Haptoglobin for use in the treatment or prevention of hyper-erectile response or erectile dysfunction - Patent Application 20070122997

Abstract

The present invention relates to haptoglobin or a nucleic acid encoding haptoglobin for use in the treatment or prevention of an excessive erectile response and / or the prevention of permanent erectile dysfunction. Furthermore, the present invention relates to a pharmaceutical composition for use in the treatment or prevention of an excessive erectile response and / or the prevention of permanent erectile dysfunction, the pharmaceutical composition comprising an adeno-associated virus (AAV) vector carrying a transgene encoding a haptoglobin gene. The excessive erectile response may be, for example, priapism associated with sickle cell disease (SCD).

Description

[Technical Field]

[0001] The present invention relates to haptoglobin or a nucleic acid encoding haptoglobin for use in the treatment or prevention of an excessive erectile response and / or the prevention of permanent erectile dysfunction. Furthermore, the present invention relates to a pharmaceutical composition for use in the treatment or prevention of an excessive erectile response and / or the prevention of permanent erectile dysfunction, the pharmaceutical composition comprising an adeno-associated virus (AAV) vector carrying a transgene encoding a haptoglobin gene. The excessive erectile response may be, for example, priapism associated with sickle cell disease (SCD). [Background technology]

[0002] Sickle cell disease (SCD) is the most common single-gene disorder and is caused by a point mutation in the β-globin gene. This mutation promotes hemoglobin polymerization under conditions of reduced oxygen concentration, resulting in rigid, sickle-shaped red blood cells and hemolytic disease. SCD affects millions of people worldwide, approximately 100,000 Americans, and is most prevalent in sub-Saharan Africa (Non-Patent Document 1). Patients with SCD may exhibit exaggerated erectile responses, such as priapism.

[0003] Priapism is characterized by a prolonged, painful penile erection (Non-Patent Document 2). A full or partial erection may last for several hours or more, but it is not caused by sexual stimulation. Erection typically occurs in response to physical or psychological stimulation. This stimulation causes certain smooth muscles to relax, increasing blood flow to the corpus cavernosum of the penis. As a result, the blood-filled penis becomes erect. When stimulation ends, blood flows out, and the penis returns to its non-rigid (flaccid) state. Priapism occurs when parts of this system—blood, blood vessels, smooth muscle, and nerves—alter normal blood flow, resulting in a sustained erection. The underlying cause of priapism is often unidentifiable, but several conditions may be involved. The main types of priapism are ischemic and non-ischemic. Ischemic priapism is a medical emergency. Priapism is rare overall but is more common in certain groups, including those with sickle cell disease, blood disorders such as leukemia, and blood (hematological) disorders such as thalassemia and multiple myeloma. Prompt treatment for priapism is usually necessary to prevent tissue damage that can lead to the inability to achieve or maintain an erection (erectile dysfunction). Priapism is most common in men over the age of 30, but it can begin in childhood in men with sickle cell disease.

[0004] Recurrent priapism promotes fibrosis of erectile tissue, resulting in permanent erectile dysfunction (Non-Patent Document 3). Erectile dysfunction (impotence) is the inability to achieve or maintain an erection rigid enough for sexual intercourse. However, if erectile dysfunction is a persistent problem, it can cause stress, affect self-confidence, and contribute to relationship problems.

[0005] Clinical studies have shown that 30-45% of SCD patients develop ischemic priapism, and 30% of these patients progress to erectile dysfunction (Non-Patent Documents 4-6). Despite the high incidence of priapism, no major treatments can prevent it (Non-Patent Document 2).

[0006] In SCD, reduced NO bioavailability is associated with increased intravascular hemolysis and oxidative stress (Non-Patent Document 7). Increased production of reactive oxygen species in the penis is associated with increased levels of the gp91phox and p47phox subunits of NADPH oxidase, uncoupled eNOS, and xanthine oxidase (Non-Patent Documents 8-10). Previous studies have shown that intravascular hemolysis may promote endothelial dysfunction associated with decreased eNOS activity in SCD mice (Non-Patent Document 11). Reduced NO bioavailability and increased oxidative stress in the penis have been identified as one of the major causes of priapism in men and mice with SCD (Non-Patent Documents 8, 12-14). Experimental evidence suggests that reduced NO / cGMP bioavailability leads to a compensatory decrease in PDE5 activity and expression in the corpus cavernosum, thereby impairing the mechanisms controlling excessive penile erections (Non-Patent Documents 8, 12, 15).

[0007] Previous studies have investigated the relationship between increased oxidative stress and the pathophysiology of priapism (Non-Patent Documents 8, 13, 16, 17). Excess superoxide anion reacts with NO to form peroxynitrite, a potent oxidant (Non-Patent Document 18). Furthermore, in men and mice with SCD, the expression and activity of the RhoA-Rho-kinase (ROCK) pathway, which is involved in contraction of corpus cavernosum smooth muscle and maintaining penile flaccidity, is reduced (Non-Patent Documents 13, 19). Several previous studies have examined the relationship between priapism and the level of intravascular hemolysis in SCD men (Non-Patent Documents 20-22). Haptoglobin administration to SCD mice and its effects on blood vessels have also been previously studied (Non-Patent Documents 23-25, 26, 27). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Telen MJ, Malik P, Vercellotti GM. Therapeutic strategies for sickle cell disease:towards a multi-agent approach. Nat Rev Drug Discov. 2019 February;18(2):139~58. [Non-patent document 2] Salonia A, Eardley I, Giuliano F, Hatzichristou D, Moncada I, Vardi Y, et al. European Association of Urology guidelines on priapism. Eur Urol. 2014 February;65(2):480~9. [Non-patent document 3] Musicki B, Burnett AL. Mechanisms underlying priapism in sickle cell disease: targeting and key innovations on the preclinical landscape. Expert Opin Ther Targets. 2020 May;24(5):439~50. [Non-patent document 4] Adeyoju AB, Olujohungbe ABK, Morris J, Yardumian A, Bareford D, Akenova A, et al. Priapism in sickle-cell disease; incidence, risk factors and complications - an international multicentre study. BJU Int. 2002 December;90(9):898~902. [Non-Patent Document 5] Mantadakis E, Cavender JD, Rogers ZR, Ewalt DH, Buchanan GR. Prevalence of priapism in children and adolescents with sickle cell anemia. J Pediatr Hematol Oncol. Dec 1999;21(6):518~22.

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[0009] To date, no definitive treatment strategy has been established to specifically treat patients with hypererectile response or permanent erectile dysfunction, particularly priapism associated with sickle cell disease. Therefore, it is highly desirable to provide an innovative approach for treating or preventing hypererectile response and / or permanent erectile dysfunction, particularly associated with sickle cell disease. [Means for solving the problem]

[0010] The present invention relates to haptoglobin or a nucleic acid encoding same for use in the treatment or prevention of hyper-erectile response and / or the prevention of permanent erectile dysfunction, preferably in humans.

[0011] The haptoglobin may be a naturally occurring haptoglobin, such as human haptoglobin, or may be recombinant haptoglobin.

[0012] In this context, the hyper-erectile response may be a prolonged erectile response.Preferably, the hyper-erectile response is priapism, for example, priapism associated with (caused by) sickle cell disease (SCD).Optionally, haptoglobin increases the expression of PDE5 protein.Alternatively or additionally, haptoglobin reduces hyper-cavernosal relaxation.

[0013] The permanent erectile dysfunction may be an exacerbation of priapism associated with sickle cell disease (SCD). Preferably, the permanent erectile dysfunction occurs in patients suffering from SCD.

[0014] Preferably, the haptoglobin is human plasma haptoglobin. Optionally, the haptoglobin or nucleic acid encoding haptoglobin is administered parenterally, preferably by injection or infusion. The dosage of the haptoglobin or nucleic acid encoding haptoglobin may be 0.5 to 20 g per subject. It may also be administered based on the subject's body weight. Preferably, the haptoglobin or nucleic acid encoding haptoglobin is administered three times a week for a period of one month or more.

[0015] According to another aspect of the present invention, there is provided a pharmaceutical composition for use in treating or preventing hypererectile response and / or preventing permanent erectile dysfunction. The hypererectile response is preferably priapism, such as priapism associated with or caused by sickle cell disease (SCD). The permanent erectile dysfunction may be an exacerbation of priapism associated with or caused by sickle cell disease (SCD). Preferably, the permanent erectile dysfunction occurs in patients suffering from SCD. The pharmaceutical composition comprises an adeno-associated virus (AAV) vector carrying a transgene encoding a haptoglobin gene. Optionally, the adeno-associated virus (AAV) vector comprises the AAV2 serotype, the AAV5 serotype, the AAV9 serotype, or a combination thereof.

[0016] The present invention also relates to a method for treating or preventing hypererectile response and / or a method for preventing permanent erectile dysfunction, which method comprises administering a therapeutically effective amount of haptoglobin, or a nucleic acid encoding haptoglobin, or a pharmaceutical composition as described above to a subject. Preferably, the subject is a human.

[0017] Specific embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0018] [Figure 1] Concentration-response curves for acetylcholine (ACh; A-B) and sodium nitroprusside (SNP; C-D) in cavernosal strips from WT and SCD mice treated with or without haptoglobin (400 mg / kg) or vehicle every Monday, Wednesday, and Friday for one month. Data were calculated relative to the maximum change from the contraction produced by phenylephrine (10-5 M) in each tissue, defined as 100%. Data represent the mean ± SEM of six mice per group. *P<0.05 vs. WT-vehicle. [Figure 2] Figure 1 shows the relaxation response induced by electrical field stimulation (EFS) in corpus cavernosum strips from WT and SCD mice treated with or without haptoglobin (400 mg / kg) or vehicle every Monday, Wednesday, and Friday for one month. Data were calculated relative to the maximum change from the contraction produced by phenylephrine (10-5 M) in each tissue, which was set as 100%. Data represent the mean ± SEM of six mice per group. *P<0.05 vs. WT-vehicle. [Figure 3]Concentration-response curves for phenylephrine (A-B) and KCl (C-D) in cavernosal strips from WT and SCD mice treated with or without haptoglobin (400 mg / kg) or vehicle every Monday, Wednesday, and Friday for one month. Data are shown in mN and represent the mean ± SEM of six mice per group. *P<0.05 vs. WT-vehicle. [Figure 4] Representative Western blotting images (top) and protein values ​​(bottom) for p-eNOS (Ser-1177; A), eNOS (B), nNOS (C), and PDE5 (D) in penile homogenates from WT and SCD mice treated with or without haptoglobin (400 mg / kg) or vehicle every Monday, Wednesday, and Friday for one month. Data are shown in mN and represent the mean ± SEM of six mice per group. *P<0.05 vs. WT-Vehicle. Single bands for eNOS, nNOS, and PDE5 were normalized to β-actin. Single bands for p-eNOS (Ser-1177) were normalized to eNOS. [Figure 5] Representative Western blotting images (top) and protein values ​​(bottom) of ROCK2 (A) and ROCK1 (B) in penile homogenates from WT and SCD mice treated with or without haptoglobin (400 mg / kg) or vehicle every Monday, Wednesday, and Friday for one month. Data are shown in mN and represent the mean ± SEM of six mice per group. *P<0.05 vs. WT-vehicle. Single bands of ROCK1 and ROCK2 were normalized to β-actin. [Figure 6]Representative Western blotting images (top) and protein values ​​(bottom) for gp91phox (A), 3-NT (B), and 4-HNE (C) in penile homogenates from WT and SCD mice treated with or without haptoglobin (400 mg / kg) or vehicle every Monday, Wednesday, and Friday for one month. Data are shown in mN and represent the mean ± SEM of six mice per group. *P<0.05 vs. WT-vehicle. Single bands for gp91phox, 3-NT, and 4-HNE were normalized to β-actin. [Figure 7] Basal cGMP content in the penis of WT mice and SCD mice with or without haptoglobin treatment. Data represent the mean ± SEM of six mice per group. *P<0.05 vs. WT-vehicle; #P<0.05 vs. SCD-Hap. DETAILED DESCRIPTION OF THE INVENTION

[0019] During intravascular hemolysis, hemoglobin is released into the plasma (16). Under physiological conditions, haptoglobin is a plasma protein that protects the body from the accumulation of free hemoglobin (17). In plasma, haptoglobin binds to free hemoglobin to form hemoglobin-haptoglobin complexes, which are metabolized by macrophages of the reticuloendothelial system. Macrophages express the CD163 receptor, which mediates the endocytosis and internalization of hemoglobin-haptoglobin complexes (17). However, in SCD, high concentrations of hemoglobin are released into the plasma, and haptoglobin is depleted, resulting in the accumulation of free hemoglobin in the plasma (16, 18, 19). Hemoglobin in the plasma or interstitial space reacts with NO to produce nitrate and methemoglobin (16). Elevated plasma hemoglobin and heme levels promote vascular and oxidative damage in SCD ( 37 – 39 ).

[0020] Without wishing to be bound by theory, the inventors of the present invention have surprisingly found that treatment with haptoglobin can reverse hyper-erectile responses, particularly hyper-erectile responses induced by stimulation of the NO-cGMP pathway, by normalizing the expression of eNOS and PDE5 and normalizing the increase in corpus cavernosum contractile activity and oxidative stress.

[0021] Without wishing to be bound by theory, it is believed that the improvement was achieved by upregulation of eNOS-PDE5 expression and downregulation of the gp91phox subunit of NADPH oxidase and oxidative / nitrosative stress in the penis.

[0022] Haptoglobin may reduce excessive corpus cavernosum relaxation. Alternatively or additionally, haptoglobin may increase PDE5 protein expression. Phosphodiesterase 5 (PDE5) is a multidomain protein that functions as a dimer to hydrolyze cGMP. PDE5 expression is positively regulated by basal levels of cGMP in the penis (30). Basal NO production is reduced in the penis of men and mice with SCD due to decreased eNOS expression and activity (9, 10, 12, 31). Reduced NO bioavailability reduces the activation of sGC, a heme-containing heterodimeric enzyme that catalyzes the synthesis of the second messenger cGMP (32). Low PDE5 expression in the penis may contribute to increased corpus cavernosum relaxation (8, 29). The present inventors surprisingly found that haptoglobin treatment increases PDE5 protein expression, thereby potentially helping to treat or prevent excessive erectile responses.

[0023] Haptoglobin and its variants Haptoglobin (Hp) is a tetrameric plasma glycoprotein with a molecular weight of approximately 90,000 kDa. It is produced in hepatocytes and its primary function is to prevent iron loss through red blood cell hemolysis by its affinity and binding capacity to free hemoglobin (Hb). Hp is synthesized as a single chain and post-translationally cleaved into an amino-terminal α-chain and a carboxy-terminal β-chain. The basic structure of Hp found in most mammals is a homodimer, with two Hp molecules linked by a single disulfide bond via their respective 9 kDa α-chains. In humans, a variant with a longer α-chain is also present in all populations. This variant apparently arose by an early intragenic duplication resulting from unequal crossover of two basic alleles, resulting in Hp with a 14 kDa α-chain. The short and long α-chains are designated α1 and α2, respectively. Because the cysteines that form the intermolecular disulfide bonds between the α chains are also duplicated, humans with the long variant allele exhibit a multimeric Hp phenotype. The α chain has two isoforms, determining the existence of the Hp phenotype subtypes, Hp2-1 and Hp1-1. The isoforms α-1f and α-1s differ only at amino acid positions 52 and 53: the α-1f chain contains an aspartic acid and a lysine, whereas the α-1s chain contains two residues: one asparagine and one glutamic acid (47). These different isoforms share essentially identical characteristics in hemoglobin affinity, binding, and clearance, with only minor differences in function.

[0024] In vivo, haptoglobin is synthesized as a single polypeptide precursor with a molecular weight of 38,000 kDa. All three phenotypes of the mature protein are thought to originate from a single precursor, the haptoglobin-1 precursor. The polypeptide precursor is proteolytically processed to form the α and β subunits of the native protein (48). The precursor protein contains an amino-terminal 18-residue signal sequence preceding the α chain and / or an intervening polypeptide between the α and β regions (49). In vivo, post-translational modification results in proteolytic removal of the signal sequence and incorporation of the core oligosaccharide side chain into the β region by a membrane-associated enzyme system (48). Post-translational modification can also result in cleavage of both the α and β regions of the precursor polypeptide to form the native protein (48).

[0025] It should be understood that naturally occurring or recombinant Hp are suitable for use in the present invention, so long as they are capable of forming complexes with cell-free Hb to neutralize the biological activity of cell-free Hb. Suitable naturally occurring forms of Hp are known to those of skill in the art, and illustrative examples include those described in Koch et al. (50) and Kasvosve et al. (51), the entire contents of which are incorporated herein by reference.

[0026] Optionally, the haptoglobin comprises, consists of, or consists essentially of plasma-derived Hp. Preferably, the haptoglobin is human plasma haptoglobin. Preferably, the haptoglobin is human haptoglobin, such as the protein disclosed in NCBI accession number NP_005134 or UniProt accession number P00738, which UniProt entries include various variants. Protocols for isolating Hp from its natural source (e.g., plasma) will be well known to those skilled in the art.

[0027] The sequence of the beta chain of human haptoglobin is: ILGGHLDAKGSFPWQAKMVSHHNLTTGATLINEQWLLTTAKNLFLNHSENATAKDIAPTLTLYVGKKQLVEIEKVVLHPNYSQVDIGLIKLKQKVSVNERVMPICLPSKDYAEVGRVGYVSGWGRNANFKFTDHLKYVMLPVADQDQCIRHYEGSTVPEKKTPKSPVGVQPILNEHTFCAGMSKYQEDTCYGDAGSAFAVHDLEEDTWYATGILSFDKSCAVAEYGVYVKVTSIQDWVQKTIAEN (SEQ ID NO: 3) may be.

[0028] The sequence of the α2 chain of human haptoglobin is: VDSGNDVTDIADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVYTLNDKKQWINKAVGDKLPECEADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVYTLNNEKQWINKAVGDKLPECEAVCGKPKNPANPVQ (SEQ ID NO: 4) may be.

[0029] The sequence of the α1 chain of human haptoglobin is: VDSGNDVTDIADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVYTLNNEKQWINKAVGDKLPECEAVCGKPKNPANPVQ (SEQ ID NO: 5) may be.

[0030] The reference sequence for human haptoglobin α2-β is shown as isoform 1 in P00738 (last updated: 1986-07-21). The sequence for isoform 1 is: MSALGAVIALLLWGQLFAVDSGNDVTDIADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRT EGDGVYTLNDKKQWINKAVGDKLPECEADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTE GDGVYTLNNEKQWINKAVGDKLPECEAVCGKPKNPANPVQRILGGHLDAKGSFPWQAKMV SHHNLTTGATLINEQWLLTTAKNLFLNHSENATAKDIAPTLTLYVGKKQLVEIEKVVLHP NYSQVDIGLIKLKQKVSVNERVMPICLPSKDYAEVGRVGYVSGWGRNANFKFTDHLKYVM LPVADQDQCIRHYEGSTVPEKKTPKSPVGVQPILNEHTFCAGMSKYQEDTCYGDAGSAFA VHDLEEDTWYATGILSFDKSCAVAEYGVYVKVTSIQDWVQKTIAEN (SEQ ID NO: 1) is.

[0031] The sequence of the human haptoglobin isoform (α1-β) resulting from alternative splicing is shown as isoform 2 in P00738. This sequence differs from the reference isoform 1 by the deletion of amino acids 38-96. The sequence of isoform 2 is: MSALGAVIALLLWGQLFAVDSGNDVTDIADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRT EGDGVYTLNNEKQWINKAVGDKLPECEAVCGKPKNPANPVQRILGGHLDAKGSFPWQAKM VSHHNLTTGATLINEQWLLTTAKNLFLNHSENATAKDIAPTLTLYVGKKQLVEIEKVVLH PNYSQVDIGLIKLKQKVSVNERVMPICLPSKDYAEVGRVGYVSGWGRNANFKFTDHLKYV MLPVADQDQCIRHYEGSTVPEKKTPKSPVGVQPILNEHTFCAGMSKYQEDTCYGDAGSAF AVHDLEEDTWYATGILSFDKSCAVAEYGVYVKVTSIQDWVQKTIAEN (SEQ ID NO: 2) may be.

[0032] As used herein, the term "haptoglobin" is understood to encompass all phenotypes of Hp (including all isoforms). Haptoglobin may be homogeneous (as long as it consists essentially of the same isoform of Hp) or heterogeneous (comprising a combination of different Hp isoforms, including Hp1-1, Hp1-2, and Hp2-2). Optionally, haptoglobin for use according to the present invention may be a mixture of two or more phenotypes and / or isoforms. Preferably, haptoglobin for use according to the present invention comprises Hp2-2. More preferably, haptoglobin for use according to the present invention is a mixture of Hp2-2 and Hp1-2. It should be understood that the composition of Hp ultimately depends on the phenotype of the source. For example, when extracting / purifying Hp using pooled plasma samples, it is likely that one or more isoforms of Hp will be isolated. Suitable methods for determining the Hp isoforms present in an isolate will be familiar to those skilled in the art.

[0033] In one embodiment, the Hp is selected from the group consisting of Hp1-1 homodimers, Hp1-2 multimers, Hp2-2 multimers, and combinations thereof. The Hp may be a naturally occurring Hp (e.g., plasma-derived) or produced as a recombinant protein. In some embodiments, the haptoglobin comprises naturally occurring human plasma haptoglobin. Optionally, the plasma-derived Hp comprises, consists of, or consists essentially of Hp2-2. Alternatively, the plasma-derived Hp comprises, consists of, or consists essentially of Hp1-1. In a further aspect, the Hp comprises, consists of, or consists essentially of a recombinant Hp.

[0034] As used herein, the term Hp includes naturally occurring Hp or functional analogs of naturally occurring Hp. The term "functional analog" is intended to mean an agent that shares substantially the same biological activity as naturally occurring (native) Hp, provided that the biological activity is at least the ability of the analog to form a complex with cell-free Hb and neutralize its biological activity.

[0035] The term "substantially the same biological activity" typically means that the functional analog has at least 40% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, etc.) of the binding affinity for Hb. Suitable methods for determining whether an agent is a functional analog of Hp will be familiar to those of skill in the art.

[0036] In some cases, the functional analog of Hp is a functional fragment of native Hp. The functional fragment of native Hp can be of any suitable length, so long as the fragment retains the ability to form a complex with cell-free Hb and neutralize its biological activity.

[0037] Alternatively, a functional analog is a polypeptide having an amino acid sequence that differs from that of a naturally occurring (native) Hp molecule (i.e., a control agent). A functional analog differs from the amino acid sequence of the α and / or β chains of native Hp by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more) amino acid substitutions that result in a different amino acid sequence, but the differences do not eliminate or completely eliminate the ability of the analog to form a complex with cell-free Hb and neutralize its biological activity. In some embodiments, a functional analog contains amino acid substitutions that enhance the ability of the analog to form a complex with cell-free Hb compared to native Hp. In one embodiment, a functional analog has an amino acid sequence that differs from that of the α and / or β chains of native Hp by one or more conservative amino acid substitutions. As used herein, the term "conservative amino acid substitution" refers to changing the identity of an amino acid at a given position and substituting it with an amino acid of approximately the same size, charge, and / or polarity. Examples of naturally occurring conservative substitutions for amino acids include the following eight groups of substituents (designated by common one-letter codes): (1) M, I, L, V; (2) F, Y, W; (3) K, R; (4) A, G; (5) S, T; (6) Q, N; (7) E, D; and (8) C, S.

[0038] In one embodiment, a functional analog has at least 85% or more sequence identity with the amino acid sequence of the α and / or β chain of native Hp.

[0039] References to "at least 85%" refer to 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or similarity, for example, after best alignment or best fit analysis. Thus, in one embodiment, the sequence has at least 85%, at least 86%, at least 87%, at least 87%, at least 85%, at least 86%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity or sequence homology with the amino acid sequence of the α and / or β chain of native Hp.

[0040] As used herein, the terms "percent identity" and "% identity" between two amino acid (peptide) or nucleic acid (nucleotide) sequences refer to the percentage of identical amino acid or nucleotide residues at corresponding positions in two optimally aligned sequences. Conservative substitutions are not considered as part of identity. N- or C-terminal extensions or insertions should not be construed as reducing sequence identity or homology.

[0041] To determine the "percent identity" of two amino acid or nucleic acid sequences, the sequences are aligned. To achieve optimal matching, gaps can be introduced into the sequences (i.e., deletions or insertions, which can also be placed at the ends of the sequences). The amino acid and nucleotide residues at corresponding positions are then compared. If a position in the first sequence is occupied by the same amino acid or nucleotide residue that occupies the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions divided by the sequence [i.e., % identity = (number of identical positions / total number of positions) × 100].

[0042] As used herein, the term "similarity" or "sequence similarity" indicates that at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, a leucine may be substituted with an isoleucine or valine residue. As noted elsewhere herein, this may be referred to as a conservative substitution. In one embodiment, the amino acid sequence is modified by conservative substitution of any amino acid residue contained therein, such that the modification does not affect the binding specificity or functional activity of the modified polypeptide compared to the unmodified (native) Hp polypeptide.

[0043] The percent identity can be determined using a mathematical algorithm. A non-limiting example of an algorithm used to compare two sequences is incorporated into the BLASTn and BLASTp programs of Altschul

[52] . These programs can be used to achieve alignment even with one or more gaps (in the case of insertions). For this purpose, the BLASTn and BLASTp programs can be used with default parameters (i.e., for amino acid sequences, a penalty of -12 is allowed for gaps and a penalty of -4 for each extension). When using BLAST programs, the BLOSUM62 matrix is ​​usually used.

[0044] In one embodiment, the functional analog contains amino acid substitutions and / or other modifications relative to native Hp to increase the stability of the analog or to increase the solubility of the analog.

[0045] Functional analogs may be naturally occurring polypeptides or may be synthetically produced by chemical synthesis using methods known to those skilled in the art.

[0046] Hp is preferably produced as a recombinant protein in a microorganism, isolated, and may be further purified if desired. Microorganisms suitable for producing recombinant Hp are well known to those skilled in the art, and exemplary examples include bacteria, yeast or fungi, eukaryotic cells (e.g., mammalian cells or insect cells), or recombinant viral vectors (e.g., adenovirus, poxvirus, herpesvirus, Simki Forest virus, baculovirus, bacteriophage, Sindbis virus, or Sendai virus). Bacteria suitable for producing recombinant peptides are well known to those skilled in the art, and exemplary examples include Escherichia coli, Bacillus subtilis, or any other bacterium capable of expressing a peptide sequence. Illustrative examples of yeast types suitable for producing recombinant peptides include Candida, Chiapas Pastoris (Pichia pastoris), Saccharomyces cerevisiae, Schizosaccharomyces pombe, or any other yeast capable of expressing peptides. Such methods are well known in the art. Methods for isolating and purifying recombinantly produced peptide sequences are also well known in the art and include, for example, gel filtration, affinity chromatography, ion exchange chromatography, and the like.

[0047] To facilitate isolation of the recombinant Hp described herein, the Hp peptide sequence or a functional analog thereof can be translationally fused (covalently linked) to a heterologous polypeptide to produce a fusion polypeptide that can be isolated by affinity chromatography. Illustrative examples of suitable heterologous polypeptides include His tags (e.g., His6:6 histidine residues), GST tags (glutathione transferase-S), etc.

[0048] Phage and / or peptide libraries are also suitable for the production of recombinant Hp, such as those generated by conjugation chemistry or obtained by high-throughput screening techniques for the most diverse structures.

[0049] Illustrative examples of recombinant Hp include NCBI accession number NP_005134 (described by Morishita et al. (53)) and UniProt accession number P00738.

[0050] Haptoglobin variants with extended half-lives The haptoglobin described in the present invention can be fused, conjugated, or attached to one or more heterologous moieties as part of a fusion protein. The one or more heterologous moieties may improve, enhance, or prolong the activity or stability of Hp. In one embodiment, Hp is preferably attached to a heterologous moiety to extend the half-life of Hp in vivo. Suitable half-life-enhancing heterologous moieties will be known to those of skill in the art, and illustrative examples include polyethylene glycol (PEG), glycosylated PEG, hydroxyethyl starch (HES), polysialic acid, elastin-like polypeptides, heparoic acid polymers, and hyaluronic acid. Thus, in some embodiments disclosed herein, the heterologous moiety is selected from the group consisting of polyethylene glycol (PEG), glycosylated PEG, hydroxyethyl starch (HES), polysialic acid, elastin-like polypeptides, heparoic acid polymers, and hyaluronic acid. In another embodiment, the heterologous moiety can be a heterologous amino acid sequence fused to Hp.

[0051] Alternatively, or in addition, the heterologous moiety may be chemically conjugated, e.g., covalently bound, to Hp. The half-life-extending heterologous moiety may be fused, conjugated, or otherwise attached to Hp by any suitable means known to those skilled in the art, an illustrative example of which is via a chemical linker. The principles of this conjugation technique have been described by Conjuchem LLC (see, e.g., U.S. Pat. No. 7,256,253), the entire contents of which are incorporated herein by reference.

[0052] In another embodiment, the heterologous moiety is a half-life enhancing protein (HLEP). Suitable half-life enhancing proteins will be known to those skilled in the art, and illustrative examples include albumin or a fragment thereof. Thus, in one embodiment, the HLEP is albumin or a fragment thereof. The N-terminus of albumin or a fragment thereof may be fused to the C-terminus of the α-chain and / or β-chain of Hp. Alternatively, or in addition, the N-terminus of albumin or a fragment thereof may be fused to the C-terminus of the α-chain and / or β-chain of Hp. One or more HLEPs may be fused to the N- or C-terminal portions of the α-chain and / or β-chain of Hp, as long as they do not abolish the binding of Hp to cell-free Hb. However, it should be understood that a slight reduction in the binding of Hp to cell-free Hb may be tolerated as long as the Hp component of the fusion protein is still able to form a complex with and neutralize cell-free Hb.

[0053] The fusion protein may further comprise a chemical bond or linker sequence located between the Hp and the heterologous moiety. The linker sequence comprises at least one amino acid, particularly 1 to 50, preferably 1 to 30, preferably 1 to 20, preferably 1 to 15, preferably 1 to 10, and preferably 1 to 5. It may also be a peptide linker of 5 or more preferably 1 to 3 (e.g., 1, 2, or 3) amino acids, which may be identical or different. Preferably, the linker sequence is not present at the corresponding position in wild-type Hp. Preferred amino acids present in the linker sequence include glycine and serine. In a preferred embodiment, the linker sequence is substantially non-immunogenic to a subject treated according to the methods disclosed herein. By substantially non-immunogenic, it is meant that the linker sequence does not elicit a detectable antibody response against the linker sequence in a subject to which it is administered. A preferred linker consists of alternating glycine and serine residues. Suitable linkers will be known to those skilled in the art, and illustrative examples are described in WO2007 / 090584. In one embodiment, the peptide linker between Hp and the heterologous moiety comprises, consists of, or consists essentially of a peptide sequence that serves as a native interdomain linker in human proteins. In the natural environment, these peptide sequences are located near the protein surface and are accessible to the immune system, so natural resistance to these sequences can be assumed. Illustrative examples are provided in WO2007 / 090584. Suitable cleavable linker sequences are described, for example, in WO2013 / 120939A1.

[0054] Illustrative examples of suitable HLEP sequences are described below. Similarly, fusion to the exact "N-terminal amino acid" or "C-terminal amino acid" of each HLEP, or to the "N-terminal portion" or "C-terminal portion" of each HLEP that includes an N-terminal deletion of one or more amino acids of HELP, are also described. Fusion proteins may contain one or more HLEP sequences, e.g., two or three HLEP sequences. Such multiple HLEP sequences may be fused in tandem, e.g., in consecutive repeats, to the C-terminal portions of the α- and / or β-chains of Hp.

[0055] In one embodiment, the heterologous moiety is a half-life extending polypeptide. In one embodiment, the half-life extending polypeptide is selected from the group consisting of albumin, a member of the albumin family or a fragment thereof, solvated random chains with large hydrodynamic volumes (e.g., XTEN(56), homoamino acid repeats (HAP), or proline-alanine-serine repeats (PAS), apamin, alpha-fetoprotein, vitamin D binding protein, transferrin or a variant or fragment thereof, the beta subunit of human chorionic gonadotropin carboxyl terminal peptide (CTP), neonatal Fc receptor (FcRn), in particular polypeptides capable of binding to immunoglobulin constant regions and portions thereof, such as Fc fragments. The immunoglobulin constant region or portion thereof is preferably selected from the group consisting of an Fc fragment of immunoglobulin G1 (IgG1), an Fc fragment of immunoglobulin G2 (IgG2), or an Fc fragment of immunoglobulin α (IgA). The HLEP is an Fc fragment of the HLEP. The HLEP is a full-length half-life-enhancing protein or one or more fragments thereof that can stabilize or extend the therapeutic or biological activity of Hp, particularly increasing the in vivo half-life of Hp. The fragment may be 10 amino acids or more, or at least about 15 amino acids, preferably at least about 20 amino acids, preferably at least about 25 amino acids, preferably at least about 30 amino acids, preferably at least about 50 amino acids, or more preferably at least about 100 or more contiguous amino acids from the HLEP sequence. The HLEP fragment may include part or all of a specific domain of each HLEP, so long as the HLEP fragment provides a functional half-life extension of at least 10%, preferably at least 20%, and more preferably at least 25% compared to Hp in the absence of HLEP. Methods (in vivo or in vitro) for determining whether a heterologous moiety confers a functional half-life extension to Hp will be familiar to those skilled in the art.

[0056] The HLEP portion of the fusion proteins described herein may be a variant of a wild-type HLEP. The term "variant" includes insertions, deletions, and / or substitutions, whether conservative or non-conservative, where such changes do not substantially alter the ability of Hp to complex with and neutralize cell-free Hb. HLEPs are preferably derived from vertebrates, particularly mammals such as humans, monkeys, cows, sheep, and pigs. Non-mammalian HLEPs include, but are not limited to, chicken and salmon.

[0057] The fusion proteins described herein can be produced by the in-frame ligation of at least two DNA sequences encoding heterologous moieties, such as Hp and HLEP. One skilled in the art will understand that translation of the fusion protein DNA sequences will result in a single protein sequence. In-frame insertion of a DNA sequence encoding a peptide linker according to embodiments disclosed herein can result in a fusion protein comprising Hp, a suitable linker, and a heterologous moiety.

[0058] As used herein, "albumin" collectively refers to an albumin polypeptide or amino acid sequence, or an albumin fragment or variant, having one or more functional (e.g., biological) activities of albumin. In particular, "albumin" refers to human albumin or a fragment thereof, and includes mature human albumin, or albumin from other vertebrates or a fragment thereof, or analogs or variants of these molecules or fragments thereof. In some embodiments disclosed herein, the alternative term "FP" is used to identify HLEPs, and in particular to define albumin as an HLEP.

[0059] The fusion proteins described herein may suitably comprise naturally occurring polymorphic variants of human albumin and / or fragments of human albumin. Generally, the albumin fragment or variant will be at least 10 amino acids in length, preferably at least 40 amino acids in length, or most preferably at least 70 amino acids in length.

[0060] In one embodiment, the HLEP is an albumin variant with enhanced binding to the FcRn receptor. Such an albumin variant may increase the plasma half-life of Hp or a functional analog thereof compared to Hp or a functional fragment thereof fused to wild-type albumin. The albumin portion of the fusion protein described herein may suitably comprise at least one subdomain or domain of human albumin or a conservative modification thereof.

[0061] In one embodiment, the heterologous moiety is an immunoglobulin molecule or a functional fragment thereof. The immunoglobulin G (IgG) constant region (Fc) is known in the art to extend the half-life of therapeutic proteins (57). The IgG constant region of the heavy chain consists of three domains (CH1-CH3) and a hinge region. The immunoglobulin sequence can be derived from any mammal or from the respective subclasses IgG1, IgG2, IgG3, or IgG4. IgG and IgG fragments lacking the antigen-binding domain can also be used as heterologous moieties containing HLEP. Hp or its functional analogs may be suitably linked to IgG or IgG fragments via the hinge region of the antibody or via a cleavable peptide linker. Several publications have described fusing therapeutic proteins to immunoglobulin constant regions to improve their in vivo half-life. For example, US2004 / 0087778 and WO2005 / 001025 disclose fusion proteins of biologically active peptides with at least a portion of an Fc domain or immunoglobulin constant region, which extend the half-life of the peptide and allow rapid clearance in vivo. Fc-IFN-β fusion proteins that achieve enhanced biological activity, extended circulating half-life, and higher solubility have been described (WO2006 / 000448A2). Fc-EPO proteins with extended serum half-lives and increased in vivo potency have been disclosed (WO 2005 / 063808 A1), as well as Fc fusion proteins with G-CSF (WO 2003 / 076567 A2), glucagon-like peptide-1 (WO 2005 / 000892 A2), coagulation factors (WO 2004 / 101740 A2), and interleukin-10 (U.S. Patent No. 6,403,077), all of which have half-life enhancing properties.

[0062] Illustrative examples of suitable HLEPs for use in accordance with the present invention are also described in WO2013 / 120939A1, the contents of which are incorporated herein by reference in their entirety.

[0063] Nucleic acid encoding haptoglobin As an alternative to haptoglobin, nucleic acids encoding haptoglobin may be used in the present invention. The haptoglobin may be any of the haptoglobins described above, including variants, fragments and fusion proteins thereof.

[0064] Human haptoglobin is encoded by the HP gene, with NCBI accession numbers NP_001119574, NP_001305067, and NP_005134. The Hp gene, located on chromosome 16, has two alleles, Hp1 and Hp2, which carry three possible genotypes with structural polymorphisms: homozygous (1-1 or 2-2) and heterozygous 2-1.

[0065] The term "nucleic acid" as used herein generally refers to any nucleotide molecule that encodes a haptoglobin according to the present invention and may be of variable length. Examples of nucleic acids of the present invention include, but are not limited to, plasmids, vectors, or any type of DNA and / or RNA fragment. Nucleic acid molecules for use in the present invention may be in the form of RNA, such as mRNA or cRNA, or in the form of DNA, including cDNA and genomic DNA, obtained by cloning, produced by chemical synthesis techniques, or a combination thereof. DNA may be triple-stranded, double-stranded, or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or the non-coding strand, also known as the antisense strand. As used herein, nucleic acid molecules also refer to, inter alia, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that are single-stranded or more typically double-stranded, or triple-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, as used herein, a nucleic acid molecule refers to a triple-stranded region comprising RNA or DNA, or both RNA and DNA. Furthermore, the nucleic acid molecules encoding the haptoglobin described in the present invention can be operably linked to any desired sequence, such as a control sequence, a leader sequence, a heterologous marker sequence, or a heterologous coding sequence, using standard techniques, such as standard cloning techniques, to produce a fusion protein.

[0066] The nucleic acids according to the present invention may be present in a vector, such as a plasmid or viral vector, together with appropriate promoter sequences, enhancer sequences, marker sequences, etc., to enable expression of the polypeptide or mRNA in target cells, tissues, or body fluids. The nucleic acids of the present invention may also include a nucleic acid sequence encoding a haptoglobin receptor or a haptoglobin-binding fragment thereof.

[0067] gene therapy Another aspect of the present invention relates to a pharmaceutical composition for use in treating or preventing hypererectile response and / or preventing permanent erectile dysfunction, the pharmaceutical composition comprising an adeno-associated virus (AAV) vector carrying a transgene encoding a haptoglobin gene. In the context of the present invention, a haptoglobin gene is any gene encoding a haptoglobin as described herein.

[0068] A number of (viral) delivery systems have been investigated, each with its own advantages and disadvantages. One viral delivery vehicle used in gene therapy is the adeno-associated virus (AAV).

[0069] AAV has a single-stranded DNA genome of approximately 4.8 kilobases (kb). AAV belongs to the parvovirus family and depends on co-infection with other viruses, particularly adenovirus, for replication. The genome contains the Rep (replication) gene and the Cap (capsid) gene. These coding sequences are flanked by inverted terminal repeats (ITRs) required for genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) that replicate and promote packaging of the viral genome, while expression of Cap produces the viral capsid proteins (VP; VP1, VP2, and VP3) that form the outer capsid shell.

[0070] Recombinant AAV (rAAV) for gene therapy consists of a protein capsid containing a transgene, the desired nucleic acid to be delivered to target cells. The desired nucleic acid is flanked by the AAV ITRs. rAAV-encoded transgenes can form circular concatemers that remain episomal in the nucleus of transformed cells. Because the majority of the episomes remain episomal, expression of the AAV-delivered nucleic acid sequence may be diluted over time if the target cell replicates. This dilution does not generally apply to postmitotic cells, such as neurons, which are the target cells for many neurodegenerative diseases. A review of AAV vectors for gene therapy is published in Naso et al., Biodrugs 2017 (pp. 317–334).

[0071] In some embodiments, the adeno-associated virus vector comprises AAV2 serotype, AAV5 serotype, AAV9 serotype, hybrid AAV serotype, or a combination thereof.In some embodiments, the adeno-associated virus vector comprises AAV5 serotype.In some embodiments, the adeno-associated virus vector comprises AAV9 serotype.

[0072] In some embodiments, the adeno-associated viral vector comprises a hybrid AAV serotype. By way of example, the hybrid AAV serotype may be a hybrid AAV2 / AAV5; AAV2 / AAV9; or AAV5 / AAV9 serotype.

[0073] Administration The haptoglobin or nucleic acid of the present invention is administered to a subject in the form of, for example, a lyophilized formulation or an aqueous solution. The dosage form may further comprise one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). The haptoglobin or nucleic acid of the present invention as an active ingredient may be encapsulated in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Dosage forms to be used for in vivo administration are generally sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0074] Preferably, the haptoglobin or haptoglobin-encoding nucleic acid of the present invention is administered parenterally, more preferably by injection or infusion. For example, the haptoglobin or haptoglobin-encoding nucleic acid is administered systemically, for example, by intravenous administration, such as intravenous injection or infusion. Alternatively, the haptoglobin or haptoglobin-encoding nucleic acid is administered subcutaneously. The dosage of Hp may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, weekly, or at other appropriate time intervals, or the dosage may be proportionally reduced depending on the urgency of the condition. Optionally, the haptoglobin or haptoglobin-encoding nucleic acid is administered three times a week for a period of one month or more. Alternatively, the haptoglobin or haptoglobin-encoding nucleic acid may be administered for a period of one to three months. Preferably, the haptoglobin or haptoglobin-encoding nucleic acid is administered according to a repeating pattern of two 48-hour and one 72-hour administration intervals.

[0075] The above administration routes may also be applied to the pharmaceutical composition of the present invention.

[0076] As used herein, the term "therapeutically effective amount" refers to an amount or concentration of Hp sufficient to bind to and complex with acellular Hb present in plasma, thereby neutralizing the adverse biological effects of acellular Hb. Those skilled in the art will understand that a therapeutically effective amount of a peptide may vary depending on several factors, including, by way of illustrative example, the route of administration, the health and physical condition of the subject being treated, the taxonomic group of the subject being treated, the severity of the bleeding (e.g., the extent of bleeding), the concentration and / or amount of acellular Hb in the plasma, and any of the foregoing, and any combinations thereof.

[0077] The therapeutically effective amount of Hp will typically fall within a relatively broad range that can be determined by one of ordinary skill in the art. In some cases, the therapeutically effective amount of Hp is about 0.5 g to about 50 g, about 0.5 g to about 40 g, about 0.5 g to about 30 g, about 0.5 g to about 20 g, about 0.5 g to about 15 g, or about 0.5 g to about 10 g per subject. Preferably, haptoglobin or a nucleic acid encoding haptoglobin may be administered at 1 g to 5 g, more preferably 1.5 g to 3 g, per subject. The administered Hp may be at a concentration of about 2 μM to about 20 mM, about 2 μM to about 5 mM, about 2 μM to about 300 μM, about 5 μM to about 100 μM, about 5 μM to about 50 μM, or about 5 μM to about 30 μM per subject, and the administered volume may be 1 to 50 mL, 1 to 40 mL, 1 to 30 mL, 3 to 20 mL, e.g., 5 to 10 mL. Alternatively, the therapeutically effective amount of Hp may be based on the subject's body weight. For example, the therapeutically effective amount of Hp may be 1 to 100 mg / kg, or optionally 1 to 80 mg / kg, 5 to 75 mg / kg, 10 to 50 mg / kg, 15 to 50 mg / kg, 20 to 40 mg / kg, or 25 to 35 mg / kg of the subject's body weight.

[0078] In some embodiments, haptoglobin or a nucleic acid encoding haptoglobin is administered in a volume of 5-10 mL at a concentration of 50-220 mg / dL (5.88-25.87 μM) per subject. In other embodiments, the therapeutically effective amount of Hp is sufficient to complex with about 3 μM to about 300 μM, about 5 μM to about 250 μM, about 10 μM to about 200 μM, or about 50 μM to about 150 μM of cell-free Hb in plasma. Suitable methods for measuring cell-free Hb concentrations in CSF are known to those skilled in the art, and illustrative examples are described in Cruickshank et al. (54) and Hugelshofer M. et al. (55), the contents of which are incorporated herein by reference in their entireties.

[0079] The pharmaceutical compositions of the present invention may be administered at a dose of about 1E12 to about 5E14, about 5E12 to about 1E14, or about 1E13 to about 1E14 genome copies of the adeno-associated virus (AAV) vector per subject.

[0080] Features described above in connection with any one aspect or embodiment of the present invention are also disclosed herein in connection with all other aspects and embodiments. Similarly, any combination of two or more of the individual features or elements described above may be present in any aspect or embodiment. For the sake of brevity, not all possible features and combinations are described in connection with every aspect and embodiment, but they are expressly contemplated and disclosed herein. [Example]

[0081] ethical approval All example protocols were approved by the Committee for Ethics in Animal Experimentation of the University of Campinas (IACUC / CEEA-UNICAMP, permit number 4754-1 / 2017).

[0082] material ACh, phenylephrine, guanethidine, and atropine were obtained from Sigma-Aldrich (St Louis, MO, USA). Human haptoglobin (Hp) solution was a gift from CSL Behring (Bern, Switzerland). All reagents were analytical grade. Deionized water was used as the solvent, and working solutions were diluted before use.

[0083] Animals and treatments All mouse strains were purchased from Jackson Laboratories (Bar Harbor, ME). Characterization and breeding were performed at the University of Campus's Multidisciplinary Center for the Investigation of Biological Sciences. C57BL / 6 male mice (wild-type, WT) and Berkeley transgenic SCD mice (3–4 months old) were used. Mice were housed three per cage under a 12-hour light / dark cycle. SCD male mice were treated with haptoglobin (400 mg / kg, subcutaneously) or vehicle on Mondays, Wednesdays, and Fridays for 1 month (24).

[0084] statistical analysis Statistical analysis was performed using the GraphPad Prism Program (GraphPad Software Inc.). Data are expressed as the mean ± SEM of N experiments. Statistical comparisons were performed using the Student's unpaired t-test. A value of P < 0.05 was considered statistically significant.

[0085] Functional studies and concentration-response curves of cavernosal strips Strips of mouse corpus cavernosum were mounted in a 7 ml organ system containing Krebs' solution at 37°C, continuously bubbled with a mixture of 95% O2 and 5% CO2 (pH 7.4), and suspended between two metal hooks. One hook was connected to a force transducer, while the other served as a fixed attachment point. The tissue was allowed to equilibrate for 60 min under a resting tension of 2.5 mN. Isometric muscle force was recorded using a PowerLab400™ data acquisition system (Software LabChart, version 7.0, AD Instrument, MA, USA). Phenylephrine (10 -5 In cavernosal strips pre-contracted with muscarinic agonist acetylcholine (ACh; 10 -9 ~10 -5 M) and sodium nitroprusside (SNP; 10 -8 ~10 -4A cumulative concentration-response curve was constructed for the contractile agent phenylephrine (α1-adrenergic receptor agonist, 10 M). -8 ~3×10 -4 M) and KCl (3 × 10 -4 ~3×10 -1 Cumulative concentration-response curves to pEC 50 Nonlinear regression analysis to determine was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

[0086] Electric field stimulation (EFS) EFS was applied to the cavernosal strip placed between two platinum ring electrodes connected to a Grass S88 stimulator (Astro-Med Industrial Park, RI, USA). EFS was applied at 50 V, with a 1 ms pulse width and varying frequency, lasting 10 seconds. To examine nitrergic cavernosal relaxation, phenylephrine (10 -5 Before precontraction with guanethidine (3 × 10 -5 M; to deplete catecholamine stores in adrenergic fibers) and atropine (10 -6 The rats were pretreated with 100 M (to induce muscarinic antagonism) for 30 min. Once a stable contraction level was reached, a series of EFS-induced relaxations was constructed (2–32 Hz).

[0087] Western blot analysis Corpus cavernosum tissue was homogenized in lysis buffer and centrifuged at 12,000 g for 20 minutes at 4°C. Homogenates containing 50 μg of total protein were run on a 4-20% Tris-HCl gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to a nitrocellulose membrane. To block nonspecific binding sites, 5% nonfat dry milk in Tris-buffered saline / Tween (Bio-Rad) was used for 1 hour at 24°C. The membranes were incubated for 15–16 h at 4°C with the following antibodies: monoclonal anti-3-NT (1:1000, Abcam, Cambridge, MA), polyclonal anti-4-HNE antibody (1:1000, Abcam), polyclonal anti-p-eNOS (Ser-1177) antibody (1:1000, Abcam), polyclonal anti-eNOS antibody (1:1000, Abcam), polyclonal anti-nNOS antibody (1:1000, Abcam), polyclonal anti-PDE5 (1:500, Abcam), polyclonal anti-ROCK-1 / ROCK-2 (1:1000, Abcam), gp91phox (1:1000, BD Transduction). Antibodies against eNOS phosphorylated at Ser1177 were normalized to total eNOS.

[0088] Determination of cGMP levels Quantitative assays of cGMP were performed using a commercially available enzyme immunoassay kit (Cayman Chemical Cyclic GMP EIA Kit, Ann Arbor, MI, USA). For penile cGMP content, frozen penile tissue was homogenized in 5% trichloroacetic acid and centrifuged. After three washes with water-saturated ether, TCA was extracted from the supernatant. cGMP was expressed as pmol / mg tissue. [Example]

[0089] Haptoglobin treatment corrects excessive cavernosal relaxation in SCD mice The tissue contracted by PE was treated with ACh(10 -9 ~10 -5 Cumulative addition of haptoglobin (M) induced concentration-dependent relaxation in all groups (Fig. 1A, 1B). However, the maximum response (Emax) induced by ACh was significantly higher in the SCD group compared with the WT group (P<0.05; Fig. 1A; Table 1 below). Treatment of SCD mice with haptoglobin reduced the Emax value of ACh (P<0.05; Table 1). No significant differences in the potency (pEC50) of ACh were observed between groups. The pEC50 values ​​of ACh are shown in Table 1.

[0090] [Table 1]

[0091] SNP(10 -8 ~10 -4 Cumulative addition of 100 mg of 100 M (M) resulted in concentration-dependent relaxation in all groups (Fig. 1C, 1D). The Emax produced by SNP was significantly higher in the corpus cavernosum of SCD mice compared with that of WT mice (P < 0.05) (Fig. 1C; Table 1), which was reduced by haptoglobin treatment (Fig. 2D). The pEC50 value of SNP was significantly higher in the corpus cavernosum of SCD mice compared with that of WT mice (P < 0.05) (Fig. 1C and Table 1), which was reversed by haptoglobin treatment (P < 0.05) (Fig. 1D and Table 1).

[0092] Electric field stimulation (EFS) applied to cavernous tissue pretreated with guanethidine (3 × 10 M) and atropine (10 M) induced frequency-dependent relaxation of the mouse corpus cavernosum in all groups. Cavernous relaxation in response to EFS, observed at 2 Hz to 32 Hz, was significantly lower in SCD compared with WT mice (P < 0.05) (Figure 2A). Haptoglobin treatment reduced the enhanced EFS-induced relaxation response in SCD-derived corpora cavernosum (Figure 2B).

[0093] Nitric oxide (NO), produced after erectile stimulation, is an essential molecule produced in the penis to induce penile erection. NO is produced not only by the endothelium lining the sinusoids of the corpus cavernosum but also by nitrergic neurons (26). ACh promotes relaxation of the corpus cavernosum by stimulating endothelial NO production. According to the results of Example 1, ACh-induced relaxation was greater in the corpus cavernosum of the SCD group. EFS promotes relaxation through neurogenic stimulation, which leads to NO production by nitrergic fibers (27). EFS-induced relaxation was also greater in the CC of the SCD group. SNP, an NO-donating compound, has been used as a pharmacological tool to evaluate endothelium-independent relaxation (28). Consequently, SNP-induced relaxation was also higher in the SCD group. Therefore, when NO activates GCs, cGMP is produced, but because it is not efficiently degraded by PDE5, cGMP accumulates in smooth muscle cells, promoting cavernous relaxation and excessive penile erection in SCD (3). Long-term treatment with haptoglobin reduced relaxation induced by ACh, EFS, and SNP in CC from the SCD group. Without wishing to be bound by theory, the improvement in erectile function by haptoglobin treatment may be due to normalization of PDE5 expression and reduction of oxidative stress in the penis of SCD mice. [Example]

[0094] Haptoglobin treatment corrects the impaired cavernous contractility in SCD mice Phenylephrine (10 -8 ~3×10 -4 M) induced concentration-dependent contraction of the corpus cavernosum in all groups (Fig. 3A). The maximum response (Emax) was significantly lower in the corpus cavernosum of SCD mice (0.50 ± 0.08 mN) compared with that of WT mice (0.90 ± 0.11 mN) (P < 0.05). Treatment of SCD mice with haptoglobin increased the Emax value of phenylephrine in the SCD group (0.79 ± 0.11 mN).

[0095] In the evaluation of receptor-independent stimulation, cumulative addition of KCl induced concentration-dependent contraction of the corpus cavernosum in all groups (Figures 3C and 3D). The maximum response (Emax) was significantly lower in the corpus cavernosum of SCD mice (0.16 ± 0.04 mN) compared with that of WT mice (0.45 ± 0.08 mN) (P < 0.05). Treatment of SCD mice with haptoglobin increased the Emax of KCl in the SCD group (0.30 ± 0.07 mN).

[0096] Norepinephrine released from the sympathetic nervous system induces contraction of the corpus cavernosum through postjunctional activation of α1-adrenergic receptors coupled to Gq proteins, which activate phospholipase C. Phospholipase C catalyzes the cleavage of phosphatidylinositol into inositol triphosphate and diacylglycerol, thus increasing intracellular calcium levels (34). Calcium binds calmodulin, resulting in smooth muscle contraction, which in turn activates and phosphorylates myosin light chain kinase (MLC) (27). During this process, phosphorylated MLC interacts with β-actin, resulting in smooth muscle contraction and maintaining penile flaccidity (27, 34). KCl promotes receptor-independent contraction by depolarizing the smooth muscle cell membrane and promoting calcium influx (27). Surprisingly, we found that contractions induced by α1-adrenergic receptor agonists and KCl were reduced in SCD mice. [Example]

[0097] Haptoglobin treatment corrects downregulated p-eNOS (Ser-1177) and eNOS but does not affect nNOS protein expression in the penis of SCD mice Protein expression of p-eNOS (Ser-1177) and eNOS was significantly reduced by approximately 45% and 44% in the penis of the SCD-Vehicle group compared with the WT-Vehicle group (p<0.05) (Figures 4A and 4B, respectively). Haptoglobin treatment normalized p-eNOS (Ser-1177) and eNOS protein levels in the penis of the SCD group (P<0.05). Protein expression of nNOS was not altered by SCD or haptoglobin treatment (Figure 4C).

[0098] According to the results of Example 3, haptoglobin treatment normalized the expression of total eNOS and eNOS phosphorylated at its positive regulatory site, Ser-1177, in the SCD group, but did not affect the expression of nNOS. These results indicated that basal NO production was normalized in the penis of SCD mice. [Example]

[0099] Haptoglobin treatment increased PDE5 protein expression in the penis of SCD mice PDE5 protein expression was significantly (p<0.05) reduced by approximately 50% in the penis of the SCD-Vehicle group compared with the WT-Vehicle group (Figure 4D). Haptoglobin treatment increased PDE5 protein levels in the penis of the SCD group by approximately 52% (Figure 4D).

[0100] The combined results of Examples 3 and 4 demonstrate that improved endothelial function increases PDE5 expression. Increased PDE5 expression in corpus cavernosum smooth muscle prevents excessive relaxation induced by stimulation of the NO-GC pathway by ACh, EFS, and SNP in SCD mice. [Example]

[0101] Haptoglobin treatment restores ROCK2 protein expression but not ROCK1 protein expression in the penis of SCD mice ROCK2 protein expression was significantly reduced by approximately 48% in the penis of vehicle-treated SCD mice compared with vehicle-treated WT mice (P < 0.05) (Figure 5A). Haptoglobin treatment completely restored ROCK2 protein levels in the SCD group (Figure 5A). ROCK1 protein expression was unchanged by SCD or haptoglobin treatment compared with the WT-vehicle group (Figure 5B).

[0102] Activation of the RhoA / Rho-kinase signaling pathway acts by increasing Ca2+ sensitivity and is therefore involved in the contractile mechanism of corpus cavernosum smooth muscle (35). ROCK has two isoforms, designated ROCK1 and ROCK2 (35). The results of Example 5 showed that ROCK2 expression was reduced in the corpus cavernosum of SCD mice. The reduction in ROCK2 is consistent with the results of Example 2, which showed reduced contraction in SCD mice. Haptoglobin treatment increased phenylephrine- and KCl-induced contractions and also increased ROCK2 expression. Mice lacking the eNOS enzyme exhibit reduced NO bioavailability and a priapistic phenotype, associated with reduced RhoA / Rho-kinase activity in the penis (36). Previously, haptoglobin treatment restored eNOS expression in SCD mice, indicating that normalized endothelial NO bioavailability is associated with increased ROCK2 expression in the penis. [Example]

[0103] Haptoglobin treatment corrects increased oxidative stress and increased expression of the NADPH oxidase subunit gp91phox protein in the penis of SCD mice The protein expression of gp91phox was significantly higher in the penis of vehicle-treated SCD mice by approximately 67% compared with vehicle-treated WT mice (P < 0.05) (Fig. 6A). Treatment with haptoglobin completely restored the protein level of gp91phox in the SCD group (Fig. 6A).

[0104] The protein expression of 3-nitrotyrosine and 4-HNE was significantly higher in penile tissues of SCD animals by approximately 95% and 75%, respectively, compared with that of the WT group (P < 0.05) (Figures 6B and 6C). Haptoglobin treatment reduced the protein levels of 3-nitrotyrosine and 4-HNE by approximately 27% and 28% in the SCD group (Figures 6B and 6C).

[0105] In this example, haptoglobin treatment reduced gp91phox expression and reduced superoxide anion production in the penis. Consequently, the expression of oxidative stress markers (4-HNE) and nitrosative stress markers (3-NT) was also reduced by haptoglobin treatment. In the penis, gp91phox expression is downregulated by a cGMP-dependent mechanism (42, 43), whereas exogenous NO inhibits NADPH oxidase activity in human endothelial cells through direct S-nitrosylation of the p47phox subunit (44). In this example, normalization of gp91phox subunit expression by haptoglobin treatment may be involved in improving endothelial function. [Example]

[0106] Haptoglobin treatment increased cGMP levels in the penis of SCD mice Basal cGMP content in erectile tissue was 60% lower in the penises of SCD mice compared with WT-vehicle mice (P<0.05) (Fig. 7). Haptoglobin treatment increased cGMP levels by approximately 72% in the penises of the SCD group (Fig. 7).

[0107] The above embodiments are described for illustrative purposes only. Many other embodiments within the scope of the appended claims will be apparent to those skilled in the art. Thus, while the present invention has been described in connection with the preferred embodiment in the various figures, it is understood that other similar embodiments may be used, or modifications or additions may be made to the described embodiments to perform the same functions of the present invention without departing from the scope thereof. Therefore, the present invention should not be limited to a single embodiment, but rather should be construed in breadth and scope based on the recitation of the appended claims.

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Claims

1. Haptoglobin or a nucleic acid encoding haptoglobin for use in the treatment or prevention of hyper-erectile response and / or in the prevention of permanent erectile dysfunction.

2. 2. A haptoglobin or a nucleic acid encoding a haptoglobin for use according to claim 1, wherein the hyper-erectile response is priapism such as that associated with sickle cell disease (SCD).

3. 3. A haptoglobin or a nucleic acid encoding a haptoglobin for use according to claim 1 or 2, wherein the haptoglobin increases the expression of the PDE5 protein.

4. A haptoglobin or a nucleic acid encoding a haptoglobin for use according to any one of claims 1 to 3, wherein the haptoglobin reduces excessive corpus cavernosum relaxation.

5. A haptoglobin or a nucleic acid encoding a haptoglobin for use according to any one of claims 1 to 4, wherein the haptoglobin is human plasma haptoglobin.

6. Haptoglobin or a nucleic acid encoding a haptoglobin for use according to any one of claims 1 to 5, wherein the haptoglobin or the nucleic acid encoding a haptoglobin is administered parenterally, preferably by injection or infusion.

7. The haptoglobin or nucleic acid encoding haptoglobin for use according to any one of claims 1 to 6, wherein the haptoglobin or nucleic acid encoding haptoglobin is administered to a subject in a dosage of 0.5 to 50 g per subject.

8. The haptoglobin or nucleic acid encoding haptoglobin for use according to any one of claims 1 to 7, wherein the haptoglobin or nucleic acid encoding haptoglobin is administered three times a week for a period of one month or more.

9. A pharmaceutical composition for use in the treatment or prevention of hyper-erectile response and / or the prevention of permanent erectile dysfunction, the pharmaceutical composition comprising an adeno-associated virus (AAV) vector carrying a transgene encoding a haptoglobin gene.

10. 10. The pharmaceutical composition for use according to claim 9, wherein the adeno-associated virus (AAV) vector comprises an AAV2 serotype, an AAV5 serotype, an AAV9 serotype, or a combination thereof.

11. 11. The pharmaceutical composition for use according to claim 9 or 10, wherein the hyper-erectile response is priapism such as that associated with sickle cell disease (SCD).

12. 2. The haptoglobin or nucleic acid encoding haptoglobin for use according to claim 1, wherein permanent erectile dysfunction is treated or prevented in patients suffering from sickle cell disease (SCD).

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