WO1999049317A1

WO1999049317A1 - Method for diagnosing epilepsy and related disorders, and kit therefor

Info

Publication number: WO1999049317A1
Application number: PCT/FI1999/000228
Authority: WO
Inventors: Anna-Elina Lehesjoki; Riitta Rinne; Pekka Saukko; Kimmo Virtaneva; Albert De La Chapelle; Mikko Järvinen; Ari Rinne
Original assignee: Aboatech Ltd.
Priority date: 1998-03-23
Filing date: 1999-03-23
Publication date: 1999-09-30
Also published as: FI980647A0; AU2937199A

Abstract

The object of the present invention is a method for diagnosing epilepsy or other neurodegenerative disorders in an individual, comprising measuring from a sample from said individual, one or more cathepsins selected from the group consisting of cathepsin S, cathepsin B and cathepsin L, and optionally also cystatin B inhibitor. An object of the invention is also a kit for carrying out the diagnosis.

Description

Method for diagnosing epilepsy and related disorders, and kit therefor

FIELD OF THE INVENTION

The present invention relates to a method for diagnosing epilepsy, especially progressive myoclonus epilepsy, and other neurodegenerative disorders in an individual. The invention also comprises a kit for use in said method of diagnosis.

BACKFGROUND OF THE INVENTION

Progressive myoclonus epilepsies (PME) are disorders that manifest as myoclonus, epilepsy and progressive neurological deterioration, in particular ataxia and dementia (for example Bercovic, S. F. et al, N Engl J Med 1986: 31:296-304). The onset of symptoms is usually in late childhood or adolescence but can occur at any age. Most PME: s are autosomal recessively inherited and many are caused by the accumulation of abnormal storage material. Despite a common name, PMEs differ in clinical features, aetiology and pathogenesis as well as in prognosis. The most common type of PME worldwide is the Unverricht-Lundborg disease. Typical features include onset at the age of 6-15 years, stimulus-sensitive myoclonus, tonic-clonic seizures, a progressive course and characteristic electroencephalographic findings.

A positional cloning strategy has been applied to identify the genetic defects that are responsible for the Unverricht-Lundborg disease (Penacchio, L. A., et al., Science, 1996: 271: 1731-1734, WO 97/34625). The underlying gene encodes cystatin B, a cysteine proteinase inhibitor, also termed neutral cysteine proteinase inhibitor,

NCPI.

Cysteine proteinases are proteolytic enzymes which in the active center carry cysteine. They require an external sulfhydryl reagent in order to be in active reduced form. The most and the best known of the mammal cysteine proteinases belong to the cathepsin family, and of these, at least the cathepsins B, H, L, S, O, U, N, K, W and F have been purified and classified. Cathepsins exist everywhere in the organism, but especially they are present in the kidneys, liver and macrophages 2

(Rinne, A. et al, Biomed Biochim Acta 1986:45: 11-12; 1465-1476). Cystatin B is is present in spleen, muscle and palatine tonsils (Rinne A., et a , Verh. Anat. Ges. 75, 573-574 (1981), Jarvinen, M. and Rinne A., Biochem. Biophys. Acta 1982: 708, 210-217), and has been localized intracellularly in the cytosol of cells in different tissues such as lymphocytes (Davies, M. E. and Barrett A. J. , Histochem. 80, 373-377, 1984). The cathepsins are in general proteolytically active at acid pH, but cathepsin S is active at physiological pH (Kirschke H. , et al., Biochem J 1989:264:467-473). The cysteine proteinases have activity for dissolving biological materials, which activity can be inhibited by means of cysteine proteinase inhibitors.

Also the caspase and calpaine families belong to the cysteine proteinases. The cas- pases are able to cleave their substrates at the C-terminal site of specific aspartic acid (Martin, S. J. and Green, D. R., Cell 82, 349-352, 1995). The activation of caspases is a critical step in the death-signalling process in the different cell types. Caspase-3 like proteinase activation has been observed in different processes leading to apoptosis and it has also been documented that this process can be inhibited by using caspase-3 inhibitor (Kuida, K. et al., Nature 384, 368-372, 1996).

Until recently the clinical diagnosis of myoclonus epilepsy has been uncertain, and carriers have been difficult to trace. The method referred to above based on molecular genetics developed in 1996 which is aimed at detecting a mutation in the cystatin B encoding gene, is very elaborate.

SUMMARY OF THE INVENTION

The present invention provides a method for diagnosing epilepsy, in particular, progressive myoclonus epilepsy, which method is sensitive and which is easy to carry out on any specimen, for example on lymphoblastoid cell cultures, derived from an individual, such as a mammal, to be diagnosed, or suspected of being a carrier of the disease. The object of the present invention is thus a method for diagnosing epilepsy or other neurodegenerative disorders in an individual, comprising measuring from a sample from said individual, one or more cathepsins selected from the group consisting of cathepsin S, cathepsin B and cathepsin L.

According to a preferred embodiment of the invention, the diagnosis is based on measuring specifically cathepsin S, which has proven to be a sensitive marker in the diagnosis of epilepsy, especially progressive myoclonus epilepsy (PME).

In accordance with a further embodiment of the invention, in addition to determining the said cathepsins in a sample from an individual, also cysteine proteinase inhibitor, namely cystatin B or NCPI, is measured. The absence of or decrease in inhibitor concentration in the sample can be taken as further strengthening a diagnosis of the disorder.

DETAILED DESCRIPTION OF THE INVENTION

According to a preferred embodiment of the invention, the method is carried out by using as test samples lymphoblastoid or lymphocyte cell cultures, or muscle tissue, for the determination of the various cathepsins, which samples are easily obtainable.

The method for detecting and measuring the level of cathepsins in a sample can easily be carried out using per se known methods. Typically such methods include using suitable enzyme substrates, especially chromogenic or fluorogenic substrates, for measuring the enzyme activity in a sample to be tested, or immunological methods. Inhibitor activity can be determined by using, as a test enzyme, papain or a papain like substance. Papain is known to be a cysteine proteinase and therefore able to hydrolyze the same chemical substrates as human cathepsins. Inhibitor activity can also be measured for example using immunological methods with antibo- dies against cystatin B. 4

According to one embodiment of carrying out the method according to the invention, in a first step, a screening step, the concentration of papain inhibition activity (cystatin B activity) and papain like hydrolyzing activity is determined in the sample. In addition to cultured lymphoblasts, also muscle tissue is suitable for the deter- mination of cystatin B activity, as such cells are low in cystatin A activity, which can interfere with the measurement. Before the measurement, the cells or tissue should be homogenized in a buffer and the supernatant is collected for the measurement. The inhibition of the hydrolyzing activity of papain by the cystatin B concentration in the sample is documented. The activity is measured suitably using fluoro- genie or chromogenic substrates. This method allows both the inhibitor activity and the papain like (cathepsin) activity in the sample to be determined in the same test series simultaneously.

For further diagnosis, an individual is selected which has a low or no inhibitor activity. Also an increase in papain like activity can be recognized as a screening criteria. Thereafter the specific cathepsin S, B or L activity is measured from a sample of said individual. For the said measurement, the cathepsins in the sample are activated, and the activated sample is brought into contact with a suitable enzyme substrate. The reaction products from the reaction between the enzyme and the substrate are measured, using suitable perse known techniques, such as methods based on fluorescence or chromogenic methods.

An object of the invention is also a kit for use in diagnosing epilepsy or other neurodegenerative disorders in an individual, by measuring from a sample from said individual one or more cathepsins selected from the group consisting of cathepsin S, cathepsin B and cathepsin L, the kit comprising means for measuring the said cathepsin in the sample.

According to one embodiment of the invention the means for measuring the cathep- sin comprise means for activating the said cathepsin, and a substrate for said activated cathepsin. 5

According to an embodiment of the invention, the kit also contains a means for stopping the reaction between the activated cathepsin and the substrate.

According to a further embodiment of the invention, the said kit further comprises a means for measuring cysteine proteinase inhibitor.

The invention also provides a kit which can be used for screening individuals for cathepsin S, B and/or L determination in accordance with the invention, said kit comprising - a reagent containing papain or a papain like cysteine proteinase,

- means for activating cysteine proteinase, and

- a substrate for the activated cysteine proteinase.

In addition, the kit can contain a stopper for stopping the reaction between the activated enzyme and the substrate. According to a preferred embodiment of the invention, the kit contains papain as the cysteine proteinase enzyme.

In both kits employing a substrate, the substrate is preferably a fluorogenic substrate, for example containing the fluorescing group 4-methyl coumaryl amide (-NH- Mec). When the cathepsin to be determined is cathepsin S, the enzyme substrate of choice is Z-Phe-Arg-NHMec, which is available commercially (e.g. Bachem).

In accordance with a further embodiment of the kit, the means for measuring cathepsin and/or inhibitor activity comprise immunological means preferably for use in a method based on immunofluorescence or immunoenzyme techniques.

The kit according to the invention can comprise a combination of the various components needed for carrying out the said diagnosis or identification, presented as a single pack. For such a purpose the kit can contain for example various vials or containers containing the different reagents or substrates needed in the method. 6

The present invention also allows for the treatment of an individual after having been diagnosed for epilepsy as described above by detecting and measuring from a sample from said individual one or more cathepsins selected from the group consisting of cathepsin S, cathepsin B and cathepsin L, by administering to said individual en effective amount of neutral cy stein proteinase inhibitor.

DESCRIPTION OF THE DRAWING

In the appended Figures, individuals Bl-3 mean controls, B4-8 mean carriers and B9-19 mean patients.

Figure 1 shows both papain like and papain inhibitor activity in individuals Bl to B19.

Figure 2 shows activity of cathepsin S on a Z-Phe-Arg-NHMec substrate at pH 7.5 after pre-incubation for 60 minutes. The cathepsin S activity is negative for controls and carriers, but a high cathepsin S activity is evident in almost all patient samples.

Figure 3 shows activity of cathepsin B on a Z-Arg-Arg-NHMec substrate at pH 5.5. In all patient samples, the activity was clearly increased with respect to controls and carriers.

Figure 4 shows activity of cathepsin L on a Z-Phe-Arg-NHMec substrate at pH 5.0, in the presence of cathepsin L inhibitor (Z-Phe-Tyr(rBu)-diazomethyl) (no remaining samples B13 and B14). Activity behaviour is similar to that of cathepsin B.

Figure 5 shows activity of cathepsins B + L on a Z-Phe-Arg-NHMec substrate at pH 5.0. The increase in activity in the patient samples with respect to controls and carriers is clearly evident. Figure 6 shows activity of cathepsin H on Z-Arg-NHMec substrate at pH 6.5. Contrary to the findings above, there is no clear distinction between the activities for patients, controls and carriers.

Figure 7 shows the activity of indicated cathepsins (B+L, B, L and S) as well as inhibitor activity for specific pairs of controls, carrier and patients. The trend in activities between controls, carriers and patients is clearly evident, the controls having the highest inhibitor activity and lowest cathepsin activity, the reverse being largely true for the patient group.

Figure 8 shows the caspase-3 activity in samples from individuals Bl to B19, and shows an increase in activity in patients and carriers, as compared to controls.

From the Figures shown above it is evident that increased levels of cathepsin S, B and/or L activity support a diagnosis and make it possible to distuingish between patients on the one hand and carriers and controls on the other hand. We have also documented an increased activity of caspase-3 in the samples of PME patients and also of carriers which activity is reduced by cystatin B. This information can be used for distinguishing carriers from controls.

Based on the above it is evident that measured activities of cathepsin S, B and L in samples which can be of the order of from appr. 5 to 10 times those found in controls, can be regarded to support a diagnosis of PME. In addition, by determining the inhibitor activity, as is evidenced from Figure 7 above, a supporting diagnosis can be made. It is seen that the inhibitor activity is clearly increased in the controls, less increased in the carriers and only slight inhibitor activity is evidenced in the patient samples. It has been measured that inhibitor activity in controls and carriers are of the order of appr. 2-5 times those found in patients.

Cathepsins, especially cathepsin B, and cystatin B have been measured also using immunofluorescence and immunoenzyme techniques. Such methods are known to the person skilled in the art. The results obtained support the results obtained above, 8 namely the cystatin B activity is decreased and the cathepsin activity is increased in patients, the reverse being true for controls.

The following examples illustrate the invention without restricting the same.

EXAMPLE 1

Preparation of lymphoblastoid samples

For the preparation of the lymphoblastoid samples, a concentration of appr. 100 million cells/mm³ are used for each sample. The cells were washed twice with PBS buffer and added to a suspension buffer made from 0.25 M sucrose, 0.1 M NaCl and 50 mM K-phosphate (pH 6.5-7.4) so that the concentration is at least 100 million cells/ml of buffer. The suspension is homogenized in an ice bath 2-3 x 30 seconds, after which it is left standing for 10-15 minutes prior to centrifugation. It is centrifuged for 10 minutes at + 4°C at 6000 rpm. The supernatant is collected. The samples can be stored at -20°C.

EXAMPLE 2

Determination of cysteine proteinase inhibitor and of papain like activity

A sample of 100 μl is combined with 100 μl of a papain solution. Before the assay a stock solution of 0J mg/ml 2 x crystallized papain (Sigma) is diluted 1: 10 with the assay activation buffer containing 0.1 M sodium phosphate, pH 6.2, and 2.5 mM dithiotreitol (DTT) and 2.5 mM Na₂EDTA. The reaction between inhibitor and papain is allowed to incubate for 10 min at + 20 °C in the presence of 200 μl activation buffer. Thereafter 200 μl of a substrate made from 10 μl of 10 mM Z- Phe-Arg-NHMec in 8 ml of water (= 12.5 μM) is added and the mixture incubated for 10 minutes at +37°C. The reaction is stopped exactly after 10 minutes with a solution containing 0.1 M monochloro-acetate, 0J3 mol/1 NaOH and 0J mol/1 acetic acid. The measurement is carried out spectrofluorometrically using excitation at 375 nm and measuring emission at 460 nm. As the standard, 7-aminomethyl- coumarin 0.1 μmol/1 is used.

In the control tubes there is water instead of sample. The relative inhibitor content of the sample is calculated using the formula

K - A x E_cooc = PlU/ml K

where K = the result from the control assay A = the result of the sample assay E_conc = the concentration of papain.

The amount of inhibitor is then calculated per total protein content of the sample (PlU/mg). The enzyme activity of the sample is given as units per protein concentration per time of the reaction.

The inhibitor activities as well as papain like activity are shown in the Figure 1 for individuals Bl - B19.

EXAMPLE 3

Determination of cathepsin S

A sample of 100 μl is activated by combining the same with 200 μl of an activation buffer, pH 7.5, made from 2 ml of 0.2 M Tris-HCl pH 7.5, 100 μl of each 0J M DTT and 0J M Na₂-EDTA. This mixture is preincubated for 60 min at + 37°C. Then 200 μl of an enzyme substrate solution made from 10 μl 10 mM Z-Phe-Arg- NHMec (Z-Phe-Arg-NH-(4-methyl cumaryl)) is added in 8 ml of water (= 12.5 μM). The mixture is then incubated for 10 minutes at +37 °C. Thereafter 2 ml of a stopper solution made from 4.73 g of 0J M monochloro-acetate, 65 ml of 1 M 10

NaOH (=0.13 mol/1), 50 ml of 1 M acetic acid (=0.1 mol/1) and water to 500 ml is added.

The concentration is measured spectrofluorometrically using excitation at 375 nm and measuring emission at 460 nm.

The activities so measured for cathepsin S are disclosed in the Figure 2.

Determination of cathepsin B

The determination was carried out in the same manner as for cathepsin S, except that in the activation buffer 2 ml of 0J M acetate buffer pH 5.5 was used instead of 2 ml 0.2 M Tris-HCl pH 7.5, and the substrate was Z-Arg-Arg-NHMec instead of Z-Phe-Arg-NHMec. Also the preincubation step between sample and activation solution lasted for 10 min rather than 60 minutes.

The activity results obtained are disclosed in the Figure 3.

Determination of cathepsin L

Determination of cathepsin L took place in the presence of the specific cathepsin L inhibitor Z-Phe-Tyr-(rBu)-diazomethyl. A sample of 100 μl and 100 μl of an inhibitor solution made from a 10 mM base solution by diluting the base solution immediately prior to use with water 1:20 (= 0J ml + 1.9 ml water = 500 μM), there thus being 8 μM in the reaction, is actived by combining the same with 200 μl of an activation buffer made from 2 ml of 0J M acetate buffer pH 5.0, 100 μl of each 0.1 M DTT and 0J M Na₂-EDTA. This mixture is preincubated for 10 min at + 37°C. Then 200 μl of an enzyme substrate solution made from 10 μl 10 mM Z- Phe-Arg-NHMec is added in 8 ml of water, the resulting solution being 12.5 μM with respect to substrate. The mixture is then incubated for 10 minutes at +37 °C. Thereafter 2 ml of a stopper solution made from 4.73 g of 0J M monochloro 11 acetate, 65 ml of 1 M NaOH (=0.13 mol/1), 50 ml of 1 M acetic acid (=0.1 mol/1) and water to 500 ml is added.

The control solution contains 100 μl of water instead of the inhibitor solution and represents the activity of cathepsins B and L together.

The measurement is then carried out spectrophotometrically using excitation at 375 nm and measuring emission at 460 nm.

The activities shown in Figure 4 represent the difference between the activity of the control solution and that measured in the presence of the inhibitor.

Determination of cathepsins B + L

The determination was carried out in the same manner as for cathepsin S, except that in the activation buffer 2 ml of 0J M acetate buffer pH 5.0 was used instead of 2 ml 0.2 M Tris-HCl pH 7.5, the substrate being unspecific Z-Phe-Arg-NHMec. The pre-incubation step between sample and activation solution lasted 10 minutes rather than 60 minutes. The activity results obtained are disclosed in the Figure 5.

Determination of cathepsin H

The determination was carried out in the same manner as for cathepsin S, except that in the activation buffer 2 ml of 0J M acetate buffer pH 6.5 was used instead of 2 ml 0.2 M Tris-HCl pH 7.5, the substrate being unspecific Z-Arg-NHMec. The pre-incubation step between sample and activation solution lasted 10 minutes rather than 60 minutes.

The activity results obtained are disclosed in the Figure 6. 12 Determination ofcaspase-3

A sample of 100 μl is actived by combining the same with 300 μl of an activation buffer, pH 7.5, made from 2 ml of 20 mM HEPES pH 7.5, 10 % glycerol, and 200 μl of each 0.1 M DTT and 0J M Na₂-EDTA. This mixture is pre-incubated for 30 min at + 37°C. Then 200 μl of an enzyme substrate solution made from 10 μl 10 mM Ac-DEVD-AMC (Pharmingen) is added in 5 ml of water (=20 μM). The mixture is then incubated for 30 minutes at +37 °C. Thereafter 2 ml of a stopper solution made from 4.73 g of 0J M monochloro-acetate, 65 ml of 1 M NaOH (=0.13 mol/1), 50 ml of 1 M acetic acid (=0.1 mol/1) and water to 500 ml is added.

The activities so measured for caspase-3 are disclosed in the Figure 8.

Claims

13Claims

1. A method for diagnosing epilepsy or other neurodegenerative disorders in an individual, comprising measuring from a sample from said individual, one or more cathepsins selected from the group consisting of cathepsin S, cathepsin B and cathepsin L.

2. The method according to claim 1 for diagnosing progressive myoclonus epilepsy (PME) in the individual.

3. The method according to claim 1 or 2, comprising measuring cathepsin S from said sample.

4. The method according to claim 1, comprising measuring from a sample from said individual also neutral cysteine proteinase inhibitor, cystatin B.

5. The method according to claim 1 or 4, wherein the sample is a lymphoblastoid or lymphocyte sample, or a muscle sample.

6. A method for diagnosing epilepsy or other neurodegenerative disorders in an individual, comprising

- measuring neutral cysteine proteinase inhibitor from a sample from said individual,

- selecting for further diagnosis an individual with low or no inhibitor activity, and

- measuring from a sample of such a selected individual one or more cathepsins selected from the group consisting of cathepsin S, cathepsin B and cathepsin L.

7. The method according to claim 6 comprising the further step of measuring prior to the selection, also cysteine proteinase activity in a sample, preferably the same sample used for measuring the inhibitor. 14

8. The method according to claim 1 or 6, comprising measuring in addition caspase- 3 activity from a sample of said individual.

9. The method accoridng to any one of claims 1-8, wherein the measurement of cathepsin and cystatin B inhibitor is carried out using enzyme substrate techniques.

10. The method according to any one of claims 1-8, wherein the measurement of cathepsin and cystatin B inhibitor is carried out using immunological, such as immunofluorescence or immunoenzyme techniques.

11. Kit for use in diagnosing epilepsy, preferably progressive myoclonus epilepsy, or other neurodegenerative disorders in an individual, by measuring from a sample from said individual one or more cathepsins selected from the group consisting of cathepsin S, cathepsin B and cathepsin L, the kit comprising - means for measuring the said cathepsin in the sample.

12. The kit according to claim 11, the means for measuring the said cathepsin in the sample comprising

- means for activating the said cathepsin, and - a substrate for said activated cathepsin.

13. The kit according to claim 12, comprising a means for stopping the reaction between the activated cathepsin and the substrate.

14. The kit according to claim 12 for measuring cathepsin S activity, wherein the enzyme substrate contains a -Phe-Arg-NHMec group.

15. The kit according to claim 12 comprising means for measuring caspase-3 activity.

16. The kit according to claim 11 or 12 comprising means for measuring neutral cysteine proteinase inhibitor in a sample from said individual. 15

17. A kit comprising

- a reagent containing papain or a papain like cysteine proteinase,

- means for activating cysteine proteinase, and

- a substrate for the activated cysteine proteinase.

18. The kit according to claim 17, comprising means for stopping the reaction between the activated cysteine proteinase and the substrate.

19. The kit according to claims 11 or 16, the means for measuring cathepsin and/or inhibitor activity comprising immunological means for use in a method based on immunofluorescence or immunoenzyme techniques.