RADICAL ACTIVITY

DESCRIPTION

This invention is based on the surprising identification of several substances that can be used for detecting the occurrence of a radical activity in a chemical preparation. In particular, although this invention may be applied for radical activities due to the development of any type of free radicals (hereinafter FR), it is advantageously applied in the case of radical activities due to the action of reactive oxygen species free radicals (hereinafter ROS). The fact that the development of free radicals in general, and of ROS in particular, is a risk to patient health in any type of treatment, and in particular in surgical treatments, was established years ago.

In fact, as is known, the occurrence of radical activity is correlated with many post-treatment complications. In particular, it is correlated both with transitory (more or less long) inflammatory phenomena and long-term pathologies.

In an attempt to overcome that problem, there is the known practice of attempting to neutralise FR activity in order to try to avoid the oxidative stress that may be induced by the structural and functional alteration of molecules which results in functional imbalances and many consequent pathologies. However, until now, the chemical - physical processes linked to the development of FR and to the consequent occurrence of radical activity have only been partly known. In particular, the evolution of FR over time is unknown, and there is no known method able to allow a prediction of whether or not a predetermined treatment may lead to the formation of FR, let alone able to quantify such a phenomenon.

Although if is also known that every type of FR causes a different radical activity, given the considerable instability of such molecules so far there are no known methods able to differentiate with certainty the various radical activities that may occur.

At present, other than nuclear magnetic resonance spectroscopy (NMR spectroscopy), the only system considered reliable for measuring radical activity during a predetermined period of time, is based on a fluorescence measurement and involves the use of fluorescent probes specifically developed for detecting FR. More precisely, they are probes which change the fluorescence signal emitted following a reaction with FR. Even more precisely, given that there are various types of FR with different chemical properties (to which different radical activities correspond), there are various types of fluorescent probes for FR available, each suitable for detecting and measuring the presence of only specific types of FR. Therefore, each probe is able to give indications only about a part of the total radical activity that could occur. However, in the scientific community it is thought that the indication obtainable using such probes should be considered sufficient for a correct assessment of the risks correlated with radical activity.

However, the measurement of FR using methods involving fluorescence has significant disadvantages.

First, it is in fact necessary to have available special fluorescence measuring instruments, with relatively high purchase costs. The fluorescent probes, which must be changed after each measurement, are also relatively expensive.

Second, since operation of the fluorescent probes is based on the formation of a chemical bond between the FR and the probe, they are devices that allow measurement of the actual presence of FR only until probe saturation. In contrast, as soon as a probe reaches saturation point, it is no longer possible either to detect new development of FR, much less quantify them, therefore everything must be repeated from the start using a larger quantity of fluorescent probe.

Furthermore, measurements using fluorescence cannot be used for detecting the presence of FR whenever whatever is to be examined contains substances that are able to alter or inhibit the reading by the fluorescence measuring instruments. In particular, it is not currently possible to detect the presence of FR whenever there is a dye or other substances present which interfere with the fluorescent probe (ail substances with high absorption at the fluorophore excitation and emission wavelength), as happens, for example, in the case of medical devices used in ophthalmic surgeries in order to obtain selective staining of the various parts of the eye.

In this context the technical purpose which forms the basis of this invention has been to overcome at least some of the above-mentioned disadvantages. In particular, according to a first aspect of this invention, the technical purpose of this invention has been to provide an alternative method to measurement using fluorescence, for detecting the occurrence of a radical activity in a chemical preparation during a time interval. Therefore, the radical activity to which this invention relates is, first at least the same that currently, at least in some situations, may be detected with probes that use fluorescence.

According with this innovative aspect it has been conceived the use of crystalline lens proteins for detecting the occurrence of a radical activity in a chemical preparation during a time interval, wherein a reduction in the quantity of crystalline lens proteins present in the chemical preparation between a start and an end of the time interval indicates that radical activity occurred.

!n one embodiment, the crystalline lens proteins are used for detecting the occurrence of a radical activity in a chemical preparation that is subjected to artificial lighting.

Moreover, in one embodiment, the chemical preparation comprises at least one dye or another substance that interferes with a fluorescence measurement.

In one embodiment, the crystalline lens proteins used are selected from the j group constituted of human type crystalline lens proteins obtained by extraction or by synthesis, porcine type crystalline lens proteins obtained by extraction or by synthesis and bovine type crystalline lens proteins obtained by extraction or by synthesis.

Preferably, the crystalline lens proteins used are porcine type crystalline lens proteins obtained by extraction or by synthesis.

In one embodiment, the crystalline lens proteins used are a-crystaliin proteins, b-crystallin proteins or g-crystal!in proteins. Preferably, however, the crystalline lens proteins used are g-crystailin proteins.

In one embodiment, the crystalline lens proteins are used in the chemical preparation in a concentration, by weight/volume, of between 0.01 % and 10%.

In one embodiment, a measurement is also taken of an extent of reduction in the quantity of crystalline lens proteins during the time interval and that extent of reduction is used for quantifying the radical activity that occurred.

In one embodiment, the quantity of crystalline lens proteins is measured using a liquid chromatography technique.

Moreover, also according to the first aspect of this invention, the technical purpose has been to provide a method for predicting the risk of the occurrence of a predetermined radical activity following the execution of a specific treatment in a chemical preparation.

This has been obtained with a method for verifying whether or not subjecting a chemical preparation to predetermined conditions of use causes the occurrence of a radical activity, the method comprising the operating steps of:

- taking a sample of said chemical preparation;

- adding to the sample a predetermined quantity of crystalline lens proteins;

- subjecting the sample to which the crystalline lens proteins have been added to said predetermined conditions of use;

- then measuring the residual quantity of crystalline lens proteins in the sample;

- comparing the residual quantity of crystalline lens proteins with the predetermined quantity of crystalline lens proteins; and

- determining that a residual quantity of crystalline lens proteins that is less than the predetermined quantity of crystalline lens proteins indicates that a radical activity occurred following the application of said predetermined conditions of use.

In one embodiment, said predetermined conditions of use correspond to physiological conditions or to physiopatho!ogical conditions, and/or to a specific treatment. In particular, it can been foreseen that the specific treatment comprises a step of artificial lighting of the chemical preparation.

In one embodiment, added crystalline lens proteins are selected from the group constituted of human type crystalline lens proteins obtained by extraction or by synthesis, porcine type crystalline lens proteins obtained by extraction or by synthesis and bovine type crystalline lens proteins obtained by extraction or by synthesis, but advantageously, they are porcine type crystalline lens proteins obtained by extraction or by synthesis.

In one embodiment, crystalline lens proteins are a-crystaliin proteins, b- crystallin proteins or g-crystallin proteins, preferably, g-crysfallin proteins.

In one embodiment, the crystalline lens proteins are added in a concentration, by weight/voiume, of between 0.01 % and 10%.

In one embodiment, a measurement is also taken of an extent of reduction in the quantity of crystalline lens proteins between the predetermined quantity of crystalline lens proteins and the residual quantity of crystalline lens proteins, and that extent of reduction is used for quantifying the radical activity that occurred following the application of said predetermined conditions of use.

Finally, in one embodiment, the step of measuring the residual quantity of crystalline lens proteins is carried out using a liquid chromatography technique. In contrast, according to another aspect of this invention, the technical purpose of this invention has been to provide a method able to allow monitoring of the occurrence of a predetermined radical activity in an environment which is artificial in vitro or ex vivo, physiological in situ or in vivo, or physiopathoiogicai.

According to another aspect of this invention, the technical purpose of this invention has been to provide a method for preparing a chemical preparation to be used in contact with at least one biological product in predetermined conditions of use, which allows the preparation of chemical preparations that have a reduced risk of causing the occurrence of a predetermined radical activity during their use. In particular, the predetermined conditions of use may correspond to the physiological or physiopathoiogicai conditions of the biological product or to a specific treatment carried out on/with the biological product.

This technical purpose has been reached by a method for preparing a chemical preparation to be used in contact with at least one biological product in predetermined conditions of use, the method comprising the operating steps of:

preparing a chemical preparation;

taking a sample of the chemical preparation;

adding to the sample a predetermined quantity of crystalline lens proteins in order to obtain a sample to be tested;

simulating in vitro or on an ex vivo model said predetermined conditions of use, using the sample to be tested;

after said step of simulating the predetermined conditions of use, measuring a residual quantity of crystalline lens proteins that are present in the sample to be tested; and

comparing the residual quantity of crystalline lens proteins measured with the predetermined quantity of crystalline lens proteins;

and wherein if the predetermined quantity of crystalline lens proteins is greater than the residual quantity of crystalline lens proteins measured, by a value that is greater than a reference deviation, the following further operating steps are also envisaged:

modifying the chemical preparation by adding to it at least one quantity of a substance able to neutralise the action of free radicals; and

then and in sequence, repeating the steps of taking, adding, simulating, measuring, comparing and, if necessary, even of modifying and repeating, until the predetermined quantity of crystalline lens proteins is equal to the residual quantity of crystalline lens proteins measured or greater than it by a value that is less than the reference deviation;

and wherein when the predetermined quantify of crystalline lens proteins is equal to the residual quantity of crystalline lens proteins or greater than it by a value that is less than the reference deviation, the method is terminated and it is determined that the chemical preparation to be used in contact with said at least one biological product in said predetermined conditions of use corresponds to the chemical preparation for which the predetermined quantity of crystalline lens proteins proved equal to the residual quantity of crystalline lens proteins or greater than it by a value that is less than the reference deviation.

In one embodiment, said predetermined quantify of crystalline lens proteins added to the sample to be tested is added with a concentration equal to 0.15% w/v.

In one embodiment, the crystalline lens proteins added are selected from the group constituted of human type crystalline lens proteins obtained by extraction or by synthesis, porcine type crystalline lens proteins obtained by extraction or by synthesis and bovine type crystalline lens proteins obtained by extraction or by synthesis, but preferably are porcine type crystalline lens proteins obtained by extraction or by synthesis.

In one embodiment, the crystalline lens proteins added are a-crysta!iin proteins, b-crystailin proteins or g-crystallin proteins, but preferably are y- crysfallin proteins.

In one embodiment, said step of simulating said predetermined conditions of use using the sample to be tested is performed by also placing the sample to be tested in contact with said at least one biological product.

In one embodiment, the difference between the predetermined quantity of crystalline lens proteins and the residual quantity of crystalline lens proteins is used for also quantifying a radical activity that occurred in the sample to be tested during the step of simulating the predetermined conditions of use.

In one embodiment, the step of measuring the residual quantity of crystalline lens proteins in the sample to be tested is carried out preferably using a liquid chromatography technique.

In one embodiment, the chemical preparation can comprise at least one dye or another substance that interferes with fluorescence measurements.

In one embodiment, the predetermined conditions of use correspond to physiological conditions or to physiopathological conditions, and/or to a specific treatment, for example a treatment which comprises a step of artificial lighting of the sample to be tested.

According to a further aspect of this invention, the technical purpose of this invention has been to provide a generic chemical preparation for use in a method for treatment of the bodies of humans or animals, or for use in a diagnostic method applied to the bodies of humans or animals which has a reduced risk of causing the occurrence of a predetermined radical activity during that treatment.

According to another aspect of this invention, the technical purpose of this invention has been to provide a chemical preparation for use in a method for treatment of the bodies of humans or animals, or for use in a diagnostic method applied to the bodies of humans or animals which allows easy a posteriori verification of whether or not a predetermined radical activity occurred following use of the chemical preparation in a particular treatment.

In general, the technical purpose of this invention has been to provide a method for detecting the occurrence of a predetermined radical activity, which may even be used if there is a dye or another substance present which interferes with measurements using fluorescence, unlike what currently happens with measurements using fluorescence.

At least one of the technical purposes indicated is substantially achieved by what is described in the appended claims.

Further features and the advantages of this invention are more apparent in the detailed description which follows of various inventive aspects, all part of this invention.

It should be noticed that although they are ail inventive aspects, it is possible that only some of them are the subject of the appended claims and others are protected by further, parallel patent applications or may be protected in future by means of specific divisional applications.

In any case, all of the inventive aspects of this invention come from the fact that the Applicant, following intensive research and development work, has succeeded in identifying several substances that on one hand may be used for monitoring the occurrence of a predetermined radical activity, and that on the other hand are measurable using relatively simple techniques even in the presence of any dyes, fluorescent substances or other substances that interfere with measurements using fluorescence). In particular these substances identified are substances which, as demonstrated by the tests carried out, in a predetermined time interval change their quantity in inverse proportion to the occurrence of a predetermined radical activity.

In other words, if such substances are present in a predetermined quantity in a predetermined environment, then following the occurrence of a predetermined radical activity in that environment, the quantity of those substances is reduced, and the reduction is also greater the greater the radical activity that occurred is.

The substances able to act as indicated above, which the Applicant has succeeded in identifying, are the so-called proteins of the crystalline lens of the eye.

!t should be noticed that in the context of this invention, in accordance with the scientific terminology of the sector, the term "crystalline lens proteins" means both the proteins which may be extracted directly from the crystalline lens of an eye, and the proteins which may be synthesized in vitro and which have a structure similar to that of the crystalline lens proteins obtained by extraction.

In particular, for all of the innovative aspects described below, human, porcine and bovine type crystalline lens proteins obtained by extraction or by synthesis proved advantageous. Moreover, in the context of this invention, the term "type" crystalline lens proteins (for example "porcine type") means both those of biological origin obtained by extraction from crystalline lenses (in the example, "of pigs"), and any proteins synthesized in vitro which have the same structure and chemical - physical properties as those of biological origin. However, in the particularly preferred embodiments, the crystalline lens proteins are the porcine type.

As is independently known, crystalline lens proteins comprise a-crystailin proteins, b-crysta!!in proteins or g-crystaliin proteins.

According to ail of the innovative aspects of this invention, the crystalline lens proteins usable for detecting the occurrence of a predetermined radical activity may advantageously be a-crystallin proteins and/or b-crystallin proteins and/or g-crystallin proteins. However, the use of only y-crysta!iin proteins is particularly preferred.

Moreover, advantageously, in accordance with all of the innovative aspects of this invention, the quantity of crystalline lens proteins present is preferably always measured with a liquid chromatography technique (HPLC, UPLC, etc.).

As already indicated, in order to have a scientific basis for the method for detecting the occurrence of radical activity by means of measuring the concentration of crystalline lens proteins, the Applicant carried out many tests.

Hereinafter, some experimental results are shown, by way of example, obtained by using various artificial light sources to directly treat pig crystalline lens extract samples

Those pig crystalline lens extract samples were in liquid form at all of the test temperatures, and had a concentration of crystalline lens proteins, expressed by weight/volume, equal to approximately 0.15 mg/ml.

In a first control experiment it was first verified that the artificial lighting of the samples with different light sources induces the production of FR.

In that experiment, a fluorescence probe (DHR 123) was used, which interacted in a specific way with the FR that could develop in the crystalline lens extract.

The samples were lit with different light sources in the time (understood to mean the duration of the lighting) and temperature conditions shown in table 1

Table 1 Test lighting conditions

In contrast, control samples were kept at the ambient temperature for the same time, but sheltered from any type of lighting

The appended Figures 1 to 4 show the results obtained. In particular: Figure 1 shows the increase in fluorescence following lighting of the samples with the ultraviolet lamp (UV) lamp, respectively for 15, 30 and 60 minutes and in two different temperature conditions (10°C and 23°C); the values are shown on the y-axis and expressed as the ratio of the fluorescence measured for each sample to the fluorescence measured for the control sample;

Figure 2 shows the increase in fluorescence following lighting of the samples for 60 minutes with the 22 W halogen lamp in two different temperature conditions (10°C and 23°C); the values are shown on the y- axis and expressed as the ratio of the fluorescence measured for each sample to the fluorescence measured for the control sample;

Figure 3 shows the increase in fluorescence following lighting of the samples for 60 minutes with the endoilluminator connected to the Xenon White Light in two different temperature conditions (23°C and 31 °C); the values are shown on the y-axis and expressed as the ratio of the fluorescence measured for each sample to the fluorescence measured for the control sample;

Figure 4 shows the increase in fluorescence following lighting of the samples for 60 minutes with the endoilluminator connected to the 150 W halogen lamp, in two different temperature conditions (23°C and 31 °C); the values are shown on the y-axis and expressed as the ratio of the fluorescence measured for each sample to the fluorescence measured for the control sample;

As shown in Figures 1 to 4, in all of the tests carried out an increase in fluorescence was observed, that is to say, the occurrence of radical activity: that increase in fluorescence compared with the untreated control is much greater following lighting with UV rays.

In order to also demonstrate that the occurrence of radical activity is correlated with a reduction in the quantity of crystalline lens proteins present (following their degradation), in parallel the same samples were tested with ^ HPLC to detect changes in the concentration of the crystalline lens proteins.

Figures 5 to 8 show the relative results obtained. In particular

Figure 5 shows the reduction in the concentration of crystalline lens proteins observed with HPLC as regards the samples to which Figure 1 relates, that is to say, lit for 15, 30 and 60 minutes with a UV lamp in two different temperature conditions (10°C and 23°C); the values on the y- axis represent the quantity of residual crystalline lens proteins, as a percentage compared with the control sample;

Figure 6 shows the reduction in the concentration of crystalline lens proteins observed with HPLC as regards the samples to which Figure 2 relates, that is to say, lit for 60 minutes with the 22 W halogen lamp in two different temperature conditions (10°C and 23°C); the values on the y~axis represent the quantity of residual crystalline lens proteins, as a percentage compared with the control sample;

Figure 7 shows the reduction in the concentration of crystalline lens proteins observed with HPLC as regards the samples to which Figure 3 relates, that is to say, lit for 60 minutes with an endoiliuminator connected to a "Xenon white light" in two different temperature conditions (23°C and 31 °C); the values on the y-axis represent the quantity of residual crystalline lens proteins, as a percentage compared with the control sample;

Figure 8 shows the reduction in the concentration of crystalline lens proteins observed with HPLC as regards the samples to which Figure 4 relates, that is to say, lit for 60 minutes with an endoiliuminator connected to a 150 W halogen lamp in two different temperature conditions (23°C and 31 °C); the values on the y~axis represent the quantity of residual crystalline lens proteins, as a percentage compared with the control sample.

As shown in Figures 5 to 8, a reduction in the concentration of crystalline lens proteins present in the pig crystalline lens extract was observed following artificial lighting of the samples with different light sources in different time/temperature conditions. That reduction in concentration, due to a degradation of the crystalline lens proteins, was the most intense following UV lighting, that is to say, corresponded to the greatest occurrence of radical activity observed with the fluorescence tests. The protein degradation was also greater, the longer the sample exposure time to the artificial light source was.

As already indicated, this invention, starting with the afore-said qualitatively inverse correlation between the quantity of crystalline lens proteins present and the occurrence of radical activity, further provided various innovative aspects that may be implemented in various contexts.

Each of those innovative aspects will now be described in detail. Since all of the various aspects are based on a common principle, any details described with reference to one of them shall be considered also potentially valid for the other aspects described, if technically compatible.

According to a first innovative aspect, this invention relates in general to the use of crystalline lens proteins as a tool for detecting the occurrence of radical activity. In particular, the invention relates to the use of crystalline lens proteins in a chemical preparation, in particular at temperatures at which the preparation is in liquid or gel form. According to the invention, a reduction in the quantity of crystalline lens proteins present in the chemical preparation during a predetermined time interval indicates that radical activity occurred in the chemical preparation.

Depending on requirements, it may be the case either that the crystalline lens proteins are an integral part of the chemical preparation, or that they are added specifically with the sole purpose of acting as a tool for detecting the occurrence of radical activity.

Moreover, advantageously, the crystalline lens proteins are used in the chemical preparation in a concentration, by weight/volume relative to the overall chemical preparation and crystalline lens proteins, of between 0.01 % and 10%. In any case, the choice of the actual concentration to be used will depend on the context in which this invention is to be implemented. In fact, if the crystalline lens proteins are to be used in physiological conditions, in which the possible development of free radicals is relatively limited, then the concentration of crystalline lens proteins may also be relatively low. In contrast, if they are to be used for tests or for perfecting products, with artificial conditions such that they cause a big development of free radicals and/or a long exposure time, the concentration of crystalline lens proteins will have to be greater. Furthermore, when deciding on the concentration, it is essential to take into account the measuring sensitivity of the instruments available. It may also be useful to repeat the assessments at different concentrations, in order to identify a concentration which although allowing a considerable change in the quantity of proteins during the reference time interval, allows a significant quantity of proteins to be left at the end of the same time interval.

In a particularly preferred embodiment, the extent of the reduction in the quantity of crystalline lens proteins is also measured and that extent of reduction measured is used for quantifying the radical activity that occurred. According to particularly preferred applications of this invention, the crystalline lens proteins are used in a chemical preparation subjected to artificial lighting, and/or for preparations which comprise at least one dye or another fluorescent substance which interferes with fluorescence measurements. With reference to the later aspect, the first innovative aspect of this invention is particularly advantageously applied with reference to preparations that are dyed or contain other substances which interfere with fluorescence measurements, which are used as medical devices during surgeries, in particular ophthalmic surgeries.

A second innovative aspect of this invention relates to a method for verifying whether or not predetermined conditions of use can cause the occurrence of a predetermined radical activity in a chemical preparation. In the context of this invention, the expression predetermined conditions of use may mean either a "specific treatment", that is to say, any type of physical or chemical stress, or a physiological condition, or a physiopathological condition. It should be noticed that this invention was devised both for specific physical treatments (such as exposure to a predetermined type of artificial light, exposure to a predetermined temperature, being subjected to mechanical mixing, etc.), and for specific chemical treatments (for example, contact with hydrogen peroxide, a treatment often used at the test stage with the aim of generating FR). According to all of the preferred embodiments of this invention, the predetermined conditions of use to be tested will be those to which the chemical preparation must be subjected during its use or others which simulate or reproduce those to which the chemical preparation must be subjected during its use. In particular, if the treatments to which the chemical preparation must be subjected are part of surgical or diagnostic procedures carried out on the bodies of humans or animals, the specific treatment to which the method according to the second innovative aspect of this invention relates will be a reproduction, in vitro or in any case on biological material removed and not intended for use as an implant (ex vivo procedure), of the actual treatment that is part of a surgical or diagnostic procedure.

It should also be noticed that a specific treatment, like a physiological or physiopathological condition, may in itself include a plurality of different stresses (e.g : lighting, temperature and ultrasound), which may be applied together and/or at different times and/or for different lengths of time. A particularly preferred specific treatment consists of artificial lighting of the chemical preparation with a specific light source and at a predetermined temperature (in fact, this is a stress which reproduces that to which all preparations normally used in the context of an ophthalmic surgery are subjected).

Returning to the method that is the subject matter of the second innovative aspect of this invention, it comprises first the operating steps of taking a sample of the chemical preparation to be tested and of adding to the sample a predetermined quantity of crystalline lens proteins. Advantageously, the crystalline lens proteins are added in a quantity such that it sets their concentration, by weight/voiume, at between 0 01 % and 10%. The same assessments indicated above apply concerning the choice of concentration to be used each time.

The method also comprises the operating step of subjecting the sample to the predetermined conditions of use (for example, to a specific treatment of interest for a predetermined time), followed by the operating step of measuring the residual quantity of crystalline lens proteins in the sample (measurement, as already indicated, advantageously carried out using a liquid chromatography technique, such as HPLC, UPLC).

Then the method comprises the operating step of comparing the residual quantity of crystalline lens proteins measured, with the predetermined quantity of crystalline lens proteins initially added to the chemical preparation, and the final step of determining that a residual quantity of crystalline lens proteins measured, which is less than the predetermined quantity of crystalline lens proteins, indicates that a predetermined radical activity occurred following treatment with the predetermined conditions of use. It should be noticed that whilst in most cases any development of FR will have originated from the chemical preparation used, in the case for example of more complex treatments in which the chemical preparation is brought into contact with other substances and/or with biologic material, the development of FR could also originate from those other substances and/or from the biological material.

In other embodiments of the method, there may be a measurement of the extent of the reduction in the quantity of crystalline lens proteins and that extent of reduction measured may then be used for quantifying the extent of the radical activity. In an example of application of the method described above, a test was carried out to identify the behaviour in response of ultraviolet light (the specific treatment) of two commercial preparations intended for use in the context of ophthalmic surgeries. These are BSS (Balanced Salt Solution) and BSS Pius® (Balanced Sait Solution with additives able to neutralise the action of FR), products which are both marketed under the Aicon brand by the company Novartis AG

Several samples of both of the preparations had crystalline lens proteins added to them in such a way as to obtain a concentration of 0 15% w/v; those samples were then irradiated for approximately 45 minutes with ultraviolet light. At various intervals for each sample the HPLC technique was used to measure the residual quantity of crystalline lens proteins.

Figure 9 shows the trend over time (expressed as a percentage compared with the initial quantity) of the quantity of crystalline lens proteins present in the samples.

As can be seen, whilst for the normal BSS the quantity of crystalline lens proteins present after approximately 45 minutes of treatment was practically zeroed, after the same time for the BSS Pius® there was still approximately 35% of crystalline lens proteins present, confirming the BSS Plus® effect of neutralising the FR.

Tests carried out with fluorescence measurements during the treatment and at the end of it also allowed verification that said behaviour of the crystalline lens proteins corresponded to less radical activity in the case of use of BSS Plus® than in the case of use of normal BSS.

As regards a third innovative aspect of this invention, a method was provided for preparing a chemical preparation to be used in contact with at least one biological product in predetermined conditions of use, intended to allow the obtaining of a chemical preparation such that its use in the predetermined conditions of use involves a reduced or even null risk of the occurrence of a predetermined radical activity. It should be noticed that the predetermined conditions of use may correspond to physiological conditions or to physiopatho!ogical conditions, and/or to a specific treatment (understood in the sense indicated above).

Preferably, it is a method for preparing either a chemical preparation which at least in the range of temperatures between +1 °C and +50 °C is in liquid or gel form, or a chemical preparation which in the same range of temperatures is soluble in a solvent which is also to be used in the predetermined conditions of use (advantageously water).

According to this innovative aspect of this invention, the method comprises first the operating steps of preparing a chemical preparation (which advantageously has a formula giving it the desired chemical - physical properties for the intended use in contact with at least one biological product), and of taking a sample of the chemical preparation formulated in that way.

Then the method comprises adding to the sample a predetermined quantity of crystalline lens proteins in order to obtain a sample to be tested, and of simulating in vitro or in an ex vivo model the predetermined conditions of use, using the sample to be tested substantially in the same conditions in which the chemical preparation should be used. The predetermined conditions of use may comprise both one or more physical or chemical stresses and a predetermined timing/length of time for application of each of them.

At the end of the step of simulating the predetermined conditions of use (that is to say, after a predetermined time interval), the method comprises measuring the residual quantity of crystalline lens proteins still present in the sample to be tested, advantageously using the above-mentioned liquid chromatography technique, and comparing the residual quantity of crystalline lens proteins measured in that way with the predetermined quantity of crystalline lens proteins initially added to the sample (obviously, each "quantity" may be assessed both in absolute terms and in terms of concentration).

The result of the comparison step is then used as a parameter for assessing whether or not the chemical preparation may be considered perfected, that is to say, whether or not it is such that it has a reduced or null risk of promoting the occurrence of a predetermined radical activity in the predetermined conditions of use.

In particular, there is an assessment of whether or not the predetermined quantity of crystalline lens proteins is greater than the residual quantity of crystalline lens proteins measured, and if if is, also how much greater it is.

As the discriminating factor for deciding whether or not the method may be considered concluded, advantageously a reference deviation value is set, which corresponds to the maximum tolerable value of the reduction in the quantity of crystalline lens proteins present in the sample to be tested, which may be obtained between the start and the end of the predetermined conditions of use (that value may be expressed either in absolute terms or as a percentage).

If the difference between the predetermined quantity of crystalline lens proteins and the residual quantity of crystalline lens proteins measured is less than the reference deviation, the method is terminated and it is determined that the chemical preparation to be used in contact with at least one biological product in the predetermined conditions of use corresponds to the chemical preparation initially prepared.

If, in contrast, that difference is greater than the reference deviation, the method comprises first modifying the chemical preparation by adding to it at least one quantity of a substance able to neutralise the action of the FR, obtaining a new formula for the chemical preparation. Examples of substances able to neutralise the action of FR are: ascorbic acid, glutathione, tocopherol, curcumin and others with an antioxidant action.

Then the method comprises repeating in sequence the steps described above of taking a sample of the chemical preparation (the one with the new formula obtained by adding the substance able to neutralise the action of the FR, adding to the sample the predetermined quantity of crystalline lens proteins, simulating the predetermined conditions of use, measuring the residual quantity of crystalline lens proteins and comparing that residual quantity of crystalline lens proteins with the predetermined quantity of crystalline lens proteins.

At this point, if the difference between the predetermined quantity of crystalline lens proteins and the residual quantity of crystalline lens proteins measured is less than the reference deviation, the method is terminated as described above. In contrast, if that difference is still greater than the reference deviation, the method comprises the further execution of the steps of modifying the chemical preparation and of repeating the various steps of taking, adding, simulating, measuring and comparing. It can ail be repeated multiple times until the difference between the predetermined quantity of crystalline lens proteins and the residual quantity of crystalline lens proteins measured is less than the reference deviation.

It should be noticed that with each repetition of the step of modifying the chemical preparation, the substance able to neutralise the action of the FR and/or the quantity of it that is added may be different.

In all cases, when the method is terminated, it is determined that the chemical preparation to be used in contact with at least one biological product in the predetermined conditions of use, perfected thanks to this method, corresponds to the chemical preparation for which the predetermined quantity of crystalline lens proteins is equal to the residual quantity of crystalline lens proteins, or greater than it by a value that is less than the reference deviation.

Advantageously, if required in the predetermined conditions of use for which the chemical preparation is intended, the step of simulating in vitro or ex vivo those predetermined conditions of use using the sample to be tested may be carried out by placing the sample to be tested in contact with one or more biological products. For example, if the chemical preparation is intended to be used in ophthalmic surgery, the specific treatment may be simulated on an ex vivo model using an eyeball that has been removed.

According to the preferred embodiments of the method according to this third innovative aspect of this invention, the predetermined quantity of crystalline lens proteins added to the sample to be tested, may be such that in the sample to be tested a concentration of crystalline lens proteins is obtained which, by weight/volume, is between 0.01 % and 10%. Again in this case, the same assessments indicated above apply concerning the method for selecting the concentration to be used.

Again in this third innovative aspect of this invention, the difference between the predetermined quantity of crystalline lens proteins and the residual quantity of crystalline lens proteins may be used for quantifying a predetermined radical activity that has occurred in the sample to be tested. For example, the purpose of that may be to decide the quantity of substance able to neutralise the action of the FR to be added to the chemical preparation before repeating the various steps indicated above (in fact, in general, the greater the radical activity that occurred is, the greater the amount of "substance able to neutralise the action of the FR" to be added will be).

Even the method according to the third innovative aspect of this invention can be advantageously applied for perfecting chemical preparations which are intended to be used in specific treatments which comprise a step of artificial lighting of the sample to be tested, as well as for perfecting chemical preparations which comprise at least one dye or other substances which interfere with fluorescence measurement.

A fourth innovative aspect of this invention relates to the fact that it has been possible to identify a criterion for being able to consider safe enough, in terms of the possible occurrence of a predetermined radical activity, a chemical preparation to be used in the bodies of humans or animals during a specific treatment. The expression safe enough means such that at most it causes or allows the occurrence of a predetermined radical activity which is less than a predetermined limit considered a safety limit. That criterion is based on the behaviour of the chemical preparation during execution of the specific treatment in the presence of crystalline lens proteins.

Therefore, the subject matter of the fourth innovative aspect is a chemical preparation for use in a method for the treatment of the bodies of humans or animals, or for use in a diagnostic method applied to the bodies of humans or animals. That preparation will, in particular, have to have a predetermined behaviour when, in the presence of a predetermined quantity of crystalline lens proteins, it is subjected:

- either in vivo to one or more steps of the method for the treatment of the bodies of humans or animals or of the diagnostic method applied to the bodies of humans or animals for which it is intended;

- or in vitro or ex vivo to one or more steps which reproduce steps of the method for the treatment of the bodies of humans or animals or of the diagnostic method applied to the bodies of humans or animals.

In particular, after those steps of the specific treatment have been carried out the quantity of crystalline lens proteins which are initially present together with the chemical preparation, must either remain constant or be reduced at most by a reduction quantity that is equal to 40% of the predetermined quantity of crystalline lens proteins. In other words, the subject matter of this innovative aspect of this invention is any chemical preparation for use in a method for the treatment of the bodies of humans or animals, or for use in a diagnostic method applied to the bodies of humans, which is able to pass a specific test comprising mixing the chemical preparation with a predetermined quantity of crystalline lens proteins in order to obtain a modified chemical preparation and the use of that modified chemical preparation either directly in the method for which the chemical preparation is intended or in an in vitro simulation of that method. The chemical preparation is considered capable of passing the test if at the end of the method for the treatment of the bodies of humans or animals or of the diagnostic method applied to the bodies of humans or animals, or of their in vitro or ex vivo simulation, the quantity of crystalline lens proteins present in the modified chemical preparation has been reduced, compared with the predetermined quantity of crystalline lens proteins initially added, at most by a reduction quantity which is equal to 40%.

However, in the preferred embodiments, the maximum acceptable reduction quantity is equal to 20%, more preferably to 10% and even more preferably to 5% of the predetermined quantity of crystalline lens proteins.

Advantageously, according to this fourth innovative aspect, the chemical preparation is a chemical preparation which comprises a dye and/or is intended to be used in an ophthalmic surgery method. Moreover, the method for the treatment of the bodies of humans or animals or the diagnostic method applied to the bodies of humans or animals, advantageously comprise a step of artificial lighting of the chemical preparation.

Furthermore, the chemical preparation is preferably in liquid or gel form at least in the range between +1 °C and +50°C, or, in the same temperature range, is soluble in a solvent (advantageously water) which is also to be used in the same method.

Advantageously, a chemical preparation according to the fourth innovative aspect of this invention is such that it has the behaviour described above in the presence of a predetermined quantity of crystalline lens proteins which, once mixed with the chemical preparation has a concentration by weight/volume equal to 0.15%.

The final innovative aspect of this invention is a chemical preparation for use in a method for the treatment of the bodies of humans or animals, or for use in a diagnostic method applied to the bodies of humans or animals, which comprises crystalline lens proteins in a concentration, by weight/volume, of between 0.01 % and 10%. In particular, preferably, it is a chemical preparation which, at the end of the treatment, is intended to be at least partly extracted from the bodies of humans or animals and, therefore, its quantity or concentration of residual crystalline lens proteins after use can easily be measured; thanks to that measurement, it is possible to estimate whether, and if necessary to what extent, a predetermined radical activity occurred during the treatment. In fact, the more radical activity occurred, the greater the risk that the patient may develop physiopathologicai phenomena.

However, preferably, the chemical preparation comprises crystalline lens proteins in a concentration, by weight/volume, of between 0.01 % and 0.5%. Advantageously, again according to this final innovative aspect of this invention, the chemical preparation comprises at least one dye, is a chemical preparation for use in an ophthalmic surgery method, and/or, at least in the temperature range between 1 °C and 50°C, is in liquid or gel form, or is soluble in a solvent (preferably water).

This invention brings important advantages.

First, thanks to this invention it has been possible to provide a system for detecting radical activity, which may even be used if there is a dye or another substance present which interferes with fluorescence measurement, unlike what currently happens with fluorescence measurements.

Second, according to the first and second innovative aspects described above, it has been possible to provide a method which, through the use of specific substances, allows on one hand easy detection of the occurrence of a predetermined radical activity with simple measurements of protein content, and on the other hand allows prediction of the risk of the occurrence of a predetermined radical activity, following the execution of a specific treatment, in a predetermined chemical preparation

According to the third innovative aspect of this invention, it has been possible to provide a method for preparing a chemical preparation to be used in contact with at least one biological product during a specific treatment, which allows the preparation of chemical preparations which have a reduced risk of causing the occurrence of a predetermined radical activity during that specific treatment.

In contrast, the fourth innovative aspect of this invention, has allowed the identification of a property which must be possessed by a chemical preparation for use in a method for treatment of the bodies of humans or animals, or for use in a diagnostic method applied to the bodies of humans or animals, in order to be able to be considered as having a reduced risk of causing the occurrence of a predetermined radical activity, following its use in those methods.

Finally, according to the fifth innovative aspect of this invention, it has been possible to provide a chemical preparation for use in a method for treatment of the bodies of humans or animals, or for use in a diagnostic method applied to the bodies of humans or animals, which allows easy a posteriori verification of whether or not a predetermined radical activity occurred following use of the chemical preparation in one of those methods.

Finally, it should be noticed that this invention is relatively easy to produce and that even the cost linked to implementing the invention is not very high. The invention described above may be modified and adapted in several ways without thereby departing from the scope of the inventive concept.

All details may be substituted with other technically equivalent elements and the materials used, as well as the shapes and dimensions of the various components, may vary according to requirements.