Patent Application
20240148736
The present invention relates to a combination drug for treating a renal cancer and a potentiator for therapeutic effects of a tyrosine kinase inhibitor on a renal cancer.
Renal cell carcinoma (RCC) is one of the most common tumors among varieties of a renal cancer for adults, and the RCC incidence is currently over 400,000 cases worldwide (see NPLs 1 to 2). Five-year survival of RCC patients who undergo radical excision is promising, generally 90% or greater. Conversely, 5-year survival of metastatic RCC patients is from 10% to 20% (see NPL 3). In recent years, immune checkpoint inhibitors and molecularly targeted agents including a tyrosine kinase inhibitor (TKI) etc. have been used for treatment, however, further improvement in therapeutic effects is desired to extend the mean survival of RCC patients.
From the recent development of cancer research, it has been made clear that cancer stem-like cells (CSC) have capabilities of indefinite cell divisions and self-regeneration (see NPL 4). In CSC, cancer stem-like cell related genes, such as CD44, CD133, OCT3/4, aldehyde dehydrogenase 1 (ALDH1), CXC-chemokine receptor 4 (CXCR4), and the like, are expressed, and the above-mentioned genes are often associated with progression of tumors or resistance to cancer treatment. Interleukin-6 (IL6) is a hyperactive cytokine. It has been known that treatment using a tyrosine kinase inhibitor (TKI) induces secretion of IL6 and activation of the pathway of the AKT-mammalian target of rapamycin (mTOR), and overexpression of IL6 may be a pathological factor for TKI resistance of RCC (see NPL 5).
Dipeptidyl peptidase IV (dipeptidyl peptidase-4, DPP4)/CD26 is integral membrane glycoprotein and also serine exopeptidase. DPP4 (CD26) has been recently indicated as a protein associated with formation of cancer stem-like cells of solid tumors including multiple myeloma.
[NPL 1] Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2018. CA Cancer J. Clin. 68, 7-30 (2018)
[NPL 2] Bray, F. et. al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 68, 394-424 (2018)
[NPL 3] Ghatalia, P, Zibelman, M, Geynisman, D. M., Plimack, E. R. Checkpoint Inhibitors for the Treatment of Renal Cell Carcinoma. Current treatment options in oncology 18, 7 (2017)
[NPL 4] Clevers, H. The cancer stem cell: premises, promises and challenges. Nat. Med. 17, 313-319 (2011)
[NPL 5] Ishibashi, K. et al. Overriding TKI resistance of renal cell carcinoma by combination therapy with IL-6 receptor blockade. Oncotarget 8, 55230-55245 (2017)
The present invention aims to solve the above-described various problems existing in the related art and to achieve the following object. Specifically, an object of the present invention is to provide a combination drug for treating a renal cancer, which has excellent therapeutic effects on a renal cancer, and a potentiator for therapeutic effects of a tyrosine kinase inhibitor, which can enhance therapeutic effects of a tyrosine kinase inhibitor on a renal cancer.
As a result of research diligently conducted by the present inventors to achieve the above-described object, the present inventors have found that use of a dipeptidyl peptidase-4 inhibitor and a tyrosine kinase inhibitor in combination can enhance therapeutic effects of a tyrosine kinase inhibitor on a renal cancer including a renal cancer having resistance to the tyrosine kinase inhibitor.
Means for solving the above-described problems are as follows.
According to the present invention, the above-described various problems existing in the related art can be solved; a combination drug for treating a renal cancer, which has excellent therapeutic effects on a renal cancer, and a potentiator for therapeutic effects of a tyrosine kinase inhibitor, which can enhance therapeutic effects of a tyrosine kinase inhibitor on a renal cancer, can be provided.
The combination drug of the present invention is a combination drug for treating a renal cancer. The combination drug includes a combination of a tyrosine kinase inhibitor and a dipeptidyl peptidase-4 inhibitor, and may further include other ingredients as necessary.
In the present invention, the term “treating” means improving or reducing symptoms of a cancer, and/or preventing any progress of the disease.
The renal cancer is not particularly limited, and may be appropriately selected according to the intended purpose. Examples of the renal cancer include renal cell carcinoma, a renal pelvic cancer, and the like. The renal cancer may be a metastatic renal cancer, or may not be a metastatic renal cancer. Moreover, the renal cancer may be a renal cancer having resistance (may be referred to as “invulnerability”) to a tyrosine kinase inhibitor, or may be a renal cancer not having resistance to the tyrosine kinase inhibitor. The combination drug of the present invention can be suitably used for the renal cancer having resistance to the tyrosine kinase inhibitor.
The tyrosine kinase inhibitor is not particularly limited, provided that the tyrosine kinase inhibitor can inhibit enzymatic activities of tyrosine kinase, or can suppress expression of tyrosine kinase. The tyrosine kinase inhibitor may be appropriately selected according to the intended purpose. Examples of the tyrosine kinase inhibitor include sunitinib, sorafenib, axitinib, pazopanib, cabozantinib, lenvatinib, and the like. The above-listed examples may be used alone or in combination.
As the tyrosine kinase inhibitor, a commercially available product may be used, or an appropriately synthesized product may be used.
The dipeptidyl peptidase-4 inhibitor is not particularly limited, provided that the dipeptidyl peptidase-4 inhibitor can inhibit enzymatic activities of dipeptidyl peptidase-4 or can suppress expression of dipeptidyl peptidase-4. The dipeptidyl peptidase-4 inhibitor may be appropriately selected according to the intended purpose. Examples of the dipeptidyl peptidase-4 inhibitor include sitagliptin, linagliptin, alogliptin, teneligliptin, anagliptin, vildagliptin, saxagliptin, trelagliptin, omarigliptin, gemigliptin, evogliptin, gosogliptin, dipeptidyl peptidase-4 expression inhibitors, and the like. The above-listed examples may be used alone or in combination.
As the dipeptidyl peptidase-4 inhibitor, a commercially available product may be used, or an appropriately synthesized product may be used.
The dipeptidyl peptidase-4 expression inhibitor is not particularly limited, provided that the dipeptidyl peptidase-4 expression inhibitor can suppress expression of dipeptidyl peptidase-4. The dipeptidyl peptidase-4 expression inhibitor may be appropriately selected according to the intended purpose. Examples of the dipeptidyl peptidase-4 expression inhibitor include double-stranded nucleic acid molecules inhibiting expression of a dipeptidyl peptidase-4 gene (may be referred to as “double-stranded nucleic acid molecule(s)” hereinafter), DNA including a base sequence encoding the double-stranded nucleic acid molecule (may be referred to as “DNA”), vectors including the DNA (may be referred to as “vector(s)” hereinafter), and the like. The above-listed examples may be used alone or in combination.
The double-stranded nucleic acid molecule is not particularly limited, provided that the double-stranded nucleic acid molecule can suppress expression of a dipeptidyl peptidase-4 gene. The double-stranded nucleic acid molecule may be appropriately selected according to the intended purpose. Examples of the double-stranded nucleic acid molecule include double-stranded nucleic acid molecules each including (a) a sense strand including a base sequence corresponding to a target sequence composed of a base sequence of SEQ ID NO: 1 or SEQ ID NO: 4, and (b) an antisense strand that constitutes a double stranded nucleic acid with the sense strand of (a), and includes a base sequence complimentary to the sense strand of (a), and the like. The above-listed examples may be used alone or in combination.
In the present invention, the term “double-stranded nucleic acid molecule” encompasses a double-stranded nucleic acid molecule in which a sense strand and an antisense strand are hybridized.
In the present invention, mRNA of the dipeptidyl peptidase-gene is a target of the double-stranded nucleic acid molecule so that the double-stranded nucleic acid molecule suppresses expression of the dipeptidyl peptidase-4 gene. Accordingly, the dipeptidyl peptidase-4 gene may be referred to as a “target gene” of the double-stranded nucleic acid molecule in the present specification.
The double-stranded nucleic acid molecule preferably includes (a) a sense strand including a base sequence corresponding to a target sequence composed of a base sequence of SEQ ID NO: 1 or SEQ ID NO: 4, and (b) an antisense strand that constitutes a double stranded nucleic acid with the sense strand of (a), and includes a base sequence complimentary to the sense strand.
The sense strand and the antisense strand may be RNA strands or chimeric RNA-DNA strands. The sense strand and the antisense strand may be hybridized to each other to form the double-stranded nucleic acid molecule.
The sense strand of the double-stranded nucleic acid molecule is not limited, as long as the sense strand includes a base sequence corresponding to the target sequence. The sense strand may further include other base sequences, or may be composed only of the base sequence corresponding to the target sequence.
Moreover, the antisense strand of the double-stranded nucleic acid molecule is not limited, as long as the antisense strand includes a base sequence complimentary to the sense strand, where the degree of the complementarity is sufficient enough for the antisense strand and the sense strand to hybridize. The antisense strand may further include other base sequences. The antisense strand preferably includes the base sequence complimentary to the sense strand in the proportion of 70% or greater, more preferably 80% or greater, further preferably 90% or greater, and particularly preferably 95% or greater.
A type of the double-stranded nucleic acid molecule is not particularly limited, and may be appropriately selected according to the intended purpose. Examples of the type of the double-stranded nucleic acid molecule include double-stranded RNA (dsRNA), double-stranded RNA-DNA chimeras, and the like.
In the present specification, the term “double-stranded RNA” encompasses a double-stranded nucleic acid molecule, in which both a sense strand and antisense strand are composed of RNA sequences; the term “double-stranded RNA-DNA chimera” encompasses a double-stranded nucleic acid molecule, in which both the sense strand and antisense strand are composed of chimeric RNA-DNA sequences.
The double-stranded RNA or double-stranded RNA-DNA chimera is preferably small interfering RNA (siRNA) or chimeric siRNA, more preferably siRNA.
In the present specification, siRNA is a small-molecule double-stranded RNA having a length of 18 bases to 29 bases, and has a function of cleaving a target RNA having a sequence complimentary to the antisense strand (guide strand) of the siRNA to suppress expression of the target RNA.
The terminus structure of the siRNA is not particularly limited, provided that the siRNA includes the above-described sense strand and antisense strand, and is capable of suppressing expression of a target RNA. The terminus structure of the siRNA may be appropriately selected according to the intended purpose. For example, the siRNA may have blunt ends, or sticky ends (overhangs). Among the above-listed examples, the siRNA preferably has a structure where the 3′ end of each chain is stuck out by 2 bases to 6 bases, more preferably a structure where the 3′ end of each chain is stuck out by 2 bases.
Moreover, the chimeric siRNA is a small-molecule double-stranded RNA-DNA chimera having a length of 18 bases to 29 bases, where part of the RNA sequence of siRNA is converted to DNA. Among the above-described chimeric siRNAs, the chimeric siRNA is preferably a small-molecule double-stranded RNA-DNA chimera having a length of 21 bases to 23 bases, where the bases within the 8 bases of the 3′ side of the sense strand of siRNA or the bases within the 6 bases of the 5′ side of the sense strand of siRNA are converted to DNA. Like the siRNA, the chimeric siRNA has a function of suppressing expression of a target gene. Note that, the chimeric siRNA also includes an embodiment of the chimeric siRNA, in which part of the sequence converted to DNA is again converted to RNA.
Similar to the siRNA, the terminus structure of the chimeric siRNA is not particularly limited, and may be appropriately selected according to the intended purpose. For example, the chimeric siRNA may have blunt ends, or sticky ends (overhangs).
Specific examples of the siRNA include the following.
Examples of the siRNA whose target sequence is the base sequence of the SEQ ID NO: 1 include siRNA composed of the sense strand of SEQ ID NO: 2 below and the antisense strand of SEQ ID NO: 3 below.
Moreover, Examples of the siRNA whose target sequence is the base sequence of the SEQ ID NO: 4 include siRNA composed of the sense strand of SEQ ID NO: 5 below and the antisense strand of SEQ ID NO: 6 below.
Moreover, the double-stranded RNA may be short hairpin RNA (shRNA). The shRNA is single-stranded RNA including a dsRNA region of about 18 bases to about 29 bases, and a loop region of about 3 bases to about 9 bases. As the shRNA is expressed in vivo, the shRNA forms base pairs to construct a hairpin double-stranded RNA. Then, the shRNA is cleaved by Dicer (RNase III enzyme) to become siRNA to function to suppress expression of target RNA. Similar to the siRNA and double-stranded RNA-DNA chimera, the terminus structure of the shRNA is not particularly limited, and may be appropriately selected according to the intended purpose. For example, the shRNA may have blunt ends, or sticky ends (overhangs).
Moreover, the double-stranded nucleic acid molecule may be appropriately modified according to the intended purpose. For example, 2′-O—-methylation modification, phosphorothioate (S—) modification, locked nucleic acid (LNA) modification or the like may be performed on the double-stranded nucleic acid molecule to impart resistance to nucleolytic enzymes (nuclease) to improve stability in a liquid culture medium or in vivo. For example, moreover, the 5′ end or 3′ end of the sense strand of the double-stranded nucleic acid molecule may be modified with nanoparticles, cholesterol, cell-penetrating peptides, or the like to increase transfer efficiency to cells. Note that, the method of modifying the double-stranded nucleic acid molecule to achieve any of the above-mentioned modifications is not particularly limited, and may be appropriately selected from methods known in the related art.
A method of acquiring the double-stranded nucleic acid molecule is not particularly limited, and may be produced according to any method selected from the methods known in the related art.
For example, the siRNA can be produced by chemically synthesizing two single-stranded RNA sequences each having a length of 18 bases to 29 bases corresponding to the predetermined sense strand and the predetermined antisense strand, respectively, using a known automatic DNA/RNA synthesis device, etc., and annealing the synthesized two single-stranded RNA sequences. Moreover, a commercial product of an annealed double-stranded siRNA may be acquired. Alternatively, the siRNA may be acquired by commissioning a contract siRNA synthesis service provider to synthesize the siRNA. Moreover, a suitable siRNA-expression vector, such as the below-described vector of the present invention, is constructed, followed by transferring the expression vector into a cell to produce siRNA utilizing a reaction within the cell.
Moreover, the chimeric siRNA can be produced, for example, by chemically synthesizing both a sense strand and an antisense strand, which are chimeric nucleic acid molecules, and annealing the sense strand and the antisense strand to produce a chimeric siRNA.
The DNA is not particularly limited, provided that the DNA is DNA including a base sequence encoding the above-described double-stranded nucleic acid molecule. The DNA may be appropriately selected according to the intended purpose. The DNA preferably has a sequence to which a promoter sequence is linked upstream (5′ side) of the base sequence encoding the double-stranded nucleic acid molecule. The promoter sequence is a sequence for controlling transcription of the double-stranded nucleic acid molecule. The promoter sequence is not particularly limited, and may be appropriately selected according to the intended purpose. Examples of the promoter sequence include: pol II-based promoters, such as CMV promoters; pol III-based promoters, such as H1 promoters, and U6 promoters; and the like.
Moreover, a terminator sequence to terminate transcription of the double-stranded nucleic acid molecule is more preferably linked downstream (3′ side) of the base sequence encoding the above-described double-stranded nucleic acid molecule. The terminator sequence is not particularly limited, and may be appropriately selected according to the intended purpose.
The transcription unit including the promoter sequence, the base sequence for encoding the double-stranded nucleic acid molecule, and the terminator sequence is one of preferable embodiments of the DNA. Note that, the transcription unit may be constructed using any method known in the related art.
The vector is not particularly limited, provided that the vector includes the above-described DNA. The vector may be appropriately selected according to the intended purpose. Examples of the vector include plasmid vectors, viral vectors, and the like. The vector is preferably an expression vector capable of expressing the above-described double-stranded nucleic acid molecule.
A manner of the expression of the double-stranded nucleic acid molecule is not particularly limited, and may be appropriately selected according to the intended purpose. Examples of the method of expressing siRNA as the double-stranded nucleic acid molecule include: a method of expressing two strands of short single-stranded RNA (tandem type); a method of expressing a single-stranded RNA as shRNA (hairpin type); and the like.
The tandem-type siRNA-expression vector includes DNA including a DNA sequence encoding a sense strand constituting the siRNA, and a DNA sequence encoding an antisense strand constituting the siRNA, where a promoter sequence is linked upstream (5′ side) of the DNA sequence encoding each strand, and a terminator sequence is linked downstream (3′ side) of the DNA sequence encoding each strand.
Moreover, the hairpin-type siRNA-expression vector includes DNA, in which a DNA sequence encoding a sense strand constituting the siRNA and a DNA sequence encoding an antisense strand constituting the siRNA are aligned in the reverse direction, the sense strand DNA sequence and the antisense strand DNA sequence are connected with a loop sequence, a promoter sequence is linked upstream (5′ side) of the DNA sequence encoding each strand, and a terminator sequence is linked downstream (3′ side) of the DNA sequence encoding each strand.
The above-mentioned vectors can be constructed using any method known in the related art. For example, the DNA can be constructed by linking (ligating) to a cleaved site of the vector, which is cleaved in advance by a restriction enzyme.
As the DNA of the vector is introduced (transfected) into cells, the promoter is activated to generate the double-stranded nucleic acid molecule. With the tandem vector, for example, the DNA is transcribed within a cell to generate a sense strand and an antisense strand, and the generated sense strand and antisense strand are hybridized to generate siRNA. With the hairpin-type vector, the DNA is transcribed within a cell to first generate hairpin-type RNA (shRNA), followed by processing with Dicer to generate siRNA.
Other ingredients included in the combination drug are not particularly limited, provided that such ingredients do not adversely affect the effects obtainable by the present invention. Other ingredients are appropriately selected from pharmaceutically acceptable carriers according to the intended purpose. Examples of other ingredients include additives, adjuvants, water, and the like. The above-listed examples may be used alone or in combination.
The additives or the adjuvants are not particularly limited, and may be appropriately selected according to the intended purpose. Examples of the additives or the adjuvants include bactericide, preservatives, caking additives, thickeners, adhesives, binders, colorants, stabilizers, pH regulators, buffers, tonicity agents, solvents, antioxidants, UV-blocking agents, crystal precipitation (growth) inhibitors, defoaming agents, physical property improving agents, antiseptic agents, and the like.
The amounts of other ingredients in the combination drug are not particularly limited, and may be appropriately selected according to the intended purpose.
The combination drug may be used as an embodiment including only a combination of the tyrosine kinase inhibitor and the dipeptidyl peptidase-4 inhibitor, or as an embodiment where the combination of the tyrosine kinase inhibitor and the dipeptidyl peptidase-4 inhibitor is further combined with a drug including other substances as active ingredients.
Moreover, the combination drug may be used in the state where the combination drug is blended with a drug including other substances as active ingredients.
The combination drug may contain the tyrosine kinase inhibitor and the dipeptidyl peptidase-4 inhibitor as two separate preparations, or as one preparation (compounded preparation).
A dosage form of the combination drug is not particularly limited, and may be appropriately selected according to the intended administration method. Examples of the dosage form include oral solid preparations (e.g., pills, coated pills, granules, powder, capsules, and the like), oral liquid preparations (e.g., oral solutions, syrups, elixirs, and the like), injectable preparations (e.g., solutions, suspensions, solid preparations to be used for reconstitution, and the like), ointment, patches, gel, cream, topical powder, spray, inhalant powder, and the like.
In the case where the tyrosine kinase inhibitor and the dipeptidyl peptidase-4 inhibitor prepared as separate preparations are used as the combination drug, the dosage form is not particularly limited, and may be appropriately selected according to the intended purpose. The dosage form of the tyrosine kinase inhibitor and the dosage form of the dipeptidyl peptidase-4 inhibitor may be the same or different.
The oral solid preparation can be produced by adding, to the active ingredients, a diluent, and further adding optional additives, such as binders, disintegrants, lubricants, colorants, flavoring agents, and the like, according to a commonly known method.
Examples of the diluent include lactose, refined sugar, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid, and the like. Examples of the binders include water, ethanol, propanol, simple syrup, glucose solutions, starch solutions, gelatin solutions, carboxy methyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, Shellac, calcium phosphate, polyvinyl pyrrolidone, and the like. Examples of the disintegrants include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose, and the like. Examples of the lubricants include refined talc, stearate, borax, polyethylene glycol, and the like. Examples of the colorants include titanium oxide, iron oxide, and the like. Examples of the flavoring agents include refined sugar, orange peel, citric acid, tartaric acid, and the like.
The oral liquid preparation can be produced, for example, by adding, to the active ingredients, additives, such as flavoring agents, buffers, stabilizers, and the like, according to a commonly known method.
Examples of the flavoring agents include refined sugar, orange peel, citric acid, tartaric acid, and the like. Examples of the buffers include sodium citrate, and the like. Examples of the stabilizers include tragacanth, gum arabic, and gelatin, and the like.
The injectable preparation for subcutaneous injection, intramuscular injection, intravenous injection, or the like can be produced, for example, by adding, to the active ingredients, a pH regulator, a buffer, a stabilizer, a tonicity agent, local anesthetic agents, and the like, according to a commonly known method.
Examples of the pH regulators and the buffers include sodium citrate, sodium acetate, sodium phosphate, and the like. Examples of the stabilizers include sodium metabisulfite, EDTA, thioglycolic acid, thiolactic acid, and the like. Examples of the tonicity agents include sodium chloride, glucose, and the like. Examples of the local anesthetic agents include procaine hydrochloride, lidocaine hydrochloride, and the like.
The ointment can be produced, for example, by blending, with the active ingredients, a base material, a stabilizer, a wetting agent, a preservative, and the like known in the related art according to a commonly known method.
Examples of the base material include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, paraffin, and the like. Examples of the preservative include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and the like.
The patch can be produced, for example, by applying cream, gel, paste, or the like as the ointment onto a support known in the related art according to a commonly known method.
Examples of the support include: woven fabrics and nonwoven fabrics of cotton, staple fibers, or chemical fibers; films of soft vinyl chloride, polyethylene, polyurethane, or the like; and foam sheets; and the like.
A method, dose, timing, and subject of administration of the combination drug are not particularly limited, and may be appropriately selected according to the intended purpose.
The method of administering the combination drug is not particularly limited. For example, either topical administration or systemic administration may be selected according to the dose foam of the combination drug, conditions of a patient, and the like. For the topical administration, for example, the combination drug can be administered by directly injecting the active ingredients of the combination drug into a suitable site (e.g., a tumor site). For the injection, a method known in the related art, such as injection by a syringe, can be appropriately adapted. For the systematic administration (e.g., oral administration, intravascular administration, and the like), moreover, a drug delivery technology known in the art is preferably appropriately applied to stably and efficiently deliver the active ingredients of the combination drug to a suitable site (e.g., a tumor site).
In the case where the tyrosine kinase inhibitor and the dipeptidyl peptidase-4 inhibitor prepared as separate preparations are used as the combination drug, the administration method of the tyrosine kinase inhibitor and the administration method of the dipeptidyl peptidase-4 inhibitor may be the same or different.
The administration dose is not particularly limited, and may be appropriately selected considering various factors, such as age, weight, body conditions of an administration subject, presence or absence of administration of a drug or pharmaceutical preparation including other substances as active ingredients, and the like.
The ratio between the dose of the tyrosine kinase inhibitor and the dose of the dipeptidyl peptidase-4 inhibitor in the combination drug is not particularly limited, and may be appropriately selected according to the intended purpose.
Moreover, the frequency of the administration is not particularly limited, and may be appropriately selected considering various factors, such as age, weight, body conditions of an administration subject, presence or absence of administration of a drug or pharmaceutical preparation including other substances as active ingredients, and the like.
The timing of the administration is not particularly limited, and may be appropriately selected according to the intended purpose.
As the combination drug, the tyrosine kinase inhibitor and the dipeptidyl peptidase-4 inhibitor may be administered at the same time, or may be administered at different timing.
When the tyrosine kinase inhibitor and the dipeptidyl peptidase-4 inhibitor are administered at different timing, the order of administering the tyrosine kinase inhibitor and the dipeptidyl peptidase-4 inhibitor is not particularly limited, and may be appropriately selected according to the intended purpose.
When the tyrosine kinase inhibitor and the dipeptidyl peptidase-4 inhibitor are administered at different timing, moreover, the interval between the administration of the tyrosine kinase inhibitor and the administration of the dipeptidyl peptidase-4 inhibitor is not particularly limited, and may be appropriately selected according to the intended purpose.
A subject for the administration is not particularly limited, and may be appropriately selected according to the intended purpose. Examples of the subject for the administration include humans, monkeys, pigs, cows, sheep, goats, dogs, cats, mice, rats, birds, and the like. Among the above-listed examples, the combination drug is particularly suitably used for humans.
As described in the section of Examples below, the combination drug can enhance a tumor proliferation inhibition effect (enhance therapeutic effects) of a tyrosine kinase inhibitor by using a dipeptidyl peptidase-4 inhibitor in combination. Accordingly, the present invention is also related to a method of treating a renal cancer, where the method includes administering the combination drug of the present invention to a subject.
The renal cancer is not particularly limited. Examples of the renal cancer include those described in the section of the renal cancer in association with the above-described combination drug.
The method of treating a renal cancer may further use other renal cancer drugs.
The potentiator for therapeutic effects of a tyrosine kinase inhibitor of the present invention includes at least a dipeptidyl peptidase-4 inhibitor, and may further include other ingredients as necessary.
The potentiator for therapeutic effects of a tyrosine kinase inhibitor can enhance therapeutic effects of the tyrosine kinase inhibitor on a renal cancer.
In the present invention, the meaning of the phrase “enhance therapeutic effects” includes enhancing a tumor proliferation inhibition effect, and recovering therapeutic effects of the tyrosine kinase inhibitor against tumors having resistance to the tyrosine kinase inhibitor (overturning the resistance).
Examples of the renal cancer include those described in the section of the renal cancer in association with the above-described combination drug.
Examples of the dipeptidyl peptidase-4 inhibitor include those listed in the section of the dipeptidyl peptidase-4 inhibitor in association with the above-described combination drug.
The amount of the dipeptidyl peptidase-4 inhibitor in the potentiator for therapeutic effects of the tyrosine kinase inhibitor is not particularly limited, and may be appropriately selected according to the intended purpose. The potentiator for therapeutic effects of the tyrosine kinase inhibitor may be composed only of the dipeptidyl peptidase-4 inhibitor.
Other ingredients included in the potentiator for therapeutic effects of the tyrosine kinase inhibitor are not particularly limited, and may be appropriately selected from pharmaceutically acceptable carriers according to the intended purpose. Examples of such ingredients include those listed in the section of other ingredients in association with the above-described combination drug. Those listed as examples may be used alone or in combination.
The amounts of other ingredients in the potentiator for therapeutic effects of the tyrosine kinase inhibitor are not particularly limited, and may be appropriately selected according to the intended purpose.
Examples of the tyrosine kinase inhibitor, which is a target of the potentiator for therapeutic effects of the tyrosine kinase inhibitor, include those listed in the section of the tyrosine kinase inhibitor in association with the above-described combination drug.
The potentiator for therapeutic effects of the tyrosine kinase inhibitor may be used in combination with a tyrosine kinase inhibitor, or in combination with the tyrosine kinase inhibitor and another drug including other substances as active ingredients. Moreover, the potentiator for therapeutic effects of the tyrosine kinase inhibitor may be used in the state where the potentiator is blended into a drug including a tyrosine kinase inhibitor or other substances as active ingredients.
A dosage form of the potentiator for therapeutic effects of the tyrosine kinase inhibitor is not particularly limited, and may be appropriately selected according to the intended purpose. Examples of the dosage form include those listed in the section of the dosage form of the above-described combination drug.
The method, dose, timing, and subject for the administration of the potentiator for therapeutic effects of the tyrosine kinase inhibitor are not particularly limited, and may be appropriately selected according to the intended purpose. Examples thereof include those listed in the section of the administration of the above-described combination drug.
As described in the section of Examples below, the potentiator for therapeutic effects of the tyrosine kinase inhibitor can enhance the tumor proliferation inhibition effect of the tyrosine kinase inhibitor. Therefore, the present invention is also related to a method of enhancing therapeutic effects of a tyrosine kinase inhibitor on a renal cancer, where the method includes administering the potentiator of the present invention to a subject.
The renal cancer is not particularly limited, and may be appropriately selected according to the intended purpose. Examples of the renal cancer include those listed in the section of the renal cancer in association with the above-described combination drug.
The tyrosine kinase inhibitor administered in combination with the administration of the potentiator for therapeutic effects of the tyrosine kinase inhibitor is not particularly limited, and may be appropriately selected according to the intended purpose. Examples of the tyrosine kinase inhibitor include those listed in the section of the tyrosine kinase inhibitor of the above-described combination drug.
The method of enhancing the therapeutic effects of the tyrosine kinase inhibitor on the renal cancer may further use another drug for treating a renal cancer.
Test Examples of the present invention will be described hereinafter, but Test Examples shall not be construed as limiting the scope of the present invention in any way.
Seventy-three (73) cases of renal cell carcinoma (RCC) patients who received a tyrosine kinase inhibitor (TKI) between 2008 and 2019 at Saitama Medical Center, Saitama Medical University, were retrospectively analyzed. Overall survival of the patients from the initial consultation, and the maximum tumor shrinkage defined by Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) (Eisenhauer, E. A. et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur. J. Cancer 45, 228-247 (2009)) were evaluated. For the maximum tumor shrinkage, the most recent TKI treatment was evaluated, among the evaluatable treatments. The patients who did not wish to consent with the trial, the cases where objective images could not be obtained before and after the TKI treatment, and the patients who received only immunotherapy or an mTOR inhibitor were removed from the data. If the values of clinical factors were missing, such cases were removed from statistical analysis for comparing the characteristics of the patients. This clinical analysis has been approved by the Ethics Committee of Saitama Medical Center, Saitama Medical University (No. 117, No. 2308).
After attaining the informed consent from the patients at Saitama Medical Center, Saitama Medical University, patient-derived cells (PDC) were produced from the tumors excised from the RCC patients. The tumor samples were processed with reference to the literature (Namekawa, T, et al. ALDH1A1 in patient-derived bladder cancer spheroids activates retinoic acid signaling leading to TUBB3 overexpression and tumor progression. Int. J. Cancer 146, 1099-1113 (2019)). The protocol of the research was approved by the Ethics Committee of Saitama Medical Center, Saitama Medical University (No. 1363-IV).
ACHN and 769-P, which were human RCC cell lines, were acquired from the American Type Culture Collection (ATCC), and those authenticated by short tandem repeat (STR) analysis performed by BEX CO., LTD. were used. The ACHN and 769-P cells were cultured on a DEME medium and a PRMI medium (NACALI TESQUE, INC.), respectively, to both of which 10% of FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin were added, using an incubator set at 37° C. and 5% CO2. The ACHN and 769-P cells were exposed to sunitinib (SUN) at 10 μM max., for 6 months or longer, to produce SUN resistant RCC cell lines, ACHN-R and 769-P-R, respectively.
siRNA (siDPP4 #1 and #2), whose target was dipeptidyl peptidase-4 (DPP4), and control siRNA (siControl) were acquired from RNAi Inc., and were introduced into the cells using the RNAiMAX reagent (Thermo Fisher Scientific K.K.) according to the instructions from the manufacturer.
The sequences of the siRNA are presented below.
A cell survival rate of the cells 3 days after the drug treatment and siRNA transfection was evaluated by CellTiter-Glo 3D Assay (Promega K.K.).
Extraction of RNA, synthesis of cDNA, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were carried out according to the methods disclosed in “Namekawa, T, et al. ALDH1A1 in patient-derived bladder cancer spheroids activates retinoic acid signaling leading to TUBB3 overexpression and tumor progression. Int. J. Cancer 146, 1099-1113 (2019).”
The primers used for the qRT-PCR are as follows.
The formalin-fixed tissue sample was embedded in paraffin, and the formalin-fixed, paraffin-embedded sample was sliced to prepare a cut piece. For immunohistochemical staining of DPP4, a Histofine kit (NICHIREI CORPORATION) using streptavidin-biotin amplification was used. As primary antibodies, Ki67 (dilution 1:100; MIB1) and DPP4 (dilution 1:100; AF1180-SP) were used. Secondary antibodies were acquired from Agilent Technologies and R&D Systems. The evaluation of immunostaining was carried out by a specialized pathologist.
Chromatin immunoprecipitation (ChIP) was carried out according to the method disclosed in “Namekawa, T, et al. ALDH1A1 in patient-derived bladder cancer spheroids activates retinoic acid signaling leading to TUBB3 overexpression and tumor progression. Int. J. Cancer 146, 1099-1113 (2019).”
The sequences of the primers used for the chromatin immunoprecipitation PCR are as follows.
A retinoic acid responsive element (RARE) determined by JASPAR (Wasserman W. W. & Sandelin A. Applied bioinformatics for the identification of regulatory elements. Nat. Rev. Genet. 5, 276-287 (2004)) and a mutant oligonucleotide thereof, or RARE direct repeat (DR5) (Bulens, F. et al., Retinoic acid induction of human tissue-type plasminogen activator gene expression via a direct repeat element (DR5) located at −7 kilobases. J. Biol. Chem. 270, 7167-7175 (1995)) were generated by annealing corresponding oligonucleotides (see the sequences of the primers below), and were inserted into a pGL3 promoter (Promega K.K.). A renilla luciferase signal was used as an internal standard, and the luciferase activity was analyzed by Dual-Luciferase Reporter Assay System (Promega K.K.).
The sequences of the primers used for the luciferase assay are as follows.
All of the animal tests performed was approved by Animal
Management and Use Committee of Saitama Medical University, and was carried out according to the guidelines and regulations for the management and use of test animals, set by Saitama Medical University. Male nude mice (BALB/c-nu/nu, 6 weeks old) were acquired from CLAIR Japan, Inc. The ACHN cells and the ACHN−R cells were each subjected to trypsinization, followed by washing with PBS. Then, 5×106 cells of the ACHN cells or the ACHN−R cells were dispersed in 150 μL of Matrigel (BD Biosciences), and were subcutaneously inoculated in each mouse. The size of the tumor was measured every day. The tumor volume (V) was determined by measuring the length (L) and the width (W), and inserting the measured values in the following equation.
V=(L×W2)×0.52
When the volume of the engrafted tumor reached 180 mm3, SUN (20 mg/kg) was orally administered to the mice inoculated with the ACHN cells, and SUN (20 mg/kg) alone or two drugs, SUN (20 mg/kg) and DPP4 inhibitor sitagliptin (SITA) (30 mg/kg), were orally administered to the randomly selected mice inoculated with the ACHN-R cells. As a solvent for each drug, a solution including carboxy methyl cellulose sodium (0.5% wt/vol), NaCl (1.8% wt/vol), Tween 80 (0.4% wt/vol), and benzyl alcohol (0.9% wt/vol) was used according to the literature (Stany, M. P. et al. Identification of novel therapeutic targets in microdissected clear cell ovarian cancers. PLoS One 6, e21121 (2011)). The drug was administered in a cycle consisting of 2 consecutive days of administration, followed by 1 day of washout.
For the clinical data, the long rank test was used for the Kaplan-Meier method. As the post-hoc test of multiple comparison, the Holm-Bonferroni method was used. For the partial response rate and characteristics of the patients, the Fisher's exact test was used. For the maximum tumor shrinkage, the Mann-Whitney U test was used. In the cell proliferation assay or quantitative reverse transcription PCR, the two-tailed student's t-test was used for comparison between 2 groups, and the two-way
ANOVA was used for multiple comparison. For statistical calculations, JMP 9.0.0 (SAS Institute Japan, Ltd.) was used.
In order to investigate the correlation between the expression of DPP4 and the expression of the cancer stem-like cell (CSC) related gene in the RCC stem-like cells, PDC was produced from 15 cases of clear cells RCC by 3D spheroid culture employing a technique of culturing regular tissue stem cells. The expression level of DPP4 was evaluated by quantitative reverse transcription PCR. As the internal standard, 36B4 was used. The Spearman' s rank correlation coefficient(R) and statistical significance (P) were calculated using JMP software ver. 9.0.0.0 of SAS Institute Japan, Ltd.
The results are presented in
As depicted in
Next, the hematoxylin-eosin (HE) staining and DPP4 immunohistochemical staining were performed to study the morphologies of the original tumors of RCC-A and RCC-B, which were typical PDC.
The results are presented in Table 2a.
As depicted in
In order to evaluate whether DPP4 was related to properties of cancer stem-like cells, and contributed to survival of cancer cells, an experiment was carried out using a multi-target receptor tyrosine kinase inhibitor sunitinib (SUN), which was known as a medicine for RCC. In order to investigate how inhibition of DPP4 affected therapeutic effects of SUN, DPP4 inhibitor SITA, which had been already known as a therapeutic drug for Type 2 diabetes mellitus (T2DM), or siRNA (siDPP4 #1 and #2) whose target was DPP4 was used in combination with SUN to evaluate the effect on proliferation of spheroids.
The production of the spheroid ATP in the RCC-A cells or RCC-B cells treated with SUN alone or a combination of SUN and SITA on the third day was measured with the luciferase activity using the CEllTiter-Glo. As depicted in
As depicted in
As depicted in
Next, ACHN-R cells and 769-P-R cells, which were both SUN resistant RCC cell lines, were experimentally produced using ACHN cells and 769-P cells, which were both RCC cell lines, respectively. The expression level of the mRNA of the DPP4 and the expression level of the mRNA of the CSC-related gene were compared between the ACHN-R cells and the parent cell line thereof, or between the 769-P-R cells and the parent cell line thereof.
As depicted in
Moreover, SITA or siDPP4 was used in combination with SUN in the ACHN-R cells or 769-P-R cells to evaluate an effect to the proliferation of spheroids.
First, a dose-response effect of sitagliptin (SITA) to 3D spheroid proliferation of ACHN−R, 769-P-R, or a parent cell line thereof was investigated with administration of SITA alone.
As presented in
As depicted in
As depicted in
As depicted in
As depicted in
Since the expression level of ALDH1 and the expression level of DPP4 were correlated, a hypothesis was made that the function of ALDH1 might be related to expression of DPP4. ALDH1 is an enzyme related to metabolization of retinol. ALDH1 converts retinol to retinoic acid (RA) to act as a ligand for an RA receptor, such as a retinoic acid receptor α (RARα) etc.
As depicted in
Next, in order to determine the RARα bonding site in the DPP4 gene promoter region, the promoter region of DPP4 searched from the hg19 human genome dataset was searched with the matrix profile of RARE using an open-access transcription factor binding profile database JASPAR
(http://jaspar.genereg.net/)(Wasserman W. W. & Sandelin A. Applied bioinformatics for the identification of regulatory elements. Nat. Rev. Genet. 5, 276-287 (2004)).
One RARE candidate was determined in the DPP4 promoter region that was from the position of the transcription start site (TSS) to −1619 bp, and to −1647 bp, where the threshold of the relative profile score of the JASPAR algorithm exceeded 85% (
As depicted in
Next, a luciferase reporter assay was carried out to investigate whether RA modified the DPP4 promoter activity. A luciferase reporter including wild-type (WT) RARE, and a luciferase reporter including mutated (Mut) RARE were used. Moreover, as known RARE, RARE having a direct repeat 5 (DR5) (Bulens, F. et al. Retinoic acid induction of human tissue-type plasminogen activator gene expression via a direct repeat element (DRS) located at −7 kilobases. J. Biol. Chem. 270, 7167-7175 (1995)) was inserted into a luciferase reporter, and the resulting luciferase reporter was used as a positive control.
The results are presented in
As depicted in
An effect of SITA used in combination on proliferation of SUN resistant RCC tumors was examined using a xenograft tumor model.
At the point when the volume of the ACHN or ACHN−R xenograft nude mouse tumor reached 180 mm3, oral administration of SUN alone, or a combination of SUN and SITA was started.
The proliferation curves of the ACHN xenograft tumor treated with sunitinib (SUN) (ACHN/SUN group, n=6), the ACHN−R xenograft tumor treated with SUN (ACHN-R/SUN group, n=6), and the ACHN-R xenograft tumor treated with SUN and SITA (ACHN-R/SUN+SITA group, n=6) are depicted in
Moreover, typical photographs of the xenograft nude mice models of all groups on the 13th day are depicted in
As depicted in
Ki67 immunohistochemical staining of the excised tumor of each group was carried out to determine a ratio of Ki67 positive cells (Ki67 index). The result is depicted in
As presented in
As presented in
It was confirmed from the results described above that the sitagliptin (SITA) contributed to overturn the sunitinib resistance of the RCC xenograft tumor.
To investigate how the data presented by the in vitro tests and In vivo tests above relates to the prognosis in the clinical cases or drug efficacy, a retrospective clinical study was carried out on the 73 RCC cases of the patients who received the TKI (sunitinib, sorafenib, axitinib, or pazopanib) treatment at Saitama Medical Center, Saitama Medical University. Within the 73 cases, the cases where T2DM was not present, and DPP4i (sitagliptin, linagliptin, anagliptin, teneligliptin, anagliptin, or vildagliptin) was not administered were 47 cases (T2DM−/DPP4i−), the cases where T2DM was present and DPP4i was not administered were 12 cases (T2DM+/DPP4i−), and the cases where T2DM was present and DPP4i was administered were 14 cases (T2DM+/DPP4i+).
As depicted in
Next,
aevaluation based on RECIST version 1.1
banalysis according to the Fisher's exact test
As depicted in
Moreover, an analysis was carried out on the 49 cases in which immunostaining was possible, within the 73 cases, according to immunohistochemical staining of DPP4 to evaluate the association between the immunostaining of DPP4 and the prognosis or TKI therapeutic effects.
As depicted in
Within the 49 cases, the partial response rate was analyzed on the maximum tumor change of the 20 cases of the RCC patients exhibiting low DPP4 immune response. As a result, a significant difference was not observed (see Table 2 below). On the other hand, a significant difference in the maximum tumor change by the administration of the DPP4 inhibitor was observed in the 29 cases of the RCC patient exhibiting low DPP4 immune response (see Table 3 below).
aevaluation based on RECIST version 1.1
banalysis according to the Fisher's exact test
aevaluation based on RECIST version 1.1
banalysis according to the Fisher's exact test
For example, the embodiments of the present invention include the following.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-206499 | Dec 2020 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/045772 | 12/13/2021 | WO |
