Heptoglobin for treatment or prevention of excessive erectile response or erectile dysfunction

Abstract

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

Description

Technical Field

[0001] The present invention relates to haptoglobin or a nucleic acid encoding haptoglobin for treating or preventing excessive erectile response and / or preventing permanent erectile dysfunction. In addition, the present invention also relates to a pharmaceutical composition for treating or preventing excessive erectile response and / or preventing permanent erectile dysfunction, wherein the pharmaceutical composition comprises an adeno-associated virus (AAV) vector carrying a transgene encoding a haptoglobin gene. The excessive erectile response can be priapism, such as priapism associated with sickle cell disease (SCD). Background of the Invention

[0003] Sickle cell disease (SCD) is the most common monogenic disorder and is caused by a point mutation in the β-globin gene. This alteration promotes hemoglobin polymerization under conditions of reduced oxygen, resulting in rigid sickle-shaped red blood cells and hemolytic disease. SCD affects millions of people worldwide, approximately 100,000 Americans, and is most common in sub-Saharan Africa (1). Patients with SCD may exhibit exaggerated erectile responses, such as priapism.

[0004] Priapism is characterized by a persistent, prolonged, and painful penile erection (2). Full or partial erections may last for hours, beyond or in addition to sexual stimulation. Normally, an erection occurs in response to physical or psychological stimulation. This stimulation causes certain smooth muscles to relax, increasing blood flow to the cavernous tissue of the penis. As a result, the penis, which is engorged with blood, becomes erect. When the stimulation ends, the blood flows out and the penis returns to its nonrigid (flaccid) state. Priapism occurs when some part of the system—the blood, blood vessels, smooth muscles, or nerves—alteres normal blood flow, allowing an erection to persist. The underlying cause of priapism is often not identified, but a variety of conditions may play a role. The main types of priapism are ischemic and nonischemic. Ischemic priapism is a medical emergency. Although priapism is rare overall, it is common in certain groups, such as patients with blood disorders such as sickle cell disease, leukemia, or other blood disorders (dyscrasias) such as thalassemia and multiple myeloma. Priapism usually needs to be treated promptly to prevent tissue damage that can lead to the inability to get or maintain an erection (erectile dysfunction). Priapism most often affects men 30 and older, but in men with sickle cell disease it may begin in childhood.

[0005] Repeated episodes of priapism promote fibrosis in the erectile tissue, leading to permanent erectile dysfunction (3). Erectile dysfunction (ED) is the inability to get and maintain an erection hard enough for sexual intercourse. However, if ED is a persistent problem, it can cause stress, affect self-confidence, and lead to relationship problems.

[0006] Clinical studies have shown that 30-45% of men with SCD experience episodes of ischemic priapism, of which 30% progress to erectile dysfunction (4-6). Despite the high incidence of priapism, the main treatments currently available do not prevent priapism (2).

[0007] In SCD, decreased NO bioavailability is associated with increased intravascular hemolysis and oxidative stress (7). Increased reactive oxygen species in the penis are associated with increased gp91phox and p47phox subunits of NADPH oxidase, uncoupled eNOS, and xanthine oxidase (8-10). Previous studies have shown that intravascular hemolysis promotes endothelial dysfunction associated with decreased eNOS activity in SCD mice (33). Decreased NO bioavailability and increased oxidative stress in the penis have been identified as one of the main causes of priapism in men and mice with SCD (8,11-13). Experimental evidence suggests that decreased NO / cGMP bioavailability leads to a compensatory reduction in PDE5 activity and expression in the corpus cavernosum, thereby impairing the mechanisms controlling excessive penile erections (8,11,14).

[0008] Previous studies have investigated the relationship between increased oxidative stress and the pathophysiology of priapism (8,12,29,40). Excess superoxide anions react with NO to form peroxynitrite, a strong oxidant (41). In addition, men and mice with SCD exhibit lower expression and activity of the RhoA-Rho kinase (ROCK) pathway, which is involved in contraction of the corpus cavernosum smooth muscle and maintenance of the flaccid state of the penis (12,15). Several previous studies have looked at the relationship between priapism and levels of intravascular hemolysis in men with SCD (20-22). Administration of haptoglobin to SCD mice and its effects on vasculature have also been previously investigated (23-25,39,46).

[0009] To date, no definitive therapeutic strategy has been established for the specific treatment of patients suffering from excessive erectile response or permanent erectile dysfunction, particularly priapism associated with sickle cell disease. Therefore, it would be highly desirable to provide an innovative method for treating or preventing excessive erectile response and / or preventing permanent erectile dysfunction, particularly those associated with sickle cell disease. SUMMARY OF THE INVENTION

[0011] The present invention relates to haptoglobin or a nucleic acid encoding the same for use in treating or preventing an excessive erectile response and / or preventing permanent erectile dysfunction, preferably in humans.

[0012] The haptoglobin may be naturally occurring haptoglobin, such as human haptoglobin, or recombinant haptoglobin.

[0013] In this context, the excessive erectile response may be a prolonged erectile response. Preferably, the excessive erectile response is priapism, such as priapism associated with sickle cell disease (SCD). Optionally, the haptoglobin increases PDE5 protein expression. Alternatively or additionally, the haptoglobin reduces excessive corpus cavernosum relaxation.

[0014] The permanent erectile dysfunction may be an exacerbation of priapism associated with sickle cell disease (SCD). Preferably, the permanent erectile dysfunction occurs in a patient suffering from SCD.

[0015] Preferably, the haptoglobin is human plasma haptoglobin. Optionally, the haptoglobin or the nucleic acid encoding the haptoglobin is administered parenterally, preferably by injection or infusion. The dose of the haptoglobin or the nucleic acid encoding the haptoglobin can be 0.5 to 20 g per subject. It can also be administered according to the subject's body weight. Preferably, the haptoglobin or the nucleic acid encoding the haptoglobin is administered three times a week for a period of one month or longer.

[0016] According to another aspect of the present invention, it provides a pharmaceutical composition for treating or preventing an excessive erectile response and / or preventing permanent erectile dysfunction. The excessive erectile response is preferably priapism, such as priapism associated with or caused by sickle cell disease (SCD). Permanent erectile dysfunction may be an aggravation of priapism associated with or caused by sickle cell disease (SCD). Preferably, the permanent erectile dysfunction occurs in patients with 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 an AAV2 serotype, an AAV5 serotype, an AAV9 serotype or a combination thereof.

[0017] The present invention also relates to a method for treating or preventing excessive erectile response and / or preventing permanent erectile dysfunction. The method comprises administering to a subject a therapeutically effective amount of a haptoglobin or a nucleic acid encoding a haptoglobin or a pharmaceutical composition as described above. Preferably, the subject is a human.

[0018] Attached photos

[0019] Specific embodiments of the present invention will now be described by way of example with reference to the accompanying drawings, in which:

[0020] Figure 1Concentration response curves to acetylcholine (ACh; AB) and sodium nitroprusside (SNP, CD) in corpus cavernosum strips of WT and SCD mice treated or not with haptoglobin (400 mg / kg) or vehicle on Mondays, Wednesdays, and Fridays for a period of 1 month. Data are relative to the concentration of phenylephrine (10 -5 M) The maximum contraction change produced (taken as 100%) was calculated. Data represent the mean±SEM of 6 mice per group. *P<0.05 vs WT-vehicle.

[0021] Figure 2 Relaxation responses induced by electric field stimulation (EFS) in the corpus cavernosum strips of WT and SCD mice treated or not with haptoglobin (400 mg / kg) or vehicle on Mondays, Wednesdays, and Fridays for a period of 1 month. Data are relative to the relaxation responses induced by phenylephrine (10 -5 M) The maximum contraction change produced (taken as 100%) was calculated. Data represent the mean±SEM of 6 mice per group. *P<0.05 vs WT-vehicle.

[0022] Figure 3 .Concentration-response curves for phenylephrine (AB) and KCl (CD) in corpus cavernosum strips of WT and SCD mice treated or not with haptoglobin (400 mg / kg) or vehicle every Monday, Wednesday, and Friday for a period of 1 month. Data are shown in mN and represent the mean ± SEM of 6 mice per group. *P<0.05 vs WT-vehicle.

[0023] Figure 4 Representative Western blot images (upper panel) and protein values ​​(lower panel) of p-eNOS (Ser-1177; A), eNOS (B), nNOS (C), and PDE5 (D) in penile homogenates of WT and SCD mice treated or not with haptoglobin (400 mg / kg) or vehicle every Monday, Wednesday, and Friday for a period of 1 month. Data are shown in mN and represent mean ± SEM of 6 mice per group. *P<0.05 vs WT-vehicle. Single bands of eNOS, nNOS, and PDE5 were normalized to β-actin. Single band of p-eNOS (Ser-1177) was normalized to eNOS.

[0024] Figure 5Representative Western blot images (upper panel) and protein values ​​(lower panel) of ROCK2 (A) and ROCK1 (B) in penile homogenates of WT and SCD mice treated or not with haptoglobin (400 mg / kg) or vehicle every Monday, Wednesday, and Friday for a period of 1 month. Data are shown in mN and represent the mean ± SEM of 6 mice per group. *P < 0.05 vs WT-vehicle. Single bands of ROCK1 and ROCK2 were normalized to β-actin.

[0025] Figure 6 Representative Western blot images (upper panel) and protein values ​​(lower panel) of gp91phox (A), 3-NT (B), and 4-HNE (C) in corpus cavernosum strips of WT and SCD mice treated or not with haptoglobin (400 mg / kg) or vehicle every Monday, Wednesday, and Friday for a period of 1 month. Data are shown in mN and represent the mean ± SEM of 6 mice per group. *P < 0.05 vs WT-vehicle. Individual strips of gp91phox, 3-NT, and 4-HNE were normalized to β-actin.

[0026] Figure 7 .Basal cGMP content in the penis of WT and SCD mice treated or not with haptoglobin. Data represent mean ± SEM of 6 mice per group. *P < 0.05 vs WT-vehicle; #P < 0.05 vs SCD-Hap.

[0027] Detailed Description

[0028] The process of intravascular hemolysis results in the release of hemoglobin into the plasma (16). Under physiological conditions, haptoglobin is the plasma protein responsible for preventing the accumulation of free hemoglobin in the body (17). In the plasma, haptoglobin binds to free hemoglobin to form a hemoglobin-haptoglobin complex, which is metabolized by macrophages in the reticuloendothelial system. Macrophages express the CD163 receptor, which mediates the endocytosis and internalization of the hemoglobin-haptoglobin complex (17). However, in SCD, high concentrations of hemoglobin are released into the plasma, depleting haptoglobin, thereby accumulating free hemoglobin in the plasma (16,18,19). Hemoglobin in the plasma or interstitial space reacts with NO to generate nitrate and methemoglobin (16). In SCD, elevated plasma hemoglobin and heme levels promote vascular and oxidative damage (37-39).

[0029] While not wishing to be bound by theory, the inventors of the present invention have unexpectedly discovered that haptoglobin treatment can reverse excessive erectile responses, particularly those caused by stimulation of the NO-cGMP pathway, by normalizing the expression of eNOS and PDE5, as well as normalizing the contractile activity and increased oxidative stress of the corpus cavernosum.

[0030] While not wishing to be bound by theory, it is believed that this improvement is achieved through upregulation of eNOS-PDE5 expression and downregulation of the gp91phox subunit of NADPH oxidase and oxidative / nitrative stress in the penis.

[0031] Haptoglobin can reduce excessive corpus cavernosum relaxation. Alternatively or additionally, haptoglobin can 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 cGMP levels in the penis (30). The penises of men and mice with SCD show lower basal NO production due to lower expression and activity of eNOS (9, 10, 12, 31). Low bioavailability of NO leads to reduced activation of sGC, a heterodimeric enzyme containing a heme group that catalyzes the synthesis of the second messenger cGMP (32). Low expression of PDE5 in the penis may lead to increased corpus cavernosum relaxation (8, 29). The inventors of the present invention unexpectedly discovered that haptoglobin treatment can help increase PDE5 protein expression, thereby treating or preventing excessive erectile responses.

[0032] Haptoglobin and its variants

[0033] Haptoglobin (Hp) is a tetrameric plasma glycoprotein with a molecular weight of approximately 90,000 kDa. Produced by hepatocytes, its main function is to prevent iron loss due to hemolysis of erythrocytes through its affinity and binding ability to free hemoglobin (Hb). Hp is synthesized as a single chain and then cleaved into an amino-terminal α chain and a carboxyl-terminal β chain after translation. The basic structure of Hp found in most mammals is a homodimer, in which two Hp molecules are linked by a single disulfide bond through their respective 9kDa α chains. In humans, there is also a variant with a long α chain in all populations. This variant was apparently generated by early intragenic duplication (duplication), probably by unequal crossing of two basic alleles, resulting in Hp with a 14kDa α chain. The short α chain and the long α chain are called α1 and α2, respectively. Since the cysteine ​​that forms the disulfide bond between the α chains is also duplicated, people carrying the long variant allele exhibit a multimeric Hp phenotype. The presence of two isoforms of the α chain determines the existence of subtypes within the Hp phenotype Hp2-1 and Hp1-1. The α-1f and α-1s isoforms differ only in the amino acid sequence at positions 52 and 53, with the α-1f chain containing aspartic acid and lysine, while the α-1s chain has two residues, an asparagine and a glutamic acid (47). These different isoforms are essentially identical in hemoglobin affinity, binding, and clearance properties, with only minor differences in function.

[0034] 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 believed to be derived 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 includes an amino-terminal 18-residue signal sequence preceding the α chain and / or an insertion polypeptide between the α and β regions (49). In vivo, post-translational events lead to the proteolytic removal of the signal sequence and the incorporation of core oligosaccharide side chains into the β region by a membrane-associated enzyme system (48). Post-translational modifications can also result in the cleavage of the α and β regions of the precursor polypeptide to form the native protein (48).

[0035] It should be understood that as long as naturally occurring and recombinant forms of Hp can form complexes with acellular Hb to neutralize the biological activity of acellular Hb, they are suitable for use in the present invention. Suitable naturally occurring forms of Hp are known to those skilled in the art, examples of which include Koch et al. (50) and Kasvosve et al. (51), the entire contents of which are incorporated herein by reference.

[0036] 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 a protein disclosed under NCBI accession number NP_005134 or UniProt accession number P00738. This UniProt entry has various variants. Those skilled in the art will be familiar with protocols for isolating Hp from its natural source (e.g., plasma).

[0037] The sequence of the β chain of human haptoglobin may be:

[0038] ILGGHLDAKGSFPWQAKMVSHHNLTTGATLINEQWLLTTAKNLFLNHSENATAKDIAPTLTLYVGKKQLVEIEKVVLHPNYSQVDIGLIKLKQKVSVNERVMPICLPSKDYAEVGRVGYVSGWG RNANFKFTDHLKYVMLPVADQDQCIRHYEGSTVPEKKTPKSPVGVQPILNEHTFCAGMSKYQEDTCYGDAGSAFAVHDLEEDTWYATGILSFDKSCAVAEYGVYVKVTSIQDWVQKTIAEN(SEQ ID NO:3)

[0039] The sequence of the α2 chain of human haptoglobin may be:

[0040] VDSGNDVTDIADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVYTLNDKKQWINKA VGDKLPECEADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVYTLNNEKQWINKAVGD KLPECEAVCGKPKNPANPVQ(SEQ ID NO:4)

[0041] The sequence of the α1 chain of human haptoglobin may be:

[0042] VDSGNDVTDIADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVYTLNNEKQWINKAVGDKLPECEAVCGKPKNPANPVQ(SEQ ID NO:5)

[0043] The canonical sequence of human haptoglobin α2-β is shown as isoform 1 in P00738 (last updated: 1986-07-21). The sequence of isoform 1 is: MSALGAVIALLLWGQLFAVDSGNDVTDIADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVYTLNDKKQWINKAVGDKLPECEADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVYTLNNEKQWINKAVGDKLPECEAVCGKPKNPANPVQRILGGHLDAKGSFPW QAKMVSHHNLTTGATLINEQWLLLTTAKNLFLNHSENATAKDIAPTLTLYVGKKQLVEIEKVVLHPNYSQVDIGLIKLKQKVSVNERVMPICLPSKDYAEVGRVGYVSGWGRNANFKFTDHLKYVMLPVADQDQCIRHYEGSTVPEKKTPKSPVGVQPILNEHTFCAGMSKYQEDTCYGDAGSAFA

[0044] VHDLEEDTWYATGILSFDKSCAVAEYGVYVKVTSIQDWVQKTIAEN(SEQ ID NO:1)

[0045] The sequence of another human haptoglobin (α1-β) isoform produced due to alternative splicing is shown as isoform 2 in P00738. The sequence of isoform 2 differs from the standard isoform 1 by the lack of amino acids 38-96. The sequence of isoform 2 can be:

[0046] MSALGAVIALLLWGQLFAVDSGNDVTDIADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRT

[0047] EGDGVYTLNNEKQWINKAVGDKLPECEAVCGKPKNPANPVQRILGGHLDAKGSFPWQAKM

[0048] VSHHNLTTGATLINEQWLLTTAKNLFLNHSENATAKDIAPTLTLYVGKKQLVEIEKVVLH

[0049] PNYSQVDIGLIKLKQKVSVNERVMPICLPSKDYAEVGRVGYVSGWGRNANFKFTDHLKYV

[0050] MLPVADQDQCIRHYEGSTVPEKKTPKSPVGVQPILNEHTFCAGMSKYQEDTCYGDAGSAF

[0051] AVHDLEEDTWYATGILSFDKSCAVAEYGVYVKVTSIQDWVQKTIAEN(SEQ ID NO:2)

[0052] The term "haptoglobin" as used herein should be understood to include all phenotypes of Hp (including all isotypes). Haptoglobin can be homologous (as long as it is essentially composed of Hp of the same isotype) or heterologous (including a combination of different Hp isotypes, different Hp isotypes include Hp1-1, Hp1-2 and Hp2-2). Optionally, the haptoglobin for use according to the present invention may be a mixture of two or more phenotypes and / or isotypes. Preferably, the haptoglobin for use according to the present invention includes Hp2-2. More preferably, the haptoglobin for use according to the present invention is a mixture of Hp2-2 and Hp 1-2. It should be understood that the composition of Hp ultimately depends on the phenotype of the source. For example, if a combined plasma sample is used to extract / purify Hp, one or more isotypes of Hp may be isolated. Those skilled in the art will be familiar with suitable methods for determining the Hp isotypes present in the isolate.

[0053] In one embodiment, Hp is selected from the group consisting of Hp1-1 homodimers, Hp1-2 multimers, Hp2-2 multimers, and combinations thereof. Hp may be naturally occurring Hp (e.g., derived from plasma) or it may be produced as a recombinant protein. In some embodiments, haptoglobin comprises naturally occurring human plasma haptoglobin. Optionally, plasma-derived Hp comprises, consists of, or consists essentially of Hp2-2. Alternatively, plasma-derived Hp comprises, consists of, or consists essentially of Hp1-1. In a further aspect, Hp comprises, consists of, or consists essentially of recombinant Hp.

[0054] The term Hp as used herein includes naturally occurring or functional analogs of naturally occurring Hp. The term "functional analog" is intended to refer to an agent having substantially the same biological activity as naturally occurring (native) Hp, as long as the biological activity is at least the ability of the analog to form a complex with cell-free Hb to neutralize its biological activity.

[0055] The term "substantially the same biological activity" generally means that a functional analog has at least 40% of the binding affinity to Hb (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.). Those skilled in the art will be familiar with suitable methods for determining whether an agent is a functional analog of Hp.

[0056] Optionally, the functional analog of Hp is a functional fragment of native Hp. The functional fragment of native Hp may be of any suitable length, as long as the fragment retains the ability to form a complex with cell-free Hb and neutralize its biological activity.

[0057] Alternatively, a functional analog is a polypeptide having an amino acid sequence different from that of a naturally occurring (natural) Hp molecule (i.e., a comparison object). Functional analogs differ from the amino acid sequence of the α chain and / or β chain of natural Hp in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) amino acid substitutions, and these amino acid substitutions have different amino acid sequences, wherein this difference does not cancel or completely eliminate the ability of the analog to form a complex with acellular Hb and neutralize its biological activity. In certain embodiments, a functional analog comprises an amino acid substitution that enhances the ability of the analog to form a complex with acellular Hb compared to natural Hp. In one embodiment, a functional analog has an amino acid sequence that differs from the amino acid sequence of the α chain and / or β chain of natural Hp in one or more conservative amino acid substitutions. As used herein, the term "conservative amino acid substitution" refers to changing the amino acid identity of a given position and replacing it with an amino acid that is roughly equivalent in size, charge, and / or polarity. Examples of naturally conservative amino acid substitutions include the following eight substitution groups (represented 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.

[0058] In one embodiment, the functional analog has at least 85% or higher sequence identity with the amino acid sequence of the α chain and / or β chain of native H. pylori.

[0059] Mention " at least 85% " means, for example, after optimal alignment or best fit analysis, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or similarity. Therefore, 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 chain and / or β chain of natural Hp.

[0060] 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 the two optimally aligned sequences. Conservative substitutions are not considered part of the identity. N-terminal or C-terminal extensions or insertions should not be considered to reduce sequence identity or homology.

[0061] To determine the "percent identity" of two amino acid or nucleic acid sequences, the sequences are aligned together. To obtain the best match, gaps (i.e., deletions or insertions) may be introduced into the sequence, which may also be placed at the ends of the sequence. The amino acids and nucleotide residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions divided by the total number of sequences [i.e., % identity = (number of identical positions / total number of positions) x 100].

[0062] In this article, the term "similarity" or "sequence similarity" means that at any specific position of the aligned sequences, the amino acid residues have similar types between the sequences. For example, leucine can be substituted with isoleucine or valine residues. As described elsewhere herein, this can be referred to as conservative substitutions. In one embodiment, the amino acid sequence is modified by conservative substitutions of any amino acid residues contained therein so that, compared with unmodified (natural) Hp polypeptides, the modification does not affect the binding specificity or functional activity of the modified polypeptide.

[0063] Percent identity can be obtained using a mathematical algorithm. A non-limiting example of an algorithm for comparing two sequences is contained in the BLASTn and BLASTp programs of Altschul

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

[0064] In one embodiment, the functional analog comprises 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.

[0065] Functional analogs may be naturally occurring polypeptides or may be produced synthetically by chemical synthesis using methods known in the art.

[0066] Hp can be suitably produced as recombinant protein in microorganisms, and these microorganisms can be separated and further purified when necessary. Those skilled in the art will be familiar with microorganisms suitable for producing recombinant Hp, examples of which are bacteria, yeast or fungi, eukaryotic cells (such as mammals or insect cells), or recombinant viral vectors (such as adenovirus, poxvirus, herpes virus, shimki forest fever virus, baculovirus, phage, Sindbis virus or Sendai virus). Those skilled in the art will be familiar with bacteria suitable for producing recombinant peptides, examples of which include Escherichia coli (E.coli), Bacillus subtilis (B.subtilis), or any other bacteria capable of expressing peptide sequences. Examples of yeast types suitable for producing recombinant peptides include Candida (Candida), Pichia pastoris (blood Chiapas Pastoris) (Pichia pastoris), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), or any other yeast capable of expressing peptides. These 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, and ion exchange chromatography.

[0067] In order to facilitate the separation of recombinant Hp as described herein, a fusion polypeptide can be produced in which the peptide sequence of Hp or its functional analog is translated and fused (covalently bound) to a heterologous polypeptide that allows separation by affinity chromatography. Examples of suitable heterologous polypeptides are His tags (e.g., His6: 6 histidine residues), GST tags (glutathione transferase-S-), etc.

[0068] For the production of recombinant Hp, phage libraries and / or peptide libraries are also suitable, for example generated by binding chemistry or obtained by high throughput screening techniques for the most complex structures.

[0069] Examples of recombinant Hp are NCBI accession number NP_005134 (as described by Morishita et al. (53)) and UniProt accession number P00738.

[0070] Haptoglobin variants with extended half-life

[0071] According to the present invention, the haptoglobin can be fused, coupled or attached to one or more heterologous parts as part of a fusion protein. One or more heterologous parts can improve, enhance or prolong the activity or stability of Hp. In one embodiment, Hp is appropriately attached to the heterologous part to prolong the half-life of Hp in vivo. Those skilled in the art will be familiar with heterologous parts suitable for extending the half-life, examples of which are polyethylene glycol (PEGylation), glycosylated PEG, hydroxyethyl starch (HESylation), polysialic acid, elastin-like polypeptide, heparin acid (heparo acid) polymer and hyaluronic acid. Therefore, in certain embodiments disclosed herein, the heterologous part is selected from the group consisting of polyethylene glycol (PEGylation), glycosylated PEG, hydroxyethyl starch (HESylation), polysialic acid, elastin-like polypeptide, heparin acid polymer and hyaluronic acid. In another embodiment, the heterologous part can be a heterologous amino acid sequence fused to Hp.

[0072] Alternatively or additionally, the heterologous moiety can be chemically conjugated, e.g., covalently linked to Hp. The heterologous moiety that extends half-life can be fused, conjugated, or otherwise attached to Hp by any suitable method known to those skilled in the art, exemplified by a chemical linker. The principles of this bonding technology have been described by way of example by Conjuchem LLC (see, e.g., U.S. Patent No. 7,256,253), the entire contents of which are incorporated herein by reference.

[0073] In another embodiment, the heterologous part is a half-life enhancing protein (HLEP). Suitable half-life enhancing proteins are familiar to those skilled in the art and include, for example, albumin or a fragment thereof. Therefore, in one embodiment, the HLEP is albumin or a fragment thereof. The N-terminus of albumin or a fragment thereof can be fused to the C-terminus of the α and / or β chain of Hp. Alternatively or additionally, the C-terminus of albumin or a fragment thereof can be fused to the N-terminus of the α and / or β chain of Hp. As long as the binding of Hp to acellular Hb is not disrupted, one or more HLEPs can be fused to the N-terminal or C-terminal portion of the α and / or β chain of Hp. However, it should be understood that a slight reduction in the binding of Hp to acellular Hb is acceptable as long as the Hp portion of the fusion protein can still form a complex with the acellular Hb to neutralize the acellular Hb.

[0074] The fusion protein may further comprise a chemical bond or linker sequence between the Hp and the heterologous portion. The linker sequence comprises at least one amino acid, particularly 1 to 50, preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 5. It may be a peptide linker of 5 or more preferably 1 to 3 (e.g. 1, 2 or 3) amino acids, which may be the same or different from each other. Preferably, the linker sequence does not exist at the corresponding position of the wild-type Hp. The amino acids preferably 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 method disclosed herein. Substantially non-immunogenic means that the linker sequence will not induce a detectable antibody response to the linker sequence in the subject to which it is applied. A preferred linker may consist of alternating glycine and serine residues. Suitable linkers are familiar to those skilled in the art and are described, for example, in WO 2007 / 090584. In one embodiment, the peptide linker between Hp and the heterologous part comprises, consists of or consists essentially of a peptide sequence that is a linker between natural domains of human proteins. In the natural environment, these peptide sequences can be located near the surface of the protein and can be exposed to the immune system, so it can be assumed that there is a natural tolerance to these sequences. Examples are provided in WO 2007 / 090584. Suitable cleavable linker sequences are described, for example, in WO 2013 / 120939 A1.

[0075] Examples of suitable HLEP sequences are described below. Similarly, fusions to the exact "N-terminal amino acid" or the exact "C-terminal amino acid" of the respective HLEP, or to the "N-terminal portion" or "C-terminal portion" of the respective HLEP, including N-terminal deletions of one or more amino acids of the HLEP are also described. The fusion protein may comprise one or more HLEP sequences, for example two or three HLEP sequences. These multiple HLEP sequences may be fused in series, for example in the form of a continuous repeat sequence, to the C-terminal portion of the α and / or β chain of Hp.

[0076] In one embodiment, the heterologous moiety is a half-life-extending polypeptide. In one embodiment, the half-life-extending polypeptide comprises albumin, an albumin family member or a fragment thereof, a solvated random chain with a large hydrodynamic volume (e.g., XTEN (56), a homologous amino acid repeat sequence (HAP) or a proline-alanine-serine repeat sequence (PAS), apamin, alpha-fetoprotein, vitamin D binding protein, transferrin or a variant or fragment thereof, the carboxyl terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, a neonatal Fc receptor (FcRn), in particular an immunoglobulin constant region and a portion thereof, such as a polypeptide capable of binding to an Fc fragment. It is selected from the group consisting of a family member or a fragment thereof or a polypeptide or a lipid capable of binding to an immunoglobulin constant region or a portion thereof. The immunoglobulin constant region or a portion thereof is preferably an Fc fragment of immunoglobulin G1 (IgG1), an Fc fragment of immunoglobulin G2 (IgG2) or an immunoglobulin A Fc fragment of (IgA). HLEP can be a full-length half-life enhancing protein or one or more fragments thereof that can stabilize or extend the therapeutic activity or biological activity of Hp, in particular, a fragment that increases the half-life of Hp in vivo. This fragment can be more than 10 amino acids in length, or at least about 15, preferably at least about 20, more preferably at least about 25, more preferably at least about 30, more preferably at least about 50, or more preferably at least about 100 or more consecutive amino acids from the HLEP sequence, or can contain part or all of a specific domain of a specific HLEP, as long as the HLEP fragment can provide at least 10%, preferably at least 20%, more preferably at least 25% functional half-life extension compared to Hp without HLEP. Methods (in vivo or in vitro) for determining whether a heterologous portion provides a functional half-life extension to Hp are familiar to those skilled in the art.

[0077] The HLEP portion of the fusion protein described herein can be a variant of the wild-type HLEP. The term "variant" includes insertions, deletions and / or substitutions, whether conservative or non-conservative, wherein these changes do not significantly change the ability of Hp to form a complex with acellular Hb to neutralize acellular Hb. The HLEP can be suitably derived from any vertebrate, particularly any mammal, such as a human, monkey, cattle, sheep or pig. Non-mammalian HLEPs include, but are not limited to, chicken and salmon.

[0078] The fusion proteins described herein can be produced by in-frame connection of at least two DNA sequences encoding heterologous parts such as Hp and HLEP. It will be appreciated by those skilled in the art that translation of the fusion protein DNA sequence will produce a single protein sequence. Due to the in-frame insertion of a DNA sequence encoding a peptide linker according to aspects disclosed herein, a fusion protein comprising Hp, a suitable linker and a heterologous part can be obtained.

[0079] As used herein, "albumin" collectively refers to an albumin polypeptide or amino acid sequence, or an albumin fragment or variant, that has one or more functional (e.g., biological) activities of albumin. Specifically, "albumin" refers to human albumin or a fragment thereof, including the mature form of human albumin, or albumin or a fragment thereof from other vertebrates, or an analog or variant of these molecules or fragments thereof. In certain aspects disclosed herein, the alternative term "FP" is used to identify HLEPs, particularly for defining albumin as a HLEP.

[0080] The fusion proteins described herein may suitably comprise naturally occurring polymorphic variants of human albumin and / or fragments of human albumin. Generally, the length of the albumin fragment or variant will be at least 10 amino acids, preferably at least 40, or most preferably at least 70 amino acids.

[0081] In one embodiment, HLEP is an albumin variant with enhanced FcRn receptor binding ability. Such albumin variants can result in a longer plasma half-life of Hp or its functional analogs than Hp or its functional fragment fused to wild-type albumin. The albumin portion of the fusion protein described herein can appropriately include at least one subdomain or domain of human albumin or a conservative modification thereof.

[0082] In one embodiment, the heterologous portion is an immunoglobulin molecule or a functional fragment thereof. It is known in the art that the immunoglobulin G (IgG) constant region (Fc) can increase the half-life of a therapeutic protein (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 IgG1, IgG2, IgG3 or IgG4 subclasses, respectively. IgG and IgG fragments lacking an antigen binding domain can also be used as heterologous portions, including as HLEPs. Hp or a functional analog thereof can be appropriately linked to an IgG or IgG fragment via the hinge region of an antibody or a cleavable peptide linker. Several publications describe the fusion of a therapeutic protein to an immunoglobulin constant region to enhance the half-life of a therapeutic protein in vivo. For example, US2004 / 0087778 and WO 2005 / 001025 disclose fusion proteins in which a biologically active peptide is fused to at least a portion of an Fc domain or an immunoglobulin constant region, which increases the half-life of the peptide and is otherwise rapidly cleared in vivo. Fc-IFN-β fusion proteins that achieve enhanced biological activity, extended circulation half-life, and higher solubility have been described (WO 2006 / 000448 A2). Fc-EPO proteins with extended serum half-life and enhanced in vivo efficacy have been disclosed (WO 2005 / 063808 A1), as well as Fc fusion proteins fused to G-CSF (WO 2003 / 076567 A2), glucagon-like peptide-1 (WO2005 / 000892 A2), coagulation factors (WO 2004 / 101740 A2), and interleukin-10 (U.S. Pat. No. 6,403,077), all with half-life enhancement properties.

[0083] Examples of suitable HLEPs that may be used according to the present invention are also described in WO 2013 / 120939 A1, the contents of which are incorporated herein by reference in their entirety.

[0084] Nucleic acid encoding haptoglobin

[0085] As an alternative to haptoglobin, a nucleic acid encoding haptoglobin can be used in the present invention. The haptoglobin can be any of the above-mentioned haptoglobins, including variants, fragments and fusion proteins thereof.

[0086] In humans, haptoglobin is encoded by the HP gene, e.g., NCBI accession numbers NP_001119574, NP_001305067, NP_005134. The Hp gene has two major alleles, Hp1 and Hp2, located on chromosome 16. These two alleles are responsible for three different possible genotypes and structural polymorphisms: homozygous (1-1 or 2-2) and heterozygous 2-1.

[0087] The term "nucleic acid" as used herein generally refers to any nucleotide molecule encoding the haptoglobin according to the present invention and having a variable length. Examples of nucleic acids of the present invention include, but are not limited to, plasmids, vectors, or DNA and / or RNA fragments of any kind. The nucleic acid molecules used in the present invention may be in the form of RNA, such as mRNA or cRNA, or in the form of DNA, including, for example, cDNA and genomic DNA, such as obtained by cloning, or produced by chemical synthesis techniques or a combination thereof. DNA may be triple-stranded, double-stranded, or single-stranded. Single-stranded DNA may be a coding strand, also referred to as a sense strand, or may be a non-coding strand, also referred to as an antisense strand. The nucleic acid molecules used herein also refer to 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 comprising DNA and RNA, which may be single-stranded or usually double-stranded, or triple-stranded, or a mixture of single-stranded and double-stranded regions. In addition, the nucleic acid molecules used herein also refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. In addition, the haptoglobin encoding nucleic acid molecule according to the present invention can be functionally linked to any desired sequence, such as a regulatory sequence, a leader sequence, a heterologous marker sequence or a heterologous coding sequence to produce a fusion protein by using standard techniques, such as standard cloning techniques.

[0088] The nucleic acid according to the present invention can be present in a vector, such as a plasmid or a viral vector, together with appropriate promoter, enhancer, marker and other sequences, so that the polypeptide or mRNA is expressed in the target cell, tissue or body fluid. The nucleic acid of the present invention can also include a nucleic acid sequence encoding a haptoglobin receptor or a haptoglobin binding fragment thereof.

[0089] Gene therapy

[0090] According to another aspect of the present invention, it relates to a pharmaceutical composition for treating or preventing excessive erectile response and / or preventing permanent erectile dysfunction, comprising an adeno-associated virus (AAV) vector with 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.

[0091] Many (viral) delivery systems have been investigated, each with its advantages and disadvantages. One of the viral delivery vehicles used for gene therapy is the adeno-associated virus (AAV).

[0092] AAV has a single-stranded DNA genome of approximately 4.8 kilobases (kb). AAV belongs to the Parvoviridae family and relies on co-infection with other viruses, especially adenoviruses, for replication. The genome includes Rep (replication) and Cap (capsid) genes. 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 the viral genome and promote packaging, while Cap expression produces viral capsid proteins (VP; VP1VP2VP3) to form the outer capsid.

[0093] Recombinant AAV (rAAV) for gene therapy is formed by a protein capsid containing the desired nucleic acid (transgene) to be delivered to the target cell. The desired nucleic acid is flanked by the ITR of AAV. The transgene encoded by rAAV flanked by the ITR can form a circular concatemer that remains as an episome in the transduced cell nucleus. Since the episome is mainly maintained as an episome, the expression of the nucleic acid sequence delivered by AAV may be diluted over time if and when the target cell replicates. This dilution may not generally apply to post-mitotic cells such as neurons, which are target cells for many neurodegenerative diseases. Naso et al. Biodrugs 2017 (pp. 317-334) provides a review of AAV vectors for gene therapy.

[0094] In some embodiments, the adeno-associated viral vector comprises an AAV2 serotype, an AAV5 serotype, an AAV9 serotype, a mixed AAV serotype, or a combination thereof. In some embodiments, the adeno-associated viral vector comprises an AAV5 serotype. In some embodiments, the adeno-associated viral vector comprises an AAV9 serotype.

[0095] In some embodiments, the adeno-associated viral vector comprises a mixed AAV serotype. By way of example, the mixed AAV serotype can be a mixed AAV2 / AAV5; AAV2 / AAV9; or AAV5 / AAV9 serotype.

[0096] Application

[0097] The haptoglobin or nucleic acid of the present invention can be administered to a subject in the form of a lyophilized preparation or an aqueous solution. The dosage form may further comprise one or more optional pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to: buffers such as phosphate, citric acid and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; phenethylammonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). The haptoglobin or nucleic acid of the present invention as an active ingredient can be encapsulated in microcapsules, such as microcapsules prepared by coacervation technology or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, for colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Dosage forms for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0098] Preferably, the haptoglobin or the nucleic acid encoding haptoglobin of the present invention is administered parenterally, more preferably by injection or infusion. For example, the haptoglobin or the nucleic acid encoding haptoglobin is systemically administered, for example, by intravenous administration, such as intravenous injection or infusion. Alternatively, the haptoglobin or the nucleic acid encoding haptoglobin is administered by subcutaneous injection. The dose of Hp can also be adjusted to provide the best therapeutic response. For example, several divided doses can be administered daily, once a week, or at other suitable time intervals, or the dose can be reduced proportionally according to the urgency of the situation. Optionally, the haptoglobin or the nucleic acid encoding haptoglobin is administered three times a week for a period of one month or longer. Alternatively, the haptoglobin or the nucleic acid encoding haptoglobin can be administered for a period of one to three months. Preferably, the haptoglobin or the nucleic acid encoding haptoglobin is administered in a repeated mode of two 48-hour and one 72-hour dosing intervals.

[0099] The above-mentioned administration routes are also applicable to the pharmaceutical compositions of the present invention.

[0100] The term "therapeutically effective amount" as used herein refers to an amount or concentration of Hp sufficient to neutralize the adverse biological effects of acellular Hb by binding and complexing with acellular Hb present in plasma. It will be appreciated by those skilled in the art that a therapeutically effective amount of a peptide may vary depending on a variety of factors, examples of which include 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 the bleeding), the concentration and / or amount of acellular Hb in plasma, and any combination of the above factors. Any combination is included.

[0101] The therapeutically effective amount of Hp is generally within a relatively wide range that can be determined by a person of ordinary skill in the art. Optionally, the therapeutically effective amount of Hp is about 0.5 g to about 50 g per subject, 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, about 0.5 g to about 10 g. Preferably, haptoglobin or nucleic acid encoding haptoglobin can be administered to each subject at 1 g to 5 g, more preferably 1.5 g to 3 g per subject. The concentration of Hp to be administered can be about 2 μM to about 20 mM per subject, 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, and the amount of administration can be 1 to 50 mL, 1 to 40 mL, 1 to 30 mL, 3 to 20 mL, for example 5 to 10 mL per subject. Alternatively, the therapeutically effective amount of Hp can be based on the subject's body weight. For example, the therapeutically effective amount of Hp can be 1 to 100 mg per kg of subject body weight, optionally 1 to 80 mg per kg of subject body weight, 5 to 75 mg per kg of body weight, 10 to 50 mg per kg of body weight, 15 to 50 mg per kg of body weight, 20 to 40 mg per kg of body weight, or 25 to 35 mg per kg of body weight.

[0102] In some embodiments, haptoglobin or a nucleic acid encoding haptoglobin is administered at a dose of 5 to 10 mL per subject at a concentration of 50 to 220 mg / dL (5.88 to 25.87 μM). In another embodiment, a therapeutically effective amount of Hp is an amount sufficient to form a complex with about 3 μM to about 300 μM, about 5 μM to about 250 μM, about 10 μM to about 200 μM, about 50 μM to about 150 μM cell-free Hb in plasma. Methods suitable for determining the concentration of cell-free Hb in CSF are known to those skilled in the art, examples of which are described in Cruickshank et al. (54) and Hugelshofer M. et al. (55), the entire contents of which are incorporated herein by reference.

[0103] For the pharmaceutical composition of the present invention, each subject may be administered a dose of about 1E12 to about 5E14, about 5E12 to about 1E14, or about 1E13 to about 1E14 genome copies of an adeno-associated virus (AAV) vector.

[0104] Any feature described in any one of the above aspects or embodiments is also disclosed herein as being relevant to all other aspects and embodiments. Similarly, all combinations of any two or more of the above-mentioned individual features or elements may be present in any aspect or embodiment. For the sake of brevity, all possible features and combinations are not listed in all aspects and embodiments, but they are clearly considered and disclosed in this article. Example

[0105] Ethical approval

[0106] All protocols in the examples were approved by the Animal Experimentation Ethics Committee of the University of Campinas (IACUC / CEEA-UNICAMP, license number 4754-1 / 2017).

[0107] Material

[0108] ACh, phenylephrine, guanethidine, and atropine were obtained from Sigma-Aldrich (St Louis, MO, USA). Human haptoglobin (Hp) solution was kindly provided by CSL Behring (Bern, Switzerland). All reagents were required to be analytically pure. Deionized water was used as a solvent, and the working solution was diluted before use.

[0109] Animals and handling

[0110] All mouse strains were originally purchased from Jackson Laboratories (Bar Harbor, ME). Characterization and breeding were performed at the Multidisciplinary Laboratory Animal Bioscience Research Center of the University of Campinas. C57BL / 6 male mice (wild type, WT) and Berkeley transgenic SCD mice aged 3 to 4 months were used. Mice were housed three per cage under a 12-h light-dark cycle. SCD male mice were treated with haptoglobin (400 mg / kg, subcutaneous) or vehicle every Monday, Wednesday, and Friday for a period of 1 month (24).

[0111] Statistical analysis

[0112] Statistical analysis was performed using the GraphPad Prism program (GraphPad Software Inc.). Data are presented as mean ± SEM of N experiments. Statistical comparisons were performed using the Student's unpaired t-test. P values ​​< 0.05 were considered statistically significant.

[0113] Functional studies and concentration-response curves of cavernous strips

[0114] Mouse corpus cavernosum strips 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 and the other hook served as a fixed attachment point. The tissue was equilibrated at a resting tension of 2.5 mN for 60 min. The PowerLab 400 TM The isometric force was recorded by a data acquisition system (LabChart software, version 7.0, AD Instrument, MA, USA). -5 M) precontracted corpus cavernosum strips, the muscarinic agonist acetylcholine (ACh; 10 -9 Up to 10 -5 M) and sodium nitroprusside (SNP; 10 -8 Up to 10 -4 Cumulative concentration-response curves were also obtained in the corpus cavernosum strips for the contractile agent phenylephrine (α1-adrenergic receptor agonist, 10 -8 Up to 3x 10 -4 M) and KCl (3x10 -4 Up to 3x 10 -1 Nonlinear regression analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) to determine the pEC 50 .

[0115] Electric Field Stimulation (EFS)

[0116] EFS was applied to the corpus cavernosum strips placed between two platinum ring electrodes connected to a Grass S88 stimulator (Astro-Med Industrial Park, RI, USA). EFS was performed at 50 V, 1 ms pulse width, and stimulation pulse trains at different frequencies for 10 s. To study nitroergic corpus cavernosum relaxation, tissue was treated with guanethidine (3 × 10 -5 M; to deplete catecholamine reserves in adrenergic fibers) and atropine (10 -6 M; to produce muscarinic antagonism) for 30 minutes, followed by phenylephrine (10 -5 M) Precontraction. When a stable contraction level was reached, a series of EFS-induced relaxation curves (2-32 Hz) were constructed.

[0117] Western blot analysis

[0118] The corpus cavernosum tissue was homogenized in lysis buffer and centrifuged at 12,000 g for 20 minutes at 4°C. The homogenate containing 50 g of total protein was run on a 4-20% Tris-HCl gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to a nitrocellulose membrane. Nonspecific binding sites were blocked using 5% nonfat dry milk (Bio-Rad) in Tris buffered saline / Tween at 24°C for 1 hour. The membranes were incubated for 15-16 h at 4°C with the following antibodies: monoclonal anti-3-NT antibody (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 Laboratories, San Diego, CA), and monoclonal anti-β-actin (1:7000; Sigma-Aldrich, St. Louis, MO). Densitometry was analyzed using Image J software (National Institute of Health, Bethesda-MD, USA). Quantitative densitometry results were normalized to β-actin. Quantitative densitometry results for eNOS phosphorylated at Ser1177 were normalized to total eNOS.

[0119] Determination of cGMP levels

[0120] Quantification of cGMP was performed using a commercial 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. TCA was extracted from the supernatant by three water-saturated ether washes. cGMP was expressed in pmol / mg tissue.

[0121] Example 1: Haptoglobin treatment corrects excessive corpus cavernosum relaxation in SCD mice ACh (10 -9 Up to 10 -5 Cumulative addition of M) produced concentration-dependent relaxation of PE-contracted tissues in all groups ( Figure 1However, the maximum response (Emax) of ACh in the SCD group was significantly higher than that in the WT group (P<0.05; Figure 1 A; Table 1 below). Haptoglobin treatment of SCD mice reduced the Emax value of ACh (P<0.05; Table 1). There was no significant difference in the potency (pEC50) of ACh between the groups. The pEC50 values ​​of ACh are shown in Table 1.

[0122] Table 1. Potency (pEC) obtained from concentration-response curves in cavernous strips from WT and SCD mice treated with vehicle or haptoglobin. 50 ) and maximum response (E max )value.

[0123]

[0124] SNP(10 -8 Up to 10 -4 Cumulative addition of M) also produced concentration-dependent relaxation in all groups ( Figure 1 C, 1D). Compared with WT mice, the Emax produced by SNP in the corpus cavernosum of SCD mice was significantly higher (P<0.05) ( Figure 1 C; Table 1), which was reduced by treatment with haptoglobin ( Figure 1 D). Compared with WT mice, the SNP pEC50 value in the corpus cavernosum of SCD mice was significantly higher (P<0.05) ( Figure 1 C and Table 1 ), which was reversed by treatment with haptoglobin ( P < 0.05) ( Figure 1 D and Table 1).

[0125] Phenylephrine (3x 10 -5 M) and atropine (10 -6 M) pre-treated corpus cavernosum tissue was subjected to electric field stimulation (EFS), which resulted in frequency-dependent corpus cavernosum relaxation in all groups. As observed from 2 to 32 Hz, EFS-induced corpus cavernosum relaxation was significantly lower in SCD mice compared with WT mice (P<0.05) ( Figure 2 A). Haptoglobin treatment reduced the EFS-induced increased relaxation response in the corpus cavernosum of SCD mice ( Figure 2 B).

[0126] NO, which is generated after erectile stimulation, is an essential molecule in the penis that causes penile erection. NO is produced by the endothelium lining the sinusoids of the corpus cavernosum and also by nitric oxide neurons (26). ACh promotes relaxation of the corpus cavernosum by stimulating the endothelium to produce NO. According to the results of Example 1, ACh produced greater relaxation in the corpus cavernosum of the SCD group. EFS promotes relaxation by neurogenic stimulation that causes nitric oxide fibers to produce NO (27). EFS also produced greater relaxation in the CC of the SCD group. SNP is a NO donor compound used as a pharmacological tool to evaluate endothelium-independent relaxation (28). According to the results, SNP-induced relaxation was also higher in the SCD group. Therefore, when NO activates GC, cGMP is produced, but PDE5 cannot effectively degrade cGMP, so that cGMP accumulates in smooth muscle cells, promoting relaxation of the corpus cavernosum and excessive penile erection in SCD (3). Long-term treatment with haptoglobin reduced relaxation induced by ACh, EFS and SNP in the CC of the SCD group. While not wanting to be bound by theory, the improvement in erectile function with haptoglobin treatment may be due to normalization of PDE5 expression and reduction of oxidative stress in the penis of SCD mice.

[0127] Example 2: Haptoglobin treatment corrects reduced contraction of the corpus cavernosum in SCD mice

[0128] Phenylephrine (10 -8 Up to 3x 10 -4 M) caused concentration-dependent contraction of the corpus cavernosum in all groups ( Figure 3 A). The maximum response (Emax) of the corpus cavernosum was significantly lower in the SCD group (0.50±0.08mN) compared with that in WT mice (0.90±0.11mN) (P<0.05). Haptoglobin treatment of SCD mice increased the Emax value of phenylephrine in the SCD group (0.79±0.11mN).

[0129] When evaluating receptor-independent stimulation, cumulative addition of KCl induced concentration-dependent corpus cavernosum contractions in all groups ( Figure 3 C and 3D). The maximum response (Emax) of the corpus cavernosum of SCD mice was significantly lower (0.16±0.04mN) compared with WT mice (0.45±0.08mN) (P<0.05). Haptoglobin treatment of SCD mice increased the Emax value of KCl in the SCD group (0.30±0.07mN).

[0130] Norepinephrine released from the sympathetic nervous system induces contraction of the corpus cavernosum due to postsynaptic activation of α1-adrenergic receptors coupled to Gq proteins, which activate phospholipase C, which catalyzes the cleavage of phosphatidylinositol into inositol triphosphate and diacylglycerol, thereby increasing intracellular calcium levels (34). Calcium produces smooth muscle contraction due to binding to calmodulin, which leads to activation of myosin light chain kinase (MLC), resulting in phosphorylation of MLC (27). In this process, phosphorylated MLC interacts with β-actin, leading to smooth muscle contraction and maintaining the penis in a flaccid state (27, 34). KCl promotes receptor-independent contraction, acting by depolarizing the smooth muscle cell membrane and promoting calcium influx (27). The inventors of the present invention unexpectedly found that contraction caused by α1-adrenergic receptor agonists and KCl was lower in SCD mice.

[0131] Example 3: Haptoglobin treatment corrects down-regulated p-eNOS (Ser-1177) and eNOS in the penis of SCD mice and does not affect nNOS protein expression

[0132] Compared with the WT-vehicle group, the protein expressions of p-eNOS (Ser-1177) and eNOS in the penis of the SCD-vehicle group were significantly decreased (p<0.05), by approximately 45% and 44%, respectively (see Table 2). Figure 4 A and 4B). Haptoglobin treatment normalized the protein levels of p-eNOS (Ser-1177) and eNOS in the penis of the SCD group (P<0.05). SCD or haptoglobin treatment did not alter the protein expression of nNOS ( Figure 4 C).

[0133] According to the results of Example 3, haptoglobin treatment normalized the expression of total eNOS and phosphorylated eNOS at its positive regulatory site Ser-1177 in the SCD group, while no change was observed in the expression of nNOS. These results indicate that basal NO production in the penis of SCD mice is normalized.

[0134] Example 4: Haptoglobin treatment increases PDE5 protein expression in the penis of SCD mice

[0135] Compared with the WT-vehicle group, PDE5 protein expression in the penis of the SCD-vehicle group was significantly reduced (p<0.05), with a decrease of approximately 50% ( Figure 4 D). Haptoglobin treatment increased PDE5 protein levels in the penis of the SCD group by approximately 52% ( Figure 4 D).

[0136] Combined with the results of Examples 3 and 4, it was demonstrated that the improvement of endothelial function leads to increased expression of PDE5. Increased expression of PDE5 in corpus cavernosum smooth muscle prevents excessive relaxation induced by ACh, EFS and SNP via NO-GC pathway stimulation in SCD mice.

[0137] Example 5: Haptoglobin treatment restores ROCK2 and does not affect ROCK1 protein expression in the penis of SCD mice

[0138] Compared with vehicle-treated WT mice, the protein expression of ROCK2 in the penis of vehicle-treated SCD mice was significantly decreased (P < 0.05), with a decrease of approximately 48% ( Figure 5 A). Haptoglobin treatment completely restored the protein level of ROCK2 in the SCD group ( Figure 5 A). SCD or haptoglobin treatment did not alter ROCK1 protein expression compared to the WT-vehicle group ( Figure 5 B).

[0139] Activation of the RhoA / Rho kinase signaling pathway acts by increasing Ca2+ sensitivity, thereby participating in the contractile mechanism of the corpus cavernosum smooth muscle (35). ROCK has two isoforms, named ROCK1 and ROCK2 (35). The results of Example 5 showed that ROCK2 expression was lower in the corpus cavernosum of SCD mice. The reduction in ROCK2 is consistent with the results of Example 2, in which contraction was reduced in SCD mice. Haptoglobin treatment increased contraction induced by phenylephrine and KCl, as well as the expression of ROCK2. eNOS enzyme-deficient mice showed reduced NO bioavailability associated with lower RhoA / Rho kinase activity in the penis and a penile abnormal erection phenotype (36). In the previous example, haptoglobin treatment restored eNOS expression in the SCD group, indicating that normalization of NO bioavailability in the endothelium is associated with increased ROCK2 expression in the penis.

[0140] Example 6: Haptoglobin treatment corrects increased oxidative stress and upregulated NADPH oxidase subunit gp91phox protein expression in the penis of SCD mice

[0141] Compared with vehicle-treated WT mice, the protein expression of gp91phox in the penis of vehicle-treated SCD mice was significantly higher (P<0.05), increasing by approximately 67% ( Figure 6 A). Haptoglobin treatment completely restored the protein level of gp91phox in the SCD group ( Figure 6 A).

[0142] Compared with the WT group, the protein expressions of 3-nitrotyrosine and 4-HNE in the SCD penile tissue were significantly higher (P<0.05), increasing by approximately 95% and 75%, respectively ( Figure 6 B and 6C). Haptoglobin treatment reduced the protein levels of 3-nitrotyrosine and 4-HNE in the SCD group by approximately 27% and 28%, respectively ( Figure 6 B and 6C).

[0143] In this example, haptoglobin treatment reduced the expression of gp91phox, indicating a reduction in superoxide anion production in the penis. According to the results, haptoglobin treatment reduced the expression of markers of oxidative stress (4-HNE) and nitrosative stress (3-NT). The expression of gp91phox in the penis is downregulated by a cGMP-dependent mechanism (42, 43), while exogenous NO inhibits NADPH oxidase activity by directly S-nitrosylating the p47phox subunit in human endothelial cells (44). In this example, normalization of gp91phox subunit expression by haptoglobin treatment may be related to the improvement of endothelial function.

[0144] Example 7: Haptoglobin treatment increases cGMP levels in the penis of SCD mice Basal cGMP levels in the penis of SCD mice were reduced by 60% compared to WT-vehicle mice (P<0.05) ( Figure 7 ). Haptoglobin treatment increased cGMP levels in the penis of the SCD group by approximately 72% (P<0.05) ( Figure 7 ).

[0145] The above embodiments are described as examples only. Many other embodiments will be apparent to the skilled reader within the scope of the appended claims. Therefore, although the present invention has been described in conjunction with the preferred embodiments of the various figures, it should be understood that other similar embodiments may be used or the described embodiments may be modified or added to perform the same functions of the present invention without departing from the present invention. Therefore, the present invention should not be limited to any single embodiment, but should be interpreted in breadth and scope according to the description of the appended claims.

[0146] References

[0147] 1.Telen MJ, Malik P, Vercellotti GM. Therapeutic strategies for sicklecell disease: toward a multi-agent approach. Nat Rev Drug Discov. 2019 Feb; 18(2):139–58.

[0148] 2.Salonia A,Eardley I,Giuliano F,Hatzichristou D,Moncada I,Vardi Y,etal.EuropeanAssociation of Urology guidelines on priapism.Eur Urol.2014Feb;65(2):480–9.

[0149] 3.Musicki B,Burnett AL.Mechanisms underlying priapismin sickle celldisease:targeting andkey innovations on the preclinical landscape.Expert OpinTher Targets.2020 May;24(5):439–50.

[0151] 4.Adeyoju AB,Olujohungbe ABK,Morris J,Yardumian A,Bareford D,AkenovaA,et al.Priapismin sickle-cell disease;incidence,risk factors andcomplications-an internationalmulticentre study.BJU Int.2002Dec;90(9):898–902.

[0153] 5.Mantadakis E,Cavender JD,Rogers ZR,Ewalt DH,Buchanan GR.Prevalenceof priapismin children and adolescents with sickle cell anemia.J PediatrHematol Oncol.1999 Dec;21(6):518–22.

[0155] 6.Alvaia MA,Maia HAA da S,Nelli A de M, COS,Carvalho ES de S,Netto JMB,et al.Prevalence of priapism in individuals with sickle celldisease and implications on malesexual function.Einstein Sao Paulo Braz.2020;18:eAO5070.

[0156] 7.Gladwin MT,Sachdev V.Cardiovascular abnormalities in sickle celldisease.J Am CollCardiol.2012 Mar 27;59(13):1123–33.

[0157] 8.Silva FH,Karakus S,Musicki B,Matsui H,Bivalacqua TJ,Dos Santos JL,et al.Beneficialeffect of the nitric oxide donor compound 3-(1,3-dioxoisoindolin-2-yl)benzyl nitrate ondysregulated phosphodiesterase 5,NADPHoxidase and nitrosative stress in the sickle cellmouse penis:implication forpriapism treatment.J Pharmacol Exp Ther.2016 Aug 18; 9.Bivalacqua TJ,Musicki B,Hsu LL,Berkowitz DE,Champion HC,BurnettAL.Sildenafilcitrate-restored eNOS and PDE5 regulation in sickle cell mousepenis prevents priapism viacontrol of oxidative / nitrosative stress.PloSOne.2013;8(7):e68028.

[0158] 10.Musicki B,Karakus S,Akakpo W,Silva FH,Liu J,Chen H,etal.Testosterone replacementin transgenic sickle cell mice controls priapicactivity and upregulates PDE5 expression andeNOS activity in thepenis.Andrology.2018 Jan;6(1):184–91.

[0159] 11.Champion HC,Bivalacqua TJ,Takimoto E,Kass DA,BurnettAL.Phosphodiesterase-5Adysregulation in penile erectile tissue is a mechanismof priapism.Proc Natl Acad Sci U SA.2005 Jan 2;102(5):1661–6.

[0160] 12.Lagoda G,Sezen SF,Cabrini MR,Musicki B,Burnett AL.Molecularanalysis of erectionregulatory factors in sickle cell disease associatedpriapism in the human penis.J Urol.2013 Feb;189(2):762–8.

[0162] 13.Kato GJ.Priapism in sickle-cell disease:a hematologist’sperspective.J Sex Med.2012Jan;9(1):70–8.

[0164] 14.Bivalacqua TJ,Musicki B,Kutlu O,Burnett AL.New insights into thepathophysiology ofsickle cell disease-associated priapism.J Sex Med.2012 Jan;9(1):79–87.

[0165] 15.Bivalacqua TJ,Ross AE,Strong TD,Gebska MA,Musicki B,Champion HC,etal.AttenuatedRhoA / Rho-kinase signaling in penis of transgenic sickle cellmice.Urology.2010 Aug;76(2):510.e7-12.

[0167] 16.Reiter CD,Wang X,Tanus-Santos JE,Hogg N,Cannon RO,Schechter AN,etal.Cell-freehemoglobin limits nitric oxide bioavailability in sickle-celldisease.Nat Med.2002 Dec;8(12):1383–9.

[0169] 17.Buehler PW,Humar R,Schaer DJ.Haptoglobin Therapeutics andCompartmentalization ofCell-Free Hemoglobin Toxicity.Trends Mol Med.2020 Jul;26(7):683–97.

[0170] 18.Muller-Eberhard U,Javid J,Liem HH,Hanstein A,Hanna M.Plasmaconcentrations ofhemopexin,haptoglobin and heme in patients with varioushemolytic diseases.Blood.1968 Nov;32(5):811–5.

[0172] 19.Santiago RP,Guarda CC,Figueiredo CVB,Fiuza LM,Aleluia MM,AdanhoCSA,et al.Serum haptoglobin and hemopexin levels are depleted in pediatricsickle cell diseasepatients.Blood Cells Mol Dis.2018 Sep;72:34–6.

[0174] 20.Cita K-C,Brureau L,Lemonne N,Billaud M,Connes P,Ferdinand S,etal.Men with SickleCell Anemia and Priapism Exhibit Increased Hemolytic Rate,Decreased Red Blood CellDeformability and Increased Red Blood Cell AggregateStrength.PloS One.2016;11(5):e0154866.

[0176] 21.Kato GJ,McGowan V,Machado RF,Little JA,Taylor J,Morris CR,etal.Lactatedehydrogenase as a biomarker of hemolysis-associated nitric oxideresistance,priapism,leg ulceration,pulmonary hypertension,and death inpatients with sickle cell disease.Blood.2006 Mar 15;107(6):2279–85.

[0178] 22.Nolan VG,Wyszynski DF,Farrer LA,Steinberg MH.Hemolysis-associatedpriapism in sicklecell disease.Blood.2005 Nov 1;106(9):3264–7.

[0179] 23.Belcher JD,Chen C,Nguyen J,Abdulla F,Zhang P,Nguyen H,etal.Haptoglobin andhemopexin inhibit vaso-occlusion and inflammation in murinesickle cell disease:Role ofheme oxygenase-1 induction.PloS One.2018;13(4):e0196455.

[0180] 24.Shi PA,Choi E,Chintagari NR,Nguyen J,Guo X,Yazdanbakhsh K,etal.Sustainedtreatment of sickle cell mice with haptoglobin increases HO-1 andH-ferritin expression anddecreases iron deposition in the kidney withoutimprovement in kidney function.Br JHaematol.2016 Nov;175(4):714–23.

[0181] 25.Schaer CA,Deuel JW,Schildknecht D,Mahmoudi L,Garcia-Rubio I,Owczarek C,et al.Haptoglobin Preserves Vascular Nitric Oxide Signaling duringHemolysis.Am J Respir CritCare Med.2016 May 15;193(10):1111–22.

[0183] 26.Hurt KJ,Sezen SF,Lagoda GF,Musicki B,Rameau GA,Snyder SH,etal.Cyclic AMP-dependent phosphorylation of neuronal nitric oxide synthasemediates penile erection.ProcNatl Acad Sci U S A.2012 Oct 9;109(41):16624–9.

[0185] 27.Andersson K-E.Mechanisms of Penile Erection and Basis forPharmacological Treatmentof Erectile Dysfunction.Pharmacol Rev.2011 Jan 12;63(4):811–59.

[0186] 28.Kowaluk EA,Seth P,Fung HL.Metabolic activation of sodiumnitroprusside to nitric oxide invascular smooth muscle.J Pharmacol ExpTher.1992 Sep;262(3):916–22.

[0187] 29.Musicki B,Karakus S,La Favor JD,Chen H,Silva FH,Sturny M,etal.TSPO ligand FGIN-1-27 controls priapism in sickle cell mice via endogenoustestosterone production.J CellPhysiol.2020 Sep 24;

[0188] 30.Lin C-S,Chow S,Lau A,Tu R,Lue TF.Human PDE5A gene encodes threePDE5 isoformsfrom two alternate promoters.Int J Impot Res.2002 Feb;14(1):15–24.

[0189] 31.Silva FH,Claudino MA,Calmasini FB,Alexandre EC,Franco-Penteado C,Burnett AL,et al.Sympathetic Hyperactivity,Increased Tyrosine Hydroxylase andExaggerated CorpusCavernosum Relaxations Associated with Oxidative StressPlays a Major Role in the PenisDysfunction in Townes Sickle Cell Mouse.PloSOne.2016;11(12):e0166291.

[0191] 32.Horst BG,Marletta MA.Physiological activation and deactivation ofsoluble guanylatecyclase.Nitric Oxide Biol Chem.2018 Jul 1;77:65–74.

[0192] 33.Hsu LL,Champion HC,Campbell-Lee SA,Bivalacqua TJ,Manci EA,DiwanBA,et al.Hemolysis in sickle cell mice causes pulmonary hypertension due toglobal impairment innitric oxide bioavailability.Blood.2007 Apr 1;109(7):3088–98.

[0194] 34.Mitidieri E,Cirino G,d’Emmanuele di Villa Bianca R,SorrentinoR.Pharmacology andperspectives in erectile dysfunction in man.PharmacolTher.2020 Apr 1;208:107493.

[0195] 35.Chitaley K,Wingard CJ,Clinton Webb R,Branam H,Stopper VS,Lewis RW,et al.Antagonism of Rho-kinase stimulates rat penile erection via a nitricoxide-independentpathway.Nat Med.2001 Jan;7(1):119–22.

[0197] 36.Bivalacqua TJ,Liu T,Musicki B,Champion HC,Burnett AL.Endothelialnitric oxide synthasekeeps erection regulatory function balance in thepenis.Eur Urol.2007 Jun;51(6):1732–40. 37.Rifkind JM,Mohanty JG,Nagababu E.The pathophysiology ofextracellular hemoglobinassociated with enhanced oxidative reactions.FrontPhysiol.2014;5:500.

[0198] 38.Schaer DJ,Buehler PW,Alayash AI,Belcher JD,VercellottiGM.Hemolysis and freehemoglobin revisited:exploring hemoglobin and heminscavengers as a novel class oftherapeutic proteins.Blood.2013 Feb 21;121(8):1276–84.

[0199] 39.Chintagari NReddy,Nguyen J,Belcher JD,Vercellotti GM,AlayashAI.Haptoglobinattenuates hemoglobin-induced heme oxygenase-1 in renalproximal tubules cells andkidneys of a mouse model of sickle celldisease.Blood Cells Mol Dis.2015 Mar;54(3):302–6.

[0201] 40.Musicki B,Liu T,Sezen SF,Burnett AL.Targeting NADPH oxidasedecreases oxidativestress in the transgenic sickle cell mouse penis.J SexMed.2012 Aug;9(8):1980–7.

[0202] 41.Pacher P,Beckman JS,Liaudet L.Nitric oxide and peroxynitrite inhealth and disease.Physiol Rev.2007 Jan;87(1):315–424.

[0204] 42.Nunes KP,Teixeira CE,Priviero FBM,Toque HA,Webb RC.Beneficialeffect of the solubleguanylyl cyclase stimulator BAY 41-2272 on impairedpenile erection in db / db- / -type IIdiabetic and obese mice.J Pharmacol ExpTher.2015 May;353(2):330–9.

[0205] 43.Teixeira CE,Priviero FBM,Webb RC.Effects of 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1 H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine(BAY 41-2272)on Smooth Muscle Tone,Soluble Guanylyl Cyclase Activity,and NADPHOxidase Activity / Expression in CorpusCavernosum from Wild-Type,Neuronal,andEndothelial Nitric-Oxide Synthase Null Mice.JPharmacol Exp Ther.2007 Jan 9;322(3):1093–102.

[0207] 44.Selemidis S,Dusting GJ,Peshavariya H,Kemp-Harper BK,DrummondGR.Nitric oxidesuppresses NADPH oxidase-dependent superoxide production by S-nitrosylation in humanendothelial cells.Cardiovasc Res.2007 Jul 15;75(2):349–58.

[0208] 45.Lagoda G,Sezen SF,Hurt KJ,Cabrini MR,Mohanty DK,BurnettAL.Sustained nitric oxide(NO)-releasing compound reverses dysregulated NOsignal transduction in priapism.FASEB J.2014 Jan 1;28(1):76–84.

[0211] 46.Belcher JD,Chen C,Nguyen J,Milbauer L,Abdulla F,Alayash AI,etal.Heme triggers TLR4 signaling leading to endothelial cell activation andvaso-occlusion in murine sickle celldisease.Blood.2014 Jan 16;123(3):377–90.

[0213] 47.Kimmel JR.

[70] Guanidination of proteins.Methods Enzymol.1967;11:584-589.

[0214] 48.Haugen TH,Hanley JM,Heath EC.Haptoglobin.A novel mode ofbiosynthesis of a liversecretory glycoprotein.J Biol Chem.1981;256(3):1055-1057.

[0215] 49.Misumi Y,Tanaka Y,Ikehara Y.Biosynthesis,intracellular processingand secretion ofhaptoglobin in cultured rat hepatocytes.Biochem Biophys ResCommun.1983;114(2):729-736.

[0216] 50.Koch W,Latz W,Eichinger M,et al.Genotyping of the commonhaptoglobin Hp 1 / 2 polymorphism based on PCR.Clin Chem.2002;48(9):1377–1382.

[0218] 51.Chapter 2-Haptoglobin Polymorphism and Infection;Advances inClinical Chemistry,2010,50:23-46

[0220] 52.Altschul SF,Gish W,Miller W,Myers EW,Lipman DJ.Basic localalignment search tool.JMol Biol.1990;215(3):403-410.

[0221] 53.Morishita K,Ito N,Koda S,et al.Haptoglobin phenotype is a criticalfactor in the use offucosylated haptoglobin for pancreatic cancerdiagnosis.Clin Chim Acta.2018;487:84-89.54.Cruickshank AM.ACP Best PracticeNo 166:CSF spectrophotometry in the diagnosis ofsubarachnoid haemorrhage.JClin Pathol.2001;54(11):827-830.

[0222] 55.Hugelshofer M,Sikorski CM,Seule M,et al.Cell-Free Oxyhemoglobin inCerebrospinal FluidAfter Aneurysmal Subarachnoid Hemorrhage:Biomarker andPotential Therapeutic Target.World Neurosurg.2018;120:e660-e666.

[0224] 56.Schellenberger V,Wang CW,Geething NC,et al.A recombinantpolypeptide extends the invivo half-life of peptides and proteins in atunable manner.Nat Biotechnol.2009;27:1186–1190.

[0226] 57.Dumont JA,Low SC,Peters RT,Bitonti AJ.Monomeric FcFusions.BioDrugs.2006;20:151–160.

Claims

1. Haptoglobin or a nucleic acid encoding haptoglobin for use in treating or preventing excessive erectile response and / or preventing permanent erectile dysfunction.

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

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

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

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

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

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

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

9. A pharmaceutical composition for treating or preventing excessive erectile response and / or preventing permanent erectile dysfunction, wherein the pharmaceutical composition comprises an adeno-associated virus (AAV) vector carrying a transgene encoding a haptoglobin gene.

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

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

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

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