Preventive or therapeutic agent for the side effects of anthracycline anticancer drugs

Abstract

[Problem] To provide a safe prophylactic agent or therapeutic agent for side effects of an anthracycline anticancer agent, the agent having a broad range of applications. [Solution] In a treatment of a cancer using an anthracycline anticancer agent, 5-aminolevulinic acid is used in combination.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating side effects of anthracycline anticancer drugs. [Background technology]

[0002] Anthracycline anticancer drugs, including doxorubicin, are used to treat many cancers, including leukemia, lymphoma, breast cancer, uterine cancer, ovarian cancer, and lung cancer. Anthracycline anticancer drugs are prone to cause dose-dependent side effects, and exceeding a certain dosage can cause irreversible myocardial contractile disorder called anthracycline (doxorubicin) cardiomyopathy, with extremely poor prognosis after onset. For this reason, the cumulative dosage of anthracycline anticancer drugs is strictly limited by clinical guidelines (500 mg / m for doxorubicin). 2 (See below). However, particularly for hematologic tumors that repeatedly undergo remission and relapse, it is not uncommon for this dose limit to be reached. This limitation often forces the abandonment of first-line treatment regimens, resulting in inadequate chemotherapy for malignant tumors. Furthermore, despite strict dose restrictions, it has been reported that nearly 20% of patients receiving anthracycline anticancer drugs experience cardiotoxicity, with the majority occurring within one year of the end of chemotherapy. Therefore, to ensure adequate chemotherapy for malignant tumors without the onset of cardiomyopathy, it is essential to establish a method for preventing anthracycline (doxorubicin)-induced cardiomyopathy. However, the mechanism by which cardiotoxicity manifests has not been clearly elucidated, and no effective treatment has yet been established.

[0003] Dexrazoxane is known as a therapeutic agent for treating extravasation of anthracycline anticancer drugs administered intravenously (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Langer SW Dexrazoxane for the treatment of chemotherapy-related side effects. Cancer Manag. Res. 2014;6:357-363. Summary of the Invention [Problem to be solved by the invention]

[0005] As mentioned above, dexrazoxane is known as a treatment for the side effects of anthracycline anticancer drugs, but in Japan, its application is limited to drug extravasation. Dexrazoxane is approved overseas, primarily in the United States, as a preventive drug for doxorubicin-induced cardiomyopathy. However, concerns remain that dexrazoxane may weaken the antitumor efficacy of doxorubicin. Furthermore, dexrazoxane itself has been shown to cause side effects such as bone marrow suppression, nausea, injection site fever and pain, and vomiting. It has also been reported to potentially induce secondary malignancies. Therefore, the FDA strictly restricts its use. There is a strong need for the development of drugs that suppress the cardiotoxicity of anthracycline anticancer drugs and for their safe use in combination with other drugs. Therefore, there is a need for broader and safer preventive or therapeutic agents for anticancer drug side effects. [Means for solving the problem]

[0006] The present inventors have surprisingly found that by combining an anthracycline anticancer drug with 5-aminolevulinic acids (ALAs), it is possible to reduce the side effects of the anthracycline anticancer drug while maintaining its efficacy.

[0007] That is, in one embodiment, the present invention relates to a pharmaceutical composition for reducing the toxicity of anthracycline anticancer agents, which comprises 5-aminolevulinic acids or derivatives thereof, or salts thereof as an active ingredient.

[0008] In one embodiment of the present invention, the pharmaceutical composition further comprises an iron compound.

[0009] In one embodiment of the present invention, the toxicity is a decrease in cardiac function or an increase in lipid peroxidation.

[0010] In one embodiment of the present invention, the pharmaceutical composition is administered orally or intravenously.

[0011] In one embodiment of the present invention, the 5-aminolevulinic acids or derivatives thereof, or salts thereof are prepared so as to be administered to the subject at a dose of 0.5 mg / kg to 20 mg / kg.

[0012] In one embodiment of the present invention, the pharmaceutical composition contains 5 mg to 1500 mg of 5-aminolevulinic acids or derivatives thereof, or salts thereof.

[0013] In one embodiment of the present invention, the pharmaceutical composition is administered to the subject simultaneously with or 2 hours to 7 days before the administration of the anthracycline anticancer drug.

[0014] In one embodiment of the present invention, the pharmaceutical composition is administered to the subject daily for a period of 1 to 15 days prior to administration of the anthracycline anticancer drug.

[0015] In one embodiment of the present invention, the pharmaceutical composition is administered to the subject continuously from at least 3 days (preferably 7 days) before administration of the anthracycline anticancer drug to at least 7 days (preferably 14 days) after administration of the anthracycline anticancer drug.

[0016] In one embodiment of the present invention, the administration cycle is repeated every 1 to 4 weeks.

[0017] In one embodiment of the present invention, the 5-aminolevulinic acid compound is a compound represented by the following formula (I): [ka] (In the formula, R 1 represents a hydrogen atom or an acyl group, and R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group), or a pharmacologically acceptable salt or ester thereof.

[0018] In one embodiment of the present invention, the iron compound is one or more iron compounds selected from the group consisting of ferrous citrate, ferrous sodium citrate, sodium ferrous citrate, ferrous ammonium citrate, ferric pyrophosphate, heme iron, iron dextran, ferrous lactate, ferrous gluconate, iron DTPA, sodium ferrous diethylenetriaminepentaacetate, ammonium ferrous diethylenetriaminepentaacetate, sodium ferrous ethylenediaminetetraacetate, ammonium ferrous ethylenediaminepentaacetate, triethylenetetraamineiron, sodium ferrous dicarboxymethylglutamate, ammonium ferrous dicarboxymethylglutamate, iron lactoferrin, iron transferrin, ferric chloride, ferric oxide, sodium iron chlorophyllin, iron ferritin, ferrous fumarate, ferrous pyrophosphate, saccharified iron oxide, iron acetate, iron oxalate, ferrous succinate, sodium ferrous citrate succinate, iron sulfate, and iron glycine sulfide.

[0019] In one embodiment of the present invention, the anthracycline anticancer drug is an anticancer drug selected from the group consisting of doxorubicin, aclarubicin, pirarubicin, idarubicin, epirubicin, daunorubicin, amrubicin, derivatives thereof, and pharmacologically acceptable salts thereof.

[0020] Another embodiment of the present invention relates to a pharmaceutical composition for inhibiting tumor growth, comprising an anthracycline anticancer drug as an active ingredient, wherein the pharmaceutical composition is used in combination with 5-aminolevulinic acids or derivatives thereof, or salts thereof.

[0021] In one embodiment of the present invention, the 5-aminolevulinic acid or a derivative thereof, or a salt thereof is administered to the subject together with an iron compound.

[0022] In one embodiment of the present invention, the 5-aminolevulinic acids or derivatives thereof, or salts thereof are administered to reduce the toxicity of the anthracycline anticancer drug.

[0023] In one embodiment of the present invention, the toxicity is a decrease in cardiac function or an increase in lipid peroxidation.

[0024] In one embodiment of the present invention, the 5-aminolevulinic acids or derivatives thereof, or salts thereof are administered orally.

[0025] In one embodiment of the present invention, the therapeutically effective amount of the anthracycline anticancer agent is administered in combination with 0.5 mg / kg to 20 mg / kg of the 5-aminolevulinic acids or derivatives thereof, or salts thereof.

[0026] One embodiment of the present invention is characterized in that the 5-aminolevulinic acids or derivatives thereof, or salts thereof are administered simultaneously with or 2 hours to 7 days before the administration of the anthracycline anticancer drug.

[0027] In one embodiment of the present invention, the 5-aminolevulinic acids or derivatives thereof, or salts thereof are administered daily for a period of 1 to 15 days (preferably 1 to 3 days) before the administration of the anthracycline anticancer drug.

[0028] In one embodiment of the present invention, the 5-aminolevulinic acids or derivatives thereof, or salts thereof are administered to the subject continuously from at least 3 days (preferably 7 days) before administration of the anthracycline anticancer drug to at least 7 days (preferably 14 days) after administration of the anthracycline anticancer drug.

[0029] In one embodiment of the present invention, the administration cycle is repeated every 1 to 4 weeks.

[0030] Another embodiment of the present invention relates to a method for reducing the toxicity of an anthracycline anticancer drug in a subject, the method comprising administering to the subject 5-aminolevulinic acids or derivatives thereof, or salts thereof, simultaneously or asynchronously with the administration of an anthracycline anticancer drug to the subject.

[0031] Another embodiment of the present invention relates to a method for inhibiting tumor growth in a subject, comprising the step of administering to the subject 5-aminolevulinic acids or derivatives thereof, or salts thereof, and an anthracycline anticancer drug simultaneously or at different times.

[0032] Any combination of one or more of the above-described features of the present invention is also included within the scope of the present invention. [Effects of the Invention]

[0033] 5-aminolevulinic acid (ALA) is a natural amino acid used in the body as a precursor to heme and is also used in a variety of applications, including pharmaceuticals and supplements. Because 5-aminolevulinic acid has been confirmed to be highly safe, it can be used in a wide range of subjects as a preventative or therapeutic agent for the side effects of anthracycline anticancer drugs. [Brief explanation of the drawings]

[0034] [Figure 1]FIG. 1 shows that the increase in lipid peroxides caused by doxorubicin was suppressed by administration of 5'ALA. [Figure 2] FIG. 2 shows that cell death caused by doxorubicin was suppressed by administration of 5'ALA. [Figure 3] FIG. 3 shows that the decrease in left ventricular contractility, indicated by left ventricular ejection fraction (LVEF), caused by doxorubicin was improved by administration of 5'ALA in an in vivo experimental system. [Figure 4] FIG. 4 shows that the increase in lipid peroxides (acrolein, MDA) caused by doxorubicin was suppressed by administration of 5'ALA in an in vivo experimental system. [Figure 5] FIG. 5 shows that cell death caused by various anthracycline anticancer drugs was suppressed by administration of 5'ALA. [Figure 6] FIG. 6 shows that the action of doxorubicin reduces ALAS1, the rate-limiting enzyme in the heme synthesis pathway, in cardiomyocytes and myocardial tissue. [Figure 7] FIG. 7 shows that doxorubicin reduces protoporphyrin IX (PpIX), an intermediate metabolite in the heme synthesis pathway, in cardiomyocytes. [Figure 8] FIG. 8 shows that the action of doxorubicin causes iron overload and impaired heme synthesis in cardiomyocytes, but the action of 5′-ALA normalizes this condition. [Figure 9] FIG. 9 shows that the action of doxorubicin causes iron overload and impaired heme synthesis in myocardial tissue, but the action of 5′-ALA normalizes this condition. [Figure 10] FIG. 10 shows that 5′-ALA does not attenuate the cell death-inducing effect of doxorubicin. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention is based on the discovery that the combined use of an anthracycline anticancer drug and a 5-aminolevulinic acid (ALA) can reduce side effects while maintaining the efficacy of the anthracycline anticancer drug. That is, the present invention relates to the use of ALA as a preventive or therapeutic agent for the side effects of anthracycline anticancer drugs, or to a combination of an anthracycline anticancer drug and an ALA. Alternatively, the present invention relates to the use of ALA in a standard treatment regimen using an anthracycline anticancer drug.

[0036] In the present disclosure, the anthracycline anticancer drug and the ALAs may be administered to a subject simultaneously or at different times. However, it is preferable that the ALAs be administered to a subject prior to the administration of the anthracycline anticancer drug. When the ALAs are administered once, for example, the ALAs may be administered to the subject simultaneously with the administration of the anthracycline anticancer drug to the subject, or 2 hours to 7 days before the administration. Note that, in the present disclosure, the term "simultaneously" does not mean strict simultaneousness in minutes, but substantial simultaneousness taking into consideration the clinical situation (e.g., administering the ALAs within 2 hours before or after the administration of the anthracycline anticancer drug). When the ALAs are administered multiple times, for example, the ALAs may be administered to the subject continuously starting at least 3 days (preferably 7 days) before the administration of the anthracycline anticancer drug. Furthermore, ALA may be administered to the subject for at least 7 days (preferably 14 days) after the administration of the anthracycline anticancer drug. When an anthracycline anticancer drug is administered multiple times for cancer treatment, the treatment cycle using the combination of the anthracycline anticancer drug and ALA may be repeated multiple times.

[0037] Generally, anthracycline anticancer drugs refer to a group of antitumor antibiotics derived from microorganisms of the genus Streptomyces, and are also called anthracycline antibiotics. Representative anthracycline anticancer drugs include doxorubicin, aclarubicin, pirarubicin, idarubicin, epirubicin, daunorubicin, amrubicin, etc., but the anthracycline anticancer drugs of the present invention are not limited to these and may include all drugs generally classified as anthracycline anticancer drugs. In addition, the scope of the present invention broadly includes derivatives having pharmacological activity similar to these compounds, formulations of these compounds, etc.

[0038] The present invention is applicable to any type of cancer for which the effectiveness of anthracycline anticancer drugs has been confirmed, including, but not limited to, malignant lymphoma, lung cancer, digestive tract cancer (stomach cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, hepatocellular carcinoma, colon cancer, etc.), bladder cancer, urothelial cancer, osteosarcoma, breast cancer, uterine cancer, bone and soft tissue tumors, bone tumors, multiple myeloma, and pediatric solid tumors (Ewing's sarcoma family tumors, rhabdomyosarcoma, neuroblastoma, retinoblastoma, hepatoblastoma, nephroblastoma, etc.).

[0039] Side effects of anthracycline anticancer drugs are known to include cardiotoxicity (e.g., decreased cardiac function, increased lipid peroxides in cardiomyocytes or myocardial tissue, and increased free iron in mitochondria in cardiomyocytes or myocardial tissue) and bone marrow suppression, and the present invention may be particularly applied to the prevention or treatment of cardiotoxicity.

[0040] The route of administration of the anthracycline anticancer drug to a subject is not limited, and may be, for example, the administration route in accordance with the instructions in the package insert of the drug (e.g., intravenous administration). The dose of the anthracycline anticancer drug to a subject is also not limited, and an optimal dose may be determined by a person skilled in the art (e.g., a physician) depending on the treatment stage, symptoms, etc. of the subject.

[0041] As used herein, ALA refers to 5-aminolevulinic acid, also known as δ-aminolevulinic acid, which is a naturally occurring amino acid.

[0042] In the present invention, the derivative of ALA refers to a compound that is metabolized in vivo to produce PpIX, and an example thereof is a compound represented by the following formula (I): 1 represents a hydrogen atom or an acyl group, and R 2 represents a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. 1 and R 2 corresponds to the case of a hydrogen atom. [ka]

[0043] R in formula (I) 1 Examples of the acyl group in the formula (I) include linear or branched alkanoyl groups having 1 to 8 carbon atoms, such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl, and benzylcarbonyl groups, and aroyl groups having 7 to 14 carbon atoms, such as benzoyl, 1-naphthoyl, and 2-naphthoyl groups.

[0044] R in formula (I) 2 Examples of the alkyl group in the formula (I) include linear or branched alkyl groups having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl groups.

[0045] R in formula (I) 2 Examples of the cycloalkyl group in the formula (I) include cycloalkyl groups having 3 to 8 carbon atoms, which may have a saturated or partially unsaturated bond, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclododecyl, and 1-cyclohexenyl groups.

[0046] R in formula (I) 2 Examples of the aryl group in the formula (I) include aryl groups having 6 to 14 carbon atoms, such as phenyl, naphthyl, anthryl, and phenanthryl groups.

[0047] R in formula (I) 2 In the aralkyl group, the aryl portion can be exemplified by the same aryl groups as above, and the alkyl portion can be exemplified by the same alkyl groups as above. Specific examples include aralkyl groups having 7 to 15 carbon atoms, such as benzyl, phenethyl, phenylpropyl, phenylbutyl, benzhydryl, trityl, naphthylmethyl, and naphthylethyl groups.

[0048] Among ALA compounds, salts of ALA or its derivatives include pharmacologically acceptable acid addition salts, metal salts, ammonium salts, and organic amine addition salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, and sulfate, and organic acid addition salts such as formate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, and malate. Examples of metal salts include alkali metal salts such as lithium salt, sodium salt, and potassium salt, alkaline earth metal salts such as magnesium and calcium salt, and metal salts such as aluminum and zinc. Examples of ammonium salts include ammonium salts and alkyl ammonium salts such as tetramethylammonium salt. Examples of organic amine salts include triethylamine salts, piperidine salts, morpholine salts, and toluidine salts.

[0049] Examples of esters of ALA include, but are not limited to, methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, hexyl ester, and heptyl ester.

[0050] Since ALAs are highly safe substances, there are no limitations on the route of administration to a subject, and various administration routes can be selected. Examples include oral administration including sublingual administration, inhalation administration, direct administration to target tissues or organs via a catheter, intravenous administration including intravenous drip infusion, transdermal administration using patches, suppositories, and parenteral administration such as forced enteral nutrition using a nasogastric tube, nasoenteric tube, gastrostomy tube, or enterostomy tube. Oral administration is the simplest administration method.

[0051] The optimal dosage of ALAs to be administered to a subject may be determined by a person skilled in the art (e.g., a physician) depending on the treatment stage, symptoms, etc. Specifically, the dosage may be 0.1 mg to 1000 mg / day, preferably 0.2 mg to 500 mg / day, 0.3 mg to 250 mg / day, even more preferably 0.4 mg to 100 mg / day, and most preferably 0.5 mg to 20 mg / day in terms of ALA per kg of subject body weight.

[0052] In the present disclosure, ALAs may be administered to a subject together with an iron compound. Non-limiting examples of iron compounds include ferrous citrate, sodium ferrous citrate (SFC), sodium iron citrate, ammonium iron citrate, ferric pyrophosphate, heme iron, iron dextran, iron lactate, ferrous gluconate, iron DTPA, sodium iron diethylenetriaminepentaacetate, ammonium iron diethylenetriaminepentaacetate, sodium iron ethylenediaminetetraacetate, ammonium iron ethylenediaminepentaacetate, triethylenetetraamineiron, sodium iron dicarboxymethylglutamate, ammonium iron dicarboxymethylglutamate, iron lactoferrin, iron transferrin, ferric chloride, ferric oxide, sodium iron chlorophyllin, iron ferritin, ferrous fumarate, ferrous pyrophosphate, saccharified iron oxide, iron acetate, iron oxalate, ferrous succinate, sodium iron citrate succinate, iron sulfate, and iron glycine sulfide.

[0053] The terms used in this specification, unless otherwise defined, are used to describe particular embodiments and are not intended to limit the invention.

[0054] Furthermore, the term "comprise" used in this specification intends that the described items (components, steps, elements, numbers, etc.) are present, unless the context clearly dictates otherwise, and does not exclude the presence of other items (components, steps, elements, numbers, etc.).

[0055] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in this specification and the related technical field, and should not be interpreted in an idealized or overly formal sense, unless otherwise defined.

[0056] The present invention will be described in more detail below with reference to examples. However, the present invention may be embodied in various forms and should not be construed as being limited to the examples set forth herein. [Example]

[0057] Hearts removed from neonatal rats were isolated using trypsin and collagenase treatment and plated on petri dishes. After several days of culture, the medium was replaced with serum-free medium (DMEM). 24 hours later, doxorubicin (2 μM final concentration) was added, and the cells were harvested in RIPA buffer. Aminolevulinic acid (5'ALA, 1 mM) or ferrostatin-1 (Fer-1, 50 μM) was added 1 hour before doxorubicin. Malondialdehyde (MDA) was measured using the harvested samples using a thiobarbituric acid reactive substances (TBARS) assay kit (Cayman Chemical). Protein concentrations were measured using the BCA method, and MDA concentrations were corrected for protein concentration (nmol / mg protein) and normalized to the mean value of the control group.

[0058] The test results are shown in Figure 1. As shown in the figure, the addition of 5'ALA suppressed the increase in lipid peroxides in cardiac cells caused by doxorubicin. Furthermore, the inhibitory effect was similar to that of Fer-1, a known ferroptosis inhibitor.

[0059] Hearts removed from neonatal rats were isolated using trypsin and collagenase treatment and plated on petri dishes. After several days of culture, the medium was replaced with serum-free medium (DMEM). 24 hours later, doxorubicin (2 μM final concentration) was added. Aminolevulinic acid (5'ALA, 1 mM) or ferrostatin-1 (Fer-1, 50 μM) was added 1 hour before doxorubicin addition. 24 hours after doxorubicin addition, a cell death assay was performed using Cell Counting Kit-F (Dojindo) and a plate reader (Varioskan LUX Multimode Microplate Reader) based on fluorescence intensity. Data were presented as normalized values ​​for the control group.

[0060] The test results are shown in Figure 2. As shown in the figure, the addition of 5'ALA suppressed doxorubicin-induced cardiac cell death (ferroptosis). Furthermore, the inhibitory effect was similar to that of Fer-1, a known ferroptosis inhibitor.

[0061] Eight-week-old C57BL / 6J mice were administered doxorubicin (6 mg / kg / dose) via the tail vein three times on days 0, 2, and 4. Aminolevulinic acid was dissolved in the drinking water bottle at a dose of 300 mg / kg / day, assuming a daily water intake of 4 ml / day. Administration of aminolevulinic acid in the drinking water began three days before doxorubicin administration. Cardiac echocardiography (Vevo 1100, FUJIFILM VisualSonics) was performed on days 7 and 14 after the start of doxorubicin administration.

[0062] The test results are shown in Figure 3. As shown in the figure, pre-administration of 5'ALA significantly improved the decline in left ventricular contractility in the doxorubicin-induced cardiomyopathy model.

[0063] After echocardiography on day 14, mice were euthanized and tissues were collected and immediately frozen in liquid nitrogen. Proteins were extracted from the collected frozen tissues using RIPA buffer, and the extracted proteins were immunostained using acrolein (JaICA, MAR-020n) by Western blotting. Quantitative analysis of the images was performed using ImageJ (NIH). GAPDH (Santa Cruz Biotechnology Inc., sc-32233) was used as an endogenous control. Similarly, proteins were extracted from the collected frozen tissues using RIPA buffer, and the extracted samples were used to measure malondialdehyde (MDA) using a thiobarbituric acid reactive substances (TBARS) assay kit (Cayman Chemical). Protein concentrations were measured using the BCA method, and MDA concentrations were corrected for protein concentration (nmol / mg protein) and normalized to the mean value of the control group.

[0064] The test results are shown in Figure 4. As shown in the figure, pre-administration of 5'ALA significantly suppressed the increase in lipid peroxides (acrolein, MDA) in cardiac cells of the doxorubicin-induced cardiomyopathy model.

[0065] Neonatal rat hearts were isolated using trypsin and collagenase and plated on petri dishes. After several days of culture, the medium was replaced with serum-free medium (DMEM). 24 hours later, doxorubicin (DOX; 1 or 2 μM), daunorubicin (DNR; 1 or 2 μM), pirarubicin (THP; 1 or 2 μM), idarubicin (IDR; 1 or 2 μM), and epirubicin (EPI; 1 or 2 μM) were added (all final concentrations). Aminolevulinic acid (5'ALA, 1 mM) was added 2 hours before doxorubicin addition. 26 hours after doxorubicin addition, cell death was assessed by measuring fluorescence intensity using the Cell Counting Kit-F (Dojindo) on a Varioskan LUX Multimode Microplate Reader. Data were normalized to 1 for the control group.

[0066] The test results are shown in Figure 5. As shown in the figure, in all treatment groups, cardiac cell death (ferroptosis) was observed as the dose of the anticancer drug increased. However, in all treatment groups, cardiac cell death was suppressed by pre-administration of 5'ALA.

[0067] After doxorubicin (2 μM, 24 hours) was added to primary isolated rat cardiomyocytes, proteins were extracted using RIPA buffer, and the extracted proteins were immunostained using Alas1 (Abcam, ab84962) by Western blot analysis. Quantitative analysis of the resulting images was performed using Image J (NIH). β-actin (Santa Cruz, sc-47778) was used as an endogenous control. The results are shown in Figure 6A.

[0068] Next, 8-week-old C57BL / 6J mice were administered doxorubicin (6 mg / kg / dose) via the tail vein three times on days 0, 2, and 4. After euthanasia on day 7, the mice were harvested and immediately frozen in liquid nitrogen. Proteins were extracted from the harvested frozen cardiac tissue using RIPA buffer, and the extracted proteins were immunostained using Alas1 (Abcam, ab84962) by Western blotting. Quantitative analysis of the resulting images was performed using Image J (NIH). GAPDH was used as an endogenous control. The results are shown in Figure 6B.

[0069] As shown in Figures 6A and 6B, it was revealed that the action of doxorubicin reduced ALAS1, the rate-limiting enzyme in the heme synthesis pathway, in cardiomyocytes and myocardial tissue.

[0070] Doxorubicin (2 μM, 24 hours) was administered to isolated primary rat cardiomyocytes, which were then harvested in PBS. A mixture of 50% acetic acid and dimethylformamide (DMF) / 2-propanol (IPA) was added to the harvested sample and vortexed vigorously. After centrifugation, the supernatant was collected (Supernatant 1). The DMF / IPA mixture was added to the pellet and centrifuged again. The supernatant (Supernatant 2) was combined with Supernatant 1 to obtain the protoporphyrin IX extract. After deproteinization with acetanilyl, the extract was centrifuged at 20,000 g for 20 minutes. The supernatant was then analyzed for protoporphyrin IX (PPIX) in cardiomyocytes using a mass spectrometer (column: 1.8 μM, 2.1 × 100 mm; Waters, MA, USA; instrument: Q exactive Ultimate 3000; Thermo Fisher Scientific). The results are shown in Figure 7.

[0071] As shown in FIG. 7, it was revealed that the action of doxorubicin reduces protoporphyrin IX (PpIX), an intermediate metabolic substance in the heme synthesis pathway, in cardiomyocytes.

[0072] 5'-ALA (1 mM) was added to isolated primary rat cardiomyocytes, followed by doxorubicin (2 μM) 1 hour later. 24 hours after doxorubicin addition, the cells were washed once with PBS and cultured for 30 minutes in medium containing Mito-FerroGreen (5 μM), which fluorescently stains mitochondrial iron. After washing again with PBS, the cells were observed using a fluorescence microscope (BZ-X800, Keyence) (Figure 8A). The signal intensity of the images obtained was quantitatively analyzed using Image J (NIH). The results are shown in Figure 8B.

[0073] As shown in Figures 8A and 8B, it was revealed that the action of doxorubicin causes an iron overload state in cardiomyocytes, but the action of 5'-ALA normalizes this iron overload state.

[0074] 5'-ALA (1 mM) was added to isolated primary rat cardiomyocytes, followed by doxorubicin (2 μM) 1 hour later. 24 hours after doxorubicin addition, cells were harvested with PBS, and mitochondria were isolated using a mitochondria isolation kit (P507L, 101 Bio, LLC, Mountain View, CA, USA). Protein was extracted from the isolated mitochondria with RIPA buffer, and mitochondrial heme was measured using a QuantiChrom Heme Assay Kit (BioAssay Systems, Hayward, CA, USA). Five minutes after adding alkaline solution to the protein extract, absorbance at 400 nm was measured using a Varioskan LUX Multimode Microplate Reader, and heme content was estimated using a calibration curve. The measured heme content was corrected by dividing by the protein concentration of the protein extract. The results are shown in Figure 8C.

[0075] As shown in FIG. 8C, it was revealed that the action of doxorubicin causes impaired heme synthesis in cardiomyocytes, but the impairment is normalized by the action of 5′-ALA.

[0076] Eight-week-old C57BL / 6J mice received three doses of doxorubicin (6 mg / kg / dose) via the tail vein on days 0, 2, and 4. 5'-ALA was administered via drinking water (1.875 mg / mL, assuming a daily water intake of 4 mL / day per 25 g mouse) starting 3 days before the first dose. After euthanasia on day 14, the mice were harvested and immediately frozen in liquid nitrogen. Mitochondria were isolated from the harvested frozen cardiac tissue by centrifugation in HES buffer, and protein was extracted using RIPA buffer. The protein extract was adjusted to pH 2-3 with HCl, and the supernatant was centrifuged. The absorbance at 560 nm was measured using the Ferrozine method with a metalloassay kit (Metallo Assay Iron LS, Metallogenics, Chiba, Japan). Total iron content was estimated using a calibration curve. The total iron content was corrected by dividing it by the protein concentration of the protein extract. The results are shown in Figure 9A.

[0077] As shown in FIG. 9A, it was revealed that the action of doxorubicin causes an iron overload state in myocardial tissue, but the action of 5′-ALA normalizes the iron overload state.

[0078] Eight-week-old C57BL / 6J mice received three doses of doxorubicin (6 mg / kg / dose) via the tail vein on days 0, 2, and 4. 5'-ALA was administered via drinking water (1.875 mg / mL, assuming a daily water intake of 4 mL per 25 g mouse) starting 3 days before the first dose. After euthanasia on day 14, the mice were harvested and immediately frozen in liquid nitrogen. Mitochondria were isolated from the harvested frozen cardiac tissue by centrifugation using HES buffer. Protein was extracted from the isolated mitochondria using RIPA buffer, and mitochondrial heme was measured using the QuantiChrom Heme Assay Kit (BioAssay Systems, Hayward, CA, USA). After adding the alkaline solution to the protein extract, the absorbance at 400 nm was measured using a plate reader (Varioskan LUX Multimode Microplate Reader) 5 minutes later, and the heme content was estimated using a calibration curve. The measured heme content was corrected by dividing it by the protein concentration of the protein extract.

[0079] As shown in FIG. 9B, it was revealed that the action of doxorubicin causes impaired heme synthesis in myocardial tissue, but the action of 5′-ALA normalizes this impairment.

[0080] EL-4 cells, a mouse malignant lymphoma cell line, were treated with 5'-ALA (1 mM) and then doxorubicin (1 μM) 1 hour later. Cells were harvested 24 hours after doxorubicin addition, stained for dead cells with Trypan Blue, and the number of live and dead cells was counted using an automated cell counter (Countess II FL, Thermo Fisher Scientific). Cell viability was assessed as the percentage (%) of live cells out of the total cell count. The results are shown in Figure 10A.

[0081] HeLa cells, a type of human cervical cancer, were treated with 5'-ALA (1 mM) and then doxorubicin (2 μM) 1 hour later. Cell death was assessed 24 hours after doxorubicin addition using the Cell Counting Kit-F (Dojindo) and a plate reader (Varioskan LUX Multimode Microplate Reader) to measure fluorescence intensity (ex 490 nm / em 520 nm). Data were normalized to the control group as 1. The results are shown in Figure 10B.

[0082] As shown in Figures 10A and 10B, the cell death-inducing effect of doxorubicin was observed in cancer cells (EL-4 cells, which are mouse malignant lymphoma cells, and HeLa cells, which are human cervical cancer cells), but this effect was not attenuated by the application of 5'-ALA.

Claims

1. A pharmaceutical composition for reducing the toxicity of an anthracycline anticancer drug, comprising 5-aminolevulinic acid or a pharmacologically acceptable salt or ester thereof as an active ingredient, The toxicity is a decrease in cardiac function or an increase in lipid peroxidation. Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , which is administered orally.

3. 3. The pharmaceutical composition according to claim 1, wherein the 5-aminolevulinic acid or a pharmacologically acceptable salt or ester thereof is prepared so as to be administered to a subject at a dose of 0.5 mg / kg to 20 mg / kg.

4. 3. The pharmaceutical composition according to claim 1, which contains 5 mg to 1500 mg of 5-aminolevulinic acid or a pharmacologically acceptable salt or ester thereof.

5. The pharmaceutical composition according to any one of claims 1 to 4, which is administered to a subject simultaneously with or 2 hours to 7 days before administration of the anthracycline anticancer drug.

6. 5. The pharmaceutical composition of claim 1, wherein the composition is administered daily to a subject for a period of 1 to 15 days prior to administration of the anthracycline anticancer drug.

7. 5. The pharmaceutical composition according to claim 1, wherein the composition is administered to a subject continuously from at least 3 days before administration of the anthracycline anticancer drug to at least 7 days after administration of the anthracycline anticancer drug.

8. The pharmaceutical composition of claim 7, wherein the administration cycle is repeated every 1 to 4 weeks.

9. 9. The pharmaceutical composition according to claim 1, wherein the anthracycline anticancer drug is an anticancer drug selected from the group consisting of doxorubicin, aclarubicin, pirarubicin, idarubicin, epirubicin, daunorubicin, amrubicin, derivatives thereof, and pharmacologically acceptable salts thereof.

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