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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
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• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/975—Kit
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
  • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
  • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

• This invention relates to a diagnostic method for determining statin induced myopathy.
• the method is especially applicable for determining warn- ings, early signs and also symptomatic myopathy.
• the method includes collecting and comparing iipid biomarker patterns to reference lipidomic markers.
• the method also includes chemometric modelling and statistical analysis of the biomarker patterns.
• This invention further relates to a kit for performing a diagnostic method for determining statin induced myopathy.
• statins 3-hydroxy-3 ⁇ methylglutaryl-coenzyme A reductase inhibitors
• statins have been showed in large clinical trial to effectively lower cholesterol blood levels.
• High cholesterol values are very common in Europe and in USA and the use of statins to lower cholesterol values is increasing drastically.
• statin use increased from 1997 to 2002 by 30% on average.
• statin therapy is associated with adverse effects. The most prevalent and important adverse effect associated with statin therapy is myopathy.
• Myopathy is a collective term for various muscle related problems, such as muscle pain (myalgia), weakness and cramps (Paul D. Thompson et al, Am J Cardiol 2006, 97 [suppl]; 69C-76C). The exact mecan- ism for statin induced myopathy is still unclear.
• a recent study showed that clinically acceptable doses of atorvastatin and simvastatin resulted in lowered levels of plasma ubiquinone.
• Ubiquinone is a coenzyme that is involved in mithocondrial electron transport and is therefor involved in tissue energy metabolism.
• Statins such as atorvastatin and simvastatin clearly have an effect on sceletal muscle (Paiva et al, Clin Pharmacol Ther 2005; 78:60-8).
• IVletabolomics is a discipline dedicated to the systematic study of small molecules ⁇ i.e., metabolites) in cells, tissues, and biofluids. Metabolites are the end products of cellular regulatory processes, and their levels can be regarded as the amplified response of biological systems to genetic or envi- ronmenta! changes. Clinicians have relied for decades on a small part of the information contained in the metabolome, for example measuring glucose to monitor diabetes and measuring choiesteroi for cardiovascular health. New sophisticated rnetabolomic analytical platforms and informatic tools have already been developed that afford extended and sensitive measurement of the rnetabotome.
• Lipids are known to piay an important role as structural components (e.g., cell membranes), energy storage components, and as signalling molecules. Lipids are broadly defined as hydrophobic or amphipathic small molecules that may originate entirely or in part by carbanion based condensation of thioesters, and/or by carbocation based condensation of isoprene units.
• Lipi- domics can be considered as a sub-fiefd of metaboiomics which aims to elucidate the biological processes in the context of lipids by measuring and characterizing the extended lipid profiles at the molecular level (lipidomic profiles).
• Traditional clinical lipid measures quantify total amounts of triglycerides, cho- iesterol, or lipoproteins.
• lipid profile is more complex at the molecular level.
• Current lipidomics platforms enable quantitative characterization of 100s of diverse lipid molecular species across multiple lipid classes such as sphingoltpids, phospholipids, sterol esters, acylglycerols, sterols, bile acids, fatty acids, eicosanoids, and steroids.
• Myopathy is today mainly diagnosed from the symptoms of the patient, Elevated creatine kinase (CK) levels can be used for testing patients with muscle symptoms. However, CK levels can be elevated due to other reasons such as exercise, and is not a reliabie biomarker for statin induced myopathy.
• the present invention discloses a diagnostic method for determining the risk and early signs of statin induced myopathy.
• This invention discloses a method for determining statin induced myopathy comprising the steps: a) providing a biological sample from an individual prior to or during statin treatment, b) collecting a lipidomic profile from said biological sample, c) comparing said collected lipidomic profile to reference lipidomic markers, wherein said reference ⁇ pidom ⁇ c markers have been established by combining a pro- inflammatory gene expression profile with a iipidomic profile connected to high dosage statin treatment.
• the method is especially useful for determining the risk or early warning signs of statin induced myopathy.
• the method is aiso useful for de- termining statin induced myopathy in individuals showing clinical symptoms of myopathy.
• Another aspect of the invention is to provide a kit for determining statin induced myopathy.
• Figure 2 denotes loadings on LV3 for most important lipids in simvastatin (B) or atorvastatin (A) groups selected by VIP analysis from figure 1. Onfy lipids for which at least one of the two groups has VIP value greater than 2 are shown.
• the objective of the present invention is to provide an early stage biomarker for statin induced myopathy.
• the early stage biornarker can be used for determining a risk of developing myopathy as a result of cholesterol lowering treatment with statins before any symptoms of actual myopathy occurs.
• the biomarker can also be used for early warning signs of statin induced myopathy.
• the cholesterol lowering medication can be adjusted when early sign of myopathy is detected.
• the biomarkers can be used to determine statin induced myopathy, when clinical symptoms of myopathy already occurs.
• the inventors have now surprisingly found that iipidomic biomarkers can be used as biomarkers for statin induced myopathy.
• the present invention provides a method for determining the risk and early warning signs for a patient to develop statin induced myopathy.
• the method is based on comparison of the established lipid profile from an individual to a reference iipidomic markers.
• the reference Iipidomic markers are created by combining gene expression analysis data with serum iipidomics data.
• Gene expression profiles associated with high dose statin treatment have been detected by whole genome microarray analysis of muscle biopsies.
• the information from the microarray analysis and Iipidomics analyses are combined and statistically modified to provide Iipidomic markers useful for statin induced myopathy.
• This invention discloses a method for determining myopathy.
• the method is useful for determining early signs of myopathy prior to any clinically observable signs occur.
• the benefits of the current method is that the statin treatment can be adjusted or stopped before any physical myopathy symptoms occu re.
• Another aspect of the current invention is to provide a method for determining statin induced myopathy in individuals already suffering from clinical myopathy symptoms.
• the method according to the current invention can be used as a biochemical diagnostic method for myopathy in patients already experiencing muscle pains and other symptoms of myopathy.
• the method can be used as a verification diagnostic method besides other clinica! diagnosis of myopathy.
• Creatine kinase (CK) levels are usually measured in patients suspected of suffering from myopathy.
• the disclosed method can be used in parallel with CK level measurements. CK levels are not reliable biomarkers for myopathy since CK levels can be high because of e.g. small muscle injuries after exercise and physical activity.
• the current inventions provides more reli- able biomarkers for statin induced myopathy than CK levels.
• the current invention provides a method for determining statin induced myopathy comprising the steps: a) providing a biological sample from an individual prior to or during statin treatment, b) collecting a iipidomic profile from said bioiogical sample, c) comparing said collected lipid profile to reference iipidomic markers, wherein said reference iipidomic markers have been established by combining a pro-inflammatory gene expression profile with a Iipidomic profile connected to high dosage statin treatment. The difference between the collected Iipidomic profile and the reference iipidomic markers indicates or is associated with statin induced myopathy.
• the difference between the collected Iipidomic profile and the reference Iipidomic markers can also be used for determining a risk of or susceptibility for developing statin induced myopathy,
• the method according to the current invention can be used for determining a risk to develop statin induced myopathy as a result of statin treatment
• the method according to the current invention can be used for determining early warning signs of statin induced myopathy.
• the early warning signs can be determined before actual symptoms of myopathy occurs in the individual.
• the method according to the current invention can be used for determining statin induced myopathy in individuals already showing signs of myopathy.
• the current method can be a biochemical verification of clinically diagnosed myopathy.
• the biological sample can be whole blood, serum, plasma sample or a tissue sample. Taking a blood sample of a patient is a part of normal clinical practice. The blood sample can be taken in connection with e.g. measuring the cholesterol levels in the patients. The collected biood sample can be pre- pared and serum or plasma can be separated with techniques well known for a person skilled in the art.
• Collecting a Iipidomic profile from said biological sample can be performed with various chemical and high resolution analytical techniques.
• Suitable analytical techniques include but are not limited to mass spectrometry and nuclear resonance spectroscopy. Any high resolution technique capable of re- solving individual lipids or lipid classes and provide structural information of the same can be used to collect the lipid profile from the biological sample.
• Coliecting the lipidomic profile with mass spectrometry is one embodiment of the current invention.
• the MS instrument can be coupled to a high performance separation method such as HPLC or UPLC.
• the analytical technique used for collecting the lipid profile should be able to quantify or measure either the exact amount or at least a relative amount of the individual lipids or lipid classes.
• the amount of the individual lipids or lipid classes in the collected lipidomic profile is used when comparing the collected lipid profile to the reference lipidomic biomarkers.
• the reference lipidomic biomarkers can be established from the same individual receiving the statin treatment or it can be from a generalised population. If the same individual is used to create the reference lipidomic marker, then a sample is collected from the individual prior to statin treatment. The reference lipidomic marker is then created from that first lipid profile of that individual. This lipidomic marker is used as a base-Hne or starting point. A series of iipidomic profiles can be collected during statin treatment. These lipidomic profiles are then compared with the reference lipidomic marker that was created prior to statin treatment. The reference lipidomic markers can also be created from a generalized population. If a generalized population is used then several lipid profiles from a population are combined and the lipidomic marker is created from this combination.
• the reference lipidomic markers are one or more lipid(s) selected from the lipids presented in table 1 , more preferable in table 2.
• the reference iipidomic marker is created by combining gene expression data with lipidomics analysis as described below. The levels or amounts of the individual lipids or lipid classes are compared to the levels or amounts of the individual lipids or lipid classes in the reference lipidomic bio- markers for determining statin induced myopathy. Table 1
• GPCho phosphatidylcholine
• SM sphingomyelin
• GSEA gene set enrichment analysis
• Partial Least Squares Discriminant Analysis revealed drug-specific changes in lipid profiles ( Figure 1 ).
• the differences between the simvastatin and atorvastatin lipid profiles were found in the third latent variable (LV3).
• ViP variable
• the most important lipid species were identified for each intervention group.
• the list of loadings in direction of atorvastatin-simvastatin differences (LV3) for most important lipids in simvastatin and atorvastatin groups is shown in Figure 2.
• the main plasma lipid profile differences between the two statins appear itpid-ciass specific, with upregulation of several phosphati- dylethanolamines and long chain triacyiglycerols, and down regulation of ether phosphatidylcholine and cholesterol esters in the simvastatin group.
• Gene expression analysis revealed upregulated pathways in skeletal muscle associated with inflammation and mitochondrial damage in the high dose simvastatin intervention group. We investigated if any of these changes are associated with the differences observed in the serum lipidome.
• kits for performing a method for determining statin induced myopathy comprises reference lipids to form the Iipidomic reference biomarkers and necessary reagents.
• Lipids from the Iipidomic analysts were named according to Lipid Maps (http://wwwJipidmaps.org). For example, lysophosphatidylchoSine with 16:0 fatty acid chain was named as monoacyl-glycerolphosphocholine GPCho(16:0/0:0). In case the fatty acid composition was not determined, total number of carbons and double bonds was marked. For example a phosphatidylcholine species GPCho(16:0/20:4) is represented as GPCho(36:4). However GPCho(36;4) couid represent other molecular species such as GPCho(20:4/16:0) or GPCho(18:2/18:2). Such mass isomers may be sepa- rated chromatographically.
• the study patients had never been treated with statins before. They were instructed to adhere to their normal diet during the study. Patients with familial hypercholesterolemia and patients with serum total cholesterol > 7.0 mmol/L in the initial screening were excluded. Other exclusion criteria were: use of concurrent lipid altering medication or antioxidant vitamins, renal or hepatic dysfunction, and use of medication known to affect metabolism of ator- vastatin or simvastatin.
• the study protocol was accepted by the Ethics Committee of the Tampere University Hospital and written informed consents were obtained from ail participants.
• Microarray experiments were performed by using Sentrix® Human- 6 Expression BeadChips analyzing over 46 000 known genes, gene candi- dates and spiice variants ⁇ lllumina, San Diego, CA, USA) according to given instructions.
• the biopsy samples were homogenized using Ultra-Turrax (IKA Turrax T8 / S8N-5G, iKA-Werke, Staufen, Germany).
• the total RNA was extracted using TRIzol (#15596-018, Invitrogen Corporation, Carisbad, CA) 1 DNase treatment and a second RNA purification by Qiagen kits (#74106, and, #79254, Qiagen GmbH, Hifden, Germany), ail by given instructions.
• RNA from each sample were amplified to cDNA using Ambion's lilumina RNA Amplification kit following the instructions (cat no 11755, Ambion, Inc., Austin, TX, USA).
• In vitro transcripiton (iVT) reaction of cDNA to cRNA was performed overnight (14h) including biotin-11-dUTP (PerkinElmer, cat no PC 3435-0402-Biotin-11-dUTP, >95%, NEL539001 EA, PerkinElrner Life And Analytical Sciences, Inc., Boston, MA, USA) for labelling the cRNA product.
• biotin-11-dUTP PerkinElmer, cat no PC 3435-0402-Biotin-11-dUTP, >95%, NEL539001 EA, PerkinElrner Life And Analytical Sciences, Inc., Boston, MA, USA
• RNA/cRNA concentrations were checked with Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) and RNA/cRNA quality was controlled by BioRad's Experion Automated Electrophoresis System and RNA StdSens Analysis Kit (BioRad Laboratories, Inc., Hercules, CA 1 USA).
• Raw intensity data obtained from the lllumina piatfrom were normal- ized with inforsense Knowledge Discovery Environment (inforsense, London, UK) using non-linear cubic-sp ⁇ ne normalization.
• the lnforsense KDE platform was also used to conduct singte-gene analysis including fold-change calculations and filtering the probes.
• microarray expression results recorded in the simvastatin group were verified by RealTime Quantitative TaqMan PCR.
• Previously purified cRNA was used as starting material for cDNA synthesis.
• a 1000 ng-18 ⁇ l aliquote of cRNA was mixed with 1 ⁇ ! Promega Random Primer (C1 181 , Promega U.S., Madison, Wl, USA) and incubated in +70 0 C for 10 min.
• PCR reaction consisting of 2 ⁇ l aiiquote of 1 :10 diluted cDNA sample, and, Abgene ABsolute 2x QPCR ROX mix (AB- 1 139, Abgene, Epsom, UK).
• the primer concentrations were 300 nM, probe concentrations for Universal Probe Library (Exiqon, Vedbaek, Denmark) probes 100 nM and for ordinary long probes 200 nM.
• rtPCR system (ABI Prism 7700 Sequence Detection System, Applied Biosystems) having the following PCR procedure: 95 0 C for 15 min, and 40 cycles of 95 0 C for 15 s and 60 0 C for 1 min.
• Lipid extracts were analysed on a Waters Q-Tof Premier mass spectrometer combined with an Acquity Ultra Performance LCTM (UPLC).
• the column which was kept at 50 0 C, was an Acquity UPLCTM BEH C18 10 * 50 mm with 1.7 mm particles.
• the binary solvent system included A. water (1 % 1 M NH 4 Ac 1 0.1 % HCOOH) and B. LC/MS grade (Rathbum) aceton ⁇ trile/- isopropano! (5:2, 1 % 1 M NH 4 Ac, 0.1 % HCOOH).
• the gradient started from 65% A/ 35% B 1 reached 100% B in 6 min and remained there for the next 7 min.
• the total run time including a 5 min re-equilibration step was 18 min.
• the flow rate was 0,200 ml/min and the injected amount 0.75 mf.
• the temperature of the sample organizer was set at 10 0 C.
• the lipid profiling was carried out on Waters Q-Tof Premier mass spectrometer using ESi+ mode. The data was collected at mass range of m/z 300-1200 with a scan duration of 0.2 sec. The source temperature was set at 120 °C and nitrogen was used as desolvation gas (800L/h) at 250 0 C. The videages of the sampling cone and capillary were 39 V and 3.2 kV, respectively. Reserpine (50 mg/L) was used as the lock spray reference compound (5m!/min; 10 sec scan frequency).
• Tandern mass spectrometry was used for the identification of se- lected molecuiar species of lipids. MS/MS runs were performed by using ESI+ mode, collision energy ramp from 15 to 30 V and mass range starting from m/z 150. The other conditions were as shown above.

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