WO2010104882A1 - Procédés de modulation de la motilité des spermatozoïdes - Google Patents

Procédés de modulation de la motilité des spermatozoïdes Download PDF

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WO2010104882A1
WO2010104882A1 PCT/US2010/026705 US2010026705W WO2010104882A1 WO 2010104882 A1 WO2010104882 A1 WO 2010104882A1 US 2010026705 W US2010026705 W US 2010026705W WO 2010104882 A1 WO2010104882 A1 WO 2010104882A1
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alkyl
haloalkyl
substituted
phenyl
independently
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PCT/US2010/026705
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English (en)
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Charles Henley
David Martin
Juan Mariano Rodriquez Portillo
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Amgen Inc.
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Publication of WO2010104882A1 publication Critical patent/WO2010104882A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4402—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 2, e.g. pheniramine, bisacodyl
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/061—Sperm cells, spermatogonia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/10—Conditioning of cells for in vitro fecondation or nuclear transfer

Definitions

  • This invention relates generally to the field of medicine and, more specifically, to methods for enhancing mammalian sperm motility, mobility and vitality.
  • Male infertility is characterized by low sperm count, low motility and low proportion of sperm with normal morphology. It is a disorder with largely unknown causes, probably related to primary testicular disease or external factors such as trauma or environmental causes. It has been suggested that he chances of conception with low sperm motility are lower than the chances of conception with low sperm count because of the differences in causes between the two. The most common cause of low sperm motility is a genetic problem. In addition, there are fewer treatments that have proven to be effective in increasing the chances of conception with low sperm motility (asthenozoospermia) than there are for other causes of infertility. Analysis of sperm motility includes a description of specific motility characteristics such as speed, straight-line swimming or "hyperactivity" (a wildly gyrating swim pattern).
  • male infertility includes mostly addressing known reversible factors first. For example, discontinuing any medication known to have an effect on spermatogenesis or ejaculation, as well as decreasing alcohol intake, and treating thyroid or other endocrine disease. There are currently no available or acceptable pharmaceutical treatments for male infertility or subfertility.
  • the present invention provides methods for enhancing the motility, mobility and viability of a mammalian spermatozoon comprising contacting the mammalian spermatozoon with a therapeutically effective amount of a calcimimetic compound.
  • a plurality of mammalian spermatozoa are identified as having decreased motility.
  • mammalian spermatozoa are identified as having subnormal motility.
  • mammalian spermatozoa are identified as having normal motility, but the subject is in need of increasing sperm motility to increase chances of fertilization.
  • the contacting comprises culturing the spermatozoon in a physiological medium comprising a therapeutically effective amount of a calcimimetic compound.
  • Contacting may be, for example, from about 30 seconds to about 120 minutes or from about 1 minute to 20 minutes.
  • the spermatozoon can be substantially separated from seminal plasma.
  • the contacting may occur in a mammalian female reproductive tract.
  • the mammalian female can be a human.
  • the mammalian aspect can be a horse, a pig, a goat, a sheep, or a cow.
  • the invention further provides methods of fertilizing a mammalian egg, the method comprising contacting a mammalian egg with a mammalian spermatozoon, whose motility has been enhanced contacting the mammalian spermatozoon with a therapeutically effective amount of a calcimimetic compound.
  • the contacting of the mammalian egg occurs in vitro.
  • the method may be further comprising fertilization of the mammalian egg and placing the fertilized egg in a mammalian uterus.
  • the contacting of the mammalian egg occurs in vivo.
  • the contacting of the mammalian egg occurs in a female reproductive tract.
  • the subject can be mammal.
  • the mammal may be a horse, a pig, a goat, a sheep, or a cow.
  • the subject may be human.
  • the mammalian egg is human.
  • the mammalian egg can be from a horse, a pig, a goat, a sheep, or a cow or any other mammal.
  • calcimimetic compounds useful in the methods of the present invention are described in detail in Detailed Description below.
  • Figure 1 illustrates the expression of the calcium sensing receptor (CaSR) in as demonstrated by immunohistochemistry staining in rat testicle, a) Sertoli cells; b) spermatogonias I and II; c) spermatocyte I and II; d) spermatides; e) spermatozoa; f) Leydig cells.
  • CaSR calcium sensing receptor
  • Figure 2 demonstrates that the CaSR is expressed both in basal and principal epidymal cells as demonstrated by immunohistochemistry staining of the CaSR.
  • Basal epididymal cells b) principal epididymal cells; c) spermatozoa.
  • Magnification X20 Magnification X20.
  • Figure 3 shows immunohistochemistry staining of the CaSR within the epididymal duct. The staining is more pronounced in the head of spermatozoa. Magnification X40.
  • Figure 4 demonstrates the effect of calcimimetic Compound A (N-((6-(methyloxy)-4'- (trifluoromethyl)-l, l'-biphenyl-3-yl)methyl)-l-phenylethanamine) on rat sperm motility after 60 and 120 minutes of incubation. Results are presented as the mean ⁇ SE of a semiquantitative score (0-4).
  • Figure 5 illustrates the effect of calcimimetic Compound A on pig sperm motility after 60 and 240 minutes of incubation. Influence of Compound A on the percentage of cells that reach a mean velocity (VAP) > 45 ⁇ m/s.
  • Treating" or “treatment” of a disease includes: (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a subject that may be or has been exposed to the disease or conditions that may cause the disease, or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or any of its clinical symptoms, or (3) relieving the disease, i.e., causing regression of the disease or any of its clinical symptoms.
  • Administration in combination with or “together with” one or more further therapeutic agents includes simultaneous or concurrent administration and consecutive administration in any order.
  • terapéuticaally effective amount is the amount of the compound of the invention that will achieve the goal of improvement in disorder severity and the frequency of incidence.
  • the improvement in disorder severity includes the reversal of the disease, as well as slowing down the progression of the disease.
  • CaSR calcium sensing receptor
  • Activation of the CaSR produces rapid, transient increases in cytosolic calcium concentration by mobilizing calcium from thapsigargin-sensitive intracellular stores and by increasing calcium influx though voltage-insensitive calcium channels in the cell membrane (Brown et ah, Nature 366: 575-580, 1993; Yamaguchi et al, Adv Pharmacol 47: 209-253, 2000).
  • spermatozoon and “spermatozoa” are used interchangeably with the term “sperm”.
  • Asthenospermia is defined as less than 60% motility, frequently with subnormal sperm velocity.
  • Calcimimetic compounds and pharmaceutical compositions comprising them, administration and dosage
  • Calcimimetic compounds definitions
  • the term "calcimimetic compound” or “calcimimetic” refers to a compound that binds to calcium sensing receptors and induces a conformational change that reduces the threshold for calcium sensing receptor activation by the endogenous ligand Ca + .
  • These calcimimetic compounds can also be considered allosteric modulators of the calcium receptors.
  • a calcimimetic can have one or more of the following activities: it evokes a transient increase in internal calcium, having a duration of less that 30 seconds (for example, by mobilizing internal calcium); it evokes a rapid increase in [Ca 2+ J, occurring within thirty seconds; it evokes a sustained increase (greater than thirty seconds) in [Ca 2+ J (for example, by causing an influx of external calcium); evokes an increase in inositol-1,4,5- triphosphate or diacylglycerol levels, usually within less than 60 seconds; and inhibits dopamine- or isoproterenol-stimulated cyclic AMP formation.
  • the transient increase in [Ca J can be abolished by pretreatment of the cell for ten minutes with 10 mM sodium fluoride or with an inhibitor of phospholipase C, or the transient increase is diminished by brief pretreatment (not more than ten minutes) of the cell with an activator of protein kinase C, for example, phorbol myristate acetate (PMA), mezerein or (-) indolactam V.
  • a calcimimetic compound can be a small molecule.
  • a calcimimetic can be an agonistic antibody to the CaSR.
  • Calcimimetic compounds useful in the present invention include those disclosed in, for example, European Patent No. 637,237, 657,029, 724,561, 787,122, 907,631, 933,354, 1,203,761, 1,235 797, 1,258,471, 1,275,635, 1,281,702, 1,284,963, 1,296,142, 1,308,436, 1,509,497, 1,509,518, 1,553,078; International Publication Nos.
  • the calcimimetic compound is chosen from compounds of Formula I and pharmaceutically acceptable salts thereof:
  • Xi and X 2 which may be identical or different, are each a radical chosen from CH 3 , CH 3 O, CH 3 CH 2 O, Br, Cl, F, CF 3 , CHF 2 , CH 2 F, CF 3 O, CH 3 S, OH, CH 2 OH, CONH 2 , CN, NO 2 , CH 3 CH 2 , propyl, isopropyl, butyl, isobutyl, t-butyl, acetoxy, and acetyl radicals, or two of Xi may together form an entity chosen from fused cycloaliphatic rings, fused aromatic rings, and a methylene dioxy radical, or two of X 2 may together form an entity chosen from fused cycloaliphatic rings, fused aromatic rings, and a methylene dioxy radical; provided that X 2 is not a 3 -t-butyl radical; n ranges from 0 to 5; m ranges from 1 to 5; and the alkyl radical is chosen
  • the calcimimetic compound may also be chosen from compounds of Formula II:
  • R 1 is aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, cycloalkyl, or substituted cycloalkyl;
  • R 2 is alkyl or haloalkyl
  • R 3 is H, alkyl, or haloalkyl
  • R 4 is H, alkyl, or haloalkyl
  • R 6 is aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, cycloalkyl, or substituted cycloalkyl; each R a is, independently, H, alkyl or haloalkyl; each R b is, independently, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl, each of which may be unsubstituted or substituted by up to 3 substituents selected from the group consisting of alkyl, halogen, haloalkyl, alkoxy, cyano, and nitro; each R c is, independently, alkyl, haloalkyl, phenyl or benzyl, each of which may be substituted or unsubstituted; each R is, independently, H, alkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl wherein the alkyl , aryl, aralkyl, heterocyclyl, and hetero
  • the calcimimetic compound can be N-((6-(methyloxy)-4'- (trifluoromethyl)-l,r-biphenyl-3-yl)methyl)-l-phenylethanamine, or a pharmaceutically acceptable salt thereof.
  • the calcimimetic compound can be (lR)-N-((6- chloro-3'-fluoro-3-biphenylyl)methyl)-l-(3-chlorophenyl)ethanamine, or a pharmaceutically acceptable salt thereof.
  • the calcimimetic compound can be (IR)-I -(6- (methyloxy)-4'-(trifluoromethyl)-3-biphenylyl)-N-((lR)-l-phenylethyl)ethanamine, or a pharmaceutically acceptable salt thereof.
  • the calcimimetic compound can be chosen from compounds of Formula III
  • R 1 is R b ;
  • R is Ci_ 8 alkyl or Ci_ 4 haloalkyl
  • R 3 is H, Ci- 4 haloalkyl or C 1-8 alkyl
  • R 4 is H, Ci- 4 haloalkyl or C 1-4 alkyl
  • — is a single bond then Y is -CR a R 6 - or -NR d - and Z is -CR a R 7 - or -NR d -;
  • R a is, independently, at each instance, H, Ci- 4 haloalkyl or Ci- ⁇ alkyl
  • R b is, independently, at each instance, phenyl, benzyl, naphthyl or a saturated or unsaturated 5- or 6-membered ring heterocycle containing 1, 2 or 3 atoms selected from N, O and S, with no more than 2 of the atoms selected from O and S, wherein the phenyl, benzyl or heterocycle are substituted by 0, 1, 2 or 3 substituents selected from Ci- ⁇ alkyl, halogen, Ci_ 4 haloalkyl, -OCi- ⁇ alkyl, cyano and nitro;
  • a calcimimetic compound is N-(3-[2-chlorophenyl]-propyl)-R----methyl- 3-methoxybenzylamine.
  • a calcimimetic compound is N-((6-(methyloxy)-4'- (trifluoromethyl)- 1 , 1 '-biphenyl-3 -yl)methyl)- 1 -phenylethanamine.
  • the calcimimetic compound of the invention can be chose from compounds of Formula IV
  • R 1 is phenyl, benzyl, naphthyl or a saturated or unsaturated 5- or 6-membered heterocyclic ring containing 1, 2 or 3 atoms selected from N, O and S, with no more than 2 of the atoms selected from O and S, wherein the phenyl, benzyl, naphthyl or heterocyclic ring are substituted by 0, 1, 2 or 3 substituents selected from Ci- ⁇ alkyl, halogen, Ci_ 4 haloalkyl, -OCi- ⁇ alkyl, cyano and nitro;
  • R is Ci- 8 alkyl or Ci- 4 haloalkyl
  • R 3 is H, Ci_ 4 haloalkyl or d_ 8 alkyl
  • R 4 is H, Ci_ 4 haloalkyl or C 1-8 alkyl
  • R a is, independently, at each instance, H, Ci_ 4 haloalkyl, Ci_ 6 alkyl, Ci_ 6 alkenyl, Ci- ⁇ alkylaryl or arylCi- ⁇ alkyl:
  • R is, independently, at each instance, Ci_salkyl, Ci_ 4 haloalkyl, phenyl, benzyl, naphthyl or a saturated or unsaturated 5- or 6-membered heterocyclic ring containing 1, 2 or 3 atoms selected from N, O and S, with no more than 2 of the atoms selected from O and S, wherein the phenyl, benzyl, naphthyl or heterocyclic ring are substituted by 0, 1 , 2 or 3 substituents selected from Ci- ⁇ alkyl, halogen, -OCi- ⁇ alkyl, cyano and nitro;
  • R c is, independently, at each instance, Ci_ 6 alkyl, Ci_ 4 haloalkyl, phenyl or benzyl;
  • the calcimimetic compound can be N-(2-chloro-5-((((lR)-l- phenylethyl)amino)methyl)phenyl)-5-methyl-3-isoxazolecarboxamide or a pharmaceutically acceptable salt thereof.
  • the calcimimetic compound can be N-(2-chloro-5- ((((lR)-l-phenylethyl)amino)methyl)phenyl)-2-pyridinecarboxamide or a pharmaceutically acceptable salt thereof.
  • Calcimimetic compounds useful in the methods of the invention include the calcimimetic compounds described above, as well as their stereoisomers, enantiomers, polymorphs, hydrates, and pharmaceutically acceptable salts of any of the foregoing.
  • compounds binding at the CaSR-activity modulating site can be identified using, for example, a labeled compound binding to the site in a competition-binding assay format.
  • Calcimimetic activity of a compound can be determined using techniques such as those described in International Publications WO 93/04373, WO 94/18959 and WO 95/1121 1. Other methods that can be used to assess compounds calcimimetic activity are described below.
  • HEK 293 cells engineered to express human parathyroid CaSR have been described in detail previously (Nemeth EF et al. (1998) Proc. Natl. Acad. Sci. USA 95:4040-4045). This clonal cell line has been used extensively to screen for agonists, allosteric modulators, and antagonists of the CaSR (Nemeth EF et al. (2001) J. Pharmacol. Exp. Ther. 299:323-331).
  • the cells are recovered from tissue culture flasks by brief treatment with 0.02% ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS) and then washed and resuspended in Buffer A (126 mM NaCl, 4 mM KCl, 1 mM CaCl 2 , 1 mM MgSO 4 , 0.7 mM K 2 HPO 4 ZKH 2 PO 4 , 20 mM Na-Hepes, pH 7.4) supplemented with 0.1% bovine serum albumin (BSA) and 1 mg/ml D-glucose.
  • BSA bovine serum albumin
  • the cells are loaded with fura-2 by incubation for 30 minutes at 37°C in Buffer A and 2 ⁇ M fura-2 acetoxymethylester.
  • the cells are washed with Buffer B (Buffer B is Buffer A lacking sulfate and phosphate and containing 5 mM KCl, 1 mM MgCl 2 , 0.5 mM CaCl 2 supplemented with 0.5% BSA and 1 mg/ml D-glucose) and resuspended to a density of 4 to 5 x 10 6 cells/ml at room temperature.
  • Buffer B is Buffer A lacking sulfate and phosphate and containing 5 mM KCl, 1 mM MgCl 2 , 0.5 mM CaCl 2 supplemented with 0.5% BSA and 1 mg/ml D-glucose
  • Excitation and emission wavelengths are 340 and 510 nm, respectively.
  • the fluorescent signal is recorded in real time using a strip- chart
  • HEK 293 cells are maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 200 ⁇ g/ml hygromycin.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • the cells are trypsinized and plated in the above medium at 1.2 x 10 5 cells/well in black sided, clear-bottom, collagen 1-coated, 96-well plates. The plates are centrifuged at 1,000 rpm for 2 minutes and incubated under 5% CO 2 at 37°C overnight. Cells are then loaded with 6 ⁇ M fluo-3 acetoxymethylester for 60 minutes at room temperature.
  • the ECso's for the CaSR-active compounds can be determined in the presence of 1 mM Ca + .
  • the EC 50 for cytoplasmic calcium concentration can be determined starting at an extracellular Ca 2+ level of 0.5 mM. FLIPR experiments are done using a laser setting of 0.8 W and a 0.4 second CCD camera shutter speed.
  • Bovine Parathyroid Cells are challenged with calcium, CaSR-active compound or vehicle (20 ⁇ l) and fluorescence monitored at 1 second intervals for 50 seconds. Then a second challenge (50 ⁇ l) of calcium, CaSR-active compound, or vehicle can be made and the fluorescent signal monitored. Fluorescent signals are measured as the peak height of the response within the sample period. Each response is then normalized to the maximum peak observed in the plate to determine a percentage maximum fluorescence.
  • Bovine Parathyroid Cells The effect of calcimimetic compounds on CaSR-dependent regulation of PTH secretion can be assessed using primary cultures of dissociated bovine parathyroid cells.
  • Dissociated cells can be obtained by collagenase digestion, pooled, then resuspended in Percoll purification buffer and purified by centrifugation at 14,500 x g for 20 minutes at 4°C.
  • the dissociated parathyroid cells are removed and washed in a 1 : 1 mixture of Ham's F- 12 and DMEM (F- 12/DMEM) supplemented with 0.5% BSA, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 20 ⁇ g/ml gentamicin.
  • the cells are finally resuspended in F-12/DMEM containing 10 U/ml penicillin, 10 ⁇ g/ml streptomycin, and 4 ⁇ g/ml gentamicin, and BSA was substituted with ITS+ (insulin, transferrin, selenous acid, BSA, and linoleic acid; Collaborative Research, Bedford, MA).
  • ITS+ insulin, transferrin, selenous acid, BSA, and linoleic acid
  • the cells are removed from flasks by decanting and washed with parathyroid cell buffer (126 mM NaCl, 4 mM KCl, 1 mM MgSO 4 , 0.7 mM K 2 HPO 4 /KH 2 PO 4 , 20 mM Na-Hepes, 20; pH 7.45 and variable amounts Of CaCl 2 as specified) containing 0.1% BSA and 0.5 mM CaCl 2 .
  • the cells are resuspended in this same buffer and portions (0.3 ml) are added to polystyrene tubes containing appropriate controls, CaSR-active compound, and/or varying concentrations Of CaCl 2 . Each experimental condition is performed in triplicate.
  • Rat MTC 6-23 cells (clone 6), purchased from ATCC (Manassas, VA) are maintained in growth media (DMEM high glucose with calcium/15% HIHS) that is replaced every 3 to 4 days. The cultures are passaged weekly at a 1 :4 split ratio. Calcium concentration in the formulated growth media is calculated to be 3.2 mM. Cells are incubated in an atmosphere of 90% O 2 /10% CO 2 , at 37°C. Prior to the experiment, cells from sub-confluent cultures are aspirated and rinsed once with trypsin solution. The flasks are aspirated again and incubated at room temperature with fresh trypsin solution for 5-10 minutes to detach the cells.
  • DMEM high glucose with calcium/15% HIHS calcium/15% HIHS
  • the detached cells are suspended at a density of 3.0 x 10 5 cells/mL in growth media and seeded at a density of 1.5 x 10 5 cells/well (0.5 mL cell suspension) in collagen-coated 48 well plates (Becton Dickinson Labware, Bedford, MA).
  • the cells are allowed to adhere for 56 hours post- seeding, after which the growth media was aspirated and replaced with 0.5 mL of assay media (DMEM high glucose without/2% FBS).
  • the cells are then incubated for 16 hours prior to determination of calcium-stimulated calcitonin release.
  • the actual calcium concentration in this media is calculated to be less than 0.07 mM.
  • calcitonin release 0.35 mL of test agent in assay media is added to each well and incubated for 4 hours prior to determination of calcitonin content in the media. Calcitonin levels are quantified according to the vendor's instructions using a rat calcitonin immunoradiometric assay kit (Immutopics, San Clemente, CA). Inositol phosphate Assay
  • CHO(CaSR) Chinese hamster ovarian cells transfected with an expression vector containing cloned CaSR from rat brain [CHO(CaSR)] or not [CHO(WT)] (Ruat M., Snowman AM., J. Biol. Chem 271, 1996, p 5972).
  • CHO(CaSR) has been shown to stimulate tritiated inositol phosphate ([ 3 H]IP) accumulation upon activation of the CaSR by Ca 2+ and other divalent cations and by NPS 568 (Ruat et ah, J. Biol. Chem 271, 1996).
  • [ 3H ]IP accumulation produced by 10 ⁇ M of each CaSR-active compound in the presence of 2 mM extracellular calcium can be measured and compared to the effect produced by 10 mM extracellular calcium, a concentration eliciting maximal CaSR activation (Dauban P. et ah, Bioorganic & Medicinal Chemistry Letters, 10, 2000, p 2001).
  • Calcimimetic compounds useful in the present invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids.
  • the salts include, but are not limited to, the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxy-ethanesulfonate, lactate, maleate, mandelate, methansulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, palm
  • salts for the carboxy group are well known to those skilled in the art and include, for example, alkaline, alkaline earth, ammonium, quaternary ammonium cations and the like.
  • suitable pharmaceutically acceptable salts see Berge et al. J. Pharm. Sci. 66: 1, 1977.
  • salts of hydrochloride and salts of methanesulfonic acid can be used.
  • the calcium-receptor active compound can be chosen from cinacalcet, i.e., N-(l-(R)-(l-naphthyl)ethyl]-3-[3-(trifluoromethyl)phenyl]-l- aminopropane, cinacalcet HCl, and cinacalcet methanesulfonate.
  • the calcimimetic compound such as cinacalcet HCl and cinacalcet methanesulfonate, can be in various forms such as amorphous powders, crystalline powders, and mixtures thereof.
  • the crystalline powders can be in forms including polymorphs, psuedopolymorphs, crystal habits, micromeretics, and particle morphology.
  • the compounds useful in this invention are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • the compounds useful in this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
  • the pharmaceutical compositions may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • the pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
  • Solid dosage forms for oral administration may include capsules, tablets, pills, powders, suppositories, and granules.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch.
  • Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate.
  • the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
  • Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting, sweetening, flavoring, and perfuming agents.
  • the therapeutically effective amount of the calcium receptor-active compound in the compositions useful in the invention can range from about 0.1 mg to about 180 mg, for example from about 5 mg to about 180 mg, or from about lmg to about 100 mg of the calcimimetic compound per subject.
  • the therapeutically effective amount of calcium receptor-active compound in the composition can be chosen from about 0.1 mg, about 1 mg, 5 mg, about 15 mg, about 20 mg, about 30 mg, about 50 mg, about 60 mg, about 75 mg, about 90 mg, about 120 mg, about 150 mg, about 180 mg. While it may be possible to administer a calcium receptor-active compound to a subject alone, the compound administered will normally be present as an active ingredient in a pharmaceutical composition.
  • a pharmaceutical composition of the invention may comprise a therapeutically effective amount of at least one calcimimetic compound, or an effective dosage amount of at least one calcimimetic compound.
  • an "effective dosage amount” is an amount that provides a therapeutically effective amount of the calcium receptor-active compound when provided as a single dose, in multiple doses, or as a partial dose.
  • an effective dosage amount of the calcium receptor-active compound of the invention includes an amount less than, equal to or greater than an effective amount of the compound; for example, a pharmaceutical composition in which two or more unit dosages, such as in tablets, capsules and the like, are required to administer an effective amount of the compound, or alternatively, a multidose pharmaceutical composition, such as powders, liquids and the like, in which an effective amount of the calcimimetic compound is administered by administering a portion of the composition.
  • a pharmaceutical composition in which two or more unit dosages, such as in tablets, capsules and the like, are required to administer an effective amount of the calcium receptor-active compound may be administered in less than an effective amount for one or more periods of time (e.g., a once-a-day administration, and a twice-a-day administration), for example to ascertain the effective dose for an individual subject, to desensitize an individual subject to potential side effects, to permit effective dosing readjustment or depletion of one or more other therapeutics administered to an individual subject, and/or the like.
  • the effective dosage amount of the pharmaceutical composition useful in the invention can range from about 1 mg to about 360 mg from a unit dosage form, for example about 5 mg, about 15 mg, about 30 mg, about 50 mg, about 60 mg, about 75 mg, about 90 mg, about 120 mg, about 150 mg, about 180 mg, about 210 mg, about 240 mg, about 300 mg, or about 360 mg from a unit dosage form.
  • the compositions disclosed herein comprise a therapeutically effective amount of a calcium receptor-active compound for the enhancement of sperm motility.
  • a calcium receptor-active compound for the enhancement of sperm motility.
  • the calcimimetic compound such as cinacalcet HCl can be present in an amount ranging from about 1% to about 70%, such as from about 5% to about 40%, from about 10% to about 30%, or from about 15% to about 20%, by weight relative to the total weight of the composition.
  • compositions useful in the invention may contain one or more active ingredients in addition to the calcium sensing receptor-active compound.
  • the additional active ingredient may be another calcimimetic compound, or it may be an active ingredient having a different therapeutic activity.
  • the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • the pharmaceutical compositions useful for methods of the invention may include additional compounds as described in more detail below.
  • the compounds used to practice the methods of the instant invention can be formulated for oral administration that release biologically active ingredients.
  • the invention provides methods for enhancing the motility of a mammalian spermatozoon.
  • the methods provide contacting a spermatozoon from a mammalian subject with a calcimimetic compound.
  • the spermatozoon can be derived from a male subject of the relevant species by methods known in the art including as a component of semen ejaculated by the male subject.
  • sperm can be removed by aspiration from the vas deferens, the epididymis, or the testis of a male subject.
  • the semen can be optionally washed by the standard procedures so that the contacting of the spermatozoon with the calcimimetic compounds of the invention occurs in a solution that is substantially free from seminal fluid (e.g., free from seminal fluid-derived components such as proteins, lipids or nucleic acids that occur in the seminal fluid).
  • seminal fluid e.g., free from seminal fluid-derived components such as proteins, lipids or nucleic acids that occur in the seminal fluid.
  • the mammalian male subjects can be any mammal including a human, a primate (e.g., a monkey, a gorilla, a chimpanzee), an equine subject (e.g., a horse, a donkey, a zebra), a pig, a goat, a bull, a sheep, a dog, a cat, a rabbit, a guinea pig, a hamster, a gerbil, a rat, or a mouse.
  • a mammalian subject used in the present invention may be one whose spermatozoa have lower than normal progressive (straight line) velocity.
  • normal straight line velocity or "normal progressive velocity” is the straight line velocity exhibited by spermatozoa from a fertile male subject of the same species (i.e., a subject whose spermatozoa have no evident compromised ability to fertilize and egg of the same species).
  • the methods of the invention can be applied to spermatozoa from normal subjects, e.g., subjects whose spermatozoa have no evident compromised ability to fertilize an appropriate egg but whose chances to conceive need to be increased.
  • spermatozoa of the subject Prior to treatment using the methods of the invention, spermatozoa of the subject can be optionally tested for motility.
  • the spermatozoa tested in this way may be obtained from the same semen sample used to obtain spermatozoa to be contacted with the compounds of the invention or from a separate sample of semen from the subject.
  • Spermatozoa can be tested for motility after the treatment to assess the efficacy of the methods of the invention.
  • spermatozoa can be cultured in a physiological medium (e.g., tissue culture medium) at a variety of temperatures (e.g., from about 15°C to 39°C).
  • physiological medium e.g., tissue culture medium
  • the physiological medium can be any culture medium in which mammalian spermatozoa can remain viable and retain their fertilizing potential. Examples of appropriate media include BWW medium or Dulbecco's modified Eagle's Medium (DMEM).
  • medium supplements can be added to the medium, for example, bacterial and fungicidal antibiotics (e.g., penicillin, streptomycin, gentamicin, amphotericin B); blood serum from the same species but different individuals as the semen donor (i.e., allogenic blood serum), blood semen from the semen donor (i.e., autologous blood serum), blood serum from one or more individuals of a species other than that of the semen donor (i.e., xenogeneic blood serum).
  • Xenogeneic blood serum can be, for example, fetal bovine serum, equine serum, goat serum, sheep serum, or pig serum.
  • Sera used to supplement tissue culture medium can be allogenic or autologous blood serum.
  • Sera to be used as culture medium supplement can be screened for the presence of anti-spermatozoa antibodies prior to use, and those sera containing detectable levels of such antibodies would be excluded from use.
  • Additional media supplements include various essential or non-essential amino acids (e.g., glutamine), proteins (e.g., human or bovine serum albumin, insulin, and transferrin), nucleic acids, nucleotides, nucleosides, and lipids.
  • the sperm sample from a subject optionally washed to substantially remove seminal fluid, is combined with the culture medium.
  • the compounds of the invention can be added to the medium prior to combining with the spermatozoa or it can be added after the mixing.
  • the resulting mixture can be incubated for a various periods of time, e.g., from 1 minute to 180 minutes. After the incubation, this mixture can be used for a number of fertilization procedures.
  • IVF in vitro fertilization
  • the methods of the invention can be practiced in vivo by delivering a composition comprising a calcimimetic compound of the invention and a sperm sample containing a spermatozoon into a female reproductive tract.
  • the composition containing a calcimimetic compound can be delivered into a female reproductive tract prior to (for example, from 1 to 60 minutes) or soon after (for example, from 1 minute to 180 minutes) sexual intercourse between a male and a female subject of a species of interest.
  • a composition comprising a calcimimetic compound and spermatozoa can be artificially inserted or infused into a female reproductive tract.
  • the compounds of the invention can be used with carriers that protect the compound of the invention against rapid elimination from the body, such as controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Liposomal suspension can also be used. These can be prepared as described, for example, in U.S. Pat. No. 4,522,81 1.
  • Rats were sacrificed by aortic puncture and exsanguination under general anesthesia (ip sodium tiopenthal). Immediately following sacrifice, both testes, epididymis, parathyroid glands and kidneys were removed: one sample was used for measurement of CaSR mRNA and the other for CaSR protein expression.
  • RNA isolation and real time RT-PCR Fresh testicle, epididymus, parathyroid gland and kidney were dry- frozen in liquid nitrogen and stored at -8O 0 C until RNA isolation.
  • 1 ml of phenol- guanidine isothiocyanate solution Tri-Reagent; Sigma, St Louis, MO, USA
  • Tri-Reagent Tri-Reagent
  • Tissues were ultrasonicated for 5 min at 4 0 C to allow for complete cell rupture. Thereafter, total RNA was extracted following a modification of the Chomczynski and Sacchi protocol. Extracted total RNA was dissolved in nuclease-free water (Promega, Madison, WI, USA) and heated for 10 min at 6O 0 C.
  • CaSR versus ⁇ -actin were amplified with a RT-PCR kit (QuantiTect SYBR green, Qiagen, Hilden, Germany) using specific primers and 100 ng of total RNA per sample. The following primers were used: CaSR (sense) 5'-TGG AGA GAC AGA TGC GAC TG-3', (antisense) 5'-GTC CAC GCC AGA AAC TCA AT-3'; ⁇ -Actin (sense) 5'-TGT CAC CAA CTG GGA CGA TAT GGA G-3', (antisense) 5'-ACA ATG CCA GTG GTA CGA CCA GA-3'. DNA amplifications were processed by real time PCR (Lightcycler; Roche, Basel, Switzerland. CaSR protein expression
  • the avidin-biotin-peroxidase (ABC) method (Vector, Burlinghame, USA) was used for the immunohistochemical study. Endogenous peroxidase activity was inhibited by incubation in 3% hydrogen peroxyde in methanol for 30 min. After 30 min incubation in 10% normal goat serum in phosphate buffered saline (PBS) pH 7.6, tissue sections were incubated with the primary antibodies overnight at 4°C (Acris antibodies GmBH, Hiddenhausen, Germany).
  • tissue sections were incubated with the secondary antibody biotinilated goat anti-rabbit immunoglobulins for 30 min (Dako, Glostrup, Denmark). After two 10 min rinses in PBS, tissue section were incubated with the ABC diluted in PBS for 1 hour. After three 10 min rinses in PBS, tissue sections were incubated with for several seconds in Vector ® NovaRed (Vector laboratories, CA, USA), rinsed in tap water, lightly counterstained with Mayer's haematoxylin, dehydrated and mounted. Assessment of CaSR function
  • the sperm was diluted in basal Tyrode's medium (TBM) (13), which consisted of 96 mM NaCl, 4.7 mM KCl, 0.4 mM MgSO 4 , 0.3 mM NaH 2 PO 4 , 5.5 mM glucose, 1 mM sodium pyruvate, 21.6 mM sodium lactate, 20 mM Hepes and 3 mg/ml BSA.
  • TCM basal Tyrode's medium
  • TBM basal Tyrode's medium
  • All media were prepared on the day of use and maintained at an osmolality of 290-310 mOsm/kg and pH 7.45 at 39 0 C.
  • Sperm was obtained from Male Wistar rats under anesthesia (i.p. sodium tiopenthal). Rat spermatozoa were retrieved from the distal cauda epididymis.sperm cells were dispersed in 3 ml of PBS. After culturing (37 0 C, 5% CO 2 ) for 15 min, the concentration of spermatozoa was determined by haemocytometer. Pooled semen from 3 rats was used in each experiment. Samples were diluted with the corresponding incubation medium to give a final concentration of about 3xlO 4 spermatozoa per ml.
  • Rat sperm motility was visually evaluated after 1 and 2 h of incubation. 7 ⁇ l of TBM diluted rat sperm were placed on a slide-cover and the sperm motility was video-recorded using a microscope (Zeiss Axiophot, Carl Zeiss S.A, Germany) and a videocamara (DS Camera Head DS-5M, Nikon Instruments Europe, Netherlands). Ten high-power fields (x 450) were recorded in each sample. All materials were thermostatized at 37 0 C. Video recordings were blindly evaluated for motility (number of motile cells and spermatozoa speed) by two independent observers. A semiquantitative score (graded 0 to 4), with the higher number reflecting higher sperm motility was assigned to each sample. Pig sperm
  • Objects with velocities between 11 and 45 ⁇ m/s were considered as medium speed objects; those with a velocity > 45 ⁇ m/s were considered rapid objects.
  • Spermatozoa deviating ⁇ 10% from a straight line were designated linear motile.
  • the sperm motility descriptors obtained by CASA analysis were: VCL, curvilinear velocity; VSL, linear velocity; VAP, mean velocity; LIN, linearity coefficient; STR, straightness coefficient; WOB, Wobble coefficient; ALH, mean lateral head displacement and BCF, frequency of head displacement (18).
  • 5 ⁇ l of the sperm suspension were placed on prewarmed (38 0 C) glass slides. All slides were covered with a 22 x 22 coverslip. Analyses were then immediately performed.
  • SYBR 14 100 nM were added to 1 ml of a dilute semen sample (containing 15OxIO 6 spermatozoa per ml) in HEPES-buffered saline solution (10 mM HEPES, 150 mM NaCl, pH 7.4) with 10% BSA and incubated at 38 0 C. After 10 min incubation, 5 ml of propidium iodide (final concentration 12 mM) were added and incubated for 8 additional min at 38 0 C. The cells were immediately acquired by the flow cytometer (FACScan, Becton- Dickinson, San Jose, CA, USA).
  • Example 2 This experiment demonstrates CaSR localization and distribution.
  • CaSR immunostaining was more marked in principal epididymal cells in which a high staining was noted in their apical pole ( Figure 2).
  • the mature sperm cells present inside the epididymis also expressed CaSR.
  • Immunostaining for CaSR was present mostly in the head of the spermatozoa ( Figure 3).
  • CaSR was also detected by immunohistochemistry in parathyroid and renal tissue for comparison (data not shown).
  • Table 2 summarizes sperm motility parameters obtained by CASA analysis at 240 min.
  • VAP mean velocity
  • VCL curvilinear velocity
  • VSL linear velocity
  • LIN linearity coefficient
  • STR straightness coefficient
  • WOB wobble coefficient
  • ALH mean lateral head 5 displacement
  • BCF frequency of head displacement.
  • Figure 4 depicts the percentage of sperm cells that reached VAP > 45 ⁇ m/s in rat sperm motility after 60 and 120 minutes of incubation. Results are presented as the mean ⁇ SE of a semiquantitative score (0-4). Although addition of calcium ImM did not influence this parameter, calcimimetic Compound A resulted in increases (up to 12%) which, again, were more accentuated in the range 10-300 nM.
  • Figure 5 illustrates the effect of calcimimetic Compound A on pig sperm motility after 60 and 240 minutes of incubation. Influence of Compound A on the percentage of cells that reach a mean velocity (VAP) > 45 ⁇ m/s.

Abstract

Cette invention concerne des procédés propres à favoriser la motilité, la mobilité et la vitalité des spermatozoïdes chez les mammifères par l'emploi de calcimimétiques.
PCT/US2010/026705 2009-03-10 2010-03-09 Procédés de modulation de la motilité des spermatozoïdes WO2010104882A1 (fr)

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