Ergothioneine, S-methyl-ergothioneine and their uses

Abstract

The present invention provides S-methyl-L-ergothioneine for use in diagnosis and / or prognosis. The present invention also provides a method for diagnosing and / or prognosing renal disease comprising the step of determining the amount of S-methyl-L-ergothioneine in an isolated test sample of a subject, and a method for determining or recommending whether to initiate a therapeutic intervention or determining the efficacy of a therapeutic intervention. Additionally, the invention provides ergothioneine for use in the treatment and / or prevention of renal calculosis or aminoaciduria, and ergothioneine for use in combination therapy.

Description

Technology Field

[0001] The present application claims priority to European patent application EP19382644.3 filed on July 26, 2019.

[0002] The present invention belongs to the field of medicine. In particular, the present invention belongs to renal disease. The present invention provided herein is particularly useful for the diagnosis and treatment of cystine lithiasis. Background Technology

[0003] Cystinuria is a disorder involving an autosomal recessive inheritance pattern, involving the amino acid carrier rBAT / b 0,+ It is characterized by problems with the renal reabsorption and intestinal absorption of cystine and dibasic amino acids caused by defects in AT. Cystinuria progresses to cystine calculi, which are caused by the precipitation of cystine in the urinary system to form calculi. These calculi can lead to obstruction, infection, and ultimately, renal failure.

[0004] So far, the two genes that cause cystinuria, SLC3A1 and SLC7A9 This was sympathized with, and these are b 0,+ rBAT / b, responsible for the amino acid transport system 0,+ It encodes the AT heteromeric complex. This is the major upstream reabsorption system for cystine in the kidney. This transporter belongs to the heteromeric amino acid transporter (HAT) family, and it is formed into a light subunit by disulfide (in the case of rBAT, b 0,+ It is formed as a heavy subunit (rBAT or 4F2hc) connected to the range of AT).

[0005] Cystinuria is diagnosed by demonstrating selective hyperexcretion of cystine and dibasic amino acids in the urine. Since hexagonal crystals are found in the urine of 20 to 25% of patients with cystinuria, cystine stones are the only proven clinical sign of the condition. In fact, cystinuria is the cause of up to 10% of all urinary stones in young children. More than 80% of patients with cystinuria develop their first cystine stones in both kidneys within 20 years of birth, with 75% developing them in both kidneys. Most patients suffer from recurring stone formation and the need for repeated interventions throughout their lives. Currently, there is no way to predict when a patient with cystinuria will develop cystine stones. Furthermore, the only way to detect whether stones have already formed in patients is through complex imaging techniques such as KUB X-rays, computed tomography (CT), and ultrasound.

[0006] Human cystinuria is treated by preventing the formation of cystine stones through high fluid intake, a low-salt diet (less than 2 g NaCl per day), a moderate reduction in protein intake (less than 0.8 g per day), and urine alkalinization to pH values ​​of at least 7.5 to maximize cystine solubility. Even with medical management, long-term outcomes are poor due to insufficient efficacy and low patient compliance.

[0007] If prevention fails, patients receive treatment to dissolve or crush the stones. Stones are removed by surgical nephrectomy (for large stones), percutaneous nephrectomy, intracorporeal lithotripsy, and, in the case of recently formed stones, extracorporeal shockwave lithotripsy. These procedures carry a risk of progressive renal impairment.

[0008] The pharmaceutical approach is based on the oral intake of thiol-based agents such as penicillamine and thiopronin, which can exhibit a redox equilibrium between cystine (insoluble) and cysteine ​​(soluble) by forming complexes with cysteine. Although these agents are highly effective, they have multiple side effects that lead to treatment discontinuation and disease recurrence (Halperin EC et al., "The use of D-penicillamine in cystinuria: efficacy and untoward reactions", Yale J Biol Med., 1981, vol. 54(6), pp. 439-46).

[0009] Therefore, there is still a need for simple and inexpensive methods to determine the onset of cystine stones in patients with cystinuria, as well as for effective and non-toxic treatments to prevent or delay the formation of cystine stones.

[0010] The inventors have developed a novel method for diagnosing cystine stone disease based on the detection of S-methyl-L-ergothioneine (S-met-L-Erg) in urine samples from a patient.

[0011] As shown in the examples below, the inventors surprisingly discovered that the level of the amino acid derivative S-met-L-ergothioneine in urine can determine the presence of renal cystine stones with strong statistical power. This was quite unexpected because S-met-L-ergothioneine is a metabolite with no known association with any human disease and has never been detected in samples from patients with cystinuria. Accordingly, the inventors developed a method that enables the precise diagnosis of cystine stones in patients with cystinuria through a simple analysis of a single metabolite in readily available samples.

[0012] Prior to the present invention, the presence of cystine stones in patients with cystinuria could only be determined by complex imaging techniques. Consequently, the clinical management of patients with cystinuria consisted of increased fluid intake and urine alkalinization until urinary cystine levels or renal pain indicated a potential for calculous events. Only then was the presence of cystine stones determined by imaging techniques such as CT scans or ultrasound. These techniques require expensive equipment and cannot even distinguish cystine from the chemical composition of other stones.

[0013] The simplicity and efficiency of the method provided in this specification enable routine screening of urolithiasis in patients with cystinuria. This significant improvement facilitates the detection of cystine stones at the early stages of onset before the urolithiasis causes any syndrome in the patient, thereby reducing the patient's suffering and enabling the optimization of treatment.

[0014] Accordingly, in a first embodiment, the present invention provides S-methyl-L-ergothioneine for use in diagnosis and / or prognosis.

[0015] In a second aspect, the present invention provides a method for diagnosing and / or prognosing a renal disease, the method comprising the step of determining the amount of S-methyl-L-ergothioneine in an isolated test sample of a subject.

[0016] As described in the cystinuria animal model in the example below, the amount of S-methyl-L-ergothioneine in the urine was lower compared to control animals (wild-type-WT). Therefore, S-methyl-L-ergothioneine is a useful tool for determining or recommending the initiation of therapeutic interventions to prevent or reduce urolithiasis.

[0017] In a third aspect, the present invention provides a method for determining or recommending whether to initiate a therapeutic intervention in a subject suspected of suffering from renal disease, wherein the method comprises (a) determining the amount of S-methyl-L-ergothioneine in an isolated test sample of the subject; and

[0018] (b1) Decide or recommend initiating therapeutic intervention when the amount of S-methyl-L-ergothioneine is within the reference range for subjects suffering from renal disease; or alternatively,

[0019] (b2) The above criteria for the subject, which does not suffer from renal disease, includes the step of comparing an amount in an isolated test sample of the subject with a reference value; wherein if the amount of S-methyl-L-ergothioneine measured in step (a) is lower than the reference value, the subject is to initiate a therapeutic intervention.

[0020] In a fourth aspect, the present invention provides a method for determining the efficacy of a therapeutic intervention in a subject already diagnosed with renal disease, wherein the method comprises: (a) measuring the amount of S-methyl-L-ergothioneine in a test sample isolated from the subject prior to the therapeutic intervention; (b) measuring the amount of S-methyl-L-ergothioneine in a biological sample isolated from the subject once the therapeutic intervention has been initiated; and (c) comparing the amounts measured in steps (a) and (b) in such a manner that if the amount of S-methyl-L-ergothioneine measured in step (b) is higher than the amount of S-methyl-L-ergothioneine measured in step (a), the medical therapy is effective in treating the renal disease; or, alternatively, the present method comprises: (i) measuring the amount of S-methyl-L-ergothioneine in a test sample isolated from the subject once the therapeutic intervention has been initiated; and (ii) comparing the amount measured in step (i) with a reference value of S-methyl-L-ergothioneine, wherein the reference value is that of a subject not suffering from renal disease, or the reference value is the amount of S-methyl-L-ergothioneine in an isolated test sample of a subject suffering from renal disease at a previous test moment; wherein if the amount of S-methyl-L-ergothioneine measured in step (i) is not lower than the reference value, it indicates that the medical therapy is effective for the treatment of renal disease.

[0021] In a fifth aspect, the present invention provides the use of means for determining the amount of S-methyl-L-ergothioneine in a method as defined above.

[0022] In a sixth aspect, the present invention provides the use of S-methyl-L-ergothioneine as a marker for the diagnosis and / or prognosis of renal disease in isolated test samples of a subject.

[0023] In a further embodiment, the present invention provides an S-methyl-L-ergothioneine / L-ergothioneine ratio for use in diagnosis and / or prognosis.

[0024] The inventors have also developed a novel treatment for cystine stone disease. Surprisingly, as shown in the example below, the inventors discovered that the administration of ergothioneine (Erg) prevents or delays kidney stone formation in a mouse model of cystinuria.

[0025] Current pharmaceutical treatments for cystine stones are based on thiol-based agents that solubilize cystine stones by breaking disulfide bonds and thereby forming mixed cysteine ​​disulfide compounds that are more soluble in urine. These known drugs exhibit highly variable levels of efficacy and strong secondary effects that limit the use of the drugs to short durations (Halperin EC et al., cit.). As a result, patients with cystinuria show a high rate of disease recurrence, which can lead to infection and renal failure.

[0026] Unexpectedly, the inventors discovered that the administration of ergothioneine not only prevents and delays the formation of cystine stones but also produces no adverse effects in mice with cystinuria. This allows for the long-term treatment of patients with cystinuria, thereby significantly reducing the occurrence and recurrence of stones. Without wishing to be bound by theory, the different mechanism of action of ergothioneine discovered by the inventors may be the cause of ergothioneine's exceptional properties. In contrast to currently used thiol-based agents, ergothioneine does not act directly on cystine stones. However, ergothioneine promotes the synthesis of glutathione, increases the intracellular levels of cysteine, methionine, and gamma-glutamyl-cysteine ​​(γ-glutamyl-cysteine), increases the intracellular ratio of cysteine ​​to cystine in renal cells, and reduces the redox potential of urine.

[0027] The distinct advantage demonstrated by the novel treatment provided in this specification is a clear indication that it can be used for the prevention or even treatment of cystine stones in human patients. Indeed, as shown in the example below, ergothioneine reduced the number of stone-forming mice by 50%; delayed the formation of stones in the remaining 50% by one month compared to untreated mice, and furthermore, the growth rate of the formed stones was lower.

[0028] In light of the above, the novel treatment for cystine stone disease provided in this specification represents a significant advancement in the field of medicine, particularly in the treatment of such genetic disorders.

[0029] Accordingly, in the seventh aspect, the present invention provides ergothioneine for use in the treatment and / or prevention of kidney stones or aminoaciduria.

[0030] This embodiment may also be expressed as the use of ergothioneine for the manufacture of a drug for the treatment and / or prevention of a disease selected from renal calculus and aminoaciduria. This embodiment may also be expressed as a method for treating and / or preventing a disease selected from renal calculus and aminoaciduria, comprising administering a therapeutically effective amount of ergothioneine to a subject requiring treatment and / or prevention.

[0031] The mechanism of action of ergothioneine is particularly suitable for complementing currently used treatments involving cystine-solubilizing agents. Their combined use can remove existing stones while simultaneously preventing the formation of new stones. Furthermore, the administration of ergothioneine can reduce the toxicity of current treatments.

[0032] Accordingly, in the eighth aspect, the present invention provides ergothioneine for use in combination therapy with a compound selected from the group consisting of a separate cystine-solubilizing agent, L-cystine dimethyl ester, L-cystine methyl ester, L-cystine diamide, lipoic acid, and combinations thereof in the treatment and / or prevention of renal calculosis or aminoaciduria.

[0033] This embodiment may also be expressed as the use of ergothioneine for the preparation of a drug to be used in combination therapy with a compound selected from the group consisting of a separate cystine-solubilizer, L-cystine dimethyl ester, L-cystine methyl ester, L-cystine diamide, lipoic acid, and combinations thereof in the treatment and / or prevention of a disease selected from renal calculus and aminoaciduria. This embodiment may also be expressed as a method for treating and / or preventing a disease selected from renal calculus and aminoaciduria, comprising administering a therapeutically effective amount of ergothioneine in combination with a compound selected from the group consisting of a separate cystine-solubilizer, L-cystine dimethyl ester, L-cystine methyl ester, L-cystine diamide, lipoic acid, and combinations thereof to a subject requiring treatment and / or prevention. Brief explanation of the drawing

[0034] Fig. 1. Determination of L-Erg and S-met-L-Erg in urine of 6-month-old male mice. 6-month-old wild-type and S / c7a9 - / - (KO) L-Erg and S-met-L-Erg concentrations in the urine of male mice. Each point represents a single sample, and bars represent the mean ± SEM (standard error). Mann-Whitney probability test values ​​are displayed at the top of each chart. Fig. 2. Differences in L-Erg and S-met-L-Erg concentrations in urine of male mice at different ages. 3-month and 6-month-old wild-type and S / c7a9 - / - (KO) L-Erg and S-met-L-Erg concentrations in the urine of male mice. Bars represent mean ± SEM. Mann-Whitney probability test values ​​were expressed as **, P≤0.01 for 3-months vs. 6-months, ***, P≤0.001, and +++, P≤0.001 for wild type vs. KO. Fig. 3. Sex-associated differences in L-Erg and S-met-L-Erg concentrations in mouse urine. 6-month-old wild-type and S / c7a9 - / - (KO) L-Erg and S-met-L-Erg concentrations in the urine of mice. Bars represent mean ± SEM. Mann-Whitney probability test values ​​were denoted as *, P≤0.05, **, P≤0.01, ***, P≤0.001, ****, P≤0.0001 versus male mice, and +, P≤0.05, +++, P≤0.001 versus female mice. "M" indicates male, and "F" indicates female. Fig. 4. L-Erg and S-met-L-Erg urine concentrations in stone-former cystinuria mice and stone-non-stone-former cystinuria mice. 3-month-old (a) and 6-month-old (b) cystinuria mice associated with stone-former (SF) or stone-non-stone-former (NSF) distinguished by sex ( S / c7a9 - / - L-Erg and S-met-L-Erg concentrations in the urine of ). Stone-forming (SF) mice were subdivided by the time of stone detection: ESF, stones detected before 3 months of age, and LSF, stones detected after 3 months of age. Bars represent mean ± SEM. Mann-Whitney probability test values ​​are denoted as ~, P≤0.1, *, P≤0.05, **, P≤0.01, ***, P≤0.001 versus non-stone-forming mice, and +, P≤0.05, ++, P≤0.05, +++, P≤0.001 versus stone-forming mice. "M" indicates male, and "F" indicates female. Fig. 5. Urinary S-Met-L-Erg / L-Erg ratio in cystinuria mice. a. Stone-forming (SF) and non-stone-forming (NSF) S / c7a9 - / - Urinary S-met-L-Erg / L-Erg ratio in mice. Bars represent mean ± SEM. Mann-Whitney probability test values ​​are indicated as *, P≤0.05, ***, 10, P≤0.001, ****, P≤0.0001 versus stones-free mice. b. Receiver operator characteristic curve showing the performance of the urinary S-met-L-Erg / L-Erg ratio in distinguishing SF from NSF. AUC, area under the curve. The y-axis represents the true positive fraction and the x-axis represents the false positive fraction. Fig. 6. (In relation to Example 2). Metabolic fluctuations due to L-Erg treatment. Water intake normalized to mouse surface area (a), pH (b), and redox potential (c) before (initial, left bar, "B") and after (boil, right bar, "A") treatment with different concentrations of L-Erg supplemented in drinking water. Each dot represents one mouse. Wilcoxon test results. pH and redox potentials were not determined in all treated mice due to urine volumes below the lower limits of the pH and redox electrodes. Urinary L-Erg concentration (d) and S-met-L-Erg concentration (e) on a semi-log scale for visualization purposes before (B) (initial, left bar) and after (A) (boil, right bar) treatment with different concentrations of S-Erg supplemented in drinking water. Each dot represents one mouse, and bars represent mean ± SEM. In all panels, ns, not significant; * p<0.05 by Wilcoxon signed-rank test. Initial values ​​prior to treatment correspond to the left bars in each figure (a to e) and / or for each L-Erg test dose. Final values ​​after treatment correspond to the right bars in each figure (a to e) and / or for each L-Erg test dose. The x-axis represents L-Erg (mg / L), and the y-axis represents fluid intake / body weight. 2 / 3 Ratio (ml / kg) 2 / 3 )(a); pH(b); ORP (redox potential)(mV)(c); [L-ergothioneine](μM) in urine(d); and [S-Methyl-L-ergothioneine](μM) in urine(e). Fig. 7. (In relation to Example 2). Stone growth rate in lithic mice treated with L-Erg. Male and female mice were treated with 60 mg / L L-Erg in drinking water (ERG) or left untreated for 3 months (C). The y-axis represents the stone growth rate (mg / day). The bars represent the mean ± SEM of the stone growth rate determined monthly from X-ray images of 13 to 15 mice. Each dot represents the cystine stone growth rate determined for each mouse. Fig. 8. (In relation to Example 2). Effects of long-term (6-month) L-Erg treatment on the development of cystine stone disease and metabolic parameters. (a) Effect on the development of cystine stone disease. The percentage of stone-causing mice and necropsy during 6 months of treatment are shown for L-Erg-treated mice (ERG) and untreated mice (Control). The y-axis represents the percentage of cystine stones, and the x-axis represents the time under treatment in months. (b) Effect on stone growth rate. The stone growth rate was calculated using a linear regression model for each mouse with two or more data points for stones. The y-axis represents the stone growth rate (mg / day). Bars represent untreated mice (Control, n = 7 (7 individuals)) and L-Erg-treated mice (ERG, n = 3). The results of the statistical analysis are shown at the top. (c) Effect on urine pH. Urinary pH was monitored at the end of the treatment period for L-Erg treated mice (ERG) and untreated mice (Control). (d) Effect on urinary redox potential. Urinary ORP was monitored at the end of the treatment period for L-Erg treated mice (ERG) and untreated mice (Control). The results of the statistical analysis are shown at the top. In (c) and (d), the large black dots represent the mean, and the small black dots above and below the boxes represent specific values. Fig. 9 (in relation to Example 2). L-Erg increases the intracellular concentrations of components of the transulfuration pathway in kidney cells. This figure shows the intracellular content of these metabolites of the transulfuration pathway in the kidneys of long-term treated and untreated mice. The y-axis represents nmol / mg protein. (a) glutathione (GSH); (b) oxidized glutathione (GssG); (c) GSH / GssG ratio; (d) cysteine ​​(Cys); (e) cystine (CssC); (f) Cys / CssC ratio; (g) S-adenosylhomocysteine ​​(SAM); (h) S-adenosylmethionine (SAM); (i) SAM / SAH ratio; (j) methionine (Met); (k) gamma-glutamylcysteine ​​(gammaGluCys); And (l) is cystathionine. Each point represents one individual mouse, and bars represent mean ± SEM. In all panels; Wilcoxon signed-rank test * p<0.05, **, P≤0.01, ***, P≤0.001, ****, P≤0.0001. Fig. 10 (in relation to Example 3). Two different mouse models of cystinuria on a C57BL6 / J genetic background ( S / c7a9 - / - or S / c3a1 D140G Urinary ORP (mV) in cystinuria mice from ) . Type I cystinuria mouse model 129S2 / SvPasCrl( S / c3a1 E383K Preliminary data from ). Wilcoxon signed-rank test * p<0.05, **, P≤0.01. Specific details for implementing the invention

[0035] All terms as used herein as in this specification shall be understood in their general sense as known in the art, unless otherwise stated. Other more specific definitions for specific terms as used herein are set forth below and are intended to apply consistently throughout this Detailed Description and Claims, unless otherwise explicitly defined provides a broader definition.

[0036] As used herein, the indefinite articles "a" and "an" are synonyms for "at least one" or "one or more." Unless otherwise indicated, definite articles used herein, such as "the," also include the plural of nouns.

[0037] As used herein, "diagnosis" is understood as recognizing the complications or risks of a subject's specific medical condition; determining the attributes of a disease or condition; or distinguishing one disease or condition from another. This implies both the process of attempting to determine or identify a possible disease or disorder and the opinion reached by this process. In the sense of a diagnostic procedure, a diagnosis can be considered an attempt to classify an individual's condition into distinct and clear categories from which medical decisions regarding treatment and prognosis can be made. Thus, a diagnostic opinion is often described in terms of a disease or other condition. However, a diagnosis can take various forms. It may be a matter of detecting and naming the presence of a disease, lesion, dysfunction, or disability. It may be an event that contributes to a category for management or prognosis. It may indicate the degree of anomaly in a continuum or the type of anomaly in classification.

[0038] As used herein, "prognosis" means the prediction of the probable progression and outcome of a disease. In this case, prognosis means, in a specific embodiment, a distinction between patients who may develop calculosis and those who may not.

[0039] In the present invention, the term "reference value" as referred to in the methods of the present invention should be understood as a predetermined value of S-met-L-erg derived from the amount of the molecular marker in a single sample or group of samples. The sample is taken from a subject or group of subjects in which the presence, absence, stage, histological subtype or grade, or process of the disease has previously been appropriately performed. This value is used as a threshold value to distinguish a subject in which the condition to be analyzed is present from a subject in which such condition is absent. This reference value is also useful for determining whether a subject should initiate medical therapy and how effective the therapy is. The subjects or subjects from which the reference value is derived may include subjects / s in which the condition is absent, subjects / s in which the condition is present, or both. A person skilled in the art may, using general knowledge, select a subject or group of subjects that is more suitable for obtaining the reference value for each of the methods of the present invention. Methods for obtaining reference values ​​from a selected group of subjects are well known in the art (Burtis CA et al., 2008, Chapter 14, section "Statistical Treatment of Reference Values"). In certain cases, the "reference value" is a cut-off value defined by means of conventional ROC analysis (recipient operational characteristics analysis). As a person skilled in the art may recognize, the optimal cut-off value may be defined according to the specific application of the following diagnosis or prognosis: the purpose, the target population for the diagnosis or prognosis, the balance between specificity and susceptibility, etc. As used herein, the term "reference value" may be an absolute value; a relative value; a value having an upper or lower limit; a value within a certain range; an average value; a median, an average value, or a value compared to a specific control or baseline value.

[0040] A biomarker (any one of L-Erg or S-met-L-erg in the present invention) is considered to be higher than the reference value of the biomarker when the biomarker is at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%: at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or higher than the reference value. Similarly, in the context of the present invention, if the level of the biomarker in the sample is lower than a reference value, the level of the biomarker is reduced. The level of the biomarker is considered to be lower than the reference value if the biomarker is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%; at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or higher than the reference value.

[0041] As used herein, “treatment” means to fully or partially relieve or restore a patient’s health. “Therapeutic intervention” means administering a therapeutic agent suitable for a specific disease. In a specific embodiment, the therapeutic intervention includes the administration of ergothioneine.

[0042] The term "cystine-solubilizing agent" refers to a compound capable of increasing the solubility of cystine in the urinary tract. In particular, these compounds are thiol-based compounds, such as penicillamine, that break the disulfide bonds of cystine and form mixed cysteine ​​disulfide compounds. "Any cystine-solubilizing agent" means including any cystine-solubilizing agent other than ergothioneine.

[0043] The expression “pharmaceutical composition” encompasses both compositions intended for humans and compositions intended for non-human animals (i.e., veterinary compositions). In particular, ergothioneine is also useful for the treatment of livestock, such as cats and dogs, which are known to develop cystine stones. The pharmaceutical composition of the present invention comprises a therapeutically effective amount of ergothioneine. The expression “therapeutically effective amount” as used herein means an amount of compound sufficient to prevent or alleviate to some extent the development of one or more syndromes of the disease or disorder being resolved upon administration. The specific dose of the agent administered according to the present invention may be determined by the specific circumstances surrounding the case, including the route of administration, the specific condition being treated, and similar considerations.

[0044] The expression "pharmaceuticalally acceptable carriers or excipients" means pharmaceutically acceptable substances, compositions, or vehicles. Each component must be pharmaceutically acceptable in the sense that it is compatible with other components of the pharmaceutical composition. It must also be suitable for use in contact with tissues or organs of humans and non-human animals without causing excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or sequelae corresponding to a reasonable benefit-risk ratio.

[0045] Examples of suitable pharmaceutically acceptable excipients include solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. The use of excipient media is considered to be within the scope of the present invention, except where any conventional excipient media is incompatible with the substance or its derivatives, such as by producing undesirable biological effects or otherwise interacting in a harmful manner with any other component(s) of the pharmaceutical composition.

[0046] The relative amount of ergothioneine in the pharmaceutical composition of the present invention, pharmaceutically acceptable excipients and / or additional components may vary depending on the identity, size and / or condition of the subject being treated and additionally depending on the route by which the composition is administered.

[0047] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inactive diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Colorants, coating agents, sweeteners, and flavoring agents may be present in the composition at the discretion of the formulationr.

[0048] A pharmaceutical composition containing ergothioneine may be provided in any dosage form, e.g., solid or liquid, and may be administered by any appropriate route, e.g., oral, parenteral, topical, intranasal, or sublingual, and for this purpose, the pharmaceutical composition may include pharmaceutically acceptable excipients necessary for formulation in the desired dosage form.

[0049] It is obvious to a person skilled in the art that the composition can be manufactured using state-of-the-art excipients and applying conventional pharmaceutical techniques.

[0050] The dosage form may be a tablet or coated tablet containing ergothioneine, powder, fine granules, granules, capsules, e.g., hard gelatin capsules or soft gelatin capsules, troches (pastilles), bolus, and chewable preparations.

[0051] Alternatively, the pharmaceutical composition may be in the form of semi-solid or liquid dosages such as gels, e.g., hydrogels, creams, ointments, lotions, water-in-oil emulsions or oil-in-water emulsions, suspensions, aerosols, and liquid formulations such as syrups, including solutions, elixirs, and dry syrups.

[0052] The ergothioneine-containing pharmaceutical composition of the present invention may be administered to a patient as a daily dose in amounts once a day or several times a day, when the pharmaceutical composition is in the form of an orally administered solid preparation such as a tablet or a liquid preparation administered orally or nasally.

[0053] In formulations of compositions containing ergothioneine, various currently used additives such as fillers, thickeners, gelling agents, binders, disintegrants, surfactants, lubricants, coating agents, sustained-release agents, diluents, and / or one or more excipients may be employed. In addition to the foregoing, the formulation of the present invention may further include, if necessary, other additives such as solubilizers, buffers, preservatives, isotonic agents, emulsifiers, suspending agents, dispersants, curing agents, absorbents, adhesives, elasticizing agents, adsorbents, perfumes, coloring agents, correctors, antioxidants, humectants, light-screening agents, gloss agents, viscosity enhancers, oils, tableting adjuvants, and / or antistatic agents.

[0054] More specifically, examples of these additives include lactose, corn starch, mannitol, D-sorbitol, crystalline cellulose, erythritol, and sucrose; binders such as hydroxypropyl cellulose (HPC-L), hydroxypropyl methylcellulose, polyvinylpyrrolidone, methylcellulose, and gelatinized starch; disintegrants such as calcium carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose, and cross-linked polyvinylpyrrolidone (crospovidon); and lubricants such as magnesium stearate and talc; One or more excipients are included, such as fragrances, e.g., apple essence, honey flavor, l-menthol (1-menthol), vanillin, lemon oil, cinnamon oil, mentha oil, or peppermint oil; and / or adsorbents such as synthetic aluminum silicate and light anhydrous silicic acid. Furthermore, it is also possible to manufacture coated pharmaceutical formulations through the use of currently used coating agents such as hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose, or polyvinylpyrrolidone.

[0055] If necessary, sweeteners may be used, as in lozenges, syrups, and chewable formulations, among others. Specific examples of such sweeteners include mannitol, glucose, maltose, starch syrup, malt extract, maltitol, sorbitol, sucrose, unrefined sugar, fructose, lactose, honey, xylitol, hydrangea tea, saccharin, aspartame, cyclamate, Sunett®, aspartyl phenylalanine ester and other malto-oligosaccharides and oligosaccharides such as maltosyl sucrose, reduced isomaltylose and raffinose, any type of sugar alcohol such as acesulfame potassium or sorbitol, mannitol and / or xylitol, or mixtures thereof.

[0056] As a solubilizing agent, any known solubilizing agent suitable in the medical field, e.g., polyethylene glycol, polyoxyethylene-polyoxypropylene copolymer (e.g., poloxamer 188), glycofurol, arginine, lysine, castor oil, propylene glycol, solketal, polysorbate, glycerol, polyvinylpyrrolidone, lecithin, clesterol, 12-hydroxystearic acid-PEG660-ester, propylene glycol monostearate, polyoxy-40-hydrogenated castor oil, polyoxyl-10-oleyl-ether, Polyoxyl-20-ceto-stearylether and polyoxyl-40-stearate or a mixture thereof may be used.

[0057] Any preservative known for use in the pharmaceutical field, e.g., ethanol, benzoic acid and sodium or potassium salts of benzoic acid, sorbic acid and sodium or potassium salts of sorbic acid, chlorobutanol, benzyl alcohol, phenylethanol, methyl-para-hydroxybenzoate, ethyl-para-hydroxybenzoate, propyl-para-hydroxybenzoate or butyl-para-hydroxybenzoates (butyl-p-hydroxybenzoates), phenol, meta-cresol (m-cresol), para-chloro-meta-cresol (p-chloro-m-cresol), selected from the group of PHB (polyhydroxybutyrate) esters, e.g., a mixture of PHB-methyl ester and PHB-propyl ester, quaternary ammonium compounds such as benzalkonium chloride, Phenyl-mercury salts such as thiomersal, phenyl-mercury nitrate, and phenyl-mercury borate can be used.

[0058] A buffer system used to achieve a predetermined pH value may be, for example, glycine, a mixture of glycine and hydrochloric acid (HCl), a mixture of glycine and a sodium hydroxide solution and a sodium glycine salt and a potassium glycine salt, a mixture of potassium hydrogen phthalate and hydrochloric acid, a mixture of potassium hydrogen phthalate and a sodium hydroxide solution, or a mixture of glutamic acid and a glutamate.

[0059] For example, suitable gelling agents are cellulose and derivatives such as methyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose, poly(vinyl) alcohol, polyvinylpyrrolidone, polyacrylate, poloxamer, tragacanth, carrageenan, starch and its derivatives, or any other gelling agent used in pharmaceutical technology.

[0060] Thickening agents that may be mentioned are, for example, the previously mentioned small amount of gelling agent, glycerol, propylene glycol, sorbitol, and other sugar alcohols, polyethylene glycol or polyol.

[0061] The emulsifying agent used may include a polyoxyethylene derivative of castor oil or a polyoxyethylene alkyl ether, excluding emulsifying agents known in the prior art.

[0062] Suitable synthetic or natural coloring agents known in the pharmaceutical field, such as indigo carmine, may be used.

[0063] Suitable oil components that may be present are, for example, vegetable oils, in particular, for example, cottonseed oil, groundnut oil, peanut oil, maize oil, rapeseed oil, sesame oil and soybean oil, or any oil material among oil materials known in the prior art for the manufacture of medicines, such as medium-chain length triglycerides, for example, refined coconut oil or fractionated coconut oil, or isopropyl myristate, isopropyl palmitate, or mineral oils or ethyl oleate.

[0064] The antioxidant used may be any antioxidant known in the prior art, for example, alpha-tocopherol (α-tocopherol), butylhydroxytoluene (BHT), or butylhydroxyanisole (BHA).

[0065] Pharmaceutical compositions containing these additives may be prepared according to any method known in the pharmaceutical field, depending on the dosage form. It is obvious that additional additives not explicitly discussed may be used in the formulations used according to the present invention.

[0066] As described above, the inventors have for the first time discovered that S-methyl-L-erg is associated with medical diseases or conditions. Accordingly, the present invention provides S-methyl-L-ergothioneine for use in diagnosis and / or prognosis.

[0067] S-methyl-L-ergothioneine is a methyl derivative of the known ergothioneine. Ergothioneine has the IUPAC name [1-carboxy-2-(2-sulfanylidene-1,3-dihydroimidazol-4-yl)ethyl]-trimethylazanium, CAS number 497-30-3, and chemical formula (I):

[0068]

[0069] S-methyl-L-ergothioneine has the IUPAC name (2-{S})-3-(2-methylsulfanyl-1-{H}imidazol-5-yl)-2-(trimethylazaniumyl)propanoate ((2-{S})-3-(2-methylsulfanyl-1-{H}imidazol-5-yl)-2-(trimethylazaniumyl)propanoate) and chemical formula (II):

[0070]

[0071] In a specific embodiment, optionally in combination with any one of the embodiments provided above or below, S-methyl-L-ergothioneine is intended for use in the diagnosis and / or prognosis of renal disease. In a more specific embodiment, the renal disease is renal calculosis or aminoaciduria. In another specific embodiment, the renal calculosis is cystine calculosis. In a more specific embodiment, the aminoaciduria is cystinuria.

[0072] As mentioned above, a second aspect of the present invention provides a method for the diagnosis or prognosis of a kidney disease, comprising the step of determining the amount of S-methyl-L-ergothioneine in an isolated test sample of a subject.

[0073] In a specific embodiment of the method described above, optionally in combination with any one of the embodiments provided above or below, the method further comprises the step of comparing an amount of S-methyl-L-ergothioneine in a subject with a reference value, wherein if the amount determined in the subject is within the range of reference values ​​for a subject suffering from kidney disease, it indicates that the subject is suspected of suffering from kidney disease; and wherein if the amount is within the range of values ​​for a healthy subject not suffering from any kidney disease, in particular not suffering from renal calculi or aminoaciduria, it indicates that the subject is not suffering from said kidney disease.

[0074] In a specific embodiment of the method described above, optionally in combination with any one of the embodiments provided above or below, the method further comprises the step of comparing the amount of S-methyl-L-ergothioneine in a subject to a reference value, wherein if the amount determined in the subject differs from the reference value of a healthy subject or a subject not suffering from any renal disease, this is indicative. In a specific embodiment, if the amount determined in the subject is below the reference value of a subject not suffering from renal disease, particularly not suffering from renal calculi or aminoaciduria, it indicates that the subject is suspected of suffering from renal disease. In another more specific embodiment, if the amount determined in the subject is below the reference value, it indicates a poor prognosis.

[0075] As shown in the examples below, the inventors found that the ratio between the amounts of S-methyl-L-ergothioneine and L-ergothioneine in urine samples provides better statistical results in distinguishing between calculous mice and non-calculous mice (Fig. 5b). In fact, the S-methyl-L-ergothioneine / L-ergothioneine ratio was able to classify mice with 100% specificity and sensitivity using two different analytical methodologies.

[0076] Accordingly, in a specific embodiment of the method described above, optionally in combination with any one of the embodiments provided above or below, the method further comprises the step of determining the amount of ergothioneine in an isolated test sample of the subject. Even in a more specific embodiment, the method further comprises the step of determining the amount of ergothioneine in an isolated test sample of the subject and calculating the ratio between the amounts of S-methyl-L-ergothioneine and L-ergothioneine.

[0077] In a specific embodiment of the method described above, optionally in combination with any one of the embodiments provided above or below, the method further comprises the step of determining the amount of L-ergothioneine in an isolated test sample of a subject, calculating the ratio between the amount of S-methyl-L-ergothioneine and the amount of L-ergothioneine, and comparing the ratio to a reference value, wherein if the ratio determined in the subject is within the range of reference values ​​(reference value) for a subject suffering from kidney disease, it indicates that the subject is suspected of suffering from said kidney disease; and wherein if the ratio is within the range of values ​​for a healthy subject or a subject not suffering from any kidney disease, in particular not suffering from renal calculi or aminoaciduria, it indicates that the subject is not suspected of suffering from said kidney disease.

[0078] In a specific embodiment of the method described above, optionally in combination with any one of the embodiments provided above or below, the method further comprises the steps of determining the amount of L-ergothioneine in an isolated test sample of a subject, calculating the ratio between the amounts of S-methyl-L-ergothioneine and L-ergothioneine, and comparing the ratio to a reference value, wherein if the ratio determined in the subject differs from the reference value of a healthy subject or a subject not suffering from any kidney disease, particularly a subject not suffering from renal calculi or aminoaciduria, it indicates that the subject is suspected of suffering from kidney disease. In a more specific embodiment, if the ratio determined in the subject is less than the reference value, it indicates that the subject is suspected of suffering from kidney disease.

[0079] The levels of S-methyl-L-ergothioneine and L-ergothioneine can be determined according to routine techniques in the diagnostic field. A person skilled in the art can adjust the parameters of the technique for optimal results. Accordingly, in a specific embodiment of the method described above, the amount of S-methyl-L-ergothioneine and / or L-ergothioneine is determined by mass spectrometry, high-performance liquid chromatography (HPLC), derivatization, chemodetection, and combinations thereof.

[0080] As previously mentioned, a further aspect of the present invention provides a means for determining the amount of S-methyl-L-ergothioneine in a method as defined above.

[0081] In a specific embodiment, optionally in combination with any one of the embodiments provided above or below, the means forms part of the kit.

[0082] The term “kit” as used herein refers to a product comprising different reagents (or reagent means) necessary for carrying out the method of the present invention, packaged for transport and storage. Suitable materials for packaging the components of the kit include crystal, plastic (e.g., polyethylene, polypropylene, polycarbonate), bottles, vials, paper, or envelopes.

[0083] In a specific embodiment of the present invention, means for determining the amount of S-methyl-L-ergothioneine comprises isotope-labeled S-methyl-L-ergothioneine and / or L-ergothioneine, in particular S-methyl-L-ergothioneine and / or L-ergothioneine containing deuterium, and the method is carried out by adding S-methyl-L-ergothioneine and / or labeled L-ergothioneine labeled as a spike-in compound or mixture to a test sample. In a more specific embodiment, the means comprises a mixture of isotope-labeled S-methyl-L-ergothioneine and isotope-labeled L-ergothioneine.

[0084] Where isotope-labeled S-methyl-L-ergothioneine and / or L-ergothioneine is used, additional means comprising isotope-unlabeled S-methyl-L-ergothioneine and / or L-ergothioneine used as a positive control means in the present method are included.

[0085] Accordingly, a specific kit for carrying out the method of the present invention comprises or consists of: a first vial containing a composition containing isotope-labeled S-methyl-L-ergothioneine and / or L-ergothioneine; and a second vial containing a composition containing S-methyl-L-ergothioneine and / or L-ergothioneine. In these kits, the first vial includes means for spiking an isolated sample tested with a standard curve of a Quality Control (QC) test. The second vial includes means for preparing a standard curve. Additionally, the kit of the present invention may include instructions for the simultaneous, sequential, or independent use of different means for detecting the amount of S-methyl-L-ergothioneine and / or L-ergothioneine present in the kit. The above instructions may exist in the form of printed materials or in the form of electronic supports capable of storing readable or understandable instructions, such as, for example, electronic storage media (e.g., magnetic discs, tapes) or optical media (e.g., CD-ROMs, DVDs) or audio materials. Additionally, or alternatively, the medium may include an internet address providing the above instructions.

[0086] As mentioned above, in additional embodiments, the present invention provides the use of S-methyl-L-ergothioneine as a marker for the diagnosis and / or prognosis of renal disease in isolated test samples of a subject. In a specific embodiment, S-methyl-L-ergothioneine is used together with L-ergothioneine as a marker for the diagnosis and / or prognosis of renal disease in isolated test samples of a subject. In a more specific embodiment, the ratio between the amounts of S-methyl-L-ergothioneine and L-ergothioneine is used as a marker for the diagnosis and / or prognosis of renal disease in isolated test samples of a subject.

[0087] In a specific embodiment of the methods and uses defined above, optionally in combination with any one of the embodiments provided above or below, the isolated test sample is selected from serum, plasma, saliva, pleural fluid, cerebrospinal fluid (CSF), blood, amniotic fluid, urine, feces, mucus, cell extracts, and pus. In a more specific embodiment, optionally in combination with any one of the embodiments provided above or below, the isolated test sample is a urine sample.

[0088] In specific embodiments of the methods and uses defined above, optionally in combination with any one of the embodiments provided above or below, the subject suffers from aminouria. In a more specific embodiment, aminouria is cystinuria. As shown in the examples below, the method of the present invention is particularly useful for the diagnosis of cystine stone disease in subjects suffering from cystinuria.

[0089] It is known that several mammalian species develop cystine stones in the kidneys as a result of a condition causing an unusual accumulation of cystine in the urine. The method of the present invention may be applied to all of these mammalian species. Accordingly, in other specific embodiments of the method and use defined above, optionally in combination with any one of the embodiments provided above or below, the subject is a mammal. In a more specific embodiment, the mammal is a domestic mammal. In another specific embodiment, optionally in combination with any one of the embodiments provided above or below, the mammal is a human.

[0090] S-methyl-L-ergothioneine for the use as described above, in a specific embodiment of the methods and uses defined above, optionally in combination with any one of the embodiments provided above or below, the renal disease is renal calculosis or aminoaciduria. In a more specific embodiment, the renal calculosis is cystine calculosis. In another specific embodiment, the aminoaciduria is cystinuria.

[0091] Accordingly, in a specific embodiment, optionally in combination with any one of the embodiments provided above or below, the present method is for the diagnosis of cystine stone disease. More specifically, the present method is for the diagnosis of cystine stone disease in patients suffering from cystinuria. In another specific embodiment, the present method is for the prognosis of cystinuria. The method of the present invention may be useful for detecting the presence of cystine stones and also for predicting the appearance of cystine stones in subjects suffering from cystinuria.

[0092] As mentioned above, it is particularly important to diagnose urolithiasis as soon as possible to provide appropriate treatment and avoid complications caused by large kidney stones (i.e., obstruction, infection, and renal failure). The method of the present invention enables such timely diagnosis.

[0093] All of the methods and uses provided herein can be carried out by determining the amount of S-methyl-L-ergothioneine or the amounts of S-methyl-L-ergothioneine and L-ergothioneine in an isolated test sample and calculating the S-methyl-L-ergothioneine / L-ergothioneine ratio.

[0094] Accordingly, as mentioned above, in additional embodiments, the present invention provides an S-methyl-L-ergothioneine / L-ergothioneine ratio for use in diagnosis and / or prognosis. It means that all embodiments provided above also apply to these additional embodiments.

[0095] The inventors also recognized that the urinary redox potential or redox status (ORP) in a mouse model of cystinuria that does not form cystine stones was lower than the urinary ORP in mice that form stones. Therefore, this parameter has also been suggested for in vitro differential diagnosis or prognosis in animals with cystinuria, including humans.

[0096] Redox potential (also known as oxidation / reduction potential, ORP) is a measure of the tendency of a chemical species (i.e., isolated sample; urine) to gain electrons from an electrode or lose electrons to the electrode, thereby being reduced or oxidized, respectively. Redox potential is measured in volts (V) or millivolts (mV). Each chemical species has its own inherent redox potential; for example, the more positive the reduction potential (reduction potential is more frequently used due to general formalism in electrochemistry), the greater the affinity for electrons of the chemical species and the greater the tendency to be reduced.

[0097] In a more specific embodiment of any of the methods for the diagnosis or prognosis of the renal diseases described above, particularly renal calculi or aminoaciduria, more particularly cystine calculi and cystinuria provided together herein, the present method further comprises the following:

[0098] (a) a step of determining the redox potential (ORP) in an isolated sample of the subject, particularly a urine sample;

[0099] (b) A step of comparing the ORP of (a) with a reference value; and

[0100] (c) (c1) A step of diagnosing the subject as a non-stone-forming subject when the ORP is within the reference value range of the non-stone-forming subject, or alternatively (c2) when the ORP is lower than the reference value obtained from the subject previously classified as a stone-forming subject.

[0101] The term "non-forming stones subject" means that the subject has a low or no tendency to develop into stones in the urine due to environmental conditions in the urine, or due to any other more complex causes (genetic background, diet, etc.).

[0102] Therefore, ORP measurements can provide additional reliable information regarding the diagnosis or prognosis of the disease, including the detection of subjects with a higher tendency to form stones, in addition to the amounts of S-methyl-L-ergothioneine or S-methyl-L-ergothioneine and L-ergothioneine. Thus, this information is useful for recommending or deciding on the initiation of appropriate therapeutic interventions.

[0103] The present invention also relates to the use of ORP as a single diagnostic or prognostic marker for renal disease, more particularly for the diagnosis or prognosis of renal calculosis or aminouria, and even more particularly as a marker for cystine calculosis or cystinuria. Accordingly, the present invention comprises an in vitro method for the diagnosis or prognosis of renal disease comprising determining the redox potential in isolated samples of a subject, particularly in urine. In a more specific embodiment of the method, the ORP of the isolated sample is subsequently compared with a reference value to classify the subject as a non-calculous subject or a calculous subject as described above.

[0104] The in vitro methods of the present invention provide diagnostic and / or prognostic information. In one embodiment, the methods of the present invention further comprise the steps of (i) collecting diagnostic and / or prognostic information and (ii) storing the information in a data carrier.

[0105] In the context of the present invention, the term "data carrier" should be understood as any means containing meaningful information data regarding the differential diagnosis and / or prognosis of renal calculosis and aminouria, such as paper. The carrier may also be any entity or device capable of carrying prognostic data. For example, the carrier may include a storage medium such as a ROM, e.g., a CD-ROM or a semiconductor ROM, or a magnetic recording medium such as a floppy disk or a hard disk. Furthermore, the carrier may be a transmittable carrier, such as an electrical signal or an optical signal, which may be transmitted via an electrical cable or an optical cable, or by wireless or other means. If the diagnostic / prognostic data is implemented as a signal that can be transmitted directly by a cable or other device or means, the carrier may be composed of such cable or other device or means. Other carriers include USB devices and computer archives. Examples of suitable data carriers are paper, CD, USB, computer archives on a PC, or sound registrations containing the same information.

[0106] The present invention also provides a method for treating a patient suffering from renal calculosis or aminoaciduria, particularly cystinuria, comprising the following steps:

[0107] (a) determining the amount of S-methyl-L-ergothioneine and optionally the amount of L-ergothioneine in the isolated test sample of the subject, and calculating the ratio between the amounts of S-methyl-L-ergothioneine and L-ergothioneine;

[0108] (b) a step of comparing the amount or ratio determined in (a) with a reference value; and

[0109] (c1) where the level determined in (a) is below the reference value for subjects not suffering from renal calculus or aminoaciduria, or alternatively (c2) where the level determined in (a) is within the reference value range for subjects suffering from renal calculus or aminoaciduria, a step of administering a pharmaceutically effective amount of cystine-solubilizing agent to subjects in need.

[0110] The present invention also provides a method for treating a patient suffering from renal calculus or aminoaciduria, comprising the following steps:

[0111] (a) determining the amount of S-methyl-L-ergothioneine and optionally the amount of L-ergothioneine in the isolated test sample of the subject, and calculating the ratio between the amounts of S-methyl-L-ergothioneine and L-ergothioneine;

[0112] (b) a step of comparing the amount or ratio determined in (a) with a reference value; and

[0113] (c1) where the level determined in (a) is below the reference value for subjects not suffering from renal calculus or aminoaciduria, or alternatively (c2) where the level determined in (a) is within the reference value range for subjects suffering from renal calculus or aminoaciduria, a step of administering a pharmaceutically effective amount of ergothioneine to subjects in need.

[0114] As previously mentioned, the inventors have also developed an effective and safe treatment for aminoaciduria and associated renal stones.

[0115] In a specific embodiment of ergothioneine for the use described above, optionally in combination with any one of the embodiments provided above or below, the kidney stone is a cystine stone.

[0116] In other specific embodiments of ergothioneine for the use described above, optionally in combination with any one of the embodiments provided above or below, aminoaciduria is cystinuria.

[0117] In a specific embodiment of ergothioneine for the use described above, ergothioneine is administered in the form of a pharmaceutical composition with one or more pharmaceutically acceptable excipients and / or carriers, optionally in combination with any one of the embodiments provided above or below. In a more specific embodiment, the pharmaceutical composition is an oral pharmaceutical composition.

[0118] In a specific embodiment, optionally in combination with any one of the embodiments provided above or below, ergothioneine is administered at a dose of 0.01 to 500 mg / kg body weight per day, in a more specific embodiment, at a dose of 0.05 to 300 mg / kg body weight per day, and even in a more specific embodiment, at a dose of 0.1 to 200 mg / kg body weight per day.

[0119] As previously mentioned, the present invention also provides ergothioneine for use in combination therapy with a compound selected from the group consisting of additional cystine-solubilizing agents, L-cystine dimethyl ester, L-cystine methyl ester, L-cystine diamide, lipoic acid, and combinations thereof in the treatment and / or prevention of renal calculosis or aminoaciduria.

[0120] All embodiments of ergothioneine for the uses defined above also apply to ergothioneine for use in combination therapy.

[0121] In a specific embodiment of ergothioneine for use in the combination therapy defined above, an additional cystine solubilizer is optionally selected from the group comprising penicillamine, thiopronin, captopril, and bucillamine in combination with any one of the embodiments provided above or below. In a more specific embodiment, the cystine solubilizer is selected from penicillamine and thiopronin.

[0122] In a specific embodiment of ergothioneine for use in a combination therapy as defined above, ergothioneine is administered simultaneously, sequentially, or independently with a compound selected from the group consisting of additional cystine-solubilizers, L-cystine dimethyl ester, L-cystine methyl ester, L-cystine diamide, lipoic acid, and combinations thereof.

[0123] In a specific embodiment of ergothioneine for the use defined above, the ergothioneine is L-ergothioneine, optionally in combination with any one of the embodiments provided above or below.

[0124] Accordingly, in a specific embodiment, optionally in combination with any one of the embodiments provided above or below, the present invention provides L-ergothioneine for use in the treatment and / or prevention of renal calculi or aminoaciduria. More particularly, L-ergothioneine is intended for use in the treatment and / or prevention of cystine calculi or cystinuria.

[0125] Throughout the detailed description and claims, the word “comprise” and variations thereof are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” includes cases of “consisting of.” Further objects, advantages, and features of the invention may become apparent to those skilled in the art by reviewing the detailed description or by learning from the practice of the invention. The following embodiments and drawings are provided for illustrative purposes only and are not intended to limit the invention. References placed in parentheses in connection with the drawings and in the claims are solely intended to increase the intelligibility of the claims and are not intended to limit the scope of the claims. Furthermore, the invention includes all possible combinations of the specific and preferred embodiments described herein.

[0126] Examples

[0127] Example 1: Urinary markers of cystine stones in a mouse model of cystinuria

[0128] method

[0129] Mouse management

[0130] All animal protocols were approved by the Animal Experimentation Ethics Committee of IDIBELL (AAALAC accredited facility, 89900010) and by the relevant department of the Generalitat de Catalunya in accordance with EU Directive 2010 / 63 / EU. Experiments were conducted in accordance with the highest scientific, human, and ethical principles. All animals were of pure genetic background C57BU6J and were maintained in humidity and temperature-controlled rooms on a 12-hour light-dark cycle. The animals were housed in sterile cages with free access to food (Teklad Global 14% protein diet, Harlan Laboratories) and water.

[0131] Type B cystinuria mouse model ( S / c7a9 - / - Knockout S / c22a4 in )

[0132] Single-function-loss mouse model S / c7a9 - / - (mouse model for cystinuria) and S / c22a4 - / - (Feliubadalo et al., "S / c7a9-deficient mice develop cystinuria non-I and cystine urolithiasis" Hum Mol Genet 2003; vol 12; pp. 2097-2108; Kato Y. et al., "Gene knockout and metabolome analysis of carnitine / organic cation transporter OCTN1", Pharm. Res., Double heterozygous mice were obtained by crossing (2010; vol 27, pp. 832-40), and double KOs were obtained by backcrossing them. S / c7a9 - / - S / c22a4 - / - Three predicted genotypes were obtained, including (dKO).

[0133] For genotyping analysis, genomic DNA was isolated from tail tissue. Based on a 3'-primer strategy capable of distinguishing genotypes (F: 5'-gggtgtggtccagaggact-3', sequence identification number: 1; R wild-type (wt)-specific: 5'tagttgccagccatctgttg-3', sequence identification number: 2; R KO-specific: 5'-gactgacataccattgaagc-3', sequence identification number: 3) by generating 255 bp and 313 bp fragments from wild-type and KO alleles, respectively S / c22a4 - / - The genotype was confirmed by PCR (polymerase chain reaction; 30 cycles at an annealing temperature of 60°C). S / c7a9 - / - For this, 452 bp and 311 bp fragments were generated from the wild type and KO alleles, respectively, and the genotype was confirmed by PCR (30 cycles at a 60°C softening temperature) based on a 3'-primer strategy (F: 5'-gcattcgccacaggctcttc-3', sequence identification number: 4; R-wild type: 5'-ctgtgttggccagcacagac-3', sequence identification number: 5; R-KO-specific: 5'-cgcagcgcatcgccttctat-3', sequence identification number: 6) capable of distinguishing genotypes.

[0134] Sample Collection

[0135] Mice from each genotype were individually housed in metabolic cages for 4 days starting from day 1 as an acclimatization period. Mouse body weight, water and food intake, and secreted feces and urine were monitored daily. 24-hour urine samples were collected and stored at -80°C with 50 µl of 10% thymol in isopropanol as a preservative until further analysis. Blood was obtained by intracardiac perforation using an EDTA-coated syringe, transferred to a Microvette EDTA-tube (Sarstedt), incubated at room temperature for 10 minutes, and then centrifuged in a minifuge at 3000 rpm for 10 minutes at 4°C. Subsequently, plasma was separated into a new tube and stored on ice. Plasma absorbance at 414 nm was determined using a NanoDrop spectrophotometer to quantify hemolysis, and only those with an OD < 0.2 were considered for further analysis. Plasma samples were stored at -80°C and centrifuged to collect red blood cells (RBCs), which were also stored at -80°C.

[0136] Determination of L-Erg and S-met-L-Erg in plasma, blood, and red blood cells

[0137] L-Erg in plasma and red blood cells is Sotgia S. et al., ("Plasma L-ergothioneine measurement by high-performance liquid chromatography and capillary electrophoresis after a pre-column derivatization with 5-iodoacetamidofluorescein (5-IAF) and fluorescence detection". Antopolsky M, ed. PLoS OneWhile plasma creatinine is measured as described by Zinellu A. et al., ("Assay for the simultaneous determination of guanidinoacetic acid, creatinine and creatine in plasma and urine by capillary electrophoresis UV-detection", 2013; vol. 8: e70374), J. Sep. Sci., It was measured as described by 2006.

[0138] For the measurement of plasma ergothioneine, 100 µl of acetonitrile was added to 100 µl of the sample, and after vigorous vortex mixing, the mixture was centrifuged at 17,000xg for 10 minutes at room temperature. To 150 µl of the clear supernatant, 50 µl of a solution consisting of 5-iodoacetamidofluorescein (770 μmol / L) and sodium phosphate tribasic dodecahydrate (150 mmol / L) at pH 13 was added. After vigorous vortex mixing, the reaction mixture was left in a light-protected area at room temperature for 30 minutes. Finally, the sample was diluted 50-fold and analyzed by capillary electrophoresis coupled with a laser-induced fluorescence detector.

[0139] For the measurement of erythrocyte ergothioneine, 100 µL of water was added to 100 µL of erythrocytes and thoroughly mixed by vigorous vortex mixing; subsequently, 400 µL of acetonitrile was added and thoroughly vortex mixed for 5 minutes. After centrifugation at 17,000xg for 10 minutes at room temperature, 2 µL of the clear supernatant was analyzed by ultra-performance liquid chromatography coupled to a photodiode array detector set to 262 nm. For the measurement of plasma creatinine, the sample was filtered through a microcon-10 device at 3,000xg for 5 minutes and subsequently analyzed directly by capillary electrophoresis coupled to a photodiode array detector set to 190 nm.

[0140] Analysis of compound concentrations in urine

[0141] Creatinine concentrations in thawed urine samples were filtered through 10 kDa MWCO spin filters (Amicon Ultra 0.5 ml, Millipore) and then determined using a Creatinine Assay Kit (Sigma) according to the manufacturer's instructions.

[0142] For the analysis of L-Erg and S-met-L-Erg in urine, thawed urine samples were centrifuged at 1000xg for 5 minutes at 4°C to remove any debris. Subsequently, the supernatant was diluted 1 / 10 in Milli-Q water supplemented with L-Erg and S-met-L-Erg containing deuterium (final concentrations of 50 ng / mL each). The samples were then filtered through eXtremeFV PVDF 0.2 µm filter vials (Thomson Instrument Company), and L-Erg and S-met-L-Erg were quantified by UPLC-MS / MS. LC-MS / MS was performed using a Dionex LPG-3400SD LC System coupled to a Thermo L TQ-XL ESI tandem mass spectrometer. The sample was placed in an autosampler at 15°C. 20 µl of the diluted sample and standard were injected into a ZORBAX Eclipse Plus C18 (3.5 µm, 75 x 4.6 mm; Agilent) and maintained at 35°C. Solvent A was 0.05% formic acid in ultrapure water, and solvent B was acetonitrile in 0.05% formic acid. Chromatography was performed under isolytic conditions (99% A : 1% B) at a flow rate of 0.9 ml / min for 3 minutes.

[0143] Mass spectrometry was performed under cation, electron spray ionization mode using multiple reaction monitoring (MRM) for the quantification of specific target ions. The source voltage was set to 3.0 kV, and the capillary temperature was maintained at 375°C. The nitrogen sheath gas flow was 90 AU, the auxiliary gas flow was 10 AU, and the sweep gas flow was 6 AU. Alphagaz 2 helium (air liquid) was used as the collision gas. The precursor-to-product ion changes for each compound are as follows: Ergothioneine: 230.0 → 186.0; d3-ergothioneine: 233.0 → 189.0; S-methyl-ergothioneine: 244.0 → 200.0; d3-S-methyl-ergothioneine: 247.0 → 203.0. In all cases, the isolation width (m / z) and CID collision energies were 2.0 and 20%, respectively.

[0144] result

[0145] Gender and age differences in L-Erg concentrations in blood and urine

[0146] The inventors first [identified] wild type and to find differences that can be explained by differential expression. S / c7a9 - / - The L-Erg content in the blood and urine of male mice was analyzed. L-Erg concentrations in blood, plasma, or red blood cells (RBCs) did not show significant differences between wild-type and cystinuria male mice, and S / c7a9 - / - Concentrations in plasma and blood in male mice were higher than in RBCs (data not shown). Since L-Erg concentrations in RBCs were double-order higher than in plasma, and to explain the estimated effect of hemolysis on the determination of L-Erg in plasma, the contribution of hemolysis to plasma L-Erg concentration was analyzed, and no correlation was found between the two variables (data not shown). Urinary concentration of L-Erg also S / c7a9 - / - Although it showed an insignificant increase in male mice, unexpectedly, the concentration of S-met-L-Erg (a metabolite of L-Erg) S / c7a9 - / - It showed a significant decrease in male mice (Fig. 1). These results indicate that S-met-L-Erg in urine can be used as a biomarker for cystine stone disease.

[0147] To find any age-related differences as reported in humans, the inventors continued to investigate whether the conditions described above persisted at other ages. At 3 months of age, no differences were detected in L-Erg concentrations in blood, plasma, or RBCs between wild-type and cystinuria male mice; however, when comparing 6 months of age versus 3 months of age, it was detected that L-Erg concentrations in RBCs increased significantly by more than twofold for all mice (data not shown). Similarly, L-Erg concentrations in urine were also significantly higher at 6 months of age, and no differences were detected in age-related urinary S-met-L-Erg concentrations (Fig. 2).

[0148] To identify sex-related differences, the inventors continued to investigate the concentrations of L-Erg in blood and urine and S-met-L-Erg in urine as described above. At 3 months of age, no sex-related differences were observed in L-Erg concentrations in blood, plasma, or RBCs, except for a 33% decrease in L-Erg concentration in RBCs (p = 0.093) (data not shown). However, the urinary concentration of S-met-L-Erg was wild-type and S / c7a9 - / - It was significantly reduced by nearly twofold in both female mice. Furthermore, female S / c7a9 - / - A significant decrease in the concentration of these metabolites and a significant increase in L-Erg were detected in mice versus female wild-type mice (data not shown).

[0149] In contrast, sex-associated significant changes at 6 months of age were detected in the blood, plasma, and RBC concentrations of L-Erg in female cystinuria mice. Nearly a twofold decrease in blood and RBCs and a 30% decrease in plasma were observed. A similar situation was observed in the urine of female mice, where the concentration of S-met-L-Erg decreased nearly twofold and L-Erg decreased by 30% (Fig. 3). Furthermore, compared to female wild-type mice, the RBC concentration of L-Erg in female cystinuria mice was 30% lower, and the urine concentration of S-met-L-Erg in female cystinuria mice was significantly 30% lower.

[0150] Differences in urinary L-Erg concentration associated with the presence of cystine stones

[0151] One of the characteristics of cystinuria is the presence of cystine stones in patients and mice with cystinuria. Accordingly, the inventors analyzed the urine concentrations of L-Erg and S-met-L-Erg in stone-forming cystinuria mice and stone-non-stone-forming cystinuria mice at 3 and 6 months of age (Fig. 4). While L-Erg urine concentrations were significantly lower in early stone former (ESF) female mice (Fig. 4a), S-met-L-Erg was higher in ESF females at 3 months of age (Fig. 4a) and higher in ESF males at both ages (Figs. 4a to 4b, P = 0.062 for 3-month-old males). Differences between the two sexes can also be identified for both L-Erg and S-met-L-Erg at both ages, with generally lower concentrations in females, except for L-Erg concentration at 3 months of age (Figs. 4a to 4b). The inventors investigated whether urinary concentrations of S-met-L-Erg could be used to distinguish calculous phenotypes in mice by the recipient operating characteristic (ROC) curve and obtained an area under the curve greater than 0.65, which indicates that S-met-L-Erg can be used as a calculous biomarker.

[0152] Since S-met-L-Erg is a byproduct of L-Erg metabolism, the inventors continued to investigate whether the ratio between S-met-L-Erg and L-Erg exhibited differences associated with the calculous phenotype. As shown in Figure 5a, the ratio in stone-forming (SF) mice was generally significantly 2 to 3 times lower than in stone-non-forming (NSF) mice. Because these results were very clear, the inventors investigated whether the ratio between the urine concentrations of S-met-L-Erg and L-Erg could be used to distinguish the calculous phenotype in mice using the Receiver Operational Characteristic (ROC) curve. As shown in Figure 5b, an area under the curve greater than 0.8 suggests the potential for using this ratio as a calculous biomarker in cystinuria mice. The inventors wondered if there were differences in the performance of the ratio according to age or sex, or if it was very similar or slightly better in all cases.

[0153] The inventors also investigated whether there is any correlation between the size (determined as dry weight) and ratio of cystine stones and observed a weak correlation between the two variables (r = 0.28, p = 0.084).

[0154] L-Erg is OCTN1( S / c22a4 It was found that it can be transported by ) S / c22a4 - / - Because the mice lacked L-Erg in their kidneys, the inventors S / c22a4 - / - mouse S / c7a9 - / - Double KO by crossbreeding with a mouse ( S / c7a9 - / - S / c22a4 - / - We produced ) and observed whether there was a difference in the proportion of mice exhibiting the calculous phenotype.

[0155] S / c7a9 - / - Similar to mice, the percentage of female calculous mice is all S / c22a4 For genotypes, it was significantly higher in males than in females. The percentage of lithic mice was S / c22a4 - / - The percentage of KO mice is 8% higher, suggesting that OTN1 is a genetic modulator of cystine calculosis in mice. This gene is also present in humans (NCBI Gene ID 6583 or UniProt KB database ID Q9H015, version 3 of the sequence dated May 1, 2007), and therefore the same mechanism may also apply herein. A notable result is that the percentage of calculous males is S / c22a4 At 40 weeks of age, showing no difference compared to genotype S / c7a9 - / - S / c22a4 - / - ( S / c22a4 Except for heterozygosity, the percentage of females with calculosis was higher than that of males.

[0156] To better understand these results, the inventors [found] a background of cystinuria at 3 months of age ( S / c7a9 - / - Mice lacking OCTN1 in ) S / c22a4 The amounts of L-Erg and S-met-L-Erg in the urine of ) were analyzed. L-Erg is S / c7a9 - / - Similar levels were detectable in mouse urine in mice and wild-type mice, but the amount of S-met-L-Erg was significantly 50% lower in mice (P = 0.0022). S / c7a9 - / - In the background S / c22a4 If you knock out L-Erg in urine S / c22a4 + / - For , P = 0.0382 and S / c22a4 - / - For this, P = 0.0138) and S-met-L-Erg concentration was significantly reduced, and S-met-L-Erg concentration was below the quantification limit of the method used.

[0157] As expected, S / c22a4 - / - Mice S / c22a4 + / - It showed lower L-Erg concentrations than mice. These data suggest that OCTN1 is required to transport L-Erg into cells, as other transporters may be involved in the absorption of L-Erg from the diet, but metabolites (S-met-L-Erg) could not be detected in the urine.

[0158] Example 2: Treatment of cystine stones in a cystinuria mouse model

[0159] method

[0160] Mouse management was as shown in Example 1.

[0161] L-Erg treatment

[0162] Three different treatments were applied to mice: 1 month, 3 months to gallstone mice (subchronic), and 6 months (long-term exposure). In all cases, L-Erg was administered into drinking water.

[0163] For the 1-month treatment, L-Erg was administered to 8 male and female mice aged 3 months at a dose of 15 or 60 mg / L for 4 weeks in a standard cage with free access to water and food.

[0164] To evaluate the effect of L-Erg on cholelithic mice, 15 mice (8 males and 7 females) were treated with 60 mg / L L-Erg in drinking water, and 13 mice (4 males and 9 females) were left untreated as controls. The mice were 10.9–26.9 weeks old at the start of the treatment period, with mean ages of 15.3±1.4 and 16.9±1.01 weeks for the control and L-Erg treatments, respectively. Water intake and mouse body weight were monitored weekly during the 3-month treatment period.

[0165] For long-term exposure (6-month treatment), 4 to 6 mice per cage were treated after 6 months of weaning. To adjust the L-Erg dosage, water intake and mouse body weight were monitored, and the concentration of L-Erg in the drinking water was adjusted to a daily dose of 16 mg / kg·day every 3 days during the first month and then every week thereafter. During the last week of treatment, all mice were individually housed in metabolic cages to collect urine, where daily water intake and mouse body weight were monitored.

[0166] Detection of cystine stones by X-ray in vivo imaging

[0167] For the detection of calculi, isoflurane-anesthetized mice were X-ray-imagingd at ages indicated in the corresponding figure legends, accompanied by calibration curves for cystine stones of known weight, and subsequently applied to the IVIS Lumina XR Series III (Caliper Lifescience - Vertex Techniques) according to the manufacturer's imaging parameters. Calcium quantification was performed by manually defining the stone area using the Living Image Software provided with the instrument, and the estimated weight was interpolated from the calibration curve.

[0168] For the analysis of growth rates, a linear regression model was used when two or more data points were available. The slope of the regression line in the model was used as the growth rate.

[0169] Sample Collection

[0170] During the last week of the randomized treatment period, mice were housed individually in metabolic cages for 4 days, with the first day serving as the acclimatization period. Mouse body weight, water and food intake, and secreted urine were monitored daily. 24-hour urine samples were collected and stored at -80°C with 50 µl of 10% thymol in isopropanol as a preservative until further analysis. pH was determined using a pH meter (cat. no. 5209, Crison), and redox potential was determined using an ORP electrode (cat. no. 5265, Crison) at room temperature at micropH 2000 (Crison).

[0171] On the last day, mice were anesthetized with isoflurane, blood was removed through cardiac perforation, and the kidneys were harvested, weighed, and stored at -80°C until further use. Cystine stones, if present, were removed, dried, weighed, and stored at room temperature.

[0172] Analysis of compound concentrations in urine

[0173] Creatinine concentrations in thawed urine samples were filtered through 10 kDa MWCO spin filters (Amicon Ultra 0.5 ml, Millipore) and then determined using a Creatinine Assay Kit (Sigma) as directed by the manufacturer.

[0174] L-Erg and S-met-L-Erg in urine were analyzed as in Example 1. To determine the light sulfurization pathway metabolites, thawed kidneys were ground using a pre-cooled mortar and pestle over dry ice. Subsequently, 100 mg of ground kidneys were homogenized in 400 µl of PBS supplemented with 10 mM NEM. To induce protein precipitation, 4% PCA was added, and the sample was centrifuged in a microfuge at 10,000 rpm for 15 minutes at 4°C. The supernatants were transferred to new tubes and stored at -80°C until further analysis. The intracellular content of light sulfurization pathway metabolites was determined by UPLC-MS / MS (Escobar J et al., Development of a reliable method based on ultra-performance liquid chromatography coupled to tandem mass spectrometry to measure thiol-associated oxidative stress in whole blood samples. J. Pharm. Biomed. Anal. 2016; 123: 104-112). Protein pellets were resuspended in 400 µl of 1 M NaOH, and the supernatant was used to determine the total protein concentration using a BCA Protein Assay Kit (ThermoScientific).

[0175] L-Erg-Cys In Vitro Binding Analysis

[0176] The reactivity of L-Erg itself and with Cys was analyzed in vitro for 17 hours at room temperature under two conditions: in 0.2 M Na2HP04 at pH = 7.2 and in boric acid buffer (Reference 33650-1L, Fluka) at pH = 11. The presence of L-Erg-L-Erg and L-Erg-Cys dimers and cystine was determined by LC / MS-MS.

[0177] Statistical analysis

[0178] Non-parametric analysis (Wilcoxon-Mann-Whitney test) was used to evaluate significance using Rstudio. Statistical significance is considered positive when p < 0.05.

[0179] result

[0180] First, the optimal working dose for treating mice was determined by investigating the effects of 1-month L-Erg treatment at two different concentrations in drinking water, 15 and 60 mg / L, on water intake, urine pH, ORP, and L-Erg concentration. The following observations were performed:

[0181] i) a statistically insignificant increase in surface-normalized water intake (Fig. 6a), particularly in females (sex data not shown), at any tested L-Erg concentrations at the endpoint of treatment (p = 0.11 and p = 0.08, respectively); ii) a statistically insignificant increase in pH at 60 mg / L (Fig. 6b) and at 15 mg / L (p = 0.052), particularly in males (data not shown); iii) a statistically insignificant decrease in urinary redox status or redox potential (ORP) (Fig. 6c); and iv) a statistically significant increase in urinary L-Erg and S-met-L-Erg concentrations (Figs. 6d and 6e, respectively). Interestingly, differences in urinary concentrations of L-Erg and S-met-L-Erg were found between the two tested conditions. At 15 mg / L, L-Erg and S-met-L-Erg concentrations increased by 3-fold (3.4±0.4) and 6-fold (5.9±0.6), respectively. At 60 mg / L, the increase was 175-fold (174.6±38.4) and 15-fold (14.7±1.9) for L-Erg and S-met-L-Erg, respectively. These differences suggest that at 60 mg / L, the endogenous pools and metabolism of L-Erg may approach saturation. To maximize the amount of L-Erg obtainable from urine, a concentration of 60 mg / ℓ of L-Erg in drinking water corresponding to an average ± SEM calculated dose of 13.6 ± 1.6 mg / kg·day was taken for further analysis (data not shown).

[0182] L-Erg treatment did not alter cystine stone growth (3 months)

[0183] The effect of L-Erg treatment (60 mg / L in drinking water) on the progression of cystine stones in cystinuric mice was first checked by monthly monitoring of cystine stone growth by X-ray for 3 months in both treated and untreated mice. No effect of treatment on cystine stone growth (p = 0.61) was observed in the tested conditions (Figure 7). Since cystinuric mice can have multiple or single stones, an analysis was also conducted to determine if there were effects for each type. No statistically significant effects were observed for single or multiple stones (p = 0.78 and p = 0.41, respectively; (data not shown)). To confirm the effect of treatment on different metabolic parameters, the effects on urine pH, redox status, and surface area-corrected water intake were also analyzed, and no differences were observed in L-Erg-treated animals before and after treatment (data not shown). The expected mean ± SEM L-Erg dose during the experiment was (17.24 ± 0.69 mg / kg·day).

[0184] L-Erg treatment prevents or delays the onset of cystine stones (6 months of Erg treatment)

[0185] We continued to investigate the effect of L-Erg on cystine stone formation by treating cystinuria mice for 6 months after weaning. Based on previous experiments in adult mice, a target dose of 16 mg / kg·day was established. To achieve this in growing mice, mouse growth and water intake were monitored throughout the entire experiment, and Tordoff et al. (Tordoff MG, Bachmanov AA, Reed DR. Forty mouse strain survey of water and sodium intake. Physiol. Behav.Based on 2007; 91: 620-31, the concentration of L-Erg in drinking water was adjusted for water intake corrected for body surface area. The expected mean ± SD dose over a 6-month period was 16.25 ± 6.29 (data not shown). To analyze the effect on the development of calculi, treated and untreated mice were X-rayed monthly during the 6-month treatment period. In the L-Erg treatment group, a 50% reduction in the number of calculous mice, delayed onset of calculi regardless of mouse sex (data not shown) (Fig. 8a), and a nearly statistically significant reduction in stone growth (Fig. 8b) were observed.

[0186] To analyze the effects of long-term treatment, urine pH and redox potential were determined; there was no difference in urine pH in L-Erg treated mice (Fig. 8c), and statistically significant lower urine output was observed in L-Erg treated mice (Fig. 8d). This long-term treatment did not affect water intake or mouse body weight (data not shown).

[0187] L-Erg increases the intracellular concentration of compounds of the hardening pathway.

[0188] The antioxidant capacity of L-Erg may explain the decrease in urinary redox potential, but it does not, in itself, explain the differences observed in reduced calculi and stone growth between the two different treatments performed. To gain some insight into the mode of action of L-Erg on cystine calculi, the formation of in vitro dimeric L-ErgCys was first analyzed by LC / MS-MS under two conditions, pH = 7.2 and pH = 11 (data not shown). Neither L-Erg-Cys nor L-Erg-L-Erg dimeric

[0189] The lower ratio between urinary concentrations of S-met-L-Erg (L-Erg metabolite) and L-Erg in cystic mice compared to non-cystic mice (refer to data in Example 1) indicates that intracellular mechanisms are involved in cystine calculosis. In this sense, it has been reported that L-Erg activates glutathione (GSH) synthesis by upregulating Nrf2 (Kerley RN and his colleagues The potential therapeutic effects of ergothioneine in pre-eclampsia. Free Radie. Biol. Med. 2018; 117: 145-157), and, in a proteomic approach to identify differential protein expression between cystinuria mice and wild-type mice, Anpep( Anpep ) , Gpx1( Gpx1 ) and Gstt1( Gstt1 GSH synthesis such as ) and Cdo1( Cdo1Some enzymes involved in cystine metabolism, such as [ ], were found to be downregulated in the renal brush borders of cystinuria mice. Based on this, the intracellular content of these metabolites of the sulfation pathway in the kidneys of long-term treated and untreated mice was analyzed. The data are depicted in Figures 9(a to l), where the intracellular concentrations of GSH (1.4x), Cys (1.6x), gamma-glutamylcysteine ​​(1.7x), and Met (1.7x) were significantly increased by about 1.5x, and the intracellular concentrations of cystine (11.1x) and S-adenosylhomocysteine ​​(SAH) (1.2x) were significantly decreased in L-Erg treated mice. Interestingly, the GSH / GSSG, Cys / CssC, and SAM / SAH ratios were increased in L-Erg treated mice (2.1-, 6.1-, and 1.8-, respectively). GSSG is glutathione oxide; CssC is cystine; and SAM is S-adenosylmethionine.

[0190] comment

[0191] The selected drug delivery system may be considered less suitable for dose control. While it is true that more variability is required, it is difficult to argue that a reduction in the dose of L-Erg taken by mice could be the cause of the calculosis observed in the treated animals, because L-Erg blood and plasma concentrations remained significantly higher for 6 weeks and urine concentrations for 1 week after the endpoint of administration of the 5 mg / kg·day equivalent.

[0192] S / c7a9 - / - Preliminary results in mice showed significant differences in the concentrations of metabolites of the sulfation pathway in the kidney. Among those with decreased concentrations were: GSH (4.5x), GssG (17.9x), SAM (2.4x), Met (23.8x), and cystathionine (10.3x). Among those with increased concentrations were Cys (6.7x), cystine (CssC, 3.8x), SAH (9x), and gamma-glutamylcysteine ​​(2.1x). Both the GSH / GssG and Cys / CssC ratios increased approximately 6-fold in male mice with cystinuria, while the SAM / SAH ratio decreased 21-fold. These data suggest reduced methylation capability in the kidney and decreased activity within the sulfation pathway in the presence of cystinuria. The unexpectedly elevated Cys content in the kidney may imply a mechanism compensating for reduced GSH content in an effort to overcome limitations in inhibiting any oxidative damage, as suggested by Banjac et al. ("The cystine / cysteine ​​cycle: a redox cycle regulating susceptibility versus resistance to cell death"; 2008; Oncogene; vol. 27(11); pp. 1618-28). Long-term treatment with L-Erg has been shown to increase the expression of glutathione reductase, catalase, and superoxide dismutase in human vascular endothelial cells and to induce Nrf2 / ARE-mediated antioxidant genes in UVA-irradiated human keratinocytes. Under these circumstances, comprehensive induction of the pyloric pathway was observed in L-Erg-treated mice, excluding GssG, CssC, and SAH.It is still unknown whether increased reducing capacity in the kidneys is associated with lower redox potential in urine, and this is subject to further experimentation.

[0193] Unexpectedly, differences in the effect of L-Erg treatment on the progression of cystine stones were observed between the tested treatments: after the onset of stones or during prevention (before the onset of stones). Since the rate of cystine stone growth in control mice differs between the two experiments (approx. 2 mg / day and approximately 3 mg / day, respectively), and the amount of cystine stones that may follow during the preventive experiment is low, or differences due to intersibling variability may have specific weights, any statistical artifact cannot be completely ruled out.

[0194] Example 3. Urinary redox status or potential (ORP) as a prognostic marker in animals with cystinuria

[0195] The inventors recognized that the urinary ORP of cystinuria mice that did not form cystine stones was lower than the urinary ORP of mice that did form stones. Therefore, these parameters (measured as indicated in Example 2) were also suggested for differential prognosis in vitro in cystinuria animals, including humans.

[0196] method

[0197] Mouse management was as shown in Example 1.

[0198] Sample Collection

[0199] Mice were individually housed in metabolic cages for 4 days starting from the first day as an adaptation period. Mouse body weight, water and food intake, and secreted urine were monitored daily. 24-hour urine samples were collected and stored at -80°C with 10 mM sodium azide as a preservative until further analysis. Redox potential was determined in fresh urine using an ORP electrode (Crison) at room temperature at micropH 2000 (Crison).

[0200] result

[0201] ORP is higher in stone-forming mice

[0202] Two different mouse models of cystinuria on a C57BL6 / J genetic background ( S / c7a9 -1- (Feliubadalo et al., above) or S / c3a1 D140G From ), urinary ORP was significantly higher in cystinuria mice. S / c3a1 D140G The model was described by Peter et al., ("A mouse model for cystinuria type I", Hum Mol Genet.- 2003 vol. 1;12(17), pp.: 2109-20). Type I cystinuria mouse model 129S2 / SvPasCrl( S / c3a1 E383K Preliminary data from ) showed similar results, considering the very limited amount of sporadic cystine stones produced by the model when analyzing. All of these data are depicted in Figure 10, clearly indicating that urinary ORP may be useful for the diagnosis or prognosis of cystine stone disease. When considering both sexes, although the trend was maintained in all groups, only C57BL6 / J S / c7a9 -1- males from and S / c3a1 D140G Only females from showed a significant decrease in urine ORP (sex data not shown).

[0203] Interestingly, although the same gene affects both models, the urinary ORP in the non-calcifying group of the 129S2 / SvPasCrl mouse model is S / c3a1 D140G Significantly lower than the urine ORP of the mouse model (p = 1.9e -09 ) suggests the existence of genetic factors associated with urinary ORP.

[0204] List of cited literature

[0205] Halperin EC et al., "The use of D-penicillamine in cystinuria: efficacy and untoward reactions", Yale J Biol Med., 1981, vol. 54(6), pp. 439-46.

[0206] Burtis CA et al., 2008, Chapter 14, section “Statistical Treatment of Reference Values”.

[0207] Kato Y. et al., “Gene knockout and metabolome analysis of carnitine / organic cation transporter OCTN1”, Pharm. Res., 2010; vol 27, pp. 832-40.

[0208] Sotgia S. et al. ."Plasma L-ergothioneine measurement by high-performance liquid chromatography and capillary electrophoresis after a pre-column derivatization with 5-iodoacetamidofluorescein (5-IAF) and fluorescence detection". Antopolsky M, ed. PLoS One2013;vol.8:e70374

[0209] Zinellu A. et al., "Assay for the simultaneous determination of guanidinoacetic acid, creatinine and creatine in plasma and urine by capillary electrophoresis UV-detection", J.Sep. Sci., 2006

[0210] Escobar J et al., "Development of a reliable method based on ultra-performance liquid chromatography coupled to tandem mass spectrometry to measure thiol-associated oxidative stress in whole blood samples$. J. Pharm. Biomed. Anal. 2016; 123: 104-112 Tordoff MG, Bachmanov AA, Reed DR. Forty mouse strain survey of water and sodium intake. Physiol. Behav. 2007; 91: 620-31

[0211] Kerley RN et al., The potential therapeutic effects of ergothioneine in pre-eclampsia. Free Radie. Biol. Med. 2018; 117: 145-157

[0212] Feliubadalo et al. "Slc7a9-deficient mice develop cystinuria non-I and cystine urolithiasis" Hum Mol Genet 2003; vol 12; pp. 2097-2108

[0213] Peter et al., "A mouse model for cystinuria type I", Hum Mol Genet.-2003 vol. 1;12(17), pp.: 2109-20.

[0214] Banjac et al., "The cystine / cysteine cycle: a redox cycle regulating susceptibility versus resistance to cell death"; 2008; Oncogene; vol. 27(11); pp. 1618-28.

Claims

Claim 1 A pharmaceutical composition comprising ergothioneine for use in the treatment and / or prevention of renal stones or aminoaciduria. Claim 2 A pharmaceutical composition comprising ergothioneine for use in the treatment and / or prevention of renal calculi or aminoaciduria in combination therapy with a compound selected from the group consisting of a separate cystine-solubilizing agent, L-cystine dimethyl ester, L-cystine methyl ester, L-cystine diamide, lipoic acid, and combinations thereof. Claim 3 A pharmaceutical composition according to claim 1 or 2, wherein the renal calculus is cystine calculus or the aminoaciduria is cystinuria. Claim 4 A pharmaceutical composition according to claim 1 or 2, further comprising one or more pharmaceutically acceptable excipients and / or carriers. Claim 5 A pharmaceutical composition according to claim 1 or 2, wherein the ergothioneine is L-ergothioneine. Claim 6 A pharmaceutical composition according to claim 1 or 2, wherein the ergothioneine is administered at a dosage of 0.01 to 500 mg / kg body weight per day. Claim 7 A composition for use in the diagnosis and / or prognosis of a kidney disease comprising S-methyl-L-ergothioneine, wherein the kidney disease is renal calculus or renal aminouria. Claim 8 A composition for use in the diagnosis and / or prognosis of kidney disease, comprising S-methyl-L-ergothioneine in isolated urine test samples. Claim 9 A method for providing information for the diagnosis and / or prognosis of a renal disease, which is renal calculosis or renal aminouria, comprising the following steps: - determining the amount of S-methyl-L-ergothioneine in an isolated test sample of a subject; and - comparing the amount of S-methyl-L-ergothioneine of a subject with a reference value, wherein if the amount determined in the subject is lower than the reference value of a healthy subject or a subject not suffering from renal disease, the subject is suspected of suffering from said renal disease. Claim 10 A method for providing information for the diagnosis and / or prognosis of kidney disease, comprising the following steps: - determining the amount of S-methyl-L-ergothioneine in an isolated urine test sample of a subject; and - comparing the amount of S-methyl-L-ergothioneine of a subject with a reference value, wherein if the amount determined in the subject is lower than the reference value of a healthy subject or a subject not suffering from kidney disease, the subject is suspected of suffering from said kidney disease. Claim 11 A method for providing information according to claim 9 or 10, further comprising the following steps: - determining the amount of L-ergothioneine in an isolated test sample of the subject, and - optionally, calculating the ratio between the amount of S-methyl-L-ergothioneine and the amount of L-ergothioneine. Claim 12 An apparatus for determining the amount of S-methyl-L-ergothioneine for use in a method as defined in paragraph 9 or 10. Claim 13 In paragraph 12, the device forms part of the kit. Claim 14 A marker for the diagnosis and / or prognosis of a renal disease in an isolated test sample of a subject comprising S-methyl-L-ergothioneine, wherein the renal disease is renal calculus or renal aminoaciduria. Claim 15 A marker for the diagnosis and / or prognosis of renal disease in isolated test samples of a subject, comprising S-methyl-L-ergothioneine in isolated urine test samples. Claim 16 In paragraph 9, a method of providing information in which the isolated test sample is a urine sample. Claim 17 A composition according to claim 8, wherein the kidney disease is renal calculus or renal aminouria. Claim 18 A composition according to claim 7 or 17, wherein the renal calculus is cystine calculus or the aminoaciduria is cystinuria. Claim 19 In paragraph 12, a device in which the isolated test sample is a urine sample.

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