FIELD OF THE INVENTION

The present invention relates to pharmacological agents useful with regard to renal lithiasis resulting from the precipitation of calcium salts, in particular, the invention relates to a new use of catechin, or derivatives thereof, for treating renal lithiasis. Also, the invention relates to a new use of catechin, or derivatives thereof for chelating calcium ions and/or soiubilizing stones containing calcium salts.

BACKGROUND OF THE INVENTION

Renal lithiasis is the presence of kidney stones, or renal calculi in the kidney. Kidney stones are made of small mineral deposits usually referred to as crystals. Kidney stones may be of different compositions, and may result from the precipitation in the kidney, ureter or bladder of calcium oxalate salts, calcium phosphate salts, struvite, uric acid, cystine or a mixture of these chemical species. Most of the kidney stones comprise crystals of calcium salts.

The type of kidney stone will affect the type of treatment that will be effective. Renal lithiasis has a prevalence above 10% in western countries, and usually relies upon a plurality of environmental factors, in particular the daily diet. The composition of renal stones from patients affected by lithiasis is almost always of calcium nature, for example made of calcium oxalate and/or calcium phosphate. Within the calcium oxalate crystals, one can distinguish whewellite crystals, a calcium oxalate monohydrate crystalline form (or CI), from weddellite crystals, a calcium oxalate dihydrate crystalline form (or C2). Hence, calcium and oxalate intakes from the diet of individuals are of major importance.

Treatment of lithiasis includes increasing diuresis by increasing drinks, and in particular water consumption, adjusting the pH or the mineral content of the urines by administration of potassium citrate, or diuretic such as thiazide or hydrochlorothiazide, or hypouricemia inducing agent, such as allopurinol, or renal surgery, such as extracorporeal shock wave lithotripsy, ureteroscopy and percutaneous nephrolithotomy. However, treatment of renal stones remains a challenge as thiazide and allopurinol have numerous side effects, such, as acute kidney failure, hypokalemia, hyponatremia, cutaneous Lyell and Stevens- Johnson syndromes, hepatic cytolysis, and kidney lesions. Besides, surgery bears the numerous drawbacks of any invasive techniques. Tea, and particularly green tea, has proven to be beneficial health since centuries. Beneficial effect of tea on health is presumed to be attributed to its diuresis effect and to the large amounts of antioxidant components contained therein, such as, for example, catechins.

Tea has been proposed by some as possibly having a preventive effect on kidney stones formation.

Indeed, Itoh et al. (2005), Grases et al. (2009), and Jeong et al. (2006) suggested, on the basis of results obtained from animal models into which kidney stones formation is induced, that antioxidants from tea or from some herbal preparations could have a preventive effect on calcium urinary stone formation. It is suggested that this beneficial effect could be due to a decrease of the excretion of urinary oxalate.

Chen et al. (2010) showed that when catechins extracted from tea are present during calcium oxalate crystallization, the crystallisation tends to be oriented toward the calcium oxalate dihydrate crystalline form (weddellite or C2 crystalline form).

However, tea is also known to contain high amount of oxalate, and consequently its consumption is regarded by others as a risk factor for individuals prone to renal stones formation.

Indeed, Honow et al. (2010) suggested that patients at risk for recurrent stone formation should take into account the oxalate content of green tea, and that daily intake of green tea is not generally advisable for calcium oxalate stone patients.

Finally, Charrier et al. (2002) reported that addition of milk to tea tends to reduce the amount of soluble oxalate ingested, as calcium from the milk will bind to and precipitate oxalate, which therefore will continue down to the digestive tract without being adsorbed. Therefore, addition of milk to tea would be proposed to prevent the possible negative effect associated with the ingestion of tea on the formation of kidney stones.

Taken together, those data tend to show that consumption of tea, or catechins, could be helpful for preventing renal stones formation, but should be avoided once renal stones are formed as it may favour precipitation of calcium salts.

On the other hand, Rode et al. (April 2013 and June 2013) confirmed that green tea has diuretic properties, and further showed that patients having kidney stones and consuming green tea display a higher prevalence of weddellite crystals.

Therefore, it remains a need for a novel active agent useful for treating renal lithiasis resulting from the precipitation of calcium salts.

There is also a need for a novel active agent useful for treating renal lithiasis with no or less side-effects than the current available treatments. There is also a need for a novel active agent useful for treating renal lithiasis which may be more easily administered to patients.

There is also a need for a novel active agent useful for treating renal lithiasis which may be cheap and efficient.

It also remains a need for a novel active agent useable for favouring the removing of already formed kidney stones comprising crystals of calcium salts.

There is also a need for a novel active agent which may be used as a complement of surgery treatment of renal lithiasis to remove the remaining small kidney stones, or fragments thereof, comprising crystals of calcium salts.

There is also a need for novel active agent for chelating calcium ion or solubilizing crystal containing calcium salts.

SUMMARY OF THE INVENTIO

The present invention has for purpose to meet those needs.

Therefore, according to a first object, the present invention relates to a catechin for use as a pharmaceutical active agent for treating, reducing the presence of, and/or favouring a removing of, a renal lithiasis, in an individual in need thereof, wherein said renal lithiasis results at least from a crystallisation of at least one calcium salt.

Unexpectedly, as detailed in the examples below, the inventors have observed that a green tea extract, and more particularly catechins, and in particular epigaliocatechin (EGC), are able to chelate calcium, or to solubilize, or to favour solubilisation of, calcium crystals or kidney stones made of calcium crystals, and therefore to reduce the size of calcium crystals or kidney stones made of calcium crystals. The chelating and/or solubilizing effect of catechins on calcium crystals or kidney stones comprising calcium crystals make them useful for helping to reduce the size and/or to remove calcium crystals or kidney stones comprising calcium crystals in case of renal lithiasis.

Also, it has been observed that once a calcium crystal of the whewellite type (CI crystalline form) is formed, it has been observed that administration of catechins, and in particular of EGC, allows for the transformation of the whewellite crystalline form into the weddellite crystalline form (or C2). Without being bound by any theory, it is believed that given that weddellite crystals tend to adhere less to surrounding biological tissue in a kidney than whewellite crystals, they tend to be eliminated more easily. Therefore, the transformation of a whewellite type crystal into a weddellite type crystal by catechins, and in particular by EGC, allows favouring the removal and/ov removing kidney stones comprising of calcium salts.

Within the meaning of the invention, the expressions "'treating ¹ ', "reducing the presence of or "favouring the removing of intend to refer to the effects achieved in individual suffering from renal lithiasis with kidney stones made of crystals of calcium salts following administration of catechin or a composition containing a catechin, and resulting in the partial or total alleviating of the symptoms linked to a renal lithiasis. Further to a use in accordance with the invention, the kidney stones made of crystals of calcium salts present in the kidney may be partially or totally removed.

The invention does not relate to preventing renal lithiasis, or, otherwise said, does not relate to reducing the risk of occurrence of renal lithiasis.

Within the meaning of the invention, the terms an "ίηώ ^' νϊώια and a "patient" intend to refer to any subject comprising at least one kidney stone comprised of calcium salts. Preferably, an individual may be a mammal, and more preferably an animal of economic importance, for example farms, laboratories or food industries animals, such as sheep, swine, cattle, goats, dogs, cats, horses, poultry, mice, rats. More preferably, an individual according to the invention may be a human.

According to another of its aspects, the present invention relates to a catechin for use as a pharmaceutical active agent for treating, reducing and/or favouring a removing of renal stone(s) resulting at least from a crystallisation of at least one calcium salt, in an individual in need thereof, wherein said renal stone(s) is/are residual stone(s), and/or fragmeni(s) thereof, following a surgical treatment of a renal lithiasis.

Within the meaning of the invention, the expression "residual stone" with regard to surgical treatment intends to mean any stone, or a fragment thereof, which may remain as a kidney stone after any surgical intervention performed to remove kidney stones and which do not eliminate by itself.

Within the meaning of the invention, the expression "stone resulting at least from a crystallisation of at least one calcium salt" intends to refer to any stone containing a sufficient amount of calcium salt for seeing, when the calcium salt is totally or partially removed further to the action of a catechin, its structure substantially altered. Preferably, the alteration of the structure of the kidney stone is such that, the stone may be naturally removed from the kidney. The expression "naturally removed ^' ' intends to mean, within the invention, that stone is eliminated from the body through the urinary tract, with the urine flow. A stone resulting at least from a crystallisation of at least one calcium salt considered with the invention may contain at 10% or more of a calcium salt, preferably 15% or more, more preferably 20% or more, still preferably 30% or more, still preferably 40% or more, still preferably 50% or more, still preferably 60% or more, still preferably 70% or more, still preferably 80% or more, still preferably 90% or more, still preferably 95% or more, and still preferably 99% or more of a calcium salt.

According to another of its aspects, the instant invention relates to a use, preferably a non-therapeutic use, of a catechin for chelating calcium ions.

According to another of its aspects, the present invention relates to a use, preferably a non-therapeutic use, for solubilizing a crystal comprising one calcium salt or a stone comprising at least one calcium salt.

According to another of its aspects, the present invention relates to a method for treating, reducing the presence of, and/or favouring a removing of, a renal lithiasis, in an individual in need thereof, wherein said renal lithiasis results at least from a crystallisation of at least one calcium salt, the method comprising at least a step of administering to the individual an efficient amount of at least one catechin.

According to another of its aspects, the present invention relates to a method for treating, reducing the presence of, and/or favouring a removing of, an ureteral lithiasis, in an individual in need thereof, wherein said ureteral lithiasis results at least from a crystallisation of at least one calcium salt, the method comprising at least a step of administering to the individual an efficient amount of at least one catechin.

According to another of its aspects, the present invention relates to a method for treating, reducing the presence of, and/or favouring a removing of, a bladder lithiasis, in an individual in need thereof, wherein said bladder lithiasis results at least from a crystallisation of at least one calcium salt, the method comprising at least a step of administering to the individual an efficient amount of at least one catechin.

According to another of its aspects, the instant invention relates to a method for treating, reducing and/or favouring a removing of renal stone(s) resulting at least from a crystallisation of at least one calcium salt, in an individual in need thereof, wherein said renal stone(s) is/are residual stone(s), and/or fragment(s) thereof, following a surgical treatment of a renal lithiasis, the method comprising at least a step of administering to the individual an efficient amount of at least one catechin. The invention has, at least, for advantage, that the novel proposed active agent is easy and cheap to prepare, making more cost-effective the now proposed treatment for renal lithiasis.

The invention has also for advantage that the novel active agent may be more easily administered than the current known treatments, and may be devoid or present less side- effect than the current known treatments.

LEGENDS OF THE FIGURES

FIGURE 1 : represents weddellite crystals seen by Scanning Electron Microscopy (SEM) from: Daily green tea drinkers, magnification x500 (a, c) and xlOOO (b, d); Control patients, magnification x500 (e, g) and xlOOO (f, h). The arrow indicates the magnification increase from x500 (left panels) to xlOOO (right panels).

FIGURE 2: represents weddellite crystals seen by SEM from: Daily green tea drinkers ("the vert", upper panels), magnification x25()0 (a, b); Control patients ("controle" lower panels), magnification x2500 (c, d). Square pores ("PC") are indicated by an arrow (upper panels).

FIGURE 3: represents weddellite crystals from control patients treated for 24 hours at room temperature with: (a) no incubation (x2500); (b) sterile water (x2500); (c) EDTA 0.1 M (x2500); (d) green tea extracts (x2500); (e) and (f) EGC 300 μΜ (x2500 and x5000). Square pores ("PC") are indicated by an arrow in panels c, d and f. Panel f is a magnified view (x5000) of the region delimited by a white square insert from panel e (x2500).

FIGURE 4: represents weddellite crystals (x2500) from control patients treated for 48 hours at room temperature with: (a) no incubation; (b) sterile water (x2500); (c) EGC 10 μΜ; (d) EGC 50 μΜ; (e) EGC 100 μΜ; (f) EGC 300 μΜ; (g and h) EDTA 300 μΜ. Square pores are indicated by an arrow in panels c-h,

DETAILED DESC RIPTION OF THE INVENTION

Uses & methods

The instant invention relates to renal lithiasis resulting at least from a crystallisation of at least one calcium salt.

The formation of stones or calculi is named lithiasis.

A renal lithiasis may manifest by different symptoms or conditions, which may occur alone or in combination. For example, symptoms linked to renal litliiasis may be irritation of nearby tissues, causing pain, swelling and inflammation, obstruction of an opening or duct, interfering with normal flow and disrupting the function of the kidney, predisposition to pyelonephritis, hydronephrosis (swollen kidneys) and renal failure. Renal Hthiasis can progress to urolithiasis, which manifests itself by the presence of urinary bladder stones, which may end by a bladder outlet obstruction.

In one embodiment, a renal lithiasis considered within the invention may result from a presence of at least one calcium stone in at least one kidney, or in at least one ureter or in a bladder. Preferably, a renal lithiasis considered within the invention may result from a presence of at least one calcium stone in at least one kidney.

In one embodiment of the invention, a catechin may allow for a partial or a total removing of at least one stone comprised of at least one calcium salt in an individual suffering from renal lithiasis. A partial removing of a stone may result in a reduction of its size further to the calcium solubilizing and/or chelating effect of the catechin in accordance with the invention.

In one embodiment of the invention, a catechin may allow for a reduction of the size of at least one stone comprised of at least one calcium salt in an individual suffering from, renal lithiasis. The reduction of the size of a kidney stone may allow for the alleviating of the symptoms associated with a renal lithiasis or the natural removing of the stone.

In another embodiment, a catechin may allow for a total removing of at least one stone comprised of at least one calcium salt in an individual suffering from renal lithiasis.

Preferably, a catechin may allow for a partial removing of at least one stone comprised of at least one calcium salt in an individual suffering from renal lithiasis.

In one embodiment, a catechin may allow for treating and/or alleviating a renal lithiasis.

It belongs to a skilled person in the art to diagnose a renal lithiasis and to evaluate the evolution of the symptoms further to the administration of a treatment. For example, to evaluate a renal lithiasis or symptoms thereof, a skilled man may rely on urinary sediment/dipstick test ; serum creatinine level ; serum electrolyte assessment in vomiting patients (e.g. measure of sodium, potassium, calcium, PTH, phosphorus) ; serum and urinary pH level ; microscopic urinalysis ; 24-hour urine profile. These assays may be combined with imaging studies, as for the evaluation of nephrolithiasis: non-contrast abdominopelvic computed tomography (CT) scan; renal ultrasonography; plain abdominal radiograph (flat plate or KUB); intravenous pyelogram (IVP), or urography; plain renal tomography; retrograde pyelography; or nuclear renal scanning. In one variant of realization, the invention may allow for a partial or a total alleviating of at least one symptom cited above for a renal lithiasis.

In one embodiment, the invention may allow for a partial alleviating of at least one symptom cited above for a renal lithiasis.

In one embodiment, the invention may allow for a total alleviating of at least one symptom cited above for a renal lithiasis.

In one embodiment, the invention relates to a catechin for use as a pharmaceutical active agent for treating, reducing and/or favouring a removing of renal stone(s) resulting at least from a crystallisation of at least one calcium salt, in an individual in need thereof, wherein said renal stonc(s) is/are residual stone(s). and/or fragment(s) thereof, following a surgical treatment of a renal lithiasis.

Surgical treatment of a renal lithiasis, which may be considered within the invention, may be extracorporeal shock wave lithotripsy, ureteroscopy and percutaneous nephrolithotomy.

One defect associated with renal surgery for removmg calcium stone(s) is that those stones must have a certain size, preferably more than 6 mm in diameter.

Therefore, in numerous cases, patients having undergone surgery procedure for removing kidney stones may still comprise kidney stones. Catechins used as active agent in accordance with the invention may allow for a removing of the remaining calcium stone and/or a fragment thereof after a renal surgery procedure.

In one embodiment, the invention may allow for the treating, reducing and/or favouring a removing of residual renal stone(s) and/or fragment(s) thereof resulting at least from a crystallisation of at least one calcium salt, following a surgical treatment of a renal lithiasis, those residual renal stone(s), and/or fragment(s) thereof, having a size below 6 mm in diameter, preferably having a size from 1 to 6 mm in diameter, preferably having a size from 2 to 6 mm in diameter, preferably having a size from 3 to 5 mm in diameter, and preferably again having a size from 5 to 6 mm in diameter.

The present invention also relates to a method for treating, reducing the presence of, and/or favouring a removing of, a renal lithiasis, in an individual in need thereof, wherein said renal lithiasis results at least from a crystallisation of at least one calcium salt, the method comprising at least a step of administering to the individual an efficient amount of at least one catechin.

Also, the instant invention relates to a method for treating, reducing and/or favouring a removing of renal stone(s) resulting at least from a crystallisation of at least one calcium salt, in an individual in need thereof, wherein said renal stone(s) is/are residual renal stone(s), and/or fragment(s) thereof, following a surgical treatment of a renal lithiasis, the method comprising at least a step of administering to the individual an efficient amount of at least one catechin.

The method of the invention may comprise the steps of further observing a treatment, a reducing of the presence of, and/or a favouring of a removing of a renal lithiasis.

Within the scope of the invention, a catechin may be administered to an individual, in need thereof by any suitable route, such as by oral, lingual, sub-lingual, cutaneous, percutaneous, sub-cutaneous, intradermal, intravenous, intra-arterial, intra-cardiac, intraperitoneal, or rectal route.

In one embodiment, a catechin may be administered by oral route or a parenteral route.

In one preferred embodiment, a catechin may be administered by oral ro te.

A catechin in accordance with the invention may be implemented alone or in combination with a second treatment. Such second treatment may be for preventing and/or treating renal lithiasis.

Such a second treatment may be a surgical treatment of a renal lithiasis as above- indicated, an intravenous hydration, or an alimentary diet or a therapeutic agent for preventing and/or treating renal lithiasis.

In one embodiment, a catechin may be administered simultaneously, sequentially or separately with an alimentary diet or a therapeutic agent for preventing and/or treating renal lithiasis.

A therapeutic agent for preventing and/or treating renal lithiasis suitable for the invention may be chosen from a diuretic agent, a hypouricemic agent, and a urine aikalinisation agent.

A hypouricemic agent may be allopurinol, or a xanthine oxidase inhibitor, such as febuxostat.

A mine aikalinisation agent may be potassium citrate, or sodium bicarbonate.

A diuretic agent may be a thiazide, and in particular a benzothiadiazine, a hydrochlorothiazide, or a chlorothiazide.

A diet for preventing and/or treating renal lithiasis for the invention may be high fluid, low salt, low protein, low fructose intakes, and any combination thereof. A catechm in accordance with the invention may be administered to an in individual in need thereof once, twice or three times a day, for one, two, three, four, five or six days, or one, two, three or four weeks, or one, two, three, four, five, six, or more months.

The regimen of administration will be adapted by the skilled man according to the usual parameters taken into account in tire field for setting a regimen of administration, such as, for example, the weight, the size, the age and/or the gender of the individual to be treated, the intensity of the symptoms linked to the renal lithiasis, the pharmaceutical form, the possible co-administration of others treatments, in particular as above- indicated.

in another aspect, the invention relates to a use, preferably non-therapeutic use, of a catechm for chelating calcium ions and/or for solubilizing a crystal comprising at least one calcium salt or a stone comprising at least one calcium, salt.

Preferably, those uses are non- therapeutic uses such as biological fluids conservation.

In such embodiments, the crystal or stone may comprise a whewellite crystalline form; a weddellite crystalline form; a calcium phosphate crystalline form, in particular selected from a calcium apatite crystalline form, a brushite crystalline form, or an octa calcium phosphate crystalline form; a calcium carbonate crystalline form; or mixtures thereof, and preferably comprising a whewellite or apatite crystalline form.

Calcium stones

A renal lithiasis retained within the invention is condition resulting from the formation of at least one stone comprising at least one calcium salt. Accordingly, a renal stone considered within the invention comprises at least one precipitated calcium salt.

A stone considered in the invention may comprise at least one calcium salt in amount ranging from 10 to 100% by weight relative to the total weight of the stone. A stone considered in the invention may have a size, in its longer size, rangmg from 0.5 to 10 mm, preferably ranging from i mm to 6 mm (4 to 900 mnr), preferably ranging from 2 to 5 mm. and more preferably ranging from 3 to 4 mm.

Preferably, a renal stone within the scope of the instant invention may have a volume ranging from 0.5 to 1500 mm ³ , preferably ranging from 4 to 900 mm ³ , more preferably ranging from 50 to 500 mm ³ .

The size and volume of a renal stone may be determined by any methods usual in the art. In renal lithiasis resulting from calcium salts precipitation, at least 3 types of crystals may be generated. The ty e of crystal may be linked to the nature of the calci m salt.

In one embodiment, a calcium salt liable to be considered within the invention may be chosen from calcium oxalate(s), calcium phosphate(s), calcium carbonate(s), and mixtures thereof.

Calcium oxalate crystals may crystalize in a whewellite crystalline form or in a weddellite crystalline form. Whewellite (or C I crystalline form) is a mineral, hydrated calcium oxalate of formula CaC ₂ 04'H ₂ 0. Weddellite (or C2 crystalline form), of formula CaC ₂ 0 ₄ '2H ₂ 0, is a di-hydrate mineral form of calcium oxalate. The weddellite or calcium oxalate di-hydrate crystallizes in the tetragonal system.

Calcium phosphate crystals may include hydroxyapatite, brushite and octacalcium phosphate ciystalline forms. Hydroxyapatite is a hexagonal and monoclinical crystal system, having the following formula Ca ₅ (Ρ0 ₄ ) ₃ (ΟΗ). Brushite, or dicalcium phosphate mono- hydrate, is a mineral of formula CaHP0 ₄ 2H ₂ 0. Octacalcium phosphate is a calcium phosphate crystalline form with a formula Ca _s ¾(P0 ₄ ) ₆ 5H ₂ 0.

Finally, calcium carbonate crystals are crystalline forms comprising a chemical compound with the formula CaCC

In one embodiment, the crystallisation of at least one calcium salt in a kidney may result at least in calcium stone comprising a whewellite ciystalline form (or CI); a weddellite ciystalline form (or C2); a calcium phosphate ciystalline form, in particular selected from a calcium apatite crystalline form, a brushite ciystalline form, or an octacalcium phosphate crystalline form; a calcium carbonate crystalline form; and mixtures thereof.

In a preferred embodiment, the crystallisation of at least one calcium salt in a kidney may result at least in a crystal comprising a whewellite or apatite crystalline form.

A renal lithiasis which may be retained for the invention may result from the presence in a kidney of at least one calcium stone as above-indicated.

Catechins

A catechin in accordance with the invention is used as a pharmaceutical active agent.

A catechin suitable for the invention may be any catechin, or a derivative thereof, known in the art. In one embodiment, a catechin suitable for the invention may be chosen from epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin, and a mixture thereof,

A derivative of catechin suitable for the invention may be chosen from a glucuronide, a sulfated or a methylated form of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin, and a mixture thereof.

Whenever applicable, the present invention encompasses isomers (+) and isomers (-) of the catechins or derivatives thereof.

Glucuronide catechins suitable for the present invention may be (-)- epigallocatechin-3'-0-glucuronide, (-)-epigallocatechin gallate-4"-0-glucuronide.

Sulfated catechin suitable for the present invention may be (-)-epicatechin 4'-0- sulfate.

Methylated catechins suitable for the present invention may be selected in a group comprising (-)-epigallocatechin-3-0-(3-0-methyl) gallate, (-)-epigailocatechin-3-0-(4-0- methyl) gallate, (-)-epigallocatechin-3-0-(3,5-0-dimethyl)-gaIlate, and 3-O-methyl- epigallocatechin- 3 -O- (3 , 5-0-dimethyl)-galIate .

A catechin, or a derivative thereof, is implemented in the invention in an amount sufficient to allow for the treating, reducing the presence of, and/or favouring a removing of, a renal lithiasis.

An amount of c techin suitable for the invention may be determined and adapted by a skilled man according to the usual parameters taken into account in the field for setting a dosage and/or regimen of administration. As example of parameters that one may take into account, one may cite the weight, the size, the age and/or the gender of the individual to be treated, the intensity of the symptoms linked to the renal, lithiasis, the pharmaceutical form, the possible co-administration of others treatments, in particular as above-indicated.

A dosage suitable for the invention may be determined according to any routine work usually implemented in the art.

A catechin, or a derivative thereof, suitable for the invention may be administered in an amount ranging from 5 mg/kg/d to 15 mg/kg/d, preferably from 6 mg/kg/d to 12 mg/kg/d, and more preferably from 7 to 10 mg/kg/d.

In one embodiment, a catechin, or a derivative thereof, may be administered under the form of an extract obtained from a tea extract, a wine extract, a dark chocolate extract, or under the form of a recombinant molecule, or a mixture thereof. An extract containing at least one catechin, or a derivative thereof, suitable for the invention may be obtained by any method routinely applied in the field by a skilled man. A suitable method may be selected according to the usual parameters considered in the field for such extraction. As examples of parameters to be considered, one may cite the nature of the material from which the extract is to be obtained, the amount of catechin, or a derivative thereof, to obtain the yield, the storage conditions of the extract, the route of administration of the extract, etc.

Extraction of a catechin, or a derivative thereof, from a plant material, such as tea leaves, wine grapes, or cocoa bean, may be carried out as reported in Ziaedini et al. (2010); Vuong et al. (201.0); Monrad et al. (2012); Casazza et al (20.1 1) or Subagio et al. (2001).

In one embodiment, tea leaves are contacted with a solvent and heated at a temperature comprised between 85 and 100°C for 5 to 20 minutes, in particular" for 5 to 10 minutes. Suitable solvents may be water, organic solvent such as alcoholic solvent, and a mixture thereof. The solvent comprising the solubilized catechin may be collected and further processed by lyophilisation to obtain a dry powder.

in one embodiment, an extract which may be particularly considered within the invention may be a tea extract, and more preferably a green tea extract.

A recombinant catechin, or a derivative thereof, suitable for the invention may be obtained by any method routinely applied in the field by a skilled man. A suitable method may be selected according to the usual parameters considered in the field for such extraction. As examples of parameters to be considered, one may cite the nature of the microorganism from which the recombinant molecule is to be obtained, the amount of catechin, or a derivative thereof, to obtain the yield, the storage conditions of the recombinant molecule, the route of administration of the recombinant molecule, etc.

Recombinant catechins, or derivatives thereof, may be obtained by genetically modifying Escherichia coli. As method to which one may refer to obtain recombinant catechins from E. coli one may cite the work of Umar et al. (2012).

A catechin, a derivative thereof, an extract containing thereof, or a recombinant molecule thereof, may be administered in a pharmaceutical composition suitably formulated for a route as above indicated. Preferably, such a pharmaceutical composition may be formulated for oral route or parenteral route.

In a still preferred embodiment, a catechin, a derivative thereof, an extract containing thereof, or a recombinant molecule thereof, may be administered by oral route. These compositions can be provided in the form of injectable solutions or suspensions, tablets, coated tablets, capsules, syrups, suppositories, creams, ointments and lotions. These pharmaceutical forms are prepared according to the usual methods. The active ingredient can be incorporated into excipients normally used in these compositions, such as aqueous or non-aqueous carriers, talc, arabic gum, lactose, starch, magnesium stearate, cocoa butter, fatty substances of animal or plant origin, paraffin derivatives, glycols, various wetting agents, dispersants or emulsifiers, or preserving agents.

For oral use of a catechin in accordance with the invention, the use of an ingestible support is preferred. The ingestible support may be of diverse nature depending on the type of composition under consideration.

In one embodiment, when a catechin suitable for the invention, or a derivative thereof, is in the form of an extract, a suitable pharmaceutical composition is a diet supplement.

A diet supplement may be formulated as milk or milk-based fermented products, products based on fermented cereals, milk-based powders, food products of candy type, chocolate, cereals, tablets, gel capsules or lozenges, oral supplements in dry form and oral supplements in liquid form are especially suitable for use as pharmaceutical or food supports.

A catechin according to the invention, and/or a fraction thereof, may moreover be formulated with excipients and components that are commonly used for such oral compositions or food supplements, as for example, fatty and/or aqueous components, humectants, thickeners, preserving agents, texture agents, taste agents and/or coating agents, antioxidants, preserving agents and dyes that are common in the food sector.

The formulating agents and excipients for an oral composition, and. especially for food supplements, are known in this field and will not be the object of a fully detailed description herein. Many embodiments of oral compositions and especially of food supplements are possible, for ingestion. They are formulated via usual processes for producing coated, tablets, gel capsules, gels, coiitrolled-release hydrogels, emulsions, tablets and capsules.

In particular, the catechin according to the invention may be incorporated into any suitable form of food supplement or enriched food, for example food bars, or compacted, or loose powders. The powders may be diluted with water, in soda, dairy products or soybean derivatives, or may be incorporated into food bars.

A catechin or a derivative thereof may be formulated in a pharmaceutical composition suitable for a systemic or a parenteral administration and in particular for an administration by injection. Formulations for injection may be presented in single-unit dosage form, such as ampoules or in multi-dose containers. The compositions may be formulated as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain additional agents, such as preservatives, emulsifying and/or stabilizing agents. Alternatively, a catechin or a catechin derivative may be formulated as a dispersible powder, which may be prepared as a liquid composition, with a suitable vehicle, for example sterile water, just before use.

A catechin or a derivative thereof may also be formulated as a rectal composition, such as for example a suppository, containing conventional suppository bases, such as cocoa butter or other glycerides.

The present invention will be better understood by referring to the following examples and figures which are provided for illustrative purpose only and should not be interpreted as limiting in any manner the present invention.

EXAMPLE

Effects of catechin consumption on renal stones

The purpose of the study was to observe potential effects of catechin consumption on lithiasis. Two groups of patients were compared: one consuming daily green tea and one not consuming green tea.

1) Material and ethods

1.1) Population

459 hypercalciuric renal stone formers referred to Hopital TENON (Laboratoire d' Explorations fonctionnelles renales) between 2009 and 201 1 for further investigations underwent a calcium load. A careful clinical examination was also performed including a survey related to diet and fluid intakes, noteworthy daily green tea intakes.

After applying exclusion criteria (i.e., patients with a diagnosis of primary hyperparathyroidism, sarcoidosis, bowel resection, on-going steroid, bisphosphonate or antiviral treatment, vegetarian diet, "exotic" infusions, food supplements (in tablets or powder) or intermittent green tea intake), 316 patients remained in the analysis.

The green tea group was defined as patients drinking daily at least one bowl (250- 300 ml) of green tea (n=74) and the control group drinking no green tea at all (n=242).

Among this selected population (n=316), renal stone composition, performed by infrared spectroscopy was available for 1 17 patients (37%). 1.2) Ex vivo renal stone analysis

The major components of the samples were collected and also when available the nuclei were analysed separately.

57 renal stones {49% of this patient subgroup) with weddellite as the major component and without significant amount of catechin intake (daily red wine, dark chocolate or black tea intake), were available and further analysed by scanning electron microscopy (SEM): 26 samples were obtained from green tea drinkers and 31 from "non drinkers". Morphological analysis was performed using a 0,5 a 30 kV Zeiss microscope SUPRA55-VP SEM. High resolution was obtained using SE and Everhart-Thornley SE detectors. All samples selected on the presence of weddellite crystals detected by low magnification microscope were systematically analysed at different magnifications (xlOO, x250, x500, xlOOO, x2500) in the range of 1 ,2 and 1,6 kV. For each sample, different criteria were collected including the presence or absence of pores, their shape and size, the size of the crystals, the presence of sharp or erased edges.

1.3) In vitro renal stone analysis

Renal stones were selected from our stone bank upon the following criteria: 1) weddellite as the major component (infrared spectroscopy (IRS) analysis) and 2) regular weddellite crystals on the surface with no pores and sharp edges (SEM analysis). Samples were analysed by SEM after a 24 hours incubation with different solutions containing either EDTA 0.1 M, green tea infusion, catechin 10, 50 and 100 μΜ.

2) Results

2.1) Biological survey

The biological data obtained from the population under study are summarized in the following Tables 1 to 5. Table 1. Demographic and clmical data fi-om the population within the scope of the study.

Table 2. Daily fluid, and mineral intake data from the population within the scope of the study.

Population Women (n=I 25) Men (162)

Groups Non Green P Non Green P

Drinkers tea Drinkers tea

Fluid intake > 2 L/d

13 44 0.001 21 26 NS (%)

Coca Cola (%) 19 27 NS 1 1 28 NS

Salt (mmol/d) 123 108 NS 153 181 0.04

Proteins 1.2 1 .1 NS 1.2 1.2 NS

Acidic charge (NH4/d) 40 36 NS 50 45 NS

Magnesium 3.9 4.7 NS 4.9 4.9 NS Table 3. Biological data from the population within the scope of the study.

Population Women Men

Groups Noo Green P Non Green P

drinkers tea drinkers tea

Na+ 139.1 137.8 0.005 139.0 138.7 NS

K+ 4.1 4.1 NS 4.1 4.1 NS

Glycemia 5.5 5.4 NS 5.8 5.8 NS

CO, 27 27 NS 28.2 29.3 0.02

Creatinine 63 59 NS 75 78 NS

CI creatinine i n 121 NS 348 120 0.01

Table 4. Biological data from the population within the scope of the studv.

Population Women Men

Groups on Green P NOR Green P

drinkers tea drinkers tea

Calcemia (mmol/L) 2.28 2.27 NS 2.28 2.23 NS

Ionised calcium

1.17 1.17 NS 1.17 1.17 NS (mmol/L)

P0 ₄ (mmol/L) 0.97 0.94 NS 0.90 0.91 NS

PTH (pg/mL) 36 36 NS 39 37 NS

250H (ng/mL) 27 24 NS 25 24 NS

1-250H D3 (pg/mL) 74 71 NS 70 66 NS

BALP 14 14 NS 16 14 NS

Deoxypyridinolin 7.7 7.5 NS 5.0 4.6 NS Table 5. Biological data from, the population within the scope of the study.

Demographic, clinical and biological data were similar between the two groups "drinkers" and "non drinkers" in male and female populations except for hyponatremia that was encountered more frequently in women and metabolic alkalosis with a lower creatinine clearance in men (Tables 1 , 2, and 3).

Altogether, these data favour a diuretic property of green tea intake. In female but not in male population, 24 hours diuresis was significantly higher in "drinkers" suggesting that this habit was probably more related to a healthy concern in women than in men.

Of interest the groups were similar for other stone risk factors including 24 hours ealciuria and bone remodelling biomarkers (Tables 4 and 5).

2.2) Analysis of collected renal stones

1 17 renal stones collected from women (49) and men (68) were analysed by InfraRed Spectroscopy (IRS).The major stone component identified by IRS analysis revealed a significant higher prevalence of calcium oxalate di-hydrates (weddellite) in female "drinkers" whereas in male "drinkers", weddellite major compound was found in stone nuclei lib (Table 6). Table 6, i S analysis of the stones obtained from the « non drinker » and the « tea drinker » populations.

Nuclei la, lb refer to whewellite subtype crystalline forms; Nuclei ila and lib refer to weddellite subtype crystalline forms, nucleus ilb may also comprise whewellite crystalline form; Nucleus IVa refers to carbapatite subtype crystalline form.

Scanning Electronic Microscopy (SEM) analysis of collected renal stones (57) was performed. Three types of pores could be observed, namely square pores, round pores and trapezoid pores. The numbers of renal stones presenting pores within the women population were identical to the numbers of renal stones presenting pores within the men population. However, the analysis of stones in "drinliers" and "non drinkers" detected a significant higher prevalence of square pores in "drinkers" with no difference for size (92% versus 40% p<0.05) (see Figures 1 and 2; Table 7). Table 7. SEM analysis of the stones obtained from the « non drinker » and the « tea drinker » populations.

The presence of pores in greater number in the stones from "drinkers" than in the stones from "non drinkers" shows that the consumption of green tea favours renal stones dissolution.

2.3) In vitro solubilisatioti of weddellite crystals

As green tea seems to induce renal stones sohibilisation in vivo, as evidence by IRS and SEM analysis, further evidences were needed to provide an unambiguous correlation of the effect of green tea on renal stones sohibilisation.

Hence, the capability of green tea to induce renal stones sohibilisation was assessed in vitro.

Incubation of renal stones from weddellite crystalline form, for 24 hours, in green tea solutions, EDTA (0.1 mol/1) or EGC (300 ,umol/l), results in the apparition of square pores at the surface of weddellite crystals.

Incubation for 24 hours at room temperature of weddellite stones with regular weddellite crystals on the surface and no pores (checked by SEM analysis) with green tea infusion or catechin 10, 50, 100, and 300 μΜ solutions revealed the occurrence of square pores at the surface that appeared very similar to pores detected after 24 hours incubation with EDTA 0.1 M solution, a well-known calcium chelator (Figures 3 and 4).

3) Conclusions

The collected data provide evidences that green tea infusion and catechin solution in vitro solubilize calcium oxalate dihydrated stones as 24 hours incubation provides the same appearance as the calcium chelator EDTA (i.e. square pores). As shown, this effect seems to occur also in vivo as we observed in weddellite (calcium oxalate di-hydrate, or C2 crystalline form) stones from "drinkers" compared to "non drinkers", indeed, in "non drinkers", most weddellite deposits are within months transformed into whewellite (calcium oxalate monohydrates, or CI crystalline form), a phenomenon called crystalline conversion" . Drinking daily green tea seems to prevent this conversion from weddellite into whewellite, seemingly through catechin ligation to calcium molecules, resulting in weddellite major content as shown.

In this regard, one may note that the data show that 10 μΜ catechin concentration is able to induce renal stone square pores in vitro.

Of notice, urine catechin concentration found after green tea consumption is about the same order of magnitude, thus suggesting that daily intake of catechin may be a potential useful drug to favour renal stones removing after formation, in particular following renal surgery, and to prevent renal stone recuiTence especially following renal stone surgery.

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