Diagnostic marker compounds and their use

Abstract

The present Invention relates to the monitoring of biological substances, such as non-invasive monitoring of such substances in animal, for examples biomarkers and metabolites. Specifically, the invention further relates to such monitoring using rare earth tagged marker compounds. The invention further relates to such monitoring using laser spectroscopy or Raman spectroscopy. The invention further relates to the use of such monitoring in disease states, such as stroke, neurological disorders and cardiovascular disorders. The invention further relates to novel rare-earth conjugated marker compounds and processes for preparing said rare-earth conjugated compounds

Claims

1. A method for the measurement of a metabolites in an animal comprising: (a) using spectroscopy, detecting a rare earth metal-conjugated marker formed from a rare earth metal conjugated with the metabolite, wherein the spectroscopy is laser spectroscopy or Raman spectroscopy, and wherein the spectroscopy uses an excitation wavelength and a measurement wavelength, each being in the range of about 600 nm to about 2100 nm; and (b) measuring the metabolite based on the detected rare earth metal-conjugated marker.

2. A method according to claim 1 wherein the measured-metabolite correlates with a disease condition.

3. A method according to claim 2 wherein the disease condition is cardiovascular diseases, neuropsychiatric diseases, neurological diseases or cancer.

4. A method according to claim 3 wherein the rare earth metal-conjugated marker is Eu.sup.3+-conjugated lactic acid.

5. A method according to claim 2 wherein the rare earth metal-conjugated marker is Eu.sup.3+-conjugated lactic acid.

6. A method according to claim 1 wherein the detecting is non-invasive.

7. A method according to claim 6 wherein the rare earth metal-conjugated marker is Eu.sup.3+-conjugated lactic acid.

8. A method according to claim 1 wherein the metabolite is (i) a small molecule metabolite; (ii) a lipid; (iii) a peptide; (iv) a protein; or (v) an enzyme.

9. A method according to claim 8 wherein the metabolite is: (i) an amino acid or related compounds selected from taurine, glutamine, N-acetyl-L-asparate (NAA), and homocysteine; (ii) Lipids and related intermediates selected from phosphatidylcholine and phosphocholine; (iii) lipid binding proteins selected from lipoprotein A, HDL and LDL; (iv) Peptides/Proteins selected from PARK 7, Nucleoside Diphosphate Kinase A (NDKA), amyloid beta peptide, Tau (e.g. hyperphosphorylated Tau), CD68, CD64, carcino-embryonic antigen (CEA), tumor-associated glycoprotein 72 (Tag72), folate receptor-, Alpha actin, Toll-like receptors (TLRs) Creatine, Creatinine, amyloid precursor protein (APP), troponin, C-reactive protein, Fibrinogen and B-type natriuretic peptide (BNP) (v) Enzymes selected from phospholipases, -secretase, -secretase, succinate dehydrogenase (SDH), fumarate hydratase (FH), neprilysin (NEP), endothelin-converting enzyme (ECE), insulysin (IDE), angiotensin-converting enzyme (ACE) and matrix metalloproteinase 1-9 (MMP 1-9), Creatine kinase (CK) and creatine kinase isoenzyme MB (CKMB) (vi) Cytokines selected from IL(1-6) and TNF; or (vii) small molecule metabolites selected from lactate, glucose, acetyladehyde hydrate, acetate, choline, inositol.

10. A method according to claim 9, wherein the metabolite is lactic acid.

11. A method according to claim 8 wherein the rare earth metal is Cerium, Dysprosium, Erbium, Europium, Gadolinium, Holmium, Neodymium Praseodymium, Samarium, Terbium, Thulium or Ytterbium.

12. A method according to claim 1 wherein the rare earth metal is Cerium, Dysprosium, Erbium, Europium, Gadolinium, Holmium, Neodymium Praseodymium, Samarium, Terbium, Thulium or Ytterbium.

13. A method according to claim 12 wherein the rare earth metal is Ce.sup.4+,3+, Yb.sup.3+, Eu.sup.3+, Sm.sup.3+, Tm.sup.3+, Tb.sup.3+ or Nd.sup.3+.

14. A method according to claim 13, wherein the rare earth metal is Eu.sup.3+.

15. A method according to claim 1 wherein the rare earth metal-conjugated marker is Eu.sup.3+-conjugated lactic acid.

16. A method according to claim 1, wherein a photoluminescence intensity ratio (PLIR) imaging technique is used to calculate the level or a change in level of the metabolite.

Description

(1) The invention will now be illustrated with the following non-limiting examples with reference to the following figures.

(2) FIG. 1 shows the emission spectrum of Eu.sup.3+ doped lactic acid in two different concentration ranges: (a) the fluorescence emission spectra of 0.5M europium nitrate alone and in the presence of a variation of concentrations of lactic acid when excited at 395 nm. The mole fraction of lactic acid is of the order of 10.sup.6 (b) the fluorescence emission spectra of 0.5M europium nitrate in the presence of a variation of concentrations of lactic acid when excited at 395 nm. The mole fraction of lactic acid is of the order of 10.sup.3

(3) FIG. 2 These graphs represent the cell viability of neuroblastoma cells using the MTT assay (Experiment 2) in the presence of a variation of concentrations of ytterbium nitrate for 24 hours (a) and 5 days (b) compared to a control average. The concentrations are plotted on the x-axis where the control population were not administered any ytterbium nitrate. The bar represents the Mean for the viability compared to the control average, with the Standard Error of the Mean also shown above the bar. For (a) n=32 and for (b) n=16. All groups were compared with the Mean at a level of significance p<0.05. **=p<0.01.

(4) FIG. 3 shows cytoprotective test results for SH-SY5Y cells treated with cerium or europium nitrate solution for 24 hours after treatment with hydrogen peroxide for 24 hours.

(5) FIG. 4 shows fluorescence microscopy images of untreated SH-SY5Y cells (1), previously treated with 1000 M cerium nitrate for 24 hours (2), and previously treated with 1000 M europium nitrate for 24 hours (3).

(6) FIG. 5 shows (a) confocal image of 100 M aq europium nitrate mixed with phospholipids when excited with diode laser at 405 nm (b) fluorescence emission spectra for 100 M aq europium nitrate from a confocal image of 100 M aq europium nitrate mixed with phospholipids when excited with diode laser at 405 nm.

(7) FIG. 6. shows confocal laser scanning microscope images of a phospholipid control (a), Eu.sup.3+ conjugated phospholipid (b) and Eu.sup.3+ conjugated lactic acid (c). Images were taken in the wavelength range 570 to 650 nm with a diode laser at 405 nm. The image of phospholipids conjugated with Eu.sup.3+ is predominantly yellow-orange and that of Eu.sup.3+ conjugated lactic acid is predominantly reddish in colour.

(8) FIG. 7; shows the photoluminescence intensity ratiometric (PLIR=I.sub.616/I.sub.591) determination principle. (a) Photoluminescence intensity ratio corresponding to PLIR>1 for mole fraction of lactic acid in solution with lactic acid in lower concentration (left Y-axis) and for mole fraction of europium nitrate in solution (right Y-axis). (b) Photoluminescence intensity ratio corresponding to PLIR<1 case for mole fraction of lactic acid in solution with lactic acid in higher concentration (left Y-axis) and for mole fraction of europium nitrate in solution (right Y-axis).

(9) FIG. 8 shows (a) confocal image of (0.2 ml) 100 M europium nitrate with mouse artery when excited with diode laser at 405 nm. (b) Fluorescence emission spectra for 100 M aq europium nitrate with mouse artery

(10) FIG. 9 shows Cerebellum sections following IP administration of europium nitrate.

(11) FIG. 10 shows a mouse carotid artery treated with 100 M europium as seen under: (a) bright field microscopy; and (b) under fluorescence microscopy using a DAPI filter (which allows emission in the range 470 nm-500 nm) The fluorescent areas (white in the black and white image) in (b) are the areas where europium has conjugated with the lipid regions of the artery.

(12) FIG. 11 shows confocal laser scanning microscope images of mouse brain tissue exposed to different concentrations of europium nitrate at 20 magnification. E represents the presence of europium ions. (a) 0.1 M europium nitrate (also showing 6 magnification of image) (b) 1 mM europium nitrate (c) control (no europium nitrate)

ABBREVIATIONS USED

(13) DAPI 4,6-diamidino-2-phenylindole

(14) MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide

(15) PBS Phosphate-buffered saline

(16) RE Rare earth metal

(17) UV ultraviolet

(18) VIS visible

Example 1Preparation of Rare-Earth Metal Tagged Molecules

(19) For conjugation studies using ytterbium nitrate Yb(NO.sub.3).sub.3 this was first dissolved in distilled water (0.01M) which provided a pH of 5.66, outside of the desirable range of 6.5 to 7.5 which is close to physiological ranges. Titration between ytterbium nitrate solution and NaOH was unsuccessfully performed in an attempt to neutralize the acidity.

(20) Yb(NO.sub.3).sub.3 solutions were prepared in phosphate buffered saline (PBS, pH7.2) varying from 0.01M to 1M without any significant change from the initial pH of the buffer solution. When analysed for UV/VIS, the wavelength of maximum absorption of Yb(NO.sub.3).sub.3 dissolved in 7.2 pH buffer was consistent for all the concentrations analysed, with 7 concentrations having a maximum peak at 970 nm and the remaining 3 at 971 nm. It was clear from the initial pH trial that Yb(NO.sub.3).sub.3 is a hydrogen donor, which is why as its concentration increases in water, the pH of its solutions decreases.

(21) The results from the final trial suggest that Yb(NO.sub.3).sub.3 is adequately buffered in the 7.2 pH buffer to remain within the target range (6.5-7.5 pH) for the highest concentration of ytterbium solution that is intended to be used (1M). For conjugation with phospholipid (L--Lecithin, Egg Yolk (from Merck Chemicals Ltd)), 100 M solutions were prepared either in distilled deionised water or in a cell culture medium of supplemented Ham's F12 medium and Eagle's minimal essential medium as, described as SH, by Webster et al. (2004, Brain Res Mol Brain Res, 130: 161-169). to maintain the physiological environment of the cells and added to 10 ml Yb(NO3)3 solution prepared in PBS (7.2 pH). After 24 hr 1 g of 8.4% phospholipase A2 powder from bovine pancreas (Sigma) was manually mixed into Yb(NO3)3 and phospholipid solutions after 10 minutes of thawing from a frozen state at room temperature. This mixing occurred within minutes of the analysis to prevent any adaptation, including denaturing, of the enzyme's molecular structure prior to use.

(22) Europium was also couple to lactate. Measurements were taken and analysed at two different lactic acid concentration ranges. FIG. 1(a) shows the Fluorescence emission spectra of 0.5M europium nitrate alone and when coupled with lactic acid with lactic acid in lower concentration when excited at 395 nm. FIG. 1(b) shows the Fluorescence emission spectra of 0.5M europium nitrate alone and when coupled with lactic acid where the lactic acid is in higher concentration when excited at 395 nm. The photoluminescence intensity ratio (PLIR), defined by the ratio of the fluorescence intensity at 616 nm to the fluorescence intensity at 591 nm (I.sub.616/I.sub.591) and may represent the chemical environment of the Eu.sup.3+ ion. As shown in FIGS. 1(a) and 1(b), the PLIR reverses when the mole fraction of lactic acid is very high compared to when the mole fraction is low, such that the PLIR>1 at low lactic acid concentrations and the PLIR<1 at high lactic acid concentrations.

(23) FIG. 1(a) shows that the main peak emission of Eu.sup.3+ conjugated-lactic acid is at 616 nm when the concentration of lactic acid expressed in mole fraction is less than about 510.sup.6. Therefore, at low concentrations of lactic acid the PLIR is greater than 1, as plotted in FIG. 7(a). FIG. 7(a) also shows the corresponding PLIR variation with the europium nitrate mole fraction which is around 910.sup.3. The plot shown in FIG. 7(a) can therefore be used for calibration of the amount of lactic acid based on the measured PLIR under these conditions. When the fluorescence image is recorded under these conditions the image will be predominately reddish as shown in FIG. 6(c). FIG. 1(b) shows the photoluminescence intensity when the mole fraction of lactic acid is at higher concentrations and the corresponding PLIR is plotted in FIG. 7(b). As shown in FIG. 1(b) the main peak fluorescence emission of Eu.sup.3+ conjugated-lactic acid is at 593 nm when the concentration of lactic acid expressed in mole fraction is of the order of 10.sup.3. Therefore, at high concentrations of lactic acid the PLIR is less than 1, as plotted in FIG. 7(b). The plot shown in FIG. 7(b) can therefore be used for calibration of the amount of lactic acid based on the measured PLIR under these conditions. The image will be yellowish as shown in FIG. 6(b) and the spectrum peaks at 593 nm instead of 616 nm as shown in FIG. 1(b).

Example 2Viability of 5SH5Y Cells in the Presence of Ytterbium Nitrate

(24) We confirmed the cell viability of 5SH5Y cells in a variation of concentrations of ytterbium nitrate using the MTT assay. The MTT Cell Proliferation Assay is a colorimetric assay system which measures the reduction of a yellow tetrazolium component (MT) into an insoluble purple formazan product by the mitochondria of viable cells which are solubilized by the addition of a detergent. The colour can then be quantified by spectrophotometric means. For each cell type a linear relationship between cell number and absorbance is established, enabling accurate, straightforward quantification of changes in proliferation. Cells derived from a sympathetic tumour were used as they proliferate rapidly (labelled human neuroblastoma cells from the SH-SY5Y cell-line (European Collection of Cell Cultures, Porton Down, Wiltshire, UK), of passage 8 to 10). These were cultured in SH medium (see Example 1) in 500 ml tissue culture flasks under incubation at 37 C. until the confluence was deemed at least 75% under microscopic analysis. Twenty-four hours before the cells were needed for treatment, cells were cultured in 96-well plates to approximately 50% confluency or greater. Experiments were carried out when all of the cell groups showed a similar confluency when viewed under the microscope. Cells were then treated with various concentrations (1 M, 10 M, 1000 M, 1000 M and 10,000 M) of ytterbium nitrate solutions prepared in SH medium for 24 hours and 5 days. The final volume of each well after any treatment was kept at 100 L. 11 L Thiazolyl Blue Tetrazolium Bromide (5 mg/mL, MTT, Sigma) made up in sterile PBS was added to each well (10% by volume) and the cells were incubated at 37 C. for 3 hours. An equal volume (111 l per well) of solubilizing solution (24 ml propan-1-ol/isopropyl alcohol (Sigma)+1.0 ml 1 M HCl) was added to each well to lyse the cells, and the contents thoroughly mixed by pipetting. Absorbance was measured at 570 nm.

(25) The effect on cell viability was recorded after 24 hours and also over 5 days using various concentrations of ytterbium nitrate solutions. Over a 24 hour period all concentrations of ytterbium nitrate solution had a notable effect on the viability of the cell population compared to the control. The trail involving 10,000 M showed this level of ytterbium to be detrimental to cell survival and proliferation, whereas all the others were shown to be beneficial.

(26) One-way ANOVA (and nonparametric) statistical test with a Dunnett post test was completed on the data, with significance defined as p<0.05. This test was chosen as it is able to make multiple group comparisons and was performed through the software GraphPad Prism. The cell viability results are shown in FIG. 2 (a, b). We also performed the cytotoxicity test for europium and cerium. Our results show that both lanthanides are significantly non-toxic when compared with control (untreated cells). However cytotoxic effects of both compounds were observed at 10,000 M concentration at 5 days of treatment.

Example 3Cytoprotective Test

(27) Cytoprotective effect tests were performed on SH-SY5Y cells that had previously been exposed to hydrogen peroxide for 24 hours. Hydrogen peroxide (H.sub.2O.sub.2) is a strong oxidant, and its activity is based on hydroxide radical's action on DNA strand ((Jonas et al, 1989, Biochem. J., 264, 651-655.). Cells were cultured in 96-well plates to approximately 50% confluency or greater. Experiments were carried out when all of the cell groups showed a similar confluency when viewed under the microscope. To kill the cells, 500 M of hydrogen peroxide (dissolved in SH medium) were added to cell culture 24 hours prior to additions of cerium and europium nitrate (dissolved in SH medium), and this concentration is deemed adequate to kill a considerable number of cells to measure good cytoprotective test result (Glden et al, 2010, Free Radic Biol Med. 49:1298-305). Cell viability was measured after 24 hours exposure to cerium and europium nitrate using the MTT assay as described above.

(28) Further data interpretation and statistical analysis by using one-way ANOVA and Dunnett post-test (n=80, 95% confidence) shows that both lanthanides especially cerium significantly inhibiting hydrogen peroxide's cytotoxicity. Based on the cytoprotective test results, cerium nitrate is a more potent antioxidant-based cytoprotective agent compared to europium nitrate. The results are shown in FIG. 3.

Example 4Confocal Laser Scanning Microscopy Results

(29) In Vitro Study

(30) For confocal laser scanning microscopy SH-SY5Y cells were grown as above, harvested in PBS without Ca.sup.2+ or Mg.sup.2+ and subcultured on glass coverslips. After 3-4 days in culture cells were washed in PBS then fixed in 4% paraformaldehyde. After three 10 minute washes in PBS cells were permeabilized with 0.02% Triton-X100 in PBS supplemented with 10% goat serum. The cells were then incubated at 4 C., overnight in the presence of 1000 M cerium nitrate and 1000 M europium nitrate solution. Cells untreated with either cerium nitrate or europium nitrate were used as a control. After 24 hours coverslips were mounted on slides using Vectashield (Vector Laboratories Ltd, Burlingame, Calif., USA) and examined using a Zeiss laser scanning confocal microscope (LSM 510). The results are shown in FIG. 4.

(31) For in vitro tissue and in vivo experiments, 10- to 12-week-old (25-30 g) C57BL/6J mice (Harlan-Olac, Bicester, UK) were used under appropriate United Kingdom Home Office personal and project licenses and adhered to regulations as specified in the Animals (Scientific Procedures) Act (1986) and according to institutional ethical guidelines.

(32) The mice were killed by decapitation under appropriate anaesthesia. Brains, arteries and blood were removed, rapidly frozen on dry ice and stored at 80 C. for conjugation study. Conjugation of cerium and europium with mouse brain tissue and atherosclerotic tissue was obtained by homogenising 50 mg of tissue in Tris saline buffer (pH7.4) and mixing with different dilutions (1 M, 10 M, 100 M) of the europium nitrate and cerium nitrate. The mixers were kept on a shaker table for 24 hours. The tissue was then mounted onto a slide and coverslipped using Vectashield mounting medium and examined using a Zeiss laser scanning confocal microscope (LSM 510).

(33) Fluorescence emission spectrum of europium nitrate was obtained by exciting with a diode laser of 405 nm and it gives 3 emissions at 448 nm, 535 nm and 588 nm. Thereafter conjugation of the europium with phospholipids was assessed by first mixing phospholipids with 100 M europium nitrate analysing under confocal microscope. Two emissions were observed i.e. at 448 nm and 674 nm shown in FIG. 5. Therefore, europium conjugation with phospholipids will give two emission peaks near 448 nm and 674 nm.

(34) Fluorescence Microscopy Results

(35) Brain tissue exposed to 0.1 M concentration of europium nitrate showed a large number of fluorescent peaks (10 peaks in a 100 m.sup.2 area) as shown in FIG. 11(a). The white circles indicate Eu.sup.3+ ions which are conjugated to lipid regions of the brain. This was more than that seen in brain tissue exposed to 1 mM of europium nitrate solution (FIG. 11(b)). The brain tissue controls did not show any evidence of fluorescence under the microscope (FIG. 11(a)).

(36) In Vivo Study

(37) For this 10- to 12-week-old (25-30 g) C57BL/6J mice (Harlan-Olac, Bicester, UK) received intraperitoneal injection of 02. ml of 100 M europium nitrate in distilled water). After 24 hours mice were perfused transcardially with 0.9% saline followed by 4% paraformaldehyde. Brains, arteries, heart, liver kidneys were removed, post-fixed and 50 m sections were cut on a vibratome (Leica Microsystems, Germany) The sections were mounted on slides, dried at 4 C., cover slipped using vectashield mounting medium and examined using a Zeiss laser scanning confocal microscope (LSM 510).

(38) The confocal image of mouse artery showed some spots with blue emissions.

(39) Fluorescence spectra were obtained and the spectra was compare with aq europium nitrate as shown in the FIG. 8 and similar peak emission are observed which shows that europium is conjugating to the endothelial tissue in artery.

(40) Following intra peritoneal injection of rare earth ions, all the mice appeared completely healthy and behaved normally. For the mice injected with europium nitrate solution, a very low level of europium ions were found to be present in the cardiac and lung, tissue with the highest number of peaks seen in a 100 m.sup.2 area being 1 for both the heart and lung tissue. The liver tissue showed a much higher concentration of fluorescence than the heart and lung tissue, with the highest number of fluorescent peaks in a 100 m.sup.2 area being <10. At higher magnifications it was evident that the europium ions had entered the hepatocytes in the liver tissue. The brain tissue also showed a large amount of fluorescence under the microscope, with the highest number of fluorescent peaks per 100 m.sup.2 area being 20.

(41) Fluorescence Microscopy Results for In Vivo Study

(42) The carotid artery of an atherosclerotic mouse was conjugated with europium nitrate solution (1000 M) dissolved in Tris saline buffer, mounted onto a slide and a coverslip added using vectashield mounting medium. Sections were viewed on an Axiolmager Z.1 epifluorescence microscope (Carl Zeiss, Welwyn Garden City, UK). FIG. 10(b) shows a fluorescent image with DAPI filter which allows the emission range of 470-500 nm. The white circles seen in FIG. 10(a) indicate the conjugation of the europium with lipid regions of the artery which can be compared to the blue marks in the Fluorescence microscope image shown in FIG. 10(b). Following IP injection of europium and cerium nitrate solution we have found europium and cerium labelling in the brain tissue (FIG. 9) suggesting that these lanthanides cross the blood brain barrier.