IE55441B1 - Test device and method of testing for thiols - Google Patents

Description

- ia - TEST DEVICE AND METHOD OF TESTING FOR THIOLS The present invention relates to a test device and to a method o£ testing for thiols in a body fluid.

While devices for testing body fluids for 5 the presence or concentration of various substances ace known, an example being dipping papers for testing urine for sugars, such devices are not available for testing thiols such as sodium 2-raercapto-ethanesulphonate (hereinafter MESNA), penicillamine, 10 cysteine, etc.

In cancer therapy, MESNA is administered as a uroprotecting agent in conjunction with oxazaphosp-horines such as cyclophosphamide, ifosfamide and tciphosphamide. The oxazaphosphorines are alkylating 15 agents whose urotoxic side effects limit their usefulness. The metabolites of the oxazaphosphorines such as acrolein, when present in urine in the bladder cause harmful and painful effects from increased frequency and dysuria to severe haemorrhagic 20 cystitis and appear to lead to fibrosis, reduced capacity and even cancers of the bladder. Forced diuresis and bladder flushing are thus routinely carried out to minimise such complications. However, such treatment requires the patient to be non-ambulatory 25 and is not only time consuming but is uncomforable for and may even be dangerous to the patient.

MESNA however is effective as a uroprotecting agent to counteract the side effects of these oxazaphos-phorines when present in the urine in sufficient 30 concentration.

MESNA does not impair the alkylating and cytotoxic effect of the oxazaphosphorines and furthermore does not react with other cytotoxic compounds such as BCNO, Doxorubicin, 5-FU, MTX, VCR or Cis-platinum. 2 MESNA is rapidly oxidised in vivo into the biologically inactive bisulphide DIMESNA which is reduced into the free thiol in the kidneys. MESNA reacts in __ the urine With acrolein, which is the metabolite 5 of the oxazaphosphorines most responsible for the urotoxic effects, to produce a non-toxic, stable thioether that is then eliminated. Furthermore, MESNA acts to reduce the rate of the spontaneous dissociation of 4-hydroxy-oxazaphosphorine to 10 form acrolein in the urine by forming a relatively stable non-urotoxic condensation product and so minimising the rate of acrolein liberation.

As the half lives within the patient's body of the oxazaphosphorines and their metabolites 15 are much greater than that of MESNA, and as the degree of protection depends on the level of free thiol in the urine it is extremely important that that MESNA concentration in the urine should at all times be at a uroprotecting level (estimated 20 at in excess of approximately 300 pg/ml). However, although MESNA dosages for normal patients are such as to give a concentration of about 900 ^ug/ml of urine in the bladder, it has been found that a significant number of patients on normal MESNA 25 dosages will show low MESNA concentrations in the urine and so be at risk from the oxazaphosphorine metabolites unless the MESNA level in the urine is monitored and increased.

Possible causes of low MESNA levels in the 30 urine are high fluid intake, which results in the flushing of MESNA from the bladder, and kidney malfunction.

Where low MESNA levels in the urine are detected the MESNA dosage can be increased until a uroprotecting 35 level isattained. However, since large excesses of MESNA may possibly result in undesired side effects such as nausea, dosage increase is only 3 indicated in those cases where normal dosages are shown to be insufficient.

The thiols, such as MESNA, within a urine sample are susceptible to oxidation and unless 5 the thiol content of the urine sample is to be determined within about i hour of sample collection, inaccurate results will be obtained. In order to use the available laboratory methods for thiol determination, the oxidation must be chemically 10 arrested before the samples are taken to the laboratory for analysis. The thiol determination can then be carried out in the laboratory by the addition to the urine sample of Ellman's reagent, a liquid solution the active component of which is 5',5'-15 dithio-bis-(2-nitrobenzoic acid) (hereinafter "5,5-DNA"), and the measurement of the change in absorbence of the sample. In the presence of thiols, Ellman's reagent takes on a bright yellow colour.

The above-described laboratory procedure 20 for determining urine thiol levels requires the use of sophisticated laboratory facilities and is thus generally time-consuming with a possible time-lag in the order of hours between urine sampling and thiol dosage variation following a finding 25 of unsatisfactory thiol concentration levels.

During such a time-lag the harmful and painful effects referred to above as being caused by the presence of oxazaphosphorine metabolites in the bladder can occur. If the MESNA level in the urine 30 is to be monitered and when necessary increased in order to avoid the occurence of such harmful and painful effects, there is a need for a new method of testing for thiols in body fluids such as urine which is adapted for ready monitoring 35 of the levels of thiols therein.

It is thus an object of the present invention to provide a method and a device for testing for thiols in body fluids which may be used quickly 4 and without the need for laboratory equipment or trained laboratory personnel.

According to one aspect of the present invention there is thus provided a device for testing for thiols in a body fluid, the device comprising an absorbent substrate having absorbed therein a reagent capable of acting as an indicator for thiols, preferably a reagent capable of undergoing a visible colour change in the presence of thiols, a buffer and a non-thiol group containing anti-oxidant.

According to a further aspect of the present invention there is provided a method of testing for thiols in a body fluid, the method comprising obtaining a sample of the said body fluid, contacting therewith the absorbent substrate of a device comprising an absorbent substrate having absorbed therein a reagent capable of acting as an indicator for thiols, a buffer and a non-thiol group containing anti-oxidant, and thereafter observing said absorbent substrate to determine whether or not said reagent indicates the presence of thiols.

The method of the invention conveniently comprises obtaining a sample of the body fluid, within i hour thereafter contacting a device comprising an absorbent substrate having absorbed therein a reagent capable of undergoing a visible colour change in the presence of thiols, a buffer and non-thiol group containing atji-oxidant with the said sample, and visually observing, generally within 5 to 10 seconds of the said contacting, the absorbent substrate of the said device to detect whether or not the visible colour change has occurred.

The device of the present invention has absorbed within its absorbent substrate an anti-oxidant.

In the absence of this anti-oxidant it was found that the absorbent substrate, which conveniently is paper such as filter paper, is discoloured making the determination of the colour change in the prsence 5 of thiols imprecise. The anti-oxidant must be a non-thiol group containing material and suitable substances include flavonoids, + catechin, ascorbic __ acid, quinol and quinol derivatives. Quinol is 5 particularly preferred as it appears to act as an anti-oxidant without affecting the effectiveness of the colour changing reagent. Suitable quinol concentrations within the absorbent substrate, where this is filter paper, are achieved by dipping 10 the substrate into a solution containing at least 3.5 g/litre of quinol, preferably at least 4 g/litre quinol.

As the reagent which is capable of undergoing a colour change in the presence of thiols such 15 as MESNA, penicillamine, cysteine etc, the active component of Ellman's reagent is suitable. The use of this reagent, 5,5-DNA, results in the absorbent substrate turning bright yellow immediately on contacting urine containing a sufficient concentration 20 of a thiol. By varying the concentration of the reagent in the absorbent substrate, the sensitivity of the test device can also be varied to ensure that the colour change is observed for levels of the particular thiol of interest, for example MESNA, 25 above a particular minimal acceptable concentration.

Thus for example where the absorbent material is Whatman No. 1 filter paper dipped into a solution containing, inter alia, 2 g/1 of 5,5-DNA and dried, the colour change to bright yellow with MESNA-containing 30 urine will occur for MESNA concentrations of about 400 fig/ml or greater.

Thus, the normal thiol levels of less than about 50 ^ig/ml in the urine of a patient receiving no thiol treatment would not result in a positive 35 result with the device of the present invention.

The absorent substrate of the device of the present invention has absorbed therein a buffer which acts to ensure that the microenvironment 6 of the thiol/reagent reaction is at the correct pH for the colour change to occur. The buffering pH will be dependent upon the particular reagent but for 5,5-dna a slightly alkaline pH, conveniently 5 about 7.4, is suitable.

The device of the present invention may conveniently be in the form of a plastics strip having mounted on one end thereof the absorbent substrate. In this manner, the fingers of the person performing 10 the test need not contact the sample or the absorbent substrate. Elution may cause the colour generated on the absorbent substrate to migrate across the substrate surface and to minimise this effect the absorbent substrate, which is suitably in the form 15 of a disc, strip or sheet of up to about 0.5mm thickness, may be covered on its large area faces by a plastics film leaving only the edges exposed.

Test devices in this form may be packaged in a conventional manner, for example in bottles 20 which also contain a silica gel sachet which prevents the absorbent substrate from becoming damp.

The device and the method of the present invention are non-invasive and may quickly and readily be used for bed-ridden or ambulatory patients 25 by the patients themselves or by nurses or others attending the patients. With the use of the device and method of the invention, it is a simple matter to monitor the urine thiol level and and if necessary to modify the MESNA dosage immediately if the thiol 30 level is found to be low thus minimizing the time for which urotoxic effects occur as a result of low urine MESNA levels.

MESNA has a broad therapeutic index and can be administered intravenously or orally. The use 35 of the device and method of the invention permits the adjustment of the MESNA dosage and thus provides protection against the urothelial toxicity of oxazaphosp-horines. Thus the invention assists the successful 7 ocal administration o£ MESNA as pact of oxazaphosphocine therapy o£ ambulatory patients. By means of the device and method of the invention the free thiol concentration in the urine can be monitored permitting 5 any necessary modification of MESNA dosage to be made.

The use of the device and method of the invention in oxazaphosphorine cytostatic therapy of bed-ridden and ambulatory patients is thus of significant 10 clinical value. By allowing the MESNA - dosage to be maintained at the correct level not only is the therapy made more effective but the patients’ condition is also made more tolerable.

A preferred embodiment of the test device 15 of the invention will now be described by way of example with reference to the accompanying drawing in which:- Figure 1 is a perspective view of a preferred embodiment of the test device of the invention.

Referring to Figure' 1, the test device comprises a thin plastics strip 1 having affixed to one end thereof a sheet 2 of filter paper having absorbed therein a reagent capable of undergoing a visible colour change in the presence of thiols, a buffer 25 and a non-thiol group containing anti-oxidant.

In use, the strip 1 is held by the end remote from the sheet 2 and the sheet is dipped into a body fluid such as urine, removed and examined visually to determine whether or not the reagent 30 colour change has taken place.

The preparation, use, sensitivity and stability of test devices according to the present invention will now be described in the following Examples: Example 1: Preparation 35 A reagent solution was prepared having the following concentration: 5',5'-dithio-bis-(2-nitrobenzoic acid) 0.05g tripotassium citrate 2.5g 8 quinol O.lg phosphate buffer (0.25M, pH 7.4) ad 25ml Whatman No. 1 filter paper was dipped into the solution, allowed to absorb the solution fully, removed from the solution and dried slowly at ambient temperature overnight.

Example 2: Use Urine samples can be tested using the device of the invention either on being taken or some time afterward. If there is to be a delay before samples are tested then, to reduce the spontaneous formation of MESNA - disulphide which is not detected by the device of the invention, a stabilizer such as EDTA should be added, suitably to achieve a concentration of about lmg/ml of EDTA in the urine sample.

In the following tests, small sections of impregnated filter paper prepared according to Example 1 above were dipped into the urine samples.

A bright yellow colour developing in less than 30 seconds was taken to indicate the presence of MESNA within.the urine samples.

As a control, urine samples from 8 people (4 males and 4 females) not receiving MESNA or other thiols except as normal dietary components were tested by dipping with the impregnated test paper. All gave negative results.

A series of decreasing concentrations of MESNA were tested with the impregnated test paper prepared according to Example 1 above. The lower limit of detection for MESNA was approximately 0.015mg/ml. The concentration above which no further colour developed was approximately 2mg/ml, The visually observed colour change for the impregnated paper varied from pale yellow at concentrations of 0.015 to 0.062mg/ml through yellow at concentrations of 0.025 to 0.08mg/ml, to orange at concentrations of 0.07 to 0.6mg/ml and above. The overlapping 9 of these semi-quantitative categories arises as a result of different colour perception by the various observers. A trial test is thus recommended before MESNA is used.

Example 3; Stability Impregnated papers prepared according to Example 1 above were exposed to a normal laboratory atmosphere, in the light, for one month. A slight browning of the paper occurred, it is believed 10 as a result of quinol oxidation, during this time; however this did not adversely affect the detection of thiols in urine samples or the sensitivity of the technique. Nevertheless, storage over silica gel in dark containers appeared to reduce the paper discolouration.

Claims (5)

1. A device for testing for thiols in a body fluid, the device comprising an absorbent substrate having absorbed therein a reagent capable of acting 5 as an indicator for thiols, a buffer and a nonthiol group containing anti-oxidant,

2. A device as claimed in claim 1 wherein said reagent is a reagent capable of undergoing a visible 10 colour change in the presence of thiols.

3. A device as claimed in either of claims 1 and 2 wherein said reagent is 5',5'-dithio-bis-(2-nitrobenzoic acid). 15

4. A device as claimed in any one of claims 1 to 3 wherein said anti-oxidant is selected from flavonoids, + catechin, ascorbic acid, quinol and quinol derivatives. 20 5. A device as claimed in any one of claims 1 to 4 wherein said anti-oxidant is quinol or a quinol derivative. 25 6. A device as claimed in any one of claims 1 to 5 comprising a plastics strip having mounted at one end thereof a disc, strip or sheet of up to about 0.5mm thickness of said absorbent substrate. 30 7. A device as claimed in any one of claims 1 to 6 wherein said absorbent substrate comprises filter paper. 8. A device as claimed in claim 7 wherein said 35 anti-oxidant and said reagent comprise quinol and 5',5'-dithio-bis-(2-nitrobenzoic acid) absorbed into said filter paper from a solution containing 11 - them at concentrations of at least 3.5mg/ml and about 2mg/ml respectively. 9. A device as claimed in any one of the preceding 5 claims comprising a flexible plastics stip having mounted at one end thereof a sheet of filter paper having absorbed therein 5',5'-dithio-bis-(2-nitrobenzoic acid) and quinol. 10 10. A device for testing for thiols in a body fluid substantially as hereinbefore disclosed with particular reference to the Examples and the accompanying drawing. 15 11. A method of testing for thiols in a body fluid the method comprising obtaining a sample of the said body fluid, contacting therewith the absorbent substrate of a device comprising an absorbent substrate having absorbed therein a reagent capable 20 of acting as an indicator for thiols, a buffer and a non-thiol group containing anti-oxidant, and thereafter observing said absorbent substrate to determine whether or not said reagent indicates the presence of thiols. 25 12. A method as claimed in claim 11 wherein said reagent is a reagent capable of undergoing a visible colour change in the presence of thiols and after contact with said body fluid said substrate is 30 visually observed to determine whether or not said visible colour change occurs. 13. A method as claimed in either of claims 11 and 12 wherein said absorbent substrate is contacted , 35 with said body fluid within 30 minutes of the obtaining of said body fluid from the body. 14. A method as claimed in either of claims 11 and 12 wherein a stabilizer is added to said sample of body fluid before said absorbent substrate is contacted therewith. 15. A method as claimed in claim 14 wherein EDTA is added to said sample of body fluid as said stabilizer to a concentration of about lmg/ml. 16. A method as claimed in any one of claims 11 to 15 wherein the observation of said absorbent substrate to determine whether or not the presence of thiols is indicated is performed within 10 seconds of the contacting of said aborbent substrate with said body fluid. 17. A method is claimed in any one of claims 11 to 16 wherein said body fluid is urine. 18. A method as claimed in any one of claims 11 to 17 wherein said device is a device as claimed in any one of claims 1 to 10. 19. A method of testing for thiols in a body fluid substantially as hereinbefore disclosed with particular reference to the Examples and the accompanying drawing. Dated this 30th day of April 1984. BY: /fotodNS & CO., Apflicants' Agents, (Signed) '

5. , T&rtmouth Road, Dublin 6.