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WO2010086382A1 - Cible pour le traitement d'une insuffisance cardiaque aiguë - Google Patents

Cible pour le traitement d'une insuffisance cardiaque aiguë Download PDF

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Publication number
WO2010086382A1
WO2010086382A1 PCT/EP2010/051019 EP2010051019W WO2010086382A1 WO 2010086382 A1 WO2010086382 A1 WO 2010086382A1 EP 2010051019 W EP2010051019 W EP 2010051019W WO 2010086382 A1 WO2010086382 A1 WO 2010086382A1
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WIPO (PCT)
Prior art keywords
quiescin
agent
ahf
protein
peptide
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English (en)
Inventor
Koen Kas
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Pronota NV
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Pronota NV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/03—Oxidoreductases acting on sulfur groups as donors (1.8) with oxygen as acceptor (1.8.3)
    • C12Y108/03002—Thiol oxidase (1.8.3.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

Definitions

  • the invention relates to new targets for treating diseases or conditions in subjects. More particularly, the application discloses new target for treating acute heart failure; methods for treating acute heart failure and methods for identifying agents modulating the activity or of said target for treatment of acute heart failure; and kits and devices for performing said methods.
  • Heart failure is a major public health issue in developed countries and is the cause of considerable morbidity and mortality among older adults. It is usually a chronic disease characterised by frequent recurrent decompensation leading to worsening breathing problems. Moreover, 5 years after diagnosis 50% of heart failure patients will have died from the disease.
  • Acute heart failure (AHF) is a sudden inability of the heart to pump efficiently and where it can no longer foresee the bodily demands for oxygen. AHF is the cause of over two million hospitalisations annually in US and Europe, and displays a mortality rate of about 20-40% within one year of hospital discharge in many populations. About 90% of AHF admissions are typically from patients with chronic heart disease, the remaining about 10% are de novo patients. The clinical signs of heart disease and AHF are often non-specific which can make unambiguous diagnosis demanding.
  • a common symptom of AHF is the shortness of breath (dyspnea or dyspnoea).
  • dyspnea or dyspnoea
  • a rapid, proper and effective treatment of AHF requires to adequately distinguish AHF patients from patients having dyspnea due to other causes.
  • BNP B-type natriuretic peptide
  • BNP levels vary with age, sex, weight and other medical conditions, thereby confounding the diagnosis. Notably, BNP levels tend to be elevated in patients with medical history of heart failure. For example, Chung et al. 2006 (supra) have shown that BNP performance for diagnosing AHF in patients presenting with dyspnea is significantly reduced in patients with a history of heart failure.
  • Such novel AHF biomarkers may be comparable to or improved over previously existing markers, such as over BNP, in one or more of their characteristics, such as, for example, in their sensitivity and/or specificity, in their reliability in patients presenting with a symptom potentially indicative of AHF such as with dyspnea, in their reliability in patients with history of heart disease, in their ability to discriminate AHF from chronic heart failure, etc.
  • the present invention addresses the above needs in the art by identifying a new target for treatment of AHF, and methods of identifying agents modulating the activity of said target for treatment of AHF.
  • Treatment of AHF using the Quiescin Q6 protein or gene as a target can be done in analogy to another known marker for AHF, Brain natriuretic peptide (BNP) has been shown to exert an important compensatory role in sustaining cardiac output in heart failure (for review see e.g. John C. Burnett in European Heart Journal Supplements, Volume 7, Suppl B, Pp. B25-B30).
  • Great elevation of plasma brain natriuretic peptide is observed in patients with acute congestive heart failure suggesting a compensatory mechanism of brain natriuretic peptide release in heart failure.
  • the present invention addresses the above needs in the art by identifying a new target for treatment of AHF, and methods of identifying agents modulating the activity of said target for treatment of AHF.
  • the polypeptide Quiescin Q6 represents a newly realised biomarker particularly advantageous for predicting, diagnosing and/or prognosticating acute heart failure (AHF).
  • the Applicant has first identified and subsequently validated Quiescin Q6 as a biomarker displaying a significantly altered level in dyspneic patients having AHF, when compared to dyspneic patients not having AHF, CHF patients or healthy controls.
  • Quiescin Q6 may be a useful biomarker for monitoring the progression of AHF, since the amount of Quiescin Q6 significantly differed between dyspneic AHF patients upon admission (i.e., before treatment) and upon discharge (i.e., following treatment).
  • Quiescin Q6 can outperform BNP and NT-proBNP in a number of relevant respects.
  • the AUC value (area under the ROC curve; "ROC” stands for receiver operating characteristic) is higher for Quiescin Q6 (0.89) than for each one of BNP (0.84) and NT-proBNP (0.80).
  • the AUC value is a combined measure of sensitivity and specificity and a higher AUC value (i.e., approaching 1 ) in general indicates an improved performance of the test.
  • the AUC value for Quiescin Q6 (0.83) is substantially higher than that for BNP (0.65) and NT-proBNP (0.72).
  • the Quiescin Q6 test performs more reliably in patients with a history of heart failure.
  • the Applicant has identified and validated Quiescin Q6 as a further and improved biomarker for predicting, diagnosing and/or prognosticating AHF and consequently as a promising therapeutic target for treatment of AHF.
  • the invention provides an agent that is able to modulate the level and/or the activity of Quiescin 6, for use as a medicament and, more preferably, for use in treating acute heart failure (AHF).
  • said agent is able to increase the level and/or the activity of Quiescin 6.
  • the agent for use according to the invention is able to specifically bind to Quiescin 6, or to its gene promotor.
  • the agent for use according to the invention preferably is an antibody or a fragment or derivative thereof; a polypeptide; a peptide; a peptidomimetic; an aptamer; or a chemical substance, preferably an organic molecule, more preferably a small organic molecule.
  • the agent for use according to the invention is able to increase the expression of Quiescin 6, preferably wherein said agent is a recombinant nucleic acid encoding Quiescin 6 or a functional variant or functional fragment thereof.
  • the agent for use according to the invention able to increase the expression of Quiescin 6 can be a transcription factor or an agent increasing the endogenous expression of the Quiescin Q6 gene.
  • the agent for use according to the invention is an activator of Quiescin 6, preferably chosen from the group consisting of FAD-linked sulfhydryl oxidase activators, chaperones or cofactors, oxidoreductase activators, chaperones or cofactors.
  • the agent for use according to the invention is a recombinant or isolated Quiescin 6 polypeptide, a functional variant, a functional fragment, or a constitutively active fragment thereof.
  • the agent for use according to the invention may be an inhibitor of proteolysis and/or degradation of Quiescin Q6.
  • the agent for use according to the invention may enhance the stability of Quiescin Q6.
  • the invention provides for an assay to select, from a group of test agents, a candidate agent potentially useful in the treatment of AHF, said assay comprising determining whether a tested agent can increase the level and/or activity of Quiescin 6.
  • agents that bind to the promoter of the Quiescin Q6 gene are screened.
  • agents that increase or mimic the enzymatic activity of the Quiescin Q6 protein are screened.
  • the assay of the invention preferably further comprises the use of the selected candidate agent for the preparation of a composition for administration to and monitoring the prophylactic and/or therapeutic effect thereof in a non-human animal model, preferably a non- human mammal model, of AHF.
  • the invention further provides for the agents and or compounds identified or isolated by the assays according to the invention.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a prophylactically and/or therapeutically effective amount of one or more agents as defined herein, or a pharmaceutically acceptable ⁇ /-oxide form, addition salt, prodrug or solvate thereof, and further comprising one or more of pharmaceutically acceptable carriers.
  • the invention also provides for a method of treatment of a patient suffering from acute heart failure comprising the step of administering to said patient a therapeutically effective amount of one or more agents as defined herein, or a pharmaceutically acceptable ⁇ /-oxide form, addition salt, prodrug or solvate thereof.
  • the invention provides a method of inducing cardioprotection in an individual in need of such treatment, comprising administering a pharmacologically effective of one or more agents as defined herein, or a pharmaceutically acceptable ⁇ /-oxide form, addition salt, prodrug or solvate thereof.
  • the denotations prot22 and Pro-M22 refer to Quiescin Q6.
  • Figure 1 illustrates sequences of isoforms 1 (A) and 2 (B) of Quiescin Q6.
  • Figure 2 illustrates the differences between isoforms 1 and 2 of Quiescin Q6.
  • the C-terminal portion missing in isoform 2 is indicated in small letters.
  • MASStermind discovered peptides pept10012 - bold, underlined with full line: SEQ ID NO: 7; pept10014 - bold, underlined with pointed line: SEQ ID NO: 8; pept10055 - bold, underlined with dashed line: SEQ ID NO: 9) and the selected MASSterclass quantified peptide (pept110 - bold, double underlined: SEQ ID NO: 10).
  • MASStermind and MASSterclass peptides can quantify both isoforms of Quiescin Q6.
  • Figure 3 illustrates sequences of preproBNP and peptides derived there from.
  • FIG 4 provides a schematic overview of the acute heart failure discovery experimental setup.
  • Protein profiles of populations A AHF on admission to emergency department (ED)
  • B standard patients at discharge
  • H healthy controls
  • Quantitation in populations C chronic heart failure
  • D Diaspnea patients without HF on admission
  • Figure 5 shows MASStermind discovery results for Quiescin Q6. Boxplots show the relative peptide levels measured according to the MASStermind reference for the 3 analyzed populations for 3 different pro-M22 specific peptides detected using MASStermind.
  • Figure 6 shows absolute levels of BNP as measured by ELISA in populations A, C, D and H (left panel), and corresponding MASSterclass quantitation of Quiescin Q6 pept110 (right panel).
  • FIG. 7 illustrates that Quiescin Q6 shows superior performance than B-type natriuretic peptides in discriminating AHF from dyspneic non-acute heart failure patients.
  • A Receiver operating characteristic curve of BNP compared to Quiescin Q6 and NT-proBNP compared to Quiescin Q6 respectively for diagnosis of heart failure cause of dyspnea in the ED. Calculated area under the curve (AUC) and 95% confidence intervals are given in the table.
  • AUC area under the curve
  • B Accuracy plot for BNP and Quiescin Q6 with calculated sensitivity and specificity at different cut-off concentrations for diagnosis of AHF in dyspneic patients in the ED.
  • Figure 8 shows ROC curve of BNP compared to Quiescin Q6 and NT-proBNP compared to Quiescin Q6 respectively for discriminating acute decompensated heart failure from chronic stable heart failure patients.
  • the present invention derives from the highly innovative realisation of the Applicant that Quiescin Q6 is a valuable biomarker and therapeutic target particularly for acute heart failure (AHF).
  • biomarker is widespread in the art and may broadly denote a biological molecule and/or a detectable portion thereof whose qualitative and/or quantitative evaluation in a subject is predictive or informative (e.g., predictive, diagnostic and/or prognostic) with respect to one or more aspects of the subject's phenotype and/or genotype, such as, for example, with respect to the status of the subject as to a given disease or condition.
  • heart failure means of further guidance, the term “heart failure” as used herein broadly refers to pathological conditions characterised by an impaired diastolic or systolic blood flow rate and thus insufficient blood flow from the ventricle to peripheral organs.
  • heart failure means of further guidance, the term “heart failure” as used herein broadly refers to pathological conditions characterised by an impaired diastolic or systolic blood flow rate and thus insufficient blood flow from the ventricle to peripheral organs.
  • Acute heart failure or also termed “acute decompensated heart failure” may be defined as the rapid onset of symptoms and signs secondary to abnormal cardiac function, resulting in the need for urgent therapy.
  • AHF can present itself acute de novo (new onset of acute heart failure in a patient without previously known cardiac dysfunction) or as acute decompensation of CHF.
  • the cardiac dysfunction may be related to systolic or diastolic dysfunction, to abnormalities in cardiac rhythm, or to preload and afterload mismatch. It is often life threatening and requires urgent treatment.
  • AHF includes several distinct clinical conditions of presenting patients: (I) acute decompensated congestive heart failure, (II) AHF with hypertension/hypertensive crisis, (III) AHF with pulmonary oedema, (IVa) cardiogenic shock / low output syndrome, (IVb) severe cardiogenic shock, (V) high output failure, and (Vl) right-sided acute heart failure.
  • chronic heart failure or “congestive heart failure” may generally refer to a case of heart failure that progresses so slowly that various compensatory mechanisms work to bring the disease into equilibrium.
  • Common clinical symptoms of CHF include inter alia any one or more of breathlessness, diminishing exercise capacity, fatigue, lethargy and peripheral oedema. Other less common symptoms include any one or more of palpitations, memory or sleep disturbance and confusion, and usually co-occur with one or more of the above recited common symptoms.
  • CHF population may differ from the AHF population in that CHF patients do not have an acute decompensation and hence do not represent themselves to the ED at the time the clinical sample used in such a study or research is taken.
  • Chronic heart failure patients may, however, easily decompensate leading to "acute heart failure".
  • a population of dyspneic patients without heart failure may comprise for example patients who present themselves to the ED with similar symptoms as AHF population but where the cause of dyspnea is unrelated to acute decompensated heart failure.
  • Typical examples are COPD or pneumonia patients.
  • Such patients may or may not have underlying heart failure history, which may particularly complicate the final diagnosis using conventional diagnostic means.
  • subject typically denotes humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, more preferably mammals, such as, e.g., non-human primates, rodents, canines, felines, equines, ovines, porcines, and the like, in suffering or at risk of suffering from acute heart failure.
  • the subjects may be at risk of having AHF or having or being at risk of having chronic heart failure (CHF).
  • CHF chronic heart failure
  • the subject may suffer from one or more symptoms and/or signs potentially indicative of AHF.
  • the subject may have dyspnea due to AHF.
  • the subject may have a medical history of heart failure, such as, for example AHF and/or CHF.
  • the subject may have a renal failure or may display one or more risk factors for AHF, such as, for example, a genetic predisposition or one or more developmental, environmental or behavioural risk factors, such as, e.g., insulin resistance (impaired blood glucose), truncal obesity, high serum low density lipoprotein (LDL) cholesterol levels, low serum high density lipoprotein (HDL) cholesterol levels, high serum triglyceride levels, and high blood pressure (hypertension).
  • AHF blood glucose
  • LDL high serum low density lipoprotein
  • HDL low serum high density lipoprotein
  • high serum triglyceride levels high blood pressure (hypertension).
  • Quiescin Q6 and “Sulfhydryl oxidase 1” are synonymous and refer to proteins and polypeptides commonly known under these designations in the art.
  • the terms encompass such proteins and polypeptides of any organism where found, and particularly of animals, preferably vertebrates, more preferably mammals, including humans and non-human mammals, even more preferably of humans.
  • the terms particularly encompass such proteins and polypeptides with a native sequence, i.e., ones of which the primary sequence is the same as that of Quiescin Q6 found in or derived from nature.
  • native sequences of Quiescin Q6 may differ between different species due to genetic divergence between such species.
  • the native sequences of Quiescin Q6 may differ between or within different individuals of the same species due to normal genetic diversity (variation) within a given species.
  • the native sequences of Quiescin Q6 may differ between or even within different individuals of the same species due to post-transcriptional or post-translational modifications. Accordingly, all Quiescin Q6 sequences found in or derived from nature are considered "native".
  • the terms encompass Quiescin Q6 proteins and polypeptides when forming a part of a living organism, organ, tissue or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources.
  • the terms also encompass proteins and polypeptides when produced by recombinant or synthetic means.
  • Exemplary Quiescin Q6 includes, without limitation, human Quiescin Q6 having primary amino acid sequence as annotated under Uniprot/Swissprot (http://www.expasy.org/) accession number 000391 (entry version 69 revised on January 20, 2009; sequence version 3 created on June 1 , 2001 ), including isoform 1 (ace. no. 000391 -1 ) and isoform 2 (000391- 2) generated due to alternative splicing.
  • the sequence of said isoforms 1 and 2 of Quiescin Q6 is shown in Fig. 1A (SEQ ID NO: 1 ) and Fig. 1B (SEQ ID NO: 2), respectively.
  • Figure 2 illustrates the differences in the C-terminal region between said isoforms 1 and 2.
  • sequences are of precursor of Quiescin Q6 and may include parts which are processed away from mature Quiescin Q6.
  • the Uniprot/Swissprot entry specifies a signal peptide composed of amino acids 1-29.
  • Exemplary human Quiescin Q6 has been also described inter alia by Coppock et al. 1998 (Genomics 54: 460-468).
  • the reference herein to Quiescin Q6 may also encompass fragments of Quiescin Q6.
  • the reference herein to measuring Quiescin Q6, or to measuring the quantity of Quiescin Q6, may encompass measuring the Quiescin Q6 protein or polypeptide (such as, e.g., measuring the mature isoform 1 and/or isoform 2 of Quiescin Q6) and/or measuring one or more fragments of Quiescin Q6.
  • Quiescin Q6 and/or one or more fragments thereof may be measured collectively, such that the measured quantity corresponds to the sum amounts of the collectively measured species.
  • Quiescin Q6 and/or one or more fragments thereof may be measured each individually.
  • Quiescin Q6 and fragments thereof may generally also encompass modified forms of said Quiescin Q6 and fragments such as bearing post-expression modifications including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, and the like.
  • fragment of a protein, polypeptide or peptide generally refers to N-terminally and/or C-terminally deleted or truncated forms of said protein, polypeptide or peptide.
  • the term encompasses fragments arising by any mechanism, such as, without limitation, by alternative translation, exo- and/or endo-proteolysis and/or degradation of said protein or polypeptide, such as, for example, in vivo or in vitro, such as, for example, by physical, chemical and/or enzymatic proteolysis.
  • a fragment of a protein, polypeptide or peptide may represent at least about 5%, or at least about 10%, e.g., > 20%, > 30% or > 40%, such as > 50%, e.g., > 60%, > 70% or > 80%, or even > 90% or > 95% of the amino acid sequence of said protein, polypeptide or peptide.
  • a fragment of Quiescin Q6 may include a sequence of > 5 consecutive amino acids, or > 10 consecutive amino acids, or > 20 consecutive amino acids, or > 30 consecutive amino acids, e.g., >40 consecutive amino acids, such as for example > 50 consecutive amino acids, e.g., > 60, > 70, > 80, > 90, > 100, > 200, > 300, > 400, > 500 or > 600 consecutive amino acids of Quiescin Q6.
  • a fragment of Quiescin Q6 may be N-terminally and/or C-terminally truncated by between 1 and about 20 amino acids, such as, e.g., by between 1 and about 15 amino acids, or by between 1 and about 10 amino acids, or by between 1 and about 5 amino acids, compared to mature, full-length Quiescin Q6 (e.g., isoforms 1 or 2).
  • a fragment of proBNP, NTproBNP or BNP may be N-terminally and/or C- terminally truncated by between 1 and about 20 amino acids, such as, e.g., by between 1 and about 15 amino acids, or by between 1 and about 10 amino acids, or by between 1 and about 5 amino acids, compared to proBNP, NTproBNP or BNP.
  • proBNP, NTproBNP and BNP fragments useful as biomarkers are disclosed in WO 2004/094460.
  • fragments of a given protein, polypeptide or peptide may be achieved by in vitro proteolysis of said protein, polypeptide or peptide to obtain advantageously detectable peptide(s) from a sample.
  • proteolysis may be effected by suitable physical, chemical and/or enzymatic agents, e.g., proteinases, preferably endoproteinases, i.e., protease cleaving internally within a protein, polypeptide or peptide chain.
  • suitable endoproteinases includes serine proteinases (EC 3.4.21 ), threonine proteinases (EC 3.4.25), cysteine proteinases (EC 3.4.22), aspartic acid proteinases (EC 3.4.23), metalloproteinases (EC 3.4.24) and glutamic acid proteinases.
  • Exemplary non-limiting endoproteinases include trypsin, chymotrypsin, elastase, Lysobacter enzymogenes endoproteinase Lys-C, Staphylococcus aureus endoproteinase GIu-C (endopeptidase V8) or Clostridium histolyticum endoproteinase Arg-C (clostripain). Further known or yet to be identified enzymes may be used; a skilled person can choose suitable protease(s) on the basis of their cleavage specificity and frequency to achieve desired peptide forms.
  • the proteolysis may be effected by endopeptidases of the trypsin type (EC 3.4.21.4), preferably trypsin, such as, without limitation, preparations of trypsin from bovine pancreas, human pancreas, porcine pancreas, recombinant trypsin, Lys-acetylated trypsin, trypsin in solution, trypsin immobilised to a solid support, etc. Trypsin is particularly useful, inter alia due to high specificity and efficiency of cleavage.
  • the invention also contemplates the use of any trypsin-like protease, i.e., with a similar specificity to that of trypsin. Otherwise, chemical reagents may be used for proteolysis. For example, CNBr can cleave at Met; BNPS-skatole can cleave at Trp.
  • the conditions for treatment e.g., protein concentration, enzyme or chemical reagent concentration, pH, buffer, temperature, time, can be determined by the skilled person depending on the enzyme or chemical reagent employed.
  • variant of a protein or polypeptide such as Quiescin Q6 refers to proteins and polypeptides the amino acid sequence of which is substantially identical (i.e., largely but not wholly identical) to a native sequence of said protein or polypeptide such as Quiescin Q6.
  • Substantially identical refers to at least about 85% identical, e.g., preferably at least 90% identical, e.g., at least 91 % identical, 92% identical, more preferably at least 93% identical, e.g., 94% identical, even more preferably at least 95% identical, e.g., at least 96% identical, yet more preferably at least 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical.
  • the length of an alignment window in which the variant shows the above sequence identity with Quiescin Q6 may correspond to the entire Quiescin Q6 protein sequence (i.e., overall sequence identity) or may refer to sequence identity in an alignment window corresponding to at least 20%, or at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, yet more preferably at least 70%, very preferably at least 80%, and still more preferably at least 90% or at least 95% of the entire sequence of Quiescin Q6; such as, e.g., the length of the alignment window may be > 50 consecutive amino acids, e.g., > 60, > 70, > 80, > 90, > 100, > 200, > 300, > 400, > 500 or > 600 consecutive amino acids of Quiescin Q6.
  • Sequence identity between two polypeptides can be determined by optimally aligning (optimal alignment of two protein sequences is the alignment that maximises the sum of pair-scores less any penalty for introduced gaps; and may be preferably conducted by computerised implementations of algorithms, such as "Gap”, using the algorithm of Needleman and Wunsch 1970 (J MoI Biol 48: 443-453), or "Bestfit”, using the algorithm of Smith and Waterman 1981 (J MoI Biol 147: 195—197), as available in, e.g., the GCGTM v.
  • binding agents capable of specifically binding to any one or more of the isolated fragments of Quiescin Q6 as taught herein. Further provided are binding agents capable of specifically binding to only one of the isolated fragments of Quiescin Q6 as taught herein.
  • binding agents may include inter alia an antibody, aptamer, photoaptamer, protein, peptide, peptidomimetic or a small molecule.
  • telomere binding agent binds to one or more desired molecules or analytes, such as to one or more proteins, polypeptides or peptides of interest or fragments thereof substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related.
  • an agent may be said to specifically bind to protein(s) polypeptide(s), peptide(s) and/or fragment(s) thereof of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold or more greater, than its affinity for a non-target molecule.
  • Specific-binding agents as used throughout this specification may include inter alia an antibody, aptamer, photoaptamer, protein, peptide, peptidomimetic or a small molecule.
  • antibody is used in its broadest sense and generally refers to any immunologic binding agent.
  • the term specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., 2-, 3- or more-valent) and/or multi-specific antibodies (e.g., bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest), as well as multivalent and/or multi-specific composites of such fragments.
  • antibody is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one complementarity-determining region (CDR) capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro or in vivo.
  • CDR complementarity-determining region
  • an antibody may be any of IgA, IgD, IgE, IgG and IgM classes, and preferably IgG class antibody.
  • the antibody may be a polyclonal antibody, e.g., an antiserum or immunoglobulins purified there from (e.g., affinity-purified).
  • the antibody may be a monoclonal antibody or a mixture of monoclonal antibodies.
  • Monoclonal antibodies can target a particular antigen or a particular epitope within an antigen with greater selectivity and reproducibility.
  • monoclonal antibodies may be made by the hybridoma method first described by Kohler et al. 1975 (Nature 256: 495), or may be made by recombinant DNA methods (e.g., as in US 4,816,567).
  • Monoclonal antibodies may also be isolated from phage antibody libraries using techniques as described by Clackson et al. 1991 (Nature 352: 624-628) and Marks et al.
  • the antibody binding agents may be antibody fragments.
  • Antibody fragments comprise a portion of an intact antibody, comprising the antigen-binding or variable region thereof.
  • Examples of antibody fragments include Fab, Fab', F(ab')2, Fv and scFv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multivalent and/or multispecific antibodies formed from antibody fragment(s), e.g., dibodies, tribodies, and multibodies.
  • the above designations Fab, Fab', F(ab')2, Fv, scFv etc. are intended to have their art-established meaning.
  • antibody includes antibodies originating from or comprising one or more portions derived from any animal species, preferably vertebrate species, including, e.g., birds and mammals.
  • the antibodies may be chicken, turkey, goose, duck, guinea fowl, quail or pheasant.
  • the antibodies may be human, murine (e.g., mouse, rat, etc.), donkey, rabbit, goat, sheep, guinea pig, camel (e.g., Camelus bact ⁇ anus and Camelus dromaderius), llama (e.g., Lama paccos, Lama glama or Lama vicugna) or horse.
  • an antibody can include one or more amino acid deletions, additions and/or substitutions (e.g., conservative substitutions), insofar such alterations preserve its binding of the respective antigen.
  • An antibody may also include one or more native or artificial modifications of its constituent amino acid residues (e.g., glycosylation, etc.).
  • aptamer refers to single-stranded or double-stranded oligo-DNA, oligo-RNA or oligo-DNA/RNA or any analogue thereof, that can specifically bind to a target molecule such as a peptide.
  • aptamers can display fairly high specificity and affinity (e.g., K A in the order 1x10 9 M "1 ) for their targets.
  • photoaptamer refers to an aptamer that contains one or more photoreactive functional groups that can covalently bind to or crosslink with a target molecule.
  • peptidomimetic refers to a non-peptide agent that is a topological analogue of a corresponding peptide.
  • Preferred small organic molecules range in size up to about 5000 Da, e.g., up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g., up to about 900, 800, 700, 600 or up to about 500 Da.
  • an elevated quantity of Quiescin Q6 in the sample from the subject compared to a reference value representing the prediction or diagnosis of no AHF or representing a good prognosis for AHF indicates that the subject has or is at risk of having AHF or indicates a poor prognosis for AHF in the subject.
  • the Applicant has also observed and verified that methods using Quiescin Q6 as a biomarker, and particularly but without limitation the methods for discriminating between the dyspneic patients with and without AHF, can achieve sensitivity of 80% or more and/or specificity of 80% or more.
  • the sensitivity and/or specificity (and preferably, the sensitivity and specificity) of the methods is at least 80%, e.g., > 81 %, > 82%, > 83%, > 84%, > 85%, > 86%, or > 87%, or > 90% or >95%, e.g., between 80% and 100%, or between 81 % and 95%, or between 83% and 90%, or between 84% and 89%, or between 85% and 88%.
  • fragments and/or variants of a given protein or polypeptide such as Quiescin Q6 denotes that such fragments and variants at least partly retain the biological activity or functionality of said corresponding protein or polypeptide.
  • functional fragments and/or variants may retain at least about 20%, e.g., at least 30%, or at least 40%, or at least 50%, e.g., at least 60%, more preferably at least 70%, e.g., at least 80%, yet more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95% or even 100% of the activity of the corresponding protein or polypeptide.
  • Such functional fragments and/or variants may even have higher activity than the corresponding protein or polypeptide.
  • Functional fragments and/or variants of a given protein or polypeptide such as Quiescin Q6 may be functionally equivalent to said polypeptide in at least one and preferably more or all aspects of its biological activity or function. Relevant aspects of biological function of Quiescin Q6 are explained below.
  • the biological activity of a given fragment and/or variant of Quiescin Q6 can be determined by standard tests, such as for example the enzyme activity tests.
  • quiescin Q6 family was created by the fusion of the flavin- linked sulfhydryl oxidase fragment of the yeast essential for respiration and vegetative growth (ERV)I prototype (an orthologue of hepatopoietin (HPO)) and thioredoxin (TRX)/disulfide isomerase domain during evolution (Hoober et al., J Biol Chem. 1999 Nov 5;274(45):31759- 62).
  • the flavin-linked sulfhydryl oxidase activity domain is involved in catalysis of the formation of disulfide bridges in several proteins.
  • Many secreted proteins contain disulfide bonds, which are required for their proper folding, function and stability.
  • disulfide bond formation usually occurs while the protein folds in the lumen of the ER (endoplasmic reticulum). While disulfide bonds can be formed spontaneously in vitro, an intermediary, such as a transition metal or flavin, is required to overcome this kinetically sluggish reaction.
  • the thioredoxin domain has an oxidoreductase activity, i.e. catalysis of an oxidation-reduction (redox) reaction, a reversible chemical reaction in which the oxidation state of an atom or atoms within a molecule is altered.
  • redox oxidation-reduction
  • One substrate acts as a hydrogen or electron donor and becomes oxidized, while the other acts as hydrogen or electron acceptor and becomes reduced.
  • Augmenter of Liver Regeneration is an important secondary hepatic growth factor, shown to be critically important for the survival of hepatocytes by its association with mitochondria and regulation of ATP synthesis (Thirunavukkarasu et al., J. Hepatol. 2008 Apr;48(4):578-88).
  • Augmenter of liver regeneration protein has been shown to control mitochondrial gene expression and the lytic activity of liver- resident Natural Killer cells through the levels of interferon-gamma, but the precise enzymatic function of this protein is unknown.
  • rat and human augmenter of liver regeneration protein are flavin-linked sulfhydryl oxidases that catalyze the formation of disulfide bonds in reduced protein substrates.
  • a flavin moiety is firmly but not covalently attached to the protein.
  • augmenter of liver regeneration protein is expressed in a long and short form that both exist as covalently linked dimers.
  • the active site of the enzyme is associated with a conserved CXXC motif in the carboxy-terminal domain, that is present in the homologous proteins from yeast to humans and also in the human quiescin Q6 growth regulator protein.
  • Erv2p is a FAD-binding QSOX (sulfhydryl oxidase) and belongs to the ERV (essential for respiration and vegetative growth)/ALR (augmenter of liver regeneration) family of proteins. Proteins containing an Erv2p homologous domain, known as the QSOX, have been detected in all multicellular plants and animals for which complete genome sequences exist.
  • the longer protein has a potential C-terminal transmembrane domain which has been spliced out to generate the short form of the enzyme.
  • a second gene was identified as sharing similarity with hQSOXI and encoding another member of the QSOX family.
  • the corresponding protein, SOXN or hQSOX2 has been studied in human neuroblastoma cells. All members of the QSOX family have a thioredoxin domain fused to their C-terminal Erv2p homologous domain.
  • a second more weakly scoring thioredoxin domain lacking any redox-active disulfides adjacent to the first thioredoxin domain has been identified in human and avian QSOX sequences.
  • hQSOXI a contains a putative N-terminal signal peptide, a conserved N-glycosylation site and a C-terminal transmembrane domain. It was shown that, when expressed in mammalian cells, the longer version of human QSOX1 protein (hQSOXIa) is a transmembrane protein localized primarily to the Golgi apparatus.
  • hQSOXIa can act in vivo as an oxidase.
  • Overexpression of hQSOXIa suppresses the lethality of a complete deletion of ERO1 (endoplasmic reticulum oxidase 1 ) in yeast and restores disulfide bond formation, as assayed by the folding of the secretory protein carboxypeptidase Y.
  • Candidate substrates for QSOX enzymes would be components of the extracellular matrix which are typically of a high molecular mass containing several disulfide bonds which mature at later stages of the secretory pathway, often forming higher order structures stabilized by disulfide bonds.
  • One characteristic of the disulfides that would be formed by QSOX in these proteins is that they would be either between separate polypeptide chains or between already folded protein domains. Hence the chances of formation of the incorrect disulfide bonds would be minimal and therefore so would be the need for an isomerase.
  • hQSOXIa may be involved in the formation of disulfide bonds within the cell or on the cell surface
  • the shorter QSOX1 form (hQSOXI b) may function as a secreted oxidase to counteract the effects of extracellular reducing agents.
  • the overall homology between the QSOX2 and the QSOX1 proteins is -40% with 68% identity in functional regions such as the thioredoxin-like or the ERV-1 domain. While initial studies indicate that hQSOX2 plays an important role in inducing apoptosis in neuroblastoma cells, it will be interesting to determine whether it can also act as an oxidase.
  • the inventors show here for the first time that the Quiescin Q6 protein level is increased in patients suffering from acute heart failure when compared to subjects suffering from chronic heart failure, dyspnea without heart failure and healthy subjects, and that targeting AHF and in particular increasing the level and/or activity is beneficial in AHF.
  • Drug screening assays of the invention show here for the first time that the Quiescin Q6 protein level is increased in patients suffering from acute heart failure when compared to subjects suffering from chronic heart failure, dyspnea without heart failure and healthy subjects, and that targeting AHF and in particular increasing the level and/or activity is beneficial in AHF.
  • the invention provides means, assays and methods to screen for drugs, lead compounds or agents that can increase the level (e.g., increase the expression, translation, and maturation level of the protein, increased the stability thereof, reduce its degradation, etc.), or that increase its activity in any possible way.
  • the invention thus provides means, assays and methods to screen for agents that can modulate the expression, translation, and maturation level of the protein, or that modulate its activity in any possible way.
  • the invention further provides the use of the Quiescin Q6 protein or gene to screen for agents that can modulate the expression, translation, and maturation level of the protein, or that modulate its activity in any possible way.
  • the invention further provides the use of the Quiescin Q6 protein or gene to screen for agents that can increase the expression, translation, and maturation level of the protein, or that increase its activity in any possible way.
  • the invention further provides compounds, agents, activators or agonists identified by any one of the assays defined herein and the use of said compounds, agents, activators or agonists for treating AHF in a subject.
  • the term "compound” or “agonist” means any molecular entity of natural, semi-synthetic or synthetic origin that either activates the Quiescin Q6 protein or mimicks its activity or increases its expression or presence.
  • molecular entities are small molecules, organic or inorganic compounds, peptides, proteins, enzymes, ligands, cofactors, transcription factors and the like.
  • Examples of possible compounds or agonists of the invention are plasmids encoding Quiescin Q6 peptides as defined herein, Quiescin Q6 polypeptides or fragments thereof as defined herein, transcription factors targeting the promoter of the endogenous Quiescin Q6 gene, interaction partner(s) of the Quiescin Q6 protein, aiding in its activity (e.g. chaperones or co-factors), activating proteins (e.g. involved in protein maturation) or ligands, agents mimicking the activity of the Quiescin Q6 protein and the like.
  • a typical assay for identifying putative therapeutic agents for treating AHF comprises the steps of: a) providing a cell or cell-line expressing or overexpressing the Quiescin Q6 protein b) contacting said cell with a candidate agent c) measuring the binding of the candidate agent to the Quiescin Q6 protein, wherein a candidate agent binding to the Quiescin Q6 protein is a putative therapeutic agent for treating AHF.
  • Another assay for identifying putative therapeutic agents for treating AHF comprises the steps of: a) providing a cell or cell-line expressing or overexpressing the Quiescin Q6 protein b) measuring the enzymatic activity of said Quiescin Q6 protein, b) contacting said cell with a candidate agent c) measuring the effect of the candidate agent on the activity of the Quiescin Q6 protein, wherein the candidate agent activates or increases the activity of the Quiescin Q6 protein if the activity in step c) is higher than the activity in step b).
  • oxidoreductase activity measuring assays or sulfhydryl oxidase activity measuring assays known in the art can be used.
  • Non-limiting example are the assays as used by Hoober et al., (J Biol Chem. 1999 Nov 5;274(45):31759-62) and Chakravarthi et al., (Biochem J. 2007 June 15; 404(Pt 3): 403-411 ).
  • Another exemplary system can be based on the use of human breast cancer MCF-7 cell lines overexpressing the QSOX1 protein, which are submitted to apoptosis inducing treatments.
  • the Quiescin Q6 overexpressing MCF-7 cells have been shown to be less sensitive to cell death than the MCF-7 control cells (Moral et al., Exp. Cell Res. 2007, Nov. 15;313(19):3971- 82).
  • a typical assay would then be: a) provide MCF-7 cells or other suitable tumor cell-lines, as a reference b) provide MCF-7 cells or other suitable tumor cell-lines overexpressing Quiescin Q6, c) inducing apoptosis through oxidative stress in cell populations of a) and b) in the presence of a candidate test agent d) inducing apoptosis through oxidative stress in cell populations of a) and b) in the absence of a candidate test agent, and e) comparing the effect of the test agent on apoptosis protection in MCF-7 cells overexpressing Quiescin Q6, wherein a test agent is activating or increasing the Quiescin Q6 activity when the apoptosis protective effect is increased when the candidate agent is added.
  • MCF-7 cells In stead of MCF-7 cells, other cell-lines may be used. Examples are tumor cell-lines and CHO cells and PC12 cells could be used.
  • Quiescin Q6 peptide formulations Quiescin Q6 polypeptides or peptides of the invention are as defined above. They can be recombinantly made or synthetically made.
  • the Quiescin Q6 peptide used in the medicament is full-length Quiescin Q6 protein; in other cases, the Quiescin Q6 peptide may be the mature processed fragment of Quiescin Q6, or a fragment of Quiescin Q6 retaining at least 50, 60, 70, 80, 90 or 95% of the activity of the mature Quiescin Q6 protein.
  • Quiescin Q6 peptide used in the medicament can be a truncated constitutively active form of the Quiescin Q6 protein.
  • the medicament can be formulated for continuous intravenous administration over a time period of at least 24 hours, more preferably from 24 hours to 120 hours.
  • the medicament is formulated for a sustained release of the Quiescin Q6 peptide over a period of at least 24 hours.
  • the time period for administration may be 24 to 72 hours, 48 to 72 hours.
  • the administration of the Quiescin Q6 peptide- containing medicament may last 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 96 hours, 120 hours, or any desirable time duration within this range.
  • the medicament is administered in a manner such that the patient is receiving the active ingredient (e.g., Quiescin Q6 peptide as defined herein) at a rate of at least
  • the administration rate is 15 ng/(kg.min.), 30 ng/(kg.min.), or 1 mg/(kg.min.).
  • Quiescin Q6 peptide is administered at the rate of 15 ng/(kg.min.).
  • one or more different cardiac medicines is also administered to the patient. These one or more different cardiac medicines may be given to the patient in the same composition as the Quiescin Q6 peptide, or may be given to the patient in a separate composition.
  • the medicament often further comprises a pharmaceutically acceptable excipient or carrier, such as mannitol.
  • a pharmaceutically acceptable excipient or carrier such as mannitol.
  • the concentration of mannitol in the medicament is 10 times of the concentration of Quiescin Q6 peptide.
  • the medicament is an aqueous solution of 0. 9% NaC1 in which Quiescin Q6 peptide and mannitol axe dissolved.
  • the medicament administered to the patient is an aqueous solution of 0.9% NaC1 in which Quiescin Q6 peptide and mannitol are dissolved, and the medicament is infused into the patient at a rate of 15 ng/(kg.min.) Quiescin Q6 peptide continuously over the time period of 24 hours.
  • the administration of a Quiescin Q6 peptide according to the present invention is preferably achieved by intravenous injection, subcutaneous injection, or oral ingestion.
  • the composition comprising a Quiescin Q6 peptide may be formulated with an aqueous diluent, suitably mixed with other optional additives such as a surfactant and/or a preservative for proper fluidity, stability, and sterility of the composition, necessary for easy storage and injection.
  • the injectable solution containing a Quiescin Q6 peptide may be prepared using a solvent or dispersion medium including water, ethanol, polyol (e.
  • Proper fluidity may be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the proliferation of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chiorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the injectable solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • the injectable solution once prepared by incorporating the active ingredients in the required amount in the appropriate solvent with optional excipients, is sterilized using a method that does not inactivate the active ingredient(s) of the composition, e.g., by filtered sterilization.
  • the composition comprising a Quiescin Q6 peptide may be formulated with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard- or soft-shell gelatin capsule, or it may be compressed into tablets.
  • the active ingredients e. g., Quiescin Q6 peptide
  • the orally ingestible formulation preferably contains high molecular weight polymers or gel- forming agents that allows sustained release of the Quiescin Q6 peptide over an extended period of time, for example, at least 24 hours.
  • This sustained release system achieves the slow release of the active ingredient over a period of time, either as a controlled release system, which is effective in maintaining substantially constant level of the Quiescin Q6 peptide in the blood, or as a prolonged prolonged release system, which, although unsuccessful at achieving substantially constant blood level of a Quiescin Q6 peptide, but nevertheless extends the duration of action of the Quiescin Q6 peptide over that time period.
  • the identification of the biomarker of the present invention could be of use in the treatment or amelioration of the AHF condition of the subject.
  • biomarker of the invention it is possible to increase the expression level or abundance of a protein in a subject by administrating such a purified, synthetically or recombinantly produced biomarker of the invention to a subject having a reduced level of said biomarker in comparison to a subject having CHF, Dyspnea or a healthy subject.
  • Administering agents that increase the expression or activity of said biomarker may also be beneficial to the patient.
  • the invention also contemplates activator(s) of Quiescin Q6 for treating AHF, or a pharmaceutical composition comprising an activator of said Quiescin Q6 for treating AHF and the use of an activator of Quiescin Q6 for the manufacture of a medicament for treating AHF.
  • the invention provides method for treating AHF in a subject in need of such treatment, comprising administering to said subject a therapeutically or prophylactically effective amount of an activator of Quiescin Q6.
  • Such activators may be administered and/or may be in a composition for combined administration, simultaneously, separately or sequentially in any order.
  • composition described herein can be administered independently, either systemically or locally, by any method standard in the art.
  • Dosage formulations of the composition described herein may comprise conventional non-toxic, physiologically or pharmaceutically acceptable carriers or vehicles suitable for the method of administration and are well known to an individual having ordinary skill in this art.
  • composition described herein may be administered independently or in combination with an agonist or antagonist and may comprise one or more administrations to achieve, maintain or improve upon a therapeutic effect. It is well within the skill of an artisan to determine dosage or whether a suitable dosage of the composition comprises a single administered dose or multiple administered doses. An appropriate dosage depends on the subject's health, the treatment or prevention of effects of the stimulant drug, the route of administration and the formulation used.
  • administer encompasses various methods of delivering a composition containing a pharmaceutical composition according to the invention to a patient.
  • Modes of administration may include, but are not limited to, methods that involve delivering the composition intravenously, intraperitoneal ⁇ , intranasally, transdermal ⁇ , topically, subcutaneously, parentally, intramuscularly, orally, or systemically, and via injection, ingestion, inhalation, implantation, or adsorption by any other means.
  • the preferred means of administering a composition according to the invention is intravenous injection, where the composition is formulated as a sterile solution.
  • the pharmaceutical composition can be formulated in the form of a syrup, an elixir, a suspension, a powder, a granule, a tablet, a capsule, a lozenge, a troche, an aqueous solution, a cream, an ointment, a lotion, a gel, or an emulsion.
  • the pharmaceutical composition for oral ingestion is formulated for sustained release over a period of at least 24 hours.
  • a Quiescin Q6 peptide according to the invention can be achieved by subcutaneous injection of a Quiescin Q6 peptide-containing composition, which is prepared as a sustained release system comprising microspheres or biodegradable polymers, such that the Quiescin Q6 peptide can be released into a patient's body at a controlled rate over a period of time, e.g., at least 24 hours or 48 hours.
  • an “effective amount” refers to the amount of an active ingredient, e.g., Quiescin Q6 peptide or an agent activating the Quiescin Q6 protein or increasing the Quiescin Q6 protein expression level, in a pharmaceutical composition that is sufficient to produce a beneficial or desired effect at a level that is readily detectable by a method commonly used for detection of such an effect.
  • such an effect results in a change of at least 10% from the value of a basal level where the active ingredient is not administered, more preferably the change is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or an even higher percentage from the basal level.
  • the effective amount of an active ingredient may vary from subject to subject, depending on age, general condition of the subject, the severity of the condition being treated, and the particular biologically active agent administered, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.
  • the term "cardiac medicine” refers to a therapeutic agent that is useful for treating a cardiac condition.
  • a "cardiac medicine” includes but is not limited to the Quiescin Q6 protein or its agonists, acitvators or expression increasing agents as claimed herein, one or more other relevant agents such as preferably BNP, proBNP, NTproBNP and/or fragments of any one thereof, ACE inhibitors (ACEIs), beta-adrenergic blocking agents (beta-blockers), vasodilators, diuretics, digitalis preparations (e.g., digoxin), dopamine, dobutamine, levosimendan, nesiritide, blood thinners, angiotensin Il receptor blockers, calcium channel blockers, nitrates, and potassium.
  • ACEIs ACE inhibitors
  • beta-adrenergic blocking agents beta-blockers
  • vasodilators diuretics
  • digitalis preparations e.g., digoxin
  • dopamine dobutamine
  • levosimendan e.g., levosimendan
  • pharmaceutically acceptable excipient or carrier refers to any inert ingredient in a composition that may act, for example, to stabilize the active ingredient.
  • a pharmaceutically acceptable excipient can include, but is not limited to, carbohydrates (such as glucose, sucrose, or dextrans), antioxidants (such as ascorbic acid or glutathione), chelating agents, low molecular weight proteins, high molecular weight polymers, gel- forming agents, or other stabilizers and additives.
  • Other examples of a pharmaceutically acceptable carrier include wetting agents, emulsifying agents, dispersing agents, or preservatives, which are particularly useful for preventing the growth or action of microorganisms.
  • Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).
  • either a normal or a modified gene can be inserted into the genome to replace an existing gene, being either an abnormal or a disease-causing gene or a normal gene which is e.g. prone to proteolytic cleavage by a protease.
  • This disease-causing gene is then replaced by a functional recombinant gene.
  • the expression of the Suiescin Q6 peptide can be increased using gene therapy e.g. by introducing an additional copy of the gene into the genome of the patient.
  • a carrier or vector is used to deliver the therapeutic gene to the subject's target cells.
  • the most common type of vectors are viruses that have been genetically altered to carry a specific DNA or RNA molecule.
  • Target cells are infected with the vector, which then unloads its genetic material containing the specific DNA or RNA molecule into the target cell.
  • the generation of a functional protein product from the RNA or DNA can e.g. restore the functioning of the target cell to a normal state.
  • the specific DNA or RNA molecule may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.
  • a gene could be swapped for a modified gene through homologous recombination.
  • the gene could be repaired through selective reverse mutation, which returns the gene to its normal function.
  • the regulation (the degree to which a gene is turned on or off) of a particular gene could also be altered.
  • carrier viruses are retroviruses, adenoviruses, Adeno-associated viruses, from the parvovirus family and Lentiviruses.
  • the method of the invention uses the so called integrase-defective lentiviral vectors (IDLV), for sequence specific gene editing (cf. Lombardo et al., Nat Biotechnol. 2007 Nov;25(11 ): 1298-306).
  • IDLV integrase-defective lentiviral vectors
  • Non-viral methods can also be used such as administering of naked DNA in the form of plasmids or naked PCR products encoding the Quiescin Q6 peptide as defined herein.
  • More efficient methods for delivery of the naked DNA are electroporation and the use of a so called gene gun, which shoots DNA coated gold particles into the cell using high pressure gas.
  • the DNA must be protected from damage and its entry into the cell must be facilitated.
  • molecules such as lipoplexes, polyplexes and dendrimers can be used that have the ability to protect the DNA from undesirable degradation during the transfection process.
  • the gene therapy can be effected through the ex vivo transfection of target cells isolated form a patient (e.g. white blood cells, endothelial cells, islet cells, stem cells, heart cells, cardiomyocytes, etc.) with the plasmid of choice, which are then transplanted back into the subject after modification and can start producing the (modified) protein of choice in the subject.
  • target cells e.g. white blood cells, endothelial cells, islet cells, stem cells, heart cells, cardiomyocytes, etc.
  • the term "target cells” implies all possible cell types that express the protein of interest.
  • the therapy can be effected in a non-cell-specific way or can be directed to a specific target cell-type, depending on the disease or condition of the subject.
  • Putative Quiescin Q6 activators may be any modulator acting on either the FAD-linked sulfhydryl oxidase domain, or to the oxidoreductase domain of the Quiescin Q6 protein, or to both domains.
  • Examples of possible agonists of the invention are transcription factors activating the promoter of the endogenous Quiescin Q6 gene, interaction partner(s) of the Quiescin Q6 protein, aiding in its enzymatic activity (e.g. chaperones or co-factors), activating proteins (e.g. involved in protein maturation), ligands, or agents mimicking the activity of the Quiescin Q6 protein and the like.
  • Agonists or activators of Quiescin Q6 can be agents, transcription factors or proteins that increase protein expression or protein activity in any possible manner.
  • One known agonist of Quiescin Q6 is gefitinib, an EGFR inhibitor (Yano, S et al., Oncol. Rep. 2006 Jun; 15(6): 1453- 60). The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
  • Example 1 MASSTERMIND discovery platform for discovery of new biomarkers for AHF
  • MS spectra were analyzed using in-house developed bioinformatics tools, such as tools for peak recognition and deisotoping, ratio determination between analyte and reference, clustering, inter-sample alignment and extensive sample quality control. Once all the samples were aligned and quality controlled, statistical analysis was initiated. MASSTERMIND statistical analysis
  • the method was originally developed for use in microarray experiments and proves applicable in Pronota's data matrices.
  • the main advantage of this method over the one-rule classifier is that it will still allow to pick up useful trends when the difference in ratios between both classes start to diminish and random noise from the experiment starts to obscure the actual levels of the candidate markers.
  • SAM calculates the relative difference in the ratio of features between two classes of samples. To estimate the significance of this score, a null distribution is estimated by permuting the class assignments of all samples and re-scoring. This gives us a confident estimation of the false discovery rat (FDR), that is the percentage of proteins or gene products that were identified by chance.
  • FDR false discovery rat
  • Example 2 MASSterclass targeted protein quantitation for early validation of candidate markers derived from discovery
  • MASSterclass assays use targeted tandem mass spectrometry with stable isotope dilution as an end-stage peptide quantitation system (also called Multiple Reaction Monitoring (MRM) and Single Reaction Monitoring (SRM)).
  • MRM Multiple Reaction Monitoring
  • SRM Single Reaction Monitoring
  • the targeted peptide is specific (i.e., proteotypic) for the specific protein of interest, i.e., the amount of peptide measured is directly related to the amount of protein in the original sample.
  • peptide fractionations precede the end-stage quantitation step.
  • a suitable MASSTE RCLASS assay includes the following steps:
  • This peptide has the same amino acid sequence as the proteotypic peptide of interest, typically with one isotopically labelled amino acid built in to generate a mass difference.
  • the labelled peptide has identical chemical and chromatographic behaviour as the endogenous peptide, except during the end-stage quantitation step which is based on molecular mass.
  • the proteins in the depleted serum/plasma sample are digested into peptides using trypsin. This enzyme cleaves proteins C-terminally from lysine and argninine, except when a proline is present C-terminally of the lysine or arginine. Before digestion, proteins are denatured by boiling, which renders the protein molecule more accessible for the trypsin activity during the 16h incubation at 37°C.
  • Free Flow Electrophoresis is a gel-free, fluid separation technique in which charged molecules moving in a continuous laminar flow are separated through an electrical field perpendicular to the flow. The electrical field causes the charged molecules to separate in the pH gradient according to their isoelectric point (pi). Only those fractions containing the monitored peptides are selected for further fractionation and LC-MS/MS analysis. Each peptide of interest elutes from the FFE chamber at a specific fraction number, which is determined during protein assay development using the synthetic peptide homologue. Specific fractions or fraction pools (multiplexing) proceed to the next level of fractionation.
  • FFE Free Flow Electrophoresis
  • Phenyl HPLC XBridge Phenyl; Waters
  • Phenyl HPLC XBridge Phenyl; Waters
  • pH is by far the most drastic parameter to alter peptide selectivity in RP-HPLC.
  • Each peptide of interest elutes from the Phenyl column at a specific retention time, which is determined during protein assay development using the synthetic peptide homologue.
  • LC-MS/MS based quantitation including further separation on reversed phase (C18) nanoLC (PepMap C18; Dionex) and MS/MS: tandem mass spectrometry using MRM (4000 QTRAP; ABI)/SRM (Vantage TSQ; Thermo Scientific) mode.
  • the LC column is connected to an electrospray needle connected to the source head of the mass spectrometer. As material elutes from the column, molecules are ionized and enter the mass spectrometer in the gas phase.
  • the peptide that is monitored is specifically selected to pass the first quadrupole (Q1 ), based on its mass to charge ratio (m/z).
  • the selected peptide is then fragmented in a second quadrupole (Q2) which is used as a collision cell.
  • the resulting fragments then enter the third quadrupole (Q3).
  • Q2 a second quadrupole
  • Q3 the third quadrupole
  • transition The combination of the m/z of the monitored peptide and the m/z of the monitored fragment of this peptide is called a transition. This process can be performed for multiple transitions during one experiment. Both the endogenous peptide (analyte) and its corresponding isotopically labelled synthetic peptide (internal standard) elute at the same retention time, and are measured in the same LC-MS/MS experiment.
  • the MASSterclass readout is defined by the ratio between the area under the peak specific for the analyte and the area under the peak specific for the synthetic isotopically labelled analogue (internal standard). MASSterclass readouts are directly related to the original concentration of the protein in the sample. MASSterclass readouts can therefore be compared between different samples and groups of samples.
  • the depleted sample (225 ⁇ l_) is denatured for 15min at 95°C and immediately cooled on ice
  • - 200 ⁇ l_ of the sample (representing 20 ⁇ l_ of serum equivalent and containing 400fmol of the labelled peptide) is prepared for FFE adding SPADNS to visualize the sample and bringing the sample in the right starting conditions, according to the manufacturer's protocol.
  • a standard mixture of 4 fluorescent peptides is also spiked in the sample for standardization and QC purposes.
  • the use of an internal control system, based on a spiked standard mixture of fluorescent peptides, allows correcting for possible inter- sample shifts.
  • a 3 to 10 pH gradient is set up using the manufacturer's protocol; except that glycerol and HPMC were excluded from all buffers.
  • the sample is fractionated into 96 fractions and collected in a black 96-well plate.
  • method contains the transitions for the analyte as well as for the synthetic, labelled peptide.
  • the MASSterclass readout was defined by the ratio of the analyte peak area and the internal standard peak area MASSTERCLASS statistical analysis
  • the measured ratios are differential quantitations of peptides.
  • a ratio is the normalised concentration of a peptide.
  • the concentration of a peptide is proportional to the ratio measured in the mass spectrometer.
  • a statistical analysis is conducted in order to determine the diagnostic accuracy of a specific protein. To do so, sample classes are compared pairwise. The analysis defines the ability of a protein to discriminate two sample populations.
  • the diagnostic accuracy of a specific protein was determined by measuring the area under the Receiver-Operating-Characteristics (ROC) curves (AUC).
  • ROC Receiver-Operating-Characteristics
  • the estimated and confidence intervals for AUCs were also computed using a non-parametric approach, namely bootstrapping (Efron B, Tibshirani RJ. Nonparametric confidence intervals. An introduction to the bootstrap. Monographs on statistics and applied probability. 1993; 57:75-90 Chapman & Hall New York).
  • the MASStermind proteomic discovery platform was used to discover novel low abundance AHF protein biomarker candidates directly in patient plasma.
  • Serial plasma samples collected prospectively from 10 patients with AHF on admission to the emergency department and just prior to their discharge from hospital were analyzed alongside age and gender matched control samples collected from healthy individuals ( Figure 4). Comparing protein profiles of AHF patients at admission versus at discharge yields biomarker candidates for treatment monitoring and discharge decisions while a comparison of AHF patients with healthy matched controls provides with new biomarker candidates for improving diagnostic accuracy.
  • the three specific Quiescin Q6 peptides are all localized in the N-terminal extracellular region of the protein, and hence can correspond to both Quiescin isoforms ( Figures 1 and 2).
  • Figure 6 illustrates relative levels of Quiescin Q6 as measured by MASSterclass in 40 samples of each population.
  • the levels at discharge of AHF patients are less relevant and are therefore excluded for the remaining analysis.
  • Median Quiescin Q6 levels among patients with AHF were 1.5 fold higher than dyspneic patients without AHF.
  • the interquartile range, representing 50% of the samples, in all patients populations is less than 1.5 fold.
  • the median fold difference for BNP between AHF and D patients is much larger, up to 8 fold, but also the interquartile range is 8-fold in these populations.
  • the levels of Quiescin Q6 in D patients are very much comparable to levels in stable CHF patients and healthy controls, while for BNP there is no significant difference between AHF and CHF patients.
  • Receiver-operating characteristics (ROC) analysis demonstrated Quiescin Q6 to be highly sensitive and specific for diagnosing AHF in dyspneic patients presenting to the ED, as indicated by an overall median AUC of 0.89 with 95% Cl 0.79-0.96 ( Figure 7A). Compared to the B-type natriuretic peptides this is an improvement of 5-9%, where BNP performs slightly better than NT-proBNP. At a single ratio or concentration cut-off point where sensitivity equals specificity, Quiescin Q6 has calculated sensitivity and specificity of 87%, an increase of 7% to rule in AHF compared to BNP.

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Abstract

L'invention concerne la Quiescine Q6 en tant que nouvelle cible pour le traitement d'une insuffisance cardiaque aiguë ; des procédés de criblage d'agents qui peuvent moduler le niveau et/ou l'activité de ladite cible et des procédés de traitement d'une insuffisance cardiaque aiguë fondés sur la modulation du niveau et/ou de l'activité de ladite cible ; et des kits et des dispositifs pour réaliser lesdits procédés.
PCT/EP2010/051019 2009-01-30 2010-01-28 Cible pour le traitement d'une insuffisance cardiaque aiguë Ceased WO2010086382A1 (fr)

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