Inspection method of expression risk of hepatic disorder by abemaciclib or pharmaceutically acceptable salt thereof

Abstract

To provide an inspection method for predicting an expression risk of hepatic disorder by Abemaciclib.SOLUTION: An inspection method of an expression risk of hepatic disorder by Abemaciclib or a pharmaceutically acceptable salt thereof includes: (a) a step of determining whether or not a sample has one or more HLA allele selected from a group consisting of dHLA-DQA1*05:05, HLA-DPB1*05:01 and HLA-A*31:01; and (b) a step of, in the case where the sample has one or more HLA allele selected from a group consisting of HLA-DQA1*05:05, HLA-DPB1*05:01 and HLA-A*31:01, determining that the sample has high expression risk of hepatic disorder by administration of Abemaciclib or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for examining the risk of developing liver damage caused by abemaciclib or a pharmaceutically acceptable salt thereof. [Background technology]

[0002] Abemaciclib (LY2835219, brand name Verzenio, Eli Lilly Japan K.K.), or N-{5-[(4-Ethylpiperazin-1-yl)methyl]pyridin-2-yl}-5-fluoro-4-[4-fluoro-2-methyl-1-(1-methylethyl)-1H-benzimidazol-6-yl]pyrimidin-2-amine, is a small-molecule oral anticancer drug that selectively inhibits cyclin-dependent kinase (CDK) 4 and CDK 6, inhibiting tumor growth by halting cell cycle progression. It was approved in Japan in 2018 for the treatment of hormone receptor-positive, HER2-negative, inoperable or recurrent breast cancer. In 2021, it was approved for use as adjuvant chemotherapy for hormone receptor-positive, HER2-negative, high-risk breast cancer. Hormone receptor-positive, HER2-negative breast cancer is the most common subtype of breast cancer, with more than 50,000 new cases diagnosed annually in Japan. The structure of abemaciclib is as follows:

[0003] [ka]

[0004] Hepatic dysfunction (hereinafter referred to as "liver damage") is listed as a serious side effect of abemaciclib in the drug's package insert (AST increase 7.9%, ALT increase 8.1%), and liver damage is also listed as an "important identified risk" in the drug's risk management plan. Furthermore, the review report by the Pharmaceuticals and Medical Devices Agency (PMDA) states that the incidence of liver damage was higher in clinical trials conducted in Japan than in clinical trials conducted overseas. However, no biomarkers have yet been reported to predict the onset of abemaciclib-induced liver damage.

[0005] Meanwhile, a method is known for predicting the risk of hepatotoxicity caused by lapatinib (GW572016, trade name "Tykerb", Novartis Pharma K.K.), a tyrosine kinase inhibitor indicated for the treatment of inoperable or recurrent breast cancer with confirmed HER2 overexpression, by examining allelic polymorphisms of HLA (Human Leukocyte Antigen) (see Patent Document 1). * 02:01, HLA-DQB1 * 02:02, HLA-DRB1 * Patients with one or more of the 07:01 HLA alleles can be identified as being at high risk for lapatinib-induced hepatotoxicity.

[0006] HLA is distributed in almost all cells and body fluids except for red blood cells, and plays an important role in immune responses, such as the recognition of self and non-self. HLA is composed of a combination of multiple antigens, which are classified into class I (HLA-A, -B, -C, etc.), class II (HLA-DR, -DQ, -DP, etc.), and class III based on differences in protein structure and function. The genes encoding HLA are located on the short arm of human chromosome 6, and each antigen has multiple alleles, making it highly polymorphic. More than 35,000 alleles have been identified for HLA-A, -B, -C, -DR, -DQ, and -DP. Genotype analysis is performed using DNA, and allele names are written in four sections separated by a colon (:). For example, the allele name "HLA-C" is *07:02:01:17" is the gene name "HLA-C" based on the antigen encoded by the gene, followed by an asterisk indicating that it is an allele marking, and then the first section (which identifies the antigen type that can be serologically classified) is " * The first region is ":07", then the second region (which distinguishes alleles with amino acid mutations within the same first region group) is ":02", then the third region is ":01" (which distinguishes alleles with synonymous base mutations without amino acid mutations), and then the fourth region is ":17" (which distinguishes alleles with base mutations outside of exons that encode HLA molecules). Because the structure and function of HLA are determined by the amino acid sequence of the genes that encode each antigen, the research that led to this disclosure examined classifications up to region 2. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 5876827 specification Summary of the Invention [Problem to be solved by the invention]

[0008] The abemaciclib package insert states that patients who experience an increase in liver enzyme levels due to abemaciclib, i.e., aspartate aminotransferase (AST) or alanine aminotransferase (ALT), are at or above Grade 2 (more than three times the upper limit of institutional normal range) according to the National Cancer Institute - Common Terminology Criteria for Adverse Events (NCI-CTCAE) severity classification, should be advised to suspend or discontinue administration depending on the grade (Table 1).

[0009] [Table 1] Note 1) Grades are based on NCI-CTCAE ver. 4.0. Note 2) When no obvious cholestasis is observed

[0010] Therefore, if the risk of developing liver damage caused by abemaciclib can be predicted before administration begins, it will be possible to select a treatment other than abemaciclib for patients at high risk of developing the disease, or for patients who have no other treatment options and are forced to administer abemaciclib, to strive for early detection by conducting frequent blood tests to monitor liver enzyme levels (AST and ALT), thereby enabling individualized treatment and preventing the development and worsening of liver damage.

[0011] An object of the present disclosure is to provide a testing method for predicting the risk of developing liver damage caused by abemaciclib. [Means for solving the problem]

[0012] To achieve the above-mentioned objectives, the present inventors conducted extensive research, focusing on the relationship between the risk of liver damage and HLA alleles in patients receiving abemaciclib, using elevated ALT levels, an enzyme found only in the liver, as an indicator of liver damage. As a result, they discovered that possession of specific HLA alleles is associated with the risk of liver damage during abemaciclib administration. Furthermore, the present inventors discovered that by determining the presence of these HLA alleles in advance, it is possible to predict the risk of liver damage caused by abemaciclib. Based on these findings, the present disclosure has been completed.

[0013] That is, the present disclosure provides a method for detecting HLA-DQA1 * 05:05, HLA-DPB1 * 05:01 and HLA-A * (b) determining whether the subject has one or more HLA alleles selected from the group consisting of HLA-DQA1 * 05:05, HLA-DPB1 * 05:01 and HLA-A *and determining that the subject has a high risk of developing liver damage due to administration of abemaciclib or a pharmaceutically acceptable salt thereof, when the subject has one or more HLA alleles selected from the group consisting of: 31:01; [Effects of the Invention]

[0014] The present disclosure can provide a testing method for predicting the risk of developing abemaciclib-induced liver damage. In the present disclosure, the above-mentioned specific HLA alleles are used as biomarkers. Because HLA genotype analysis can be performed using a subject's blood sample, the above-mentioned specific HLA alleles can serve as non-invasive biomarkers. The present disclosure makes it possible to predict the risk of developing abemaciclib-induced liver damage using a simple method, and further enables personalized treatment that provides highly safe treatment based on the prediction results. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a graph comparing the proportion of patients with HLA-DQA1*05:05 in patients with NCI-CTCAE grade 2 or higher liver damage (case group) and those without (control group). [Figure 2] 1 is a graph comparing the proportion of patients with HLA-DPB1*05:01 in patients with NCI-CTCAE grade 2 or higher liver damage (case group) and those without (control group). [Figure 3] 1 is a graph comparing the proportion of patients with HLA-A*31:01 in patients with NCI-CTCAE grade 2 or higher liver damage (case group) and those without (control group). DETAILED DESCRIPTION OF THE INVENTION

[0016] The testing method according to this embodiment is a method for testing the risk of liver damage caused by abemaciclib or a pharmaceutically acceptable salt thereof, comprising the following steps: (a) The subject is HLA-DQA1 * 05:05, HLA-DPB1 * 05:01 and HLA-A * determining whether the individual has one or more HLA alleles selected from the group consisting of: 31:01; (b) the subject is HLA-DQA1 * 05:05, HLA-DPB1 * 05:01 and HLA-A * and determining that the subject has a high risk of developing liver damage due to administration of abemaciclib or a pharmaceutically acceptable salt thereof when the subject has one or more HLA alleles selected from the group consisting of:

[0017] In the present disclosure, "onset of liver damage" can be defined based on the grade of the severity classification of ALT increase by NCI-CTCAE. For example, when the ALT value in a blood test is more than three times the upper limit of the reference value at the institution, i.e., when the ALT value is NCI-CTCAE grade 2 or higher, it can be determined that liver damage has occurred. Alternatively, when the ALT value in a blood test exceeds the upper limit of the reference value at the institution, i.e., when the ALT value is NCI-CTCAE grade 1 or higher, it can be determined that liver damage has occurred.

[0018] In the present disclosure, the "risk of developing liver damage" may refer to the risk of developing liver damage due to administration of abemaciclib or a pharmaceutically acceptable salt thereof. In the present disclosure, the "'high' risk of developing liver damage" does not mean that the development of liver damage can be predicted with 100% accuracy.

[0019] In the present disclosure, the "test" may be a test for predicting whether or not there is an increased risk of developing liver damage due to administration of abemaciclib or a pharmaceutically acceptable salt thereof.

[0020] In the present disclosure, a "subject" may be any human, and preferably includes a cancer patient, particularly a cancer patient who may soon start drug treatment with abemaciclib or a pharmaceutically acceptable salt thereof, or who is scheduled to start such treatment, or who is currently undergoing such treatment, or who is currently discontinuing such treatment.

[0021] In the method of determining the HLA alleles of a subject in step (a) of the present disclosure, for example, DNA is first extracted from the subject's biological sample by any method, and the HLA gene region is specifically amplified by PCR (Polymerase Chain Reaction), followed by detection of the nucleotide sequence or amino acid sequence by various methods. Examples of biological samples that can be used include blood, blood-derived products (buffy coat, serum, and plasma), lymph, urine, tears, saliva, cerebrospinal fluid, oral mucosa, nasal mucosa, feces, body hair, and tissue. The above-mentioned allele determination techniques can be routinely performed by researchers based on common technical knowledge in the field. Multiple of the above-mentioned allele determination techniques may also be combined. Furthermore, the above-mentioned allele determination only requires that the first two regions be determined; it is not necessary to determine the third and subsequent regions.

[0022] In the present disclosure, the HLA alleles to be determined whether a subject has HLA-DQA1 * 05:05, HLA-DPB1 * 05:01 and HLA-A * 31:01, in particular HLA-DQA1 * 05:05 is preferred. These HLA alleles are registered in the IPD-IMGT / HLA database (http: / / www.ebi.ac.uk / ipd / imgt / hla / allele.html), and their nucleotide sequences and amino acid sequences are also available from the same database.

[0023] In step (a) of the present disclosure, methods for determining whether a subject has the above-mentioned specific HLA allele include, for example, DNA extraction from a biological sample, DNA amplification of the HLA gene region, and detection of the nucleotide sequence or amino acid sequence; more specifically, the PCR-SSP (Sequence-Specific Primer) method, which utilizes specific amplification by PCR; the PCR-SSO (Sequence-Specific Oligonucleotide Probes) method, which uses hybridization with a detection probe complementary to the sequence specificity; the PCR-rSSO (reverse SSO) method, which is an improved version of the PCR-SSO method; the PCR-Luminex method, which is an advanced version of the PCR-SBT method; or the PCR-SBT (Sequence-Based Typing) method, which determines the sequence using a sequencer; and the NGS (Next Generation Sequencer)-SBT method.

[0024] The PCR-SSP method is based on the principle that only primers with a perfectly matched sequence to the target sequence will produce an amplification product under controlled PCR conditions. Sequence-specific primer pairs are designed to selectively amplify target sequences specific to one or more of the specific HLA alleles listed above. The presence or absence of PCR amplification can be determined by the presence or absence of a band in electrophoresis. A control primer pair with a nonspecific sequence present in all samples serves as an internal PCR control to verify the efficiency of PCR amplification. Those skilled in the art can perform the PCR-SSP method using commercially available reagent kits, such as the MicroSSP™ from One Lambda, Inc. (California, USA). Data obtained using the MicroSSP method are interpreted using One Lambda's HLA Fusion™ software. Reagent kits, such as One Lambda's LinkSeq™, are also commercially available, which perform the conventional SSP method using a real-time PCR device, eliminating the need for electrophoresis and visual confirmation of amplification. Data obtained using LinkSeq are interpreted using the dedicated analysis software SureTyper.

[0025] The PCR-SSO method involves, for example, PCR amplification targeting a specific region, hybridization of the PCR product to a panel of sequence-specific oligonucleotides (probes) immobilized on beads, detection of the probe-bound PCR product by color development, and data analysis. These probes may be labeled with fluorescent or other labels for detection. Alternatively, labeled molecules that specifically bind to these probes can be contacted with the probes. Those skilled in the art can perform the PCR-SSO method using commercially available reagent kits, such as One Lambda's LABType®. LABType is a DNA typing reagent kit for the rSSO method that uses sequence-specific oligonucleotide (SSO) probes and color-coded microspheres to identify HLA alleles. The SSO method using this kit involves PCR amplification of target DNA in the HLA gene region and subsequent hybridization with a bead probe array. This assay is performed in each well of a 96-well PCR plate, allowing for simultaneous testing of 96 samples. Data obtained using LABType is analyzed using One Lambda's HLA Fusion™ software.

[0026] The PCR-SBT method, for example, involves PCR amplification of a specific region, sequencing of the PCR product by the Sanger method, and data analysis. The NGS-SBT method, for example, involves PCR amplification of a specific region, sequencing of the PCR product by the NGS method, and data analysis. The presence or absence of the above-mentioned specific HLA alleles can be determined by comparing the base sequence with the HLA alleles registered in a database. For example, HLA-DQA1 * The base sequence for 05:05 is SEQ ID NO: 1 (HLA-DQA1 * 05:05:01:01; see IMGT / HLA Accession No. HLA00619). The amino acid sequence encoded by SEQ ID NO: 1 is shown in SEQ ID NO: 2. The specific allele derived by the present disclosure is defined up to the second region, and therefore, HLA-DQA1 *The base sequence of 05:05 may be a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2. For example, HLA-DPB1 * The base sequence of 05:01 is SEQ ID NO: 3 (HLA-DPB1 * 05:01:01:01; see IMGT / HLA Accession No. HLA00523). The amino acid sequence encoded by SEQ ID NO: 3 is shown in SEQ ID NO: 4. Since the specific allele derived by the present disclosure is defined up to the second region, HLA-DPB1 * The base sequence of 05:01 may be a base sequence encoding the amino acid sequence shown in SEQ ID NO: 4. For example, HLA-A * The base sequence for 31:01 is SEQ ID NO: 5 (HLA-A * 31:01:02:01; see IMGT / HLA Accession No. HLA00097). The amino acid sequence encoded by SEQ ID NO: 5 is shown in SEQ ID NO: 6. Since the specific allele derived by the present disclosure is defined up to the second region, * The base sequence used as the reference for 31:01 may be a base sequence encoding the amino acid sequence shown in SEQ ID NO:6.

[0027] Those skilled in the art can perform the PCR-SBT method using commercially available reagent kits such as SeCore™ from Thermo Fisher Scientific (Massachusetts, USA). Data obtained using SeCore is analyzed using One Lambda's uTYPE™ software. Those skilled in the art can perform the NGS-SBT method using commercially available reagent kits such as One Lambda's ALLType™ NGS. Data obtained using ALLType NGS is analyzed using Thermo Fisher Scientific's TypeStream™ Visual software.

[0028] In other embodiments, the presence of a particular HLA allele as described above is determined, for example, by measuring RNA levels. Here, the HLA allele of interest can be detected using a PCR-based assay or reverse transcriptase PCR (RT-PCR). In RT-PCR, RNA is enzymatically converted into cDNA using reverse transcriptase, and the cDNA is used as a template for the PCR reaction. PCR products can be detected by any suitable method, including, but not limited to, gel electrophoresis and staining with a DNA-specific stain or hybridization to a labeled probe. In a further embodiment, quantitative RT-PCR using a standardized mixture of competitive templates can be utilized.

[0029] In another embodiment, the presence of a specific HLA allele is determined, for example, by detecting a protein or polypeptide expression product encoded by the HLA allele. Preferably, the presence of the HLA allele is determined by identifying the amount of one or more polypeptides encoded by one of the specific HLA alleles. For example, immunoassays and devices therefor can be used to detect or quantify proteins or polypeptides in biological samples. These assays and devices can utilize various labeled molecules to generate signals related to the presence or amount of the analyte protein or polypeptide. Alternatively, certain methods and devices, such as optical immunoassays and biosensors, can determine the presence or amount of the analyte without the need for labeled molecules.

[0030] In a preferred embodiment, the presence or amount of a protein or polypeptide can be determined by detecting specific binding using a specific antibody, e.g., using any suitable immunoassay, particularly an enzyme-linked immunoassay (ELISA), a radioimmunoassay (RIA), a competitive binding assay, or the like. Specific immunological binding of an antibody to a protein or polypeptide can be detected by direct or indirect labeling. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. Indirect labels include various enzymes (e.g., alkaline phosphatase), hydrogen peroxide, and the like, which are well known in the art. Furthermore, antibodies can be immobilized on a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay location (e.g., a microtiter well), or a strip of solid substrate material (e.g., a strip of plastic, nylon, or paper). As an example, an assay strip can be used, prepared by coating one or more antibodies onto a solid support. After immersion of the strip in a biological sample, it is washed to produce a measurable signal, such as a colored spot, and through further steps of detecting the signal, the presence or amount of a protein or polypeptide can be rapidly detected or measured.

[0031] The method for determining the HLA alleles of a subject in step (a) of the present disclosure is not limited to the specific methods described above, and these are merely examples, as long as the method can accurately determine up to the second zone. Furthermore, it goes without saying that the present disclosure also includes embodiments in which HLA alleles are determined by new methods developed after the filing of the present application.

[0032] Step (b) of the present disclosure is a step of determining whether the subject is at high risk of developing liver damage due to the administration of abemaciclib or a pharmaceutically acceptable salt thereof based on the HLA allele of the subject. * 05:05, HLA-DPB1 * 05:01 and HLA-A *31:01, the subject can be determined to be at high risk of developing liver damage due to administration of abemaciclib or a pharmaceutically acceptable salt thereof. * 05:05, the subject can be determined to be at particularly high risk of developing liver damage due to administration of abemaciclib or a pharmaceutically acceptable salt thereof. * 05:05, HLA-DPB1 * 05:01 and HLA-A * 31:01, it can be determined that the subject is not at high risk of developing liver damage due to administration of abemaciclib or a pharmaceutically acceptable salt thereof. * When the subject does not have 05:05, it can be determined that the subject is not at high risk of developing liver damage due to administration of abemaciclib or a pharmaceutically acceptable salt thereof.

[0033] If the subject is determined to be at high risk of developing liver damage due to the administration of abemaciclib or a pharmaceutically acceptable salt thereof in step (b), a treatment that does not use abemaciclib or a pharmaceutically acceptable salt thereof can be selected for the subject, or, even if there is no other treatment option and abemaciclib or a pharmaceutically acceptable salt thereof must be administered, individualized measures can be implemented, such as frequent blood tests (for example, weekly during the first two months of administration, which is the most common period, and once every two weeks from the second month onwards) to monitor liver enzyme levels (AST and ALT) in an effort to detect the disease early.

[0034] The testing method of the present disclosure may be performed, for example, before the subject starts treatment with abemaciclib or a pharmaceutically acceptable salt thereof, during treatment, or while treatment is discontinued. [Example]

[0035] The clinical study that led to this disclosure involved 82 patients with inoperable advanced or recurrent breast cancer who were treated with abemaciclib and whose ALT levels were collected at the start and during treatment. This study was conducted in accordance with the ethical guidelines for life science and medical research involving human subjects and was approved by the Showa University Ethics Review Board.

[0036] Two milliliters of blood was collected from each patient, and DNA was extracted and used for HLA allele analysis. HLA allele analysis was performed at the HLA Research Institute, a public interest incorporated foundation, using the NGS-SBT method to determine the alleles of HLA-A, -B, -C, -DRB1, -DQA1, -DQB1, and -DPB1 (a total of seven genes).

[0037] Of the 82 patients mentioned above, 13 patients developed liver damage of NCI-CTCAE grade 2 or higher (ALT levels more than three times the upper limit of the facility's reference range = "case group"), and 47 patients did not develop liver damage of NCI-CTCAE grade 0 (ALT levels within the facility's reference range = "control group"), and Fisher's test was used to examine whether there was a significant difference in the frequency of HLA alleles. Note that no patients developed grade 4 liver damage, so the cases of grade 2 or higher in the case group were those with grades 2 and 3.

[0038] The results are shown in Figures 1 to 3. Figure 1 shows the relationship between HLA-DQA1 in patients with liver damage of grade 2 or higher (case group) and those without liver damage of grade 0 (control group). * Figure 2 shows the HLA-DPB1 expression in patients with grade 2 or higher liver damage (case group) and those without grade 0 liver damage (control group). * Figure 3 shows the HLA-A activity in patients with grade 2 or higher liver damage (case group) and those without grade 0 liver damage (control group). * 1 is a graph comparing the proportions of those with 31:01.

[0039] In the 13 case group, 28 alleles with an allele frequency of 0.1 or higher were analyzed, and the significance level was set at 0.05 / 28 alleles = 0.00178 using Bonferroni correction. As shown in Figure 1, HLA-DQA1 * The proportion of patients with 05:05 was 30.8% in those with grade 2 or higher liver damage and 0.0% in those without grade 0 liver damage, a significant difference was observed (P = 0.00147), with an odds ratio of 45.0 (95% CI = 2.23-907). * The allele possession rate at 05:05 was 8.5% (out of 3078 people), which was not significantly different from the allele possession rate in the control group, suggesting that sampling bias in the control group was small.

[0040] In addition, HLA-DPB1 * 05:01 (P = 0.00930; odds ratio 11.5; 95% CI = 1.38-95.7, Figure 2) and HLA-A * Although the significance level was not met, 31:01 (P = 0.0152; odds ratio 5.86; 95% CI = 1.46-23.4, Figure 3) was suggested to be a useful biomarker related to the risk of liver damage. * 05:01, HLA-A * The allele prevalence rates for 31:01 were 47.5% (out of 2966 people) and 16.0% (out of 40902 people), respectively. No significant difference was observed between the allele prevalence rates in the control group and those in the control group, suggesting that sampling bias in the control group was small.

[0041] These results suggest that HLA-DQA1 may be a biomarker for liver injury induced by abemaciclib or its pharmaceutically acceptable salts. * 05:05 was identified. HLA-DPB1 * 05:01 and HLA-A * 31:01 was also identified as a useful biomarker candidate.

[0042] Although the embodiments of the present disclosure have been described in detail above, the above embodiments are merely examples of the present disclosure, and the present disclosure is not limited to the configurations of the above embodiments. Of course, any design changes that do not deviate from the gist of the present disclosure are also included in the present disclosure. [Industrial Applicability]

[0043] The drug package insert states that if abemaciclib causes liver damage of NCI-CTCAE grade 2 or higher, administration should be suspended or discontinued. Therefore, for patients scheduled for treatment with abemaciclib, HLA-DQA1 should be checked before starting treatment. * 05:05, HLA-DPB1 * 05:01, HLA-A * Liver damage can be prevented by checking for 31:01 carrier status and switching to other treatments for patients with one or more HLA alleles. For patients who cannot switch to other treatments, early detection of liver damage and prevention of its worsening can be achieved by shortening the intervals between outpatient visits and monitoring liver enzyme levels (AST and ALT) through frequent blood tests.

[0044] Based on the above, prior assessment of patients' HLA alleles will improve safety in patients receiving abemaciclib.

Claims

[Claim 1] (a) The subject is HLA-DQA1 ＊ 05:05, HLA-DPB1 ＊ 05:01 and HLA-A ＊ determining whether the individual has one or more HLA alleles selected from the group consisting of: 31:01; (b) the subject is HLA-DQA1 ＊ 05:05, HLA-DPB1 ＊ 05:01 and HLA-A ＊ determining that the subject has a high risk of developing liver damage due to administration of abemaciclib or a pharmaceutically acceptable salt thereof, when the subject has one or more HLA alleles selected from the group consisting of: A method for testing the risk of liver damage caused by abemaciclib or a pharmaceutically acceptable salt thereof, comprising:

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