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WO2012010291A1 - Méthodes et compositions de diagnostic et de traitement d'une maladie métabolique - Google Patents

Méthodes et compositions de diagnostic et de traitement d'une maladie métabolique Download PDF

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Publication number
WO2012010291A1
WO2012010291A1 PCT/EP2011/003604 EP2011003604W WO2012010291A1 WO 2012010291 A1 WO2012010291 A1 WO 2012010291A1 EP 2011003604 W EP2011003604 W EP 2011003604W WO 2012010291 A1 WO2012010291 A1 WO 2012010291A1
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WIPO (PCT)
Prior art keywords
conjugate
antibodies
reactivity
levels
metabolic disease
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PCT/EP2011/003604
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English (en)
Inventor
Johan FROSTEGÅRD
Hans GRÖNLUND
Ingrid Dahlbom
Knut Pettersson
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Athera Biotechnologies Ab
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Publication of WO2012010291A1 publication Critical patent/WO2012010291A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]

Definitions

  • the present invention relates to the treatment, prevention and diagnosis of metabolic diseases.
  • the metabolic syndrome is the clustering of a number of symptoms that relates to the consequences of disturbances in energy metabolism, that is the metabolism of lipids, carbohydrates and proteins. Obesity, insulin resistance, diabetes, hypertension and hyperlipidemia are the components of the syndrome.
  • NECP/ATP III criteria Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). JAMA, 2001.
  • 285(19), 2486-97) is one definition, according to which at least three of the following five criteria should be fulfilled: Blood pressure >130/85 mmHg or antihypertensive treatment, fasting plasma glucose > 6.1 mmol/l, serum triglycerides >1.7 mmol/l, waist circumference > 102 cm in men and >88 cm in women, HDL-cholesterol ⁇ 1.0 mmol/l in men and ⁇ 1.3 in women.
  • the individual components of the syndrome are themselves associated to increased morbidity and mortality, especially for premature cardiovascular disease (CVD), in individuals suffering from metabolic syndrome this risk is greatly increased (Bonora, E., The metabolic syndrome and cardiovascular disease. Ann Med, 2006. 38(1), 64-80).
  • PCOS Polycystic ovary syndrome
  • PCOS pathophysiology of PCOS also involves disturbances in energy metabolism, with symptoms similar to the metabolic syndrome (Lindholm, A., L. Andersson, M. Eliasson, M. Bixo, and I. Sundstrom-Poromaa, Prevalence of symptoms associated with polycystic ovary syndrome. Int J Gynaecol Obstet, 2008. 102(1 ), 39-43).
  • patients suffering from PCOS do not only suffer the well known fertility related morbidities, but also suffers the same health problems as other victims of the metabolic syndrome not having the PCOS, including increased risk for CVD etc (Wild, S., T. Pierpoint, P. McKeigue, and H.
  • Diabetes mellitus is a group of diseases resulting in elevated levels of plasma glucose. Diabetes is currently defined (WHO/ADA) as symptoms of diabetes plus: random plasma glucose concentration above 1 1.1 mmol/L [200mg/dl], or fasting plasma glucose above 7.0 mmol/L [126mg/dl], or 2-h plasma glucose concentration after 75 g anhydrous glucose in an oral glucose, tolerance test above 11.1 mmol/L [200mg/dl].
  • T1 DM is an autoimmune disease where pancreatic beta cells are destroyed, and the patients are thus dependent on exogenous insulin administration.
  • T1 DM is characterized by elevated glucose levels and low insulin levels, as the pancreas is unable to secrete insulin in response to the elevation in plasma glucose.
  • T2DM there is a relation between elevated markers for ongoing systemic inflammatory processes and disease development (Devaraj, S., U. Singh, and Jialal, Human C-reactive protein and the metabolic syndrome. Curr Opin Lipidol, 2009.
  • insulin resistance is diagnosed as elevated fasting insulin levels with normal fasting glucose levels, or as increased HOMA-IR, the product of fasting glucose and fasting insulin levels. Also insulin resistance in pre-diabetic individuals is associated to low-grade systemic inflammation.
  • PC antibodies are natural antibodies that belong to the innate immune system (Binder, C.J., P.X. Shaw, M.K. Chang, A. Boullier, K. Hartvigsen, S. Horkko, Y.I. Miller, D.A. Woelkers, M. Corr, and J.L. Witztum, The role of natural antibodies in atherogenesis. J Lipid Res, 2005. 46(7), 1353-63.). Natural antibodies have scavenging functions and are a part of the first line defence against certain infections. Thus, these antibodies can recognize PC - containing epitopes of certain infectious agents such as some parasites and bacteria, e.g. streptococcus bacteria.
  • PC neuropeptide
  • these immunogenic PC epitopes are generated by oxidative and/or enzymatic modification of the membrane phospholipid phosphatidylcholine. It is shown that membranes containing immunogenic PC induce inflammation in other cells, and that this inflammation can be blocked by anti-PC (Chang, M.K., C.J. Binder, Y.J. Miller, G. Subbanagounder, G.J. Silverman, J.A. Kirk, and J.L. Witztum, Apoptotic cells with oxidation-specific epitopes are immunogenic and proinflammatory. J Exp Med, 2004.
  • the present invention is based on the surprising findings that low levels of antibodies reactive with a PC-conjugate are related to an increased risk of developing metabolic diseases.
  • the present inventors have shown that the progression of metabolic diseases, such as insulin resistance, can be reduced by administration of a composition that increases the levels of anti-PC antibodies, and that risk of metabolic diseases, such as polycystic ovary syndrome, can be identified by low levels of anti-PC antibodies.
  • a first aspect of the present invention provides a composition comprising at least one phosphorylcholine (PC) conjugate, or an antibody preparation with reactivity to PC or a PC conjugate, for use in the immunization or prophylaxis against, or the prevention or treatment of, metabolic diseases in mammals.
  • the first aspect of the present invention provides for the use of a composition comprising at least one PC conjugate, or an antibody preparation with reactivity to PC or a PC conjugate, in the manufacture of a medicament for the immunization or prophylaxis against, or the prevention or treatment of, metabolic diseases in mammals.
  • a method for the immunization or prophylaxis against, or the treatment of, metabolic diseases in a mammal comprising the step of administering to the mammal a pharmaceutical composition comprising at least one PC conjugate, or an antibody preparation with reactivity to PC or a PC conjugate.
  • the method may thus include administration of a therapeutically effective amount of a composition comprising at least one PC conjugate or an antibody preparation with reactivity to PC or a PC conjugate is administered to the mammal
  • any mammal may be treated, although in one embodiment the mammal may be a human.
  • any metabolic disease may be addressed.
  • exemplary metabolic diseases include a condition selected from the group consisting of metabolic syndrome, insulin resistance, glucose intolerance, hyperglycemia, type I diabetes, type II diabetes, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycystic ovary syndrome (PCOS).
  • PCOS polycystic ovary syndrome
  • the composition comprising at least one PC conjugate may, for example, be a pharmaceutical composition comprising at least one PC conjugate, and may optionally include an adjuvant.
  • Any suitable adjuvant for example aluminium hydroxide, may be used.
  • the antibody preparation with reactivity to PC or a PC conjugate may, for example, comprise a polyclonal antibodies, or a monoclonal antibody, with reactivity to PC or a PC conjugate.
  • the first aspect of present invention may provide, for example, for the therapeutic treatment of a mammal suffering from metabolic disease, or for the prophylactic treatment of a mammal facing the risk of developing metabolic disease.
  • the mammal may be identified as being of risk of developing metabolic disease by a method according to the second aspect of the present invention, as discussed further below.
  • the first aspect of the invention provides the use of at least one PC conjugate, or an antibody preparation, for example a monoclonal antibody, with reactivity to PC or a PC conjugate, in the manufacture of a medicament for immunization and prophylaxis, prevention or treatment of mammals, including humans, against metabolic diseases, such as metabolic syndrome, insulin resistance (IRS), glucose intolerance, hyperglycemia, type I diabetes, type II diabetes, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycystic ovary syndrome (PCOS).
  • the medicament is intended to provide immunization having immunogenic or therapeutic properties against metabolic diseases.
  • the first aspect of the invention provides a method for immunization and treatment of a mammal, including a human, against metabolic diseases, such as metabolic syndrome, insulin resistance (IRS), glucose intolerance, hyperglycemia, type I diabetes, type II diabetes, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycystic ovary syndrome (PCOS), the method comprising the step of administering to the mammal a pharmaceutical composition comprising at least one PC conjugate, or an antibody preparation, for example a monoclonal antibody, with reactivity to PC or a PC conjugate.
  • the pharmaceutical composition is intended to provide immunization having immunogenic or therapeutic properties against metabolic diseases.
  • the first aspect of the invention provides the use of one or more of the PC conjugates as defined in relation to the preceding aspects of the invention, in the manufacture of a pharmaceutical composition, optionally in combination with an adjuvant, for immunotherapy or therapy for the prevention, prophylaxis and/or treatment of metabolic diseases.
  • the first aspect of the invention provides a method of prophylactic or therapeutic treatment of a mammal, which may be a human being, suffering from metabolic disease or facing the risk of developing metabolic disease, whereby a therapeutically effective amount of at least one PC conjugate or an antibody preparation, for example a monoclonal antibody, with reactivity to PC or a PC conjugate is administered.
  • a second aspect of the present invention provides a method for diagnosing metabolic disease, or assessing a patient's risk of developing or progression of metabolic disease, the method comprising the steps of -
  • the method of the second aspect of the present invention may assess the level of all of the patient's antibodies with reactivity to PC or a PC conjugate, or may comprise the assessment of a particular isotype, such as the patient's level of IgM, IgG or IgA antibodies with reactivity to PC or a PC conjugate.
  • the patient's level of antibodies with reactivity to PC or a PC conjugate are assessed by analysis of an ex vivo sample taken from the patient.
  • the sample may be a blood, plasma or serum sample that has been obtained from the patient.
  • the method of the second aspect of the present invention may be employed to diagnose metabolic disease, or assess a patient's risk of developing or progression of metabolic disease, in any mammalian patient, although in one embodiment the patient is human.
  • lower levels of antibodies with reactivity to PC or a PC conjugate are indicative of the presence of metabolic disease and/or the risk of developing or progression of metabolic disease. Accordingly, in the method of the second aspect of the present invention, the patient's level of antibodies with reactivity to PC or a PC conjugate may correlate negatively with the patient's risk of developing or progression of the metabolic disease.
  • the method of the second aspect of the present invention may be employed to diagnose any metabolic disease, or assess a patient's risk of developing or progression of any metabolic disease.
  • exemplary metabolic diseases include a a condition selected from the group consisting of metabolic syndrome, insulin resistance, glucose intolerance, hyperglycemia, type I diabetes, type II diabetes, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycystic ovary syndrome (PCOS).
  • PCOS polycystic ovary syndrome
  • the second aspect of the invention provides a method of diagnosing the presence or absence of antibodies, for example IgM, IgG or IgA antibodies, related to increased or decreased risk of developing metabolic diseases, using PC or a PC conjugate.
  • the second aspect of the present invention also provides for the use of PC or a PC conjugate in a method for diagnosing metabolic disease and/or for assessing a patient's risk of developing or progression of metabolic disease in which the patient's levels of antibodies (for example, all antibodies, or a particular isotype, such as IgM, IgG or IgA antibodies) with reactivity to PC or the PC conjugate are assessed.
  • antibodies for example, all antibodies, or a particular isotype, such as IgM, IgG or IgA antibodies
  • the invention relates to pharmaceutical compositions comprising a PC conjugate, or an antibody preparation, for example a monoclonal antibody, with reactivity to PC or a PC conjugate, and the use of these compositions in the treatment, prophylaxis or prevention of metabolic diseases, such as metabolic syndrome, insulin resistance (IRS), glucose intolerance, hyperglycemia, type I diabetes, type II diabetes, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycystic ovary syndrome (PCOS).
  • the invention also relates to the use of PC conjugates or said antibody preparation, for example monoclonal antibody to produce a pharmaceutical composition optionally with an adjuvant.
  • the invention relates to diagnosing the absence, presence and/or levels of antibodies, for example IgM, IgG or IgA antibodies, reactive with a PC- conjugate (such as PC-BSA) related to increased or decreased risk of displaying or developing metabolic diseases.
  • a PC- conjugate such as PC-BSA
  • PC phosphorylcholine according to the formula.
  • a PC conjugate is meant a PC moiety linked to a carrier, optionally via a spacer.
  • the PC moiety can be covalently or non-covalently linked to the carrier.
  • the PC moiety may be linked to the carrier via the phosphate group.
  • the carrier may be selected from the group consisting of a protein, a carbohydrate, a polymer, latex beads, or colloid metal.
  • the PC conjugate may for example be a protein-PC conjugate, such as a human serum albumin (HSA)-PC conjugate, a transferrin-PC conjugate, a keyhole limpet hemocyanin (KLH)-PC conjugate or a bovine serum albumin (BSA)-PC conjugate.
  • HSA human serum albumin
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Examples of PC-conjugates and generation of anti-PC antibodies are, e.g., described in WO 2005/100405 and U.S. Patent 5,455,032, the contents of both of which are incorporated by reference.
  • any suitable spacer may be used.
  • Non-limiting examples of spacers include coupling agents (typically, bi-functional compounds), such as a di-carboxylic acids like succinic and glutaric acid, the corresponding di-aldehydes, di-amines such as 1 ,6 diaminohexane, di-substituted phenols such as p-amino-phenol, p-diazo-phenol, p- phenylenediamine, p-benzoquinone, and the like.
  • coupling agents typically, bi-functional compounds
  • di-aldehydes di-amines such as 1 ,6 diaminohexane
  • di-substituted phenols such as p-amino-phenol, p-diazo-phenol, p- phenylenediamine, p-benzoquinone, and the like.
  • Metabolic diseases that can be treated, prevented and/or diagnosed according to the first or second aspects of the present invention are exemplified, but not limited to, metabolic syndrome, insulin resistance (IRS), glucose intolerance, hyperglycemia, type I diabetes also referred to as insulin-dependent diabetes mellitus or IDDM, type II diabetes also referred to as noninsulin-dependent diabetes mellitus or NIDDM, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycystic ovary syndrome (PCOS).
  • metabolic syndrome insulin resistance
  • IDDM insulin-dependent diabetes mellitus
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM noninsulin-dependent diabetes mellitus
  • hyperlipidemia hypertriglyceridemia
  • hypercholesterolemia hypercholesterolemia
  • dyslipidemia dyslipidemia
  • PCOS polycystic ovary syndrome
  • individual for treatment according to the method or use of the present invention may be an individual that is identified as suffering from, or being at risk of suffering from, a metabolic disease, such as, but not limited to, metabolic syndrome, insulin resistance (IRS), glucose intolerance, hyperglycemia, type I diabetes also referred to as insulin-dependent diabetes mellitus or IDDM, type II diabetes also referred to as noninsulin-dependent diabetes mellitus or NIDDM, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycystic . ovary syndrome (PCOS).
  • a metabolic disease such as, but not limited to, metabolic syndrome, insulin resistance (IRS), glucose intolerance, hyperglycemia, type I diabetes also referred to as insulin-dependent diabetes mellitus or IDDM, type II diabetes also referred to as noninsulin-dependent diabetes mellitus or NIDDM, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycystic .
  • the individual for treatment is an individual that is not suffering from a disease or condition as discussed in WO 2005/100405 or WO 2010/003602, the contents of both of which are incorporated by reference.
  • the individual to be treated may be an individual who is not suffering from, and/or has not been diagnosed as suffering from or being at risk of the progression or development of, conditions selected from one or more of Alzheimer's disease, atherosclerosis, atherosclerosis related disease, cardiovascular disease, or ischemic cardiovascular diseases.
  • the individual to be treated may be an individual who is suffering from, and/or has been diagnosed as suffering from or being at risk of the progression or development of, conditions selected from one or more of Alzheimer's disease, atherosclerosis, atherosclerosis related disease, cardiovascular disease, or ischemic cardiovascular diseases, and who is receiving therapy or prophylaxis for that condition by administration of a therapeutic or prophylactic agent other than a composition comprising at least one phosphorylcholine (PC) conjugate, or an antibody preparation with reactivity to PC or a PC conjugate.
  • a therapeutic or prophylactic agent other than a composition comprising at least one phosphorylcholine (PC) conjugate, or an antibody preparation with reactivity to PC or a PC conjugate.
  • PC phosphorylcholine
  • the medicament is for administration by injection.
  • it can be administered by any suitable means that allows the PC-conjugate to provoke an immune response in, or allows efficient delivery of the antibody preparation to, the subject to which it is administered.
  • a clinician can determine the most appropriate administrative regimen for an individual based on factors such as the individual's weight, age, gender, diagnosis or prognosis, and the half-life of the administered therapeutic molecule.
  • it may be suitable to treat an individual with a single dose, or multiple doses, of the composition comprising at least on PC conjugate and/or an antibody preparation with reactivity to PC or a PC conjugate.
  • multiple administrations may be made at a rate of, for example, once, twice, three times, four times or more often per day, week or month, and may be continued for a period of time necessary and effective to increase the levels of anti-PC antibodies in the individual and thereby obtain a therapeutically or prophylactically-beneficial effect in respect of metabolic disease.
  • treatment may continue for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or more days, weeks, months or years, or even for the rest of the life of the subject.
  • administration would most typically be made weekly, or once or twice per month, and continue for as long as is clinically beneficial.
  • the treatment may involve an initial immunisation, followed by a further administration as a booster (for example, within about one month of the initial immunisation), and optionally followed by yearly further administrations, continued for as long as is clinically beneficial.
  • the first aspect of the invention provides active (where the composition comprises at least one PC-conjugate) or passive (where the composition comprises the defined antibody) immunization having immunogenic or therapeutic properties against metabolic diseases.
  • the invention provides at least one PC-conjugate, or an antibody preparation (for example a monoclonal antibody) with reactivity to PC and/or a PC- conjugate, for use in the prophylaxis, prevention and/or treatment of metabolic diseases, and provides a method for immunization and treatment against metabolic diseases.
  • the method may comprise the step of administering to a subject a pharmaceutical composition comprising at least one PC-conjugate, or an antibody preparation (for example a monoclonal antibody) with reactivity to PC and/or a PC- conjugate.
  • the pharmaceutical composition is intended to provide active or passive immunization having immunogenic or therapeutic properties against metabolic diseases.
  • the prophylaxis, prevention and/or treatment of metabolic diseases according to the present invention is for the treatment of humans.
  • the human subject may, for example, be aged at least 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85 of more years.
  • the human subject may be one that has been diagnosed as being at increased risk of development or progression, of one or more metabolic diseases, for example by using a method of diagnosis based on the assessment of anti-PC levels according to the other aspects of the present invention.
  • the present invention provides a method for the prophylaxis, prevention and/or treatment of polycystic ovary syndrome (PCOS) in females belonging to older age groups, such as human females aged at least 40, 50, 60, 65, 70, 75, 80, 85 of more years and/or females that are pre-, peri-, and/or post-menopausal.
  • PCOS polycystic ovary syndrome
  • One embodiment of the present invention is thus to use a PC-conjugate for the preparation of a pharmaceutical composition to be used in the treatment, prophylaxis and/or prevention of metabolic diseases, such as metabolic syndrome, insulin resistance (IRS), glucose intolerance, hyperglycemia, type I diabetes, type II diabetes, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycystic ovary syndrome (PCOS).
  • metabolic diseases such as metabolic syndrome, insulin resistance (IRS), glucose intolerance, hyperglycemia, type I diabetes, type II diabetes, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycystic ovary syndrome (PCOS).
  • the conjugate can, for example, be PC linked to a pharmaceutically acceptable carrier such as a protein, carbohydrate, or polymer.
  • a pharmaceutically acceptable carrier such as a protein, carbohydrate, or polymer.
  • the pharmaceutical composition is preferably given by injection, but can in practice be administered by any suitable means that allows the PC-conjugate to provoke an immune response in the subject to which it is administered.
  • one or more PC-conjugate molecules are prepared in an immunogenic formulation, optionally containing suitable adjuvants and carriers and administered to the patient in known ways.
  • suitable adjuvants include Freund's complete or incomplete adjuvant, muramyl dipeptide, the "Iscoms" of EP 109 942, EP 180 564 and EP 231 039, aluminium hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pluronic polyols or the Ribi adjuvant system (see, for example GB-A-2 189 141 ).
  • "Pluronic" is a Registered Trade Mark.
  • active immunization will modulate (preferably, increase) the titre of anti-PC antibodies which in turn will have a positive effect on the development of metabolic diseases (that is, the development of metabolic disease will be reduced).
  • active immunisation may be used to increase the titre of anti-PC antibodies to a level that, when assessed by the methods of diagnosis according to the present application, would not be said to be "low” or indicative of an increased risk of development, or progression, of metabolic diseases.
  • a method of active immunisation according to the present invention may be used to increase anti-PC levels, such as IgM anti-PC levels, in an individual to a level that is greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 U/ml when tested by the methods described below.
  • anti-PC levels such as IgM anti-PC levels
  • the method of active immunisation according to the present invention may be used to increase anti-PC levels to a level that is above the mean and/or median average, or above a particular percentile value determined with reference to the wider population, such as above the 5 th , 10 th , 20 th or 25 th percentile, such as to a level wherein the odds ratio is below one, the p-value is ⁇ 0.05 and the upper limit of the odd ratio confidence interval is less than one, indicating a statistically significant level of low risk for a metabolic disease.
  • Another embodiment of the invention is the use an antibody preparation, for example a monoclonal antibody, recognizing PC or a PC-conjugate for the preparation of a pharmaceutical composition to be used in the treatment, prophylaxis and/or prevention of metabolic diseases, such as metabolic syndrome, insulin resistance (IRS), glucose intolerance, hyperglycemia, type I diabetes, type II diabetes, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, and polycystic ovary syndrome (PCOS).
  • metabolic syndrome insulin resistance
  • I diabetes insulin resistance
  • type II diabetes hyperlipidemia
  • hypertriglyceridemia hypercholesterolemia
  • dyslipidemia dyslipidemia
  • PCOS polycystic ovary syndrome
  • Monoclonal antibodies can be produced using methods known in the art and/or as discussed further below.
  • Other antibody preparations may be used, such as anti- PC enriched preparations obtained from Intravenous immunoglobulin preparations, recombinantly produced anti-PC antibodies and/or other artificially created anti-PC antibody derivatives, as discussed above.
  • passive immunisation may be used to increase the titre of anti-PC antibodies in an individual to a level that, when assessed by the methods of diagnosis according to the present application, would not be said to be "low” or indicative of an increased risk of development, or progression, of metabolic diseases.
  • a method of passive immunisation according to the present invention may be used to increase anti-PC levels, such as IgM anti-PC levels, in an individual to a level that is greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 U/ml when tested by the methods described above.
  • the method of active immunisation according to the present invention may be used to increase anti-PC levels to a level that is above the mean and/or median average, or above a particular percentile value determined with reference to the wider population, such as above the 5 th , 10 th , 20 th or 25 th percentile, such as to a level wherein the odds ratio is below one, the p-value is ⁇ 0.05 and the upper limit of the odd ratio confidence interval is less than one, indicating a statistically significant level of low risk.
  • Monoclonal antibodies reactive against PC and /or PC-conjugate can be produced using any standard method known in the art. See for example Briles et al., 1982. J Exp Med 156, 1177-1185 or Spira et al., 1988. J Immunology 140. 2675-2680.
  • antibodies against a phosphorylcholine and/or its conjugate can be prepared using methods well known to those skilled in the art.
  • a sub- fraction with anti-PC activity of a human immunoglobulin preparation can be prepared, for example as described below, for example by affinity purification using a phosphorylcholine conjugate.
  • Intravenous immunoglobulin preparations e.g., IGIV; Baxter and others
  • IgG intravenous immunoglobulin preparations
  • Immunoglobulin preparations include those available from the following manufacturers: Baxter (US), e.g., Gammagard ® , Isiven (Antimo Naples, Italy), Omrix (Tel-Hashomer, Israel), Miles (Biological Products Division, West Heaven, CT), Sclavo (Lucca, Italy), Sandoz (Nonachis, Basel, Swizerland), e.g., Sandoglobulin ® , Biotest Diagnostic Corporation (Deville, NJ).
  • immunoglobulin preparations are GammagardS/D ® , GammarlV ® , Gaimnar-PIV ® , Gammimune N ® , Iveegam ® , Panglobulin ® , Polygam S/D ® , Sandoglobulin ® , Venoglobulin ® .
  • Immunoglobulin preparations typically contain some IgM as well as IgG. Trace amounts of IgM are present in Gammagard ® .
  • Pentaglobin (Biotest) is an enriched IgM preparation which has been used for treatment of SARS.
  • the subfraction with anti-PC activity may comprise both IgG and IgM, or may be selected to comprise mainly IgG (for example by starting with an IgG-rich preparation such as Gammagard ® and/or by selecting for IgG); or mainly IgM (for example by starting with an IgM-rich preparation such as Pentaglobin and/or by selecting for IgM).
  • the present invention contemplates the use of recombinantly produced anti-PC antibodies and/or other artificially created anti-PC antibody derivatives, such as CDR-grafted and/or humanised antibodies, scFv, dAb, Fab, or Fv or other molecules which comprise or consists of PC-binding fragments of an antibody.
  • An antibody preparation with specificity to a PC-conjugate binds to unconjugated
  • a second aspect of the present invention provides a method for diagnosing metabolic disease, or assessing a patient's risk of developing or progression of metabolic disease, the method comprising the steps of -
  • the method of the second aspect of the invention comprises exposing PC or the PC conjugate to a sample (for example, an ex vivo sample) from an individual and detecting antibodies which have bound to PC or the PC conjugate.
  • a sample for example, an ex vivo sample
  • the individual is a human.
  • the sample is blood, serum or plasma. Serum may be preferred in one embodiment.
  • PC is linked to a carrier via a spacer.
  • the carrier may, for example, be a protein, such as KLH (keyhole limpet hemocyanin), transferrin, human serum albumin (HSA) or bovine serum albumin (BSA).
  • the carrier may be latex beads.
  • antibodies which have bound to the PC conjugate are determined by an assay, preferably an immunoassay.
  • the patient's levels of antibodies e.g., of all or a particular isotype such as IgM, IgG or IgA antibodies, with reactivity to PC or the PC conjugate may be assessed using an immunoassay. Examples of suitable immunoassays are described below and will in any case be apparent to those skilled in the art.
  • low levels of antibodies with reactivity to PC or the PC conjugate are indicative of the presence of metabolic disease and/or an increased risk of developing metabolic disease.
  • high levels of antibodies with reactivity to PC or the PC conjugate are indicative of the absence of metabolic disease and/or a reduced risk of developing metabolic disease.
  • antibodies are determined in a sample of patient blood, plasma or serum.
  • levels of antibodies with reactivity to PC or a PC conjugate are likely to vary.
  • the level of antibodies with reactivity to PC or a PC conjugate determined for any given individual may be categorised as high or low by reference to the range observed in the wider population.
  • a level of such antibodies below a particular percentile value determined with reference to the wider population may be categorised as a low level.
  • a low level may correspond to a value below the 25 th percentile, or below the 20 th , 10 th or 5 th percentile.
  • a high level may correspond to a value of above the 5 lh , 10 lh , 20 th , or 25 th percentile, for example.
  • this information may assist in the diagnosis or prognosis of the presence of, or the increased risk of development or progression of, metabolic disease.
  • a clinician may take other factors into account in arriving at a diagnosis or prognosis. It may be desirable to measure antibodies reactive with platelet activating factor
  • PAF also known as 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine
  • a PAF conjugate as well as measuring antibodies, e.g., IgM, IgG and/or IgA antibodies, with reactivity to the PC conjugate.
  • oxLDL oxidised low density lipoprotein
  • MD-LDL maiondialdehyde modified LDL
  • LpPLA 2 lipoprotein associated phospholipase A 2
  • CRP C- reactive protein
  • HSP70 high density lipoprotein
  • TNF TNFa
  • HSP60 antibodies, e.g., IgM, IgG and/or IgA antibodies, with reactivity to the PC conjugate. Assaying for such factors may assist in the diagnosis or prognosis of increased risk of development or progression of metabolic disease. Other factors presented by a patient may also be taken into account clinician in arriving at a diagnosis or prognosis.
  • a clinician may take into account any one, two, three or more of the presence of obesity, insulin resistance, diabetes, hypertension and hyperlipidemia in the patient.
  • the clinician may also take into account whether the patient presents at least one, two, three or more of the following features (i) blood pressure >130/85 mmHg or antihypertensive treatment, (ii) fasting plasma glucose > 6.1 mmol/l, (iii) serum triglycerides >1.7 mmol/l, (iv) waist circumference > 102 cm in men and >88 cm in women, and (v) HDL-cholesterol ⁇ 1.0 mmol/l in men and ⁇ 1.3 in women.
  • the clinician may also take into account whether the female patient presents at least one, two or more of the following features: (i) oligoovulation or anovulation, (ii) excess androgen activity or (iii) the presence of polycystic ovaries.
  • the clinician may also take into account whether the individual (such as male or female) patient presents and one, two or more of the following features: (i) random plasma glucose concentration above 11.1 mmol/L [200mg/dl], or (ii) fasting plasma glucose above 7.0 mmol/L [126mg/dl], or (iii) 2-h plasma glucose concentration after 75 g anhydrous glucose in an oral glucose, tolerance test above 11.1 mmol/L [200mg/dl].
  • treatments including treatment in accordance with the first aspect of the present invention
  • life-style changes may be recommended.
  • prophylactic treatments including prophylactic treatment in accordance with the first aspect of the present invention
  • life-style changes may be recommended.
  • his or her clinician may recommend treatments and/or life-style changes tailored to the individual.
  • levels of antibodies may be characterised by assaying for all antibodies with reactivity to PC or a PC conjugate, or for only antibodies of a particular isotype, such as IgM, IgG or IgA, or for a combination of two or more antibody isotypes.
  • a particular isotype such as IgM, IgG or IgA
  • the level of IgM is determined.
  • Immunoassays can be competitive or noncompetitive.
  • a typical competitive immunoassay the antibody in the sample competes with labeled antibody to bind with the PC conjugate. The amount of labeled antibody bound to the PC conjugate is then measured. There is an inverse relationship between concentration of antibody in the sample and the quantity of labeled antibody detected.
  • noncompetitive immunoassays antibody in the sample is bound to the PC conjugate, then a labeled detection reagent, typically an anti-immunoglobulin antibody, is bound to the antibody. The amount of labeled detection reagent bound to the antibody is then measured. Unlike the competitive method, the results of the noncompetitive method will be directly proportional to the concentration of the antibody.
  • a labeled detection reagent typically an anti-immunoglobulin antibody
  • a labeled detection reagent is used to detect antibody bound to the PC conjugate.
  • a suitable anti-immunoglobulin antibody must bind specifically to immunoglobulin of the species from which the sample is obtained. It may bind to all immunoglobulin isotypes of that species, or only a subset of isotypes. For example, it may bind only to IgA, IgD, IgE, IgG or IgM, or combinations of two or more of these isotypes.
  • the anti-immunoglobulin antibody may bind specifically only to certain subtypes of any given isotype.
  • Subtypes of human IgA are lgA1 and lgA2.
  • the antiimmunoglobulin antibody may bind to one or both of these subtypes.
  • Subtypes of human IgG are lgG1 , lgG2, lgG3 and lgG4.
  • the anti-immunoglobulin may bind to one or more of these human IgG subtypes. It will be appreciated that there are different isotypes and subtypes in different vertebrate species.
  • the antibody or detection reagent is labeled with a radioisotope, such as 3 1 or 125 l.
  • a radioisotope such as 3 1 or 125 l.
  • the antibody or detection reagent is labeled with an enzyme. Suitable enzymes are capable of being detected with the use of a chromogenic substrate.
  • a chromogenic substrate is a substance which, as a result of the reaction with the enzyme, gives rise to a coloured product which can thus be detected spectrophotometrically.
  • Enzymes such as horse radish peroxidase, alkaline phosphatase, beta-galactosidase, and pyrophosphatase from E.coli have been widely employed.
  • Chemi-luminescent systems based on enzymes such as luciferase can also be used.
  • Other labels include fluorescent labels such as fluorophores of the Alexa series.
  • the sample to be analyzed is placed in contact and incubated with the PC conjugate adsorbed on a solid substrate. Any anti-PC conjugate antibodies that are possibly present in the sample are thus specifically bound by the PC conjugate adsorbed on the solid substrate, producing a PC conjugate /anti- PC conjugate antibody complex.
  • the sample is then separated from the solid substrate so as to eliminate non-bound materials, for example, by washing.
  • an indicator antibody capable of binding any anti-PC conjugate antibodies that are present on the substrate in the form of a PC conjugate /anti-PC conjugate antibody complex is added to the solid substrate, thus producing a PC conjugate /anti-PC conjugate antibody/indicator antibody complex.
  • the indicator antibody may, for example, be an anti-human IgG immunoglobulin raised in a non-human animal species.
  • the solid substrate is a micro-titration plate, for example, of the type commonly used for performing ELISA immunological assays.
  • the micro-titration plate is preferably a polystyrene plate.
  • suitable solid substrates are latex particles, beads and coated red blood cells.
  • the PC conjugate is adsorbed to the solid substrate by incubating the PC conjugate in a buffer with the solid substrate.
  • Suitable buffers include carbonate buffer or phosphate buffered saline.
  • the PC conjugate may be covalently linked to the solid substrate.
  • the solid substrate is incubated with a blocking agent to reduce non-specific binding of matter from the sample to the solid substrate.
  • Suitable blocking agents include bovine serum albumin.
  • a quantitative estimate of antibody which can bind to PC or the PC conjugate is obtained by one or more of the above techniques.
  • a linear relationship between the measured variable, whether it be optical density or some other read-out, and antibody concentration is assumed. For example, if sample A has double the optical density of sample B in the assay (background having been subtracted from both), it is assumed that the concentration of antibody is double in A compared to B.
  • CVDefineTM assay kit available from Athera Biotechnologies AB.
  • the CVDefineTM assay has been discussed in numerous publications, such as Gronlund, H., Hallmans, G.; Jansson, J.H., Boman, K., Wikstrom, M., de Faire, U., Frostegard, J. Low levels of IgM antibodies against phosphorylcholine predict development of acute myocardial infarction in a population-based cohort from northern Sweden. Eur J Cardiovasc Prev Rehabil. 2009.
  • the CVDefineTM assay is an indirect non-competitive enzyme immunoassay for quantitative determination of anti-phosphorylcholine (anti-PC) IgM antibodies in human serum or plasma.
  • the wells of a microplate are coated with PC antigen.
  • PC-specific IgM antibodies present in the patient sample bind to the antigen.
  • an enzyme labelled second antibody conjugates to the antigen-antibody complex which leads to the formation of an enzyme labelled conjugate-antibody-antigen complex.
  • the enzyme labelled antigen-antibody complex converts the added substrate to form a coloured solution.
  • the rate of colour formation from the chromogen is a function of the amount of conjugate complexed with the bound antibody and thus is proportional to the initial concentration of the respective antibodies in the patient sample.
  • the CVDefineTM kit contains the following reagents -
  • Microplate Strips 12 strips * 8 wells (for 96 determinations) coated with PC conjugated to bovine serum albumin (BSA), maintained in a foil pouch containing desiccant.
  • BSA bovine serum albumin
  • Control High a vial of buffer containing BSA, 0.095% (w/v) sodium azide, detergent and human serum, 1.5 ml, ready to use.
  • the level of IgM anti-PC in this control should be high enough to provide an optical density of greater than 0.6 when assayed by the following procedure.
  • Control Low a vial of buffer containing BSA, 0.095% (w/v) sodium azide, detergent and human serum, 1.5 ml, ready to use.
  • the level of IgM anti-PC in this control should be low enough to provide an optical density of less than 0.2 when assayed by the following procedure.
  • Wash Buffer Concentrate a vial of 20x PBS concentrate and detergent, 75 ml
  • Sample Diluent a vial of PBS containing BSA, with ⁇ 0.1% (w/v) sodium azide and detergent (Tween 20), 100 ml (yellow coloured), ready to use.
  • IgM HRP Conjugate a vial of anti-human IgM HRP (horse-radish peroxidase) from goat, 20 ml, ready to use.
  • Substrate TMB a vial of TMB (3, 3', 5, 5' Tetramethylbenzidine), ⁇ 0.05 % (w/v) in 20 ml water, ready to use,
  • the direct results of the CVDefineTM assay are initially expressed in arbitrary units. Calibrators and controls are adjusted and traceable to an in-house human reference serum preparation established at Athera Biotechnologies AB. The assay has a measuring range of 6.25 U/ml - 100 U/ml and a detection limit of 0.5 U/ml. For quantitative evaluation of the results from this type of assay, an individual calibration must be performed for each run. Quantitative evaluation can be performed manually or by the use of data reduction software.
  • the manual procedure requires calculation of the mean absorbance (OD) value of each calibrator, which is plotted against the calibrator concentrations of 0, 6.25, 12.5, 25, 50, 100 U/ml on suitable graph paper. A smooth curve is then drawn considering all calibrator points, and the concentrations of the samples can then be read from the calibration curve.
  • OD mean absorbance
  • a suitable computer program When using data reduction software, a suitable computer program is selected that uses a 4 parameter or Cubic Spline curve fitting algorithm. In case the implemented curve fitting algorithm is not able to automatically handle standards with 0 U/ml, assign a minimal value to Calibrator 1 (0 U/ml), which is derived one log scale below the Calibrator 2 (e.g. 0.625 U/ml for Calibrator 1). Samples which give absorbances above that of the highest Calibrator are out of range of this assay and should be stated as > 100 U/ml. Such samples should be diluted as appropriate and reassayed.
  • Quantile cut-offs can be based on the anti-PC distribution in control groups.
  • the associations between serum levels of anti-PC and risk of developing a metabolic disease can be determined by conditional logistic regression models with calculation of odds ratios (ORs) and 95% confidence intervals (CI).
  • Test samples and controls may be age and gender matched for by design of the study. To test for differences in means/medians between cases and controls, the t-test may be used for normally distributed variables and the Wilcoxon Rank sum test for non-normally distributed variables.
  • the Chi-Square test (and Fisher's exact for small samples) may be used to test for differences in proportions.
  • the non-parametric Spearman Rank Correlation Coefficient may be used to test for correlations.
  • Linear trend in proportions may be assessed through the Cochran-Armitage trend test.
  • Linear trend in ORs over quantiles may be assessed by the Score-test (a Cochran-Armitage trend test) of quantile included as a continuous variable in SAS PROC LOGISTIC.
  • a two-tailed p-value ⁇ 0.05 may be considered as significant.
  • SAS may be used for the statistical analyses (release 9.2, SAS Institutet Inc.Cary.NC ).
  • the level of antibodies with reactivity to PC and/or a PC- conjugate determined for any given individual may be categorised as high or low by reference to the range observed in the wider population or test cohort. It may be appropriate to assess anti-PC levels blood samples taken from individuals in a cohort before the onset of metabolic disease (incident cases) compared to three unrelated age- and sex-matched controls at blood draw (+/- 1 year), and/or to assess anti-PC levels blood samples taken from individuals in a cohort after the onset of disease (prevalent cases) compared to three unrelated age- and sex-matched controls at blood draw (+/- 1 year).
  • the total number of test and controls individuals in a suitable cohort may be greater than 100, such as about 200, 300, 400, 500, 600, 700, 800, 900 or 1000.
  • a test case shows a level of anti-PC antibodies below the mean average, or below a particular percentile value determined with reference to the wider population or cohort, it may be categorised as a low level.
  • a low level may correspond to a value below the 25 th percentile, or below the 20 th , 10 th or 5 th percentile.
  • a high level may for example, correspond to a value of above the 5 th , 10 th , 20 th , or 25 th percentile, or above the mean average level.
  • any percentile value cut-off point can be used to indicate a low level of anti-PC that is associated with the presence of, or the increased risk of developing, a metabolic disease, so long as, when conditional logistic regression analysis is performed on the anti-PC levels generated from a test cohort:-
  • the calculated odds ratio for all individuals within that percentile group is greater than 1 (indicating that a person having an anti-PC level within the levels associated with that percentile is more likely to develop a metabolic disease than a person having an anti-PC level above that percentile); and ⁇ the p-value calculated from the anti-PC values for individuals within that percentile group is less than 0.05 and the 95% odds ratio confidence interval for that group provides a range in which the lower limit is above 1 (wherein such p- values and CI values indicate that the odds ratio value ascribed to individuals with anti-PC levels falling within that percentile group is statistically significant).
  • the skilled person can readily determine by statistical analysis of the data from a cohort, the highest percentile value for which anti-PC levels indicate a statistically significant risk of developing a metabolic disease, and can also calculate associated (and incrementally higher) hazard ratios for individuals with anti- PC levels falling within lower percentile values. Additionally, or alternatively, the level of antibodies with reactivity to PC and/or a PC-conjugate determined for any given individual may be categorised as high or low by reference to the absolute level of anti-PC antibody within a sample taken from that individual, in view of the inventors' findings.
  • mean levels of anti-PC IgM levels in a population are typically about 40-50 U/ml (corresponding to about 4-5 ⁇ g/ml), and median levels in female humans are around 68 U/ml (corresponding to about 6.8 ⁇ g/ml) in PCOS patients and around 84 U/ml (corresponding to about 8.4 ⁇ g/ml) in a control group.
  • Values in a sample at or below any one or more of these levels may be considered as being low.
  • anti-PC IgM levels of, or below, about 25-20 U/ml (which corresponds to about 2.5-3 ⁇ g/ml) are typically representative of values below the about the 25 th percentile, and values under about 17 U/ml (which corresponds to about 1.7 ⁇ g/ml) antibody are typically representative of values below about the 10 th percentile.
  • anti-PC levels such as IgM anti-PC levels
  • IgM anti-PC levels in a sample at or below about 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10 or less U/ml (wherein 1 U/ml is equal to about 100 ng of antibody per ml) may be particularly associated with an increased risk of developing a metabolic disease, such as PCOS, and/or as a marker of actually having an already developed metabolic disease.
  • an aspect of the invention is to provide a method of diagnosing the absence, presence and/or levels of antibodies, for example IgA, IgM or IgG antibodies, with reactivity towards PC and/or a PC-conjugate (that is, anti-PC antibodies) which factor is related to an increased or decreased risk of developing metabolic diseases, using a PC-conjugate and to the use of this information to determine whether an individual is at risk of developing metabolic diseases.
  • a PC-conjugate that is, anti-PC antibodies
  • a preferred method is an immunoassay.
  • the method may be used in assessing an individual's risk of developing or progression of metabolic diseases and/or may be used to monitor the efficacy of the treatment methods of the invention by active or passive immunisation, insofar as they are directed at increasing the anti-PC titre in an individual in order to effect prophylaxis, prevention and/or treatment of a metabolic disease.
  • the method of diagnosis will be performed on a sample, such as an ex vivo sample, taken from the test subject.
  • the sample may, for example, be an ex vivo serum sample or an ex vivo plasma sample.
  • the test subject will be human.
  • the human subject from which the test sample is taken may, for example, be aged at least 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85 of more years.
  • PCOS polycystic ovary syndrome
  • PCOS polycystic ovary syndrome
  • any method or composition described herein can be implemented with respect to any other method or composition described herein.
  • any embodiment discussed with respect to one aspect of the invention may be used in the context of any other aspect of the invention.
  • the term "about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. Alternatively, it may be used to signify a value that is ⁇ 20, 10, 5, 4, 3, 2, 1 or less than 1% of the stated value.
  • Figure 1 shows a graph illustration the IgM anti-PC levels in patients with PCOS and in healthy controls.
  • Figure 2 shows a graph illustrating the plasma levels of anti-PC after PC-BSA vaccination.
  • Figure 3 shows a graph illustrating the association between anti-PC levels and waist circumference.
  • PCOS was defined according to the Rotterdam criteria and two of the following three features had to be present for the PCOS diagnosis 1) oligomenorrhea with eight or fever menstruations in the previous 12 months or amenorrhea; 2) clinical and/or biochemical signs of hyperandrogenism such as testosterone > 2.7 nmol/l, elevated DHEAS, free androgen index > 5.0, or hirsutism (> 7 on the Ferriman and Gallway scale); 3) polycystic ovaries on ultrasound examination (> 12 follicles 2 to 9 mm in diameter and/or increased ovarian volume (> 10 ml)).
  • PCOS diagnosis also implied no evidence of thyroid disease, adrenocortical dysfunction or, hyperprolactinemia. All women with PCOS were premenopausal and were currently not using any hormonal compounds.
  • Body mass index (BMI) was calculated as the weight in kilogram (kg) divided by the square of height in meters.
  • Waist circumference (to the nearest 0.5 cm) was measured horizontally around the smallest circumference between the ribs and iliac crest.
  • Hip circumference (to the nearest 0.5 cm) was measured around the maximal girth of the hips. Seated blood pressure was taken (average of three readings) after five minutes rest. Ovarian morphology was assessed in each subject, including the control subjects, by transvaginal ultrasound.
  • Insulin, SHBG, and testosterone were analyzed on a Modular E170 (Roche Diagnostics, Mannheim, Germany).
  • the total coefficients of variation of the instrument were 1.6% at 7.0 mU/L for insulin, 1.5% at 43 nmol/L for SHBG, and 6.8% at 3.9 nmol/L for testosterone.
  • Triglycerides and glucose were analyzed on an Architect ci8200 (Abbott Laboratories, Abbott Park, IL, USA).
  • the total imprecision (coefficient of variation, CV) of the instrument were 1.1 % at 0.9 mmol/L for triglycerides, 1.0% at 4.4 mmol/L for glucose.
  • IgM anti-PC Detection of IgM anti-PC was performed with ELISA (CVDefine ® , Athera Biotechnologies AB, Sweden) according the manufacturer's instruction. Serum samples were tested in dilution 1 : 01 and antibody levels were expressed as arbitrary units (U/ml) calculated from a six-point calibrator curve containing 0, 6.25, 12.5, 25, 50, and 100 U/ml of IgM anti-PC. The between CV of the assay was 5.2% and the within CV was 1.9%.
  • Statistical methods were performed with ELISA (CVDefine ® , Athera Biotechnologies AB, Sweden) according the manufacturer's instruction. Serum samples were tested in dilution 1 : 01 and antibody levels were expressed as arbitrary units (U/ml) calculated from a six-point calibrator curve containing 0, 6.25, 12.5, 25, 50, and 100 U/ml of IgM anti-PC. The between CV of the assay was 5.2% and the within CV was 1.9%.
  • IgM anti-PC levels were also categorized according to the median level for all participants and the proportion of subjects with IgM anti-PC levels below the median level were compared between groups by Chi-Square test. Stepwise logistic regression analysis with below-median IgM anti-PC as outcome measure, and group (PCOS patients vs. control subjects), age, BMI, testosterone, and HOMA-IR as independent confounders was performed. Nagelkerke pseudo R 2 was used as a measure of the goodness of fit between the logistic regression models.
  • SPSS statistical package was used for all analyses (SPPS, Inc., Chicago, IL, USA). A p-value ⁇ 0.05 was considered statistically significant.
  • PCOS polycystic ovaries syndrome
  • Triglycerides mmol/L 1.26 ⁇ 0.81 ** 0.99 ⁇ 0.44
  • the median IgM anti-PC level in PCOS patients (68 U/ml, 32 - 283 U/ml) was not significantly different compared to control subjects (84 U/ml, 33 - 316 U/ml).
  • the proportion of PCOS patients with low IgM anti-PC levels defined as number of individuals below the median level (70.8 U/ml), was significantly higher than among healthy controls (65.3% of subjects with low values IgM anti-PC levels had PCOS vs. 34. 7% of the control subjects, p ⁇ 0.05).
  • the median age of PCOS patients and control subjects were in same range but the age distribution between the two groups differed slightly.
  • Model 1 The relation for low IgM anti-PC and PCOS, adjusted for age.
  • Model 2-4 Adjustments of Model 1 by adding the explanatory variables body mass index (BMI), testosterone levels, and homeostasis model assessment of insulin resistance index (HOMA-IR) in a stepwise fashion.
  • BMI body mass index
  • HOMA-IR homeostasis model assessment of insulin resistance index
  • IgM anti-PC As low levels of IgM anti-PC have been reported to be associated with both inflammatory responses involved in atherosclerosis (19) and increased risk for CVD (17-19), low levels of IgM anti-PC might be a pathway through which part of the increased risk for CVD reported among PCOS patients operate.
  • the generally lower levels of IgM anti-PC among the oldest PCOS patients might also support further investigations into the use of IgM anti-PC as a marker for risk of developing CVD in this specific group of women. Although direct comparisons of IgM anti-PC levels in-between studies may be limited, it was evident that our study population of relatively young and healthy women displayed higher median IgM anti-PC levels than previous studies have reported.
  • IgM anti-PC levels in our cohort are presumably attributed to the female study population, the relatively low age of our subjects and the fact that cardiovascular events are rare in premenopausal women (21).
  • IgM anti-PC levels decrease with increasing age (20) and are higher in women compared to men (16, 19).
  • the IgM anti-PC autoantibodies are suggested to have an atheroprotective function, by reducing development of atherosclerosis in mice and humans (14-16).
  • significantly higher anti- PC IgM levels were found as compared to a Swedish age- and sex-matched population (20).
  • Cibula D Cifkova R, Fanta M, Poledne R, Zivny J, Skibova J. Increased risk of non- insulin dependent diabetes mellitus, arterial hypertension and coronary artery disease in perimenopausal women with a history of the polycystic ovary syndrome. Hum Reprod 2000; 15(4):785-9.
  • Binder CJ Shaw PX, Chang MK, Boullier A, Hartvigsen K, Horkko S, Miller Yl, Woelkers DA, Corr M, Witztum JL. The role of natural antibodies in atherogenesis. J Lipid Res 2005;46(7): 1353-63.
  • PC-BSA will be added to alum (5 mg/ml in NaCI), and the mixture gently shaken for an hour before administration. 200 ⁇ _ will be injected subcutaneously in the neck at each occasion.
  • PC-BSA will be replaced by BSA, and in the controls, only NaCI will be administered. Starting when the mice were placed on the Western diet, they will receive injections every second week until termination of the experiment after 16 weeks on the diet. Fasting blood samples (4 hours fasting) will be taken from the tail vein or v saphena before the experiment starts and before each immunisation.
  • Fasting insulin F insu
  • FPG Fasting blood glucose
  • Insulin sensitivity will be estimated as HOMA-IR, obtained as the product of FPG and F lnsu ii n , or as elevated F insu ii n .
  • the active immunisation is expected to result in markedly elevated levels of IgM antibodies against PC, while levels in the other groups will be low. After approximately two weeks, antibody levels will begin to rise, and reach maximal levels after 6-8 weeks in actively immunized mice. After this they remain high. IgG antibodies against PC will appear after 6 to 10 weeks in the actively immunized mice, and remain low in the other groups. Although the methods used to measure the IgM and IgG antibodies are not quantitative, the levels of IgG anti-PC will appear to be lower than levels of the IgM anti-PC. As the PC was linked to BSA, we will also measure if antibodies towards BSA are formed.
  • mice actively immunized with PC-BSA will have better peripheral insulin sensitivity than the other groups, as shown by significantly lower increases in fasting insulin and HOMAir, without major differences in fasting glucose levels. Mice receiving BSA and adjuvant will not show any improvement in fasting insulin or glucose levels compared to saline treated animals.
  • mice fed an atherogenic diet a so called Western diet containing 21.2% fat and 0.15 % cholesterol
  • atherosclerosis develops rapidly.
  • these mice will show features of the metabolic syndrome (hypertension, hyperlipidemia, insulin resistance and obesity) as a consequence of the lipid laden diet (de Roos, B., G. Rucklidge, M. Reid, et al., Divergent mechanisms of cis9, trans11-and transW, cisl 2-conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach. FASEB J, 2005. 19(12): p.
  • mice were placed on the Western diet, they received injections every second week until termination of the experiment after 16 weeks on the diet.
  • Fasting blood samples (4 hours fasting) were taken from the tail vein or vena saphena before the experiment started and before each immunisation.
  • the levels of antibodies against PC were measured using the ELISA method as described above. Fasting insulin and glucose were measured before the mice were placed on the western diet, and after 16 weeks on the diet. Fasting blood glucose levels were measured using an Accu-Chek Compact glucometer (Roche Diagnostics Corp., Indianapolis; Indiana, USA), and insulin was determined by a ELISA kit specific for mouse insulin (Ultra Sensitive Mouse Insulin ELISA kit #90080, Crystal Chem Inc.).
  • Obesity is one of the components of the metabolic syndrome, and increased waist circumference is one of the symptoms of the metabolic syndrome.
  • CV cardiovascular
  • Figure 3 show that individuals in the decentile of subjects with the lowest IgM anti-PC also had a significantly higher waist circumference.
  • anti-PC levels roughly corresponding to the lowest decentile is also associated to an increased risk for premature CVD (Sjoberg, B.G., J. Su, I. Dahlbom, et al., Low levels of IgM antibodies against phosphorylcholine-A potential risk marker for ischemic stroke in men. Atherosclerosis, 2008. 203(2): p. 528-32).
  • no previous association between anti-PC levels and the metabolic syndrome has been observed.
  • the present findings indicate that increasing anti-PC levels in subjects with the metabolic syndrome and/or obesity is beneficial.

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Abstract

La présente invention concerne un procédé d'immunisation ou de prophylaxie contre, ou de traitement de maladies métaboliques chez un mammifère. La méthode consiste à administrer au mammifère une composition pharmaceutique comprenant au moins un conjugué de la phosphorylcholine (PC) ou une préparation d'anticorps réactive à la PC ou à un conjugué de la PC. La maladie métabolique peut être, par exemple, une affection choisie dans le groupe constitué par le syndrome métabolique, la résistance à l'insuline, l'intolérance au glucose, l'hyperglycémie, le diabète insulino-dépendant, le diabète non insulino-dépendant, l'hyperlipidémie, l'hypertriglycéridémie, l'hypercholestérolémie, la dyslipidémie, et le syndrome des ovaires polykystiques (SOPK). L'invention concerne également une méthode de diagnostic d'une maladie métabolique, ou d'évaluation du risque de développement ou de progression d'une maladie métabolique chez un patient, la méthode consistant à: a) évaluer chez le patient le taux des anticorps réactifs à la PC ou à un conjugué de la PC; et b) diagnostiquer une maladie métabolique ou déterminer chez le patient le niveau de risque de développement ou de progression d'une maladie métabolique, sur la base de l'évaluation du taux des anticorps réactifs à la PC ou à un conjugué de la PC.
PCT/EP2011/003604 2010-07-21 2011-07-15 Méthodes et compositions de diagnostic et de traitement d'une maladie métabolique WO2012010291A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013022968A1 (fr) 2011-08-09 2013-02-14 Athera Biotechnologies Ab Anticorps aptes à se lier à la phosphorylcholine (pc) et/ou à un conjugué de pc
WO2013020995A1 (fr) 2011-08-09 2013-02-14 Athera Biotechnologies Ab Nouveaux anticorps contre la phosphorylcholine
WO2021097379A1 (fr) * 2019-11-14 2021-05-20 The Regents Of The University Of California Méthodes et compositions permettant de déterminer des maladies et des troubles associés à oxpl
WO2023217787A1 (fr) 2022-05-10 2023-11-16 Inflavona Ab Compositions, procédés et utilisations

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013022968A1 (fr) 2011-08-09 2013-02-14 Athera Biotechnologies Ab Anticorps aptes à se lier à la phosphorylcholine (pc) et/ou à un conjugué de pc
WO2013020995A1 (fr) 2011-08-09 2013-02-14 Athera Biotechnologies Ab Nouveaux anticorps contre la phosphorylcholine
US9796786B2 (en) 2011-08-09 2017-10-24 Athera Biotechnologies Ab Antibodies binding to phosphorylcholine (PC) and/or PC conjugates
US9803028B2 (en) 2011-08-09 2017-10-31 Athera Biotechnologies Ab Antibodies against phosphorylcholine
WO2021097379A1 (fr) * 2019-11-14 2021-05-20 The Regents Of The University Of California Méthodes et compositions permettant de déterminer des maladies et des troubles associés à oxpl
WO2023217787A1 (fr) 2022-05-10 2023-11-16 Inflavona Ab Compositions, procédés et utilisations

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