Methods for the prevention or treatment of erectile dysfunction (ED) by administering slpis (secretory leukocyte protease inhibitors)

Abstract

The present invention provides methods for the prevention or treatment of Erectile dysfunction (ED) by administering SLPIs to animals, such as male humans. These methods can be administer together with vitamin D and / or phosphodiesterase 5 inhibitors.

Description

[0001]Methods for the prevention or treatment of Erectile Dysfunction (ED) by administering SLPIs (Secretory Leukocyte Protease Inhibitors)Technical field The present invention provides methods for the prevention or treatment of ErectileDysfunction (ED) by administering to animals, such as male humans, of SLPIs, or nucleotidesequences that can be transcribed into SLPI in the human or animal bodies. These methodscan be administered together with phosphodiesterase 5 inhibitors.Background art Erectile dysfunction (ED) is characterized by the inability to achieve or maintain an erection ofthe penis during sexual activity. It is a highly prevalent condition, affecting 30% of European(40-79 years) and 52% of US (40-70 years) males. Moreover, it adversely impacts the quality of life and is considered a sentinel marker for poor general health. In fact, ED has independent predictive value for future myocardial infarction and stroke. Obesity, metabolic diseases, and diabetes are well-recognized risk factors for sexual dysfunction. Impaired bioactivity of NO released by nerve and endothelial cells in the corpora cavernosa (CC) of the penis is a major pathogenic mechanism in ED. Contemporary treatment algorithms for ED involve the use of phosphodiesterase 5 inhibitors (PDE5i) as first choice agents to potentiate the NO / cyclic GMP pathway. It is estimated that 30% of patients with ED are non-responders to PDE5i and intracavernosal injection of prostaglandins or other vasoactive agents remains as an alternative therapy. Vitamin D, mainly synthesized in skin after UV exposure, has physiological functions beyond calcium and phosphorus homeostasis, including the regulation of cellular growth, intracellular metabolism, or innate and adaptive immunity. Vitamin D status is an important health issue since more than the half of the world population shows vitamin D deficiency (usually defined as 25-hydroxyvitamin D plasma levels below 20ng / mL). Epidemiological studies have reported a higher prevalence of vitamin D deficiency in ED patients, an association between the severity of ED with 25-hydroxyvitamin D plasma levels and an improved response to PDE5i after vitamin D replacement. However, the cause-effect relationship is unclear, and the potential mechanisms are unknown. Increased superoxide, i.e. oxidative stress, leading to NO / cyclic GMP is the most recognized mechanism involved in vascular ED. Herein, we show for the first time that isolated human CCfrom organ donors with vitamin D deficiency exhibit impaired erectile function and that this isreproduced ex vivo and in vivo in rats with vitamin D deficiency, and in vitamin D receptorknockout mice. Our data indicates that this is due to increased radical superoxide via downregulation of the anti-inflammatory and antioxidant protein SLPI. We found that reduced SLPI is associated with human vascular ED. We show that, SLPI gene downregulation leads to impaired erectile function. Moreover, under conditions of increased superoxide, even in the presence of normal vitamin D, administration of SLPI can restore the impaired erectile function. Altogether, the data shows that SLPI plays a key role in the maintenance of penile oxidative status and normal erectile function and impaired SLPI leads to ED. Exogenous SLPI can restore erectile function. SLPI, by itself or in combination with other therapies, including PDE5 inhibitors, may be an effective treatment for ED. Brief description of the figures Fig.1.25-hydroxyvitamin D plasma levels correlate with human corpus cavernosum (CC) and human penile resistance artery (PRA) function ex vivo. 25-hydroxyvitamin D plasma levels were measured in donors and CC were divided into two groups, those from patients with 25- hydroxyvitamin D above or those below the median value (14.2 ng / mL). A) Original recordings of human CC stimulated by EFS from a patient with normal (left) or low (right) 25- hydroxyvitamin D levels. B) Relaxant responses of human CC induced by EFS in the two groups. C) Relaxant responses of human PRA induced by ACh in the two groups. Results are means ± standard error of the mean (sem) and n is indicated in parenthesis. *, ** P<0.05 and P<0.01,respectively, vs low 25-hydroxyvitamin D by two-way (deficit x frequency or deficit xconcentration) ANOVA test followed by a Sidak's multiple comparisons test. Fig. 2. Vitamin D deficiency induces ED in rats in vivo and ex vivo. A) Original traces of the mean arterial pressure (MAP) and intracavernosal pressure (ICP) recordings after electrical stimulation of the cavernous nerve in anaesthetized rats at 10Hz of frequency. B) Averaged increases in ICP normalized with MAP. C and D) Effects of vitamin D deficiency on the relaxant responses of CC induced by C) EFS including original traces and D) sildenafil. E) Representative images of cross-sections of penises stained with Masson trichrome. Extracellular collagen deposition was quantified as the percentage of blue over the total area, 4-7 images per animal were used. Results are means ± sem and n is indicated in parenthesis. F) VDR localization by immunofluorescence labelling and confocal imaging in penis rat slices. VDR expression is shown in green, α-actin in red and nuclei are shown in blue (DAPI). DPA = Dorsal penile artery; DPV = Dorsal penile vein. Arrows indicate colocalization of VDR and α-actin. *,** P<0.05 and P<0.01, respectively, vs control using t-student test (for panel E) or otherwise using two-way (deficit x frequency or deficit x concentration) ANOVA test followed by a Sidak's multiple comparisons test. Fig.3. Vitamin D receptor knockout induces ED in mice ex vivo and in vivo. A) Original traces of intracavernosal pressure recording after electrical stimulation of the cavernous nerve inanaesthetized WT and Vdr- / - mice with increasing frequencies. B) Averaged increases inpressure. C, D, E) Effects of Vdr deletion on the relaxant responses of CC induced by C) EFS, D)sildenafil, E) Riociguat. A full frequency- or concentration-response curve was performed forEFS and riociguat but only two concentrations of sildenafil were tested because the responses were slower to this drug. Results are means ± sem and n is indicated in parenthesis. *,** indicates P<0.05 and P<0.01, respectively, vs WT using two-way (deficit x frequency or deficit x concentration) ANOVA test followed by a Sidak’s multiple comparisons test.Fig. 4. Vitamin D deficiency- and Vdr deletion- induced erectile dysfunction is mediated byincreased superoxide. A) Dihydroethidium (DHE) staining in the CC of a donor with high (above) and low (below) 25-hydroxyvitamin D. B) Correlation between DHE intensity normalised by DAPI (DHE / DAPI) and the maximal effect of electrical field stimulation (EFS Max) (left) and between 25-hydroxyvitamin D plasma levels and DHE / DAPI (right) in CC of human donors. C) Levels of superoxide (O2•-) measured by lucigenin chemiluminescence in rat CC strips in the absence (basal) or in the presence of NAPDH or NADPH plus TEMPOL. D) Rat penis slices showing the blue fluorescence of the nuclear stain DAPI (left), the red fluorescence produced when DHE is oxidized to ethidium by superoxide (middle), and the merged images (right). E) Values of DHE / DAPI.4-10 images were taken from each animal. F, G) Effects of the relaxant responses of CC induced by EFS in presence or absence of the antioxidant PegSOD from F) vitamin D deficient rats, G) Vdr knockout mice. The results from figures 2C and 3C are superimposed in light grey and discontinuous lines just for reference. Results are means ± sem and n is indicated in parenthesis. ** indicates P<0.01, ***P<0.001 vs basal or control; ƴP<0.05, ƴƴƴP<0.001 vs NADPH, δδδP<0.01 vs control NADPH using one way ANOVA test followed by a Bonferroni post hoc test or student’s t test in panel C. Fig. 5. Transcriptomic changes in CC from vitamin D deficient rats. A) Principal component analysis (PCA), B) Heat-map of significant down and up-regulated genes. FC=Fold change, C) Slpi mRNA expression in rats CC by qRT-PCR. D) Averaged densitometric protein expression of SLPI in homogenized penis analysed by Western blot and normalized to vinculin expression. E) SLPI localization by immunofluorescence labelling and confocal imaging in penis rat slice.SLPI expression is shown in green, α-actin in red and nuclei are shown in blue (DAPI). F) SLPIlevels in plasma from human donors with 25-hydroxyvitamin D levels below and above the median of the cohort (14.2). G) Maximal response to EFS in human donor CC with SLPI levels below and above the median of the cohort (67.6). Results are means ± sem. * P<0.05, ** P<0.01 vs control by t-student test. Fig.6. Recombinant SLPI rescues superoxide-induced ED and Slpi gene knockdown induces EDin a superoxide-dependent manner. (A-C) Effects of the superoxide generator pyrogallol onthe relaxant CC responses induced by the NO donor DEA-NO in CC from healthy animals incubated in the presence or absence of recombinant SLPI (0.166µg / µL, for 24h) and in the presence or absence of pyrogallol (0.1mM), and in the absence (A) or presence (B) of pegSOD (30U / mL). Panel C shows the areas under the curve (AUC) calculated from the data in panels A and B. (D-F) Effects of Slpi knockdown using Slpi siRNA or scramble siRNA in the absence (D) or presence (E) of pegSOD. The RT-PCR verification of Slpi silencing is shown as inset in panel D. Panel F shows the AUC calculated from the data in panels D and E. Results are means ± sem and n is indicated in parenthesis. * P<0.05, ** P<0.01, ***P<0.001 vs control or scramble, # P<0.05, ## P<0.01, ### P<0.001 vs control or scramble using student t test (for mRNA) or two-way (treatment x concentration) ANOVA test followed by a Sidak's multiple comparisons test. AUCs were compared by two or three-way ANOVA followed by Bonferroni test.Fig. 7. Vitamin D levels does not correlate with testosterone levels in human plasma donors.Pearson correlation coefficient was calculated.Fig. 8. Vitamin D deficiency induces ex vivo erectile dysfunction in male Sprague Dawley rats.Effect of vitamin D deficit on the relaxant responses in corpora cavernosa induced by electrical field stimulation (EFS, A), acetylcholine (ACh, B), sildenafil (C), and riociguat (D). Results are means ± standard error of the mean. * indicates P<0.05, **P<0.01 vs Control using two-way (deficit x frequency or deficit x concentration) ANOVA test followed by a a Sidak's multiple comparisons test. The number of experiments from different rats is indicated in parenthesis. Fig.9. Potentiation by sildenafil and inhibition by L-nitro-Arg of EFS-induced relaxation of CC. Effect of sildenafil (0.3 µM) and L-nitro-Arg (100 µM) on the curve of relaxation induced by EFS in CC from control (A) and vitamin D deficit (B) Sprague Dawley rats. Results are means ± standard error of the mean. * indicates P<0.05, **P<0.01 vs Control using two-way (treatment x frequency) ANOVA test followed by a Sidak's multiple comparisons test. The number of experiments from different rats is indicated in parenthesis. Fig. 10. Vitamin D deficiency induces endothelial dysfunction in penile dorsal arteries from male Sprague Dawley rats. Effects of vitD deficiency on relaxant responses in penile dorsal arteries induced by A) acetylcholine (ACh) and B) sildenafil. Results are means ± standard error of the mean. * indicates P<0.05 using two-way (deficit x concentration) ANOVA test followed by a Bonferroni post hoc test. The number of experiments from different rats is indicated in parenthesis. Fig.11. Vitamin D levels does not correlate with testosterone levels in male Wistar rats. A,B) Effect of a 5-months vitD-free diet in A) 25(OH)VitD plasma levels and B) testosterone plasma levels. C) Pearson correlation was performed between vitD and testosterone plasma level. Results are means ± standard error of the mean. ** indicates P<0.01 using two-way (deficit x concentration) ANOVA test followed by a Bonferroni post hoc test. n=6-7 animals. Fig. 12. A) Negative control for VDR immunohistochemistry in the absence of primary antibody anti-VDR, α-actin in red and nuclei are shown in blue (DAPI). B) Negative control for SLPI immunohistochemistry in the absence of both anti-VDR and anti-α-actin. Nuclei are shown in blue (DAPI). Summary of the inventionThe present invention refers to a pharmaceutical composition for use in a method of treatingerectile dysfunction in a male human subject in need thereof, the composition comprising asan active ingredient a human secretory leukocyte protease inhibitor, or a polynucleotidecoding for the same, wherein the pharmaceutical composition is administered, in a manner which can affect thecorpora cavernosa of the penis of the male human subject in need thereof.In a preferred embodiment, the method is for treating erectile dysfunction wherein thedamage results from increased superoxide and downregulation of SLPI. In another preferred embodiment, the erectile dysfunction is vascular erectile dysfunction. In another preferred embodiment, the composition is administered together, subsequently or simultaneously, with vitamin D, or a salt thereof. In another preferred embodiment, the active agent is the recombinant human secretory leukocyte protease inhibitor. In another preferred embodiment, the active ingredient is: a. the human secretory leukocyte protease inhibitor of any one of SEQ ID NO 1 to3 or any other sequence comprising variations in the amino acid sequences of any one of SEQ ID NO 1 to SEQ ID NO 3, provided that the variations in theamino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% or more sequence identity and the molecule retains bioactivity; and / orb. a polynucleotide sequence that may be transduced and / or transcribed in thehuman or animal body leading to the expression of any one of SEQ ID NO 1 to 3 or any other sequence comprising variations in the amino acid sequences of anyone of SEQ ID NO 1 to SEQ ID NO 3, provided that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% or more sequence identity and the molecule retains bioactivity. In another preferred embodiment, the pharmaceutical composition is administered prior to, or simultaneously to the administration of a selective phosphodiesterase 5 inhibitor (PDE5i)to the subject in need thereof. Preferably, the PDE5i is selected from the group consisting ofavanafil, lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, zaprinast, icariin and synthetic derivatives thereof, and benzamidenafil. Description of embodiments Here, we report for the first time that vitamin D levels correlate with EFS-induced relaxation in isolated CC and with endothelial function in isolated resistance penile arteries from humandonors. This invention also demonstrates, in rats and mice, a causal relationship betweenexposure to vitamin D-free diet or Vdr (vitamin D receptor) knockout and ED both ex vivo andin vivo. Moreover, we suggest that increased superoxide is involved in vitamin D deficiency-mediated ED and that downregulation of SLPI is a novel underlying mechanism to induce ED.Vitamin D deficiency may potentially impact most aetiologies of ED. In this regard, low levels of 25-hydroxyvitamin D are linked to depressive disorders, a possible source of psychogenic ED. Optimal testosterone levels, essential for correct erectile function, have also been correlated with adequate 25-hydroxyvitamin D levels. Our results indicate that the cause of the failure in penile erection may be intrinsic to the CC and that neither human nor rat testosterone levels are influenced by 25-hydroxyvitamin D levels. Consequently, vitamin D deficiency is associated with ED of vascular origin, although psychogenic and hormonal aetiologies cannot be completely ruled out. In rats exposed to the standard diet or to vitamin D-free, 25-hydroxyvitamin D levels are comparable to those found in the two categories of human subjects studied, above and below the median level of 25-hydroxyvitamin D levels, respectively. Therefore, rats exposed to vitamin D-free diet is a suitable model to study ED. In addition, when the active form of vitamin D, calcitriol, binds VDR, it acts as a transcription factor and interacts with the VDRE in the promoter region of target genes regulating theirexpression. In the present invention, we observed ubiquitous VDR expression in the penis,and, in particular, in the smooth muscle cells of the vessels and the CC. Interestingly, thepresent results indicate that the absence of VDR causes erectile dysfunction in vivo and exvivo. Furthermore, impairment of the NO pathway is recognized as a major pathogenetic factor inanimal models and patients with ED. The relaxation induced by EFS in both experimentalgroups is NO-dependent since the NO synthesis inhibitor L-NAME suppressed it. Likewise, sildenafil, that inhibits the degradation of cyclic GMP, the downstream signal of NO, potentiates EFS in both groups. Moreover, vitamin D deficiency significantly reduces theresponses to Ach (acetylcholine) and sildenafil in both the CC and the penile dorsal artery.Accordingly, the relaxation of the sGC stimulator riociguat in CC, which does not require endogenous NO, was unaffected by vitamin D deficiency or Vdr ablation. This is consistent with a reduced bioavailability of NO. The finding that vitamin D deficiency induces a reduced response to PDE5i in both the CC and the penile arteries may have additional clinical implications. For unclear reasons, manypatients with ED are non-responders to PDE5i. The present results suggest that vitamin Ddeficiency, may contribute to the lack of response to this first-line treatment of ED. Likewise, addition of vitamin D to the PDE5i tadalafil increased the erectile function in patients with ED. Similarly, vitamin D deficiency reduces the effect of sildenafil in rat pulmonary arteries and is a good predictor of poor therapeutic response to PDEi in patients with pulmonary arterial hypertension. Oxidative stress is an important factor in the development of ED and NADPH oxidase has been reported to be the main generator of superoxide in the CC in vascular ED. Accordingly, thepresent results of increased NADPH-stimulated lucigenin luminescence and dihydroethidiumfluorescence indicate an increase in superoxide penile production after chronic vitamin D deficiency in both rats and humans. The functional experiments with pegSOD confirm thecausative role of superoxide in vitamin D deficiency- or Vdr knockout-induced ED.Finally, we wondered which potential pathways are involved in the redox imbalance causedby vitamin D deficiency in the penis. A large number of genes are potentially regulated by vitamin D and these are tissue specific. We performed a transcriptomic analysis in CC from control and vitamin D deficiency animals. We found 46 downregulated and 17 upregulated genes in vitamin D deficient rat CC. Notably, there was an upregulation of actin-binding rho activating protein (Abra) gene that has been related with smooth muscle cell proliferation and aortic endothelial dysfunction, and whose expression has been reported to be controlled by vitamin D in the rat aorta. Myoglobin gene (Mb) expression was also upregulated. Free myoglobin and haemoglobin are well known quenchers of NO and thus, a possible cause for ED. Nevertheless, we focused on the downregulation of the Slpi gene for further study due to the following reasons. First, SLPI reduces reactive oxygen species production and increases superoxide dismutase-2 expression (SOD-2). SLPI expression is positively regulated by the antioxidant system Nrf2, which, in turn, is a target of VDR and can restore erectile function in rats and humans. Second, SLPI also has multiple roles in inflammation, the resolution ofinflammation, atherosclerosis and fibrosis. Interestingly, SLPI is downregulated specifically inthe smooth muscle cells of CC from diabetic patients with ED. Therefore, we first find that SLPI is expressed specifically in carvernosal smooth muscle cells by immunofluorescence and confirm that it is downregulated in vitamin D deficient CC by both RT-PCT and Western blot. We demonstrate the functional role of SLPI by 1) the protective effect of recombinant SLPI on exogenous superoxide-induced ED and 2) the superoxide-dependent deleterious effect of Slpisilencing on erectile function.In conclusion, we demonstrate for the first time that vitamin D deficiency or Vdr knockout induced-ED is mediated by increased superoxide and downregulation of SLPI. We further suggest that vitamin D deficiency is an aetiological factor for vascular ED and for thetherapeutic failure of PDE5i. Moreover, our results show that restoring SLPI in patients withED improves their sexual performance and / or the efficacy of the treatments for ED.Therefore, a first aspect of the invention is directed to one or more compositions for use in amethod of treating erectile dysfunction in a male subject in need thereof, preferably a malehuman subject, wherein the one or more compositions comprised as an active ingredient atleast a human secretory leukocyte protease inhibitor (SLPI) alone or in combination withvitamin D or a salt thereof. It is noted that vitamin D or a salt thereof and the human secretory leukocyte proteaseinhibitor (SLPI) might be formulated in a single composition or in different compositions andcan, therefore, be administered to a male human subject in need thereof simultaneously,alternatively, or subsequently if both active ingredients are used and these are formulated indifferent compositions. Human SLPI is a 11.7 kDa protein found in parotid saliva, and in seminal plasma, cervical, nasal, and bronchial mucus. In human epithelial cells, SLPI is constitutively expressed, and its expression is increased by phorbol ester, TNF-α, and LPS at supraphysiologic concentrations, as well as by synergistic combinations of elastase and corticosteroids. SLPI is composed of two cysteine-rich domains with a protease inhibitory site situated at leucine 72 (human form) in the carboxy-terminal domain.As already indicated, we have discovered the protective effect of recombinant SLPI onexogenous superoxide-induced ED and the superoxide-dependent deleterious effect of Slpi silencing on erectile function. An SLPI used in the present methods can be a wildtype SLPI protein from mammals such as humans, rats, and mice, or its various homologs, allelic variants, and isoforms. The amino acid sequences of a human SLPI, a rat SLPI and a mouse SLPI are described in Wang et al., Nature 417:941-944 (2002). See also Grütter et al, for a full length human SLPI sequence and its X-raycrystal structure. For the present invention we have used a recombinant SLPI produced inE.Coli, which is a single, non-glycosylated polypeptide chain containing 128 amino acids (26- 132 a.a.) and having a molecular mass of 14 kDa. The SLPI is fused to a 21 amino acid His-Tag at N-terminus and purified by proprietary chromatographic techniques. The recombinant sequence is reproduced herein below as SEQ ID NO 2:SEQ ID NO 1 (SLPI): LVPRGSH MSGKSF KAGV CPPKKSAQCL RYKKPECQSD WQCPGKKRCCPDTCGIKCLD PVDTPNPTRR KPGKCPVTYG QCLMLNPPNF CEMDGQCKRD LKCCMGMCGK SCVSPVKA It is noted that SEQ ID NO 1 is usually fused to the following sequence: MGSSHHH HHH SSG;which is a 21 amino acid His-Tag fused at N-terminus for purification purposes. This gives raiseto SEQ ID NO 2:SEQ ID NO 2: MGSSHHH HHH SSG LVPRGSH MSGKSF KAGV CPPKKSAQCL RYKKPECQSDWQCPGKKRCC PDTCGIKCLD PVDTPNPTRR KPGKCPVTYG QCLMLNPPNF CEMDGQCKRD LKCCMGMCGK SCVSPVKAIt is further noted that the SLPI Homo sapiens sequence as indicated in NP_003055, isreproduced herein below as SEQ ID NO 3:mkssglfpfl vllalgtlap wavegsgksf kagvcppkks aqclrykkpe cqsdwqcpgk krccpdtcgi kcldpvdtpnptrrkpgkcp vtygqclmln ppnfcemdgq ckrdlkccmg mcgkscvspv kaSEQ ID NO 2 and SEQ ID NO 3 are closely similar as indicated below:All of SEQ ID NO 1 to 3 are included in the present invention as SLPIs.Also minor variations in the amino acid sequences of SLPI (such as any of SEQ ID NO 1 to SEQID NO 3) are considered to be part of the present invention, provided that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% or more sequence identity and the molecule retains bioactivity. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are an aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting molecule. Whether an amino acid change results in a functional peptide or protein can readily be determined by assaying the specific activity of the peptide or protein derivative using, e.g., the assays described in detail herein. However, the SLPIs of this invention need not show high degrees of homology or sequence identity to the wildtype sequences. In fact, rat, mouse and human SLPIs do not share a high degree of homology. Rat SLPI shares only about 80% and 60% amino acid sequence identity with its mouse and human counterparts, respectively. However, these SLPIs share striking structural similarities. Thus, when the SLPI proteins useable in this invention share a relatively low degree of sequence identity (e.g., 50%, 60%, or 70%) to known wildtype sequences, they preferably preserve amino acid residues at positions critical to the overall protein structureand function (e.g., neuro-stimulatory function, serine protease inhibition function and NF-κBinhibitory function). For example, the highly conserved cysteine and proline residues in rat,mouse and human SLPIs is known. Mulligan et al. (Am. J. of Path. 156:1033-39 (2000)) furthershows examples of SLPI variants useful in this invention. SLPIs useable in this invention also include fragments of a full-length SLPI that preserve the desired SLPI functions. For example, Masuda et al. (British J. of Pharma.115:883-888 (1995)) describes a SLPI protein containing the C-terminal domain of the full length SLPI and having significant levels of the full length molecule's serine protease and NF-κB inhibitory activities. SLPIs of this invention also include fusion proteins containing SLPI linked to a functional moiety. The functional moiety can be used to direct SLPI to the desired neuronal site, to enhance the function, including the in vivo half-life, of SLPI, or to facilitate production and purification of SLPI. For example, the moiety can be genetic, enzymatic or chemical or immunological markers such as epitope tags, myc, hemagglutinin (HA), GST, immunoglobulins, β-galactosidase, biotin trpE, protein A, β-lactamase, α-amylase, maltose binding protein,alcohol dehydrogenase, polyhistidine (for example, six histidine at the amino and / or carboxylterminus of the polypeptide), lacZ, green fluorescent protein (GFP), yeast a mating factor, GAL4 transcription activation or DNA binding domain, luciferase, and serum proteins such as ovalbumin, albumin and the constant domain (e.g., Fc) of IgG. See, e.g., Godowski et al., 1988, and Ausubel et al., supra. Immunoglobulin Fc regions are especially useful fusion partners for making secreted fusion proteins as immunoglobulin molecules are secreted at high levels fromthe mature plasma cell, and the Fc region appears to be well suited as a “surrogate mother,”accepting domains from other proteins and efficiently directing them through the endoplasmic reticulum and secretory pathway. Fusion proteins may also contain sites for specific enzymatic cleavage, such as a site that is recognized by enzymes such as Factor XIII, trypsin, pepsin, or any other enzyme known in the art. Fusion proteins will typically be made by either recombinant nucleic acid methods, as described above, chemically synthesized using techniques such as those described in Merrifield, 1963, herein incorporated by reference, or produced by chemical cross-linking. Tagged fusion proteins permit easy localization, screening and specific binding via the epitope or enzyme tag. Some tags allow the protein of interest to be displayed on the surface of a phagemid, such as M13, which is useful for panning agents that may bind to the desired protein targets. Another advantage of fusion proteins is that an epitope or enzyme tag can simplify purification. These fusion proteins may be purified, often in a single step, by affinity chromatography. For example, a His6 tagged protein can be purified on a Ni affinity column and a GST fusion protein can be purified on a glutathione affinity column. Similarly, a fusion protein comprising the Fc domain of IgG can be purified on a Protein A or Protein G column and a fusion protein comprising an epitope tag such as myc can be purified using an immunoaffinity column containing an anti-c-myc antibody. It is preferable that the epitope tag be separated from the protein encoded by the essential gene by an enzymatic cleavage site that can be cleaved after purification. A second advantage of fusion proteins is that the epitope tag can be used to bind the fusion protein to a plate or column through an affinity linkage for screening targets. The SLPI proteins of this invention can be derivatized, e.g., pegylated, acetylated, carboxylated, phosphorylated, glycosylated or ubiquitinated. In some embodiments, the derivative has been labeled with, e.g., radioactive isotopes such as 125I, 32P, 35S, and 3H. In other embodiments, the derivative has been labeled with fluorophores, chemiluminescent agents, enzymes, and antiligands that can serve as specific binding pair members for a labeled ligand. In some embodiments, the methods of this invention use peptide analogs and mimetics which mimic the three-dimensional structure of an SLPI protein in lieu of SLPI proteins. Such peptide mimetics can compete with SLPI for NF-κB and serine protease inhibitory functions. Peptide mimetics may be superior to naturally-occurring peptides for a variety of reasons, including greater chemical stability, enhanced bioactivity and pharmacological properties (half-life,absorption, potency, efficacy, etc.), the potential for altered specificity (e.g., a broad-spectrumof biological activities), reduced antigenicity, and economic considerations with regard to production. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a desired biochemical property or pharmacological activity), but have one or more peptide linkages optionally replaced by a linkage selected from the groupconsisting of: —CH2NH—, —CH2S—, —CH2—CH2—, —CH═CH— (cis and trans), —COCH2—, —CH(OH)CH2—, and BCH2SO—, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may also be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem.61:387 (1992)), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide. In one embodiment, mimetics of the invention are peptide-containing molecules that mimic elements of protein secondary structure by orienting chemical structural motifs to facilitate desired molecular interactions similar to the natural molecule (see, e.g., Johnson et al., (1993) Peptide Turn Mimetics, in Biotechnology and Pharmacy, Pezzuto et al., (editors) Chapman and Hall). In another embodiment, peptide analogs of the invention are non-peptide compounds with properties analogous to those of a template peptide, also referred to as “peptide mimetics” or “peptidomimetics” and may be developed with the aid of computerized molecular modeling, as described (see, e.g., Fauchere, (1986) Adv. Drug Res. 15, 29-69; Veber & Freidinger, (1985) Trends Neurosci.8, 392-396; Evans et al., (1987) J. Med. Chem.30, 1229- 1239, which are incorporated herein by reference). In some embodiments, the methods of this invention use agonists of SLPI proteins and positive regulators of the SLPI protein or gene (including those that can up-regulate SLPI transcription in a mammal) in lieu of SLPI proteins. In some embodiments, the method of this invention use nucleotide sequences that may be transduced and / or transcribed in the human or animal body leading to the expression of SLPI. In some embodiments, the active agent is the human secretory leukocyte protease inhibitor,preferably a recombinant human secretory leukocyte protease inhibitor comprising any ofSEQ ID NO 1 to 3 or any other sequence comprising variations in the amino acid sequences of anyone of SEQ ID NO 1 to SEQ ID NO 3, provided that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% or more sequence identity and the molecule retains bioactivity. In some embodiments, thepresent invention further includes nucleotide sequences that may be transduced and / ortranscribed in the human or animal body leading to the expression of any one of SEQ ID NO 1 to 3 or any other sequence comprising variations in the amino acid sequences of anyone of SEQ ID NO 1 to SEQ ID NO 3, provided that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% or more sequence identity and the molecule retains bioactivity. The present invention thus provides a method of treating or preventing erectile dysfunction,preferably vascular erectile dysfunction, in a male subject in need thereof, comprising the stepof administering, in any manner which can affect the corpora cavernosa, an SLPI moleculealone or in combination with a vitamin D or a salt thereof of this invention. In a preferredembodiment, the damage results from increased superoxide and downregulation of SLPI.It is further noted that SLPIs of this invention or the vitamin D, or a salt thereof, may beformulated into pharmaceutical compositions and administered in vivo at an effective dose totreat the particular clinical condition addressed. Administration of one or more of the pharmaceutical compositions according to this invention will be useful for treating orpreventing erectile dysfunction, preferably vascular erectile dysfunction, in a male subject inneed thereof.As already indicated, the compositions of this invention may be administered alone or incombination, also with one or more therapeutic agents. For example, the compositions of thisinvention may be administered together with but not limited to, e.g., anti-inflammatoryagents, anticoagulants, antithrombotics, or tissue plasminogen activators. Preferably, thecompositions of this invention may be administered alone or in combination together with a selective phosphodiesterase 5 inhibitor (PDE5i) to the male subject in need thereof. Preferably, the PDE5i is selected from the group consisting of avanafil, lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, zaprinast, icariin and synthetic derivatives thereof, and benzamidenafil. The male patient to be treated may be a human or a veterinary animal. Determination of a preferred pharmaceutical formulation and a therapeutically efficient dose regiment for agiven application is within the skill of the art taking into consideration, for example, thecondition and weight of the patient, the extent of desired treatment and the tolerance of the patient for the treatment.Administration of the SLPIs of this invention alone or in combination with a vitamin D,including isolated and purified forms, their salts or pharmaceutically acceptable derivatives thereof, may be accomplished using any of the conventionally accepted modes of administration of agents which are used to treat neuronal injuries or disorders. The pharmaceutical compositions of this invention may be in a variety of forms, which may be selected according to the preferred modes of administration. These include, for example, solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories, and injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application. Modes of administration may include oral, parenteral, subcutaneous, intravenous, intralesional or topical administration.The SLPIs molecules of this invention or the vitamin D, or a salt thereof, may, for example, beplaced into sterile, isotonic formulations with or without cofactors which stimulate uptake or stability. The formulation is preferably liquid, or may be lyophilized powder. For example, the SLPI molecules may be diluted with a formulation buffer comprising 5.0 mg / ml citric acid monohydrate, 2.7 mg / ml trisodium citrate, 41 mg / ml mannitol, 1 mg / ml glycine and 1 mg / ml polysorbate 20. This solution can be lyophilized, stored under refrigeration and reconstituted prior to administration with sterile Water-For-Injection (USP). The compositions also will preferably include conventional pharmaceutically acceptable carriers well known in the art (see for example Remington's Pharmaceutical Sciences, 16th Edition, 1980, Mac Publishing Company). Such pharmaceutically acceptable carriers may include other medicinal agents, carriers, genetic carriers, adjuvants, excipients, etc., such as human serum albumin or plasma preparations. The compositions are preferably in the form of a unit dose and will usually be administered one or more times a day. The pharmaceutical compositions of this invention may also be administered usingmicrospheres, liposomes, nanoparticles and nano- or micro-particulate delivery systems orsustained release formulations placed in, near, or otherwise in communication with affected tissues or the bloodstream. Suitable examples of sustained release carriers include semipermeable polymer matrices in the form of shaped articles such as suppositories or microcapsules. Implantable or microcapsular sustained release matrices include polylactides (U.S. Pat. No. 3,773,319; EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L- glutamate (Sidman et al., Biopolymers 22:547-56 (1985)); poly(2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981); Langer, Chem. Tech.12:98-105 (1982)). Liposomes containing SLPIs of the invention can be prepared by well-known methods (See, e.g. DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A.82:3688-92 (1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A. 77:4030-34 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545). Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol. The proportion of cholesterol is selected to control the optimal rate of SLPI molecule release. The SLPIs of this invention may also be attached to liposomes, which may optionally contain other agents to aid in targeting or administration of the compositions to the desired treatment site. Attachment may be accomplished by any known cross-linking agent such as heterobifunctional cross-linking agents that have been widely used to couple toxins or chemotherapeutic agents to antibodies for targeted delivery. Conjugation to liposomes can also be accomplished using the carbohydrate-directed cross-linking reagent 4-(4- maleimidophenyl) butyric acid hydrazide (MPBH) (Duzgunes et al., J. Cell. Biochem. Abst. Suppl.16E 77 (1992)). The SLPIs of the present invention may also be delivered by nanoparticle delivery. Numerousnanoparticle delivery methods are known in the art, including but not limited to nanocapsules.Further aspects of the present invention relate to:An in vitro method for identifying a subject at risk of suffering from erectile dysfunction,preferably vascular erectile dysfunction, the method comprising:a. measuring the expression level of 25-hydroxyvitamin D and / or SLPI levels in abiological sample obtained from the subject thereby obtaining an expressionprofile of said sample; and b. identifying the subject as a subject at risk of suffering from erectile dysfunction,preferably vascular erectile dysfunction by a predictive model which correlatesthe expression levels identified in step (i) with representative expression levelsfrom samples obtained from subjects previously identified as suffering from erectile dysfunction, preferably vascular erectile dysfunction, said predictive model having been generated by training a computer with a plurality of expression levels from previously identified subjects suffering from erectiledysfunction, preferably vascular erectile dysfunction, by machine learning onsaid plurality of expression levels so as to obtain representative expressionlevels associated with erectile dysfunction, preferably vascular erectiledysfunction. An in vitro method for identifying a subject at risk of suffering from erectile dysfunction, preferably vascular erectile dysfunction, the method comprising: 1. measuring the expression level of SLPI levels in a biological sample obtainedfrom the subject thereby obtaining an expression profile of said sample;2. comparing said level of expression with a reference value, wherein a deviationin the level of expression of said SLPI levels with respect to said reference value,is indicative that the subject is at risk of having erectile dysfunction; and3. selecting said patient as a candidate to receive therapy with SLPI, vitamin Dsupplements, a selective phosphodiesterase 5 inhibitor (PDE5i) or a combination of two or three of these treatments. An in vitro method for selecting a subject suspected of suffering from erectile dysfunction, preferably vascular erectile dysfunction, as a candidate to receive therapy to treat erectile dysfunction, preferably vascular erectile dysfunction, with a selective phosphodiesterase 5 inhibitor (PDE5i), the method comprising:1. measuring the expression level of 25-hydroxyvitamin D and / or SLPI levels in abiological sample obtained from the subject thereby obtaining an expressionprofile of said sample; and 2. identifying the subject as a candidate to receive therapy to treat erectiledysfunction, preferably vascular erectile dysfunction, with a selective phosphodiesterase 5 inhibitor (PDE5i) by a predictive model which correlates the expression levels identified in step (i) with representative expression levels fromsamples obtained from subjects previously identified as suffering from erectile dysfunction, preferably vascular erectile dysfunction, said predictive model having been generated by training a computer with a plurality of expression levels frompreviously identified subjects suffering from erectile dysfunction, preferably vascular erectile dysfunction, responsive to phosphodiesterase 5 inhibitor (PDE5i)or not responsive to phosphodiesterase 5 inhibitor (PDE5i) by machine learning on said plurality of expression levels so as to obtain representative expression levelsassociated candidates responsive to selective phosphodiesterase 5 inhibitor (PDE5i);wherein, preferably, the PDE5i is selected from the group consisting of avanafil, lodenafil,mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, zaprinast, icariin and synthetic derivatives thereof, and benzamidenafil. Finally, in some further embodiments, the present invention also encompasses apharmaceutical composition for use in a method of treating erectile dysfunction in a malesubject in need thereof, preferably a male human subject, wherein the composition comprises a selective phosphodiesterase 5 inhibitor (PDE5i), and wherein the composition is administered to a subject identified as responsive to selective phosphodiesterase 5 inhibitor(PDE5i) according to the above method. Again, the PDE5i can be preferably selected from thegroup consisting of avanafil, lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, zaprinast, icariin and synthetic derivatives thereof, and benzamidenafil. The following terms, unless otherwise indicated, shall be understood to have the following meanings: The term “percent sequence identity” or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1990, (herein incorporated by reference). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factorfor the scoring matrix) or using Gap with its default parameters as provided in GCG Version6.1, herein incorporated by reference. The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 50%, more preferably 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above. Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions. “Stringent hybridization conditions” and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization. In general, “stringent hybridization” is performed at about 25° C. below the thermal melting point (Tm) for the specific DNA hybrid under a particular set of conditions. “Stringent washing” is performed at temperatures about 5° C. lower than the Tm for the specific DNA hybrid under a particular set of conditions. The Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., supra, page 9.51, hereby incorporated by reference. For purposes herein, “high stringency conditions” are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65° C. will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing. The term “fusion protein” refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein. The term “non-peptide analog” refers to a compound with properties that are analogous to those of a reference polypeptide. A non-peptide compound may also be termed a “peptidemimetic” or a “peptidomimetic”. See, e.g., Jones, (1992) Amino Acid and Peptide Synthesis,Oxford University Press; Jung, (1997) Combinatorial Peptide and Nonpeptide Libraries: A Handbook John Wiley; Bodanszky et al., (1993) Peptide Chemistry—A Practical Textbook, Springer Verlag; “Synthetic Peptides: A Users Guide”, G. A. Grant, Ed, W. H. Freeman and Co., 1992; Evans et al. J. Med. Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and references sited in each of the above, which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to useful peptides of the invention may be used to produce an equivalent effect and are therefore envisioned to be part of the invention. A “polypeptide mutant” or “mutein” refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a native or wild type protein. A mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and / or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and / or truncations of the amino acid sequence at either or both the amino or carboxy termini. A mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein. For instance, a mutein may have an increased or decreased serine protease activity A mutein has at least 70% overall sequence homology to its wild-type counterpart. Even more preferred are muteins having 80%, 85% or 90% overall sequence homology to the wild-type protein. In an even more preferred embodiment, a mutein exhibits 95% sequence identity, even more preferably 97%, even more preferably 98% and even more preferably 99% overall sequence identity. Sequence homology may be measured by any common sequence analysis algorithm, such as Gap or Bestfit. Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs. As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ- carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, s-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right hand direction is the carboxy-terminal direction, in accordance with standard usage and convention. A protein has “homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences). In a preferred embodiment,a homologous protein is one that exhibits 60% sequence homology to the wild type protein,more preferred is 70% sequence homology. Even more preferred are homologous proteins that exhibit 80%, 85% or 90% sequence homology to the wild type protein. In a yet more preferred embodiment, a homologous protein exhibits 95%, 97%, 98% or 99% sequence identity. As used herein, homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted as implying similarity in function. When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson et al., 1994, herein incorporated by reference). The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T);2) Aspartic Acid (D), Glutamic Acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis.53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. A preferred algorithm when comparing an SLPI sequence to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn. The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1990, herein incorporated by reference). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference. As used herein the phrase “therapeutically effective amount” means an amount of a molecule of the invention, such that a subject shows a detectable improvement in erectile dysfunction,preferably in vascular erectile dysfunction, after being treated under the selectedadministration regime (e.g., the selected dosage levels and times of treatment). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting. Throughout this specification and paragraphs, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The following are examples which illustrate the compositions and methods of this invention.These examples should not be construed as limiting: the examples are included for thepurposes of illustration only. Examples Vitamin D deficiency is associated with ED in vitro in human donors The aetiologies of ED include vascular, hormonal, neurologic, and / or psychologicaldysfunctions which are often interlinked. We interrogated whether the relationship betweenED and vitamin D deficiency reported in epidemiological studies could be also observed ex vivo, i.e., in the absence of hormonal or psychological influences. Therefore, we analysed 25- hydroxyvitamin D levels in the plasma from organ donors and compared them with the relaxant responses induced by electrical field stimulation (EFS) in their isolated human CC. Donor characteristics are shown in Table below. Table 1. Characteristics of human subjects. Data from all subjects as well as segregated data from subjects with plasma vitamin D (vitD) levels below the median (<14.2 ng / ml) or above the median (>14.2 ng / ml) are presented. Numerical variables are expressed as mean±SEM and were compared by unpaired Mann-Whiney U-test while categorical variables were compared by Fisher’s exact test. EFS induces the release of NO from nitrergic nerves leading to arterial vasodilation andrelaxation of the trabecular smooth muscle cells of the CC, causing an increase inintracavernosal pressure and, hence, penile engorgement. Endothelium- and NO-dependentvasodilation of penile arteries is also an important contributor to penile erection. Thus, EFS- induced relaxation represents an ex vivo surrogate test for erectile function while acetylcholine-(ACh) induced vasodilation represents a standard test to analyse endothelial function. In agreement with the high prevalence of both ED and vitamin D deficiency, many of the donors show moderately or severely reduced 25-hydroxyvitamin D levels and / or reduced responses to EFS in CC (Fig. 1). Both parameters were normally distributed in the cohort. Interestingly, we found a significant direct correlation (r2=0.41, p=0.03) between both parameters, and, therefore, erectile function was markedly different in patients with 25- hydroxyvitamin D levels above vs. below the median value (14.2 ng / ml) of the cohort (Fig.1Aand 1B). A similar correlation (r2=0.62, p=0.04) was found between 25-hydroxyvitamin Dlevels and the endothelial response stimulated with ACh in human penile resistance artery(HPRA). Again, the median 25-hydroxyvitamin D levels clearly discriminated between those with high and low endothelial-dependent response (Fig.1C). However, testosterone in plasma did not correlate with 25-hydroxyvitamin D levels (Fig.7A). Thus, even when the sample size was small, the results strongly suggest that the association of vitamin D deficiency with poor erectile function remains in isolated human CC. Vitamin D deficiency induces in vivo and ex vivo ED and penile fibrosis To analyse the cause-effect relationship, we evaluated the erectile function in wild type Sprague rats exposed to a vitamin D-free diet for 5 months in comparison with a standard diet. These preliminary results indicate that a decrease in 25-hydroxyvitamin D levels results in a significant reduction in EFS-induced relaxation (Fig.8A). In addition to neuronal-derived NO, NO released from the endothelium of sinusoids and blood vessels may also be involved in the penile erection. Vitamin D deficiency also induces a significant decrease in the endothelium- and NO-dependent relaxation induced by ACh (Fig.8B). Moreover, the relaxation induced by the PDE5i sildenafil, which potentiates endogenous NO, is also reduced (Fig. 8C). However, the relaxations to the soluble guanylyl cyclase (sGC) stimulator riociguat are unchanged (Fig. 8D). The relaxation induced by EFS is almost entirely inhibited by the NO synthesis inhibitor L- Nitro-Arg and potentiated by sildenafil in both groups (Fig.9). In addition, the relaxation to ACh (Fig.10A) and sildenafil (Fig.10B) in the dorsal penile arteries is also reduced in animals with vitamin D deficiency. We designed a new set of experiments with Wistar rats exposed for 5 months to a vitamin D- free diet to analyse the erectile function in vivo and to gain insights into the mechanisms involved in ED. This intervention reduces significantly the 25-hydroxyvitamin D plasma levels but does not affect those of testosterone (Fig. 11). Notably, the increase in intracavernosal pressure induced by electrical stimulation of the cavernosal nerve in anaesthetized rats is markedly decreased by vitamin D deficiency (Fig.2A and 2B). In addition, CC strips isolated from vitamin D deficient rats also show reduced relaxation induced by EFS (Fig.2C) and by the PDE5i sildenafil (Fig.2D). Therefore, vitamin D deficiency induces erectile dysfunction ex vivo, confirming the pilot study, and in vivo, which was associated with reduced response to sildenafil, the first-choice therapeutic drug for ED.Penile cavernous fibrosis is considered an important factor leading to ED. Therefore, weanalysed the collagen deposition in paraffin penis sections by Masson trichrome staining, as a marker of fibrosis. We find that vitamin D deficiency significantly increases the blue stained area indicating excess deposition of collagen in the CC (Fig.2E). The active form of vitamin D, calcitriol, activates vitamin D receptor (VDR), a member of the nuclear receptor superfamily of transcription factors that regulate gene expression. To confirm that VDR is present in the rat penis, its expression is analysed in sections of penises from Wistar rats by immunohistochemistry. Fig. 2F shows a positive staining for VDR in cavernosal smooth muscle cells, the dorsal penile arteries and the dorsal penile vein where itcolocalizes with smooth muscle α-actin. Vdr knock-out mice exhibit erectile dysfunction ex vivo and in vivo Then, we analyse whether the ED induced by vitamin D deficiency could be also mimicked by genetic deletion of its receptor. In vivo and ex vivo erectile function was explored in Vdr knockout (Vdr- / -) and WT mice. As shown in the original recordings (Fig. 3A) and in the averaged data (Fig.3B), the increase in ICP in response to electrical stimulation is lower at allelectrical frequencies in Vdr- / - mice compared to WT, confirming the erectile dysfunction invivo. Furthermore, CC isolated from Vdr- / - mice show decreased relaxation induced by EFS(Fig.3C) and by the PDE5i sildenafil (Fig.3D), also corroborating the ED ex vivo. However, asin vitamin D deficient rats, there are no changes in the response to the sGC stimulator riociguatin Vdr- / - mice (Fig. 3E).Excess superoxide mediates vitamin D deficiency- and Vdr deletion-induced EDThe reduced responses to EFS and sildenafil in vitamin D deficient rats and Vdr- / - mice, whichare dependent of endogenous NO, and the unaffected response to riociguat, which mimics NO but does not require endogenous NO, gave us a clue of a possible mechanism: a reduced bioavailability of NO due to excess levels of superoxide anion. Thus, we measured superoxide production in human penile sections by DHE fluorescence and compare these data with the ex vivo function of the CC and the 25-hydroxyvitamin D levels in each donor. As expected, donor tissue sections with higher intensity of DHE fluorescence exhibit reduced maximal relaxant response to EFS (Fig. 4A), i.e., the higher the superoxide the worse the erectile function. Interestingly, DHE signal also correlates with the levels of 25-hydroxyvitamin D (Fig. 4B). To analyse the causal relationship between 25-hydroxyvitamin D and superoxide, we measured superoxide levels by both lucigenin luminescence and DHE staining in CC isolated from control and vitamin D deficient rats. In CC strips from vitamin D deficient rats, the basal lucigenin signal is not significantly different from the control strips (Fig.4C), but the NADPH- induced increase is significantly higher in CC from vitamin D deficient rats. In both experimental groups the antioxidant tempol prevents the NADPH-induced signal, indicating that it is specific for superoxide production. Similarly, the penises from vitamin D deficient rats show marked red DHE nuclear staining (Fig. 4D,E), again indicating high superoxide levels, compared to controls. To explore whether increased superoxide generation is involved in the ED, we analysed theresponse to EFS of CC strips from control and vitamin D deficient rats and from Vdr- / - and WTmice incubated with the pegylated superoxide dismutase (PegSOD). This antioxidant reverted the in vitro ED induced by both vitamin D deficiency in rats (Fig.4F) and by genetic ablation of the receptor in mice (Fig. 4G). Therefore, these data strongly suggest that vitamin Ddeficiency- and VDR ablation-induced ED is mediated by increased superoxide production.Transcriptomic analysis of rat CC with vitamin D deficiency VDR is a nuclear receptor that interacts with multiple VDR response elements (VDRE) present in the promoter region of its target genes, inducing or repressing their expression in a tissue specific manner. To search for potential target genes and proteins involved in the mechanism of vitamin D deficiency-induced ED, we compared the transcriptome of CC from control and vitamin D deficient Wistar rats analysed by RNA-seq. Despite no significant differences are observed in the principal component analysis (PCA) between the vitamin D deficit and controlCC transcriptome (Fig. 5A), several genes are significantly down- or up-regulated by vitamin Ddeficiency (Fig. 5B). We focused on Slpi because it encodes for the proteinantileukoproteinase, also called secretory leukocyte peptidase inhibitor (SLPI), an inhibitor of serine proteases with antioxidant effects. We confirm that SLPI is downregulated in the CC from the vitamin D deficient rats at the level of mRNA by RT-PCR (Fig. 5C) and protein by Western blot (Fig.5D). We also find that SLPI is expressed in the smooth muscle cells of the rat CC (Fig.5E) by IHC. To analyse whether altered SLPI could also be present in humans with vitamin D deficiency and / or reduced erectile function, we measured SLPI levels in plasma from the human donors. Remarkably, donors with reduced vitamin D also show reduced SLPI (Fig. 5F). Moreover, CC from donors with SLPI levels below the median of the cohort (67.6 ng / ml) show reduced relaxant response to EFS (Fig.5G). SLPI rescues superoxide-induced ED and Slpi gene knockdown induces ED in a superoxide- dependent manner To evaluate whether SLPI can modulate the erectile function and its relationship withsuperoxide, we incubated healthy rat CC strips with recombinant SLPI or vehicle for 24 h. Next, these CC were then exposed to vehicle or the superoxide generator pyrogallol in the presence or absence of PegSOD (Figs.6A and 6B and calculated AUC for data in both panels in Fig.6C). When CC are incubated for ≥ 24 h, the relaxant response to EFS is almost absent due to neural degeneration in culture and, therefore, in these experiments we only analysed the concentration-response curves to the NO donor DEA-NO. As expected, the superoxide generator pyrogallol reduces the response to DEA-NO due to the rapid reaction of NO with superoxide. Interestingly, the pre-incubation with SLPI does not modify the response to DEA-NO but partially reverts the pyrogallol-induced ED. Furthermore, PegSOD also prevents the in vitro EDinduced by pyrogallol and SLPI does not further increase DEA-NO relaxation in the presenceof PegSOD plus pyrogallol. Thus, exogenous SLPI mimics the effects of PegSOD. To test whether endogenous SLPI modulates erectile function, we silenced Slpi in CC strips from healthy rats using a specific siRNA and a scramble siRNA as a negative control. We achieved a >60% Slpi mRNA downregulation (Fig.6D, inset), i.e., levels similar to those found in vitamin D deficient rats. Slpi silencing results in a significant decrease in the relaxant response to DEA-NO (Fig. 6D) indicating that SLPI is necessary for a proper erectile function. Moreover, thesuperoxide scavenger PegSOD reverts the inhibitory effect of Slpi siRNA (Fig. 6E and 6F) indicating that superoxide is involved in this effect. MATERIALS AND METHODS Human tissuesCavernosal specimens and blood samples were obtained from 12 organ donors at the time oforgan collection for transplantation in the Hospital Universitario Doce de Octubre, Madrid, Spain. In addition to provide consent for organ transplantation, relatives provided informed consent for tissue procurement specifically for research. The project protocol was approved by the Ethics Committees of the Hospital Universitario Doce de Octubre, Madrid, Spain (Ethics Approval procedure 16 / 045) and the Hospital Universitario Ramón y Cajal, Madrid, Spain (Ethics Approval procedure 363-15). Subjects were only excluded when suffering any infectious disease. Tissues were maintained in sterilized M-400 solution (composition per 100 ml: mannitol, 4.19 g; KH2PO4, 0.205 g; K2HPO4·H2O, 0.97 g; KCl, 0.112 g; NaHCO3, 0.084 g; pH 7.4, at 4-6˚C). Time elapsed between harvesting of cavernosal specimens from organ donors to their functional evaluation ranged between 16 and 24 hours. Within this time range, tissues are totally viable and are adequate for functional evaluation42. Plasma was obtained from the 12 donors by centrifugation at 4ºC of blood collected in EDTA-containing tubes. Animal model Experimental procedures were approved by the Animal Welfare Body of Universidad Complutense and Consejo Superior de Investigaciones Científicas and the regional committee of Comunidad de Madrid (Ref. PROEX-016 / 019 and PROEX-396.1 / 21) in accordance with the guidelines on the ethical use of animals from the European Community Council Directive of September 22, 2010 (2010 / 63 / EU). All investigators understand the ethical principles. All animals were kept under standard conditions of temperature 22±1°C and 12:12 hour dark / light cycle with free access to food and water. A pilot study was performed in CC from wild-type male Sprague-Dawley (SD) rats with / without vitamin D-free diet from a previous protocol12. In addition, another batch of two-month old male Wistars rats was randomly allocated into two groups: rats fed with a standard diet (Teklad Global 18% Protein Rodent Diet, Envigo, n=20) or vitamin D-free (Teklad Custom Diet TD.120008, Envigo, n=20) diet during 5 months. Ex vivo incubation experiments were performed with CC obtained from control male Wistar rats (n = 14). Vdr knockout mice were originally generated by Dr. Marie Demay (Harvard Medical School, Boston, MA, USA)50 and kindly donated to us to breed our own colony. The Vdr knockout (Vdr- / -, n=14) and wild-type (WT, n=14) mice were obtained by crossing heterozygous mice (Vdr+ / -), genotyped as described (protocol 22517 in http: / / www.jax.org), and used at 4months of age. Vdr- / - were fed a g-irradiated diet (TD96348, Teklad, Madison, WI) containing2% calcium, 1.25% phosphorus, and 20% lactose to normalize the blood mineral ion levels51. Functional evaluation of CC and penile resistance arteries All functional experiments were performed in Krebs-Henseleit solution at 37° C, pH 7.4 and bubbled with 95% O2 and 5% CO2. Strips of human CC tissue from eleven patients were mounted in organ chambers and stretched to optimal isometric tension as described. Neurogenic relaxations to EFS (0.5 to 16 Hz) were obtained in CC strips contracted with U46619 (10-30 nM). Penile small helicine arterial rings (lumen diameter 300-500 µm, 1.7-2.0 mm long), were dissected from seven CC specimens and mounted on microvascular wire myographs for isometric tension recordings as described52. The arteries were contracted with 1-3 µM noradrenaline and the relaxation was evaluated by cumulative addition of acetylcholine (ACh). CC strips from rats and mice were suspended between two electrodes allocated in a wire myograph, stretched to 3 mN and incubated with guanethidine (10 µM) and atropine (1 µM) to block adrenergic neurotransmission and muscarinic receptors, respectively. The preparations were contracted with phenylephrine (0.3-1 µM). Relaxations were induced by EFS stimulation or by vasoactive agents. Rat dorsal penile arterial segments (2 mm long) were mounted in a wire myograph and stretched to give an equivalent transmural pressure of 100 mmHg. Arteries were contracted with phenylephrine (1–3 μM) and relaxations were induced by ACh or sildenafil. All drugs were incubated for 10 min except PEGSOD for 30 minutes and SLPI for 24 h as described below. Relaxations were expressed as a percentage of the reduction in vasoconstrictor-induced contraction. In some experiments, rat CC strips were incubated with / without 166 µg / mL Secretory Leukocyte Protein Inhibitor (SLPI, Cat #MBS144051, My BioSource, Vancouver, Canada) in Dulbecco’s modified Eagle’s medium supplemented with 1% antibiotic / antimycotic solution, pyruvate solution and non-essential aminoacids (Merck, Darmstadt, Germany) at 37ºC, 95% humidity and 5% CO2 for 24 h before mounting them in the myograph. Slpi mRNA silencing CC strips were transfected with specific siRNA against Slpi (Slpi Rat siRNA Oligo Duplex (Locus ID 84386), Origene, Rockville, USA) and control scramble non-targeting siRNA using LipofectamineTM RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. The complex of siRNA with a final concentration of 50 nM and lipofectamine were mixed with Opti-MEM Reduced Serum Media (Thermo Fisher Scientific) supplemented with 1% antibiotic / antimycotic solution and transfected into rat strips. Twenty-four hours after transfection, strips were mounted in the myograph or immediately frozen at -80ºC. Intracavernosal pressure recording Rats and mice were anaesthetized with ketamine (60 mg·kg-1) and diazepam (4 mg·kg-1). The right cavernous nerve was dissected and intracavernosal pressure (ICP) was recorded by insertion a 25-gauge needle into the right crus. Electrical stimulation was applied by a platinum bipolar hook electrode connected to a stimulator and frequency-response curves were performed by applying stimulation at increasing frequencies at 3 min intervals26,52. Left carotid artery was catheterized in rats for constant blood pressure measurement. Histology Tissue samples from the penis were either fixed in 4% paraformaldehyde in 0.1 M phosphate- buffered saline (PBS) and embedded in paraffin, or cryoprotected in 30% sucrose in PBS,immersed in OCT, snap frozen in liquid nitrogen, and stored at −80°C. Paraffin transversalpenis sections were stained with Mason trichrome techniques and examined by light microscopy. Quantification of blue staining was performed with Image-Pro plus 2D Image Analysis Software in the whole section. For immunostaining, OCT-embedded penis sections were permeabilized in PBS with 0.3% Triton X-100 at room temperature for 10 min following by 1h incubation with blocking buffer containing 10% normal goat serum (Life Technologies, 16210-072) and 0.1% Tween20 in PBS at room temperature. Then, slices were incubated with primary antibodies overnight at 4°C. The following duplex immune fluorescence staining were performed: 1) anti-SLPI (1:50, sc- 374575, mouse monoclonal, Santa Cruz, Dallas, USA) followed by Alexa488–conjugated goatanti-mouse (1:200, A21042, Thermo Fisher Scientific, Massachusetts, USA) and anti-α-smoothmuscle actin CY3 (1:200, C6198, mouse monoclonal, Merck, Darmstadt, Germany) and; 2) anti- VDR (1:50, sc-13133, mouse monoclonal, Santa Cruz, Dallas, USA) followed by Alexa594- conjugated anti-mouse (1:200, A11032, Thermo Fisher Scientific, Massachusetts, USA) and anti-α-smooth muscle actin Alexa488 (1:200, 53-9760-82, Thermo Fisher Scientific, Massachusetts, USA). Anti-α-smooth muscle actin was added after a series of PBS washes following the first staining. Nuclei were stained with DAPI (1.24653, Merck, Darmstadt, Germany) and slides were mounted with SlowFade Antifade mounting medium (S36937, Thermo Fisher Scientific, Massachusetts, USA). Images were captured Zeiss (LSM 710, Unidad de Citometría de Flujo y Microscopía de Fluorescencia, Universidad Complutense de Madrid) laser-scanning confocal microscopefrom each section. For Z stack images, 4-8 consecutive XY images were obtained on the Z axis by Zeiss confocal microscope (LSM 710). ImageJ software was used to analyse the collected images. Superoxide measurements by DHE and lucigenin chemiluminescence OCT-embedded penis sections from humans and rats of 6-8 μm were cut in a cryostat and incubated with or without 4-Hydroxy-TEMPO (4-TEMPOL; Merck, Darmstadt, Germany) 10mM for 30 minutes. Afterwards, slices were exposed to 3 µM (rats) or 4 µM (human) dihydroethidium (DHE, Merck, Darmstadt, Germany) for 30 minutes at 37°C42. Nuclei were stained with 1 µM DAPI for 5 minutes. All images were taken in a fluorescence microscope (Leica microsystems, Wetzlar, Alemania). DHE intensity was obtained through ImageJ software and normalized with DAPI intensity. CC strips were dissected and then transferred to microtiter plate wells containing 5 μM bis-N- methylacridinium nitrate (Lucigenin; Merck, Darmstadt, Germany), some strips were stimulated with NADPH (100 μM; Merck, Darmstadt, Germany) and 4-TEMPOL (1 mM) was used as negative control. Chemiluminescence was measured in a luminometer (BMG FluostarOptima). Baseline values were subtracted from the counting values under thedifferent experimental conditions and superoxide production was normalized to dry tissueweight. Measurement of 25-hydroxyvitamin D, testosterone and SLPI in plasma 25-hydroxyvitamin D, testosterone and SLPI levels were measured in rat citrated plasma and human EDTA plasma using a General 25-Hydroxyvitamin D3 (HVD3) ELISA Kit (Reddot Biotech Inc., Kelowna, Canada), Testosterone ELISA DE1559 (Demeditec, Kiel, Germany) and SLPI kit (DY1274-05, Bio-techne R&D systems, Minnesota, USA) following manufacturer’s instructions. RNA-seq Library preparation and sequencing were carried out in Fundación Parque Científico de Madrid. Briefly, Monarch Total RNA Miniprep Kit (New England BioLabs) was used for total RNA extraction following the manufacturer recommendations (including DNase treatment). Once extracted, 100 pg of total RNA from each sample were used as input for library preparation with NEBNext Single Cell / Low Input RNA Library Prep Kit for Illumina (New England BioLabs) following the manufacturer recommendations. The so-obtained libraries were validated and quantified in a 2100 Bioanalyzer (Agilent) and an equimolecular pool was made, purified using AMPure XP beads (Beckman Coulter) and titrated by quantitative PCR using the Kapa-SYBR FAST qPCR kit forLightCycler480 and a reference standard forquantification. The library pool was denatured and loaded on a NovaSeq6000 (Illumina)where clusters were formed and sequenced using a single read format of 1x100 nt. The number of pass-filter clusters obtained per sample was 16-22 million, with an average of 19.3 million. RNA extraction and quantitative RT-PCR Total RNA was extracted from CC strips at the Unidad de Genómica (Parque Científico de Madrid). RNA quantity and quality were assessed with a NanoDropTM 1000 Spectrophotometer (Thermo Scientific, Massachusetts, USA). One μg of RNA was reverse transcribed into cDNA using iScriptTM cDNA Synthesis Kit (Biorad, California, USA) following manufacturer’s instructions. Slpi gene expression was determined by quantitative real-time PCR (qRT-PCR) using TaqMan Gene Expression Master Mix (Thermo Fisher Scientific, Massachusetts, USA) with specific primers and probe from Thermo Fisher Scientific databases (Rn07312880_g1). Amplifications, detections, and analysis were performed in CFX384 Touch Real-Time PCR Detection System (Biorad, California, USA). The delta-delta Ct method was used to quantify relative changes in mRNA expression. Gene expression was normalized to the geometrical mean of Actb (Rn00667869_m1; Thermo Scientific, Massachusetts, USA) and B2m(Rn00560865_m1; Thermo Scientific, Massachusetts, USA) expression.Western Blot Penis tissues were homogenized with RIPA (Merck, Darmstadt, Germany) and supplemented with protease (Protease inhibitor cocktail tablets, Roche Diagnosis GmbH) and phosphatase inhibitor (PhosSTOP, Roche Diagnostics GmbH) cocktail in a glass potter homogenizer. Homogenates were run on a sodium dodecyl sulphate-polyacrylamide electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes, incubated with primary mouse antibodies against SLPI (1:500, sc-374575, Santa Cruz Biotechnology), and vinculin (1:1000, Sc-25336, Santa Cruz Biotechnology) overnight and then with the secondary peroxidase conjugated antibodies. Antibody binding was detected by an ECL system (Amersham Pharmacia Biotech, Amersham, UK). Blots were imaged using an Odissey Fc System (LiCOR, Biosciences) and were quantified by densitometry using Quantity One software. Samples were normalized through expression of vinculin. The relative abundance of the protein of interest was normalized to the mean of the controls. Statistics Analysis was performed using GraphPad Software v8 (GraphPad Software Inc., USA). All data were tested for normal distribution using the Shapiro-Wilk test and parametric or non- parametric statistics were used as appropriate. Data are presented either as scatter plots and means or as means ± SEM. Two-way ANOVA analysis and for multiple comparisons the Sidak method or the Bonferroni post hoc test were used. P values of less than 0.05 were considered statistically significant. Pearson correlations were applied when relevant.

Claims

CLAIMS 1. A pharmaceutical composition for use in a method of treating erectile dysfunction in amale human subject in need thereof, the composition comprising as an activeingredient a human secretory leukocyte protease inhibitor or a polynucleotide codingfor the same, wherein the pharmaceutical composition is administered, in a manner which can affect thecorpora cavernosa of the penis of the male human subject in need thereof.

2. The pharmaceutical composition for use according to claim 1, wherein the method isfor treating erectile dysfunction wherein the damage results from increasedsuperoxide and / or downregulation of SLPI.

3. The pharmaceutical composition for use according to any one of claims 1 or 2, whereinthe erectile dysfunction is vascular erectile dysfunction.

4. The pharmaceutical composition for use according to any one of claims 1 to 3, whereinthe composition is administered together, subsequently or simultaneously, with vitamin D, or a salt thereof.

5. The pharmaceutical composition for use according to any one of claims 1 to 4, whereinthe active agent is the recombinant human secretory leukocyte protease inhibitor.

6. The pharmaceutical composition for use according to any one of claims 1 to 4, whereinthe active ingredient is:a. the human secretory leukocyte protease inhibitor of any of SEQ ID NO 1 to 3 orany other sequence comprising variations in the amino acid sequences of anyone of SEQ ID NO 1 to SEQ ID NO 3, provided that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%,and most preferably 99% or more sequence identity and the molecule retains bioactivity; and / or b. a polynucleotide sequence that may be transduced and / or transcribed in thehuman or animal body leading to the expression of any one of SEQ ID NO 1 to 3 or any other sequence comprising variations in the amino acid sequences of anyone of SEQ ID NO 1 to SEQ ID NO 3, provided that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% or more sequence identity and the molecule retains bioactivity.

7. The pharmaceutical composition for use according to any one of claims 1 to 6, whereinthe composition is administered prior to, or simultaneously to the administration of a selective phosphodiesterase 5 inhibitor (PDE5i) to the subject in need thereof.

8. The pharmaceutical composition for use according to claim 7, wherein the PDE5i isselected from the group consisting of avanafil, lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, zaprinast, icariin and synthetic derivatives thereof, andbenzamidenafil.

Images