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WO2025191291A1 - Méthodes de prévention ou de traitement d'un dysfonctionnement érectile (de) par administration de slpi (inhibiteurs de protéase leucocytaire sécrétoire) - Google Patents

Méthodes de prévention ou de traitement d'un dysfonctionnement érectile (de) par administration de slpi (inhibiteurs de protéase leucocytaire sécrétoire)

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Publication number
WO2025191291A1
WO2025191291A1 PCT/IB2024/000094 IB2024000094W WO2025191291A1 WO 2025191291 A1 WO2025191291 A1 WO 2025191291A1 IB 2024000094 W IB2024000094 W IB 2024000094W WO 2025191291 A1 WO2025191291 A1 WO 2025191291A1
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WO
WIPO (PCT)
Prior art keywords
slpi
vitamin
erectile dysfunction
seq
pharmaceutical composition
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PCT/IB2024/000094
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English (en)
Inventor
Francisco Pérez Vizcaíno
Miguel Ángel OLIVENCIA PLAZA
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Universidad Complutense de Madrid
Centro de Investigacion Biomedica en Red CIBER
Original Assignee
Universidad Complutense de Madrid
Centro de Investigacion Biomedica en Red CIBER
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Priority to PCT/IB2024/000094 priority Critical patent/WO2025191291A1/fr
Publication of WO2025191291A1 publication Critical patent/WO2025191291A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • 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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/592—9,10-Secoergostane derivatives, e.g. ergocalciferol, i.e. vitamin D2
    • 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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence

Definitions

  • Erectile Dysfunction by administering S LPIs (Secretory Leukocyte Protease Inhibitors)
  • S LPIs Stecretory Leukocyte Protease Inhibitors
  • the present invention provides methods for the prevention or treatment of Erectile Dysfunction (ED) by administering to animals, such as male humans, of SLPIs, or nucleotide sequences that can be transcribed into SLPI in the human or animal bodies. These methods can be administered together with phosphodiesterase 5 inhibitors.
  • Background art Erectile dysfunction (ED) is characterized by the inability to achieve or maintain an erection of the 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.
  • 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.
  • PDE5i phosphodiesterase 5 inhibitors
  • 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.
  • 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.
  • 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).
  • Vitamin D deficiency induces ED in rats in vivo and ex vivo.
  • MAP mean arterial pressure
  • ICP intracavernosal pressure
  • 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.
  • Vitamin D receptor knockout induces ED in mice ex vivo and in vivo.
  • DHE Dihydroethidium staining in the CC of a donor with high (above) and low (below) 25-hydroxyvitamin D.
  • E SLPI localization by immunofluorescence labelling and confocal imaging in penis rat slice. SLPI expression is shown in green, ⁇ -actin in red and nuclei are shown in blue (DAPI).
  • F SLPI levels 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 ED in a superoxide-dependent manner.
  • A-C Effects of the superoxide generator pyrogallol on the relaxant CC responses induced by the NO donor DEA-NO in CC from healthy animals incubated in the presence or absence of recombinant SLPI (0.166 ⁇ g/ ⁇ L, 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.
  • 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.
  • the present invention refers to a pharmaceutical composition for use in a method of treating erectile dysfunction in a male human subject in need thereof, the composition comprising as an active ingredient a human secretory leukocyte protease inhibitor, or a polynucleotide coding for the same, wherein the pharmaceutical composition is administered, in a manner which can affect the corpora cavernosa of the penis of the male human subject in need thereof.
  • the method is for treating erectile dysfunction wherein the damage results from increased superoxide and downregulation of SLPI.
  • the erectile dysfunction is vascular erectile dysfunction.
  • the composition is administered together, subsequently or simultaneously, with vitamin D, or a salt thereof.
  • the active agent is the recombinant human secretory leukocyte protease inhibitor.
  • the active ingredient is: a . the human secretory leukocyte protease inhibitor of any one of SEQ ID NO 1 to 3 or any other sequence comprising variations in the amino acid sequences of a ny one 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.
  • 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.
  • PDE5i selective phosphodiesterase 5 inhibitor
  • the PDE5i is selected from the group consisting of avanafil, lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, zaprinast, icariin and synthetic derivatives thereof, and benzamidenafil.
  • avanafil lodenafil
  • mirodenafil sildenafil
  • tadalafil vardenafil
  • udenafil zaprinast
  • icariin synthetic derivatives thereof
  • benzamidenafil benzamidenafil.
  • Vitamin D deficiency may potentially impact most aetiologies of ED.
  • 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.
  • vitamin D deficiency is associated with ED of vascular origin, although psychogenic and hormonal aetiologies cannot be completely ruled out.
  • 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.
  • 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 their expression.
  • vitamin D deficiency significantly reduces the responses 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, many patients with ED are non-responders to PDE5i.
  • vitamin D deficiency may contribute to the lack of response to this first-line treatment of ED.
  • addition of vitamin D to the PDE5i tadalafil increased the erectile function in patients with ED.
  • 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.
  • 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.
  • Nrf2 antioxidant system
  • SLPI also has multiple roles in inflammation, the resolution of inflammation, atherosclerosis and fibrosis.
  • SLPI is downregulated specifically in the 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.
  • a first aspect of the invention is directed to one or more compositions for use in a method of treating erectile dysfunction in a male subject in need thereof, preferably a male human subject, wherein the one or more compositions comprised as an active ingredient at least a human secretory leukocyte protease inhibitor (SLPI) alone or in combination with vitamin D or a salt thereof.
  • SLPI human secretory leukocyte protease inhibitor
  • vitamin D or a salt thereof and the human secretory leukocyte protease inhibitor (SLPI) might be formulated in a single composition or in different compositions and can, 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 in different 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- ⁇ , 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.
  • 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-ray crystal structure.
  • SLPI recombinant SLPI produced in E.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.
  • SEQ ID NO 2 SEQ ID NO 1 (SLPI): LVPRGSH MSGKSF KAGV CPPKKSAQCL RYKKPECQSD WQCPGKKRCC PDTCGIKCLD 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.
  • SEQ ID NO 2 MGSSHHH HHH SSG LVPRGSH MSGKSF KAGV CPPKKSAQCL RYKKPECQSD WQCPGKKRCC PDTCGIKCLD PVDTPNPTRR KPGKCPVTYG QCLMLNPPNF CEMDGQCKRD LKCCMGMCGK SCVSPVKA
  • SEQ ID NO 3 mkssglfpfl vllalgtlap wavegsgksf kagvcppkks aqclrykkpe cqsdwqcpgk krccpdtcgi kcldpvdtpn ptrrkpgkcp vtygqclmln ppnfcemdgq ckrdlkccmg mcgkscvs
  • amino acid sequences of SLPI 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.
  • 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.
  • 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.
  • 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
  • phenylalanine, tryptophan, and tyrosine are an aromatic family.
  • SLPIs of this invention need not show high degrees of homology or sequence identity to the wildtype sequences.
  • 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.
  • these SLPIs share striking structural similarities.
  • 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 structure and function (e.g., neuro-stimulatory function, serine protease inhibition function and NF- ⁇ B inhibitory function).
  • amino acid residues at positions critical to the overall protein structure and function (e.g., neuro-stimulatory function, serine protease inhibition function and NF- ⁇ B inhibitory function).
  • 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)
  • SLPIs useable in this invention also include fragments of a full-length SLPI that preserve the desired SLPI functions.
  • 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- ⁇ B 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.
  • the moiety can be genetic, enzymatic or chemical or immunological markers such as epitope tags, myc, hemagglutinin (HA), GST, immunoglobulins, ⁇ -galactosidase, biotin trpE, protein A, ⁇ -lactamase, ⁇ -amylase, maltose binding protein, alcohol dehydrogenase, polyhistidine (for example, six histidine at the amino and/or carboxyl terminus 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.
  • markers such as epitope tags, myc, hemagglutinin (HA), GST, immunoglobulins, ⁇ -galactosidase, biotin trpE, protein A, ⁇ -lactamase,
  • Immunoglobulin Fc regions are especially useful fusion partners for making secreted fusion proteins as immunoglobulin molecules are secreted at high levels from the 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.
  • 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.
  • 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.
  • the derivative has been labeled with, e.g., radioactive isotopes such as 125I, 32P, 35S, and 3H.
  • the derivative has been labeled with fluorophores, chemiluminescent agents, enzymes, and antiligands that can serve as specific binding pair members for a labeled ligand.
  • 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- ⁇ B 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-spectrum of biological activities), reduced antigenicity, and economic considerations with regard to production.
  • 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 group consisting of: —CH2NH—, —CH2S—, —CH2—CH2—, —CH ⁇ CH— (cis and trans), —COCH2— , —CH(OH)CH2—, and BCH2SO—, by methods well known in the art.
  • a paradigm polypeptide i.e., a polypeptide that has a desired biochemical property or pharmacological activity
  • one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: —CH2NH—, —CH2S—, —CH2—CH2—, —CH ⁇ CH— (cis and trans), —COCH2— , —CH(OH)CH2—, and BCH2SO
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type may also be used to generate more stable peptides.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the active agent is the human secretory leukocyte protease inhibitor, preferably a recombinant human secretory leukocyte protease inhibitor comprising any 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 further includes nucleotide sequences that may be transduced and/or transcribed 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 step of administering, in any manner which can affect the corpora cavernosa, an SLPI molecule alone or in combination with a vitamin D or a salt thereof of this invention.
  • the damage results from increased superoxide and downregulation of SLPI.
  • SLPIs of this invention or the vitamin D, or a salt thereof may be formulated into pharmaceutical compositions and administered in vivo at an effective dose to treat the particular clinical condition addressed.
  • compositions of this invention may be administered alone or in combination, also with one or more therapeutic agents.
  • the compositions of this invention may be administered together with but not limited to, e.g., anti-inflammatory agents, anticoagulants, antithrombotics, or tissue plasminogen activators.
  • the compositions 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.
  • PDE5i selective phosphodiesterase 5 inhibitor
  • 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 a given application is within the skill of the art taking into consideration, for example, the condition and weight of the patient, the extent of desired treatment and the tolerance of the patient for the treatment.
  • SLPIs of this invention may be administered 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, be placed into sterile, isotonic formulations with or without cofactors which stimulate uptake or stability.
  • the formulation is preferably liquid, or may be lyophilized powder.
  • 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).
  • 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 using microspheres, liposomes, nanoparticles and nano- or micro-particulate delivery systems or sustained release formulations placed in, near, or otherwise in communication with affected tissues or the bloodstream.
  • 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.
  • 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)).
  • MPBH carbohydrate-directed cross-linking reagent 4-(4- maleimidophenyl) butyric acid hydrazide
  • the SLPIs of the present invention may also be delivered by nanoparticle delivery.
  • Numerous nanoparticle 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 a biological sample obtained from the subject thereby obtaining an expression profile of said sample; and b .
  • An in vitro method for identifying a subject at risk of suffering from erectile dysfunction, preferably vascular erectile dysfunction comprising: 1 . measuring the expression level of SLPI levels in a biological sample obtained from the subject thereby obtaining an expression profile of said sample; 2. comparing said level of expression with a reference value, wherein a deviation in 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; and 3. selecting said patient as a candidate to receive therapy with SLPI, vitamin D supplements, a selective phosphodiesterase 5 inhibitor (PDE5i) or a combination of two or three of these treatments.
  • PDE5i selective phosphodiesterase 5 inhibitor
  • PDE5i selective phosphodiesterase 5 inhibitor
  • the present invention also encompasses a pharmaceutical composition for use in a method of treating erectile dysfunction in a male subject 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.
  • PDE5i can be preferably selected from the group consisting of avanafil, lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, zaprinast, icariin and synthetic derivatives thereof, and benzamidenafil.
  • sequence identity 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.
  • 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).
  • percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.
  • 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.
  • 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.
  • “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.
  • “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.
  • 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.
  • a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.
  • 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 “peptide mimetic” 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.
  • polypeptide mutant 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.
  • muteins having 80%, 85% or 90% overall sequence homology to the wild-type protein.
  • 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.
  • the twenty conventional amino acids and their abbreviations follow conventional usage.
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ - carboxyglutamate, ⁇ -N,N,N-trimethyllysine, ⁇ -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).
  • 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.
  • a protein has homology to a second protein if the two proteins have “similar” amino acid sequences.
  • the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences).
  • 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.
  • a homologous protein exhibits 95%, 97%, 98% or 99% sequence identity.
  • homology between two regions of amino acid sequence is interpreted as implying similarity in function.
  • 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.
  • 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), and 6) 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.
  • GCG Genetics Computer Group
  • Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • 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.
  • database searching using amino acid sequences can be measured by algorithms other than blastp known in the art.
  • 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).
  • 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.
  • 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 selected administration regime (e.g., the selected dosage levels and times of treatment).
  • selected administration regime e.g., the selected dosage levels and times of treatment.
  • Vitamin D deficiency is associated with ED in vitro in human donors
  • the aetiologies of ED include vascular, hormonal, neurologic, and/or psychological dysfunctions which are often interlinked.
  • 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.
  • acetylcholine-(ACh) induced vasodilation represents a standard test to analyse endothelial function.
  • Vitamin D deficiency induces in vivo and ex vivo ED and penile fibrosis
  • a decrease in 25-hydroxyvitamin D levels results in a significant reduction in EFS-induced relaxation (Fig.8A).
  • 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).
  • the relaxation induced by the PDE5i sildenafil which potentiates endogenous NO, is also reduced (Fig. 8C).
  • 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).
  • the relaxation to ACh (Fig.10A) and sildenafil (Fig.10B) in the dorsal penile arteries is also reduced in animals with vitamin D deficiency.
  • 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, we analysed the collagen deposition in paraffin penis sections by Masson trichrome staining, as a marker of fibrosis.
  • 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.
  • VDR vitamin D receptor
  • VDR knock-out mice exhibit erectile dysfunction ex vivo and in vivo
  • ED induced by vitamin D deficiency could be also mimicked by genetic deletion of its receptor.
  • erectile function was explored in Vdr knockout (Vdr-/-) and WT mice. As shown in the original recordings (Fig.
  • DHE fluorescence exhibit reduced maximal relaxant response to EFS (Fig. 4A), i.e., the higher the superoxide the worse the erectile function.
  • DHE signal also correlates with the levels of 25-hydroxyvitamin D (Fig. 4B).
  • Fig. 4C 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.
  • SLPI secretory leukocyte peptidase inhibitor
  • 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
  • SLPI can modulate the erectile function and its relationship with superoxide, we incubated healthy rat CC strips with recombinant SLPI or vehicle for 24 h.
  • 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.
  • the superoxide scavenger PegSOD reverts the inhibitory effect of Slpi siRNA (Fig. 6E and 6F) indicating that superoxide is involved in this effect.
  • 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.
  • Vdr+/- heterozygous mice
  • genotyped as described (protocol 22517 in http://www.jax.org), and used at 4 months of age.
  • Vdr-/- were fed a g-irradiated diet (TD96348, Teklad, Madison, WI) containing 2% calcium, 1.25% phosphorus, and 20% lactose to normalize the blood mineral ion levels51.
  • TD96348, Teklad, Madison, WI g-irradiated diet
  • lactose 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.
  • 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 ⁇ M) and atropine (1 ⁇ M) to block adrenergic neurotransmission and muscarinic receptors, respectively.
  • the preparations were contracted with phenylephrine (0.3-1 ⁇ M). 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 ⁇ M) and relaxations were induced by ACh or sildenafil.
  • rat CC strips were incubated with/without 166 ⁇ g/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 Secretory Leukocyte Protein Inhibitor
  • 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.
  • ICP intracavernosal pressure
  • 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.
  • duplex immune fluorescence staining were performed: 1) anti-SLPI (1:50, sc- 374575, mouse monoclonal, Santa Cruz, Dallas, USA) followed by Alexa488–conjugated goat anti-mouse (1:200, A21042, Thermo Fisher Scientific, Massachusetts, USA) and anti- ⁇ -smooth muscle 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- ⁇ -smooth muscle actin Alexa488 (1:200, 53-9760-82, Thermo Fisher Scientific, Massachusetts, USA).
  • Anti- ⁇ -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 Citometr ⁇ a de Flujo y Microscop ⁇ a 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 ⁇ m 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 ⁇ M (rats) or 4 ⁇ M (human) dihydroethidium (DHE, Merck, Darmstadt, Germany) for 30 minutes at 37°C42. Nuclei were stained with 1 ⁇ M 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 ⁇ M bis-N- methylacridinium nitrate (Lucigenin; Merck, Darmstadt, Germany), some strips were stimulated with NADPH (100 ⁇ M; 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 the different experimental conditions and superoxide production was normalized to dry tissue weight.
  • RNA-seq Library preparation and sequencing were carried out in Funda Terms Parque Cient ⁇ fico de Madrid.
  • RNA Miniprep Kit 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 for quantification.
  • 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.
  • 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.

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Abstract

La présente invention concerne des méthodes pour la prévention ou le traitement d'un dysfonctionnement érectile (DE) par administration de SLPI à des animaux, tels que des êtres humains mâles. Ces méthodes peuvent être administrées conjointement avec de la vitamine D et/ou des inhibiteurs de phosphodiestérase.
PCT/IB2024/000094 2024-03-14 2024-03-14 Méthodes de prévention ou de traitement d'un dysfonctionnement érectile (de) par administration de slpi (inhibiteurs de protéase leucocytaire sécrétoire) Pending WO2025191291A1 (fr)

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