Inhibition of cyclodextrin-induced renal injury

Abstract

An object of the present invention is to provide a means for inhibiting renal damage induced by cyclodextrin or a derivative thereof.SOLUTION: The present invention employs cilastatin or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the suppression of renal damage induced by cyclodextrin or its derivatives. [Background technology]

[0002] Among cyclodextrins (hereinafter referred to as "CD"), natural cyclodextrins are cyclic oligosaccharides consisting of six, seven, and eight glucopyranose molecules, commonly referred to as α-CD, β-CD, and γ-CD, respectively. These have a donut-shaped three-dimensional structure. The hydrogen bonds between the C2- and C3-hydroxyl groups of adjacent glucopyranose molecules are located in the large opening, while the C6-hydroxyl groups are located in the small opening. This allows oxygen bonds to be located close together within the cavity, resulting in an electron-rich, hydrophobic interior. CDs can incorporate hydrophobic compounds of a size that matches the cavity size as guest molecules and form inclusion complexes with the guest molecules. The size of this hydrophobic cavity is determined by the number of glucopyranose molecules forming the CD.

[0003] The inclusion of guest molecules by CDs and their derivatives has been applied in various fields such as food, pharmaceuticals, cosmetics, and industry for the purposes of improving solubility, stabilization, powdering oily and low-melting substances, preventing evaporation, masking flavors and odors, reducing local irritation, and improving bioavailability.

[0004] For example, Patent Document 1 describes a technique for improving the pharmacokinetics or bioavailability of drugs using cyclodextrin derivatives such as sulfobutyl ether β-cyclodextrin, and Patent Documents 2 and 3 describe the use of cyclodextrin derivatives such as hydroxypropyl-β-cyclodextrin to solubilize or stabilize drugs.

[0005] On the other hand, toxicity problems have been reported regarding cyclodextrin and its derivatives. For example, the package insert of a medicine containing a cyclodextrin derivative (Non-Patent Document 1) states that the derivative may accumulate in patients with renal dysfunction, causing deterioration of renal function, etc.

[0006] It has been reported that cilastatin can suppress renal damage caused by several drugs by antagonizing drugs that bind to megalin expressed in proximal tubule cells at the megalin receptor (Non-patent document 2, Patent document 4). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2020 / 069138 [Patent Document 2] WO2008 / 133982 [Patent Document 3] US2002 / 0192273 [Patent Document 4] WO2019 / 208777 [Non-patent literature]

[0008] [Non-Patent Document 1] Noxafil IV Infusion 300mg Package Insert, MSD, Revised December 2021 (5th Edition) [Non-patent document 2] Hori et al., Journal of the American Society of Nephrology, 2017, Vol. 28, No. 1783-1791 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a new means for inhibiting nephropathy induced by cyclodextrin or a derivative thereof. In another aspect, an object of the present invention is to provide a new means for inhibiting the uptake of cyclodextrin or a derivative thereof into renal tissue. [Means for solving the problem]

[0010] As a result of extensive research aimed at solving the above problems, the present inventors have found that cilastatin is effective in suppressing renal damage induced by cyclodextrin or its derivatives, and also that cilastatin suppresses the uptake of cyclodextrin or its derivatives into renal tissue.

[0011] The present invention includes, but is not limited to, the following aspects. 1. An agent for suppressing renal damage induced by cyclodextrin or its derivatives, which comprises cilastatin or a pharmaceutically acceptable salt thereof as an active ingredient. 2. The inhibitor according to 1, wherein the cyclodextrin or a derivative thereof is β-cyclodextrin or a derivative thereof. 3. The inhibitor according to 2, wherein the β-cyclodextrin or a derivative thereof is selected from the group consisting of methyl-β-cyclodextrin, dimethyl-β-cyclodextrin, trimethyl-β-cyclodextrin, partially methylated β-cyclodextrin, ethyl-β-cyclodextrin, diethyl-β-cyclodextrin, triethyl-β-cyclodextrin, (2-hydroxyethyl)-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, (3-hydroxypropyl)-β-cyclodextrin, (2,3-dihydroxypropyl)-β-cyclodextrin, (2-hydroxyisobutyl)-β-cyclodextrin, sulfobutyl ether β-cyclodextrin, glucosyl-β-cyclodextrin, and maltosyl-β-cyclodextrin. 4. The inhibitor according to any one of 1 to 3, which is in the form of an injection. 5. A pharmaceutical composition comprising cilastatin or a pharmaceutically acceptable salt thereof and a cyclodextrin or a derivative thereof. 6. An inhibitor of the uptake of cyclodextrin or its derivatives into renal tissue, which comprises cilastatin or a pharmaceutically acceptable salt thereof as an active ingredient. [Effects of the Invention]

[0012] The present invention can suppress renal damage induced by cyclodextrin or its derivatives. It is believed that cyclodextrin or its derivatives are taken up into renal tissue via megalin, causing renal damage. In the present invention, cilastatin inhibits the binding between megalin and cyclodextrin or its derivatives, thereby inhibiting the uptake of cyclodextrin or its derivatives into renal tissue, thereby suppressing renal damage.

[0013] The term "suppress" used in relation to renal damage in this specification means that symptoms do not appear at all, that the symptoms are alleviated, etc. The "alleviation" includes reducing the amount of the symptoms that appear, reducing symptoms that have already appeared, and completely eliminating symptoms that have already appeared. In this specification, preventing disease symptoms from appearing at all or reducing the amount of their appearance is referred to as "prevention." Furthermore, a pharmaceutical agent for "suppression" is referred to as an "inhibitor." These explanations regarding "suppression" also apply, with appropriate modifications, to "suppression" of uptake.

[0014] The above-mentioned effects of the present invention are extremely useful in the pharmaceutical industry. For example, in the formulation of pharmaceuticals that require the incorporation of cyclodextrin or its derivatives, renal damage caused by cyclodextrin or its derivatives may limit the amount of cyclodextrin or its derivatives that can be incorporated. However, by using cilastatin in combination, it is possible to incorporate a larger amount of cyclodextrin or its derivatives while avoiding renal damage. Therefore, the present invention is expected to have an effect of promoting pharmaceutical development. For example, in situations where a cyclodextrin or its derivative is required in an amount that would have a significant effect on the kidneys due to the poor solubility of the active ingredient, the present invention facilitates the development of pharmaceuticals containing high doses of cyclodextrin or its derivatives while avoiding the effect on the kidneys. Therefore, the possibility of clinical application of currently unknown poorly soluble active ingredients is expanded.

[0015] Furthermore, if the present invention reduces the risk of kidney damage, it will be possible to ensure that all patients, including those with concerns about kidney function, have the opportunity to receive drug treatment with pharmaceuticals containing cyclodextrin or its derivatives. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows fluorescence microscopy images showing megalin-dependent uptake of HPβCD into renal proximal tubule epithelial cells. [Figure 2] FIG. 2 shows fluorescence microscopy images showing megalin-dependent uptake of SBECD into renal proximal tubule epithelial cells. [Figure 3] FIG. 3 shows fluorescence microscopic images showing the inhibitory effect of cilastatin on the uptake of HPβCD into renal proximal tubular epithelial cells. [Figure 4] FIG. 4 shows fluorescence microscopic images showing the inhibitory effect of cilastatin on the uptake of SBECD into renal proximal tubular epithelial cells. [Figure 5] FIG. 5 is a graph showing the inhibitory effect (quantitative evaluation) of cilastatin on the renal uptake of HPβCD (inner layer of renal cortex). [Figure 6] FIG. 6 is a graph showing the inhibitory effect (quantitative evaluation) of cilastatin on renal uptake of SBECD (inner layer of renal cortex). [Figure 7] FIG. 7 shows PAS staining images showing the inhibitory effect of cilastatin on HPβCD-induced renal damage. [Figure 8] FIG. 8 shows PAS staining images showing the inhibitory effect of cilastatin on SBECD-induced renal damage. [Figure 9] FIG. 9 is a graph showing the inhibitory effect of cilastatin on SBECD-induced renal damage (quantitative evaluation of damage markers). DETAILED DESCRIPTION OF THE INVENTION

[0017] (cilastatin) In the present invention, cilastatin or a pharmaceutically acceptable salt thereof is used. Cilastatin is (Z)-7-[[(R)-2-Amino-2-carboxyethyl]thio]-2-[[[(S)-2,2-dimethylcyclopropyl]carbonyl]amino]-2-heptenoic acid. For clarity, when cilastatin forms a hydrate, the use of the hydrate is also within the scope of the present invention.

[0018] Examples of pharmaceutically acceptable salts of cilastatin include alkali metal salts, such as lithium salt, sodium salt, and potassium salt; alkaline earth metal salts, such as magnesium salt and calcium salt; zinc salt and aluminum salt; organic amine salts, such as choline salt, ethanolamine salt, trimethylamine salt, triethylamine salt, dicyclohexylamine salt, dibenzylamine salt, phenethylbenzylamine salt, procaine salt, morpholine salt, pyridine salt, piperidine salt, piperazine salt, and N-ethylpiperidine salt; ammonium salt; basic amino acid salts, such as lysine salt and arginine salt; and the like. A particularly preferred salt is cilastatin sodium. It should be noted that the scope of pharmaceutically acceptable salts also includes salt hydrates.

[0019] Cilastatin or a pharmaceutically acceptable salt thereof may be, for example, a commercially available product, or may be produced or obtained by a known method or a method similar to a known method. (Cyclodextrin or its derivatives) As used herein, the term "cyclodextrin" refers to unmodified cyclic oligosaccharides composed of (α-1,4)-linked α-D-glucopyranose units, examples of which are α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0020] The term "cyclodextrin derivative" as used herein refers to a compound in which one or more of the hydroxyl groups at positions 2, 3, and 6 of the α-D-glucopyranose unit in the cyclodextrin molecule have been chemically modified. Such derivatives also include derivatives in the form of pharmaceutically acceptable salts.

[0021] The cyclodextrin or a derivative thereof used in the present invention is preferably β-cyclodextrin or a derivative thereof, and more preferably methyl-β-cyclodextrin, dimethyl-β-cyclodextrin, trimethyl-β-cyclodextrin, partially methylated β-cyclodextrin (RM-β-CD: randomly methylated beta-cyclodextrin), ethyl-β-cyclodextrin, diethyl-β-cyclodextrin, triethyl-β-cyclodextrin, (2-hydroxyethyl)-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, (3-hydroxypropyl)-β-cyclodextrin, (2,3-dihydroxypropyl)-β-cyclodextrin, (2-hydroxyisobutyl)-β-cyclodextrin, sulfobutyl ether β-cyclodextrin, glucosyl-β-cyclodextrin, and maltosyl-β-cyclodextrin, and more preferably, selected from the group consisting of (2-hydroxypropyl)-β-cyclodextrin and sulfobutyl ether β-cyclodextrin.

[0022] Cyclodextrin or a derivative thereof may be, for example, a commercially available product, or may be produced or obtained by a known method or a method similar to a known method. (Kidney damage inhibitor) In one aspect, the present invention relates to an agent for suppressing nephropathy induced by cyclodextrin or a derivative thereof, comprising cilastatin or a pharmaceutically acceptable salt thereof as an active ingredient. The cilastatin or a pharmaceutically acceptable salt thereof, and the cyclodextrin or a derivative thereof, as well as preferred examples and specific examples thereof, are as described above.

[0023] The inhibitor of the present invention contains cilastatin or a pharmaceutically acceptable salt thereof in an amount effective for inhibiting nephropathy induced by cyclodextrin or a derivative thereof. For example, the daily dose of cilastatin or a salt thereof for an adult is 1.0 to 2.0 g or 1.0 to 1.5 g. The inhibitor can be administered once or in divided doses so that the dose falls within the range. Intermittent administration, such as every other day or every other day, can also be used.

[0024] The kidney damage can be induced by cyclodextrin or its derivatives via megalin. Because megalin is thought to be primarily expressed in renal proximal tubule epithelial cells (mainly the luminal membrane) in vivo, the present invention is particularly useful for suppressing proximal tubule epithelial cell damage and the resulting kidney damage.

[0025] Such renal disorders include osmotic nephropathy, osmotic nephropathy, nephropathy, renal injury, nephritis, renal failure, kidney disease, acute nephropathy, acute kidney injury, acute nephritis, acute renal failure, acute kidney disease, chronic nephropathy, chronic kidney injury, chronic nephritis, chronic renal failure, chronic kidney disease, tubular nephropathy, tubular nephropathy, tubular nephritis, tubular renal failure, tubular kidney disease, tubulointerstitial nephropathy, tubulointerstitial nephropathy, tubulointerstitial nephritis, tubulointerstitial renal failure, tubulointerstitial kidney disease, obstructive nephropathy, obstructive nephritis, obstructive renal failure, obstructive kidney disease, acute nephritic syndrome, rapidly progressive nephritic syndrome, chronic nephritic syndrome, nephrotic syndrome, renal vasospasm, and acute tubular necrosis.

[0026] The reason cyclodextrin or its derivatives are incorporated into pharmaceuticals is due to the poor solubility of the physiologically active ingredients in the pharmaceuticals, but the true essence of cyclodextrin or its derivatives is the formation of inclusion complexes. The properties brought about by inclusion complexes include not only improved solubility of poorly soluble substances but also improved stability of the inclusion complexes. Therefore, it is reasonably expected that cyclodextrin or its derivatives will be incorporated into pharmaceutical formulations containing various physiologically active substances, both now and in the future. The present invention can also be used as a renal damage inhibitor in these pharmaceuticals.

[0027] Furthermore, the use of cyclodextrin or its derivatives as active ingredients in the treatment of lysosomal storage diseases and Alzheimer's disease, which are caused by abnormalities in lipid metabolism in the body, rather than as additives, is also under development (Ory et al., Lancet, 2017, Vol. 390, pp. 1758-1768; www.ClinicalTrials.gov NCT03624842 https: / / clinicaltrials.gov / ct2 / show / NCT05607615). The present invention can also be used as an agent for suppressing kidney damage caused by the therapeutic administration of cyclodextrin or its derivatives.

[0028] (Pharmaceutical composition) In one aspect, the present invention is a pharmaceutical composition, which comprises cilastatin or a pharmaceutically acceptable salt thereof, and a cyclodextrin or a derivative thereof.

[0029] The cilastatin or a pharmaceutically acceptable salt thereof, and the cyclodextrin or a derivative thereof contained in the composition, as well as specific and preferred examples thereof, are as described above. The content and dosage of cilastatin or a pharmaceutically acceptable salt thereof contained in the composition are the same as those described above for the inhibitor.

[0030] Cyclodextrins or their derivatives are used for a wide range of purposes, such as solubilizing or stabilizing drugs and exerting specific pharmacological effects. Therefore, the content and dosage of cyclodextrin or its derivatives in the composition of the present invention can be appropriately determined by those skilled in the art based on publicly known information, depending on the intended use. For example, the daily dosage of cyclodextrin or its derivatives for an adult is 0.1 to 5,000 mg / kg or 0.1 to 3,000 mg / kg.

[0031] The pharmaceutical composition of the present invention can be administered once a day or in divided doses several times a day. It can also be administered intermittently, such as every other day or every other day. The pharmaceutical compositions of the present invention may contain other active ingredients, including, but not limited to, posaconazole, voriconazole, remdesivir, itraconazole, letermovir, and carfilzomib.

[0032] In recent years, clinical development is underway to investigate the efficacy of hydroxypropyl-β-CD itself as a therapeutic agent for Niemann-Pick disease type C (NPC), a genetic disease characterized by abnormal intracellular lipid accumulation and a type of lysosomal storage disorder (Ory et al., Lancet, 2017, Vol. 390, pp. 1758-1768).

[0033] Additionally, Alzheimer's disease (AD) has been shown to share biological similarities with NPC, including cholesterol accumulation in certain brain regions, elevated tau concentrations in the cerebrospinal fluid, and amyloid plaques in the brain. Clinical development of hydroxypropyl-β-CD as a treatment for AD is also underway (www.ClinicalTrials.gov NCT03624842 https: / / clinicaltrials.gov / ct2 / show / NCT05607615 as of November 2022).

[0034] Thus, there is a possibility that development of pharmaceuticals using cyclodextrin or its derivatives as active ingredients rather than as additives will progress in the future. However, even in such cases, there is a possibility that kidney damage may occur due to cyclodextrin or its derivatives.

[0035] Therefore, a preferred example of the pharmaceutical composition of the present invention is a pharmaceutical composition for treating NPC, and another preferred example of the pharmaceutical composition of the present invention is a pharmaceutical composition for treating AD.

[0036] (Inhibitor of CD or its derivatives uptake into renal tissue) In one aspect, the present invention relates to an inhibitor of the uptake of cyclodextrin or a derivative thereof into renal tissue, which comprises cilastatin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0037] Cilastatin is thought to directly or indirectly inhibit the binding of cyclodextrin or its derivatives to megalin, and this inhibitory effect is thought to lead to the suppression of the uptake of cyclodextrin or its derivatives into renal tissue and further to the suppression of renal damage.

[0038] The inhibitor of the present invention contains an effective amount of cilastatin or a pharmaceutically acceptable salt thereof. The effective amount can be determined by referring to the amounts described above for the inhibitor of nephropathy of the present invention.

[0039] (Dosage form) The form of the inhibitor and composition of the present invention is not particularly limited, and may be in the form of solid preparations such as powders, granules, capsules, tablets, chewable preparations, etc., liquid preparations such as solutions and syrups, injections, sprays, etc. Injections are preferred.

[0040] (Other ingredients) The inhibitor and composition of the present invention may contain a pharmaceutically acceptable carrier depending on the formulation requirements. Examples of the carrier include excipients and solvents. Examples of additional components that may be contained in the inhibitor of the present invention include binders, pH adjusters, disintegrants, chelating agents, solubilizers, suspending agents, emulsifiers, isotonicity agents, stabilizers, soothing agents, preservatives, antioxidants, lubricants, flavoring agents, and coloring agents.

[0041] Examples of excipients include sugars such as lactose, glucose, and D-mannitol; organic excipients such as starches; and celluloses such as crystalline cellulose; and inorganic excipients such as dicalcium phosphate, calcium carbonate, and kaolin. Examples of solvents include purified water and physiological saline. Examples of binders include pregelatinized starch, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol. Examples of pH adjusters include hydrochloric acid and sodium hydroxide. Examples of disintegrants include low-substituted hydroxypropyl cellulose, chemically modified celluloses and starches, and alginic acid. Examples of chelating agents include calcium disodium edetate hydrate and calcium sodium edetate hydrate. Examples of solubilizing agents include polyethylene glycol, propylene glycol, trehalose, benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate. Examples of suspending or emulsifying agents include celluloses such as sodium lauryl sulfate, gum arabic, gelatin, lecithin, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, and sodium carboxymethylcellulose; polysorbates; and polyoxyethylene hydrogenated castor oil. Examples of isotonic agents include sodium chloride, potassium chloride, sugars, glycerin, and urea. Examples of stabilizers include polyethylene glycol, sodium dextran sulfate, and other amino acids. Examples of soothing agents include glucose, calcium gluconate, and procaine hydrochloride. Examples of preservatives include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid. Examples of antioxidants include sulfites and ascorbic acid.

[0042] When the inhibitor or composition of the present invention is in the form of an injection, the injection may be in the form in which the active ingredient is pre-dissolved, or in the form of a solid to be dissolved upon use. These injection solutions preferably contain, for example, 0.001 to 10% by mass, 0.01 to 5% by mass, or 0.1 to 1% by mass of cilastatin or a pharmaceutically acceptable salt thereof as the active ingredient, based on the mass of the entire solution. When the inhibitor is in the form of a solid to be dissolved upon use, the content of cilastatin or a pharmaceutically acceptable salt thereof in the inhibitor is, for example, 0.001 to 100% by mass, 0.001 to 99.9% by mass, or 0.01 to 90% by mass. Suitable solvents or diluents for injections may be those commonly used, and examples thereof include aqueous media such as physiological saline, distilled water for injection, aqueous glucose solution, aqueous lidocaine hydrochloride solution (for intramuscular injection), intravenous injection liquids (e.g., aqueous solutions of citric acid and sodium citrate), and electrolyte solutions (for intravenous drip infusion and intravenous injection); organic media such as ethanol, polyethylene glycol, and propylene glycol; and mixtures thereof.

[0043] In a preferred embodiment, the inhibitors and compositions of the present invention are imipenem-free. In another preferred embodiment, the inhibitors and compositions of the present invention do not contain bicarbonates such as sodium bicarbonate.

[0044] (Method and Use) Another aspect of the inhibitor of the present invention is use of cilastatin or a pharmaceutically acceptable salt thereof in suppressing nephropathy induced by cyclodextrin or a derivative thereof, or in suppressing the uptake of cyclodextrin or a derivative thereof into renal tissue. Cilastatin or a pharmaceutically acceptable salt thereof, cyclodextrin or a derivative thereof, and nephropathy, as well as specific and preferred examples thereof, are as described above. This use may also be combined with the administration of cyclodextrin or a derivative thereof. The cyclodextrin or a derivative thereof may be administered simultaneously with cilastatin or a pharmaceutically acceptable salt thereof, separately, or at intervals. For example, the cyclodextrin or a derivative thereof may be administered simultaneously with, before, or after the administration of cilastatin or a pharmaceutically acceptable salt thereof, or two or more thereof may be combined.

[0045] In another aspect, the inhibitor of the present invention is a method for suppressing nephropathy induced by cyclodextrin or a derivative thereof, or suppressing the uptake of cyclodextrin or a derivative thereof into renal tissue, the method comprising administering an effective amount of cilastatin or a pharmaceutically acceptable salt thereof to a subject in need thereof. Cilastatin or a pharmaceutically acceptable salt thereof, cyclodextrin or a derivative thereof, and nephropathy, as well as specific and preferred examples thereof, are as described above. The method may further comprise administering an effective amount of cyclodextrin or a derivative thereof to the subject. The cyclodextrin or a derivative thereof can be administered simultaneously with, separately from, or at an interval with respect to cilastatin or a pharmaceutically acceptable salt thereof. For example, the cyclodextrin or a derivative thereof can be administered simultaneously with, before, or after the administration of cilastatin or a pharmaceutically acceptable salt thereof, or two or more thereof can be combined.

[0046] Within such combined administration, the individual components or agents may be administered in separate or single formulations. The effective amount or dosage of cilastatin or a pharmaceutically acceptable salt thereof and cyclodextrin or a derivative thereof can be determined based on the above explanations regarding the inhibitor and pharmaceutical composition of the present invention.

[0047] As used herein, subjects requiring various suppressions include, for example, subjects with or at risk of having symptoms of kidney damage, and subjects for whom the symptoms are present prior to administration of cyclodextrin or a derivative thereof and administration of cyclodextrin or a derivative thereof is contraindicated. The subject is preferably a mammal, for example, a human; a domestic animal such as a mouse, rat, rabbit, guinea pig, hamster, monkey, sheep, horse, cow, pig, donkey, dog, or cat; or other laboratory animal, with humans being particularly preferred.

[0048] (Numerical range) For clarity, any numerical range expressed herein by lower and upper limits is inclusive of those limits. [Example]

[0049] The present invention will be described below based on examples, but the present invention is not limited to these examples. (material) The following materials were used in the tests in this example.

[0050] (1) Test animals The kidney-specific megalin knockout (KO) mice (kidney-specific conditional megalin knockout mice) bred and maintained at Niigata University are Ndrg1- CreER T2 / + megalin lox / lox was used, and its littermate [megalin (lox / lox); Cre(-)] was used as a control.

[0051] C57BL / 6J wild-type mice (Charles River Laboratories Japan, Yokohama, Japan) were used. (2) Reagents Hydroxypropyl-β-cyclodextrin (HPβCD) was purchased from Tokyo Chemical Industry Co., Ltd. (Japan) (Hydroxypropyl-β-cyclodextrin, H0979). Sulfobutylether β-cyclodextrin (SBECD) was purchased from Med Chem Express, USA (SBE-β-CD (sulfobutylether β-cyclodextrin), No. HY-17031).

[0052] Fluorescently labeled CDs were also purchased. Specifically, fluorescein-labeled HPβCD (6-deoxy-6-[(5 / 6)-fluoresceinylthioureido]-HPBCD, CY-F-2005.1) was purchased from CycloLab, Hungary, and rhodamine-labeled SBECD (rhodamine-labeled sulfobutylated-BCD sodium salt, CY-RL-2041.2) was purchased from CycloLab, Hungary.

[0053] Cilastatin was purchased from ACS DOBFAR, an Italian company (CILASTATIN SODIUM (sterile)). (Test Example 1) Megalin dependence of CD uptake into kidney tissue We investigated whether the uptake of CD into kidney tissue depends on megalin by administering fluorescently labeled CD to kidney-specific megalin knockout (KO) mice and control mice.

[0054] (1) Method 100 μL of a saline solution of fluorescein-conjugated HPβCD (1 mg / 2 mL) or a saline solution of rhodamine-conjugated SBECD (1 g / 2 mL) was administered via the tail vein to kidney-specific conditional megalin KO mice and littermate control mice (both male, 12-14 weeks old).

[0055] The manufacturer's instructions indicated that the labeling efficiency of rhodamine-conjugated SBECD was less than 1%, and therefore the required dose differed from that of fluorescein-conjugated HPβCD. Thirty minutes after administration, both kidneys were excised, and a sample for microscopic observation was prepared from the right kidney by the following procedure.

[0056] -Cut 3 mm thick sections including the renal pedicle Permeation fixation overnight in 4% PFA (paraformaldehyde / phosphate buffer (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) Immerse in PBS for 10-30 minutes Immerse the tissue in 15% sucrose solution until it sinks Immerse the tissue in 30% sucrose solution until it sinks Immersed in OCT compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan) -Freeze in liquid nitrogen and store at -80℃ Section the tissue into 4 μm-thick sections using a cryo-ultramicrotome (Leica CM1850; Leica Biosystems, Nussloch, Germany). Vapor fixation with 4% PFA for 10 minutes Observation was performed using a fluorescence microscope (BZ-X810; Keyence, Osaka, Japan). (2) Results Fluorescence microscopy images are shown in Figures 1 and 2. Figure 1 shows the results obtained using fluorescein-labeled HPβCD, and Figure 2 shows the results obtained using rhodamine-labeled SBECD.

[0057] In both cases, increased fluorescence indicating CD uptake was observed in the proximal tubules of control mouse tissues (indicated by arrowheads in Figures 1 and 2), whereas no uptake was observed in kidney-specific megalin knockout mice. This finding demonstrated that CD uptake is dependent on megalin.

[0058] (Test Example 2) Inhibition of CD uptake into renal tissue by cilastatin We examined whether cilastatin administration inhibited CD uptake into renal tissue using wild-type mice.

[0059] (1) Method 1 (Animal medication, sample preparation) The drugs were administered to wild-type mice (C57BL / 6J, male, 10-12 weeks old) under the following conditions: All reagents were dissolved in physiological saline.

[0060] a) 100 μL of rhodamine-conjugated SBECD solution (1 g / 2 mL) (approximately 2 g / kg) + 100 μL of saline b) 100 μL of rhodamine-conjugated SBECD solution (1 g / 2 mL) (approximately 2 g / kg) + 100 μL of cilastatin solution (manufactured by ACSD, Italy) (400 mg / kg) c) 100 μL of fluoresceinized HPβCD solution (1 mg / 2 mL) (approximately 2 mg / kg) + 100 μL of saline d) 100 μL of fluoresceinized HPβCD solution (1 mg / 2 mL) (approximately 2 mg / kg) + 100 μL of cilastatin solution (400 mg / kg) Any of the above a) to d) was administered via the tail vein, and 30 minutes later, the uptake of SBECD in kidney sections was assessed.

[0061] (2) Method 2 (Quantitative evaluation of CD uptake) CD uptake was strongly observed in the inner cortical layer, which mainly included proximal tubule segments (S) 2-3. Using a fluorescence microscope BZ-X810 (KEYENCE), photographs of the inner cortical layer (S2-S3) were taken in five fields per case (magnification 400x, size 960 × 760 pixels).

[0062] In addition, the area ratio of the CD-incorporated area was measured using a hybrid cell counter (BZ-H4C) of the BZ-X800Analyzer (BZ-H4A). (3)Result-1 Fluorescence microscopy images of the test using fluorescein-conjugated HPβCD are shown in Figure 3, and fluorescence microscopy images of the test using rhodamine-conjugated SBECD are shown in Figure 4. In both cases, the fluorescence indicating CD uptake was suppressed in the proximal tubules of mice treated with cilastatin, confirming that CD uptake was inhibited by cilastatin.

[0063] (4)Result-2 FIG. 5 shows the results of the test using fluorescein-labeled HPβCD, and FIG. 6 shows the results of the test using rhodamine-labeled SBECD.

[0064] When CD uptake was quantitatively evaluated in the S2-3 region as described above, the cilastatin-treated group showed significantly reduced CD uptake compared to the control group, demonstrating that cilastatin inhibits CD uptake in renal tissue.

[0065] (Test Example 3) Suppression of CD-induced renal damage in mice by cilastatin The inhibitory effect of cilastatin on renal damage was examined using CD-administered mice. (1) Method 1 (Animal medication, sample preparation) The left kidney was removed from wild-type mice (male, 10-12 weeks old) by conventional methods.

[0066] Two weeks later, drugs were administered intraperitoneally for seven consecutive days under one of the following conditions (food and water intake). a) 100 μL of SBECD solution (500 mg / kg) + 100 μL of cilastatin solution (400 mg / kg) b) 100 μL of SBECD solution (500 mg / kg) + 100 μL of saline c) HPBCD solution 100μL (500mg / kg) + Cilastatin solution 100μL (400mg / kg) d) HPBCD solution 100 μL (500 mg / kg) + saline 100 μL (2) Method 2 (pathological evaluation by PAS staining) After administration, the right kidney was removed from the animals. For histological evaluation, 3-mm-thick sections, including the renal pedicle, were prepared from the removed right kidney and fixed in 4% paraformaldehyde phosphate buffer. Then, they were sectioned to a thickness of 4 μm using a microtome (REM-710; Yamato Kohki Industrial Co., Ltd.). The resulting sections were stained with Periodic Acid-Shiff (PAS) and observed under a microscope to evaluate the effects of cilastatin administration.

[0067] (3) Method 3 (Quantitative evaluation using biochemical markers of renal damage) Urine was collected using metabolic cages, and each animal was dissected before the removal of the right kidney. Blood was collected from the inferior vena cava at the time of dissection. The collected blood was centrifuged at 800 g for 30 minutes at room temperature, and serum was separated and collected. The obtained urine and serum were stored at -80°C until analysis. Urinary NAG and Cr were measured by SRL Co., Ltd. to evaluate the effect of cilastatin on suppressing renal damage.

[0068] (4)Result-1 The results of the test using HPβCD are shown in Figure 7, and the results of the test using SBECD are shown in Figure 8. Histopathological evaluation using PAS staining revealed vacuolation of proximal tubular epithelial cells in the control group (indicated by arrowheads in Figures 7 and 8), but vacuolation was suppressed in the cilastatin-treated group.

[0069] (5)Result-2 The results of quantitative evaluation of biochemical markers of renal damage in a test using SBECD are shown in Figure 9. Compared to the control group, the increase in urinary NAG, a marker of renal damage, was significantly suppressed in the cilastatin-administered group.

[0070] These findings demonstrate that cilastatin has a protective effect against renal injury induced by cyclodextrins such as HPβCD and SBECD or their derivatives. [Industrial Applicability]

[0071] The present invention makes it possible to suppress kidney damage in pharmaceuticals containing cyclodextrin or its derivatives as an additive or active ingredient, and ensures opportunities for drug treatment with pharmaceuticals containing cyclodextrin or its derivatives.

[0072] Furthermore, in situations where cyclodextrin or its derivatives are required in amounts that would have a significant effect on the kidney due to the poor solubility of the active ingredient, the present invention avoids the effect of cyclodextrin or its derivatives on the kidney, thereby facilitating the development of pharmaceuticals.

Claims

1. 1. An agent for suppressing kidney damage induced by cyclodextrin or a derivative thereof, comprising cilastatin or a pharmaceutically acceptable salt thereof as an active ingredient.

2. 2. The inhibitor according to claim 1, wherein the cyclodextrin or a derivative thereof is β-cyclodextrin or a derivative thereof.

3. 3. The inhibitor according to claim 2, wherein the β-cyclodextrin or a derivative thereof is selected from the group consisting of methyl-β-cyclodextrin, dimethyl-β-cyclodextrin, trimethyl-β-cyclodextrin, partially methylated β-cyclodextrin, ethyl-β-cyclodextrin, diethyl-β-cyclodextrin, triethyl-β-cyclodextrin, (2-hydroxyethyl)-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, (3-hydroxypropyl)-β-cyclodextrin, (2,3-dihydroxypropyl)-β-cyclodextrin, (2-hydroxyisobutyl)-β-cyclodextrin, sulfobutyl ether β-cyclodextrin, glucosyl-β-cyclodextrin, and maltosyl-β-cyclodextrin.

4. The inhibitor according to any one of claims 1 to 3, which is in the form of an injection.

5. A pharmaceutical composition comprising cilastatin or a pharmaceutically acceptable salt thereof and a cyclodextrin or a derivative thereof.

6. An inhibitor of the uptake of cyclodextrin or a derivative thereof into renal tissue, comprising cilastatin or a pharmaceutically acceptable salt thereof as an active ingredient.

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