ES2564426B2 - Marker of eye diseases - Google Patents

Abstract

Marker of ocular pathologies. # The present invention relates to an in vitro method for diagnosing or determining the degree of an ocular surface pathology, to an in vitro method for monitoring the effect of a therapy administered to a patient diagnosed with a pathology of ocular surface and an in vitro method to select a subject to undergo corneal refractive surgery based on the determination of heparanase activity in a sample of said subject. The invention also relates to the use of heparanase activity as a diagnostic marker of an ocular surface pathology, as a marker to determine the degree of said pathology, as a marker to monitor the effect of a therapy administered to a patient diagnosed with a pathology of ocular surface and the use of heparanase activity to select a subject to undergo corneal refractive surgery.

Description

Marker of eye diseases

FIELD OF THE INVENTION

The present invention falls within the field of diagnostic methods of ocular pathologies.

BACKGROUND OF THE INVENTION

The cornea is a transparent part of the eye that serves to let light through and focus the images of the outside world on the retina.

Keratoconus is a degenerative disease of the cornea that leads to thinning and alteration of the corneal form, thereby losing its function of letting light in and focusing images on the retina, causing the patient to have poor vision even with the better correction of glasses or contact lenses of the cornea. In many cases the patient needs surgery (cross-linking of the collagen with ultraviolet light, segments of the intra-stromal rings or cornea transplant) and despite this the patient may have a permanent decrease in vision that affects their quality of life and work activity.

The diagnosis of keratoconus is currently made with image analysis techniques (videokeratosocopy) but there are no biological markers that allow early diagnosis even before the patient has symptoms of impaired vision. Early diagnosis is very important in order to carry out preventive measures and treatments to avoid the evolution of the disease. It is also very important since the vast majority of patients with keratoconus are myopic and suffer from astigmatism so they are possible candidates for corneal refractive surgery, which if performed would aggravate the disease and accelerate the progression of keratoconus.

There is therefore a need in the state of the art to provide biological markers for the diagnosis and detection of the development of an ocular pathology, especially keratoconus.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to an in vitro method for diagnosing or determining the degree of a pathology of the ocular surface in a subject comprising:

(i)

determining heparanase activity in a sample comprising a tear of said subject; Y

(ii)

compare the heparanase activity obtained in step (i) with a reference value;

wherein, an increase in said heparanase activity with respect to the reference value is indicative that said subject undergoes a pathology of the ocular surface or an advance in the degree of a pathology of the ocular surface.

In a second aspect, the invention relates to an in vitro method for monitoring the effect of a therapy administered to a patient diagnosed with a pathology of the ocular surface comprising:

(i)

determining heparanase activity in a sample comprising a tear of said patient; Y

(ii)

compare the heparanase activity obtained in step (i) with a reference value;

wherein, a stagnation and / or a decrease in said heparanase activity with respect to the reference value is indicative of the positive effect of said therapy.

In a third aspect, the invention relates to an in vitro method for selecting a subject to undergo corneal refractive surgery comprising:

(i)

determining heparanase activity in a sample comprising a tear of said subject; Y

(ii)

comparing said heparanase activity obtained in step (ii) with a reference value;

where, an increase in heparanase activity with respect to the reference value

it is indicative that said subject is not a candidate to undergo refractive surgery

corneal

In a fourth aspect, the invention relates to the use of heparanase activity as a diagnostic marker of an ocular surface pathology, as a marker to determine the degree of an ocular surface pathology, as a marker to monitor the effect of a therapy administered to a patient diagnosed with a pathology of the ocular surface or as a marker to select a patient to undergo corneal refractive surgery!

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Model of the structure in domains of the heparan sulfate molecule.

Figure 2: Superase 12 column molecular exclusion chromatography of [3H] _HS

Fractions 23 to 29, corresponding to molecules with molecular weights greater than 20 kDa, were isolated and concentrated for use.

Figure 3: Analysis of reactions by molecular exclusion chromatography in Superase 12 column. (E) Negative control. (.) Enzymatic reaction carried out with EBNA-293 cell extract as a positive control.

Figure 4: Analysis of reactions by ultrafiltration.

Figure 5: Heparanase activity in tear as a function of the degree of keratoconus according to Rabinowitz. Vertical bars indicate confidence intervals of 0.95.

DETAILED DESCRIPTION OF THE INVENTION The authors of the present invention have developed a method for diagnosing ocular surface pathologies, namely, keratoconus. The authors of the invention have observed that, surprisingly, there is a greater heparanase activity in samples of patients suffering from said pathology when compared to healthy subjects as demonstrated in the examples. This activity can also be used in methods to monitor the effect of a therapy in patients previously diagnosed with keratoconus as well as select patients who are candidates for corneal refractive surgery.

First method of the invention

In a first aspect, the invention relates to an in vitro method for diagnosing or determining the degree of a pathology of the ocular surface in a subject, hereinafter "first method of the invention", comprising:

(i)

determine the activity heparanase in a sample that understands a

tear of said subject; Y

(ii)

compare heparanase activity obtained in stage (i) with a value of

reference;

in

where a increase  in said heparanase activity with With respect to value of

reference is indicative that said subject undergoes a pathology of the ocular surface or an advance in the degree of a pathology of the ocular surface.

The term "diagnosis" as used herein, refers both to the process of trying to determine and / or identify a possible disease in a subject, that is to say the diagnostic procedure, and to the opinion reached by this process, that is to say , the diagnostic opinion. As such, it can also be considered as an attempt to classify the status of an individual into separate and distinct categories that allow medical decisions to be made about treatment and prognosis. As the person skilled in the art will understand, such a diagnosis may not be correct for 100% of the subjects to be diagnosed, although it is preferred that it be. However, the term requires that a statistically significant part of the subjects can be identified as suffering from a disease, particularly a pathology of ocular supertice in the context of

invention, or having a predisposition to it. The person skilled in the art can

determine if a part is statistically significant using different tools of

well-known statistical evaluation, for example, by determining intervals

of confidence, the determination of the value of p, the Student's t test, the Mann-

S

Whitney, etc. (see, Dowdy and Wearden, 1983). Preferred confidence intervals are

at least 50%, at least 60%, at least 70%, at least 80%, at least

90% or at least 95%. P values are preferably 0.05, 0.025, 0.001 or

minors

10

The term "pathology of the ocular surface" as used herein, includes the processes

chronic and progressive that are characterized by an alteration of the corneal structure

or of the sclerocorneal limbus causing a deterioration of its transparency which entails a

visual damage Such pathologies include but are not limited to: keratoconus, opacity

corneal, dry eye, conjunctivitis, corneal ulcer and blepharitis.

fifteen

In a particular and preferred embodiment of the invention, said surface pathology

Eye is keratoconus. The term "keratoconus ~, as used herein

document, refers to a progressive disease of the cornea that causes poor vision in

which the cornea adopts an irregular conical shape due to the alteration of the fibers of

twenty

collagen in its internal structure. It is bilateral in most cases and its

Progression is asymmetric. The main anatomical symptom of keratoconus is the

thinning of the cornea in its central or paracentral zone, that is, the cornea becomes

finer as keratoconus develops, which leads to worsening

of vision as keratoconus advances. Keratoconus can be classified as

25

degrees according to the corneal curvature: grade 1: when the corneal curvature is less or

equal to 45D (diopters); Grade 2: when the corneal curvature is between 45 and

52D; grade 3: when the corneal curvature is between 52 and 60D; And grade 4:

when the corneal curvature is greater than 60D. Corneal curvature can be determined

using a cornea tomograph l. Corneal tomographs have built-in algorithms

30

which allow to determine the degree of corneal curvature by calculating the index of

Maloney, the measured index of corneal irregularity, average toric keratometry, the index

of irregularity of the surface, the Rabinowitz index, the KISA index, the index of

Keratoconus prediction, the Z3 index or combinations thereof.

In an even more particular and preferred embodiment of the invention, said keratoconus is grade 3.

The term "subject", as used herein, refers to a person, such as a being

5 human, a non-human primate (eg, chimpanzees and other apes and species of monkeys), farm animals, such as birds, fish, cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs. The term does not denote a certain age or sex. In a particular embodiment of the invention, the subject is a

10 mammal In a preferred embodiment of the invention, the subject is a human.

The expression "sample comprising a tear" as used herein, refers to any sample that can be obtained from a subject in which there is body fluid produced by the tear gland of said subject. The sample can be obtained through

15 different extraction methods such as pipette or capillary extraction.

The first method of the invention comprises in step (i) determining heparanase activity in a sample comprising a tear of a subject. The term "heparanase", also referred to as HPSE, as used herein, refers to the protein with which it acts both in the cell's superstructure and within the extracellular matrix degrading polymers, preferably heparan polymers. sulfate, in oligosaccharides. The protein is initially synthesized in a 65 kDa pro-heparanase as an inactive form in the Golgi apparatus and is transferred through the endosomes / lysosomes to cell supertice. In the lysosome it is proteolytically processed in its active form. 25 By proteolytic processing of heparanase, three products are obtained: a linker peptide, an 8 kDa fragment (pro-heparanase) and a 50 kDa fragment (proheparanase). The 8 kDa and 50 kDa fragments form a heterodimer that forms the active heparanase molecule. In a preferred embodiment, the term "heparanase" refers to the active molecule. The sequence of human heparanase is found

30 deposited in the GenBanK database (May 11, 2014 version) under accession number NP 001092010.

The term "heparanase activity", as used herein, refers to the enzymatic process regulated by heparanase in particular to the cleavage between glycosaminoglycan residues and heparan sulfate glucosamine.

The person skilled in the art will readily recognize that the determination of heparanase activity according to the first method of the invention can be carried out by any method or technique known in the state of the art appropriate to determine said enzymatic activity8. Non-limiting illustrative examples for determining heparanase activity in a sample are described in WO 2000/03306 and WO 2000/77241. In a particular and preferred embodiment of the invention, the heparanase activity is determined according to the method for determining the heparanase activity defined in the present invention and which is detailed hereinafter.

Step (ii) of the first method of the invention comprises comparing the heparanase activity obtained in step (i) of said first method of the invention with a reference value.

The term "reference value ~, as used herein, refers to a laboratory value used as a reference for the values / data obtained from samples obtained from the subjects. The reference value (or level reference) can be an absolute value, a relative value, a value that has an upper and / or lower limit, a series of values, an average value, a median, an average value, or a value expressed by reference to a value reference control A reference value may be based on the value obtained from an individual sample, such as a value obtained from a sample of the subject under study but obtained at an earlier point in time. of reference may be based on a large number of samples, such as the values obtained in a population of the subjects of the chronological age group coinciding with that of the subject under study or based on a set of samples of inclusion or exclusion of the sample to be analyzed. The reference value may be based on the expression values of the markers that are to be compared obtained from samples of healthy subjects that do not have a disease state or a particular phenotype. In the context of the present invention, the reference value refers to the value of heparanase activity obtained from samples comprising tears of subjects who do not suffer from an ocular surface pathology (particularly control subject), of subjects who do not suffer keratoconus Methods to obtain said sample have been mentioned above. It is preferable that the technique or method used to determine heparanase activity in the sample obtained from said control subject is the same as the technique used to determine heparanase activity in the subject whose diagnosis is to be determined.

Once the reference value has been established, the value obtained from said heparanase activity in the sample of the subject under study is compared with the reference value. As a consequence of this comparison, the heparanase activity determined in the subject's sample may be "increased", "decreased" or "equal to" said reference value. In the context of the present invention, heparanase activity is considered to be "increased" in the subject's sample with respect to the reference value when heparanase activity in the subject's sample increases, for example, 5%, 10% , 25%, 50%, 100% or even more when compared to the reference value, or when it increases, for example, at least 1.1 times, 1.5 times, 2 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even more when compared to the reference value. Also, in the context of the present invention, heparanase activity is considered to be "decreased" in the subject's sample when heparanase activity in the subject's sample decreases, for example, 5%, 10%, 25% , 50%, 75%, or even 100% when compared to the reference value for said activity in the control subject.

Also, in the context of the present invention, the heparanase activity in the subject's sample is considered to be "equal to" the reference value for said activity when the value obtained in determining the heparanase activity in the subject under study is substantially unchanged with respect to the reference value; for example, the heparanase activity determined in the sample of the subject under study is considered to be "equal to" the reference value when the levels differ by no more than 0.1%, not more than 0.2%, no more than 0.3%, no more than 0.4%, no more than 0.5%, no more than 0.6%, no more than 0.7%, no more than 0.8% no more than 0.9%, no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, or no more than the percentage value that is the same as the error associated with the experimental method used in the determination.

Once the comparison is made between the value obtained in the determination of the heparanase activity in the subject's sample and the reference value for said activity, the first method of the invention allows to diagnose if a subject suffers from a pathology of the ocular surface, particularly keratoconus. , based on whether the heparanase activity determined in the sample of the subject under study is increased with respect to the reference value.

In another particular embodiment, the first method of the invention makes it possible to determine the degree of a pathology of the ocular surface in a subject, particularly keratoconus. In this case, the reference value used for this purpose is the value of the heparanase activity determined in a sample of a subject that does not suffer from an ocular surface pathology, (control subject), particularly keratoconus, or, preferably, the value of The heparanase activity determined in a sample of a subject suffering from said ocular surface pathology at an early stage of its development. In this case, once the comparison is made between the value obtained in the determination of the heparanase activity in the subject's sample and the reference value for said activity, the first method of the invention allows to determine an advance in the degree of a pathology of The ocular surface, particularly keratoconus, based on whether the heparanase activity determined in the sample of the subject under study is increased with respect to the reference value.

In a particular and preferred embodiment of the invention, the heparanase activity determined in step (i) of the first method of the invention, is determined by a method, hereinafter "method for determining the heparanase activity of the invention ~, which understands:

(i)

contacting said sample with a heparanase substrate;

(ii)

keeping the sample of step (i) under conditions suitable for degradation of said substrate by heparanase; Y

(iii) detect the reaction products obtained in step (ii).

Step (i) of said method comprises contacting the sample where it is desired to determine heparanase activity, that is, the sample comprising a tear of a subject under study, with a heparanase substrate. The term "heparanase substrate», as used herein, refers to polymers containing glycosaminoglycan units. The term "glycosaminoglycans" or "mucoplisaccharides", as used herein, refers to to long and unbranched chains of heteropolysaccharides, generally composed of a repetitive unit of disaccharide with the general formula (sugar-amino sugar) n, where n refers to the number of repetitions. The amino sugar may be D-glucosamine or Dgalactosamine, in which the amino group is normally acetylated in order to eliminate its positive charge, and can also carry a sulfate group in carbon 4 or 6 or in a non-acetylated nitrogen.The most common are N-acetylglucosamine or Nacetylgalactosamine. The acid sugar can be a D-glucuronic acid or L-iduronic acid, except for keratan sulfate, where it is normal for there to be a galactose. These acidic sugars prevail over the others. Each dimer is linked to the next by another glycosidic bond, but in this case in beta 1-4 position. This type of conformation confers a resistant structure.

In a particular and preferred embodiment of the invention, said substrate is heparan sulfate. The term "heparan sulfate" as used herein, refers to a heteropolysaccharide whose structure is based on the repetition of the disaccharide formed by glucuronic acid (GlcA) and N-acetyl-glucosamine (GlcNAc), giving rise to long and unbranched chains with varying sizes, between 5 and 70 kDa. In different positions, the molecule is modified by a series of interdependent enzymatic reactions that include N-deacetylation of GlcNAc, usually followed by Nsulfatation, to produce GlcNSOJ, which causes sulphated domains in the molecule (NS), within which the GlcA can be epimerized to iduronate (ldoA), and O-sulfate groups can be added to glucosamine e-6 and IdoA C-2. Also, although less frequently, additions of O-sulfate groups can occur to C-3 of GlcN residues and C-2 of GlcA residues. The modification of the chain causes flexible, highly sulfated regions, rich in IdoA (NS regions), separated by more rigid regions of low sulfation (NA regions). At the confluence of both types of domains there are mixed NAlNS regions.

The term "contacting", as used herein, refers to any process by which a sample comprising a tear of a subject comes into contact with a heparanase substrate. This expression includes any in vitro process of contacting the sample with said substrate according to the invention. In the context of the present invention, it is sufficient to add said substrate to the sample comprising a tear of a subject and whose heparanase activity wishes to be determined.

Step (ii) of the method for determining the heparanase activity of the invention comprises maintaining the sample of step (i) under conditions suitable for degradation of said substrate by heparanase. The term "suitable conditions for degradation of said substrate by heparanase", as used herein, refers to the conditions required for the interaction between said substrate and said heparanase to occur and hydrolysis of the substrate. substrate by heparanase action. The person skilled in the art will understand that the conditions suitable for the heparanase to hydrolyze said substrate include an adequate ratio between the substrate and the enzyme in terms of concentration, and suitable conditions of salt, temperature, pH and reaction time.

Suitable salt conditions for degradation of said substrate by the heparanase according to the invention include, without limitation, any commonly known buffer such as phosphate buffer, a bicarbonate buffer, a carbonate buffer, Tris buffer, a borate buffer, a succinate buffer, a histidine buffer or a citrate buffer at concentrations of at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 200 mM, at least 300 mM, or more. In a preferred embodiment, degradation of the substrate by heparanase is carried out in 100 mM citrate buffer.

Suitable pH values for degradation of said substrate by heparanase according to the invention include, without limitation, values of 4.5, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.5, 7, 7, 5, 8, 8, 5. In a preferred embodiment, said pH is 5.3.

Suitable temperature conditions for degradation of said substrate by the heparanase according to the invention include, without limitation, about 20 ° C, about 25 ° C, about 30De, about 35 ° C, about 37 ° C. In a preferred embodiment said temperature is 37 ° C. Suitable reaction times for degradation of said substrate by the heparanase according to the invention include, without limitation at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours , at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours or more. In a preferred embodiment said reaction time is 16 hours.

Suitable substrate concentrations for degradation of said substrate by the heparanase according to the invention include, without limitation, approximately 1 nM, approximately 5 nM, approximately 10 nM, approximately 20 nM, approximately 30 nM, approximately 40 nM, approximately 50 nM, about 100 nM, about 500 nM, about 1 ~ M, about 10 ~ M, about 20 ~ M, about 30 ~ M, about 40 ~ M, about 50 ~ M, about 100 ~ M, about 500 ~ M, about mM , about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 100 mM or more. In a preferred embodiment, said substrate concentration is 500 nM.

Once the adequate time has elapsed for the heparanase to degrade the substrate, for example after 16 hours, it is preferable to stop the reaction to avoid obtaining nonspecific reaction products that may interfere with the result. The stopping of said reaction can be carried out by altering the optimum conditions in which the mime is carried out, for example by altering the temperature and / or by adding to the reaction mixture any suitable agent that interferes with the reaction. The agents used to stop a reaction alter the optimal conditions in which said reaction is carried out but, preferably, without modifying the products obtained in the reaction. For example, agents that modify the optimal reaction pH, that modify the optimal concentration of salts in the reaction, agents that precipitate the enzyme carrying out the reaction, agents that sequester the reaction substrate, etc. can be employed.

In a preferred embodiment of the invention, the reaction is stopped by adding a reaction volume of a 2M NaCl solution and a chloroform reaction volume to precipitate protein residues present in the sample.

In a particular embodiment, the method for determining the heparanase activity of the invention comprises the use of a substrate labeled with a detectable label selected from a group consisting of a fluorochrome labeled molecule, an enzymatically labeled molecule and a radioactively labeled molecule. . The term "detectable label", as used herein, refers to a molecular label whose purpose is to allow the visualization or detection of the molecules to which it is anchored by appropriate procedures and equipment for the detection of labeling. Substrate labeling can be done by conventional methods. Said labeling can be direct, for which fluorophores can be used, for example, Cy3, Cy5, fluorescein, alexa, etc., enzymes, for example, alkaline phosphatase, peroxidase, etc., radioactive isotopes, for example, JJp, 3H 1251 , etc., or any other marker known to the person skilled in the art.

In an even more particular embodiment, said substrate, in particular, heparan sulfate is radioactively labeled with tritium eH). The labeling of heparan sulfate with tritium can be carried out by any conventional procedure known to the person skilled in the art. Illustratively, said marking can be carried out by the procedure detailed in the examples section of this document.

Preferably, after labeling the reaction substrate (for example, heparan sulfate), said substrate is subjected to a molecular exclusion process to purify the substrate and select fractions with a suitable average molecular weight. In an even more particular embodiment of the method for determining the heparanase activity of the invention, the heparan sulfate molecules employed as the reaction substrate have an average molecular weight of at least 20kDa. In another particular embodiment, the heparan sulfate molecules used as the reaction substrate have an average molecular weight of at least 25kDa, at least 30kDa, at least 35kDa, at least 40kDa, at least 45KDa, at least 50kDa , at least 60 kDa, at least 70 kDa, at least 80kDa, at least 90kDa or more.

Step (jii) of the method for determining the heparanase activity of the invention comprises detecting the reaction products obtained in step (ii) of said method. The detection of reaction products (oligosaccharides) can be determined by any technique appropriate for this known in the art. Methods known to the person skilled in the art and which allow to detect the product of an enzymatic reaction include but are not limited to spectrophotometric tests, tests coupled to another enzymatic reaction where the obtained reaction product is used as a substrate for a second enzymatic reaction whose Reaction product is determined, fluorometric tests, calorimetric tests, chemiluminescent tests, radiometric tests or chromatographic tests.

In a particular embodiment, the detection of the reaction products obtained in step (ii) of the method for determining the heparanase activity of the invention is carried out by a molecular fractionation technique selected from ultrafiltration and molecular exclusion chromatography. Only, the detection can be carried out by the procedures shown in the examples section of this document.

As a consequence of heparanase activity, the substrate, in particular, heparan sulfate, is hydrolyzed into oligosaccharides containing reducing carbonyl groups. Therefore, alternatively, the detection of the reaction products obtained in step (ii) of the method for determining the heparanase activity of the invention is carried out by the detection of reducing ends in said reaction products. Said reducing groups can be detected by any appropriate technique. State-of-the-art techniques that allow the detection of reducing groups include but are not limited to assays using Fehling reagent, the reaction with Tollens reagent, the Maillard reaction and the Benedict reaction.

Second method of the invention

In a second aspect, the invention relates to an in vitro method for monitoring the effect of a therapy administered to a patient diagnosed with a pathology of the ocular surface comprising:

(i)

determining heparanase activity in a sample comprising a tear of said patient; Y

(ii)

compare the heparanase activity obtained in step (i) with a value of

reference; where, a stagnation and / or a decrease in heparanase activity with respect to the reference value is indicative of the positive effect of said therapy.

The term "therapy" or "treatment", as used herein, refers to the tentative recovery of a health problem, usually after a diagnosis. As such, it is not necessarily a cure, that is, a complete reversal of a disease. Such therapy may or may not be known to have a positive effect on an ocular surface pathology or, in other words, to be useful for treating an ocular surface pathology. Therapies include, without limitation the use of eye drops with appropriate drugs for the treatment of such pathologies or the use of eye drops with immunosuppressants, surgery or a combination of therapies. The term "surgery", as used herein, means any therapeutic procedure that involves a methodical action of the hand or hand with an instrument, in the body of a human or other mammal, to produce a healing or recovery.

The term "patient", as used herein, refers to a subject as defined above, who suffers and is known to suffer from a pathology of the ocular surface, ie the subject has previously been diagnosed of said surface pathology. ocular. The diagnosis can be made by any conventional method known in the state of the art suitable for the detection of an ocular surface pathology. Preferably, the diagnosis is carried out according to the first method of the invention.

The term "ocular surface pathology" has been defined in the context of the first method of the invention and is used here with the same meaning. In a particular embodiment, said ocular surface pathology is keratoconus and even more particularly, said pathology is grade 3 keratoconus. The term "keratoconus" has been defined in the first method of the invention.

Step (i) of the second method of the invention comprises determining heparanase activity in a sample comprising a tear of the subject under study. The terms "sample comprising a tear" and "heparanase activity» as well as the particular embodiments thereof have been defined above and are used here with the same meaning.

Step (ii) of the second method of the invention comprises comparing said heparanase activity obtained in step (i) with a reference value. According to this second method of the invention, as previously defined in the context of the first method of the invention, the reference value refers to the value of the heparanase activity determined at the beginning of the therapy and / or previously determined during said treatment. The term "beginning of therapy", as used herein, means a point of time just before therapy is applied, preferably immediately before, for example, before and on the same or the previous day. The term "previously", as described herein, refers to any previous time point, preferably however, it means at least 1 day, at least 2 days, at least 4 days, at least 5 days , at least 6 days, at least 7 days, at least 10 days, weeks at least 2, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 2 months or at least 3 months before, it is emphasized that the heparanase activity determined in stage (i) can be compared with more than one heparanase activity value of stage (ii), wherein said more than one stage value (ii) are preferably determined at different time points, for example, any of the points of time mentioned above.

In a particular embodiment, the determination of heparanase activity in the second method of the invention is carried out by the "method for determining the heparanase activity of the invention" and which has been detailed above in the context of the first method of the invention. . Particular embodiments of said method have been detailed in the context of the first method of the invention and are applied here in the same manner.

Once the comparison between the heparanase activity in the patient sample and the reference value for said activity has been made, the second method of the invention allows to determine the effect of the therapy being administered to the patient under study. Specifically, a stagnation and / or a decrease in heparanase activity with respect to the reference value in said patient is indicative of the positive effect of said therapy.

The term "decrease" preferably refers to a level of at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, or at least 20% less than a level determined before, for example previously as defined above. Similarly, the decrease may refer to a level of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least% 100 (i.e. absent) below a predetermined level, for example previously as defined above. In a preferred embodiment, this "level determined before" is the value determined at the start of therapy.

The term "stagnation", as used herein, preferably means a level within 3%, 5%, 8%, 10%, 15%, or a 20% range of a level determined before, for example previously as defined above. In a preferred embodiment, this "level determined before" is the value determined at the start of therapy.

The term "positive effect of a therapy" means that such treatment slows the progression of the disease or prevents the progression of the disease, and / or cure of the patient.

Third method of the invention

In a third aspect, the invention relates to an in vitro method for selecting a subject to undergo corneal refractive surgery, hereinafter, "third method of the invention" comprising:

(i)

determining heparanase activity in a sample comprising a tear of said subject; Y

(ii)

compare the heparanase activity obtained in step (i) with a value of

reference; wherein, an increase in heparanase activity with respect to the reference value is indicative that said subject is not a candidate to undergo corneal refractive surgery.

The term "select" as used herein refers to the action of choosing a subject to undergo corneal refractive surgery.

The term "corneal refractive surgery, as used herein, refers to a set of surgical procedures intended to correct ocular refractive errors that reduce image quality on the retina and, therefore, is focused on reducing and , eventually, eliminate the absolute dependence on the use of correction means (glasses or contact lenses).

The terms "subject", "shows that it comprises a tear", "heparanase activity" have been described in detail in the context of the first method of the invention and are used with the same meaning in the third method of the invention.

In a particular embodiment, the determination of heparanase activity in the third method of the invention is carried out by the "method for determining the heparanase activity of the invention" and which has been detailed in the context of the first method of the invention. Particular embodiments of said method have been detailed in the context of the first method of the invention and are applied here in the same manner.

Once the comparison between the value of the heparanase activity in the sample of the subject and the reference value for said activity is made, the third method of the invention allows the subject under study to be selected. Specifically, an increase in heparanase activity with respect to the reference value is indicative that said subject is not a candidate to undergo corneal refractive surgery.

In a particular embodiment of the third method of the invention, said subject is characterized in that it has an eye defect selected from myopia and astigmatism. The term "myopia," as used herein, refers to a refractive defect in the eye in which parallel rays of light from infinity converge at a focal point located in front of the retina, instead in the same retina, as would be normal. The term Uastigmatism, "as used herein, refers to an eye defect characterized by a different refraction between two ocular meridians, which prevents the clear focus of objects and, generally, is due to an alteration in the anterior curvature of the cornea.

Uses of the invention

In a fourth aspect, the invention relates to the use of heparanase activity as a diagnostic marker of an ocular surface pathology, as a marker to determine the degree of an ocular surface pathology, as a marker to monitor the effect of a therapy administered to a patient diagnosed with a pathology of the ocular surface or as a marker to select a patient to undergo corneal refractive surgery In a particular embodiment, said ocular surface pathology is keratoconus.

The invention is described by means of the following examples that are to be interpreted as merely illustrative and not limiting the scope of the invention.

EXAMPLES

Example 1. Method of detection of heparanase activity

The detection of human heparanase was based on measuring the fragmentation of high molecular weight HS chains in small oligosaccharides by the catalytic action of the enzyme. To facilitate the quantification of HS, it was radioactively labeled using eH] acetic anhydride. The separation of the high molecular weight HS chains from the fragments was carried out by molecular exclusion chromatography or by ultrafiltration.

1. 1. Reaction controls

In the first place, the necessary negative and positive controls were chosen to normalize the results obtained in the different reactions with tears.

to.

NEGATIVE: The negative reaction control lacked cell extract, using H20 minia instead.

b.

POSITIVE: As a positive control, EBNA cell extract overexpressing heparanase was used. To obtain this clan, the heparanase gene was cloned and the enzyme overexpressed in EBNA-293 cells.

1.2. Heparanase cloning

to. Heparanase amplification The complete sequence of heparanase was amplified from ESTs from human pancreatic duet carcinoma (GenBank accession no. BQ691142 (IMAGE Consortium ».) Two consecutive nested PCR amplifications were carried out in the first one. oligonucleotides were used:

•

5'-GGTGAGCCCAAGATGCTGCTG-3 '(direct) (SEQ ID NO: l)

•

5'-TCAGATGCAAGCAGCAACTTIG-3 '(reverse) (SEQ ID NO: 2)

and for the second nested PCR reaction the following oligonucleotides were used:

•

5'-ATGCTGCTGCGCTCGAAGCCT-3 '(direct nested) (SEQ ID NO: 3)

•

5'-GATGCAAGCAGCAACTTIGGC-3 '(nested reverse) (SEQ ID NO: 4)

The amplification products, corresponding to a 1.7 kbp band, were identified by 0.7% agarose gel electrophoresis and extracted using the Geneclean 11 kit (QBiogene, MP Biomedicals), and cloned in pUC19 (Fermentas Life Science) previously digested with Smal. The resulting constructs were used to transform competent Escherichia coli DH10B cells. The clones were sequenced using the sequencing services of the University of Oviedo.

Using this construct in pUC19 as a template, the protein coding band was amplified with oligonucleotides that included cut sequences for Kpnl and No! 1 (shown underlined):

5 • 5'-GTIGGTACCATGCTGCTGCGCTCGAAGCCT-3 '(direct) (SEO ID NO: 5)

• 5'-TGTGCGGCCGCGATGCAAGCAGCAACTTIGGC-3 '(reverse) (SEO ID NO: 6)

10 The band obtained, analyzed by agarose gel electrophoresis and extracted, was digested with Kpnl and Notl, as was the modified expression vector pCEP4 (Invitrogen) in which the cloning was performed. The modified vector did not include a tail of six histidines at the e-terminal end to facilitate subsequent identification and purification of the recombinant protein, and digestion with Kpnl / Notl removed the signal peptide present therein,

15 replacing it with the heparanase itself.

b. Expression in EBNA-293 The resulting construct was transfected into EBNA-293 cells using the lipofectAMINE reagent (Life Technologies, Inc.). The cells were cultured in DMEM medium

20 containing 10% fetal bovine serum, and 100 units / mL of penicillin and 250 ng / mL of amphotericin B at 37 ° C and 5% CO2. Clones capable of stably expressing proteins were selected in the presence of the antibiotic puromycin at a concentration of 2 iJg / mL (Sigma-Aldrich), and their sequence verified using the sequencing services of the University of Oviedo. Among these positive clones were selected

25 those with the highest heparanase enzyme activity in in vitro activity assays.

1.3. Heparanase cloning

30 a. Obtaining the cell extracts To obtain the extracts, the culture medium is removed, after which the cells were washed twice with PBS. Next, the cells were removed from the plate using a cell scraper (SD Falcon) and subjected to four freeze / thaw cycles at -80 oC for rupture. After this, the samples were subjected to 5 pulses of 10 seconds in a Soniprep 150 sonicator with 60 seconds of rest between each of them. Finally, the samples were centrifuged at 1000 xg, and the supernatant constituted the cell-free extract.

b.

Obtaining the substrate from the reaction Monitoring of the hydrolysis of HS by the catalytic action of heparanase required prior labeling of the substrate molecule. For this, (3H] -HS was prepared following a method described by Huang (Huang KS al. 2004 Anal Biochem 333: 389-398).

C.

Radioactive labeling of HS Obtaining [3H] -HS was based on deacetylation of the molecule, followed by reacetylation using acetic anhydride- [3H). 10 mg of HS (Sigma-Aldrich) was dissolved in 0.38 mL of hydrazine hydrate containing 7.6 mg of hydrazine sulfate, and the mixture was heated for 2 hours at 100 ° C in closed glass vials. The sample was cooled and dried with argon. 0.4 mL of toluene was added and the sample was dried by rotary evaporation. This procedure was repeated twice. The residue was dissolved in 0.9 mL of 1 M NaCl and desalted in a 5 mL desalination column connected to FPLC (GE Heallhcare). The desalted material was added to a Dowex 50 column (X-8, Na + 1 form) (1 x 3 cm) and washed with 5 mL of water. The fractions corresponding to the non-bonded material and the column wash were pooled and lyophilized. The resulting N-deacetylated HS was dissolved in 0.5 mL of NaHC030.5 M containing 110% (v / v) methanol previously cooled to 0 ° C in an ice bath and 30¡JI of acetic anhydride- (3H) in toluene (GE Healthcare) The mixture was vigorously stirred for 3 h at DoC, subsequently, another 0.5 mL of NaHCOJ, 20¡JI of methanol and 25¡JI of acetic anhydride were added and stirred another 30 min at QOC after which this last step was repeated After allowing the sample to warm to room temperature, toluene was removed by argon flow and the solution was desalted again in a 5 mL desalination column connected to FPLC in ammonium acetate. mM pH 6.8 The eH] HS was collected, the radioactivity was assessed by liquid scintillation and the concentration of HS was determined by binding tests of the 1,9-dimethylene blue cationic dye.

d. Purification of high molecular weight HS molecules

The eH] -HS obtained was subjected to molecular exclusion chromatography on a Superase 12 column connected to FPLC (GE Healthcare), previously equilibrated in 25 mM phosphate lamp pH 6 containing 150 mM NaCI. Chromatography was performed at a flow of 0.5 mL! Min and 0.5 mL fractions were collected in which the radioactivity present was determined (Figure 2). Fractions 23 to 29, corresponding to molecules with molecular weights greater than 20 kDa, were isolated and subjected to precipitation for 2 hours at -20 ° C by adding ethanol until reaching a final concentration of 85%. The precipitates were sedimented by centrifugation, dried and resuspended in water; thus being concentrated to be used as a substrate of heparanase.

1.4. Analysis of the reaction products

The hydrolytic capacity of heparanase expressed in eukaryotic cells was tested by two alternative procedures: molecular exclusion chromatography and ultrafiltration.

In the case of the positive control, the reaction was carried out with 60 ~ L of cell extract in 100 mM citrate buffer pH 5.3 containing 1 mM CaCI, and 1 ~ L of ['HJ-HS (20 nCi) in a final volume of 200 ~ L.

In the case of the negative control, the volume corresponding to the cell extract was replaced by distilled H20. The reactions were incubated at 37 ° C for periods of a period of 16 hours. To stop the reaction in due course, 220 iJL of NaCI was added to avoid nonspecific HS junctions and 200 iJL of chloroform to remove protein residues. After intense stirring the sample was centrifuged at 10,000 xg and 200 µL of the aqueous phase was taken.

to. Molecule fragmentation detection

ai). FPLC For chromatographic assays, the reactions were injected into a Superase 12 column connected to FPLC (GE Healthcare), previously equilibrated in 25 mM phosphate buffer pH 6 containing 150 mM NaCI. Chromatography was performed at a flow of 0.5 mUmin and 0.5 mL fractions were collected, in which one of the aliquots was used to

the determination of the radioactivity present. 200 IJL of each of the fractions obtained in each sample at time was analyzed.

a.ii). Ultrafiltration For ultrafiltration tests, the reactions were filtered through filters with an exclusion size of 5 kDa (Vivaspin 500, Sartorius), since the heparanase would hydrolyze the [3H] _HS chains into fragments of potentially adequate size to pass through The pore of the filter. Therefore, the radioactivity in aliquots of the liquid scintillation filtrate was estimated as a measure of heparanase activity (Figure 4).

b. Detection of generation of new reducing ends The fragmentation of the heparan sulfate polysaccharide by enzymatic hydrolysis reaction catalyzed by heparanase generates, in each of the new fragments, new reducing groups.

An alternative procedure for enzyme detection would be the quantification of

new reducing ends. This can be achieved by coupling oxidation reactions reduction on the new aldehyde groups that generate color changes or electrochemical reactions, which avoid the use of radioactive material.

2. Embodiment of the invention

2. 1. Reaction with tears In all cases, the reaction was carried out with 10 J..IL of tear in 100 mM citrate buffer pH 5.3 containing 1 mM CaCI2 and 1¡L of (3H) -HS (20 nCi) in a final volume of 200

~ L.

The reactions were incubated at 37 ° C for 16 hours. To stop the reaction in due time, 220 J..IL of NaCI was added to avoid nonspecific HS junctions and 200 J..IL of chloroform to remove protein residues. After intense stirring the sample was centrifuged at 10,000 xg and 200 J..IL of the aqueous phase was taken.

2.2. Filtration

100 I-Il of the aqueous phase was subjected to filtration in filters with an exclusion size of 5 kDa (Vivaspin 500, Sartorius). Radioactivity was estimated in aliquots of the liquid scintillation filtrate.

2.3. Results The heparanase activity in keratoconus shows progressively ascending values depending on the degree of the same (Figure 5). However, the dispersion of the results obtained in tear of healthy patients concluded that the analysis of the data by ANOVA was only significant for grade 3 cases according to the Rabinowitz classification (p value = 0.0019)

In summary, this invention allows a series of advantages that clearly differentiate it from what is known by the state of the art, since it allows an early diagnosis and by means of a biological marker of the development of a keratoconus or other ocular surface pathologies. The following cases describe the use of this invention in the field of medicine, although it is not intended to be limiting with respect to the scope of this invention.

Case 1: Short-sighted (myopic) patient who chooses corneal refractive surgery to decrease the need for glasses or contact lenses. With the current diagnostic methods, keratoconus is not detected, and develops a corneal ectasia that ends in a bilateral cornea transplant. One of them with poor surgical outcome and eye loss. The use of this invention, detecting high levels of heparanase in tear could have made the early diagnosis of keratoconus and avoided corneal surgery and subsequent complications.

Case 2: Patient who looks bad even with his glasses and does not tolerate contact lenses. No ocular disease is detected with the current diagnostic means and, when he is diagnosed with keratoconus, he can no longer perform his usual work activity and leisure time activities. At that time, the optical methods already diagnose keratoconus that could have been identified early ten years earlier with the use of this invention.

Case 3: Child with Down syndrome in which the prevalence of keratoconus is much more frequent than in the general population that, due to this disease, his vision is progressively degraded. At present there is no medical treatment to prevent the progression of keratoconus, and they have to undergo successive surgical interventions that for them represent a vital risk.

Claims (11)

1. In vitro method for diagnosing or determining the degree of a pathology of the ocular surface in a subject comprising: (i) determining heparanase activity in a sample comprising a tear of said subject; Y (ii) compare the heparanase activity obtained in step (i) with a value of 10; wherein, an increase in said heparanase activity with respect to the reference value is indicative that said subject undergoes a pathology of the ocular surface or of an advance in the degree of a pathology of the ocular surface. 15 2. In vitro method for monitoring the effect of a therapy administered to a patient diagnosed with a pathology of the ocular supertice comprising: (i) determining heparanase activity in a sample comprising a tear of said patient; Y 20 (ii) compare the heparanase activity obtained in step (i) with a reference value; wherein, a stagnation and / or a decrease in said heparanase activity with respect to the reference value is indicative of the positive effect of said therapy.

3. Method according to claims 1 or 2, wherein said ocular surface pathology is keratoconus.

4. Method according to claim 3, wherein said keratoconus is grade 3

30 5. In vitro method for selecting a subject to undergo corneal refractive surgery comprising:

(i) determining heparanase activity in a sample comprising a tear of said subject; Y 35 (ii) compare the heparanase activity obtained in step (i) with a reference value; wherein, an increase in said heparanase activity with respect to the reference value is indicative that said subject is not a candidate to undergo corneal refractive surgery. 6. Method according to claim 5, wherein said subject is characterized in that it has an eye defect selected from the group consisting of myopia and astigmatism. 7_ Method according to claims 1 to 6, wherein said heparanase activity determined in step (i) is determined by a method comprising:

(i)

contacting said sample with a heparanase substrate;

(ii)

keep the sample of stage (i) in suitable conditions for the

degradation of said substrate by heparanase; Y (jii) deleting the reaction products obtained in step (ii).

8.

Method according to claim 7, wherein said substrate is labeled with a detectable label selected from a group consisting of a fluorochrome labeled molecule, an enzymatically labeled molecule and a radioactively labeled molecule.

9.

Method according to claim 8, wherein said substrate is radioactively labeled with tritium.

10.

Method according to claims 6 to 9, wherein said substrate is heparan sulfate.

eleven.

Method according to claim 10, wherein said heparan sulfate has an average molecular weight of at least 20 kDa.

12.

Method according to claims 7 to 11, wherein the detection of the reaction products obtained in step (ii) is carried out by a molecular fractionation technique selected from ultrafiltration and molecular exclusion chromatography.

13.

Method according to claims 7 to 11, wherein the detection of the reaction products obtained in step (ii) is carried out by means of a detection test of reducing ends in said reaction products.

14.

Use of heparanase activity as a diagnostic marker of an ocular surface pathology, as a marker to determine the degree of an ocular surface pathology, as a marker to monitor the effect of a therapy administered to a patient diagnosed with an ocular surface pathology or as a marker to select a patient to undergo corneal refractive surgery.

fifteen.

Use according to claim 14, wherein said ocular surface pathology is keratoconus.