초록 ▼

A novel method useful in drug screening. The method is useful for testing effects of substances on the mitochondria, notably toxic or beneficial effects of drug substances or candidate drug substances. The method is based on measurement in live human mitochondria ex vivo, but in a setting as near th

A novel method useful in drug screening. The method is useful for testing effects of substances on the mitochondria, notably toxic or beneficial effects of drug substances or candidate drug substances. The method is based on measurement in live human mitochondria ex vivo, but in a setting as near the in vivo situation as possible. The method is also useful for testing substances impact on the mitochondrial respiration. The method can be used to i) screening and selection of early or late stage drug candidates in cells derived from blood from healthy individuals or in so-called buffy coat, which is a concentrated solution of platelets and white blood cells, ii) testing a patient's sensitivity to a known mitochondrial toxicant, iii) analysing mitochondrial drug toxicity in clinical trials, and/or iv) analysing beneficial effects of drugs intended to improve mitochondrial function

대표청구항 ▼

1.-32. (canceled) 33. A method for screening and selection of potential drug candidates or for testing a person's sensitivity to a substance with effect on mitochondria, the method comprising i) subjecting a sample of human blood components containing mitochondria to high-resolution respirometry,ii)

1.-32. (canceled) 33. A method for screening and selection of potential drug candidates or for testing a person's sensitivity to a substance with effect on mitochondria, the method comprising i) subjecting a sample of human blood components containing mitochondria to high-resolution respirometry,ii) contacting the cell sample with a substance that increases the leakage of the inner mitochondrial membrane to protons,iii) adding a test sample comprising a test substance in a vehicle,iv) comparing the oxygen consumption before and after addition of the test sample and comparing the oxygen consumption of the test sample with the oxygen consumption of a control sample of the vehicle, wherein a decrease in oxygen consumption indicates a negative effect on the mitochondria,v) contacting the sample from iii) with an inhibitor of mitochondrial complex I-function, andvi) contacting the sample from v) with an inhibitor of mitochondrial complex III-function. 34. A method for investigating mitochondrial effects of drug candidates in clinical trials or in treatment regimens, the method comprising i) subjecting a sample of human blood components containing mitochondria to high-resolution respirometry, wherein the sample of cells is from a person subjected to a clinical study or to a treatment regimen, and wherein a test substance has been administered to the person during the clinical study or treatment regimenii) contacting the cell sample with a substance that increases the leakage of the inner mitochondrial membrane to protons,iii) comparing the oxygen consumption of the sample from the person subjected to the clinical study or to a treatment regimen with the oxygen consumption of a control sample, wherein a decrease in oxygen consumption indicates a negative effect on the mitochondria,iv) contacting the sample from iii) with an inhibitor of mitochondrial complex I-function, andv) contacting the sample from iv) with an inhibitor of mitochondrial complex III-function. 35. A method according to claim 33, wherein the high-resolution respirometry is performed at an oxygen concentrations in the range of 400-25 μM O2 at a constant temperature of 37° C. 36. A method according to claim 33, wherein the substance that increases the leakage of the inner mitochondria membrane to protons is added to obtain maximal capacity of the electron transport system of the mitochondria present in the sample. 37. A method according to claim 33, wherein the substance that increases the leakage of the inner mitochondria membrane to protons is selected from carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,4-Dinitrophenol (DNP) and other protonophores, and mixtures thereof. 38. A method according to claim 33, wherein the substance that increases the leakage of the inner mitochondria membrane to protons is added at a concentration in a range of from 0.1 to 10 μM such as from 0.5 to 5 μM, from 1 to 3 μM or about 2 μM. 39. A method according to claim 33, wherein the substance that increases the leakage of the inner mitochondria membrane to protons is FCCP. 40. A method according to claim 33, wherein the vehicle is an organic solvent such as e.g. ethanol, isopronol, methanol, DMSO (dimethylsulfoxide), DMF (dimethylformamide), or DMA (dimethylacetamide). 41. A method according to claim 39, wherein the test sample is in liquid form and the test sample contains a known concentration of the test substance. 42. A method according to claim 41 wherein the test sample is added in stepwise increasing concentrations. 43. A method according to claim 42, wherein the stepwise addition of test sample results in final concentrations of 1 μM to 10 mM of the test sample. 44. A method according to claim 33, wherein the control sample in iii) is identical to the test sample but without content of test substance. 45. A method according to claim 33, wherein the inhibitor of mitochondrial complex I-function is added to elucidate the cellular respiration dependent on oxidation of complex II-substrates. 46. A method according to claim 33, wherein the inhibitor of mitochondrial complex I-function is added at a concentration in a range of from 0.1 to 10 μM such as from 0.5 to 5 μM, from 1 to 3 μM or about 2 μM. 47. A method according to claim 33, wherein the inhibitor of mitochondrial complex I-function is rotenone. 48. A method according to claim 33, wherein the inhibitor of mitochondrial complex III-function is added to determine any non-mitochondrial oxygen-consuming activity, such as auto-oxidation of said sample. 49. A method according to claim 33, wherein the inhibitor of mitochondrial complex III-function is added at a final concentration of from about 0.1 to 10 μg/ml such as from about 0.5 to 5 μg/ml, from about 0.75 to 2 μg/ml or 1 μg/ml. 50. A method according to claim 33, wherein the inhibitor of mitochondrial complex III-function is antimycin A. 51. A method according to claim 34, wherein the control sample is from a control group. 52. A method according to claim 34, wherein the control sample is taken before any treatment starts. 53. A method according to claim 33 for screening of drug candidates. 54. A method according to claim 33 for testing a subject's sensitivity to a drug substance or a drug candidate. 55. A method according to claim 34 for evaluating mitochondrial toxicity of a test substance in clinical trials. 56. A method according to claim 33 for analysing the effect of a substance on mitochondrial function. 57. A method according to claim 54, wherein the sample of cells is isolated from said subject. 58. A method according to claim 33 for evaluating compound capable of stimulating mitochondrial respiration and ATP production. 59. A method for screening and selecting potential drug candidates or for testing the sensitivity of a person to a substance with effect on mitochondria, the method comprising i) subjecting a sample of human blood components containing mitochondria from a human blood sample to high-resolution respirometry,ii) contacting the sample of cell to a substance that inhibits complex I respiration,iii) adding a test sample containing a test substance in a vehicle,iv) adding a substance that permeabilizes the plasma membrane,v) adding a reference substance to the sample obtained in iv) wherein the reference substance is a complex II-linked substrate, andvi) adding a substance that inhibits complex III respiration, andcomparing the oxygen consumption obtained with the oxygen consumption obtained when the method is carried out using the reference substance in step iii) and thus omitting addition of a test sample. 60. A method according to claim 59, wherein the substance that permeabilizes the plasma membrane is digitonin. 61. A method according to claim 60, wherein digitonin is added in a concentration of from 5 μg/ml to about 250 μg/ml. 62. A method according to claim 59, wherein the reference substance is succinate. 63. A method according to claim 62, wherein the final concentration of the reference substance is from about 0.1 to about 20 mM.