Non-oxidizable bilirubin substitutes and uses therefor

Details Non-oxidizable bilirubin substitutes and uses therefor Non-oxidizable bilirubin substitutes and uses therefor

2001-02-13

2004-02-24

Wallenhorst, Maureen M. (Department: 1743)

Chemistry: analytical and immunological testing

Composition for standardization, calibration, simulation,...

Bilirubin or uric acid standard or control

C436S008000, C436S097000, C436S164000, C436S166000, C252S408100

Reexamination Certificate

active

06696297

ABSTRACT:

BACKGROUND OF THE INVENTION
The field of the invention is stable liquid control and/or calibration compositions for use in clinical diagnostic applications, particularly those used to quantitate bilirubin.
Bilirubin is a yellow-orange bile pigment primarily resulting from the degradation of heme from hemoglobin in red blood cells. The average human produces 250 to 300 mg bilirubin each day, which is transported to the liver in association with albumin. Inside the liver, bilirubin is conjugated with glucuronic acid to produce bilirubin mono- and diglucuronide, (collectively referred to as direct bilirubin) which is excreted into bile.
The concentration of bilirubin in the blood increases in response to increases in the amount of hemoglobin degraded, as well as a decrease in liver function. There are several inherited and acquired diseases which affect steps in the production, uptake, storage, metabolism and excretion of bilirubin, including physiological jaundice, Crigler-Najjar syndromes Types I and II, Gilbert's syndrome and the Dubin-Johnson syndrome. The disturbances of bilirubin metabolism associated with physiological jaundice and Crigler-Najjar syndromes Types I and II result in elevated unconjugated bilirubin levels in serum. On the other hand, total serum bilirubin levels decrease as a result of bilirubin metabolism disturbances associated with Gilbert's syndrome and the Dubin-Johnson syndrome. Therefore, quantification of the amount of bilirubin in biological fluids is a crucial clinical tool to diagnose and regulate disease.
There are commercially available calibration and control products. The most common is lyophilized serum which contains natural bilirubin. An example is LYPHOCHEK (Bio-Rad Laboratories). Lyophilized controls suffer from disadvantages in that errors can be made in reconstitution (e.g., incorrect amounts of water added, or incomplete resuspension of all lyophilized product) and they have a relatively short life due to oxidation of bilirubin when reconstituted, making them potentially wasteful and expensive to use.
Liquid controls and calibrators are also available, such as CHEMTRAK™, a liquid bilirubin control and calibration solution (Medical Analysis Systems, Inc.), and LIQUICHEK™ (Bio-Rad Laboratories). These controls have the advantage of being in liquid form and having up to an 18-month shelf life at 2 to 8° C. if unopened (CHEMTRAK™). However, once opened, such solutions have a 14-day stability if used as a control solution and a 7-day stability if used as a calibration solution, again due at least in part to the oxidation of bilirubin to biliverdin.
Accordingly, it is evident that a need exists for an improved, more stabile, bilirubin standard.
SUMMARY OF THE INVENTION
The present invention uses C10 substituted synthetic bilirubin analogs in calibration and control compositions which are stable against oxidation for periods of months to years when unopened, and weeks to months, or longer, when opened and exposed to oxidizing conditions. Compositions of the formula:
wherein:
R
1
and R
2
are each independently selected from alkyl, alkenyl, alkynyl, heterocyclic, and aryl;
R
3
and R
4
are each independently selected from hydrogen, alkyl, alkenyl, glucoronyl, tauryl, and aryl; and
R
5
, R
6
, R
7
, R
8
, R
9
and R
10
are each independently selected from hydrogen, alkyl, and alkenyl
are extremely resistant to oxidation, yet are reactive with diazo compounds commonly used in the analysis and quantification of bilirubin and unrobilinogen for clinical and research purposes. While the disclosure generally refers to detection and analysis of bilrubin, it is understood that it can apply equally well to the detection and analysis of unbilinogen.
The invention features a method of quantifying bilirubin in a sample comprising detecting bilirubin in a sample to obtain a measurement indicative of the amount of bilirubin in the sample, detecting a known quality of a compound of Formula (I) above in at least one control reagent to obtain a measurement indicative of the amount of the compound in the control reagent, and comparing the measurements to determine the amount of bilirubin in the sample compared to the amount of the compound of Formula (I) in the control reagent. In a preferred embodiment, the detection is done by measuring a diazotization reaction involving contacting the bilirubin in the sample and the compound in the control reagent with a diazonium ion source to form colored products which are measured, e.g., spectrophotometrically. This method has the advantage that it can be used in many currently available clinical analysis platforms.
The invention also features a method of calibrating a device to accurately detect bilirubin comprising detecting with the device a known quantity of a compound of Formula (I) in at least one calibration reagent to obtain a measurement indicative of the amount of the compound in the calibration reagent, and adjusting the device such that the measurement from the device is the same as or proportional to the known amount of compound in the calibration reagent. In a preferred embodiment, the detection involves measuring the products of a diazotization reaction. The diazotization reaction involves contacting the bilirubin in the sample and the compound in the control reagent with a diazonium ion source to form colored products, which are measured, e.g., spectrophotometrically. Two or more calibration or control reagents having different amounts of the bilirubin analog compounds of Formula (I) can be used to generate a standard curve, by, for example, creating measurements at the high, low, and/or intermediate ranges where sample concentrations of bilirubin are expected to fall.
Preferably, compounds used in the above methods are those where R
1
and R
2
are each independently selected from alkyl, alkenyl, alkynyl, heterocyclic, and aryl; R
3
and R
4
are each independently selected from hydrogen, alkyl, alkenyl, and glucoronyl; and R
5
, R
6
, R
7
, R
8
, R
9
and R
10
are each independently selected from hydrogen and alkyl. Particularly preferred compounds are those where R
1
and R
2
are methyl; and/or where R
3
and R
4
are each hydrogen. Preferably, the calibration and control reagents are provided as a liquid, but they can also be supplied in any other form, e.g., as a lyophilized powder.
The invention also features compositions for use in calibration or control solutions. These compositions comprise a synthetic bilirubin analog of Formula (I):
wherein
R
1
and R
2
are independently selected from alkyl, alkenyl, alkynyl, heterocyclic, and aryl;
R
3
and R
4
are indenpendently selected from hydrogen, alkyl, alkenyl, glucoronyl, tauryl, and aryl; and
R
5
, R
6
, R
7
, R
8
, R
9
and R
10
are each independently selected from hydrogen, alkyl, and alkenyl, with the proviso that when R
3
and R
4
are both hydrogen, at least one of R
5
, R
6
, R
7
, R
8
, R
9
and R
10
is not alkyl, preferably not alkyl or alkenyl.
Compositions containing the compound of Formula (I) can be provided in any form, but are preferably supplied in liquid form and contain processed serum, urine, or other biological or biologically compatible components. For example, the controls and calibrators can be supplied in any fluid or reconstitute from water or saline to a complex urine or serum matrix. The compositions can also contain preservatives, antibiotics, and/or other analytes, such as therapeutic drugs, drugs of abuse, biological enzymes, toxins, and metabolites.
Preferred compositions for use as calibration or control reagents include stable bilirubin analogs of Formula (I) wherein R
1
and R
2
are methyl, R
3
and R
4
are selected independently from the group containing hydrogen, alkyl, alkenyl, tauryl, aryl, and glucoronyl, and R
5
, R
6
, R
7
, R
8
, R
9
and R
10
are each independently selected from hydrogen and alkyl; and an additional component selected from processed serum and processed urine. The compositions are preferably provided in a liquid form, but can be lyophilized, and preferably contain at lea

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