Classifications

• • 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
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • 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
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the present invention relates to a method for the diagnosis or detection of a predisposition to developing cardiac hypertrophy, for monitoring the development of cardiac hypertrophy and for monitoring the efficiency of therapy for cardiac hypertrophy and a kit for the same.
• Cardiac hypertrophy is an important adaptive response of the heart to injury or to an increased demand for cardiac output or when there is increased resistance to blood flow.
• the heart adapts through the activation of a hypertrophic response that is characterised by the reactivation of genes normally expressed during foetal heart development, an enlargement of myocardial cells and an accumulation of sarcomeric proteins in the absence of cell division.
• LBV Left Ventricular Hypertrophy
• Kannel W.B. 1991 supra
• Other diseases associated with cardiac hypertrophy include a range of diseases of the heart and lungs which include pulmonary hypertension, cystic fibrosis and chronic obstructive airways disease, that result in hypertrophy of the right side of the heart. Once hypertrophy of the right ventricle has occurred, most of these diseases are associated with a considerable increase in morbidity and mortality. It would therefore be desirable to monitor the development of hypertrophy of the right ventricle and to assess the adequacy of management of a predisposing condition.
• a method for diagnosing or detecting a predisposition to cardiac hypertrophy comprising assaying a sample of human bodily fluid in vitro for the level of cardiotrophin- 1 contained therein.
• the invention is based on the inventor's finding that cardiotrophin- 1 levels are elevated in blood samples of patients who have been shown by classical methods such as electrocardiography and echocardiography to have cardiac hypertrophy as compared to persons not having cardiac hypertrophy
• Cardiotrophin- 1 is a cytokine of the family that includes interleukin-6 (IL-6), leukaemia inhibitory factor (LIF), ciliary neurotropic factor (CNTF), oncostatin-M (OSM) and interleukin-11 (IL-11).
• CT-1 is widely but not universally expressed in adult mouse tissues including heart, kidney and liver. In-vitro studies have demonstrated that levels of CT-1 in tissue samples from mice rise as a result of a hypertrophic response in cardiomyocytes and suggest that CT- 1 activates myocardial cell hypertrophy (Wollert, K.C. et al, J. Biol. Chem. Vol. 271, No. 16, pp9535-45, 1996).
• a gene or gene product to be suitable as a marker for a disease or predisposition for a disease it is important that the gene product is produced at the onset of, and during the complete cycle of disease progression and that the level of production does not fluctuate greatly. It is known that both ⁇ -adrenergic transmitters and endothelin are expressed in cardiac hypertrophy. However as plasma levels of these vary greatly in response to a whole range of factors over short periods of time, the use of these factors as indicators of cardiac hypertrophy is not viable. It is also important that the marker is elevated or reduced only in response to the onset or development of the disease under study and not due to other conditions. For example one suggested marker for cardiac hypertrophy is atrial natiuretic factor (ANF). The problem with ANF as a marker of cardiac hypertrophy is that ANF is raised in a number of other conditions, such as heart failure. In consequence, its use as a reliable marker of cardiac hypertrophy is flawed.
• ANF atrial natiuretic factor
• CT-1 plays a role in cardiac hypertrophy and no evidence that it is involved in cardiac hypertrophy in man and no suggestion that this cytokine would be released from human tissues if present and provide a suitable marker of cardiac hypertrophy in bodily fluid samples.
• ventricular hypertrophy is related to the pressure independent trophic effects of a number of humoral factors including hormones such as adrenaline and angiotensin II. These hormones in man influence, not only the histological pattern observed in hypertensive hypertrophy but also the degree of hypertrophy. This is in contrast to the changes seen in animal models of hypertension such as the SHRsp (Pennica, et al., 1995, PNAS 92: 1142-6) and would suggest that different mechanisms are involved in the onset and development of ventricular hypertrophy between humans and other animal models, such as the rats and mice of the prior art.
• diagnosis or detection of a predisposition to cardiac hypertrophy is determined by comparison of basal CT-1 levels in a human bodily fluid sample from a subject unaffected by cardiac hypertrophy and the level of CT-1 in a bodily fluid sample of a subject under test.
• CT-1 is present at a basal level in human bodily fluid samples of subjects unaffected by cardiac hypertrophy. This serves as background. It is this basal level which serves as the comparison with the level of CT-1 in the bodily fluid of a subject under test. Elevated CT-1 levels as compared to the basal level are indicative of a predisposition to cardiac hypertrophy or onset and development of cardiac hypertrophy. The determination of actual hypertrophy may then be confirmed by classical techniques such as electrocardiography and imaging techniques.
• the method may be used to diagnose or detect a predisposition to cardiac hypertrophy of both the right ventricle (right venticular hypertrophy - RVH) or the left ventricle (left ventricular hypertophy - LVH).
• the cause of ventricular hypertrophy may be evident before detection of the hypertrophy or ventricular hypertrophy will be detectable at a time when the patient presents with an underlying problem.
• LVH is a high risk factor for subjects with hypertension to develop cardiac problems. Diagnosis or detection of a predisposition to LVH in hypertensive persons allows such persons to be treated for hypertension to prevent the onset or development of LVH and thus a high risk of developing cardiac problems. RVH occurs in a range of disease of the heart and lungs which include pulmonary hypertension, cystic fibrosis and chronic obstructive airways disease. Diagnosis of RVH therefore provides an indicator of severity of such a condition.
• the method may be used to detect patients at risk of developing cardiac hypertrophy before any irreversible damage has occurred, i.e. before the onset of cardiac hypertrophy.
• the assay may be easily carried out on samples of bodily fluid and there is therefore not the need for a person to visit a hospital having a certain imaging machine to carry out a test for cardiac hypertrophy.
• the assay may be carried out in a doctor's surgery, by a healthcare worker, and it is even envisaged that the assay may be developed so that it can be produced as a test kit which is sufficiently simple to be used in the home by the subject at risk of cardiac hypertrophy.
• the method allows a relatively inexpensive and quick test for cardiac hypertrophy as compared to the classical techniques presently in use.
• the main advantage of the method of the present invention is its accuracy.
• the electrocardiogram has been shown to correctly identify only 3-8 % of patients at the time of diagnosis of hypertension as having cardiac hypertrophy and thus the technique does not provide for effective diagnosis.
• Echocardiography and magnetic resonance scanning have shown that the incidence of left ventricular hypertrophy at diagnosis of hypertension is of the order of 20-60 %. It is thought that the method of the present invention may prove to diagnose even more subjects with cardiac hypertrophy and also before the condition develops.
• the method of the present invention may be used to monitor the onset and progression of cardiac hypertrophy. Elevated CT-1 levels as compared to normal are indicative of the initiation or onset of cardiac hypertrophy. The method of the invention may be used to monitor the progression of cardiac hypertrophy by comparing the CT-1 level in a test sample with that of a previous sample from the same subject. Elevated levels in the subsequent sample are indicative of developing cardiac hypertrophy. Reduced levels in the subsequent sample are indicative of receding cardiac hypertrophy or optimisation of therapy.
• the method according to the present invention may be used to monitor the efficacy of therapy for cardiac hypertrophy. By carrying out repeated test over time the trend in CT-1 levels may be monitored. A reduction in CT-1 levels over time is indicative of an effective therapy.
• the method is performed yearly, more preferably, every six months, even more preferably quarterly and most preferably every six weeks.
• the number of assays taken per year would vary depending on the degree of risk associated with the subject and the results of any previous assay.
• the method according to the present invention may be carried out on whole blood, plasma, serum, urine, tears, sputum, saliva or synovial fluid samples.
• the method comprises an in vitro assay arranged to detect CT-1 protein or specific fragments thereof.
• this in vitro assay comprises radio immuno assay or enzyme-linked immunosorbant assay (ELISA).
• ELISA enzyme-linked immunosorbant assay
• a specific binding partner for CT-1 or fragments thereof can be used quantitatively to show levels of CT-1 in a sample.
• the specific binding partner is preferably functional, producing a label, or has a label attached thereto to show the presence of levels of CT-1.
• specific binding partners for CT-1 include the CT-1 receptor and anti-CT-1 antibodies, although other suitable specific binding partners will be apparent to those skilled in the art.
• antibodies raised against the whole CT-1 protein or specific fragments thereof are used in immuno assays as specific binding partners for CT-1.
• a suitable antibody for use in the assay is rabbit IgG to full length human CT-1.
• determining levels of CT-1 protein in a sample are by molecular weight or charge. Chromatography on a porous carrier or SDS PAGE may be used to show levels of CT-1 in a sample due to the distance travelled along the carrier. Isoelectric focussing may be used to identify CT-1 due to its charge.
• the method comprises an in vitro assay arranged to detect CT- 1 nucleic acid or fragments thereof.
• this in vitro assay comprises hybridisation, sequencing or amplification techniques such as PCR.
• the in vitro assay is quantitative.
• the reagents and vessels necessary for assaying CT-1 levels may be provided in a kit.
• CT-1 expression is not localised to the heart uniquely in mice but is expressed in other mouse tissues including the kidney and liver.
• a further in vitro assay for an additional marker is carried out.
• Suitable markers of cardiac function include ANF, oncostatin M, ciliary neurotrophic factor and leukaemia inhibiting factor.
• the second aspect of the invention provides the use of the method of the first aspect of the invention to determine subjects who should be treated for hypertension.
• LVH is a high risk factor for heart problems in combination with hypertension and therefore if elevated levels of CT-1 are found in a sample of bodily fluid that person should be treated for hypertension to prevent the development of heart problems.
• the efficacy of treatment for hypertension may be monitored by comparing the CT-1 level in a test sample with that of a previous sample from the same subject. A reduction in CT-1 levels over time is indicative of an effective therapy.
• a protocol for performing the method according to the first aspect of the invention involves competitive ELISA.
• the competitive ELISA involves binding by passive adsorption , the antigen (purified human CT-1) overnight.
• Plasma samples may be added with the competeing antibody which is rabbit IgG to human CT-1 or the plasma sample could be added for a period of incubation before the competing antibody.
• An enzyme-labelled antibody is then added for colour development.
• the assay may be further improved by the used of monoclonal antibodies for increased sensitivity and a reduction in the number of steps. Summary of steps:
• the chemiluminescent label 4-(2-succinimidyl-oxycarbonylethyl)phenyl-10-methyl- acridinium-9-carboxylate fluorosulfonate was used to label a peptide representing a domain in the middle section of CT-1.
• Assay of this domain of CT-1 (amino acids 105-120) in patients with heart failure revealed elevated CT-1 values compared to normal controls.
• the assay for CT-1 according to Talwar et al. is assumed to have utility according to a preferred embodiment of the present invention.

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• 2000
  • 2000-06-29 CN CN 00811583 patent/CN1370277A/zh active Pending
  • 2000-06-29 EP EP00942228A patent/EP1196781A2/de not_active Withdrawn
  • 2000-06-29 JP JP2001508865A patent/JP2003504602A/ja active Pending
  • 2000-06-29 AU AU56933/00A patent/AU5693300A/en not_active Abandoned
  • 2000-06-29 WO PCT/GB2000/002521 patent/WO2001003573A2/en not_active Application Discontinuation
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