Goldstein I, Traish A (2000) Cross- regulation of intracellular cGMP and cAMP in cultured human corpus cavernosum smooth muscle cells.Mol Cell Biol Res Commun 4(1): 10-4.
  • Kim PK Zamora R, Petrosko P, Billiar TR (2001) The regulatory role of nitric oxide in apoptosis. h t Immunopharmacol 1(8):1421-41. Review.
  • Lin CS Lau A, Tu R, Lue TF (2000a) Expression of three isoforms of cGMP- binding cGMP-specific phosphodiesterase (PDE5) in human penile cavemosum. Biochem Biophys Res Commun 268(2):628-35.
  • Magee TR, Qian A, Rajfer J, Levine L, Gonzalez-Cadavid NF (2002b) Gene expression profiles in the Peyronie's disease plaque. Urology 59:451-457.
  • Robert L Aging of the vascular-wall and atherosclerosis. Exp Gerontol. 1999; 34:491-501
  • Rogers RS Graziottin TM, Lin C-S, Kan YW, Lue TF. fritracavernosal vascular endothelial growth factor (VEGF) injection and adeno-associated vims -mediated VEGF gene therapy prevent and reverse venogenic erectile dysfunction in rats.
  • VEGF fritracavernosal vascular endothelial growth factor
  • Vemet D Bonavera JJ, Swerdloff RS, Gonzalez-Cadavid NF, Wang C (1998) Spontaneous expression of inducible nitric oxide synthase (iNOS) in the hypothalamus and other brain regions of aging rats. Endocrinology 139:3254-61.

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Abstract

L'invention concerne des procédés et des compositions utilisées pour traiter des pathologies impliquant la fibrose, telles que la plaque de la maladie de Peyronie, la fibrose corporelle du pénis, le dysfonctionnement veino-occlusif du pénis, des nodules de la maladie de Dupuytren, la fibrose vaginale, la fibrose du clitoris, un trouble d'alerte sexuelle féminin, une cicatrisation de plaie anormale, la formation de la chéloïde, une fibrose générale du rein, de la vessie, de la prostate, de la peau, du foie, du poumon, du coeur, des intestins ou tout autre état pathologique fibrotique généralisé ou localisé, la fibrose vasculaire, l'hyperplasie intime artérielle, l'athérosclérose, l'artériosclérose, la resténose, l'hypertrophie cardiaque, l'hypertension ou tout autre état caractérisé par un fibroblaste excessif ou une prolifération cellulaire du muscle souple ou un dépôt de collagène et de matrice extracellulaire dans les vaisseaux sanguins et/ou le coeur. Dans certains modes de réalisation, les compositions peuvent comprendre un inhibiteur de PDE-4, un inhibiteur de PDE-5, un composé qui augmente cGMP et/ou PKG, un stimulateur de la cyclase de guanylyl et/ou PKG, une combinaison d'un composé qui augmente cGMP, PKG, ou NO avec un antioxydant qui réduit ROS, ou un composé qui augmente l'activité de MMP. Dans d'autres modes de réalisation, la composition peut être un vecteur de thérapie génique.
PCT/US2003/033400 2002-10-22 2003-10-21 Procedes d'utilisation d'inhibiteurs de phospodiesterases et modulateurs d'acide nitrique, especes d'oxygene reactives, et metalloproteinases dans le traitement de la maladie de peyronie, de l'arteriosclerose et d'autres maladies fibrotiques WO2004037183A2 (fr)

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AU2003286555A AU2003286555A1 (en) 2002-10-22 2003-10-21 Phosphodiester inhibitors and nitric oxide modulators for treating peyronie's disease, arteriosclerosis and other fibrotic diseases
US10/779,069 US8133903B2 (en) 2003-10-21 2004-02-13 Methods of use of inhibitors of phosphodiesterases and modulators of nitric oxide, reactive oxygen species, and metalloproteinases in the treatment of peyronie's disease, arteriosclerosis and other fibrotic diseases
US13/372,094 US20120141454A1 (en) 2003-10-21 2012-02-13 Methods of Use of Inhibitors of Phosphodiesterases and Modulators of Nitric Oxide, Reactive Oxygen Species, and Metalloproteinases in the Treatment of Peyronie's Disease, Arteriosclerosis and Other Fibrotic Diseases
US14/215,444 US20140200219A1 (en) 2002-10-22 2014-03-17 Methods of Use of Inhibitors of Phosphodiesterases and Modulators of Nitric Oxide, Reactive Oxygen Species, and Metalloproteinases in the Treatment of Peyronie's Disease, Arteriosclerosis and Other Fibrotic Diseases
US15/277,550 US20170239257A1 (en) 2002-10-22 2016-09-27 Methods of Use of Inhibitors of Phosphodiesterases and Modulators of Nitric Oxide, Reactive Oxygen Species, and Metalloproteinases in the Treatment of Peyronie's Disease, Arteriosclerosis and Other Fibrotic Diseases
US15/832,918 US20180318307A1 (en) 2003-10-21 2017-12-06 Methods of use of inhibitors of phosphodiesterases and modulators of nitric oxide, reactive oxygen species, and metalloproteinases in the treatment of peyronie's disease, arteriosclerosis and other fibrotic diseases

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WO2009052630A1 (fr) * 2007-10-26 2009-04-30 Pacific Therapeutics Ltd. Compositions et procédés permettant de traiter des troubles fibroprolifératifs
WO2013011514A1 (fr) 2011-07-20 2013-01-24 Mediwound Ltd. Extrait protéolytique tiré de la bromélaïne et convenant au traitement de troubles des tissus conjonctifs
WO2014100733A1 (fr) 2012-12-21 2014-06-26 Mayo Foundation For Medical Education And Research Procédés et matériaux pour le traitement de la sténose valvulaire aortique calcifiée
WO2016111885A1 (fr) * 2015-01-06 2016-07-14 Imprimis Pharmaceuticals, Inc. Formulations pharmaceutiques à base de xanthine ou de dérivés de xanthine
WO2018167689A1 (fr) 2017-03-14 2018-09-20 Atir Holding S.A. Formulation topique destinée au traitement de problèmes de pigmentation de la peau
WO2018167687A1 (fr) 2017-03-14 2018-09-20 Atir Holding S.A. Utilisation de composés hétérocycliques dans le traitement de la peau pigmentée
WO2021093376A1 (fr) * 2019-11-11 2021-05-20 广州华真医药科技有限公司 Utilisation d'un inhibiteur de phosphodiestérase 5 dans la préparation d'un médicament permettant de résister à des maladies fibrotiques

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5824669A (en) * 1996-03-22 1998-10-20 Nitromed, Inc. Nitrosated and nitrosylated compounds and compositions and their use for treating respiratory disorders
US5981563A (en) * 1995-04-28 1999-11-09 Zonagen, Inc. Methods and formulations for modulating the human sexual response
US5990103A (en) * 1994-08-25 1999-11-23 Hoechst Aktiengesllschaft Combination preparation for use in immunological diseases

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5990103A (en) * 1994-08-25 1999-11-23 Hoechst Aktiengesllschaft Combination preparation for use in immunological diseases
US5981563A (en) * 1995-04-28 1999-11-09 Zonagen, Inc. Methods and formulations for modulating the human sexual response
US5824669A (en) * 1996-03-22 1998-10-20 Nitromed, Inc. Nitrosated and nitrosylated compounds and compositions and their use for treating respiratory disorders

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WO2006023603A2 (fr) 2004-08-17 2006-03-02 The Johns Hopkins University Compositions d'inhibiteur pde5 et méthodes de traitement des troubles cardiaques
EP1786428A2 (fr) * 2004-08-17 2007-05-23 The Johns Hopkins University Compositions d'inhibiteur pde5 et methodes de traitement des troubles cardiaques
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US8809360B2 (en) 2004-10-29 2014-08-19 Mitsubishi Tanabe Pharma Corporation Use of a pyridine compound for the preparation of a medicament for the treatment of skin lesions
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EP1888074A4 (fr) * 2005-06-10 2010-06-23 Dong A Pharm Co Ltd Agent pour la prevention et le traitement des maladies du foie contenant un derive de la pyrazolopyrimidine
EP1888074A1 (fr) * 2005-06-10 2008-02-20 Dong-A Pharmaceutical Co., Ltd. Agent pour la prevention et le traitement des maladies du foie contenant un derive de la pyrazolopyrimidine
US8796286B2 (en) 2005-06-10 2014-08-05 Mezzion Pharma Co., Ltd. Agent for treatment of liver diseases containing pyrazolopyrimidinone derivative
WO2009052630A1 (fr) * 2007-10-26 2009-04-30 Pacific Therapeutics Ltd. Compositions et procédés permettant de traiter des troubles fibroprolifératifs
US9029385B2 (en) 2007-10-26 2015-05-12 Ganesh Raghu Compositions and methods for treating fibroproliferative disorders
US10293033B2 (en) 2011-07-20 2019-05-21 Mediwound Ltd. Proteolytic extract from bromelain for the treatment of connective tissue disorders
US9511126B2 (en) 2011-07-20 2016-12-06 Mediwound Ltd. Proteolytic extract from bromelain for the treatment of connective tissue disorders
WO2013011514A1 (fr) 2011-07-20 2013-01-24 Mediwound Ltd. Extrait protéolytique tiré de la bromélaïne et convenant au traitement de troubles des tissus conjonctifs
US10238669B2 (en) 2012-12-21 2019-03-26 Mayo Foundation For Medical Education And Research Methods and materials for treating calcific aortic valve stenosis
US9789126B2 (en) 2012-12-21 2017-10-17 Mayo Foundation For Medical Education And Research Methods and materials for treating calcific aortic valve stenosis
WO2014100733A1 (fr) 2012-12-21 2014-06-26 Mayo Foundation For Medical Education And Research Procédés et matériaux pour le traitement de la sténose valvulaire aortique calcifiée
EP3470070A1 (fr) 2012-12-21 2019-04-17 Mayo Foundation for Medical Education and Research Procédés et matériaux pour le traitement de la sténose valvulaire aortique calcifiée
US10568895B2 (en) 2012-12-21 2020-02-25 Mayo Foundation For Medical Education And Research Methods and materials for treating calcific aortic valve stenosis
US11197871B2 (en) 2012-12-21 2021-12-14 Mayo Foundation For Medical Education And Research Methods and materials for treating calcific aortic valve stenosis
WO2016111885A1 (fr) * 2015-01-06 2016-07-14 Imprimis Pharmaceuticals, Inc. Formulations pharmaceutiques à base de xanthine ou de dérivés de xanthine
WO2018167689A1 (fr) 2017-03-14 2018-09-20 Atir Holding S.A. Formulation topique destinée au traitement de problèmes de pigmentation de la peau
WO2018167687A1 (fr) 2017-03-14 2018-09-20 Atir Holding S.A. Utilisation de composés hétérocycliques dans le traitement de la peau pigmentée
US11033549B2 (en) 2017-03-14 2021-06-15 Atir Holding S.A. Use of heterocyclic compounds in the treatment of pigmented skin
EP4166129A1 (fr) 2017-03-14 2023-04-19 Atir Holding S.A. Utilisation de composes heterocycliques dans le traitement de la peau agee
WO2021093376A1 (fr) * 2019-11-11 2021-05-20 广州华真医药科技有限公司 Utilisation d'un inhibiteur de phosphodiestérase 5 dans la préparation d'un médicament permettant de résister à des maladies fibrotiques

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