Agent for inhibiting progression of uremia in felids with severe renal failure

Abstract

The present invention provides an agent for inhibiting the progression of uremia in felids with severe renal failure, said agent containing an apoptosis inhibitor of macrophage (AIM), an AIM fragment having the biological activity of the AIM, or a nucleic acid that encodes the AIM or the AIM fragment.

Description

Agent for inhibiting the worsening of uremia in felines suffering from severe renal failure

[0001] The present invention relates to an agent for suppressing the worsening of uremia in a feline suffering from severe renal failure, and more particularly to an agent for suppressing the worsening of uremia in a feline suffering from severe renal failure, which comprises an apoptosis inhibitor of macrophage (AIM).

[0002] Apoptosis inhibitor of macrophage (AIM, also known as CD5 antigen-like (CD5L)) is a blood protein produced by tissue macrophages and was first identified by the present inventors as a substance that supports macrophage survival. AIM is now recognized as a molecule that induces repair processes in many diseases (Non-Patent Documents 1-3). AIM consists of three cysteine-rich domains (called SRCR domains), and the third SRCR domain at the carboxy terminus contains a unique positively charged amino acid cluster. This cluster forms a charge-based interaction with dead cells, whose surfaces are highly negatively charged due to the exposure of high levels of phosphatidylserine (Non-Patent Documents 1 and 2). AIM is efficiently internalized by phagocytes via multiple scavenger receptors, and this binding strongly enhances the phagocytosis of dead cells by phagocytes (Non-Patent Document 3).

[0003] In humans, AIM normally exists bound to IgM pentamers. However, when acute kidney injury or other conditions occur, it dissociates from the IgM pentamers to become free AIM, which then binds to dead cells and debris, promoting their clearance. This mechanism is conserved in many mammals, but in felines, the binding between AIM and IgM pentamers is so strong that it is known that this mechanism does not function adequately (Non-Patent Document 4). Therefore, felines do not adequately clear dead cell debris from renal tissue. As a result, with aging, renal function declines due to the accumulation of dead cell debris in renal tissue, ultimately leading to severe chronic renal failure, which in turn leads to uremia and death.

[0004] Taking this background into consideration, the present inventors have reported a means for treating or preventing kidney diseases in humans and felines by administering AIM (Patent Document 1).

[0005] However, in cases of severe renal failure where most of the kidney function has been lost, even if AIM is administered, recovery of kidney function cannot be expected, and therefore the progression of uremia cannot be suppressed. As a result, it was thought that there was no effective method for suppressing the onset and progression of uremia in felines suffering from severe renal failure.

[0006] International Publication No. 2015 / 119253

[0007] Arai, S. et al., Nat Med. 2016 Feb;22(2):183-93.Tomita, T. et al. Sci Rep. 2017 Jul 25;7(1):6450.Arai, S. and Miyazaki, T. Semin Immunopathol. 2018 Nov;40(6):567-575Sugisawa R. et al., Sci Rep. 2016 Oct 12;6:35251

[0008] An object of the present invention is to provide a means capable of suppressing the worsening of uremia in felines suffering from severe renal failure.

[0009] As a result of intensive research into the above-mentioned problems, the present inventors have found that when AIM is administered to cats with severe renal failure in which recovery of renal function is unlikely, the concentrations of various uremic toxins in the blood are reduced, even though renal function does not improve, and as a result, the worsening of uremia in cats suffering from severe renal failure can be suppressed. Based on this finding, further research has led to the completion of the present invention. That is, the present invention is as follows.

[0010] [1] An agent for suppressing the worsening of uremia in a feline suffering from severe renal failure, the agent comprising apoptosis inhibitor of macrophage (AIM), an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment, wherein the severe renal failure is defined as a blood indoxyl sulfate concentration of 5 μg / mL or more and a blood inorganic phosphorus concentration of 7.5 mg / dL or less. [2] An agent for reducing the blood uremic toxin concentration in a feline suffering from severe renal failure, the agent comprising AIM, an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment, wherein the severe renal failure is defined as a blood indoxyl sulfate concentration of 5 μg / mL or more and a blood inorganic phosphorus concentration of 7.5 mg / dL or less. [3] An agent for reducing the blood uremic toxin concentration in a mammal, the agent comprising AIM, an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment. [4] The agent according to [3], wherein the mammal is a human, cow, monkey, horse, pig, sheep, goat, dog, cat, guinea pig, rat, mouse, rabbit, or hamster. [5] The agent according to [3], wherein the mammal is a human. [A1] A method for suppressing the worsening of uremia in a feline suffering from severe renal failure, comprising administering to the feline suffering from severe renal failure an apoptosis inhibitor of macrophage (AIM), an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment, wherein the severe renal failure is defined as a blood indoxyl sulfate concentration of 5 μg / mL or more and a blood inorganic phosphorus concentration of 7.5 mg / dL or less. [A2] A method for reducing the concentration of uremic toxins in the blood of a feline suffering from severe renal failure, comprising administering to the feline suffering from severe renal failure AIM, an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment, wherein severe renal failure is defined as a blood indoxyl sulfate concentration of 5 μg / mL or more and a blood inorganic phosphorus concentration of 7.5 mg / dL or less.[A3] A method for reducing the concentration of uremic toxins in the blood of a mammal, comprising administering to the mammal AIM, an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment. [A4] The method of [A3], wherein the mammal is a human, cow, monkey, horse, pig, sheep, goat, dog, cat, guinea pig, rat, mouse, rabbit, or hamster. [A5] The method of [A3], wherein the mammal is a human. [B1] An apoptosis inhibitor of macrophage (AIM), an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment, for use in suppressing the worsening of uremia in a feline suffering from severe renal failure, wherein severe renal failure is defined as a blood indoxyl sulfate concentration of 5 μg / mL or more and a blood inorganic phosphorus concentration of 7.5 mg / dL or less. [B2] AIM, an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment, for use in reducing the blood concentration of uremic toxins in a feline suffering from severe renal failure, wherein severe renal failure is defined as a blood indoxyl sulfate concentration of 5 μg / mL or more and a blood inorganic phosphorus concentration of 7.5 mg / dL or less. [B3] AIM, an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment, for use in reducing the blood concentration of uremic toxins in a mammal. [B4] The AIM, an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment according to [B3], wherein the mammal is a human, cow, monkey, horse, pig, sheep, goat, dog, cat, guinea pig, rat, mouse, rabbit, or hamster. [B5] The AIM according to [B3], an AIM fragment having the biological activity of AIM, or a nucleic acid encoding said AIM or AIM fragment, wherein the mammal is a human.[C1] Use of AIM, an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment, in the manufacture of a medicament for suppressing the worsening of uremia in a feline suffering from severe renal failure, wherein the severe renal failure is defined as a blood indoxyl sulfate concentration of 5 μg / mL or more and a blood inorganic phosphorus concentration of 7.5 mg / dL or less. [C2] Use of AIM, an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment, in the manufacture of a medicament for reducing the blood uremic toxin concentration in a feline suffering from severe renal failure, wherein the severe renal failure is defined as a blood indoxyl sulfate concentration of 5 μg / mL or more and a blood inorganic phosphorus concentration of 7.5 mg / dL or less. [C3] Use of AIM, an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment, in the manufacture of a medicament for reducing the blood uremic toxin concentration in a mammal. [C4] The use according to [C3], wherein the mammal is a human, cow, monkey, horse, pig, sheep, goat, dog, cat, guinea pig, rat, mouse, rabbit, or hamster. [C5] The use according to [C3], wherein the mammal is a human.

[0011] According to the present invention, it is possible to suppress the worsening of uremia in felines suffering from severe renal failure from which renal function cannot be expected to recover. Furthermore, according to the present invention, it is possible to prolong the life of felines suffering from severe renal failure from which renal function cannot be expected to recover. Furthermore, according to the present invention, it is possible to reduce the concentration of uremic toxins in the blood of felines suffering from severe renal failure from which renal function cannot be expected to recover. Furthermore, according to the present invention, it is possible to reduce the concentration of uremic toxins in the blood of mammals.

[0012] Figure 1 shows survival curves of cats with severe renal failure (stage 3b) administered mouse AIM. (Left) Because the shortest observation period was 340 days, a survival curve up to 340 days was drawn. (Right) Survival curve up to 500 days. For cats observed for less than 500 days, a survival curve was drawn assuming they survived for 500 days. The mean age at the start of administration was 14 years for the control group (n = 9) and 16 years for the AIM-administered group (n = 6). Figure 2 shows blood Cre (creatinine) concentrations on days 0 and 70 in the AIM-administered and control groups. Error bars indicate SD. Statistical analysis was performed using two-way repeated measures ANOVA and Bonferroni's post-hoc test. Figure 3 shows blood IP concentrations on days 0 and 70 in the AIM-administered and control groups. Error bars indicate SD. Statistical analysis was performed using two-way repeated measures ANOVA and Bonferroni's post-hoc test. Figure 4 shows the blood SAA concentrations in the AIM-administered group and the control group on days 0 and 70. Error bars indicate SD. Statistical analysis was performed using two-way repeated measures ANOVA and Bonferroni's post-hoc test. Figure 5 shows the blood IS concentrations in the AIM-administered group and the control group on days 0 and 70. Error bars indicate SD. Statistical analysis was performed using two-way repeated measures ANOVA and Bonferroni's post-hoc test. Figure 6 shows the results of GC-Mass spectrometric analysis of serum from a healthy cat, serum from a cat in stage 3b, serum from a cat in stage 3b after a total of six intravenous administrations of AIM or PBS every two weeks (Day 70), and serum from a cat in stage 3b 50 days after the end of AIM administration (Day 120). In this figure, glucaric acid, mesoerythritol, thiodiglycolic acid, aconitic acid, and glucuronic acid are shown as representative uremic toxins.The vertical axis represents fold change, with the value for healthy cats set to 1. Figure 7 shows the results of LC-Mass spectrometric analysis of serum from healthy cats, serum from cats in stage 3b, serum from cats in stage 3b after a total of six intravenous administrations of AIM or PBS every two weeks (Day 70), and serum from cats in stage 3b 50 days after the end of AIM administration (Day 120). This figure shows only the results for SAA, a systemic inflammatory marker in cats, as well as apolipoprotein B, vacuolar protein sorting-associated protein 53 homolog, anionic trypsin, galectin-3-binding protein, and lipopolysaccharide-binding protein. The vertical axis represents fold change, with the value for healthy cats set to 1. Figure 8 shows a survival curve showing the survival rate of cats with severe renal failure (stage 3b) when feline AIM was administered. FIG. 9 shows time-dependent changes in blood Cre levels when feline AIM was administered to cats suffering from severe renal failure (stage 3b).

[0013] The present invention will be described in detail below.

[0014] [Definitions] [Severe renal failure] As used herein, "severe renal failure" in a feline is defined as "a feline with a blood indoxyl sulfate concentration of 5 μg / mL or more and a blood inorganic phosphorus concentration of 7.5 mg / dL or less." In one embodiment, severe renal failure in a feline may be defined as "a feline with a blood indoxyl sulfate concentration of 5 μg / mL or more and a blood inorganic phosphorus concentration of 6.8 mg / dL or less." A feline suffering from renal failure defined in this way typically has lost most (approximately 80-90%) of its kidney function. In addition, "blood indoxyl sulfate concentrations of 5 μg / mL or higher and blood inorganic phosphorus concentrations of 7.5 mg / dL or lower in felines" and "blood indoxyl sulfate concentrations of 5 μg / mL or higher and blood inorganic phosphorus concentrations of 6.8 mg / dL or lower in felines" both correspond to late stage 3 in the staging of chronic kidney disease in felines (especially domestic cats) provided by the International Renal Interest Society (IRIS). In the IRIS staging, a state in which approximately 95% of kidney function has been lost is classified as stage 4.

[0015] [Felidae] As used herein, the term "felidae" refers to animals classified in the family Felidae. Examples of felines include lions (Panthera leo), leopards (Panthera pardus), tigers (Panthera tigris), snow leopards (Panthera uncia), jaguars (Panthera onca), clouded leopards (Neofelis nebulosa), Sundaun leopards (Neofelis diardi), cougars (Puma concolor), cheetahs (Acinonyx jubatus), jaguarundi (Herpailurus yagouaroundi), African golden cats (Caracal aurata), caracals (Caracal caracal), servals (Leptailurus serval), Bornean wildcats (Catopuma badia), Asiatic golden cats (Catopuma temminckii), marbled cats (Pardofelis marmorata), colo-colo (Leopardus colocola), Geoffroy's cats (Leopardus geoffroyi), and kodokodos (Leopardus guigna), Southern tiger cat (Leopardus guttulus), Andean cat (Leopardus jacobita), ocelot (Leopardus pardalis), jaguar cat (Leopardus tigrinus), margay (Leopardus wiedii), Canadian lynx (Lynx canadensis), African lynx (Lynx lynx), Spanish lynx (Lynx pardinus), bobcat (Lynx rufus), Bengal leopard cat (Prionailurus bengalensis), Javan leopard cat (Prionailurus javanensis), Malayan leopard cat (Prionailurus planiceps), rust-spotted cat (Prionailurus rubiginosus), fishing cat (Prionailurus viverrinus), Pallas's cat (Otocolobus manul), grey cat (Felis bieti), domestic cat (FelisExamples include, but are not limited to, Felis catus, jungle cat (Felis chaus), African wildcat (Felis lybica), sand cat (Felis margarita), black-footed cat (Felis nigripes), European wildcat (Felis silvestris), and subspecies thereof.

[0016] [Uremic toxins] As used herein, the term "uremic toxins" refers to various molecules whose concentrations in the blood increase as chronic kidney disease / renal failure progresses. Examples of "uremic toxins" in this specification include indoxyl sulfate, inorganic phosphorus, glucaric acid, meso-erythritol, thiodiglycolic acid, aconitic acid, glucuronic acid, 2-hydroxyisovaleric acid, galacturonic acid, homogentisic acid, inositol, urea, phosphonic acid, arabinose, mannose, arabitol, serum amyloid A protein (SAA), apolipoprotein B, KIAA0100, and vacuolar protein sorting-associated protein 53. Examples of such proteins include, but are not limited to, trypsin homolog, anionic trypsin, IgH variable region, galectin-3-binding protein, Ig lambda variable region, coagulation factor IX, and phosphoglycerate mutase.

[0017] 1. Agent for suppressing the worsening of uremia in felines suffering from severe renal failure The present invention provides an agent for suppressing the worsening of uremia in felines suffering from severe renal failure (hereinafter, sometimes referred to as the "agent for suppressing the worsening of uremia of the present invention"), which comprises apoptosis inhibitor of macrophage (AIM), an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment.

[0018] The AIM contained in the uremia worsening suppressant of the present invention may be a protein containing an amino acid sequence identical to or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 (the amino acid sequence of a feline-derived AIM protein). AIM may be, for example, a protein isolated and purified from macrophages, which are immune cells of warm-blooded animals (e.g., cats, mice, humans, rats, rabbits, sheep, pigs, cattle, horses, dogs, monkeys, chimpanzees, birds, etc.). It may also be a protein chemically synthesized or biochemically synthesized using a cell-free translation system, or a recombinant protein produced from a transformant into which a nucleic acid containing a nucleotide sequence encoding the amino acid sequence has been introduced. The biological species from which the AIM contained in the uremia worsening suppressant of the present invention is derived may or may not be the same as the biological species of the feline to which it is applied. For example, when the uremia worsening suppressant of the present invention is applied to cats, the AIM contained in the uremia worsening suppressant of the present invention may be feline AIM or mouse AIM.

[0019] Examples of amino acid sequences substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include amino acid sequences that have an identity or similarity of about 60% or more, preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more with the amino acid sequence represented by SEQ ID NO: 1. Here, "identity" refers to the percentage (%) of identical and similar amino acid residues relative to the total number of overlapping amino acid residues in optimal alignment (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment) when two amino acid sequences are aligned using a mathematical algorithm known in the art. Furthermore, "similarity" refers to the percentage (%) of positions where identical or similar amino acid residues exist in both aligned sequences relative to the total number of amino acid residues in the two sequences. "Similar amino acids" refer to amino acids that are similar in physicochemical properties, and include, for example, amino acids classified in the same group, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), and amino acids with small side chains (Gly, Ala, Ser, Thr, Met). Substitution with such similar amino acids is expected to have no effect on the phenotype of the protein (i.e., conservative amino acid substitutions). Specific examples of conservative amino acid substitutions are well known in the art and have been described in various publications (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).

[0020] The identity or similarity of amino acid sequences herein can be calculated using the identity or similarity calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF). Other algorithms for determining identity or similarity of amino acid sequences include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993) [this algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997))], the algorithm described in Needleman et al., J. Mol. Biol., 48:444-453 (1970) [this algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4:11-17 (1988) [this algorithm is incorporated into the ALIGN program (version 2.0), which is part of the CGC sequence alignment software package], and the algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85:2444-2448 (1988) [this algorithm is incorporated into the FASTA program in the GCG software package], and the like can also be preferably used. More preferably, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence that has an identity of about 60% or more, preferably about 70% or more, even more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more with the amino acid sequence represented by SEQ ID NO: 1.

[0021] Examples of proteins comprising substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 include proteins comprising substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 and having substantially the same biological activity as that of wild-type AIM. The biological activities of wild-type AIM include, for example, endocytosis activity in macrophages (including microglia), macrophage apoptosis inhibitory activity, arteriosclerosis maintenance / promotion activity, adipocyte differentiation inhibitory activity, adipocyte lipid droplet dissolving activity, adipocyte reduction activity, CD36 binding activity, adipocyte endocytosis activity, FAS binding activity, FAS function inhibitory activity, anti-obesity activity, prophylactic or therapeutic activity for liver disease (fatty liver, NASH, cirrhosis, liver cancer), prophylactic or therapeutic activity for kidney disease (acute renal failure, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, nephropathy associated with collagen disease or IgM nephropathy), etc. However, in the present invention, endocytosis activity in macrophages can be a particularly preferred indicator. This activity can be confirmed using an in vitro macrophage (or microglia) phagocytosis test, as described in detail in the Examples of this application, but is not limited thereto. Furthermore, as used herein, "substantially the same" means that the activities are qualitatively the same (e.g., physiologically or pharmacologically). Therefore, it is preferable that the activities are equivalent, but the degree of these activities (e.g., about 0.1 to about 10 times, preferably about 0.5 to about 2 times) and quantitative factors such as the molecular weight of the protein may differ. The activity can be measured according to a method known per se.

[0022] Furthermore, the AIM used in the present invention includes, for example, (1) an amino acid sequence in which one or two or more amino acids (preferably about 1 to 100, preferably about 1 to 50, more preferably about 1 to 10, particularly preferably one to several (2, 3, 4, or 5)) have been deleted from the amino acid sequence represented by SEQ ID NO: 1; (2) an amino acid sequence in which one or two or more amino acids (preferably about 1 to 100, preferably about 1 to 50, more preferably about 1 to 10, particularly preferably one to several (2, 3, 4, or 5)) have been added to the amino acid sequence represented by SEQ ID NO: 1; (3) an amino acid sequence in which one or two or more (preferably about 1 to 50, preferably about 1 to 10, more preferably one to several (2, 3, 4, or 5)) amino acids have been inserted into the amino acid sequence represented by SEQ ID NO: 1; (4) an amino acid sequence in which one or two or more (preferably about 1 to 50, preferably about 1 to 10, more preferably one to several (2, 3, 4, or 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 1 have been substituted with other amino acids; or (5) a protein containing an amino acid sequence that is a combination thereof. When an amino acid sequence has been inserted, deleted, or substituted as described above, the position of the insertion, deletion, or substitution is not particularly limited, as long as the desired biological activity of the protein (e.g., endocytosis activity in macrophages (including microglia)) is maintained.

[0023] The AIM of the present invention is preferably a feline AIM protein having the amino acid sequence shown in SEQ ID NO: 1 or a homolog thereof in other mammals [for example, the mouse homolog (SEQ ID NO: 3) registered in GenBank under accession number AAD01445], and more preferably a feline AIM protein consisting of the amino acid sequence shown in SEQ ID NO: 1. Cats have two types of AIM (a three-domain type (SRCR1-SRCR2-SRCR3, SEQ ID NO: 1) and a four-domain type (SRCR1-SRCR1-SRCR2-SRCR3)), and either type may be used.

[0024] In this specification, proteins and peptides are described according to the convention of peptide notation, with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end. The AIM used in the present invention, including a protein comprising the amino acid sequence represented by SEQ ID NO: 1, may have a C-terminus that is any of a carboxyl group (-COOH), a carboxylate (-COO-), an amide (-CONH2), or an ester (-COOR). Here, R in the ester may be, for example, a C-terminus such as methyl, ethyl, n-propyl, isopropyl, or n-butyl. 1-6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3-8 Cycloalkyl groups such as phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl group: α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0025] When the AIM used in the present invention has a carboxyl group (or carboxylate) other than at the C-terminus, the protein of the present invention also includes those in which the carboxyl group is amidated or esterified. In this case, the ester used may be, for example, the C-terminal ester described above.

[0026] Furthermore, the AIM used in the present invention includes those in which the amino group of the N-terminal amino acid residue is protected with a protecting group (e.g., formyl group, acetyl group, etc.), those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated, and those in which the substituents on the side chains of the amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected with an appropriate protecting group (e.g., C such as formyl group, acetyl group, etc.). 1-6 C such as alkanoyl group 1-6 These include those protected by an acyl group or other suitable glycan, or conjugated proteins such as glycoproteins to which sugar chains are attached.

[0027] As used herein, the term "AIM" refers to not only wild-type AIM but also its variants and the like having substantially the same or improved biological activity as that of wild-type AIM. Here, "substantially the same activity" has the same meaning as above. Furthermore, the measurement of "substantially the same activity" can be carried out in the same manner as for AIM.

[0028] Furthermore, as a component of the agent for suppressing the worsening of uremia of the present invention, not only AIM but also an AIM fragment having the biological activity of AIM can be used. Whether or not an AIM fragment has the biological activity of wild-type AIM can be determined by the method described above.

[0029] As an example of an AIM fragment, for example, since the intact AIM protein contains three cysteine-rich scavenger-receptor cysteine-rich (SRCR) domains, each of the SRCR domains can be cited as an example of an AIM fragment having substantially the same biological activity as that of wild-type AIM. More specifically, for example, of the amino acid sequence represented by SEQ ID NO: 5 (human AIM), partial amino acid sequences containing the SRCR1 domain (amino acid numbers 24 to 125 in the amino acid sequence represented by SEQ ID NO: 5), the SRCR2 domain (amino acid numbers 139 to 239 in the amino acid sequence represented by SEQ ID NO: 5), or the SRCR3 domain (amino acid numbers 244 to 346 in the amino acid sequence represented by SEQ ID NO: 5) or partial amino acid sequences containing any combination of the SRCR domains can be used as the AIM fragment. The size of an AIM fragment having substantially the same biological activity as that of wild-type AIM is not particularly limited as long as it contains the above-mentioned functional domain, but examples thereof include those containing a partial amino acid sequence of typically 20 or more, preferably 50 or more, more preferably 100 or more, and even more preferably 200 or more. The partial amino acid sequence may be a single contiguous partial amino acid sequence, or may be a combination of multiple discontinuous partial amino acid sequences. While the SRCR domain of human AIM has been used for the explanation here, the SRCR domain of AIM from other animal species may also be used.

[0030] The AIM fragment used in the present invention has a C-terminus that is a carboxyl group (-COOH), a carboxylate (-COO -), amide (-CONH2), or ester (-COOR). Here, examples of R in the ester include those similar to those described above for AIM. When the partial peptide of the present invention has a carboxyl group (or carboxylate) other than at the C-terminus, the partial peptide of the present invention also includes those in which the carboxyl group is amidated or esterified. In this case, for example, the same ester as the C-terminal ester is used.

[0031] Furthermore, the AIM fragments used in the present invention also include, like the AIM described above, those in which the amino group of the N-terminal amino acid residue is protected with a protecting group, those in which the N-terminal glutamine residue is pyroglutamated, those in which substituents on the side chains of amino acids in the molecule are protected with appropriate protecting groups, and conjugated peptides such as so-called glycopeptides to which sugar chains are bound.

[0032] The AIM (including AIM fragments) used in the present invention may be in the form of a salt. For example, a salt with a physiologically acceptable acid (e.g., inorganic acid, organic acid) or base (e.g., alkali metal salt) is used, with physiologically acceptable acid addition salts being particularly preferred. Examples of such salts include salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

[0033] AIM (including AIM fragments) can also be produced according to known peptide synthesis methods. The peptide synthesis method may be, for example, either solid-phase synthesis or liquid-phase synthesis. The target protein can be produced by condensing a partial peptide or amino acid that can constitute AIM with the remaining portion, and if the product has a protecting group, removing the protecting group. Here, the condensation and removal of the protecting group are carried out according to known methods, for example, the methods described in (1) and (2) below. (1) M. Bodanszky and M.A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966) (2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

[0034] The AIM thus obtained can be purified and isolated by known purification methods, such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.

[0035] When AIM obtained by the above method is in a free form, the free form can be converted into an appropriate salt by a known method or a method similar thereto. Conversely, when AIM is obtained as a salt, the salt can be converted into the free form or another salt by a known method or a method similar thereto.

[0036] Furthermore, AIM can also be produced by culturing a transformant containing a nucleic acid encoding it, and isolating and purifying AIM from the resulting culture. The nucleic acid encoding AIM or an AIM fragment may be DNA or RNA, or may be a DNA / RNA chimera. DNA is preferred. Furthermore, the nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. If single-stranded, it may be the sense strand (i.e., coding strand) or the antisense strand (i.e., non-coding strand).

[0037] DNA encoding AIM (including AIM fragments) includes genomic DNA, cDNA derived from macrophages of warm-blooded animals (e.g., humans, cows, monkeys, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, mice, rabbits, hamsters, birds, etc.), synthetic DNA, etc. Genomic DNA encoding AIM or an AIM fragment can be used in any cell of the above animals [e.g., hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (e.g., macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stromal cells, or cells of these cells]. progenitor cells, stem cells, or cancer cells, etc.] or any tissue in which such cells exist [for example, brain, brain regions (e.g., olfactory bulb, amygdala, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonads, thyroid gland, gallbladder, bone marrow, adrenal gland, skin, lung, digestive tract (e.g., large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (e.g., brown adipose tissue, white adipose tissue), skeletal muscle, etc.] as a template, and polymerase cDNA encoding AIM or an AIM fragment can also be directly amplified by PCR and reverse transcriptase-PCR (hereinafter abbreviated as "RT-PCR") using total RNA or mRNA fractions prepared from macrophages as templates, respectively. Alternatively, genomic DNA and cDNA encoding AIM or a peptide fragment thereof can be cloned, respectively, by colony or plaque hybridization or PCR from genomic DNA libraries and cDNA libraries prepared by inserting the above-mentioned genomic DNA and total RNA or mRNA fragments into appropriate vectors.The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid, etc.

[0038] Examples of nucleic acids encoding AIM include nucleic acids containing a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 2. Examples of nucleic acids containing a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 2 include nucleic acids containing a nucleotide sequence having at least about 60%, preferably at least about 70%, more preferably at least about 80%, and particularly preferably at least about 90% identity or similarity to the nucleotide sequence represented by SEQ ID NO: 2, and encoding a protein having substantially the same activity as the aforementioned AIM. In one embodiment, a nucleic acid containing a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 2 is a nucleic acid containing a nucleotide sequence having at least about 60%, preferably at least about 70%, more preferably at least about 80%, and particularly preferably at least about 90% identity to the nucleotide sequence represented by SEQ ID NO: 2, and encoding a protein having substantially the same activity as the aforementioned AIM. Nucleic acids encoding AIM also include nucleic acid sequences that have been codon-optimized for the purpose of increasing expression efficiency in target organisms.

[0039] The identity or similarity of base sequences herein can be calculated using the identity or similarity calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3). Other preferred examples of algorithms for determining the identity or similarity of base sequences include the amino acid sequence homology calculation algorithms described above.

[0040] The nucleic acid encoding AIM is preferably a nucleic acid containing a base sequence encoding the feline AIM protein represented by the base sequence represented by SEQ ID NO: 2, or a homolog thereof in other mammals [e.g., the mouse homolog (SEQ ID NO: 4) registered in GenBank under accession number AF011428, etc.].

[0041] Furthermore, as a component of the agent for suppressing the worsening of uremia of the present invention, a nucleic acid encoding AIM or an AIM fragment having the biological activity of AIM can also be used.

[0042] The nucleic acid encoding AIM or an AIM fragment used in the present invention may be any nucleic acid, as long as it contains a nucleotide sequence encoding a peptide comprising an amino acid sequence identical or substantially identical to a portion of the amino acid sequence represented by SEQ ID NO: 1. Specifically, examples of nucleic acids encoding AIM fragments include (1) nucleic acids comprising a partial nucleotide sequence of the nucleotide sequence represented by SEQ ID NO: 2, or (2) nucleic acids comprising a nucleotide sequence having about 60% or more, preferably about 70% or more, more preferably about 80% or more, and particularly preferably about 90% or more identity or similarity to a nucleic acid comprising a partial nucleotide sequence of the nucleotide sequence represented by SEQ ID NO: 2, and encoding a protein having substantially the same activity as the aforementioned AIM.

[0043] Nucleic acids encoding AIM or AIM fragments can be amplified by PCR using synthetic DNA primers containing a portion of the nucleotide sequence encoding the AIM or AIM fragment, or cloned by hybridizing DNA incorporated into an appropriate expression vector with a labeled DNA fragment or synthetic DNA encoding a portion or the entire region of AIM. Hybridization can be carried out according to a method known per se or a method equivalent thereto, such as the method described in Molecular Cloning, 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When using a commercially available library, hybridization can be carried out according to the method described in the accompanying instruction manual. Hybridization is preferably carried out under stringent conditions.

[0044] Highly stringent conditions include, for example, a hybridization reaction in 6xSSC (sodium chloride / sodium citrate) at 45°C, followed by one or more washes in 0.2xSSC / 0.1% SDS at 65°C. Those skilled in the art can easily adjust the stringency to the desired level by appropriately changing the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the probe length, the number of mismatches, the hybridization reaction time, the salt concentration of the washing solution, the washing temperature, etc. When using a commercially available library, hybridization can be performed according to the method described in the instructions attached to the library.

[0045] The nucleic acid encoding AIM or an AIM fragment may be functionally linked to an expression vector or the like having a suitable promoter. In addition to the promoter, known sequences such as a Poly A addition signal, a Kozak consensus sequence, a tag sequence, a linker sequence, and an NLS may be incorporated into a viral vector or the like in combination with the nucleic acid encoding AIM or an AIM fragment, as appropriate depending on the purpose.

[0046] In a preferred embodiment, a nucleic acid encoding AIM or an AIM fragment is carried by a viral vector. Suitable viral vectors include, but are not limited to, adeno-associated viruses, adenoviruses, lentiviruses, and Sendai viruses. Considering use in gene therapy, adeno-associated viruses are preferred because they allow long-term expression of transgenes and are derived from non-pathogenic viruses, making them highly safe. Furthermore, the serotype of the adeno-associated virus is not particularly limited as long as the desired effects of the present invention are achieved; any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 may be used. The viral vectors used in the present invention also include derivatives thereof. Derivatives of viral vectors, such as those with modified capsids, are well known. In particular, AAV derivatives include, but are not limited to, those disclosed in WO2012 / 057363, etc.

[0047] A viral vector containing a nucleic acid encoding AIM or an AIM fragment can be prepared by a known method. Briefly, a viral expression plasmid vector is prepared by inserting a nucleic acid encoding AIM or an AIM fragment and, if necessary, a nucleic acid having a desired function, and this is transfected into an appropriate host cell to transiently produce a viral vector containing the polynucleotide of the present invention, which can then be recovered.

[0048] For example, when preparing an AAV vector, a vector plasmid is first prepared by retaining the ITRs at both ends of the wild-type AAV genome sequence and inserting a nucleic acid encoding AIM or an AIM fragment in place of the DNA encoding the remaining Rep and capsid proteins. Meanwhile, the DNA encoding the Rep and capsid proteins required for viral particle formation is inserted into a separate plasmid. Furthermore, a plasmid containing genes (E1A, E1B, E2A, VA, and E4orf6) responsible for the adenovirus helper function required for AAV replication is prepared as an adenovirus helper plasmid. Cotransfection of these three plasmids into host cells results in the production of recombinant AAV (i.e., an AAV vector) in the cells. It is preferable to use host cells (e.g., 293 cells) capable of supplying some of the gene products (proteins) of the genes responsible for the helper function. When such cells are used, it is not necessary to incorporate genes encoding proteins that can be supplied by the host cell into the adenovirus helper plasmid. Since the produced AAV vector is present in the nucleus, the host cells are frozen and thawed to recover the vector, and the desired AAV vector is prepared by separating and purifying it using cesium chloride density gradient ultracentrifugation or column methods.

[0049] The route of administration of the agent for suppressing the worsening of uremia of the present invention is not particularly limited, and preferred routes of administration include, but are not limited to, intravenous administration, intraarterial administration, subcutaneous administration, and intraperitoneal administration.

[0050] When the agent for suppressing the worsening of uremia of the present invention is formulated for parenteral administration, it can be formulated, for example, as an injection, suppository, etc. Injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip infusion injections. Such injections can be prepared according to known methods. For example, injections can be prepared by dissolving, suspending, or emulsifying components such as AIM, a nucleic acid encoding AIM, and / or a virus carrying a nucleic acid encoding AIM in a sterile aqueous or oily liquid typically used for injections. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and the like, which may be used in combination with an appropriate solubilizing agent, such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), or nonionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As the oily liquid, for example, sesame oil, soybean oil, etc. can be used, and a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. can be used in combination. The prepared injection solution is preferably filled into a suitable ampule.

[0051] The dose of the agent for suppressing the worsening of uremia of the present invention to be administered to a subject is not particularly limited as long as the desired effect is obtained, and may be optimized appropriately depending on the type and form of the active ingredient, the age and weight of the subject, the administration schedule, the administration method, etc.

[0052] The timing of administering the agent for suppressing the worsening of uremia of the present invention to a subject is not particularly limited as long as the desired effect can be obtained.

[0053] The agent for suppressing the worsening of uremia of the present invention may be used in combination with other therapeutic agents for treating severe renal failure, dietary therapy, etc.

[0054] The agent for suppressing the worsening of uremia of the present invention can suppress the worsening of uremia in felines suffering from severe renal failure. As used herein, "uremia" refers to a condition in which metabolic waste products (described in detail below as "uremic toxins") that should be excreted in urine accumulate in the blood due to decreased renal function. Symptoms of uremia include, but are not limited to, the following: central nervous system: impaired consciousness, insomnia, memory loss, headache, confusion, convulsions, etc. peripheral nervous system: peripheral neuropathy, discomfort in the legs, etc. gastrointestinal tract: loss of appetite, vomiting, abdominal distension, etc. lungs: difficulty breathing, pulmonary edema, hypoxemia, etc. heart or blood vessels: palpitations, high blood pressure, etc. metabolism: elevated potassium, acidosis, etc. eyes: impaired vision, etc. blood: anemia, etc. skin: subcutaneous bleeding, edema, pigmentation, itching, etc.

[0055] Furthermore, in this specification, "suppressing the worsening of uremia" means that the symptoms of uremia observed at a certain point in time in a feline suffering from severe renal failure do not progress to a worsening state after a certain period of time has passed. "Suppressing the worsening of uremia" can also be rephrased as "suppressing the progression of uremia."

[0056] Furthermore, as used herein, "suppressing the worsening of uremia" encompasses not only preventing the symptoms of uremia from worsening but also delaying the rate of worsening of uremia. Therefore, the agent for suppressing the worsening of uremia of the present invention can be used not only for the purpose of preventing the worsening of uremia in a subject, but also for the purpose of delaying the worsening of uremia in a subject. In addition, in one aspect, "suppressing the worsening of uremia" also encompasses suppressing the onset of uremia. Therefore, the agent for suppressing the worsening of uremia of the present invention can be used not only for the purpose of preventing the onset of uremia in a subject, but also for the purpose of delaying the onset of uremia in a subject.

[0057] Incidentally, it has been reported that felines develop uremia as severe renal failure progresses, and when they reach a state classified as IRIS stage 4, they die, on average, within several months (average 103 days). However, in cats in late IRIS stage 3 treated with the agent for suppressing the worsening of uremia of the present invention, the transition to IRIS stage 4 is suppressed, and as a result, the number of days they survive is significantly extended. In consideration of this situation, the present invention can also be said to enable the extension of the life of felines suffering from severe renal failure. Therefore, in one aspect, the agent for suppressing the worsening of uremia of the present invention can also be said to be a life-prolonging agent for felines suffering from severe renal failure.

[0058] Furthermore, as shown in the following examples, administration of AIM to a cat suffering from severe renal failure can reduce the concentration of uremic toxins in the blood of the cat without recovery of renal function. Therefore, the present invention also provides an agent for reducing the concentration of uremic toxins in the blood of a feline suffering from severe renal failure (hereinafter, sometimes referred to as the "uremic toxin-reducing agent of the present invention"), which comprises apoptosis inhibitor of macrophage (AIM), an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment.

[0059] The AIMs and the like used in the uremic toxin-reducing agent of the present invention and the felines and other animals to which it is applied are the same as those explained in the agent for suppressing the worsening of uremia of the present invention.

[0060] As used herein, the term "uremic toxin" refers to a substance that is typically filtered by the glomerulus of the kidney and excreted in urine. The term "uremic toxin" as used herein is a concept that encompasses not only substances that have a direct adverse effect on the living body, but also substances whose blood concentration increases as a result of decreased renal function (e.g., precursors of toxic substances). Examples of uremic toxins whose blood concentrations can be reduced by the uremic toxin-reducing agent of the present invention include indoxyl sulfate, inorganic phosphorus, glucaric acid, meso-erythritol, thiodiglycolic acid, aconitic acid, glucuronic acid, 2-hydroxyisovaleric acid, galacturonic acid, homogentisic acid, inositol, urea, phosphonic acid, arabinose, mannose, arabitol, serum amyloid A protein (SAA), apolipoprotein B, KIAA0100, and vacuolar protein sorting-associated protein (VSP). Examples of such proteins include, but are not limited to, 53 homolog, anionic trypsin, IgH variable region, galectin-3-binding protein, Ig lambda variable region, coagulation factor IX, and phosphoglycerate mutase.By administering the uremic toxin-reducing agent of the present invention, the blood concentration of at least one of these uremic toxins can be reduced, preferably at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, more preferably at least 15, at least 20, at least 25, and even more preferably all of them.

[0061] Based on the fact that administration of AIM to cats suffering from severe renal failure can reduce blood uremic toxin concentrations without improving renal function, it is clear that the reduction in blood uremic toxin concentrations by AIM administration is due to a mechanism that does not involve renal function. Therefore, it is believed that AIM directly promotes the excretion of uremic toxins via some mechanism. In consideration of this fact, it is believed that the blood uremic toxin-reducing effect of AIM does not depend on the state of renal failure or the animal species. Therefore, in one aspect, the present invention provides an agent for reducing blood uremic toxin concentrations in mammals (hereinafter sometimes referred to as the "mammalian uremic toxin-reducing agent of the present invention"), which comprises apoptosis inhibitor of macrophage (AIM), an AIM fragment having the biological activity of AIM, or a nucleic acid encoding the AIM or AIM fragment.

[0062] The mammals to which the uremic toxin-reducing agent for mammals of the present invention can be applied are not particularly limited as long as they are mammals in which uremic toxins can be reduced by AIM, including, but not limited to, humans, cows, monkeys, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, mice, rabbits, and hamsters.

[0063] The AIM and the like used in the mammalian uremic toxin-reducing agent of the present invention are the same as those explained in the agent for suppressing the worsening of uremia of the present invention.

[0064] In a preferred embodiment, the uremic toxin-reducing agent for mammals of the present invention can be applied to mammals suffering from end-stage renal failure to the extent that dialysis is required. By administering the uremic toxin-reducing agent for mammals of the present invention, the concentration of uremic toxins in the blood of the mammal can be reduced, thereby making it possible to reduce the frequency of dialysis. In this embodiment, the mammal is preferably a human or a dog, and particularly preferably a human. For humans, it is preferable to use human AIM (SEQ ID NO: 5) or a nucleic acid encoding human AIM (SEQ ID NO: 6). As mentioned above, these may be modified forms as long as they retain the biological activity of AIM.

[0065] For example, examples of modified forms of human AIM include, but are not limited to, the following. The mutant human AIM of the present invention preferably comprises any one of the following amino acid sequences (1b) to (5b): (1b) an amino acid sequence in which the cysteine ​​at amino acid number 191 in the amino acid sequence represented by SEQ ID NO: 5 is substituted with serine. (2b) an amino acid sequence in which the cysteine ​​at amino acid number 300 in the amino acid sequence represented by SEQ ID NO: 5 is substituted with serine. (3b) an amino acid sequence in which the cysteine ​​at amino acid number 191 in the amino acid sequence represented by SEQ ID NO: 5 is substituted with serine and the cysteine ​​at amino acid number 300 in the amino acid sequence represented by SEQ ID NO: 5 is substituted with serine. (4b) an amino acid sequence that is substantially identical to any one of the amino acid sequences (1b) to (3b), in which the cysteine ​​and the substituted serine present in any one of the amino acid sequences (1b) to (3b) are retained. (5b) An amino acid sequence comprising one or more amino acids deleted, added, inserted, or substituted, or a combination thereof, at a position other than the cysteine ​​and the substituted serine present in any one of the amino acid sequences (1b) to (3b). AIM variants having functions equivalent to or improved over wild-type recombinant AIM can be those disclosed in Japanese Patent No. 7231230, etc.

[0066] In another embodiment, the uremic toxin-reducing agent for mammals of the present invention can be administered to mammals suffering from mild or moderate renal failure, thereby supporting the impaired renal function.

[0067] 2. Method for suppressing the worsening of uremia in a feline suffering from severe renal failure The present invention also provides a method for suppressing the worsening of uremia in a feline suffering from severe renal failure (hereinafter, sometimes referred to as the "method for suppressing the worsening of uremia of the present invention"), which comprises administering to the feline suffering from severe renal failure an apoptosis inhibitor of macrophage (AIM), an AIM fragment having the biological activity of AIM, or a nucleic acid encoding said AIM or AIM fragment.

[0068] The method for suppressing the worsening of uremia of the present invention is achieved by administering the agent for suppressing the worsening of uremia of the present invention to a feline suffering from severe renal failure. The AIM, etc., target of application, administration timing, etc. in the method for suppressing the worsening of uremia of the present invention are the same as those explained for the agent for suppressing the worsening of uremia of the present invention.

[0069] In one embodiment, the method of the present invention for suppressing the worsening of uremia can be rephrased as a method for prolonging the life of a feline suffering from severe renal failure.

[0070] The present invention also provides a method for reducing the concentration of uremic toxins in the blood of a feline animal (hereinafter sometimes referred to as the "uremic toxin reduction method of the present invention"), which comprises administering the uremic toxin-reducing agent of the present invention to the feline animal suffering from severe renal failure.

[0071] In another aspect, the present invention provides a method for reducing the concentration of uremic toxins in the blood of a mammal, comprising administering the uremic toxin-reducing agent for mammals of the present invention to the mammal.

[0072] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0073] Example 1: Administration of AIM to cats with severe renal failure AIM was administered to cats with severe renal failure to confirm its effects. The cats used in this study (n=15) met the following conditions: (1) a blood indoxyl sulfate (IS) concentration of 5 μg / mL or more, and (2) a blood inorganic phosphorus (IP) concentration of 7.5 mg / dL or less.

[0074] Indoxyl sulfate is known as a uremic toxin and a known marker for renal failure in cats. The renal failure state defined by (1) and (2) refers to a state of renal failure equivalent to the late stage 3 of IRIS staging (in other words, a state in which most (approximately 90%) of renal function has been lost and moderate uremia has developed). In this specification, a state of renal failure that meets the conditions of (1) and (2) may be referred to as "stage 3b" or "stage 3b."

[0075] Fifteen cats classified as stage 3b were divided into a control group (n=9) and an AIM-administered group (n=6). The AIM-administered group received a slow intravenous administration of 2 mg / day of recombinant mouse AIM over one to several hours. This constituted one course of treatment, and AIM was administered once every two weeks for a total of six times until the cat died. The control group, on the other hand, did not receive AIM. The survival rates of the AIM-administered and control groups are shown in Figure 1.

[0076] As shown in Figure 1, the survival rate was significantly improved in the AIM-administered group compared with the control group.

[0077] In addition, the results of measuring Cre (renal function marker), IP (uremic toxin), SAA (inflammatory marker), and IS (uremic toxin) in each group on observation day 0 (D0) and observation day 70 (D70) are shown in Figures 2 to 5.

[0078] As shown in Figure 2, administration of AIM did not change Cre values ​​at D0 or D70. This means that administration of AIM suppresses further deterioration of renal function, but does not improve it. On the other hand, AIM administration suppressed the increase in blood concentrations of uremic toxins IP and IS, even though renal function had not recovered (Figures 3 and 5). Furthermore, administration of AIM significantly reduced the concentration of SAA, an inflammatory marker (Figure 4).

[0079] From the above, it was shown that when AIM is administered to cats suffering from severe renal failure at stage 3b, it is possible to significantly reduce the concentration of uremic toxins in the blood of cats suffering from severe renal failure, even though it is unable to improve renal function, and as a result, it is possible to suppress the worsening of uremia.

[0080] Example 2 Reduction of uremic toxins by AIM administration Serum mixtures from six healthy cats, six stage 3b cats, and six stage 3b cats (Day 70) after intravenous administration of recombinant mouse AIM (2 mg) or PBS every two weeks, as well as three cats 50 days after the end of recombinant mouse AIM administration (Day 120), were analyzed by GC-mass spectrometer (MS) and LC-MS. GC-MS and LC-MS analyses were performed using the following methods and conditions.

[0081] [GC-MS analysis method] Of the 401 compounds (568 compounds including methoxime- or TMS-derivatized isomers) listed in the database (SHIMADZU Smart Metabolites Database), compounds whose peaks were detected in the sample solution were selected, and the peak areas of these compounds were divided by the peak area of ​​IS (2-isopropylmalate-3TMS) to calculate the IS-corrected values. To compare between groups, the IS-corrected values ​​for each compound in healthy cats were set to 1, and the relative values ​​for each group were graphed.

[0082] [LC-MS TMT comparative analysis conditions] Comparative analysis was performed on peptides with unique sequences (unique peptides) and Razor Peptides (*) in each protein. The protein abundance ratio between each sample was normalized by dividing by the median abundance ratio of all proteins. To compare between groups, the value for healthy cats was set to 1, and the relative values ​​for each group were graphed. (*Peptides shared between proteins were assigned as originating from the protein with the most identified peptides, and these are called Razor Peptides.)

[0083] Representative results obtained by GC-MS and LC-MS are shown in Figures 6 and 7.

[0084] Many uremic toxins were significantly elevated (accumulated) in the blood of stage 3b cats, and this increase was suppressed and reduced after administration of recombinant mouse AIM (Day 70). It was confirmed that these uremic toxins are also elevated in the blood of human patients with end-stage renal failure undergoing dialysis (data not shown). In contrast, accumulation was maintained or even increased in the PBS-administered group (Day 70). In cats administered recombinant mouse AIM, these factors remained low even 50 days after the end of administration (Day 120). (Many stage 3b cats in the PBS-administered group died by Day 120, so analysis on Day 120 was not performed.) Similar results were obtained with SAA, a marker of systemic inflammation.

[0085] [Example 3] Administration of AIM to a cat suffering from severe renal failure 2 Recombinant feline AIM was administered to a cat suffering from severe renal failure, and the effect was confirmed.

[0086] Recombinant feline AIM was administered intravenously at 2 mg / head to six cats classified as stage 3b (AIM-administered group). This constituted one course of treatment, with AIM administered once every two weeks for a total of 12 doses. Meanwhile, no AIM was administered to the control group (the control group obtained in Example 1 was used). The survival rates (Kaplan-Meier curves) of the AIM-administered and control groups are shown in Figure 8. The changes in serum creatinine levels over time in the six cats that received AIM are shown in Figure 9.

[0087] As shown in Figure 8, the survival rate was significantly improved in the AIM-administered group compared to the control group. Furthermore, as shown in Figure 9, in most cats, administration of AIM did not change the blood Cre level. This means that administration of AIM suppresses further deterioration of renal function, but does not improve it.

[0088] These results demonstrate that administration of AIM to cats in stage 3b can reduce blood uremic toxin concentrations and suppress inflammation extremely efficiently. Without wishing to be bound by theory, because administration of AIM to cats in stage 3b did not result in improvement in renal function, it is possible that the AIM-induced reduction in blood uremic toxins is due to AIM acting directly on uremic toxins, thereby promoting their excretion from the blood. Therefore, it is highly likely that the mechanism by which AIM reduces blood uremic toxin concentrations is not limited to the stage of renal failure or to cats. Therefore, it is believed that the reduction of blood uremic toxins by AIM administration can be applied to any mammal with AIM, and is not limited to the stage of renal failure.

[0089] According to the present invention, it is possible to suppress the worsening of uremia in felines suffering from severe renal failure from which renal function is unlikely to recover. Furthermore, according to the present invention, it is possible to prolong the life of felines suffering from severe renal failure from which renal function is unlikely to recover. Furthermore, according to the present invention, it is possible to reduce the concentration of uremic toxins in the blood of felines suffering from severe renal failure from which renal function is unlikely to recover. Therefore, the present invention is extremely useful in the field of veterinary medicine. In addition, the present invention can reduce the concentration of uremic toxins in the blood of mammals. Therefore, for example, according to the present invention, it may be possible to reduce the number of dialysis sessions in human end-stage renal failure patients who require dialysis. Therefore, the present invention is also extremely useful in the field of human medicine.

[0090] This application is based on patent application No. 2022-139482 filed in Japan (filing date: September 1, 2022), the contents of which are incorporated in their entirety herein.

Claims

[Claim 1] An agent for suppressing the exacerbation of uremia in felines suffering from severe renal failure, comprising an apoptosis inhibitor of macrophage (AIM), an AIM fragment having the biological activity of AIM, or a nucleic acid encoding said AIM or AIM fragment, wherein severe renal failure is defined as a blood indoxyl sulfate concentration of 5 μg / mL or higher and a blood inorganic phosphorus concentration of 7.5 mg / dL or lower.