Treatment plan using DNase

Abstract

The present invention provides a method for inducing pregnancy in a couple in need thereof, wherein a male subject of the couple has been diagnosed with subfertility, the method comprising systemically administering to the male subject a pharmaceutical composition comprising human DNase.

Description

[Technical field]

[0001] The present invention relates generally to methods for inducing pregnancy in couples where the male subject has been diagnosed with subfertility. [Background technology]

[0002] Most couples can conceive naturally within 12 months of regular unprotected intercourse. Subfertility is defined as the delay in conceiving and can be due to pathology in either the female or male partner.

[0003] Male fertility status is assessed by semen analysis, which provides information on the number of sperm, their motility and morphology.

[0004] Idiopathic oligozoospermia, asthenozoospermia, and teratozoospermia are the most common causes of male subfertility. Sexual function is normal, but the number of spermatozoa is reduced, mainly dysfunctional. Reduced fertility is associated with increased concentrations of reactive oxygen species in semen, which can damage cell membranes. Abnormal sperm morphology, an indicator of impaired sperm production or maturation, is also associated with reduced fertility. Less common types of male subfertility are caused by infections, diseases, or abnormalities of the testes or reproductive tract. Systemic diseases, external factors (drugs, lifestyle, etc.), or a combination of these also result in male subfertility. Male subfertility is rarely caused by endocrine dysfunction (Non-Patent Document 1).

[0005] US Patent No. 5,999,633 provides a method for treating male subfertility by administering a pharmaceutical composition containing DNAse. Male subjects were administered 25 mg of bovine DNase I by intramuscular (IM) injection four times daily for 7-10 days. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 9,149,513 [Non-patent literature]

[0007] [Non-Patent Document 1] Hirsh A.BMJ 2003;327(7424):1165 Summary of the Invention [Problem to be solved by the invention]

[0008] [Means for solving the problem]

[0009] In a first aspect thereof, the present invention provides a method for inducing pregnancy in a couple in need thereof, wherein the male subject of the couple has been diagnosed with subfertility, the method comprising systemically administering to the male subject a pharmaceutical composition comprising about 50 to about 1000 μg / kg of human DNase.

[0010] In one embodiment, the pharmaceutical composition is administered once a week, once every three days, once every other day, once a day, twice a day, or three times a day.

[0011] In one embodiment, the pharmaceutical composition is administered over a course of treatment sufficient to achieve maturation of fresh sperm cells in the male subject.

[0012] In one embodiment, the administration is for a treatment course of about 10 days to about 3 months, or about 10 days to 16 days.

[0013] In one embodiment, the pharmaceutical composition is administered intravenously (IV), subcutaneously (SC), intramuscularly (IM), by inhalation, or orally.

[0014] In one embodiment, the human male subject has idiopathic poor semen quality.

[0015] In one embodiment, the human male subject has azoospermia, oligozoospermia, mild or severe isolated teratozoospermia, asthenozoospermia, or oligo-teratozoospermia-asthenozoospermia (OTA).

[0016] In one embodiment, the human male subject is the male partner of a couple who have been unable to conceive for over two years or have had a miscarriage.

[0017] In one embodiment, the human male subject is the male partner of a couple who have been unable to conceive during at least one assisted reproductive technology (ART) procedure.

[0018] In one embodiment, the couple has been unsuccessful in conceiving in at least two assisted reproductive technology (ART) procedures.

[0019] In one embodiment, the female human subject of the couple has not been diagnosed with infertility.

[0020] In one embodiment, the ART is intracytoplasmic sperm injection (ICSI), selection of good morphological sperm under a high power microscope and intracytoplasmic sperm injection (IMSI), or intrauterine insemination (IUI).

[0021] In one embodiment, the human male subject has low sperm viability and / or motility and / or concentration.

[0022] In one embodiment, the human male subject has high blood cell-free DNA levels.

[0023] In one embodiment, the sperm of the human male subject is used for ART.

[0024] In one embodiment, the ART is intracytoplasmic sperm injection (ICSI), selection of good morphological sperm under a high power microscope and intracytoplasmic sperm injection (IMSI), or intrauterine insemination (IUI).

[0025] In one embodiment, administration of the pharmaceutical composition results in at least a 35% improvement in one or more sperm quality parameters selected from the group consisting of total sperm count, sperm concentration, sperm motility, sperm motility quality or average velocity, sperm morphology, sperm viability, and sperm chromatin stability.

[0026] In one embodiment, the human male subject's sperm is sampled within 1 to 14 days after completion of the treatment course.

[0027] In one embodiment, the human male subject's sperm is sampled within 1 to 5 days after completion of the treatment course.

[0028] In one embodiment, completion of the pharmaceutical composition treatment course is synchronized with ovulation in the female subject of the couple.

[0029] In one embodiment, ovulation subsides 1-14 days after completion of the treatment course.

[0030] In one embodiment, ovulation subsides 1-5 days after completion of the treatment course.

[0031] In one embodiment, the methods of the invention further comprise administration of an additional therapeutic agent.

[0032] In another aspect, the present invention provides a pharmaceutical composition comprising DNase and a pharma- ceutical acceptable carrier for use in a method for inducing pregnancy in a couple in need thereof, wherein a human male subject of the couple has been diagnosed with subfertility, the method comprising systemically administering the pharmaceutical composition to a human subject in need thereof, the pharmaceutical composition comprising about 50 to about 1000 μg / kg of DNase.

[0033] In one embodiment, the pharmaceutical composition comprises an additional therapeutic agent.

[0034] In some embodiments, the DNase is a pegylated DNAse or a PAS-DNAse.

[0035] In some embodiments, the DNase is selected from the group consisting of DNase I, DNase I L1, DNase I L2, DNase I L3, DNase II, DNase II alpha, DNase II beta, DNase X, DNase gamma, caspase-activated DNase (CAD), endonuclease G (ENDOG), granzyme B (GZMB), phosphodiesterase I, lactoferrin, acetylcholinesterase, and variants thereof that maintain DNAse activity.

[0036] In one embodiment, the DNase is human DNase I.

[0037] In one embodiment, the human DNAse I comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, or to the mature DNAse I enzyme without the signal peptide.

[0038] In one embodiment, the DNase I is human recombinant DNase I or isolated native human DNase I.

[0039] In one embodiment, the DNase I is human DNase I comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, the human DNase I is injected intravenously at a dose of 250 μg / kg / day for 14-16 days, and the human male subject's sperm is sampled within 1-5 days after completion of the treatment course.

[0040] In one embodiment, the DNase I is human DNase I comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, the human DNase I is injected intravenously at a dose of 250 μg / kg / day for 14-16 days, and ovulation subsides 1-5 days after completion of the treatment course period.

[0041] In order to better understand the subject matter disclosed herein and to illustrate how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0042] [Figure 1] 1 is a summary of the amino acid sequence of human DNase I (represented herein as SEQ ID NO:1). [Diagram 2] 1 is a graph showing the percentage of oval sperm cells over time (days) out of the total population of cells deemed to be sperm cells as observed using high-throughput morphological examination (MSOME). The rectangle represents 16 days of DNase I treatment. [Diagram 3] 1 is a graph showing the percentage of amorphous sperm cells over time (days) out of the total population of cells considered to be sperm cells as observed using high-throughput morphological examination (MSOME). The rectangle represents 16 days of DNase I treatment. [Figure 4] 1 is a graph showing the percentage of oval sperm cells over time (days) out of the total population of cells deemed to be sperm cells as observed using high-throughput morphological examination (MSOME). The rectangle represents 16 days of DNase I treatment. [Diagram 5] FIG. 1 is a graph showing the number of round and amorphous spermatids over time (days) as observed using high-throughput morphological examination (MSOME). The rectangle represents 16 days of DNase I treatment. [Figure 6] FIG. 1 is a graph showing the number of normal sperm cells over time (days) as determined by Halosperm analysis. [Figure 7] Graph showing the percentage of normal sperm cell morphology by conventional WHO morphological criteria over time (days) out of the total population of cells considered to be sperm cells. The rectangle represents 16 days of DNase I treatment. The line represents normal levels. [Figure 8]1 is a graph showing the percentage of sperm cells with head defects by conventional WHO morphological criteria over time (days) out of the total population of cells considered to be sperm cells. The rectangle represents 16 days of DNase I treatment. The line represents normal levels. [Figure 9] FIG. 1 is a graph showing the percentage of normal sperm cells over time (days) as determined by Halosperm analysis. The rectangles represent 16 days of DNase I treatment. The line represents normal levels. [Figure 10] 1 is a graph showing the kinetics of DNase I activity mKU(Kunitz) / μl over time after injection of 125 μg / kg recombinant human DNAse I (rhDNase I) as determined by the SRED test. A Kunitz unit is defined as the amount of enzyme added to 1 mg / ml salmon sperm DNA that causes an increase in absorbance of 0.001 per minute at a wavelength of 260 nm when acting on highly polymerized DNA in 0.1 M NaOAc (pH 5.0) buffer at 25° C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The present invention provides a treatment regimen for the induction of pregnancy in couples in which the male subject has been diagnosed with subfertility, the treatment regimen comprising administering a clinical grade pharmaceutical composition comprising a human deoxyribonuclease (DNase), e.g., DNase I.

[0044] As shown in the Examples below, administration of DNase I resulted in successful conception in previously unpregnant couples. Treatment resulted in improved sperm cell DNA stability as observed in the Halosperm test, and improved sperm nuclear morphology as observed in the MSOME analysis.

[0045] Without wishing to be bound by theory, these analyses represent two processes mediated by recombinant human DNAse I (rhDNase) treatment.

[0046] Improvements in sperm cell DNA stability as measured by %DFI in the Halosperm test were rapid and transient, primarily noticeable within the 16-day treatment window (short-term effect).

[0047] Improvement in sperm morphology was observed later, reaching a maximum on day 24, 8 days after treatment was completed (long-term effect).

[0048] It is suggested that sperm cells passing through the epididymis (processing period: 8 days) are prone to cell-free DNA-induced apoptosis and that DNase treatment could prevent or limit this damage, thus improving the %DFI in the Halosperm test.

[0049] DNase also persists for 16 days and is further suggested to target sperm cells during sperm maturation in spermiogenesis, which occurs prior to transport to the epididymis.

[0050] It is assumed that in this process the sperm cells acquire their required morphology and that the presence of cell-free DNA may impair their morphology. DNase treatment may prevent or limit these effects.

[0051] Results from clinical trials in subfertile men suggest that cell-free DNA is associated with sperm cell damage and that DNase treatment may significantly improve semen quality and lead to pregnancy success.

[0052] Thus, in a first aspect thereof, the present invention relates to a method for inducing a pregnancy in a couple in need thereof, wherein the male subject of said couple has been diagnosed with subfertility, said method comprising administering from about 50 μg / kg to about 1000 μg / kg, or from about 50 μg / kg to about 350 μg / kg of human DNase, for example 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 81 The method includes systemically administering to the male subject a pharmaceutical composition comprising 50, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, or 350 μg / kg of human DNase. In one embodiment, the pharmaceutical composition comprises 125 μg / kg.

[0053] As used herein, induction of pregnancy refers to achieving a successful pregnancy in a previously unpregnant couple.

[0054] The terms "subject" or "patient" are used interchangeably herein and refer to a male subject who may benefit from the present invention. He may also be referred to as the "male partner" in a couple. In certain embodiments, the subject is a human.

[0055] The term "subject in need thereof" in the context of the present invention refers especially to a human male subject suffering from a disease or disorder resulting in subfertility, as defined herein. The term "couple in need thereof" in the context of the present invention refers especially to a human couple (male and female) who exhibit a reduced ability to conceive, as defined herein.

[0056] As used herein, the terms "male subfertility" or "subfertile male" are used in their broadest sense and refer to any condition characterized by a confirmed idiopathic decrease in semen quality.

[0057] Examples include, but are not limited to, the following: Oligospermia - Decreased sperm count. Mild to moderate: 5-20 million / ml of semen. Severe: <5 million / ml of semen. Asthenozoospermia-decreased sperm motility. Teratozoospermia (mild or severe) - an increase in sperm with abnormal morphology. Azoospermia and oligozoospermia, teratozoospermia and asthenozoospermia (OTA). Semen with high DFI (DNA Fragmentation Index) value

[0058] DNA fragmentation can be measured using methods known to those skilled in the art, such as gel electrophoresis or the Halosperm test. Halosperm is based on the sperm chromatin dispersion (SCD) technique and can be carried out using an in vitro diagnostic kit (Halotech DNA, Spain) that controls the DNA denaturation process to facilitate the subsequent removal of proteins contained in each sperm. In this way, normal sperm produce a halo formed by loops of DNA in the sperm head, which is absent in sperm with damaged DNA.

[0059] In certain embodiments, the term "reduced" is meant to refer to at least about a 50%, 60%, 70%, 80%, or 90% reduction in sperm count or motility compared to fertile males.

[0060] The term also includes male partners of couples in which female factor infertility has not been diagnosed, who have been trying to conceive for more than two years, and who have previously undergone unsuccessful assisted reproductive technology (ART).

[0061] In certain embodiments according to the invention, administration of human DNAse I results in at least a 35% improvement in one or more semen quality parameters selected from total number, concentration, motility, motility quality or average rate, morphology (Kruger or WHO), viability (eosin staining) and sperm chromatin stability (Halosperm assay).

[0062] The term "improvement" of semen quality parameters in the context of the present invention means that the pharmaceutical composition of the present invention improves semen quality parameters by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, %,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,61%,62%,63%,64%,65%,66%,67%,68%,69%,70%,71%, By this term, we mean an increase of 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100%.

[0063] Systemic administration according to the present invention can be by any of the following routes: oral administration, intravenous, intramuscular, intraperitoneal, intrathecal, or subcutaneous injection.

[0064] In certain embodiments, administration according to the present invention is performed intravenously.

[0065] The pharmaceutical composition containing the DNAse may be administered to a subject in a single dose or in multiple doses.

[0066] In some embodiments, a pharmaceutical composition according to the invention comprises from 50 μg / kg to about 1000 μg / kg of DNAse.

[0067] The pharmaceutical composition may be administered multiple times, for example from a frequency of 5 administrations per day to once every 3 days, or from a frequency of 3 administrations per day to once every 3 days, i.e., 3 times per day, or 2 times per day, or once per day, or once every 2 days, or once every 3 days, or once per week.

[0068] The pharmaceutical composition can be administered for a period of at least 10 days, or for a period of about 10 days to about 3 months, such as for a period of about 10 days to 16 days, or for 16 days. The period over which treatment is administered is also defined herein as the "treatment course period."

[0069] In one embodiment, the pharmaceutical composition is administered for a treatment course period sufficient to achieve fresh sperm cell maturation in the male subject. As used herein, the term "fresh sperm cell maturation" refers to the emergence of newly formed mature sperm. Sperm cells mature into sperm during spermatozoon formation, the final stage of spermatogenesis. Thus, in one embodiment, the pharmaceutical composition is administered for a treatment course period sufficient to achieve effective sperm levels in the male subject. Effective herein refers to a sperm level sufficient to achieve pregnancy induction in the treated couple.

[0070] In one embodiment, the method comprises administration of 250 μg of DNAse I of the present invention per kg of body weight per day (ie, 250 μg / kg / day).

[0071] In one embodiment, the pharmaceutical composition is administered at a dose of 125 μg / kg body weight twice daily (12 hours apart, for example, once in the morning and once in the evening) for 16 days.

[0072] As used herein, the term "DNase" refers to any enzyme that can cleave DNA and represents a family of proteins. Thus, this term includes, but is not limited to, any enzyme selected from the group consisting of DNase I, DNase I-like 1 (DNaseIL1), DNase I-like 2 (DNase IL2), DNase I-like 3 (DNase IL3), DNase II, DNase IIα, DNase IIβ, DNase X, DNase γ, caspase-activated DNase ("CAD"), endonuclease G ("ENDOG"), granzyme B ("GZMB"), phosphodiesterase I, lactoferrin, acetylcholinesterase, and variants thereof that maintain DNAse activity (including, but not limited to, one or more mutations in its actin-binding site).

[0073] In one embodiment, the DNAse is DNAse I. DNAse I preferentially cleaves protein-free DNA and its activity is inhibited upon binding to monomeric actin. DNAse I is sensitive to physiological salt concentrations. Furthermore, DNAse I is glycosylated at N40 (corresponding to N18 in the mature enzyme without the signal peptide) and N128 (N106), which makes the enzyme resistant to inactivation by serum proteases.

[0074] DNase I may be recombinantly produced (eg, Pulmozyme) or may be naturally occurring isolated DNAse I.

[0075] It should be understood that the term "purified" or "isolated" refers to a molecule, e.g., a DNAse, that has been removed, isolated, or separated from its natural environment. Thus, an "isolated DNAse" is a purified enzyme. As used herein, the term "purified" or "purifying" also refers to the removal of contaminants from a sample.

[0076] DNAse I according to the present invention can be obtained from a variety of species including humans, primates, and rodents. In a preferred embodiment, the DNAse I of the present invention is human DNAse I. Human DNase I can be obtained from a variety of commercial sources.

[0077] The DNAse of the present invention may also be a modified form of the enzyme having enhanced physical, enzymatic and / or pharmacodynamic properties (see, for example, US 11,046,943).

[0078] Thus, the present invention also encompasses the administration of variants of human DNAse. Variants may contain mutations that do not alter the activity of the enzyme.

[0079] The term "enzyme activity" refers to the ability of an enzyme to cleave DNA (e.g., DNA fragmentation activity) and, preferably, to affect semen quality parameters as defined herein. Enzyme activity can be measured in vivo or in vitro using methods well known in the art, for example, as described in the Examples below.

[0080] The term "variant" refers to a sequence of amino acids which differs from the sequence of DNAse I specifically identified herein by one or more amino acid residues being deleted, substituted or added.

[0081] The term "added", as used herein, should be understood to mean any addition of an amino acid residue to a sequence described herein.

[0082] Variants encompass various amino acid substitutions. An amino acid "substitution" is the result of replacing one amino acid with another having similar or different structural and / or chemical properties. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0083] Typically, variants include conservative amino acid substitutions. Conservative substitution tables resulting in functionally similar amino acids are well known in the art. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine, polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, positively charged (basic) amino acids include arginine, lysine, and histidine, and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0084] Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another. 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W), 7) Serine (S), Threonine (T), and 8) Cysteine ​​(C), Methionine (M).

[0085] Variants according to the present invention also include non-polar to polar amino acid substitutions, and vice versa.

[0086] As used herein, the term "amino acid" or "amino acid residue" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0087] Variant sequences refer to amino acid sequences that can be characterized by the percentage of identity between their amino acid sequence and the amino acid sequence of DNAse I described herein.

[0088] In some embodiments, a variant sequence as defined herein refers to an amino acid sequence of DNAse I having an amino acid sequence having at least 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when compared to the sequence of DNAse I set forth herein in SEQ ID NO:1 (also referred to herein as the wild type sequence). [ka]

[0089] Amino acid sequence similarity, i.e., percentage of sequence identity, can be determined by sequence alignment as known in the art. Such alignment can be performed using several algorithms known in the art, for example, the BLAST program.

[0090] The first 22 amino acid residues marked in bold represent the signal peptide. The mature enzyme is defined as having the sequence shown by SEQ ID NO: 1 without the signal peptide (i.e., without the first 22 amino acids as defined above). As used herein, when referring to sequence identity with a wild-type DNAse enzyme, the sequence refers to the mature enzyme lacking the signal peptide.

[0091] In some embodiments, the DNAse variant comprises an N- or C-terminal fusion to a half-life extending moiety, such as albumin, transferrin, Fc, or an elastin-like protein.

[0092] In some embodiments, the DNAse variant comprises one or more polyethylene glycol (PEG) moieties that can be conjugated at one or more positions or at the C-terminus. In such cases, the DNAse variant is referred to as PEGylated DNAse. PEGylation can be performed by any method known in the art. In other embodiments, the DNAse variant is PASylated to extend the plasma half-life of the enzyme. PASylation is performed by incorporating small amino acid residues Pro, Ala and Ser (PAS) into the enzyme (see, for example, Schlapschy et al protein Eng Des Sel 2013;26(8):489-501). In such cases, the DNAse variant is referred to as PASylated DNAse.

[0093] In one aspect, the present invention provides a pharmaceutical composition.

[0094] The term "pharmaceutical composition" according to the present invention generally comprises a DNAse as defined herein, and a buffering agent, an agent to adjust the osmolarity of the composition, and optionally one or more pharma- ceutically acceptable carriers, excipients, and / or diluents known in the art.

[0095] As used herein, the term "pharmaceutical acceptable carrier, excipient, or diluent" refers to any one of an inert, non-toxic excipient that does not essentially react with DNAse I and is added to a pharmaceutical composition to give it form or consistency, provide protection from DNAse I degradation, increase survival in and out of the body, obtain penetration into the body and delivery to bodily fluids, and facilitate distribution of the agent within the subject's body and delivery to a target site (e.g., to cell-free DNA).

[0096] "Pharmaceutically acceptable carriers, excipients or diluents" include any solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like, known in the art. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Each carrier should be pharma- ceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not deleterious to the subject. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the pharmaceutical composition is contemplated.

[0097] According to one embodiment of the present invention, the pharmaceutical composition comprising human DNase I may be administered in combination with at least one additional therapeutic agent.

[0098] As used herein, the term "additional therapeutic agent" refers to any agent that may be used to improve semen quality.

[0099] In certain embodiments, the additional therapeutic agent is a molecule that interferes with the ability of deoxyribonucleic acid molecules to bind to receptors on the surface of sperm cells. These molecules may be receptor-binding and blocking molecules, or receptor-blocking molecules. Receptor-blocking molecules can be low molecular weight, e.g., deoxyribonucleotide dimers, phosphodeoxyribosyl pyrophosphate (PdRPP), deoxyribonucleotides or metal ions, or high molecular weight, e.g., peptides, polypeptides, proteins, antibodies, or glycoproteins. In one embodiment, the molecule is glycoprotein IF-1 or its analogs, which can block the interaction of cell-free DNA with sperm cell receptors. Deoxyribonucleic acid molecules that bind to and block deoxyribonucleic acid molecules can be peptides, polypeptides, proteins, DNA-binding proteins, nuclear proteins (as histones, protamines), or antibodies. Deoxyribonucleic acid molecules can also exert a detrimental effect on sperm cells by binding to the surface of sperm cell proteins and lipids that are not receptors. In this case, the drug can be a molecule that interferes with this binding.

[0100] According to another embodiment of the present invention, the agent can be a molecule that blocks a step in the signal transduction pathway formed by the binding of a deoxyribonucleic acid molecule to a receptor on the surface of a sperm cell. The agent can be a low molecular weight compound or a polymer, for example, a caspase inhibitor such as ZVAD and the bcl-2 protein family, or a dominant negative caspase protein.

[0101] Sperm cell damage caused by cell-free DNA may be accompanied by increased DNase activity in sperm cells.According to another embodiment of the present invention, the pharmaceutical composition of the present invention may comprise a DNase inhibitor that inhibits endogenous DNase activity in sperm cells.The drug may be a low molecular weight compound or a polymer.Preferred drugs include aurintricarboxylic acid (ATA), citrate or functional analogues thereof.

[0102] In other embodiments, a pharmaceutical composition according to the present invention further comprises an additional therapeutic agent.

[0103] The term "about" as used herein refers to values ​​that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the stated value, the deviation range including integer values ​​and, where applicable, non-integer values ​​that make up a continuous range. As disclosed and described, it is to be understood that the invention is not limited to the specific examples, method steps, and pharmaceutical compositions disclosed herein, and that such method steps and pharmaceutical compositions may vary somewhat. It is also to be understood that the terminology used herein is only for the purpose of describing particular embodiments and is not intended to be limiting, since the scope of the invention is limited only by the appended claims and equivalents thereof.

[0104] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0105] Unless the context requires otherwise, throughout this specification and the examples and claims which follow, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. EXAMPLES

[0106] Example 1: Pilot study in subfertile men: DNase I Recombinant human DNase I (rhDNase I, manufactured by Catalent) was supplied as a sterile solution in a glass vial containing 1 mL of rhDNase I at a concentration of 10 mg / mL in formulation buffer (8.77 mg / mL sodium chloride, 0.15 mg / mL calcium chloride; nominal pH 6.3).

[0107] The rhDNase I enzyme is identical to the endogenous human enzyme (CAS number: 0143831-71-4).

[0108] The amino acid sequence of human DNase I (UniProt, Human DNase-I, Isoform 1, Identifier: P24855-1) is outlined in SEQ ID NO:1.

[0109] rhDNase is produced by genetically engineered Chinese Hamster Ovary (CHO) cells containing DNA encoding the human protein, deoxyribonuclease I (DNase). Purification of the product is achieved by conventional tangential flow filtration and column chromatography techniques. The purified glycoprotein contains 260 amino acids with a relative molecular weight of approximately 37,000 daltons.

[0110] Clinical Trials: This was a prospective, open-label, uncontrolled, single-center study to evaluate the efficacy of intravenous (IV) injection of rhDNase I in subfertile male patients.

[0111] Main criteria for diagnosis and inclusion: male partner of subfertile couples with no diagnosed female factor infertility, trying to conceive for more than 2 years, and previous unsuccessful assisted reproductive technology (ART). Male patients were aged 18-50 years and had confirmed idiopathic poor semen quality but not azoospermia. One subject with azoospermia was enrolled as a deviation from the study protocol.

[0112] Study endpoint: evidence of improvement in one or more semen quality parameters in subfertile men following treatment with rhDNase I. Semen quality parameters assessed: total count, concentration, motility, motility quality or average velocity, morphology (Kruger or WHO), viability (eosin staining), micromorphology by MSOME, sperm chromatin stability (Halosperm assay).

[0113] All subjects received intravenous injections of 125 μg / kg body weight of rhDNase I twice daily on days 1 to 16. Injections were given in the morning and evening, 12 h apart.

[0114] The study drug was provided as 1 mg / ml rhDNase I, 2.5 ml per vial.

[0115] Four subjects participated in the study: subject no. 001 had azoospermia, subject no. 002 had severe monomorphism teratozoospermia (100% amorphous heads), subject no. 003 had mild monomorphism teratozoospermia (12% normal morphology as defined by the World Health Organization (WHO)), and subject no. 004 had severe monomorphism teratozoospermia.

[0116] Safety results: No abnormalities were observed in vital signs or physical examination findings. No adverse events were reported. No deaths or serious adverse events occurred during the study.

[0117] Efficacy Results: Improved semen quality was observed in 3 of 4 treated subjects. 3 of 4 couples successfully conceived using semen samples collected after rhDNase I treatment. Subject number 001: Baseline characteristics: ●Azoospermic patients. • High follicle-stimulating hormone (FSH) levels. ●Trying to get pregnant for 4.5 years or having a miscarriage. ●Two intracytoplasmic sperm injection (ICSI) cycles were unsuccessful. Three high power microscopy selection and intracytoplasmic sperm injection (IMSI) cycles were unsuccessful, one of which resulted in a miscarriage at 13 weeks. He suffered from varicocele in his left testicle and underwent surgery in 2001. ●High levels of cell-free DNA in the blood.

[0118] The patient was treated with rhDNase I as described above and semen quality was analyzed. Due to the subject's azoospermia, the only semen parameter that could be tracked was a high-power (×6,000) motile sperm organelle morphology examination (MSOME, Bartoov B, et al. J Androl. 2002 Jan-Feb;23(1):1-8)) of the few sperm cells observed.

[0119] Sperm preparation for morphological observation in wet (conventional) MSOME: A 1-2 μl aliquot of sperm suspension containing several thousand spermatozoa was transferred to a microdroplet of sperm medium containing 0%-8% polyvinylpyrrolidone (PVP) solution and placed in a glass-bottom dish under paraffin oil. Morphological assessment of moving sperm cells was made possible by the formation of a small compartment extending from the edge of the droplet, which trapped the heads of motile spermatozoa.

[0120] Sperm preparation for morphological observation in dry MSOME: Fresh ejaculate after liquefaction was transferred to a 15 ml round-bottom tube at room temperature (22-25°C). A soft pellet of ejaculated sperm cells was obtained by centrifugation in a swing-out bucket at 1500 RPM for 15 minutes at room temperature. The seminal plasma supernatant was removed. The sperm pellet was suspended in 1 ml of Ferticult-IVF medium (FertiPro NVBeernem, Belgium) and then recentrifuged at 1500 rpm for 5 minutes at room temperature. The tube was then transferred to a 5 percent CO2, 37°C incubator and tilted at 45 degrees for 40 minutes of swim-up incubation. The supernatant was then carefully removed down to the interphase of the sperm pellet. The swim-up supernatant was centrifuged at 1500 RPM for 5 minutes at room temperature and the supernatant was discarded. The pellet was smeared onto a microscope slide and allowed to air dry.

[0121] Sperm MSOME observations: Sperm cells were examined at 21° C. with an inverted microscope (Olympus IX 81) equipped with Nomarski optics, an Uplan Apo ×100 / 1.35 objective, and a 0.55 NA condenser. Images were captured with a DXC-950P color video camera (Sony). Morphological evaluation was performed on a monitor screen that reached a real magnification of 6300 with the above configuration.

[0122] MSOME results are provided in Figures 2 and 3. The results show an increase in sperm cells with oval heads, indicative of normal sperm cell morphology (Figure 2), and a decrease in amorphous sperm heads, indicative of abnormal sperm cell morphology (Figure 3). The rectangle represents 16 days of DNase I treatment. Only sperm cells without gross morphological abnormalities were included in this analysis. Subject number 002: Baseline characteristics: ●Normal semen volume (about 2ml). ●Normal sperm concentration (approximately 35 million / ml). ●Normal sperm density (approximately 70 million / ejaculate). • Normal sperm motility (about 50%). • Normal survival rate (approximately 70%). • Very severe isolated teratozoospermia (100%). ●I've been trying to get pregnant for eight years. •Four ICSI cycles were unsuccessful. ●Fertilization did not occur through IVF (in vitro fertilization) - He was suffering from varicocele of the testicles. ●High levels of cell-free DNA in the blood.

[0123] Measurements of semen volume and sperm cell concentration, density, motility, and viability were performed according to WHO guidelines (See WHO Laboratory Manual on Human Semen Examination and Sperm-Cervical Mucus Interaction. Fourth edition 1999. World Health Organization. Cambridge university press).

[0124] Briefly, the volume of each ejaculate was measured using a pipette. Sperm concentration was determined by counting cells using either a Helber or Neubauer hemocytometer (Helber is used when sperm concentration is high and Neubauer when it is low). Sperm density (= number of cells in the total ejaculate) was calculated by multiplying the ejaculate volume by the sperm concentration.

[0125] Sperm motility and progressive motility: Motile and non-motile sperm cells were counted using either a Helber or Neubauer hemocytometer and the percentage of motile sperm cells was calculated accordingly. A sample of 20 random motile sperm cells was examined for progressive motility by examining their ability to progress linearly through 200 μm and the percentage of progressively motile sperm cells was calculated accordingly.

[0126] rhDNase I treatment did not affect sperm concentration, density, or sperm cell viability and motility (not shown), all of which were within normal ranges. However, as shown in Figures 4-6, rhDNase I treatment did have a beneficial effect on the following parameters: 1. Sperm morphology represented by an increase (0%-30%) in sperm cells with oval heads and a decrease in the number of sperm cells with round and amorphous heads. 2. The stability of sperm DNA was measured by the Halosperm test, which allows the measurement of DNA fragmentation. Halosperm is based on the sperm chromatin dispersion (SCD) technology. The Halosperm test is an in vitro diagnostic kit [Halotech DNA, Spain] that controls the DNA denaturation process to facilitate the subsequent removal of proteins contained in each sperm. In this way, normal sperm produce a halo formed by a loop of DNA in the sperm head, which is absent in sperm with damaged DNA. The percentage of sperm cells without DNA fragmentation, as shown by the Halosperm test, increased from 10% to 50%. 3. Three pregnancies were achieved using semen obtained after rhDNase I treatment.

[0127] DNase I treatment resulted in an increase in sperm cells with oval heads (Figure 4) and a decrease in the number of sperm cells with round and amorphous heads (Figure 5). The rectangle represents 16 days of DNase I treatment. Only sperm cells without gross morphological abnormalities were included in this analysis.

[0128] As can be seen in FIG. 6, treatment with rhDNase I (days 1-16 marked by rectangles) caused an increase in the percentage of normal sperm cells without DNA fragmentation, as demonstrated by Halosperm analysis. Subject Number 003: Baseline characteristics: ●Low semen volume (approximately 1ml) ●Normal sperm concentration (approximately 50 million / ml) ●Normal sperm cell density (approximately 50 million / ejaculate) ● Normal sperm cell motility (approximately 60%) ● Survival rate of normal sperm cells (approximately 75%) Mild isolated teratozoospermia (12% according to WHO criteria, 2% according to Kruger criteria) ●I have been trying to get pregnant for two years.

[0129] RhDNase I treatment did not induce any major changes in sperm concentration, density, and sperm cell viability and motility (not shown), all of which were within normal ranges. Rhythmic changes in semen volume were observed, probably due to reduced semen volume. However, as shown in Figures 7-9, rhDNase I treatment showed beneficial effects on the following parameters: 1. Sperm morphology, as represented by a three-fold increase in normal morphology according to WHO 4th edition criteria (reaching normal), a 30% reduction in sperm head anomalies according to WHO criteria (reaching normal), and a five-fold increase in the percentage of sperm cells with normal nuclei observed by MSOME (reaching borderline). 2. Sperm DNA stability measured by the Halosperm test (2.5-fold increase in sperm cells without DNA fragmentation, reaching normal values). 3. One pregnancy was achieved using semen obtained after rhDNase I treatment.

[0130] These changes indicate that subject #003 achieved normal semen after treatment with rhDNase I.

[0131] As can be seen in FIG. 7, treatment with rhDNase I (days 1-16 marked by rectangles) led to an improvement in sperm morphology according to WHO criteria.

[0132] As can be seen in Figure 8, treatment with rhDNase I (days 1-16 marked by the rectangle) led to a reduction in sperm head malformations according to WHO criteria.

[0133] As can be seen in FIG. 9, treatment with rhDNase I (days 1-16 marked by rectangles) resulted in an increase in the percentage of sperm cells without DNA fragmentation. Target number 004 Baseline characteristics: ●Normal semen volume (approximately 2ml) ●Low sperm concentration (approximately 13 million / ml) ●Low sperm motility (about 12%) ●Low survival rate (approximately 33%) Very severe isolated teratozoospermia (100%) ●High levels of cell-free DNA in the blood. ●We have been trying to get pregnant for 2.5 years. ●One failed intrauterine insemination (IUI) - Two unsuccessful ICSI cycles

[0134] No significant changes in sperm cells were observed after DNase I treatment. Nevertheless, one pregnancy was achieved using semen obtained in the study.

[0135] Serum DNase activity of treated subjects after injection of 125 μg / kg rhDNase was assessed using the Single Radial Enzyme Diffusion (SRED) test performed according to Nadano D, et al. Clin Chem. 1993 Mar;39(3):448-52. Analysis of one representative subject (subject no. 001) is provided in FIG. 10.

Claims

1. A pharmaceutical composition for use in a method for performing it in a couple in need of induction of pregnancy, the pharmaceutical composition comprising DNase and a pharmaceutically acceptable carrier, wherein the male subject of the couple has been diagnosed with subfertility, the method comprising systemic administration to the male subject of a pharmaceutical composition comprising about 50 to about 1000 μg / kg of human DNase over a treatment course period, the pharmaceutical composition.

2. The pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition is administered for a treatment course period sufficient to achieve maturation of fresh sperm cells in the male subject.

3. The pharmaceutical composition for use according to claim 1, wherein the administration is over a treatment course period of about 10 days to about 3 months, or about 10 days to 16 days.

4. The pharmaceutical composition for use according to claim 1, wherein the human male subject has idiopathic semen quality decline, has low sperm survival rate and / or motility and / or concentration, or has azoospermia, oligospermia, mild or severe isolated teratozoospermia, asthenozoospermia, or oligoasthenoteratozoospermia (OTA).

5. The pharmaceutical composition for use according to claim 1, wherein the human male subject is the male partner of a couple who has been unable to conceive for more than 2 years or has had miscarriages, or has been unable to conceive in at least one assisted reproductive technology (ART) procedure.

6. The pharmaceutical composition for use according to claim 1, wherein administration of the pharmaceutical composition results in at least a 35% improvement in one or more sperm quality parameters selected from the group consisting of total sperm count, sperm concentration, sperm motility, quality or average velocity of sperm motility, sperm morphology, sperm survival rate, and sperm chromatin stability.

7. The pharmaceutical composition for use according to claim 1, wherein the sperm of the human male subject is sampled within 1 to 14 days after completion of the treatment course period.

8. The pharmaceutical composition for use according to claim 1, wherein the sperm of the human male subject is sampled within 1 to 5 days after completion of the treatment course period.

9. The pharmaceutical composition for use according to claim 1, wherein completion of the treatment course period with the pharmaceutical composition is synchronized with ovulation in the female subject of the couple.

10. The pharmaceutical composition for use according to claim 9, wherein ovulation subsides 1 to 14 days after completion of the treatment course period.

11. The pharmaceutical composition for use according to claim 9, wherein ovulation ceases within 1 to 5 days after completion of the treatment course period.

12. The pharmaceutical composition for use according to claim 1, wherein the method further comprises administration of an additional therapeutic agent.

13. The pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition comprises an additional therapeutic agent.

14. The pharmaceutical composition for use according to claim 1, wherein the DNase is pegylated DNase or PASylated DNase.

15. The DNase I is a human DNase I comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, the human DNase I is intravenously injected at a dose of 250 μg / kg / day for 14 to 16 days, and the sperm of the human male subject is sampled within 1 to 5 days after completion of the treatment course period, or ovulation ceases within 1 to 5 days after completion of the treatment course period, the pharmaceutical composition for use according to claim 1.

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