25-OH vitamin D derivatives for determining vitamin D metabolites

Abstract

The present invention relates to new vitamin D compounds which are bonded to a labelling group at the C3 stereocentre by means of a linker. The present invention further relates to a method for producing these vitamin D compounds and to the use of an intermediate for producing these compounds. The present invention also relates to a method for quantitatively determining vitamin D using a vitamin D compound according to the invention as a tracer. Furthermore, the present invention relates to a reagent for determining vitamin D, containing a compound according to the invention, and to the use thereof for determining vitamin D.

Claims

1. An in-vitro method for quantitatively determining vitamin D in a sample, comprising introducing a compound having a structure according to formula (Ib) ##STR00012## as a tracer into said sample.

2. An in-vitro method according to claim 1, comprising: a) contacting a vitamin D-containing sample with the tracer, b) quantitatively determining the tracer under conditions in which the detected quantity of tracer allows a conclusion to be drawn as to the overall concentration of vitamin D in the sample.

3. An in-vitro method according to claim 1, wherein the tracer is determined by a competitive immunological method.

4. An in-vitro method according to claim 1, wherein the tracer is added to the sample in a quantity to set a final concentration of from 0.2 to 100 ng/ml of this compound.

5. An in-vitro method according to claim 1, wherein the sample to be tested is a biological fluid which is serum.

6. An in-vitro method according to claim 1, wherein the vitamin D to be determined comprises a hydroxy group at C25 and is 25-hydroxy vitamin D3.

7. An in-vitro method according to claim 1, wherein an antibody which specifically recognizes a vitamin D3 side chain having a hydroxy group at C25 is used to determine the tracer.

8. A method for determining vitamin D comprising introducing a compound having a structure according to formula (Ib) ##STR00013## into an assay for determining vitamin D.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1a: Synthesis of a biotin 25-OH vitamin D3 tracer reagent by converting 25-hydroxy vitamin D3 (1) into the epimer 3α N-phthalimido derivative (2).

(2) FIG. 1b: continuation of FIG. 1a depicting deprotecting to form the amino derivative (3) and reacting with NHS-PEG.sub.12 biotin to form 3α-[α-biotinamide-ω-dodeca(ethylene glycol)-amide]-25-hydroxy-deoxyvitamin D3 (4).

(3) FIG. 2: Characterisation of the competitive displacement of defined 25OHD3 concentrations by a biotin-25OHD3 tracer reagent. For the ELISA detection of the specific 25OHD competition, 80 μl of vitamin D release reagent were added to serial dilutions of 25OHD3 in vitamin D-free serum matrix (SerCon, Seracare Inc.) in concentrations of 0, 5, 10, 20, 20, 50, 100, 200, 300 ng/ml in the presence of 7.5 ng/ml of biotin-25OHD tracer reagent and applied to microtitre plate wells coated with an anti-25OHD antibody. The binding of the tracer on the basis of the competing 25OHD concentration was determined with peroxidase-labelled streptavidin and a tetramethylbenzidine (TMB) colour reaction by measuring the OD 450 nm.

(4) FIG. 3: The comparative analysis of serums with defined 25OHD3 and 25OHD2 concentrations (6PLUS1 Multilevel Serum Calibrator Set 25-OH-Vitamin D3/D2, Chromsystems GmbH) and dilutions thereof in vitamin D-free serum matrix (SerCon, Seracare Inc.) showed a high correspondence between the HPLC- and LC-MS/MS-defined 25-OHD3/D2 concentrations and the direct 25-OH vitamin D3/D2 determination method using the automatic Aleria™ system (ORG270, ORGENTEC).

(5) FIG. 4: The comparative determination of the recovery of dilutions of the 25-OH vitamin D derivatives 3α-[α-biotinamido-ω-dodeca(ethylene glycol)-amido]-25-hydroxy-deoxyvitamin D.sub.3 (VD1) and 3β-3′[α-biotinamido-ω-dodeca(ethylene glycol)-amido]amidopropyl-ether-25-hydroxy vitamin D.sub.3 (VD2) in vitamin D-free serum matrix after 24-hour incubation at 37° C. exhibits a high serum stability of the derivative VD1 present at the C3 atom in the (R) configuration compared with the VD2 present at the C3 atom in the (S) configuration.

EXAMPLE 1: SYNTHESIS OF 3α-[α-BIOTINAMIDE-ω-DODECA(ETHYLENE GLYCOL)-AMIDE]-25-HYDROXY-DEOXYVITAMIN D3 (FIGS. 1a AND 1b)

(6) .sup.1H-NMR (at 300 MHz) spectra were recorded on a Bruker AC300 (Bruker, Karlsruhe, Bruker) using the solvent CDCl.sub.3. The evaluation was carried out in ppm with tetramethylsilanes as the internal standard. The mass spectra were determined on a Varian MAT 311 spectrometer (70 eV, El). F.256 silica gel plates (Merck Darmstadt, Germany) were used for the analytical thin-layer chromatography. Chromatographic purification was carried out in silica gel columns, 230-400 mesh ASTM. All the reagents were obtained from Sigma Aldrich.

1.1: Synthesis of 3α-phthalimide-25-hydroxy-deoxyvitamin D3

(7) A solution of 25-hydroxy-vitamin D3 (10.0 mg, 25 μmop, phthalimide (4.5 mg, 31.25 μmop, triphenylphosphine (8.2 mg, 31.25 μmop and diethylazodicarboxylate (5.4 mg, 31.25 μmop in tetrahydrofuran (2.0 ml) was stirred at room temperature for 24 hours. After hexane (2.0 ml) was added, the mixture was filtered through a silica gel filter. After the solvent was removed, the solids were resuspended in 5 ml 10% Na.sub.2CO.sub.3 and were extracted twice using 10 ml diethyl ether each time. The combined ether phases were dried over MgSO.sub.4 and concentrated under vacuum to form a colourless oil. The product was purified by silica gel column chromatography. 3α-phthalimide-25-hydroxy-deoxyvitamin D3 (FIG. 1, formula 2) was isolated in the form of a colourless oil (7.3 mg). NMR: δ 0.58 (s, H—C(18), 3H), 0.85 (d, J=6.4 Hz, H—C(21), 3H), 1.21 (s, H—C(26 and 27), 6H), AB quartet: 4.84 (d, J=2.4 Hz, H—C(19)E, 1H) and 5.06 (d, J=1.9 Hz, H—C(19)Z, 1H), AB quartet: 6.02 (d, J=11.0 Hz, H—C(7), 1H) and 6.26 d, J=11.0 Hz, H—C(6), 1H), 7.80 (m, Ar, 4H), MS: m/e (100) 529.24 (Mt).

1.2: Synthesis of 3α-amino-25-hydroxy-deoxyvitamin D3

(8) 3α-Phthalimide-25-hydroxy-deoxyvitamin D3 (2), (7.2 mg, 13.5 mmol) was dissolved in 0.5 ml 8M methylamine in ethanol in a nitrogen atmosphere. The mixture was first stirred for 2 hours at room temperature and then for 16 hours at 4° C. The solvent was evaporated at negative pressure and the residue was resuspended in 5 ml 10% Na.sub.2CO.sub.3 and was extracted three times using 10 ml diethyl ether each time. The combined ether phases were dried over MgSO.sub.4 and concentrated under vacuum to form a colourless oil. The product was purified by silica gel column chromatography. 3α-Amino-25-hydroxy-deoxyvitamin D3 was isolated as a colourless oil (4), (5.2 mg). NMR: δ 0.57 (s, H—C(18), 3H), 0.85 (d, J=6.4 Hz, H—C(21), 3H), 1.21 (s, H—C(26 and 27), 6H), AB quartet: 4.83 (d, J=2.4 Hz, H—C(19)E, 1H) and 5.00 (d, J=1.8 Hz, H—C(19)Z, 1H), AB quartet: 5.97 (d, J=11.0 Hz, H—C(7), 1H) and 6.27 d, J=11.0 Hz, H—C(6), 1H), MS: m/e (100) 400.37 (Mt).

1.3: Synthesis of 3α-[α-biotinamide-ω-dodeca(ethylene glycol)-amide]-25-hydroxy-deoxyvitamin D3

(9) 3α-Amino-25-hydroxy-deoxyvitamin D3 (3), (5.2 mg, 12.9 μmol) triethylamine (0.02 μL), NHS-PEG.sub.12-biotin (9.4 mg, 10.0 μmol, Thermo Scientific, Prod. No. 21312) were dissolved in 0.2 ml dimethylformamide and stirred at room temperature overnight. The solvent was evaporated at negative pressure and the residue was purified by silica gel column chromatography. Here, 5.9 mg 3α-[α-biotinamide-ω-dodeca(ethylene glycol)-amide]-25-hydroxy-deoxyvitamin D3 (4) was obtained in the form of a colourless wax. NMR: δ 0.58 (s, H—C(18), 3H), 0.85 (d, J=6.4 Hz, H—C(21), 3H), 1.21 (s, H—C(26 and 27), 6H), 3.62 (m, H—C(ethylene glycol), 50H), AB quartet: 4.84 (d, J=2.4 Hz, H—C(19)E, 1H) and 5.06 (d, J=1.9 Hz, H—C(19)Z, 1H), AB quartet: 6.02 (d, J=11.0 Hz, H—C(7), 1H) and 6.26 (d, J=11.0 Hz, H—C(6), 1H), MS: m/e (100) 1247.80 (M+Na.sup.+).

EXAMPLE 2: PREPARATION OF A VITAMIN D RELEASE REAGENT

(10) Sodium toluenesulfonate was dissolved in water and adjusted with stock solutions of 1 M sodium citrate and 1 M iron(III) chloride to a final concentration of 1 M sodium toluenesulfonate, 100 mM sodium citrate and 50 mM iron(III) chloride.

EXAMPLE 3: BINDING OF A 25-OHD ANTIBODY TO A SOLID PHASE

(11) An antibody against 25-OH-vitamin D3 or 25-OH-vitamin D2 was diluted in Tris-buffered saline pH 8.0 to a concentration of 1 μg/ml. The wells of a microtitre plate (Maxisorb, Nunc) were coated with 100 μl of the diluted antibody, dried at 37° C. and stored at 4° C. until the test was carried out.

EXAMPLE 4: COMPETITIVE BINDING ANALYSIS

(12) Defined concentrations of 25-OHD3 were added to vitamin D-free serum matrix (Seracon, Seracare Inc.). In each case 80 μl of the concentration series were introduced into a microtitre plate well pre-loaded with 3α-[α-biotinamide-ω-dodeca(ethylene glycol)-amide]-25-hydroxy-deoxyvitamin D3 (25OHD-biotin tracer reagent) from example 1 as a tracer and incubated for 5 minutes at RT (20-27° C.) for resolubilisation of the tracer reagent. 80 μl of the release reagent from example 2, which dissociates vitamin D in the sample from the complex with DBP, were then added to the sample batch and mixed. 100 μl of the samples diluted with vitamin D release reagent were transferred into wells coated with 25OHD antibody and incubated for 30 minutes at RT. The 25OHD contained in the sample now competes with the 25OHD biotin tracer for binding sites on the 25OHD antibody. The sample batch was then removed and the wells were rinsed three times with 200 μl of wash buffer each time. The antibody-bound biotin-25OHD tracer was detected with peroxidase-labelled streptavidin and a tetramethylbenzidine (TMB) colour reaction. After the substrate reaction had been stopped by addition of 100 μl of 50 mM phosphoric acid, the optical density was measured at 450 nm. Here, a specific competition of the biotin-25OHD tracer was demonstrated, as shown in FIG. 2, even at a 25OHD3 concentration of 5 ng/ml.

EXAMPLE 5: DETERMINATION OF 25-HYDROXY VITAMIN D IN SERUM OR PLASMA IN THE AUTOMATED ALEGRIA™ 25-OH VITAMIN D3/D2 TEST SYSTEM

(13) In order to study the correlation between HPLC+LC-MS/MS-based 25OHD3/D2 determination methods and the automated Alegria™ 25-OH vitamin D3/D2 test system (ORG270, Orgentec Diagnostika GmbH), serum or plasma samples, the 25OHD3/D2 concentration of which was characterised by HPLC+LC-MS/MS-based methods (6PLUS1 Multilevel Serum Calibrator Set 25-OH-Vitamin D3/D2, Chromsystems GmbH), and dilutions thereof were stored in aliquots at −20° C. until the determination.

(14) For the determination, in each case 80 μl of the samples were placed in well A of the Alegria™ test strip. The 8 wells of a test strip contained the vitamin D release reagent from example 2, streptavidin-HRP conjugate, TMB substrate, a 25OHD calibrator solution and 25-OHD antibody-coated wells for in each case one sample determination. Test processing took place automatically in the Alegria™ machine. Inside a test strip, the sample and the calibrator, internal to the test strip, were in each case mixed with 25OHD-biotin tracer and vitamin D release reagent, transferred to the 25OHD antibody-coated wells and washed after being incubated for 30 minutes. The antibody-bound biotin-25OHD tracer was detected with peroxidase-labelled streptavidin and a tetramethylbenzidine (TMB) colour reaction by measuring the optical density at 650 nm.

(15) As is shown in FIG. 3, a high correspondence was observed between the HPLC and LC-MS/MS-defined 25-OHD3/D2 concentrations in serum samples and the direct 25-OH vitamin D3/D2 determination method in the automated Alegria™ system.

EXAMPLE 6: DETERMINATION OF THE SERUM STABILITY OF 25-OH VITAMIN D DERIVATIVES

(16) For comparative testing of the serum stability of biotin 25-OH vitamin D derivatives, the test compounds were incorporated in a vitamin D-free serum matrix (Seracon, Seracare Inc.). In a first batch (VD1), the 25-OH vitamin D derivative 3α-[α-biotinamido-ω-dodeca(ethylene glycol)-amido]-25-hydroxy-deoxyvitamin D.sub.3 that is in accordance with example 1 and is present at the C3 atom in the non-natural (R) configuration was set to a concentration of 3 ng/ml in a vitamin D-free serum matrix. In a second batch (VD2), the 25-OH vitamin D derivative 3β-3′[α-biotinamido-ω-dodeca(ethylene glycol)-amido]amidopropyl-ether-25-hydroxy-vitamin D.sub.3 that is in accordance with US20080317764 and is present at the C3 atom in the natural (S) configuration was set to a concentration of 3 ng/ml in a vitamin D-free serum matrix.

(17) Aliquots of 1 ml each of the batches VD1 and VD2 were stored at −20° C. and were incubated for 24 hours at 37° C. The samples were then equilibrated for 1 hour at room temperature (22° C.). 500 μl of each of the reference samples stored at −20° C. and of each of the samples incubated for 24 hours at 37° C. were added to and mixed with 500 μl release reagent according to example 2 in each case. 100 μl of the samples diluted with vitamin D release reagent were transferred into 25OHD antibody-coated wells according to example 3 and were incubated at room temperature for 30 minutes, so that the biotin-25-OH vitamin D derivatives present in the samples bound to the 25OHD antibody.

(18) The sample batches were then removed and the wells were rinsed three times with 200 μl of wash buffer each time. The antibody-bound biotin-25-OH vitamin D derivatives were detected with peroxidase-labelled streptavidin and a tetramethylbenzidine (TMB) colour reaction. After the substrate reaction had been stopped by addition of 100 μl of 50 mM phosphoric acid, the optical density was measured at 450 nm.

(19) This demonstrated, as shown in FIG. 4, a recovery of 95.2% for the 3α-[α-biotinamido-ω-dodeca(ethylene glycol)-amido]-25-hydroxy-deoxyvitamin D.sub.3 derivative in batch VD1 after incubation for 24 hours at 37° C., compared with the reference sample stored at −20° C. The 3β-3′[α-biotinamido-ω-dodeca(ethylene glycol)-amido]amidopropyl-ether-25-hydroxy vitamin D.sub.3 derivative present in batch VD2 has a significantly lower serum stability. In this case, a recovery of only 71.5% was detected after incubation for 24 hours at 37° C., compared with the reference sample stored at −20° C.