Organic compounds -- part of the class 532-570 series – Organic compounds – 9,10-seco-cyclopentanohydrophenanthrene ring system or...
Reexamination Certificate
2001-02-20
2004-09-07
Badio, Barbara P. (Department: 1616)
Organic compounds -- part of the class 532-570 series
Organic compounds
9,10-seco-cyclopentanohydrophenanthrene ring system or...
C548S303700
Reexamination Certificate
active
06787660
ABSTRACT:
The invention relates to derivatives of 25-hydroxy vitamin D, a synthesis thereof, and a method of measuring 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D in samples.
The D-vitamins or calciferols arise from their provitamins through a cleavage, catalysed by sunlight, of the B-ring in the sterane rings. Their most important representatives are vitamin D
3
(cholecalciferol) and vitamin D
2
(ergocalci-ferol), which differ slightly only in the side chains, but which—so far as known—are similarly metabolised and have identical biological effects. Whereas provitamin D
2
must be taken in with the food, the provitamin D
3
can be formed in the human organism. So far as not more specifically designated by means of indices, the term vitamin D comprehends in the following in general all vitamin D forms. Vitamin D formed in the skin or taken in with food is bound in the plasma by vitamin D binding or transport proteins (DBP), transported to the liver and there metabolised to 25-hydroxy vitamin D (25-OH-D). The vitamin D binding protein DBP is also known as Gc-globulin or group specific component (J. G. Haddad in J. Steriod Biochem. Molec. Biol. (1995) 53, 579-582). Over 95% of the 25-hydroxy vitamin D measurable in the serum is as a rule 25-hydroxy vitamin D
3
. 25-Hydroxy vitamin D
2
is only found in greater proportions if the person is receiving medication with vitamin D
2
or, as is frequently the practice in the United States, foodstuffs are supplemented with vitamin D
2
.
25-Hydroxy vitamin D is the prevailing vitamin D metabolite in the blood circulation and its concentration in the serum generally indicates the vitamin D status, i.e. the extent to which vitamin D is available to the organism. If needed, 25-hydroxy vitamin D is metabolised in the kidneys to 1&agr;,25-dihydroxy vitamin D, a hormone-like substance with great biological activity. The determination of 1&agr;,25-dihydroxy vitamin D indicates how much vitamin D is present in the activated form.
BACKGROUND OF THE INVENTION
The determination of 25-hydroxy vitamin D in a sample is preferably effected in accordance with the principle of competitive protein binding analysis, whereby on the basis of the displacement of radioactive 25-hydroxy vitamin D from the binding sites of a vitamin D binding protein, the 25-hydroxy vitamin D present in the sample can be quantified. Also, over the last several years, radioimmunoassays using
125
I-labelled vitamin D derivatives and antibodies for vitamin D derivatives have established themselves in diagnosis. The data of the normal level of 25-hydroxy vitamin D in serum vary depending on the laboratory. It is, however, agreed that the concentration of 25-hydroxy vitamin D in the serum is as a rule greater than 5 ng/ml and smaller than 80 ng/ml. The competitive protein binding analysis requires the use of a radioactive vitamin D derivative which must have the same protein binding characteristics as 25-hydroxy vitamin D. The same applies also for the competitive binding analysis for the biologically active 1&agr;,25-dihydroxy vitamin D and other vitamin D metabolites.
European patent specifications 0 312 360 and 0 363 211, and Tanabe et al. in J. Chem. Soc., Chem. Commun. 1989, 1220-1221 and J. Nutri. Sci. Vitaminol., 1991, 37, 139-147, disclose various
125
I-labelled hydroxy- and dihydroxy vitamin D derivatives and their use in binding studies. These derivatives suffer the disadvantages that they are problematic to produce and are extremely labile. Light, radioactive rays, protons, hydrogen, enzymes, free radicals or the presence of iodine in free or bound form have great effect on the configuration and the binding characteristics of the vitamin D derivatives to vitamin D binding protein DBP or specific antibodies. Above all, they can cause or catalyse a rotation of the A-ring in the sterane system. The 3&bgr;-hydroxy-group of the vitamin D molecule is thereby rotated into the pseudo-1&agr;-position, so that 5,6-trans-vitamin D is obtained. The so-called pseudo-1&agr;-hydroxy-analogs of vitamin D may be metabolised similarly to vitamin D, but they have a structure which is different in significant points and are not bound or are significantly more poorly bound by vitamin D binding proteins such as for example DBP/Gc-Globulin or anti-vitamin D antibodies.
DESCRIPTION OF RELATED ART
The above-described re-arrangement is to be understood as an example. Other chemical reactions and re-arrangements also occur. The same applies for
3
H- or
14
C-labelled vitamin b derivatives. These vitamin D derivatives are likewise not so stable that they permit a reliable binding analysis. The radioactive marking additionally increases the costs of storage, transport and disposal and is generally disadvantageous for health and the environment. Further the half-life of
125
iodine is relatively short. On the other hand, a competitive binding analysis with
3
H- and
14
C-labelled vitamin D derivatives requires particular scintillation counters and is more demanding in terms of equipment, with largely the same problems.
Ray et al., in Biochemistry, 1991, 30, 4809-4813 disclose the coupling of vitamin D
3
with various colouring groups. The detection sensitivity for dye-labelled vitamin D
3
derivatives is, however, too small that one might use them in a competitive binding analysis for natural vitamin D metabolites, apart from the fact that the dye-labelled derivatives are not stable in serum and further are particularly light-sensitive.
BRIEF SUMMARY OF THE INVENTION
It is the object of the invention to make available vitamin D derivatives which can be employed in a competitive binding analysis or quite generally in immunoassays of vitamin D metabolites such as 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D. This presumes the following properties: first, that for the vitamin D derivatives, a detection sensitivity exists which is higher than, or lies in a lower range of concentrations than, the concentration of the sought after vitamin D metabolites in the samples; second, that the derivatives are stable in serum, plasma or urine under the usual protonic conditions and are stable with the respect to serum enzymes; and finally, third, that the derivatives are sufficiently stable with regard to light and storage, over weeks and months. This object is achieved by means of vitamin D derivatives having the formula
wherein:
O represents the oxygen atom of an ether group;
X represents a substituted or non-substituted hydrocarbon group of 0.8 to 4.2 nm length, preferably a C8- to C12-group, which may have the usual heteroatoms such as S, O, N or P, most particularly preferred an hexamido-, octamido- or decamido-amidopropylether linker group;
Y represents hydrogen or a hydroxy group;
A a functional group which is bound with high affinity by a binding protein such as an antibody or vitamin D binding protein DBP;
R the side group of a vitamin D metabolite, preferably the side group of vitamin D
2
or D
3
, particularly preferably the 25-hydroxylated side group of vitamin D
2
or D
3
.
A high affinity is present when the dissociation constant (K) between the binding protein, e.g. the antibody or DBP, and the antigen or the functional group A is greater than 10
8
. A dissociation constant greater than 10
16
is advantageous for many applications. In a preferred embodiment A is selected from biotin, digoxigenin, tyrosine, substituted tyrosine, substituted amino acids, characteristic amino acid and peptide sequences, FITC, FITC-substituted tyrbsine, proteins and protein groups such as protein A and protein G or a further vitamin D derivative, most particularly preferred 25-hydroxy vitamin D.
The spacer group X is preferably selected from substituted and non-substituted C-bodies having a length of 0.8 to 4.2 nm, preferably about 0.12 nm. Particularly preferred is an amino carboxylic acid, in particular an amino undecanoic acid, peptide and keto group or a substituted or non-substituted amino polyether radical having a length of 0.8 to 4.2 nm, preferably about 0.9 to 1.5 nm. This spacing between the group A and t
Armbruster Franz Paul
Birkmayer Christian
Tampe Jens
Voelter Wolfgang
Badio Barbara P.
Immundiagnostik AG
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