Chemistry: analytical and immunological testing – Sedimentation rate or hematocrit
Reexamination Certificate
2001-01-12
2004-02-10
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Sedimentation rate or hematocrit
C436S161000, C436S164000, C436S172000
Reexamination Certificate
active
06689616
ABSTRACT:
The present invention concerns new dye-polysaccharide conjugates and their use as a diagnostic agent especially for determining the glomerular filtration rate in humans.
The glomerular filtration rate is very important for diagnostic and therapeutic measures in pre-clinical and clinical situations since it allows the determination of renal functional impairment. The glomerular filtration rate (GFR) is understood as the amount of primary urine produced by the glomeruli of the kidneys per unit of time which can be determined with any substance that is only filtered and is neither secreted by the tubules nor re-absorbed from the primary urine.
The clearance of the test substance used is examined in order to quantify the glomerular filtration rate. Clearance refers to the amount of plasma in ml which is cleared of a particular (test) substance by the kidneys in a unit of time (min).
At present various methods are used to determine this clearance. Thus endogenous creatinine clearance, inulin clearance and
51
Cr-EDTA (Na
2
51
Cr-ethylenediamine tetraacetate) clearance have become important. X-ray contrast media such as iohexol are also used to determine the renal clearance.
However, all the listed methods have serious disadvantages. Hence depending on the respective procedure for determining clearance it is necessary to continuously infuse the test substance to maintain a constant plasma level and/or to collect urine by an unpleasant catheterisation and/or to take several blood samples. Determination of the inulin concentration in the plasma or urine is a complicated method and has a relatively large margin of error. The use of radioactively labelled substances is an additional burden for the organism and should therefore be avoided if possible.
The so-called input clearance is regarded as the most elegant way of determining clearance. In this case the excretion of a marker is not measured directly but rather the plasma concentration of the marker is kept constant. The required amount of marker which is necessary to keep the plasma concentration constant corresponds to the amount of marker excreted by the kidneys and thus to the clearance. This of course requires continuous monitoring of the plasma concentration which nowadays can only be accomplished with the aid of radioactively labelled substances.
A considerable improvement would be to use non-radioactively labelled markers whose plasma concentration could be continuously monitored. In addition blood withdrawal and urine collection would not be required to determine the concentration of the marker.
The object was therefore to find a non-radioactively labelled marker which is completely eliminated by renal ultrafiltration i.e. it is neither secreted nor re-absorbed by the tubuli and whose plasma concentration can be determined continuously by an invasive or non-invasive method, the non-invasive method being preferred and whose plasma concentration is determined in particular by transcutaneous absorption measurements.
The present invention solves this problem by using polysaccharides which are covalently linked to a dye such that the plasma concentration of the whole molecule can be determined transcutaneously by absorption measurement with the aid of a detector.
Such compounds have already been described in the literature. Thus there are conjugates of the dye Cibacron Blue with amylose, glucans and cellulose (Anal. Biochem. 31, 412 (1969); Acta Chem. Scand. 25, 298 (1971)); Biochem. J. 87, 90 (1963), the so-called blue dextran (Anal. Biochem. 39, 202 (1971) and the dye Remazole Brilliant Blue R linked to amylose (Experientia 23, 805 (1967)). In all these dye-polysaccharide conjugates the polysaccharide is a polysaccharide composed of glucose units. However, these are relatively rapidly metabolically degraded and are therefore unsuitable for determining the renal clearance. On the other hand such polysaccharides are not eliminated solely by glomerular filtration through the kidney and are therefore also not suitable for this reason for determining the renal clearance.
Polysaccharides that are preferably used within the sense of the present invention are polyfructosans such as inulin, levan, asparagosine, sinistrin, fibrulin, graminin, phlein, poan, secalin and irisin which are eliminated from the kidney by glomerular filtration. These are substances with chain lengths of 10-30 fructose units some of which carry a glucose molecule at the end of the chain. Polysaccharides of the inulin type or of the more water-soluble sinistrin are preferably used.
Dyes are used as the dye components of the conjugates which carry a functional group such as a carboxyl, hydroxylsulfonyl, amino, isothiocyanato or isocyanato group. These functional residues can be directly reacted with a polysaccharide or be bound to the polysaccharide by means of a spacer.
The dye components of the conjugates used according to the invention have an absorption maximum in the range between 500 and 1300 nm, preferably between 600 and 900 nm. Phthalocyanine, phenazine, phenothiazine, phenoxazine, rhodamine, azo, triphenylmethane and cyanine dyes and derivatives thereof are particularly suitable for this. Representative dyes are for example triphenylmethanes, indocyanines, oxazines and rhodamines.
Hence the invention concerns compounds of the general formula I
F—X(spacer)
n
-Y—E—(CH
2
)
m
—PS (I)
in which
F is a dye component with an absorption maximum between 500 and 1300 nm, preferably between 600 and 900 nm,
n=0 or 1,
m=0 or 1,
if n=0, X=—CO, —NH—C═O, —NH—C═S, —NH—C═NH, —CONH—C═S
if n=1, X=NH, —CONH, —SO
2
NH, —NHCONH, —NHCSNH,
spacer=a C
2
-C
6
alkylene group which is optionally substituted by a hydroxy group or the group
in which p=0-4 and R
1
hydrogen or can be a sulfonic acid group,
Y=a valence dash, —CH
2
, —NHCOCH
2
, —NH—C═O, —NH—C═S, —NH—C═NH,
E=O or NH
and PS=a polyfructosan and if E=O and m=0, the oxygen atom via which the polysaccharide is bound is part of this polysaccharide, and if E=NH it is an aminated or aminoalkylated polyfructosan.
Preferred compounds in the sense of the invention are above all those in which X=—CO, —NH, —CONH, —SO
2
NH, —NH—C═O, —NH—C═S,
spacer=a C
2
-C
6
alkylene group or a group
in which p is preferably 0 or 1 and R
1
represents hydrogen,
n=0 or 1,
Y=valence dash, —CH
2
, —NH—COCH
2
, —NH—C═O,
m=0 or 1,
E=O or NH
and PS preferably represents a sinistrin or inulin residue or an aminated or aminoalkylated derivative of sinistrin or inulin.
In particular compounds of the general formula I are emphasized in which F represents a triphenylmethane, indocyanine, oxazine or rhodamine dye, X represents a —CO—, —CONH— group, spacer represents a C
2
-C
4
residue, n=0 or 1, m=0 or 1, Y=valence dash, CH
2
or —NHCOCH
2
and E=O or NH and the polysaccharide is sinistrin, inulin or an aminated or aminoalkylated derivative of sinistrin or inulin.
The polysaccharides can have reacted once or several times with the dyes and thus have different degrees of occupancy.
In order to improve water-solubility, the compounds of the general formula I can optionally be subsequently derivatized on the polysaccharide unit. Derivatives with 2-hydroxypropyl or carboxymethyl groups on the polysaccharide unit are especially suitable for this. Compounds of the general formula I can also be reacted with sulfones such as 1,4-butanesulfone to improve water-solubility and also to neutralize excess amino groups. These substances are also a subject matter of this invention.
Substances of the general formula I can be prepared by generally known processes, preferably by
1. reacting a dye derivative of the general formula II
F—Z (II)
in which
F represents a dye component with an absorption maximum of 500-1300 nm,
Z represents an —NCO, —NCS, —NC═NH, —CONCS or the group G-spacer-NCO, G-spacer-NCS, G-spacer-NCNH,
Bosies Elmar
Hein Heinz-Michael
Josel Hans Peter
Reiter Rudolf
Barnes & Thornburg
Cole Monique T.
Roche Diagnostics GmbH
Warden Jill
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