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WO2008056114A1 - Arginase durant la grossesse - Google Patents

Arginase durant la grossesse Download PDF

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Publication number
WO2008056114A1
WO2008056114A1 PCT/GB2007/004207 GB2007004207W WO2008056114A1 WO 2008056114 A1 WO2008056114 A1 WO 2008056114A1 GB 2007004207 W GB2007004207 W GB 2007004207W WO 2008056114 A1 WO2008056114 A1 WO 2008056114A1
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patient
arginase
arginine
activity
compound
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Pascale Kropf
Ingrid Muller
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Ip2ipo Innovations Ltd
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Imperial Innovations Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A61K31/51—Thiamines, e.g. vitamin B1
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • 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

Definitions

  • This invention is in the field of pregnancy and, in particular, the mechanism involved in maternal tolerance to the fetus.
  • a major paradox in immunology is the ability of the maternal immune system to exhibit immunological tolerance towards the semi-allogeneic fetus while maintaining responsiveness to exogenous pathogens.
  • Medawar proposed [1] three potential mechanisms that may assist maternal unresponsiveness: anatomic separation of the mother and fetus; antigenic immaturity of the fetus; and suppression or modification of the maternal immune system during pregnancy.
  • the maternal immune system is aware of fetal alloantigens during gestation, but is functionally tolerant to them until shortly after parturition [2].
  • transient suppression of the maternal immune response appears to be vital for fetal survival, and it is likely that multiple, interconnected mechanisms have evolved to prevent rejection of the fetus.
  • arginase activity is highly up-regulated in the placenta in pregnant females, but that the up-regulation is less pronounced in their peripheral blood mononuclear cells. Moreover, the up-regulation is not seen in age-matched non-pregnant controls,. Thus arginase in the placenta is believed to induce reversible and local T cell hyporesponsiveness, thereby playing a crucial role in the maintenance of normal pregnancy. This type of reversible and local immunosuppression has not previously been reported.
  • IDO indoleamine 2,3-dioxygenase
  • L-arginine depletion has been shown to down-regulate the expression of CD3 ⁇ [7], the signalling chain of the T cell receptor, through decreased CD3 ⁇ mRNA stability, and impair lymphocyte function in a reversible manner.
  • arginase's role offers several opportunities in therapy and diagnosis, including:
  • Therapeutic benefit is achieved by decreasing the anti-fetal immune response of a patient.
  • Hyporesponsiveness can be achieved in a patient by increasing the arginase activity either systemically or more preferably in targeted organs, tissues and cells.
  • Induction of targeted hyporesponsiveness can have a therapeutic benefit where there is a history of recurrent spontaneous miscarriage or infertility by interfering with localised immune response without impairing the systemic immune response of a patient.
  • Induction of hyporesponsiveness is also useful in patients to prevent rejection of the embryo or fetus, such as during conception and pregnancy.
  • Beneficial results are preferably achieved by targeting arginase or the modulators of arginase production or activity specifically to the cells or tissues in which induction of hyporesponsiveness is desired.
  • a decreased immune response can be localised to specific organs or tissues, such as the uterus, endometrium or placenta, or to specific cells in those organs or tissues, such as macrophages, neutrophils, dendritic cells or lymphocytes, to achieve beneficial results without compromising the systemic immune response.
  • the present invention provides therapeutic benefit through diminishing immune responses.
  • arginase I and II Two arginase isoenyzmes exist - arginase I and II, which differ in subcellular localization, regulation and possibly function.
  • Arginase is produced by a number of cells such as tumor cells, transplanted cells, cells undergoing autoimmune reactions and macrophages.
  • Arginase I which is inducible in macrophages and dendritic cells, is primarily responsible for the depletion of arginine in the cells and surrounding environment, resulting in suppression of an immune response.
  • the invention is aimed primarily at Arginase I.
  • arginase activity results in (a) the production or ornithine, a precursor of proline, which favours cellular regeneration, wound healing and repair [8], (b) the production of polyamines, also from ornithine, which can regulate macrophage function [9] and decrease nitric oxide production [10] and (c) reduction of arginine availability and, therefore, decreasing nitric oxide production and other arginine-mediated processes such as normal T lymphocyte proliferation [11-13].
  • the human arginase polypeptide sequences have been given accession numbers P05089 and P78540 in the Entrez protein database and their mRNA sequences are given in NM_000045.2 and NMJ)01172.3.
  • SNPs of arginase are known.
  • the following SNPs are known in the dbSNP database: rs2608898, rs2608897, rs2781664, rs2781666, rs2781665, rsl7788484, rs3756780, rsl0457573, rs2781658, rslO63493, rs2781659, rsl803151, rs2781667, rs2246012, rs2781668, rs2297637, rs3850245, rs2608937, rs7769790, rs2749935, rs9321303, rs2781621 and rs9493030.
  • rsl7104534 For ARG2, the following SNPs are known: rsl7104534, rsl885042, rs742869, rs8017597, rs7140310, rs4899215, rs2295643, rsl2884807, rs7144186 and rs4902505.
  • L-arginine The bioavailability of L-arginine is determined by two inducible intracellular enzymes, arginase and nitric oxide synthase (NOS), which share L-arginine as a common substrate [14]. These enzymes are induced by T cell derived cytokines and regulated by complex intracellular biochemical pathways and negative feedback mechanisms. The balance of L-arginine metabolism via arginase or NOS is an important determinant of the inflammatory response of murine macrophages and dendritic cells.
  • ThI cytokines such as interferon- ⁇ induce NOS, which catalyses the metabolism of L-arginine to nitric oxide
  • Th2 cytokines drive the alternative activation of macrophages including the induction of arginase, which hydrolyzes L-arginine into urea and L-ornithine.
  • arginase The importance of arginase in regulating T cell responses has been convincingly shown in vitro and in animal models, and arginase induction as a method of tumour evasion has been demonstrated in human cancers.
  • Arginine is catabolised by several enzymes including arginine decarboxylase, arginase, nitric oxide synthase and arginine:glycine amidinotransferase.
  • Arginase and NOS compete for arginine and also inhibit each other [15].
  • NOS generates hydroxy-arginine which is an inhibitor of arginase and polyamines generated by the arginase pathway are inhibitors of NOS. Feedback between the pathways regulates the level of available arginine and therefore the level of suppression of the immune system.
  • Arginine regulates p70 S6 kinase activity and phosphorylation of 4EBPI through the mTOR signalling pathway, which involves y + cationic amino acid transporters (such as Cat-1, Cat-2a, Cat- 2b and Cat-3) [16]. Activation of the p70 S6 kinase results in activation of the immune system.
  • Arginase activity may be increased either by increasing the specific activity of the enzyme, or by increasing the level of arginase.
  • any one of a number of methods to maintain or provide increased arginase activity during pregnancy in order to reduce the risk of an immunologically-mediated miscarriage or other pregnancy-related complications may be provided.
  • compounds that increase the specific activity of the enzyme may be provided.
  • the enzyme itself may be provided to a subject, or expression of endogenous arginase may be increased using a stimulator of arginase expression.
  • Increasing the expression of endogenous arginase is referred to as upregulating arginase expression.
  • gene therapy may be used to upregulate arginase expression, where a gene encoding arginase may be provided.
  • arginase inhibitors In another method to maintain or provide increased arginase activity during pregnancy to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication, the production of arginase inhibitors may be inhibited.
  • Arginase inhibitors and stimulators are further discussed in reference 17.
  • the arginase stimulator may increase the arginase expression levels or increase the activity of the arginase.
  • the arginase level and/or specific activity is increased by 10% or more (e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200% or more).
  • Arginase inhibitors which may be targeted for blocking to prevent their inhibitory action on arginase include amino acids such as NG-hydroxy-L-arginine (NOHA), N(omega)-hydroxy-nor-L-arginine (nor-NOHA), 2 (S)-amino-6-borohexanoic acid (ABH), ornithine, lysine, and norvaline.
  • Other inhibitors that downregulate arginase activity include IFN ⁇ and IL- 12 [18].
  • Arginase stimulators which may be used in the method described above to increase arginase activity include beta-adrenergic agents such as catecholamines or catecholamine analogues, epinephrine, norepinephrine, isoproterenol, prostaglandines, dopamine and salbutamol; Th2 cytokines such as IL- 4, IL-IO, IL-13 and TGF ⁇ ; and arginase substrates.
  • the stimulator may be 8-bromo- cAMP [19], 8-bromo-cAMP plus lipopolysaccharide 8-bromo-cAMP and interferon-gamma or combinations thereof.
  • An arginase enzyme obtained from a natural or synthetic source may also be provided.
  • Mn 2+ has been identified as a co-factor of arginase and so is required for arginase activity.
  • Ni 2+ and Co 2+ are also known to be activators of arginase while Hg 2+ , Ag 2+ and Zn 2+ are known to be inhibitors of arginase. Therefore, in one method to maintain or provide increased arginase activity during pregnancy to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication, specific arginase activity can be upregulated in a situation where Mn 2+ , Ni 2+ and/or Co 2+ is limiting, by providing a source of Mn 2+ , Ni 2+ and/or Co 2+ .
  • Arginine levels in the body may be decreased by any one of a number of methods described below in order to reduce the risk of a miscarriage and/or other pregnancy-related complication or to improve the chance of becoming pregnant.
  • Arginine levels may be decreased by reducing the specific activity of an arginine biosynthetic enzyme (inhibiting the enzyme) or by inhibiting the expression of the enzyme (downregulating the enzyme). For example, synthesis ofarginine from citrulline or other upstream precursors may be decreased.
  • Other precursors of arginine biosynthesis that may be targeted for reduction include carbamyl phosphate, ornithine, citrulline, aspartic acid and argininosuccinic acid (ASA).
  • Enzymes that control the metabolism of these compounds may also be targeted for inhibition or downregulation.
  • Such enzymes include ornithine transcarbamylase, ASA synthetase and ASA lyase.
  • Inhibitors may be competitive or noncompetitive.
  • antibodies may be used to bind to and sequester arginine biosynthetic enzymes.
  • the arginine levels are reduced by 10% or more (e.g. 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, 99% or more).
  • the arginine concentration in the placenta is 2000 ⁇ M or less (e.g. 1000, 750, 500, 250, 150, 75, 50, 25, 15, 10 ⁇ M or less).
  • General physiological levels of L-arginine are between 50-15OmM.
  • Other targets include ornithine transcarbamylase, ASA synthetase and ASA lyase.
  • Inhibitors may be competitive or noncompetitive.
  • Arginine is transported into mammalian cells primarily through system y + cationic amino acid transporters [20]. If the expression of these transporters is upregulated or their activity is increased, then the immune response is suppressed as less arginine is transported into cells such as macrophages.
  • Four such transporter genes have been identified which encode system y + -like activity (Cat-1, Cat-2a, Cat-2b and Cat-3). The invention therefore provides a method of reducing the risk of miscarriage and/or other pregnancy-related complications or improving the chance of becoming pregnant comprising upregulating the expression of y + cationic amino acid transporters or increasing the activity of y + cationic amino acid transporters.
  • the Cat-1 protein has been shown to have a higher affinity for arginine than the other transporters [21]. Therefore, preferably the activity of Cat-1 is targeted.
  • Rapamycin is a known suppressant of the immune system. Therefore its receptor, the molecular target of rapamycin (MTOR) is a possible target for intervention in metabolic pathways downstream of arginase, to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy- related complication or to improve the chance of becoming pregnant. Rapamycin and wortmanin inhibit the effect of arginine on the p70 S6 kinase, resulting in immune suppression. Therefore, rapamycin and wortmanin, which activate mTOR and induce immune suppression, are attractive for use in reducing the risk of miscarriage and/or other pregnancy-related complications.
  • MTOR molecular target of rapamycin
  • GCN2 kinase influences mRNA translation. Amino acid depletion can lead to the accumulation of uncharged tRNAs that bind to and activate GCN2 kinase. This kinase phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2a), which inhibits eIF2a Guanylate Exchange Factor, and the generation of eIF2a-GTP-Met-tRNAi complexes. These complexes are necessary for the initiation of translation and, in their absence, cellular protein synthesis is suppressed.
  • eIF2a eukaryotic initiation factor 2
  • the level of tRNA that is uncharged with amino acids following the local depletion of amino acids by arginase could increase and activate GCN2. Activation of GCN2 might then repress protein synthesis in T cells, therefore rendering them unresponsive.
  • CD3 ⁇ is expressed by T cells and forms part of the T cell receptor. It is essential for signal transduction by the T cell receptor. Increasing of arginase levels in the placental region during pregnancy leads to a decrease in CD3 ⁇ expression, resulting in local hyporesponsivenessand a reduced risk of miscarriage and/or other pregnancy-related complications. Therefore, the invention provides a method of intervening in a metabolic pathway downstream of arginase, to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication or to improve the chance of becoming pregnant by decreasing CD3 ⁇ expression.
  • the polyamines are organic compounds, such as putrescine, spermidine, and spermine, that are growth factors in both eukaryotic and prokaryotic cells.
  • Polyamines are synthesised by amino acid decarboxylation reactions. Arginine is broken down by arginase into ornithine, which is then converted to putrescine (a polyamine) by ornithine decarboxylase. It is believed that these polyamines play a role in the suppression of the immune system by downregulating the activity of antigen presenting cells. Thus polyamines are attractive targets for inducing suppression of the immune system.
  • Polyamines derived from proline, but not arginine, are synthesised in the porcine placenta during early pregnancy [22] and in pregnant guinea pigs polyamines are thought to be important in cell growth and development [23]. Therefore, an increase in polyamine level is likely to be beneficial during pregnancy.
  • the present invention provides a method involving intervention in metabolic pathways downstream of arginase to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication or to improve the chance of becoming pregnant, comprising increasing the activity of polyamine biosynthesis or polyamine availability. This can be done by increasing the activity of polyamine biosynthetic enzymes or by delivery of polyamines.
  • Arginine is broken down into citrulline and nitric oxide by inducible Nitric Oxide Synthase (NOS). Citrulline can then be recycled into arginine by ASS and ASL. Arginine levels can be reduced by increasing the activity of NOS, either by inducing overproduction of the enzyme or by increasing the activity of the enzyme already present.
  • a further a method involving intervention in metabolic pathways downstream of arginase to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication or to improve the chance of becoming pregnant comprises increasing NOS activity.
  • Arginine is synthesised from L- citrulline via the intermediate L-argininosuccinate by the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL).
  • ASS argininosuccinate synthase
  • ASL argininosuccinate lyase
  • arginine levels may be decreased by increasing the arginase activity.
  • the methods described above can be used from conception to parturition as a method of maintaining or providing increased arginase activity or reduction of arginine levels during pregnancy to reduce the likelihood of premature birth, or before conception as a method of increasing of arginase activity or decreasing arginine levels when attempting to become pregnant to improve the success rate.
  • Any of the methods described above may be used to increase arginase activity or decrease arginine levels systemically or may be targeted to a specific organ, tissue or cell type.
  • a composition of the invention is localised to a specific organ, tissue or cell type in order to achieve localised immunosuppression.
  • arginase activity is increased or arginine levels are decreased by altering the activity or expression of the target in the uterus. More preferably, arginase activity is increased or arginine levels are decreased by altering the activity or expression of the target in the endometrium, even more preferably arginase activity is increased or arginine levels are decreased by altering the activity or expression of the target in the region of the placenta or in the placenta itself.
  • macrophages, dendritic cells and/or neutrophils in the uterus, endometrium or placenta are targeted in order to modulate targets described above.
  • a targeted increase of arginase activity or decrease of arginine levels may be achieved by using localised delivery methods rather than systemic delivery.
  • systemic delivery may be used if the delivered material is then targeted to the desired area e.g. using immunoliposomes, etc.
  • Localised delivery may be achieved by any one of a number of methods such as hydrogels, pastes, vaginal tablets, pessaries/suppositories, particulate systems, local injection, intravaginal tampon devices, sponges and rings.
  • Intravaginal devices such as rings, tampons and sponges are either of the type where a medicament is impregnated into the device, or of the type that carries an encapsulated medicament.
  • reference 25 discloses a moist, medicated vaginal tampon that is impregnated with a contraceptive agent and a medicament for the control of venereal disease.
  • Reference 26 discloses a tampon which has a capsule of disintegratable material partially embedded in one end.
  • Such a tampon is inserted into the vagina and serves both to deliver and to retain the encapsulated medicament in the vaginal cavity.
  • Reference 26 also discloses a means for pre-wetting the tampon in order to activate the capsule.
  • Reference 27 discloses a sponge, impregnated with a liquid containing an effective amount of an active pharmaceutical agent, for insertion into the vaginal cavity.
  • Reference 28 discloses a tampon assembly that is adapted for carrying a medicament within a longitudinal bore formed within the tampon. The medicament can be selectively expelled into the vaginal cavity from the bore using a tubular inserter. Further devices designed for prolonged or improved delivery are disclosed in references 29, 30 and 31.
  • the vaginal drug delivery system preferably provides a sustained delivery of the composition to the vaginal epithelium. If a paste or hydrogel is used, it should be of sufficient thickness/viscosity to ensure prolonged vaginal epithelium contact.
  • Muco-adhesive agents are preferably used to bring the released composition in solution into prolonged, close contact with the mucosal surface.
  • the muco-adhesive agent is preferably a polymer such as an alginate, pectin or cellulose derivative.
  • the present invention also provides assays of arginase activity, both before and during pregnancy as an indicator of miscarriage and/or other pregnancy-related complication potential. Similarly, assays to determine Cat activity and/or levels of Cat expression can be used as an indicator, as can assays of CD3 ⁇ or polyamine levels. Clinical tests for evaluating the state of immune competence in a patient, and measuring the efficacy of a treatment on a condition are also provided by the present invention.
  • the present invention provides an assay comprising the step of measuring the level of arginase activity, CD3 ⁇ , Cat or polyamines in specific fluids, such as amniotic fluid or blood, in cells, such as uterine or placental cells, the peripheral blood cells or monocyte/macrophages or lymphocytes or in serum from a pregnant individual or an individual who is attempting to become pregnant.
  • the level of arginase activity has a direct correlation on T-cell function in the immune system.
  • This aspect of the invention can be performed by, for example, isolating the fluid or cells of interest from the patient and measuring the arginase, CD3 ⁇ , Cat or polyamine levels and/or activity through known assays.
  • the sample in which the arginase activity, CD3 ⁇ levels, Cat activity or polyamine levels is measured is obtained from the placenta or in the region of the placenta, for example by chorionic villi sampling (CVS).
  • Arginase activity can be measured by the conversion of L-arginine to L-ornithine or urea.
  • CD3 ⁇ levels can be measured by immunochemical techniques.
  • Cat activity can be measured by determining the levels of L-arginine taken up by cells as described in [32]
  • Arginase, CD3 ⁇ and Cat protein levels can be directly determined by western blot or by ELISA.
  • the expression of the arginase, CD3 ⁇ or Cat genes can be detected by PCR, by PCR ELISA and by Northern blot. These assays may include directly measuring the protein level or measuring a relevant activity e.g. the ability of the fluid or cells to break down arginine.
  • Polyamine levels can be measured by various techniques, including HPLC and fluoro
  • the assays described above can be used to give a single measurement of activity /level in a subject.
  • a single measurement may be sufficient to determine whether an individual is immunosuppresed, for example when arginase activity in an individual that is at least 10% (e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200% or more) greater than expected, the individual is considered to be immunosuppressed.
  • the assay can also be used to compare activity/level in an individual to a known normal baseline to determine whether activity/levels are greater than expected.
  • an individual is considered to be immunosuppressed if the arginase activity in leukocytes, such as mononuclear cells (including monocytes and lymphocytes) and/or neutrophils is at least 100 mU/mg protein (e.g. >250mU/mg).
  • leukocytes such as mononuclear cells (including monocytes and lymphocytes) and/or neutrophils is at least 100 mU/mg protein (e.g. >250mU/mg).
  • the assay can also be used to compare the activity/level in a pregnant individual to a known normal baseline in non-pregnant individuals to determine whether the pregnant individual is immunosuppressed. For example, pregnant individuals in whom arginase activity is at least 10% (e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200% or more) greater than in a non-pregnant individual are considered to be immunosuppressed.
  • the assay can also be used to compare activity/levels in a single subject before and after conception or throughout the pregnancy to determine whether the individual is immunosuppressed during pregnancy.
  • the assay can be performed in either: (a) at least one sample taken from before conception and at least one sample taken after conception (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or more samples); or (b) at least two samples (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or more samples) taken after conception.
  • the samples taken during pregnancy may be taken at different times spread throughout the entire pregnancy, the first trimester and/or the second trimester.
  • an individual is considered to be immunosuppressed if the arginase activity is at least 10% (e.g. 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200% or more) greater than at the pre-conception time point or the normal level for non-pregnant individuals.
  • arginase levels are measured, if a pregnant individual or a nonpregnant individual who is attempting to conceive is found to have a low level of arginase activity, i.e. less than 95% (e.g. 90, 80, 70, 60, 50, 40, 30, 20, 10% or less) of normal arginase activity, then fertility problems may be indicated and treatment may be used to increase arginase levels and decrease arginine levels to improve the chances of conceiving and decrease the chance of a spontaneous miscarriage and/or other pregnancy-related complication.
  • low arginase activity levels are defined as less than 100 mU/mg protein ⁇ e.g.
  • the assay provides not only a measurement of arginase activity, but also provides a solution to the problem of low arginase activity.
  • the efficacy of a treatment of a condition can be measured by determining the ability of said treatment to increase the level of arginase activity in the targeted organ, tissue or cells.
  • This assay is similar to the assay discussed immediately above, however in this assay the cells are isolated prior to and after any treatment has been administered and the level of arginase activity is measure and compared. Accordingly, a determination can then be made whether the treatment has been effective.
  • New and existing immunostimulants can be tested for their ability to prevent spontaneous miscarriage and/or other pregnancy-related complications by determining the ability of these medications to increase the level of arginase activity in the uterus, endometrium or placenta.
  • This embodiment of the invention is similar to the ones previously mentioned, the cells are isolated prior to, and after, any treatment has been administered and the level of arginase activity of the cells is measure and compared.
  • Determining arginase level systemically or in specific cells may also indicate if a patient is likely to respond to treatment. For example, some patients prone to recurrent spontaneous miscarriage and/or other pregnancy-related complications having low starting levels of arginase activity in the placenta may respond to treatment better than patients with normal starting levels of arginase activity. Low arginase levels are defined as less than 95% (e.g. 90, 80, 70, 60, 50, 40, 30, 20, 10% or less) of normal arginase activity.
  • pregnancy complications that may be indicated by low arginase activity measured by the assays mentioned above include, but are not limited to, spontaneous miscarriage, recurrent miscarriage, partic serial miscarriage, intrauterine growth retardation, preterm labour, eclampsia or preeclampsia.
  • the invention also provides a method of testing arginase genotype, before or during pregnancy as an indicator of miscarriage and/or other pregnancy-related complication potential by testing the arginase genotype of the patient for alleles of arginase I and/or arginase II, comprising the steps of detecting the presence or absence of known SNPs and/or unknown alleles of arginase and correlating the genotype to the frequency of spontaneous miscarriage and/or other pregnancy-related complication.
  • Assays of arginase genotype can be used as an indicator of the risk of miscarriage and/or other pregnancy-related complications of future offspring.
  • the method described above may be used to correlate the maternal, paternal and/or fetal arginase genotype to the risk of spontaneous miscarriage and/or other pregnancy-related complications.
  • Data gathered using the methods described above can be used to develop a genetic test that can predict the probability of having a miscarriage and/or other pregnancy-related complications by genotyping the individual and their partner for arginase.
  • testing arginase genotype, polymorphism in Cat and/or the manganese transporter can be assayed.
  • the presence/absence of known SNPs and/or unknown alleles of Cat (or the manganese transporter) can be tested, and the genotype can be correlated to the frequency of spontaneous miscarriage and/or other pregnancy-related complication.
  • a patient being treated is human.
  • the invention is not limited to humans, but includes other animals where it is desirable to maintain a successful pregnancy (such as horses, cows, sheep, pigs, dogs and cats), by providing high arginase levels.
  • compositions of the invention may be used to help prevent pregnancy or cause miscarriage in mammalian pests ⁇ e.g. rodents, marsupials, etc.) such as mice and rats, by providing low arginase levels.
  • the patient will typically be female.
  • the patient may have an arginase deficiency.
  • arginase deficiencies include argininemia, familial argininemia, and hyperargininemia.
  • the patient is a primigravida, or a multigravida but not a primiparta.
  • the patient is pregnant, wanting to get pregnant, suffers from or has suffered from preeclampsia, has previously had a spontaneous miscarriage or recurrent miscarriages, or is undergoing IVF treatment.
  • the patient is post-pubescent and pre-menopausal, i.e. is aged between 10-65 years, preferably 14-55, more preferably 16-45 e.g. 20-40.
  • sperm may contribute to low arginase activity in a fetus.
  • a sperm sample could be tested for arginase and/or spermine levels.
  • Compounds that affect arginase can be identified by screening assays, as described below.
  • the assay methods may be for use in the identification of compounds for maintaining or providing increased arginase activity or reducing the level of arginine before or during pregnancy to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication.
  • the in vivo steps may be carried out in a non-human animal, for example a non-human mammal, preferably a non-human primate.
  • the various steps of the methods may be carried out at the same or different times, in the same or different geographical locations, e.g. countries, and by the same or different people or entities.
  • the invention provides a method for identifying a compound that increases arginase activity, said method comprising a step of assessing arginase activity in the presence and absence of a candidate compound and determining whether arginase in the presence of the compound shows increased activity.
  • a compound that increases arginase activity is one that increases the specific arginase activity and/or increases the level of expression of arginase.
  • the method may also include one or more of the steps of: testing a candidate compound for its ability to reduce T-cell activation in vitro; and/or testing a candidate compound for its ability to reduce T-cell activation in vivo, wherein the T-cells may be obtained from the placenta; and/or testing a candidate compound for downregulation of alloantigen responses in vivo, for example the downregulation of CD3 ⁇ expression; and/or testing a candidate compound in vivo for unwanted side effects in pregnancy, such as teratogenic effects, premature birth and interuterine growth restriction (IUGR), and selecting compounds that do not posses any of these qualities.
  • IUGR interuterine growth restriction
  • one screening method could comprise adding arginase to arginine in the presence or absence of the candidate compound and detecting the level or ornithine and/or urea produced.
  • a further method involves mixing arginase with arginine and 2,3-butanedione and then measuring absorbance at 490nm against a blank containing no enzyme. One unit of enzyme releases one micromole of urea per minute at 37°C and pH 9.5.
  • the concentration of arginase in a sample can be detected using standard techniques such as western blot or ELISA.
  • the expression of the arginase gene can be detected by PCR, PCR ELISA and by Northern blot.
  • Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding arginase in cells.
  • an ELISA may be constructed that measures secreted or cell-associated levels of arginase using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of arginase from suitably manipulated cells or tissues. The formation of binding complexes between arginase and the compound being tested may then be measured. Screening methods for compounds that regulate other targets
  • the method described above may also be used to identify compounds that increase the activity of other polyamine biosynthetic enzymes that act downstream of L-arginine, such as L-ornithine decarboxylase.
  • polyamine synthetic enzymes can also be measured by measuring the concentration of polyamines.
  • concentration of polyamines in a sample can be detected by various methods including HPLC and GC-MS.
  • the method described above can also be used for identifying compounds that upregulate enzymes that are themselves upregulated by increased arginase activity.
  • the activity of arginine catabolic enzymes can be measured by any of the methods known in the art, for example the methods described in reference 37.
  • Arginine levels can also be altered by modulating the activity of the arginine transporters.
  • a decrease in the specific activity or expression level of any arginine transporter would lead to a decrease in the intracellular level of arginine.
  • the invention provides a method for identifying a compound that modulates the activity of any one of the arginine transporters, said method comprising the step of assessing the activity of the transporter in the presence and absence of a candidate compound and determining whether the transporter shows decreased activity in the presence of the compound.
  • the transporter is CAT-I.
  • the effect of arginase on the immune system is to decrease CD3 ⁇ expression in TCR+ cells and to suppress their biological activities (e.g. proliferation, chemokine and cytokine secretion, and cytotoxicity).
  • the invention therefore provides a further method of identifying a compound for use in suppressing the immune system during pregnancy that decrease the level of CD3 ⁇ expression in TCR+ cells, said method comprising the step of assessing the level of CD3 ⁇ expression in the presence and absence of a candidate compound and determining whether the level of expression is decreased in the presence of the compound.
  • Methods suitable for measuring the level of CD3 ⁇ expression include, but are not limited to, RT-PCR, qRT-PCR, northern blotting, western blotting, and ELISA.
  • One aspect of the present invention is the determination that compounds such as rapamycin, which act to suppress the immune system through the same pathway as arginase, are compatible with pregnancy and can prevent spontaneous miscarriage and/or other pregnancy-related complications.
  • the present invention therefore provides a method for identifying a compound for use in suppressing the immune system during pregnancy that interact with mTOR to prevent the induction of the p70 S6 kinase by arginine, and which therefore suppress the immune system via a pathway that may be compatible with pregnancy.
  • the methods described above also include the one or more of the steps of: testing a candidate compound for its ability to reduce T-cell activation in vitro; and/or testing a candidate compound for its ability to reduce T-cell activation in vivo, wherein the T-cells may be obtained from the placenta; and/or testing a candidate compound for downregulation of alloantigen responses in vivo, for example the downregulation of CD3 ⁇ expression ; and/or testing a candidate compound in vivo for unwanted side effects in pregnancy, such as teratogenic effects, premature birth and interuterine growth restriction (IUGR), and selecting compounds that do not posses any of these qualities.
  • IUGR interuterine growth restriction
  • the in vivo steps of the screening methods of the invention may be carried out in cell-free systems or in cells or tissues.
  • the cell-free system must contain all the necessary components for transcription of the reporter gene where the level of expression is detected by measuring mRNA levels, and all the necessary components for transcription and translation of the reporter gene where the level of expression is assessed by measuring protein levels.
  • the methods of screening of the invention be conducted in cell-free systems since this facilitates high-throughput screening of candidate compounds.
  • Indirect screening methods of the invention are preferably carried out in eukaryotic cells, such as mammalian ⁇ e.g. human) or yeast cells. They may also be performed in mammalian ⁇ e.g human) tissues. A typical cell is a macrophage.
  • a reference standard ⁇ e.g. a control) is typically needed in order to detect whether the arginase activity is increased.
  • the activity of arginase in the presence of a candidate compound may be compared with the activity of arginase in the absence of a candidate compound.
  • the reference may have been determined before performing the method of the invention, or may be determined during ⁇ e.g. in parallel) or after the method has been performed. It may be an absolute standard derived from previous work.
  • Typical candidate compounds for use in all the screening methods of the invention include, but are not restricted to, peptides, peptoids, proteins, lipids, metals, small organic molecules, RNA aptamers, antibiotics and other known pharmaceuticals, polyamines, antibodies (as used herein, the term “antibody” refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question) or antibody derivatives ⁇ e.g. antigen-binding fragments, single chain antibodies including scFvs, etc.), and combinations or derivatives thereof.
  • Small organic molecules have a molecular weight of about more than 50 and less than about 2,500 daltons, and most preferably between about 300 and about 800 daltons.
  • Candidate compounds may be derived from large libraries of synthetic or natural compounds. For instance, synthetic compound libraries are commercially available from MayBridge Chemical Co. (Revillet, Cornwall, UK) or Aldrich (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts may be used. Additionally, candidate compounds may be synthetically produced using combinatorial chemistry either as individual compounds or as mixtures.
  • the invention therefore provides a method of assessing the in vivo effect of a compound obtained or obtainable by any of the methods described above comprising administering the compound to a test animal and assessing the effect on pregnancy, including birth defects, changes to the gestation period and changes in birth weight.
  • Tests in non-human animals for example non-human mammals or non- human primates may be used.
  • the invention provides a compound that increases arginase activity, increases polyamine synthesis, decreases arginine biosynthesis, decreases arginine transporter activity or downregulates the expression of CD3 ⁇ , obtained or obtainable by any of the methods described above.
  • the compounds of the invention are organic compounds.
  • a compound as identified above has an established safety profile in pregnant women.
  • a compound may be necessary to conduct further work on its pharmaceutical properties. For example, it may be necessary to alter the compound to improve its pharmacokinetic properties or bioavailability.
  • the invention extends to any compounds obtained or obtainable by the methods of the invention which have been altered to improve their pharmacokinetic properties.
  • the invention provides pharmaceutical compositions comprising any of the compounds of the invention or a combination of two or more (e.g. three or more, four or more, five or more etc.) of compounds of the invention.
  • compositions should preferably comprise a therapeutically effective amount of compounds of the invention.
  • therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted disease or condition, preferably spontaneous miscarriage or preeclampsia, or to exhibit a detectable therapeutic or preventative effect.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • an effective amount for a human subject will depend upon the severity of the risk of miscarriage and/or other pregnancy-related complications, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
  • a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
  • a pharmaceutically acceptable carrier for administration of a therapeutic agent.
  • Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • the composition may be sterile.
  • compositions of the invention are preferably non-pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and preferably ⁇ 0.1 EU per dose.
  • ⁇ 1 EU endotoxin unit, a standard measure
  • compositions of the invention are preferably gluten free.
  • Compositions will generally have an osmolality of between 200 m ⁇ sm/kg and 400 m ⁇ sm/kg, preferably between 240-360 m ⁇ sm/kg, and will more preferably fall within the range of 290-300 m ⁇ sm/kg.
  • Compositions may be substantially isotonic with respect to humans.
  • Compositions may include sodium salts (e.g. sodium chloride) to give tonicity.
  • sodium salts e.g. sodium chloride
  • a concentration of 10+2mg/ml NaCl is typical.
  • Compositions of the invention may include one or more buffers.
  • Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer.
  • a phosphate buffer is typical. Buffers will typically be included in the 5-2OmM range.
  • the pH of a composition of the invention will generally be between 5.0 and 7.5, and more typically between 5.0 and 6.0 for optimum stability, or between 6.0 and 7.0.
  • compositions of the invention can be administered directly to the subject.
  • the subjects to be treated can be animals; in particular, human subjects can be treated.
  • Targeted delivery of the compositions will generally be accomplished any one of a number of methods such as hydrogels, pastes, vaginal tablets, pessaries/suppositories, particulate systems, local injection, intravaginal tampon devices, sponges and rings.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • one approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • one approach comprises administering to a subject a therapeutically effective amount of a compound that activates the target, i.e., an agonist as described above, to decrease the amount of available arginine or to suppress the immune system.
  • a compound that activates the target i.e., an agonist as described above
  • the invention includes the delivery of a protein
  • it may be modified to improve in vivo characteristics, such as improving its pharmacokinetic properties.
  • proteins may be modified by the addition of PEG.
  • PEG poly(ethylene glycol)
  • a PEG 5 kD conjugate of arginase has been shown to have an increased plasma half life in mice [39] and may be more effective than the native enzyme in vitro [40].
  • the invention provides a method of maintaining or providing increased arginase activity or reducing the level of arginine before or during pregnancy to reduce the risk of an immunologically-mediated miscarriage and/or other pregnancy-related complication.
  • Such conditions include, but are not limited to, premature labour, intrauterine growth retardation, eclampsia, preeclampsia, or other complications of pregnancy and/or fetal development.
  • the method comprises the step of administering to a patient before or during pregnancy a compound and/or composition of the invention.
  • a compound and/or composition of the invention is compatible with pregnancy e.g. has previously been certified by a regulatory authority, such as the FDA or EMEA, to be safe for administration to pregnant females.
  • the invention also provides the use of a compound and/or composition of the invention, in therapy
  • the invention also provides the use of a compound and/or compositions of the invention, in the manufacture of a medicament for treating or preventing spontaneous miscarriage and/or other pregancy-realted complication.
  • FertilityBlend contains folic acid; vitamins E, B6 and B 12; iron; magnesium; zinc; L-arginine; chasteberry, a herb that is thought to optimize ovulation; and the antioxidants green tea and selenium.
  • Materna and S-26 Mama manufactured by Wyeth contain folic acid, iron, B-complex vitamins, vitamin C, vitamin D, vitamin E, and zinc; and protein, docosahexaenoic acid, folic acid, iron, calcium and vitamins and minerals respectively.
  • the invention provides nutritional supplements suitable for use before or during pregnancy, with the aim of reducing arginine levels, comprising folate and one or more free amino acids, that is free of arginine.
  • the nutritional supplement may further comprise one or more of iron, calcium, protein, zinc, docosahexaenoic acid, vitamin A, vitamin B, vitamin C, vitamin D and vitamin E.
  • the nutritional supplement preferably also comprises a source of Mn 2+ .
  • the supplement may also comprise a source OfNi 2+ and/or Co 2+ .
  • the supplement may be zinc free.
  • spontaneous miscarriage means the spontaneous loss of a pregnancy before 24 weeks of gestation.
  • recurrent miscarriage is defined as more than one consecutive miscarriage, for example three or more consecutive miscarriages.
  • composition comprising
  • X may consist exclusively of X or may include something additional e.g. X + Y.
  • the word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
  • a process comprising a step of mixing two or more components does not require any specific order of mixing.
  • components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
  • Figure 1C shows arginase activity in sera.
  • the activity of arginase was measured in maternal (o) and non-pregnant control (D) sera, as described for Figure IA.
  • Figure ID shows urea levels.
  • Figure IE shows a western blot of arginase in various cell lysates.
  • Figure 2 shows results of CD3 ⁇ assays in arginase-expressing cells in the placenta.
  • Figures 3A shows local downregulation of CD3 ⁇ chain in the placenta.
  • the solid line shows data for PIaCs, whereas the dashed line shows data for maternal PBMCs.
  • Figure 3B shows the MFI (mean fluorescence intensity) of CD3 ⁇ in peripheral TCR+ cells (•) and paired placental TCR+ cells (o) from 13 separate patients, and % reduction in PIaCs compared to maternal PBMCs is shown in 3C.
  • Figure 4A shows CD3 ⁇ chain expression in TCR+ cells isolated from the placenta
  • Figure 4B shows the proliferation of placental T lymphocytes activated through anti-CD3/anti-CD28.
  • the X-axes show the number of days post-stimulation. Arginine was either present (•) or absent (o).
  • Figures 5A and 5B show that downregulation of CD3 ⁇ chain is restored by inhibition of arginase or by addition of L-arginine.
  • the dotted lines show results using maternal PBMCs + PIaCs.
  • the solid line shows results when nor-NOHA was included.
  • the solid line shows results when L-arginine was included.
  • the addition of nor-NOHA or L-arginine shifts the line to the right in both cases.
  • Figure 5C shows the % of dividing TCR+ cells is increased in the presence of nor-NOHA (middle bar) or exogenous L-arginine (right-hand bar) compared to controls (left).
  • the arginase enzyme activity in mononuclear cells isolated from healthy human placenta (PIaCs) immediately after parturition was determined, and compared with maternal peripheral blood mononuclear cells (PBMCs) and PBMCs from aged-match female non-pregnant controls.
  • PBMCs maternal peripheral blood mononuclear cells
  • the arginase enzyme activity in peripheral neutrophils isolated from the blood of pregnant individuals was determined, and compared the arginase activity in peripheral neutrophils from non-pregnant controls.
  • PIaCs, maternal and control PBMCs, and maternal and control neutrophils were isolated by Ficoll, the single cell suspensions were frozen and arginase activity was measured in the lysates as previously described [41]. Briefly, 25ml of the lysate was incubated with 12ml of 0.1% triton X-IOO, 12ml of 25mM Tris-HCl and 1.2ml of 2OmM MnCl 2 for 7min at 56°C. Arginase activity was conducted by adding 50ml of 0.5mM L-arginine pH9.7 and incubating for 15min at 37°C.
  • arginase activity in PIaCs was significantly higher than in paired PBMCs, or in PBMCs from non-pregnant controls (Figure IA). Although arginase activity in PBMCs from pregnant women at the time of birth was notably lower than in PIaCs, it was still significantly higher than in PBMCs from non-pregnant controls (Figure IA). Arginase activity in peripheral neutrophils isolated from pregnant women was higher than in neutrophils from non-pregnant controls ( Figure IB). Similar results were obtained when the mean fluorescent activity of arginase 1 was compared in CD14- CD15+ cells isolated from pregnant and control women by flow cytometry.
  • arginase protein Determination of arginase protein by western blot confirmed that higher arginase I protein expression was found in PIaCs ( Figure IE), but arginase II expression was not seen.
  • the type of arginase-expressing cells in the placenta was determined by a combination of intracellular protein staining and cell surface labelling.
  • the majority of arginase-expressing cells were neutrophils (CD15 + CD14- and CD15 + CD14 low , Figures 2A & 2B); CD14 high cells did not express arginase.
  • Another small population of arginase-expressing cells was identified within the CD14 low population. These were alternatively activated macrophages, as characterised by the co-expression of the mannose receptor CD206 ( Figure 2C).
  • two populations of cells express arginase in the placenta: neutrophils and alternatively-activated macrophages.
  • Figure 2D shows the percentage of alternatively-activated macrophages in three types of cell.
  • L-arginine can affect T cell functions and the consumption of this amino acid by alternatively activated macrophages leads to modulation of CD3 ⁇ chain expression in T cells [42].
  • Downregulation of CD3 ⁇ expression results in uncoupling of the TCR signal transduction pathways with consequent T cell hyporesponsiveness [43].
  • PIaCs and Jurkat cells were co-cultured; the latter have been used to assay arginase-induced modulation of T cell responsiveness [7,42].
  • PIaCs or maternal PBMCs (2xlO 5 cells) were incubated with IxIO 5 Jurkat cells in a final volume of 200 ⁇ l in the presence or in the absence of lO ⁇ l 5.6 ⁇ M nor-NOHA or 2 ⁇ l of 10OmM L-arginine.
  • MFI mean fluorescence intensity
  • CD3 ⁇ in placental TCR + cells was examined directly ex vivo.
  • Mean fluorescence intensity of CD3 ⁇ chain expression was determined in maternal TCR+ PBMCs and TCR+ PIaCs.
  • CD3 ⁇ chain expression was significantly reduced in TCR + cells derived from the placenta as compared to maternal T lymphocytes in peripheral blood (average reduction: 40.8% ⁇ 2.8) ( Figures 3A & 3C).
  • TCR + cells are likely to circulate between placenta and periphery, it was thought that this downregulation might be reversible, to allow the maternal immune system to respond to other antigenic challenges whilst maintaining a state of non-responsiveness against the fetus.
  • PIaCs (4xlO 5 ) were stimulated with plate-bound anti-CD3 and anti-CD28 mAb in a final volume of 200 ⁇ l in complete DMEM in the absence of L-arginine, or in complete DMEM containing 0.1, 0.4, 1 or 2mM L-arginine.
  • PIaCs (2x10 5 ) were stimulated with plate-bound anti-CD3 mAb and anti-CD28 mAb in a final volume of lOO ⁇ l of DMEM (O.lmM L-arginine).
  • lO ⁇ l nor-NOHA (5.6 ⁇ M) was added to some of the wells, 30min later, maternal PBMCs (IxIO 5 , labeled with CFSE to differentiate maternal TCR + from placental TCR + ) were added in a final volume of lOO ⁇ l. L-arginine (2 ⁇ l of 10OmM) was added to some of the wells. Two days later, the cells were harvested and the expression of CD3 ⁇ in maternal TCR + cells and the frequency of maternal TCR + cells undergoing division were determined.
  • the survival of the semi-allogeneic fetus is critically dependent upon the induction of unresponsiveness of the maternal immune system.
  • arginase can mediate suppression of maternal T cell responses.
  • High arginase activity is present in cells isolated from placenta, and the sources of arginase are neutrophils and a population of alternatively activated macrophages.
  • the arginase levels seen in placental cells are comparable to those in the liver, the organ with the highest arginase concentration in the human body [45].
  • High arginase levels in the placenta imply a high rate of substrate consumption and decreased levels of extracellular L-arginine.
  • This view is supported by downregulation of CD3 ⁇ chain expression in placental TCR + cells, a finding consistently associated with L-arginine depletion [7,42,46].
  • T cell hyporesponsiveness has been shown to is reversible, and T cells in the placenta retain their capacity to respond to stimulation.
  • placental cells co-cultured with peripheral blood lymphocytes induce arginase-dependent, L-arginine-mediated T cell hyporesponsiveness, indicating a novel pathway through which immune privilege can be mediated at the feto-maternal interface.

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Abstract

La présente invention concerne l'activité de l'arginase dont la régulation est fortement positive dans le placenta de femmes enceintes, alors que cette régulation positive n'est pas observée dans les tissus périphériques. La régulation positive n'est en outre pas observée dans un groupe témoin de femmes non enceintes du même âge. Par conséquent, on suppose que l'arginase présente dans le placenta induit une hyporéactivité réversible et locale des lymphocytes T et joue de ce fait un rôle crucial dans le maintien d'une grossesse normale.
PCT/GB2007/004207 2006-11-07 2007-11-06 Arginase durant la grossesse Ceased WO2008056114A1 (fr)

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EP2510356A4 (fr) * 2009-12-08 2013-05-08 Cedars Sinai Medical Center Bio-marqueur de diagnostic pour identifier des femmes qui présentent un risque d'accouchement avant terme
WO2014105985A1 (fr) * 2012-12-28 2014-07-03 NX Pharmagen Biomarqueurs de la naissance prématurée
US10545156B2 (en) 2011-03-17 2020-01-28 RPI Consulting, LLC Diagnostic biomarker to predict women at risk for preterm delivery
CN111297854A (zh) * 2020-03-24 2020-06-19 复旦大学附属妇产科医院 雷帕霉素在制备自然流产保胎药物中的应用
US10877046B2 (en) 2015-12-04 2020-12-29 Nx Prenatal Inc. Treatment of spontaneous preterm birth
US10928402B2 (en) 2012-12-28 2021-02-23 Nx Prenatal Inc. Treatment of spontaneous preterm birth
US12203946B2 (en) 2014-05-15 2025-01-21 Kellbenx Incorporated Preparation of fetal nucleated red blood cells (NRBCs) for diagnostic testing

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000067754A1 (fr) * 1999-05-12 2000-11-16 Nitromed, Inc. Activateurs nitroses et nitrosyles du canal potassique, compositions et utilisations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000067754A1 (fr) * 1999-05-12 2000-11-16 Nitromed, Inc. Activateurs nitroses et nitrosyles du canal potassique, compositions et utilisations

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BRONTE ET AL: "Regulation of immune responses by L-arginine metabolism", NATURE REVIEWS IMMUNOLOGY, vol. 5, 2005, pages 641 - 654, XP002471069 *
ESKIOCAK ET AL: "Effect of psychological stress on the L-arginine-nitric oxide pathway and semen quality", BRAZILIAN JOURNAL OF MEDICAL AND BIOLOGICAL RESEARCH, vol. 39, May 2006 (2006-05-01), pages 581 - 588, XP002471070 *
ISHIKAWA ET AL: "Identification of arginase in human placental villi", PLACENTA, vol. 28, February 2007 (2007-02-01), pages 133 - 138, XP002471068 *
KROPF ET AL: "Arginase activity mediates reversible T cell hyporesponsiveness in human pregnancy", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 37, April 2007 (2007-04-01), pages 935 - 945, XP002471014 *
KROPF ET AL: "Arginase and polyamine synthesis are key factors in the regulation of experimental leishmaniasis in vivo", THE FASEB JOURNAL, April 2005 (2005-04-01), pages 1 - 25, XP002471013, Retrieved from the Internet <URL:www.fasebj.org/cgi/reprint/04-3416fjev1> [retrieved on 20080227] *
MAKARENKOVA ET AL: "CD11b+/Gr-1+ myeloid suppressor cells cause T cell dysfunction after traumatic stress", JOURNAL OF IMMUNOLOGY, vol. 176, February 2006 (2006-02-01), pages 2085 - 2094, XP002471220 *
MUNDER ET AL: "Suppression of T-cell functions by human granulocyte arginase", BLOOD, vol. 108, September 2006 (2006-09-01), pages 1627 - 1634, XP002471219 *
WEINER ET AL: "Myometrial arginase activity increases with advancing pregnancy in the guinea pig", AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY, vol. 174, 1996, pages 779 - 784, XP005142050 *
ZEA ET AL: "Decreased expression of CD3 zeta and nuclear transcription factor kappa B in patients with pulmonary tuberculosis: Potential mechanisms and reversibility with treatment", THE JOURNAL OF INFECTIOUS DISEASES, vol. 194, 15 November 2006 (2006-11-15), pages 1385 - 1393, XP002471221 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2510356A4 (fr) * 2009-12-08 2013-05-08 Cedars Sinai Medical Center Bio-marqueur de diagnostic pour identifier des femmes qui présentent un risque d'accouchement avant terme
US10545156B2 (en) 2011-03-17 2020-01-28 RPI Consulting, LLC Diagnostic biomarker to predict women at risk for preterm delivery
WO2014105985A1 (fr) * 2012-12-28 2014-07-03 NX Pharmagen Biomarqueurs de la naissance prématurée
US10928402B2 (en) 2012-12-28 2021-02-23 Nx Prenatal Inc. Treatment of spontaneous preterm birth
US11835530B2 (en) 2012-12-28 2023-12-05 Nx Prenatal Inc. Detection of microparticle-associated proteins associated with spontaneous preterm birth
US12203946B2 (en) 2014-05-15 2025-01-21 Kellbenx Incorporated Preparation of fetal nucleated red blood cells (NRBCs) for diagnostic testing
US10877046B2 (en) 2015-12-04 2020-12-29 Nx Prenatal Inc. Treatment of spontaneous preterm birth
CN111297854A (zh) * 2020-03-24 2020-06-19 复旦大学附属妇产科医院 雷帕霉素在制备自然流产保胎药物中的应用

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