hGH Determination for Use to Guide Prevention of a Major Adverse Cardiac Event or a Cardiovascular Disease in a Subject
20170234889 · 2017-08-17
Assignee
Inventors
Cpc classification
G01N2800/52
PHYSICS
International classification
Abstract
A method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) by determining the level of hGH, and/or its isoforms in a blood sample of said subject and comparing the determined level of hGH, and/or its isoforms in said blood sample with a pre-determined threshold.
Claims
1) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) comprising the steps: determining the level of hGH, and/or its isoforms in a blood sample of said subject and comparing the determined level of hGH, and/or its isoforms in said blood sample with a pre-determined threshold and wherein in case the determined level of hGH, and/or its isoforms is above said pre-determined threshold then the subject is identified as having a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) and wherein in case the determined level of hGH, and/or its isoforms is below said pre-determined threshold then the subject is identified as not having a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins)
2) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1, wherein said subject does not have hypertension (at least >140 mmHG (systolic) to 90 mmHG (diastolic)).
3) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein the blood sample is taken from a subject that has not yet treated with antihypertensive drugs.
4) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein reducing the vascular risk by lowering blood pressure in a subject means preventing a major cardiovascular event and/or a cardiovascular disease in said subject.
5) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein said subject has never had a major cardiovascular event and has never had any cardiovascular disease.
6) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein said subject has suffered an acute myocardial infarction or acute heart failure within the last 2 weeks, preferably within the last one week, preferably within the last 36 hours.
7) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein said antihypertensive drug is selected from the group comprising Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), and Beta-Adrenoreceptor Blocker (Beta-blocker), statin or combinations thereof.
8) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein the blood sample is selected from the group comprising whole blood, serum, and plasma.
9) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein the blood sample is a fasting sample.
10) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein the measurement method that is used to determine the level of hGH, and/or its isoforms has a sensitivity of at least 50 pg/ml.
11) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein the measurement method that is used to determine the level of hGH, and/or its isoforms is an immuno assay having an assay sensitivity of at least 50 pg/ml.
12) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein the pre-determined threshold is 330 pg/ml or above, preferably 1370 pg/ml or above.
13) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein the level of hGH, and/or its isoforms is determined by an immunoassay that comprises at least two antibodies that bind to hGH, and/or its isoforms.
14) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 11 wherein each of the at least two antibodies that bind to hGH and/or its isoforms have an affinity towards hGH, and/or its isoforms that is at least 10.sup.8M.sup.−1.
15) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein said method is used for prevention of a major cardiovascular event and/or a cardiovascular disease in said subject selected from the group comprising heart failure, artherosclerosis, hypertension, cardiomyopathy, myocardial infarction and stroke.
16) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein the method further comprises: recommendation of administration of antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) to a subject whose determined level of hGH, and/or its isoforms is above said pre-determined threshold and who is identified as having a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs or recommendation of withholding with antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) to a subject whose determined level of hGH, and/or its isoforms is below said pre-determined threshold and who is identified as not having a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs
17) Method of determining whether a subject has a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs according to claim 1 wherein the method further comprises: administration of antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) to a subject whose determined level of hGH, and/or its isoforms is above said pre-determined threshold and who is identified as having a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs or withholding of antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) to a subject whose determined level of hGH, and/or its isoforms is below said pre-determined threshold and who is identified as not having a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs.
18) An antihypertensive drug (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) for use in a method of preventing a major cardiovascular event and/or a cardiovascular disease in a subject whose determined level of hGH, and/or its isoforms is above said pre-determined threshold and wherein said subject is identified according to the methods of claim 1 as having a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs.
19) An antihypertensive drug (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) for use in a method of preventing a major cardiovascular event and/or a cardiovascular disease in a subject whose determined level of hGH, and/or its isoforms is above said pre-determined threshold and wherein said subject is identified according to the methods of claim 1 as having a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs wherein said subject does not have hypertension.
20) A method for monitoring the therapy response of a subject at vascular risk that is treated with antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) comprising the steps: determining the level of hGH, and/or its isoforms in a blood sample of said subject taken at a time point X1 with antihypertensive drugs and determining the level of hGH, and/or its isoforms in a blood sample of said subject taken at a time point X2 with antihypertensive drugs, and wherein time point X1 is either before the beginning of therapy or after starting the therapy but in any event earlier than time point X2, and wherein time point X2 is after starting the therapy and in any event later than time point X1 comparing the determined level of hGH, and/or its isoforms in said blood sample taken at point X1 with the determined level of hGH, and/or its isoforms in said blood sample taken at time point X2 wherein a lower level of hGH, and/or its isoforms in said blood sample taken at time point X2 compared to the level of hGH, and/or its isoforms in said blood sample taken at time point X1 indicates a therapy response.
21) A method for monitoring the therapy response of a subject at vascular risk that is treated with antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) according to claim 20 wherein the determined level of hGH, and/or its isoforms in said blood sample taken at point X1 is above a pre-determined threshold and the subject has been identified as having a vascular risk that can be reduced by blood pressure lowering therapy with antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins).
22) A method for monitoring the therapy response of a subject at vascular risk that is treated with antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) according to claim 20 wherein the time point X1 is before starting the therapy.
23) A method for monitoring the therapy response of a subject at vascular risk that is treated with antihypertensive drugs (e.g. Angiotensin converting enzyme inhibitor (ACE inhibitor), Angiotensin Receptor Blocker (ARB), Beta-Adrenoreceptor Blocker (Beta-blocker) and/or statins) according to claim 20 wherein time point X2 is 12 months, 24 Months, or 36 months after time point X1.
Description
EXAMPLES
Example 1, us-hGH Assay
[0138] Plasma samples were measured using a hGH assay as described previously in WO/2014/108396. The measurement was made with an ultra-sensitivity chemiluminescence immunoassay (see example 1 us-hGH assay). The detection limit was 2 pg/ml, functional assay sensitivity was 8.5 pg/ml. Measurement of hGH is expressed in ng/ml (1 ng/ml=2.6 mU/L).
Chemicals
[0139] If not stated otherwise, chemicals were obtained at p.a. grade from Merck (Darmstadt, Germany).
Antigen
[0140] For immunization and for calibration we used recombinant Human Growth Hormone (NIBSC code 98/574, National Institute for Biological Standards and Control, Herfordshire, UK).
Development of Antibodies
[0141] Mouse monoclonal antibodies against hGH were developed by UNICUS (Karlsburg, Germany).
[0142] The antibodies were generated according to the following method:
[0143] A BALB/c mouse were immunized with 100 μg hGH at day 0 and 14 (emulsified in 100 μl complete Freund's adjuvant) and 50 μg at day 21 and 28 (in 100 μl incomplete Freund's adjuvant). Three days before the fusion experiment was performed, the animal received 50 μg of the conjugate dissolved in 100 μl saline, given as one intraperitonal and one intra venous injection.
[0144] Splenocytes from the immunized mouse and cells of the myeloma cell line SP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37° C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-supplement]. After two weeks the HAT medium is replaced with HT Medium for three passages followed by returning to the normal cell culture medium.
[0145] The cell culture supernatants were primary screened for antigen specific IgG antibodies three weeks after fusion. The positive tested microcultures were transferred into 24-well plates for propagation. After retesting the selected cultures were cloned and recloned using the limiting-dilution technique and the isotypes were determined. (Lane, R. D. “A short-duration polyethylene glycol fusiontechnique for increasing production of monoclonal antibody-secreting hybridomas”, J. Immunol. Meth. 81: 223-228; (1985)—Ziegler, B. et al. “Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complement-dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies”, Horm. Metab. Res. 28: 11-15, (1996))
Monoclonal Antibody Production
[0146] We selected 5 antibodies for further investigations.
[0147] Antibodies were produced via standard antibody production methods (Marx et al., Monoclonal Antibody Production (1997), ATLA 25, 121) and purified via Protein A-chromatography. The antibody purities were >95% based on SDS gel electrophoresis analysis.
Labelling and Coating of Antibodies.
[0148] All antibodies were labelled with acridinium ester according the following procedure:
[0149] Labelled compound (tracer): 100 μg (100 μl) antibody (1 mg/ml in PBS, pH 7.4), was mixed with 10 μl Acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated for 20 min at room temperature. Labelled antibody was purified by gel-filtration HPLC on Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purified labelled antibody was diluted in (300 mmol/1 potassiumphosphate, 100 mmol/1 NaCl, 10 mmol/1 Na-EDTA, 5 g/l bovine serum albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 μl. Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).
Solid Phase Antibody (Coated Antibody):
[0150] Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with antibody (1.5 μg antibody/0.3 ml 100 mmol/1 NaCl, 50 mmol/1 Tris/HCl, pH 7.8). After blocking with 5% bovine serum albumin, the tubes were washed with PBS, pH 7.4 and vacuum dried.
hGH Immunoassay:
[0151] 50 μl of sample (or calibrator) was pipetted into coated tubes, after adding labeled antibody (200 ul), the tubes were incubated for 2 h at 18-25° C. Unbound tracer was removed by washing 5 times (each 1 ml) with washing solution (20 mmol/1 PBS, pH 7.4, 0.1% Triton X-100). Tube-bound labelled antibody was measured by using the LB 953. Using a fixed concentration 1 ng/ml of hGH. The signal (RLU at ing hGH/ml) to noise (RLU without us-hGH) ratio of different antibody combinations is given in table 1. All antibodies were able to generate a sandwich complex with any other antibody. The antibody pair with strongest signal to noise ratio (best sensitivity) was subsequently used to perform the us-hGH-immunoassay: hGH G12 antibody was used as coated tube antibody and hGH H4 antibody was used as labelled antibody.
TABLE-US-00002 TABLE 1 Results of noise to ratio determinations between different pairs of hGH antibodies. Solid hGH phase antibody antibody H2 H8 G12 H4 D7 Labelled antibody H2 / 9.665 11.005 9.259 10.102 H8 8.512 / 7.833 8.446 6.384 G12 10.112 9.846 / 10.905 7.751 H4 11.213 8.675 12.225 / 6.843 D7 2.488 2.761 3.954 2.713 /
Calibration:
[0152] The assay was calibrated, using dilutions of recombinant hGH (WHO International Standard, NIBSC code 98/574), diluted in 20 mM K2PO4, 6 mM EDTA, 0.5% BSA, 50 uM Amastatin, 100 uM Leupeptin, pH 8.0. (
Assay Specifications
[0153] The analytical assay sensitivity (mean relative light units of 20 determinations of hGH free sample plus 2 S.D.) was 2 pg/ml of hGH and the functional assay sensitivity (see above) was 8.5 pg/ml. Recovery and dilution was >85% intra measurement range of 5-10.000 pg/ml hGH. The coefficient of correlation of N=997 samples between the us-hGH assay and a hGH assay specific for recombinant hGH (22KD) was r=0.98 and a r of 0.95 was found for an assay recognizing preferentially hGH isoforms naturally produced by the pituitary (Bidlingmaier M, Suhr J, Ernst A, Wu Z, Keller A, Strasburger C J, et al. High-sensitivity chemiluminescence immunoassays for detection of growth hormone doping in sports. Clinical chemistry. 2009; 55(3):445-53. Epub Jan. 27, 2009). These dataindicating the suitability of all hGH isoform measurements within the present invention.
Example 2, Study Population
[0154] We studied 953 STEMI and NSTEMI patients admitted to University Hospitals of Leicester NHS trust between August 2004 and April 2007. This observational cohort study complied with the Declaration of Helsinki and was approved by the local ethics committee; written informed consent was obtained from patients. AMI was diagnosed if a patient had a cardiac troponin I level above the 99th centile with at least one of the following: chest pain lasting >20 minutes or diagnostic serial electrocardiographic changes consisting of new pathological Q waves or ST-segment and T-wave changes. Patients with known malignancy, renal replacement therapy or surgery in the previous month were excluded. Estimated glomerular filtration rate (eGFR) was calculated from the simplified Modification of Diet in Renal Disease formula (Smilde T D, van Veldhuisen D J, Navis G, Voors A A, Hillege H L. Drawbacks and prognostic value of formulas estimating renal function in patients with chronic heart failure and systolic dysfunction. Circulation 2006; 114:1572-80). All patients received standard medical treatment and revascularisation was at the discretion of the attending physician.
[0155] Plasma samples: Blood samples (anticoagulated with EDTA and aprotinin) were drawn after 15 minutes bed rest, immediately after diagnosis and within 36 h of symptom onset. Plasma was stored at −80° C. until assayed in a single batch for blinded determination of plasma hGH.
Example 3, Analysis
[0156] The efficacy of treatment of patients with acute myocardial infarction with various drugs was analysed depending on the patients' hGH concentration, which was determined within a timeframe of maximally 36 hours after symptom onset. The drugs analysed were a) Angiotensin converting enzyme inhibitor (ACE) or Angiotensin Receptor Blocker (ARB), and b) beta-blocker. The endpoint for determining efficacy was occurrence of major adverse cardiac events (MACE) within one year after symptom onset. MACE was defined as a combination of the following endpoints: all-cause mortality, hospitalization due to heart failure, or re-acute myocardial infarction (re-AMI).
Angiotensin Converting Enzyme Inhibitor (ACE) or Angiotensin Receptor Blocker (ARB):
[0157] The patient cohort consisted of 953 cases with 245 MACE endpoints within 1 year. Various clinical variables were associated with prediction of MACE within one year, as determined by Cox regression analysis (table 1). When hGH concentrations were added to the statistical model (table 2), hGH was an independent predictor of MACE, and, surprisingly, also the interaction of hGH and treatment with ACE/ARB. Kaplan-Meier analysis for the occurrence of MACE depending on treatment with ACE/ARB was performed for the patient population after separation into hGH tertiles, that is patient with low (<330 pg/mL), medium (330-1370 pg/mL) and high concentrations (>1370 pg/mL) of hGH. The number of MACE for the subgroups are shown in table 3. As shown in table 4, the ACE/ARB treatment effect on MACE was dependent on hGH tertiles and significant only for hGH tertiles 2 and 3, that is for medium and high hGH concentrations. The effect was more pronounced in hGH tertile 3 than in hGH tertile 2.
TABLE-US-00003 TABLE 1 Multivariate Cox regression analysis for the prediction of MACE within 1 year. Variables in the Equation B SE Wald df Sig. Exp(B) age .032 .006 29.831 1 .000 1.032 sex .138 .119 1.360 1 .244 1.148 PMHmi_angina .257 .115 5.024 1 .025 1.293 pmhbp .284 .114 6.162 1 .013 1.329 PMHdiabetes .265 .119 4.957 1 .026 1.304 KillipAbove1 .493 .116 17.922 1 .000 1.637 eGFR −.015 .003 20.395 1 .000 .985 ClassSTEMI .307 .115 7.077 1 .008 1.359 DischACE_ARB −.369 .116 10.142 1 .001 .692
TABLE-US-00004 TABLE 2 Multivariate Cox regression analysis for the prediction of MACE within 1 year including hGH and interaction with ACE/ARB therapy. Variables in the Equation B SE Wald df Sig. Exp(B) age .033 .007 21.337 1 .000 1.033 sex .046 .144 .102 1 .749 1.047 PMHmi_angina .038 .143 .071 1 .789 1.039 pmhbp .123 .140 .768 1 .381 1.131 PMHdiabetes .267 .145 3.375 1 .066 1.305 killipAbove1 .553 .138 16.070 1 .000 1.739 eGFR −.019 .004 19.937 1 .000 .981 ClassSTEMI .636 .145 19.167 1 .000 1.889 DischACE_ARB −.591 .162 13.243 1 .000 .554 ZLoghGH .375 .145 6.658 1 .010 1.455 DischACE_ARB*ZLoghG −.359 .166 4.657 1 .031 .698 H
TABLE-US-00005 TABLE 3 Case Processing Summary for MACE depending on ACE/ARB treatment in patients with low (tertile 1), medium (tertile 2) and high (tertile 3) concentartions of hGH. Case Processing Summary DischACE N of Censored hGHaTertiles ARB Total N Events N Percent 1.00 0 45 9 36 80.0% 1 270 50 220 81.5% Overall 315 59 256 81.3% 2.00 0 66 27 39 59.1% 1 257 59 198 77.0% Overall 323 86 237 73.4% 3.00 0 68 37 31 45.6% 1 247 63 184 74.5% Overall 315 100 215 68.3% Overall Overall 953 245 708 74.3%
TABLE-US-00006 TABLE 4 Statistical comparison of ACE/ARB treatment effect on MACE depending on hGH tertiles. Pairwise Comparisons 0 1 DischACE Chi- Chi- hGHaTertiles ARB Square Sig. Square Sig. Log Rank 1.00 0 .070 .791 (Mantel- 1 .070 .791 Cox) 2.00 0 10.492 .001 1 10.492 .001 3.00 0 25.970 .000 1 25.970 .000
Beta-Blockers
[0158] In the same patient cohort, multivariate Cox regression analysis revealed also a significant interaction of hGH and treatment with Beta-blockers for prediction of 1 year MACE (table 5). Kaplan-Meier analysis for the occurrence of MACE depending on treatment with Beta-blockers was performed for the patient population after separation into hGH tertiles, that is patient with low, medium and high concentrations of hGH. The number of MACE for the subgroups are shown in table 6. As shown in table 7, the Beta-blockers treatment effect on MACE was dependent on hGH tertiles and significant only for hGH tertiles 2 and 3, that is for medium and high hGH concentrations. The effect was more pronounced in hGH tertile 3 than in hGH tertile 2.
TABLE-US-00007 TABLE 5 Multivariate Cox regression analysis for the prediction of MACE within 1 year including hGH and interaction with Beta-blocker therapy. Variables in the Equation B SE Wald df Sig. Exp (B) age .031 .007 17.475 1 .000 1.031 sex .086 .145 .356 1 .551 1.090 PMHmi_angina .082 .141 .337 1 .562 1.085 pmhbp .076 .140 .291 1 .590 1.079 PMHdiabetes .282 .147 3.671 1 .055 1.326 killipAbove1 .528 .139 14.540 1 .000 1.696 eGFR −.021 .004 23.658 1 .000 .979 ClassSTEMI .573 .144 15.936 1 .000 1.774 DischBetaBlocker −.383 .157 5.933 1 .015 .682 ZLoghGH .415 .153 7.348 1 .007 1.514 DischBetaBlocker*ZLogh −.309 .172 5.365 1 .021 .671 GH
TABLE-US-00008 TABLE 6 Case Processing Summary for MACE depending on Beta-blocker treatment in patients with low (tertile 1), medium (tertile 2) and high (tertile 3) concentartions of hGH. Case Processing Summary N of Censored hGHtertiles DischBetaBlocker Total N Events N Percent 1.00 0 46 10 36 78.3% 1 269 49 220 81.8% Overall 315 59 256 81.3% 2.00 0 80 30 50 62.5% 1 242 55 187 77.3% Overall 322 85 237 73.6% 3.00 0 71 39 32 45.1% 1 244 61 183 75.0% Overall 315 100 215 68.3% Overall Overall 952 244 708 74.4%
TABLE-US-00009 TABLE 7 Statistical comparison of Beta-blocker treatment effect on MACE depending on hGH tertiles. Pairwise Comparisons 0 1 DischBeta- Chi- Chi- hGHtertiles Blocker Square Sig. Square Sig. Log Rank 1.00 0 .359 .549 (Mantel- 1 .359 .549 Cox) 2.00 0 7.355 .007 1 7.355 .007 3.00 0 29.334 .000 1 29.334 .000
[0159] Characteristics of the 953 AMI patients according to hGH tertiles on admission are given in table 8.
TABLE-US-00010 TABLE 8 Characteristics of the 953 AMI patients according to hs-GH tertiles on admission. Numerical data are presented as n (%). P values are quoted for the Kruskal Wallis or Chi squared tests for continuous or categorical variables respectively. Numbers (%) or Mean ± SD are reported. hGH tertiles (pg/mL) 1 2 3 All <330 330-1370 >1370 P Value n = 953 n = 315 n = 323 n = 315 hGH pg/mL 1700 ± 2850 140 ± 80 750 ± 0.310 4240 ± 3850 <0.0005 Demographics Age (years) 66.1 ± 12.8 61.9 ± 11.9 67.3 ± 12.6 69.0 ± 12.9 <0.0005 Male (%) 687 (72) 262 (83) 221 (68) 204 (65) <0.0005 ST elevation 459 (48) 139 (44) 154 (48) 166 (53) NS AMI Previous History IUD 320 (34) 103 (33) 117 (36) 100 (32) NS Heart Failure 37 (4) 9 (3) 12 (4) 16 (5) NS Hypertension 493 (52) 149 (47) 166 (52) 178 (57) NS Diabetes 227 (24) 74 (23) 76 (24) 77 (24) NS Mellitus Killip Class >1 390 (41) 112 (36) 138 (43) 140 (45) <0.05 Glucose 8.8 ± 4.2 8.3 ± 3.4 8.8 ± 3.8 9.5 ± 4.9 <0.021 (mmol/L) Troponin I 12.5 ± 24.6 9.2 ± 20.3 14.0 ± 25.6 14.4 ± 27.2 <0.0005 (μg/L) eGFR (ml/min/ 66.2 ± 19.9 71.1 ± 16.9 64.1 ± 19.0 63.5 ± 22.4 <0.0005 1.73 m.sup.2) Treatment Aspirin 794 (83) 285 (90) 260 (80) 249 (79) <0.0005 Beta-blocker 755 (79) 269 (85) 242 (75) 244 (77) 0.004 ACE inhibitor 774 (81) 270 (86) 257 (80) 247 (78) 0.041 or ARB* Statin 824 (86) 287 (91) 278 (86) 259 (82) 0.005 Loop Diuretic 242 (25) 57 (18) 92 (29) 93 (30) 0.001 Revascularisation 241 (25) 95 (30) 76 (24) 70 (22) NS *ARB = Angiotensin 2 receptor blocker
[0160] During follow-up over 2 years, there were 281 MACE, the primary composite endpoint (comprising 117 deaths, 71 HF hospitalisations and 93 re-AMIs). Patients with MACE had higher levels of GH on presentation (median [range], 910 [40-26 280] pg/mL) compared to event free survivors (590 [20-21 600], p<0.0005 using the Mann-Whitney test). Table 8 also illustrates the higher prevalence of MACE in patients with higher GH levels.
[0161] Table 9 reports the univariate hazard ratios of various factors, therapies and biomarkers that affected the outcome of MACE at 2 years. In multivariate analysis, individual therapies and their interaction with GH levels were examined. Age, Killip class>1, eGFR were retained in all models as independent predictors, together with GH levels. Beta blocker therapy was associated with lower MACE (p=0.03) and showed a significant interaction with GH levels (p=0.047, Table 9). Therapy with ACE/ARB also showed a significant interaction with GH levels (p=0.016, Table 9).
[0162] Kaplan-Meier survival analysis was used to visualise the interactions of therapies with beta blocker or ACE/ARB according to GH tertiles (
TABLE-US-00011 TABLE 9 Cox regression analysis for MACE at 2 years post-AMI. Multivariable analysis results are reported for model 1 and 2 which included clinical variables and hGH, with interaction terms hGH with beta blockers (model 1) or ACE/ARB (model 2). Multivariable Multivariable Univariable Model 1 Model 2 HR (95% CI) P HR (95% CI) P HR (95% CI) P Age (years) 1.05 (1.04-1.06) 0.001 1.03 (1.01-1.04) 0.001 1.03 (1.02-1.04) 0.001 Male Sex 0.59 (0.46-0.75) 0.001 1.13 (0.84-1.51) NS 1.10 (0.83-1.47) NS ST elevation 0.97 (0.77-1.23) NS 1.27 (0.92-1.76) NS 1.31 (0.95-1.81) NS Killip 2.62 (2.06-3.33) 0.001 1.66 (1.26-2.19) 0.001 1.67 (1.26-2.20) 0.001 class >1 eGFR (ml 0.97 (0.96-0.97) 0.001 0.99 (0.98-0.99) 0.006 0.98 (0.97-0.99) 0.001 min.sup.−1/ 1.73 m.sup.2) Past history Ischemic 1.67 (1.32-2.11) 0.001 1.06 (0.80-1.42) NS 1.03 (0.76-1.38) NS heart disease Hypertension 1.69 (1.32-2.15) 0.001 1.13 (0.85-1.50) NS 1.18 (0.88-1.57) NS Diabetes 1.59 (1.23-2.04) 0.001 1.26 (0.94-1.69) NS 1.27 (0.95-1.69) NS Treatment Aspirin 0.54 (0.41-0.72) 0.001 excluded excluded Statin 0.38 (0.29-0.51) 0.001 excluded excluded Loop 2.30 (1.81-2.92) 0.001 excluded excluded Diuretic ACE/ARB 0.51 (0.39-0.66) 0.001 excluded 0.74 (0.53-1.04) NS β blocker 0.51 (0.39-0.65) 0.001 0.70 (0.52-0.97) 0.03 excluded Biomarkers Log 1.10 (0.97-1.26) NS 1.13 (0.93-1.36) NS 1.13 (0.93-1.37) NS Troponin (μg/L) Log hGH 1.76 (1.60-1.94) 0.001 1.43 (1.05-1.95) 0.026 1.49 (1.10-2.02) 0.01 β blocker * 0.70 (0.49-0.99) 0.047 hGH ACE/ARB * 0.65 (0.47-0.93) 0.016 hGH
Example 4. Malmö Diet and Cancer Study
[0163] The Malmö Diet and Cancer study-cardiovascular cohort (MDC-CC) is a prospective cohort examined in the early 90's. Further details about this study can be found in earlier publications (Berglund et al. 1993. Journal of Internal Medicine 233: 45-51). We performed a cross-sectional analysis of the relationship between hs-GH and intima media thickness (IMT) in MDC-CC. In multiple regression models with IMT as the dependent variable and the standardized value of the natural logarithm of hs-GH as independent we analyzed 4425 individuals from this cohort with values on both hs-GH and the mean IMT in the common carotid artery (IMT.sub.cca) and 3397 participants with values on the maximum IMT at the bifurcation (IMT.sub.bulb). Separate models were performed for IMT.sub.cca and IMT.sub.bulb and they were adjusted for either sex and age or a set of traditional cardiovascular risk factors: sex, age, current smoking, systolic blood pressure, anti-hypertensive medication, BMI, LDL-C, HDL-C and diabetes mellitus.
[0164] Carotid ultrasound was performed at baseline. In brief, a predefined extent of the right carotid bifurcation was scanned for presence of plaques and the maximum IMT at the bifurcation (IMT.sub.bulb) and the mean IMT in the common carotid artery (IMT.sub.cca) was measured. In the initial cross-sectional regression model of 3397 (1957 females, 58%) individuals in the MDC-CC hs-GH exhibited a significant positive correlation with IMT.sub.bulb in the whole cohort (P=0.003) and in males (P=0.003) (Table 10). This association was essentially unaffected when adjusting for traditional cardiovascular risk factors (P=0.002 and P=0.005 respectively). The results among females were not significant. Fasting levels of hs-GH were not associated with the IMT.sub.cca in the 4425 individuals (2658 females, 60%) that were available in this analysis.
[0165] Higher fasting levels of hs-GH were associated with an increased IMT in the carotid bulb in males. The relationship between hs-GH and IMT at the carotid bulb in males is in accordance with previous results of higher fasting values of hs-GH being associated to cardiovascular disease, which was also preferentially seen in males (Hallgren et al. 2014. JACC 64: 1452-1460; Maison et al. 1998. BMJ [Clinical Research ed] 316: 1132-1133). IMT.sub.bulb is a good measure of atherosclerosis (Naqvi and Lee. 2014. JACC Cardiovasc Imaging 7: 1025-1038), and this finding thus supports a hypothesis of the connection between hs-GH and cardiovascular diseases being mediated by atherosclerosis.
TABLE-US-00012 TABLE 10 Linear regression with IMT at baseline in the MDC-CC study as dependent variable and hs-GH as independent. One crude model adjusted for sex and age and one model in addition adjusted for traditional cardiovascular risk factors. Dependent Gender Model Beta 95% CI P IMT.sub.bulb ALL Crude 0.060 0.020 to 0.099 0.003 Adjusted 0.066 0.025 to 0.106 0.002 MALE Crude 0.075 0.025 to 0.125 0.003 Adjusted 0.074 0.022 to 0.125 0.005 FEMALE Crude 0.029 −0.014 to 0.072 0.184 Adjusted 0.038 −0.006 to 0.083 0.089 IMT.sub.cca ALL Crude −0.012 −0.046 to 0.021 0.477 Adjusted 0.017 −0.017 to 0.052 0.319 MALE Crude 0.011 −0.033 to 0.054 0.634 Adjusted 0.024 −0.020 to 0.068 0.280 FEMALE Crude −0.027 −0.063 to 0.009 0.145 Adjusted 0.001 −0.036 to 0.038 0.967
[0166] Crude models adjusted for sex and age. Adjusted model adjusted for: sex, age, systolic blood pressure, antihypertensive medication, diabetes mellitus, current smoking, BMI, LDL-C and HDL-C. The β coefficients are expressed as the increment of standardized values of the natural logarithm of IMT per 1 increment of standardized values of the natural logarithm of hs-GH. Abbreviations: IMT.sub.cca, mean value of intima media thickness in the common carotid artery. IMT.sub.bulb, max value of the intima media thickness at the carotid bifurcation.
Example 5, BCAPS
Study Description
[0167] The β-Blocker Cholesterol-Lowering Asymptomatic Plaque Study (BCAPS) was a randomized, double blind, placebo-controlled, single-center clinical trial that took place between 1994 and 1999. A detailed description of BCAPS can be found in the original paper (Hedblad et al. 2001. Circulation 103: 1721-1726), following here is a brief summary. The original study population consisted of 361 men and 432 women (total n=793) 49 to 70 years of age recruited from MDC-CC. Subjects included in BCAPS had plaque in the right carotid artery but no symptoms of carotid artery disease. Exclusion criteria were: myocardial infarction, angina pectoris or stroke within the previous 3 months; previous surgery in the right carotid artery; regular use of β-blockers or statins; blood pressure >160 (systolic) or >95 (diastolic) mm Hg; total cholesterol >8.0 mmol/l; hyperglycemia suspected to require insulin treatment; and conditions that in the view of the investigator made the participant unsuitable for the trial.
[0168] All participants provided written consent and the study was approved by The Ethics Committee of Lund University.
[0169] The study consisted of 4 different treatment groups and the participants were randomized to 1 of the following: placebo/placebo, metoprolol CR/XL (25 mg once daily)/placebo, fluvastatin (40 mg once daily)/placebo or metoprolol CR/XL (25 mg once daily)/fluvastatin (40 mg once daily).
[0170] The treatment period was 36-months. Fasting blood samples were drawn at baseline and at 12, 24 and 36 months. HDL-C, total cholesterol and TG were measured according to standard procedures at the Department of Clinical Chemistry, University Hospital of Malmö. LDL-C levels were calculated according to the Friedewald formula. GH levels were measured in stored fasting plasma samples, which were frozen immediately to −80° C. at the different examinations. A number of the samples stored in the freezer were missing or had insufficient plasma for the analysis of hs-GH. Subjects in which either the baseline-value of GH or the 12-month value of GH was missing were excluded from further analysis. After this adjustment the study consisted of 472 individuals. All samples were measured using the hs-hGH immunoassay as described above.
Ultrasound
[0171] Carotid ultrasound was performed at baseline, 18 and 36 months (Hedblad et al. 2001. Circulation 103: 1721-1726). In brief, a predefined extent of the right carotid bifurcation was scanned for presence of plaques and the maximum IMT at the bifurcation (IMT.sub.bulb) and the mean IMT in the common carotid artery (IMT.sub.cca) was measured.
Statistical Analyses
[0172] In the analyses where treatment groups are compared, the groups are either the 4 randomization groups or they are divided in to 2 groups which are fluvastatin vs non-fluvastatin (i.e placebo) or metoprolol vs non-metoprolol. Since fasting hs-GH is known to differ between men and women all models were performed separate for men and women as well as combined. All models were adjusted for age, sex (if not already gender separated) and the natural logarithm of the fasting level of hs-GH at baseline.
[0173] To investigate if fasting hs-GH was affected by treatment with fluvastatin or metoprolol we performed multiple regression analysis with ΔGH (baseline value subtracted from 12 month value) as the dependent variable and the treatment groups as independent variables.
[0174] We then analyzed if hs-GH affects the treatment effect of the different medicines on the IMT in a series of multiple regression models with IMT at 36 months as the dependent variable. In these analyses the larger randomization groups were used to gain more power. An interaction term between the treatment groups multiplied with ΔGH or the natural logarithm of hs-GH, depending on model, was created to evaluate this interaction. A small number of individuals were lost during follow-up and could not complete the ultrasound examination of IMT at 36 months. When analyzing the IMT, the latest IMT-values (i.e. at 18 months) are used for these individuals and the analysis is adjusted for time between IMT-measurements. The models were in addition adjusted for the natural logarithm of IMT at baseline.
[0175] All analyses were performed in SPSS (version 22.0.0, SPSS Inc., Chicago, Ill.). A 2-sided P-value of less than 0.05 was considered statistically significant.
Results
[0176] Baseline characteristics of the 472 individuals in the BCAPS study population are shown in table 11. The females in all groups had higher values of hs-GH at baseline compared to males. The subjects in which hs-GH could not be measured due to missing samples at either baseline or 12 months did not differ in their baseline characteristics. The missing samples were evenly distributed over the different treatment groups, but generally more men than women were in the group of missing samples. This made the male/female ratio marginally different in two of the four treatment groups (table 11).
[0177] In linear regression models the change in levels of hs-GH at 12 months compared to baseline (ΔGH) were related against the different treatment groups (Table 12). When analyzing the whole cohort none of the analyses were significant. In males treatment with fluvastatin/metoprolol had a significant negative correlation with ΔGH compared against placebo (P=0.015). There was also a significant negative correlation between ΔGH and fluvastatin treatment compared to placebo (P=0.046). Among females the correlations were positive and significant for fluvastatin/metoprolol (P=0.010) and borderline significant for fluvastatin (P=0.051).
[0178] Male subjects treated with fluvastatin had a greater reduction of hs-GH over 12 months as compared to subjects not treated with fluvastatin. In males, treatment with metoprolol/fluvastatin compared to placebo/placebo was significantly associated with a greater reduction of hs-GH over 12 months of treatment independently of baseline level of hs-GH. Moreover, when all male subjects receiving fluvastatin (metoprolol/fluvastatin and placebo/fluvastatin) were compared with all subjects with no fluvastatin treatment, fluvastatin treatment was also associated with a greater 12 months reduction of hs-GH, suggesting that the fasting value of hs-GH is lowered by treatment with fluvastatin in males. However in this context it should be noted that the change of hs-GH over 12 months in the fluvastatin/placebo-group was not significantly different when compared to the placebo/placebo group.
TABLE-US-00013 TABLE 11 Clinical characteristics of the study population in BCAPS. Variable Placebo/Placebo Metoprolol/Placebo Fluvastatin/Metoprolol Fluvastatin/Placebo Number of participants 117 118 117 120 Female (%) 78 (66.7) 78 (66.1) 70 (59.8) 72 (60.0) Age, mean (SD), years 61.5 (5.7) 60.3 (5.6) 62.3 (5.0) 61.8 (5.4) Height, mean (SD), cm 168 (10) 167 (8) 169 (8) 169 (9) Baseline Body Mass Index, Mean (SD), 25.5 (3.6) 25.5 (3.6) 25.1 (2.7) 25.7 (3.7) kg/m2 LDL-C, mean (SD), mmol/L 4.07 (0.86) 4.18 (0.92) 4.15 (0.88) 4.16 (0.82) HDL-C, mean (SD), mmol/L 1.48 (0.40) 1.39 (0.37) 1.41 (0.35) 1.37 (0.35) IMT.sub.cca, median (IQR) 0.88 (0.78-0.96) 0.88 (0.77-0.99) 0.86 (0.79-0.98) 0.86 (0.78-0.97) IMT.sub.bulb, median (IQR) 1.74 (1.46-2.09) 1.77 (1.49-2.24) 1.84 (1.52-2.29) 1.81 (1.55-2.17) Growth Hormone - males, 240 (150-390) 170 (110-270) 230 (140-380) 190 (130-260) geometric mean, 95% CI, pg/mL Growth Hormone - females, 1260 (970-1660) 1070 (780-1440) 1080 (840-1390) 1210 (940-1570) geometric mean, 95% CI, pg/mL 12 months LDL-C, mean (SD), mmol/L 4.01 (0.82) 4.17 (0.87) 3.21 (0.85) 3.27 (0.74) Growth Hormone - males, 250 (160-410) 170 (110-280) 220 (150-340) 160 (110-230) geometric mean, 95% CI, pg/mL Growth Hormone - females, 830 (640-1090) 860 (660-1130) 1090 (820-1460) 810 (610-1070) geometric mean, 95% CI, pg/mL 36 months IMT.sub.cca, median (IQR) 0.91 (0.82-1.04) 0.90 (0.83-1.02) 0.87 (0.78-0.98) 0.87 (0.77-0.99) IMT.sub.bulb, median (IQR) 1.98 (1.71-2.35) 1.89 (1.60-2.43) 1.99 (1.60-2.44) 2.02 (1.68-2.30) Abbreviations: LDL-C, Low-density lipoprotein cholesterol; HDL-C, High-density lipoprotein cholesterol; IMT, intima media thickness. Missing values in IMTbulb: placebo/placebo, n = 3; metoprolol/placebo n = 3; Fluvastatin/Metoprolol, n = 5; fluvastatin/placebo, n = 8. Missing values in HDL-C and LDL-C: placebo/placebo, n = 1; metoprolol/fluvastatin n = 1.
TABLE-US-00014 TABLE 12 Multiple linear regression models of ΔGH (12 months - baseline) vs different treatment groups. Gender Model Treatment group B 95% CI P ALL All groups Metoprolol-Placebo −0.11 −0.53 to 0.31 0.600 Fluvastatin-Metoprolol 0.25 −0.17 to 0.67 0.241 Fluvastatin-Placebo −0.11 −0.53 to 0.31 0.595 All Fluvastatin Fluvastatin 0.12 −0.18 to 0.42 0.423 All Metoprolol Metoprolol 0.13 −0.17 to 0.43 0.399 MALE All groups Metoprolol-Placebo −0.26 −0.79 to 0.28 0.342 Fluvastatin-Metoprolol −0.64 −1.16 to −0.13 0.015 Fluvastatin-Placebo −0.36 −0.88 to 0.15 0.165 All Fluvastatin Fluvastatin −0.37 −0.73 to −0.01 0.046 All Metoprolol Metoprolol −0.27 −0.63 to 0.10 0.150 FEMALE All groups Metoprolol-Placebo −0.06 −0.63 to 0.51 0.842 Fluvastatin-Metoprolol 0.77 0.185 to 1.35 0.010 Fluvastatin-Placebo 0.02 −0.56 to 0.59 0.956 All Fluvastatin Fluvastatin 0.41 0.00 to 0.83 0.051 All Metoprolol Metoprolol 0.33 −0.09 to 0.74 0.119
[0179] Models adjusted for: age and standardized values of natural logarithm of hs-GH at baseline. In addition adjusted for sex in the subgroup all.
[0180] Three different models are executed: in “all groups” the different treatment groups are each one compared against placebo. In “all fluvastatin” the individuals receiving fluvastatin are compared against individuals not receiving fluvastatin and vice versa in “all metoprolol”. B coefficients are expressed as the increment of ΔGH with treatment of the medicine in question as compared to placebo.
FIGURE DESCRIPTION
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