JP2016193848A - Agent for treating or preventing braf inhibitor resistant melanoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agent for treating or preventing BRAF inhibitor resistant melanoma.SOLUTION: The present invention provides an agent for treating or preventing BRAF inhibitor resistant melanoma comprising interferon-β as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a therapeutic or prophylactic agent for BRAF inhibitor-resistant melanoma.

  Melanoma, also called malignant melanoma, is a metastatic and highly malignant skin cancer. Melanoma patients account for only 5% of all cancer patients in the United States, but are increasing year by year, and are characterized by a higher proportion of adults in lower age groups than other cancers (Non-Patent Documents) 1).

  As therapeutic methods for melanoma, immune checkpoint inhibitors such as anticancer agents dacarbazine, interferon preparations such as interferon-α and interferon-β, anti-CTLA-4 antibody ipilimumab and anti-PD-1 antibody nivolumab BRAF inhibitors such as vemurafenib and dabrafenib are used.

  50-60% of melanomas have mutations in the protein kinase BRAF gene, particularly mutations that cause substitution of the 600th valine residue (V600 mutation). It is higher than that of cancer species (thyroid (30-50%), digestive organ (10%), lung (3%)) (Non-patent Document 2). The above-mentioned BRAF inhibitor was developed as a drug having the feature of inhibiting the kinase activity that is constitutively activated by the V600 mutation of this protein kinase BRAF, and is highly effective against melanoma having a V600 mutation in BRAF. Show.

  On the other hand, as a combination treatment of an interferon preparation and a BRAF inhibitor, cancer treatment by a combination treatment of pegylated interferon-α and vemurafenib (Patent Document 1), and a combination treatment of interferon-β and a RAS pathway inhibitor Cancer treatment (Patent Document 2) has been reported.

International Publication No. 2012/080151 International Publication No. 2012/044684

Fisher and Larkin, Cancer Management. Research, 2012, Vol. 4, p. 243-252 Lito et al., Nature Medicine, 2013, Vol. 19, p. 1401-1409

Although BRAF inhibitor is effective against melanoma having BRAF V600 mutation, melanoma that has acquired resistance to BRAF inhibitor by its long-term administration appears, and almost all patients treated with BRAF inhibitor Relapse melanoma (Non-patent Document 2). Accordingly, development of drugs that overcome BRAF inhibitor resistance is desired.
An object of the present invention is to provide a therapeutic and prophylactic agent for BRAF inhibitor-resistant melanoma.

  As a result of intensive studies in order to solve the above problems, the present inventors have found that interferon-β has an antitumor action against melanoma that has acquired BRAF inhibitor resistance, and BRAF inhibitor resistance. It has been found that it has an action of suppressing the appearance of the acquired melanoma, and has completed the present invention.

  That is, the present invention provides a therapeutic or prophylactic agent for BRAF inhibitor-resistant melanoma containing interferon-β as an active ingredient. The present invention also provides an inhibitor for the appearance of BRAF inhibitor-resistant melanoma cells, which contains interferon-β as an active ingredient.

  The interferon-β is preferably covalently bonded to polyalkylene glycol, and more preferably covalently bonded to polyethylene glycol. When administered, the covalent conjugate of interferon-β and polyalkylene glycol has a long blood half-life and continuously acts on the living body, and therefore has a superior therapeutic or preventive effect on BRAF inhibitor-resistant melanoma. I can expect.

  The present invention also provides a melanoma therapeutic drug for use in suppressing the appearance of BRAF inhibitor-resistant melanoma cells, which contains interferon-β and a BRAF inhibitor as active ingredients.

  In addition, the present invention includes an interferon-β and a BRAF inhibitor, and the interferon-β and the BRAF inhibitor are administered simultaneously or separately to suppress the appearance of BRAF inhibitor-resistant melanoma cells A combination drug for melanoma treatment is provided.

  The interferon-β used in the melanoma therapeutic drug or the melanoma therapeutic combination drug is preferably covalently bonded to polyalkylene glycol, and more preferably covalently bonded to polyethylene glycol.

  In one embodiment of the present invention, vemurafenib can be suitably used as a BRAF inhibitor.

  The therapeutic or preventive agent for BRAF inhibitor-resistant melanoma of the present invention has an antitumor action against melanoma that has acquired resistance to BRAF inhibitor, and also has an action to suppress the appearance of melanoma cells that have acquired resistance to BRAF inhibitor. . Therefore, the therapeutic or preventive agent for BRAF inhibitor-resistant melanoma of the present invention can be used for the treatment or prevention of BRAF inhibitor-resistant melanoma.

  Moreover, the pharmaceutical for melanoma treatment for use in suppressing the appearance of BRAF inhibitor-resistant melanoma cells of the present invention can be used for suppressing the appearance of BRAF inhibitor-resistant melanoma cells together with the treatment of melanoma.

It is a figure which shows the effect | action on the cell proliferation by natural type human interferon-beta or PEG-IFN-beta with respect to a BRAF inhibitor resistant melanoma cell. It is a figure which shows the effect | action on the cell proliferation by PEG-IFN- (alpha) 2a or PEG-IFN- (beta) with respect to a BRAF inhibitor resistant melanoma cell. It is a figure which shows the effect | action with respect to cell proliferation by combined use with natural type human interferon-beta or PEG-IFN-beta, and a BRAF inhibitor with respect to a melanoma cell. A photograph of A375 cells 12 days after the start of test substance treatment, showing the inhibitory action of BRAF inhibitor-resistant melanoma cells by the combined use of natural human interferon-β or PEG-IFN-β and BRAF inhibitor on melanoma cells It is. A: Control, B: VMF 0.5 μmol / L, C: VMF 1.5 μmol / L, D: PEG-IFN-β 1000 U / mL, E: VMF 0.5 μmol / L + PEG-IFN-β 1000 U / mL, F : VMF 1.5 μmol / L + PEG-IFN-β 1000 U / mL, G: PEG-IFN-β 10000 U / mL, H: VMF 0.5 μmol / L + PEG-IFN-β 10000 U / mL, I: VMF 1.5 μmol / L + PEG -IFN- [beta] 10000 U / mL, J: IFN- [beta] 1000 U / mL, K: VMF 0.5 [mu] mol / L + IFN- [beta] 1000 U / mL, L: VMF 1.5 [mu] mol / L + IFN- [beta] 1000 U / mL.

  The therapeutic or prophylactic agent for BRAF inhibitor-resistant melanoma of the present invention contains interferon-β as an active ingredient.

  The above-mentioned interferon-β is not only one having the same amino acid sequence as that of naturally occurring interferon-β (hereinafter referred to as natural interferon-β), but also one or more in the amino acid sequence of natural interferon-β. For example, 1 to 20, typically 1 or several (2 to 10) amino acids have been deleted, substituted or added, and biological activity as interferon (hereinafter referred to as interferon) , Interferon-β amino acid variants having interferon activity). Natural interferon-β may be derived from any organism, but is preferably derived from the organism to which the therapeutic or preventive agent for BRAF inhibitor-resistant melanoma of the present invention is administered. Such organisms may be mammals, for example, rodents such as mice, rats, hamsters, or primates such as humans, gorillas, chimpanzees, and cows, horses, pigs, goats. , Sheep, dogs, cats, rabbits and the like. In a preferred embodiment, native interferon-β is derived from a human. Natural human interferon-β may include, for example, the amino acid sequence represented by SEQ ID NO: 1. The above-mentioned interferon-β also includes interferon-β in which the sugar chain portion of natural type interferon-β is modified and interferon-β having no sugar chain portion. Examples of interferon-β include interferon-β1b (trade name: betaferon) in which the amino terminal (N-terminal) methionine is deleted and the 14th cysteine is substituted.

  For example, the above-mentioned interferon-β is an amino acid having the sequence identity of 70% or more, 80% or more, preferably 90% or more, more preferably 95% or more, such as 98% or more with the amino acid sequence represented by SEQ ID NO: 1. It may be a polypeptide consisting of a sequence and having interferon activity. In the present invention, “having X% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1” means that the total length of the sequence to be compared is most identical to the total length of the sequence represented by SEQ ID NO: 1. This means that the sequence identity (%) when aligned so as to be high is X% or more.

  The above-mentioned interferon-β includes recombinant interferon-β produced by gene recombination technology based on the amino acid sequence or base sequence of interferon-β including natural type interferon-β. The nucleotide sequence encoding the amino acid sequence of interferon-β is, for example, 70% or more, 80% or more, preferably 90% or more, more preferably 95% or more, for example 98% or more with the amino acid sequence represented by SEQ ID NO: 1. It may be a base sequence consisting of an amino acid sequence having sequence identity and encoding a polypeptide having interferon activity.

  The above-mentioned interferon-β is obtained by using a known method such as extraction from a tissue, protein synthesis using a gene recombination technique, biological production using natural cells or recombinant cells that express interferon-β. be able to. Moreover, commercially available interferon-beta can also be used as interferon-beta.

  It is also preferable to use the above-mentioned chemically modified interferon-β as an active ingredient of the therapeutic or preventive agent for BRAF inhibitor-resistant melanoma of the present invention. This chemical modification may be any as long as interferon-β can have interferon activity. For example, chemically modified interferon-beta includes covalent conjugates of interferon-beta and polymers, covalent conjugates of interferon-beta and lipids, and fusion proteins of interferon-beta and other proteins. To do.

  The covalent conjugate of interferon-β and polymer is preferably a covalent conjugate of interferon-β and polyalkylene glycol.

  One molecule or two or more molecules of the polyalkylene glycol can be bonded to one molecule of interferon-β. In this case, the total molecular weight of the polyalkylene glycol bonded to one molecule of interferon-β is preferably 5,000 to 240,000, more preferably 10,000 to 80,000, and more preferably 39,000 to 45,000. 000 or less is more preferable. In addition, when two molecules of polyalkylene glycol having a molecular weight of 20,000 are covalently bonded to one molecule of interferon-β, a polyalkylene glycol having a molecular weight of 40,000 is covalently bonded to one molecule of interferon-β. May be expressed.

  One molecule or two or more molecules of interferon-β can be bonded to one molecule of the above polyalkylene glycol. In this case, the molecular weight of one molecule of the polyalkylene glycol is preferably 5,000 or more and 520,000 or less, more preferably 10,000 or more and 200,000 or less, and further preferably 39,000 or more and 45,000 or less.

  Polyalkylene glycol is composed of a large number of repeating units in one molecule, and the molecular weight of polyalkylene glycol is generally different depending on the individual molecule, and therefore is represented by an average molecular weight. Therefore, the “molecular weight” of the polyalkylene glycol in the present specification means an average molecular weight.

  As the polyalkylene glycol, a polyalkylene glycol which is a high molecular compound acceptable as a drug carrier, inactive or extremely low activity to a living body, and nontoxic or extremely low toxicity can be preferably used.

  “Covalent conjugate of interferon-β and polyalkylene glycol” is one in which one molecule or two or more molecules of polyalkylene glycol is covalently bonded to one molecule of interferon-β, and has interferon activity. And those having one or more molecules of interferon-β covalently bonded to one molecule of polyalkylene glycol and having interferon activity. As a covalent conjugate of interferon-β and polyalkylene glycol, one molecule or two or more molecules of polyalkylene glycol is covalently bonded to one molecule of interferon-β, and has interferon activity. It is more preferable. In addition, the covalent conjugate of interferon-β and polyalkylene glycol may be one in which interferon-β and polyalkylene glycol are directly bonded, or may be bonded through a linker or the like.

  The dosage of a covalent conjugate of interferon-beta, preferably chemically modified interferon-beta, such as interferon-beta and polyalkylene glycol, to provide a therapeutic or prophylactic effect against BRAF inhibitor resistant melanoma is interferon It can be expressed in terms of titer (unit; hereinafter, U) or mass (g). In the covalent conjugate of interferon-β and polyalkylene glycol, the specific activity is maintained at 10% or more compared to the specific activity of interferon-β before the polyalkylene glycol is covalently bound (interferon activity per weight). It is preferable.

  Here, the binding of the polymer compound to the protein may be referred to as chemically modifying or modifying the protein with the polymer compound, and the covalent conjugate of interferon-β and polyalkylene glycol was chemically modified with polyalkylene glycol. Also referred to as interferon-β or simply modified interferon-β. In particular, a covalent conjugate of interferon-β and polyethylene glycol (hereinafter PEG) is also referred to as PEG-modified interferon-β, PEGylated interferon-β, or PEG-conjugated interferon-β.

  Examples of the covalent bond site between interferon-β and polyalkylene glycol include the amino group, thiol group, N-terminus, C-terminus or sugar chain of interferon-β. Non-natural (artificial) amino acids introduced into interferon-β using a known gene recombination technique can also be used as a covalent bond site with polyalkylene glycol. For example, the kind of non-natural amino acid and a method for introducing a non-natural amino acid into a protein are described in International Publication No. 2011/158895.

  The interferon activity of interferon-β can be measured by a known biological measurement method. Specifically, after a sample containing interferon-β is allowed to act on cells sensitive to interferon (for example, FL cells), Sindbis virus or vesicularity capable of infecting these cells. Infecting by adding a certain amount of stomatitis virus (abbreviated as VSV), etc., and measuring the degree of virus resistance induced by interferon-β (hereinafter, antiviral activity), interferon Activity can be measured. Antiviral activity is measured using suppression of virus growth as an indicator, and dilution of a sample that inhibits 50% of cytopathic effect (abbreviated as CPE) that destroys cells as a result of virus growth. Calculated as the interferon titer (U) from the magnification (Shimoho Kobayashi et al., “Immunobiochemistry Experimental Method (Second Life Chemistry Laboratory Lecture 5)”, edited by the Japanese Biochemical Society, Tokyo Chemical Dojin, 1986, p.245. ).

  A covalent conjugate of interferon-β and polyalkylene glycol can be produced by a known method. For example, methods for covalently attaching a polyalkylene glycol to the amino group of interferon are described in US Pat. No. 4,917,888 and WO 87/00056. A method for covalently bonding a polyalkylene glycol to the thiol group of interferon is described in WO 99/55377. Methods for covalently bonding a polymer to the N-terminus of interferon are described in WO 00/23114 and JP-A-9-25298. A method for covalently bonding a polyalkylene glycol to the sugar chain of interferon is described in 1978 Makromolecular Chemistry (Vol. 179, p. 301), US Pat. No. 4,101,380 or US Pat. No. 4,179,337. A method for covalently bonding a polyalkylene glycol to an unnatural (artificial) amino acid introduced into interferon is described in 2004 Bioorganic & Medicinal Chemistry Letters (Vol. 14, p. 5743-5745). The covalent bond of polyalkylene glycol to the C-terminus of interferon-β can be achieved by introducing cysteine or a non-natural (artificial) amino acid at or near the C-terminus of interferon-β.

  Depending on the method of bonding interferon-β and polyalkylene glycol, it is necessary to activate the terminal of the polyalkylene glycol used for the covalent bond reaction. For example, a polyalkylene glycol derivative having a terminal activated with hydroxysuccinimide ester, nitrobenzene sulfonate ester, maleimide, orthopyridyl disulfide, vinyl sulfone, maleimide, iodoacetamide, carboxylic acid, azide, phosphine, or amine structure, and interferon-β It can be used to form a covalent bond with polyalkylene glycol, and these polyalkylene glycol derivatives can be synthesized by a known method.

  The covalent conjugate of interferon-β and polyalkylene glycol is preferably a covalent conjugate of interferon-β and PEG. A covalent conjugate of interferon-β and PEG has a molecular weight of 5,000 to 240,000 (preferably a molecular weight of 10,000 to 80,000, more preferably a molecular weight of 39,000 or more to one molecule of interferon-β. 45,000 or less) is preferably a covalent conjugate in which one molecule of PEG is covalently bonded.

  Preferable examples of the covalent conjugate of interferon-β and PEG include a covalent conjugate of human interferon-β and PEG. For example, the covalent conjugate of interferon-β and PEG can be prepared by the method described in International Publication No. 2005/019260. it can. As the binding site of PEG in human interferon-β, the amino group of the 19th or 134th lysine in the amino acid sequence of human interferon-β is preferable, and a covalent conjugate in which one molecule of PEG is covalently bonded to this site is more preferable. preferable. Specifically, a covalent conjugate in which one molecule of PEG having a molecular weight of 40,000 or more (preferably a molecular weight of 42,000) is covalently bonded to the amino group of the 134th lysine of the amino acid sequence of human interferon-β can be exemplified. .

  Here, the “134th lysine of the amino acid sequence of human interferon-β” means that the N-terminal amino acid (methionine) of the amino acid sequence of human interferon-β consisting of 166 amino acids (SEQ ID NO: 1) is the first. Means lysine, which is an amino acid located at position 134. That is, in this specification, the position of the amino acid residue of interferon-β is represented by taking the N-terminal amino acid of interferon-β as the first.

  The covalent conjugate of interferon-β and polyalkylene glycol may also be one in which polyalkylene glycol is bonded to the amino group of lysine in the amino acid sequence of interferon-β. The lysine of the amino acid sequence of interferon-β that becomes the polyalkylene glycol binding site may be at a position corresponding to the 19th or 134th lysine of the amino acid sequence represented by SEQ ID NO: 1 in the alignment of the amino acid sequence, It does not have to be in the corresponding position.

  “Melanoma”, also called malignant melanoma, is a type of skin cancer that arises from melanocytes in the epidermis. Melanoma is characterized by anti-vimentin antibody positive and anti-cytokeratin antibody negative, and is classified into four types: superficial enlarged type, nodule type, terminal mole type, and malignant mole type.

  “BRAF inhibitor” refers to a mutation of BRAF, which is a protein kinase, in particular, a mutant BRAF that is constitutively activated by a BRAF V600 mutation that causes an amino acid mutation (V600 mutation) at the 600th valine residue. It means an agent that inhibits kinase activity. BRAF inhibitors are highly effective against melanomas with BRAF V600 mutation. Examples of BRAF inhibitors include, but are not limited to, vemurafenib (trade name: zelboraf), dabrafenib (trade name: tafinlar), encolafenib (PLX-8394, EBI-45, EBI-45, 283 or the like. As BRAF inhibitors, vemurafenib, dabrafenib and encolafenib are preferred, and vemurafenib and dabrafenib are particularly preferred.

  “BRAF inhibitor resistance” means a state in which tumor cells are resistant to suppression of tumor growth by BRAF inhibitor treatment, and such a state is also referred to as “the tumor cells have acquired BRAF inhibitor resistance”. . In a BRAF inhibitor resistant state, the dose or concentration of BRAF inhibitor that was effective when treatment with the BRAF inhibitor was initiated is no longer effective or diminished. The “BRAF inhibitor-resistant melanoma” means a melanoma that is in a BRAF inhibitor-resistant state, and is also referred to as “BRAF inhibitor-resistant melanoma”. BRAF inhibitor resistant melanoma typically appears in subjects who have received continuous administration of a BRAF inhibitor (such as vemurafenib or dabrafenib) over a period of time.

  “Therapeutic agent for BRAF inhibitor-resistant melanoma” means a drug having a therapeutic effect on BRAF inhibitor-resistant melanoma. Treatment for BRAF inhibitor-resistant melanoma is to suppress (reduce or prevent) cell growth of BRAF inhibitor-resistant melanoma. The therapeutic agent for BRAF inhibitor-resistant melanoma may be for use in combination with a BRAF inhibitor. The therapeutic agent for BRAF inhibitor-resistant melanoma of the present invention contains interferon-β as an active ingredient, and may optionally further contain a BRAF inhibitor.

  The “prophylactic agent for BRAF inhibitor-resistant melanoma” means a drug having a preventive effect on BRAF inhibitor-resistant melanoma. Prevention against BRAF inhibitor resistant melanoma is to suppress (reduce or prevent) the emergence of BRAF inhibitor resistant melanoma cells, particularly in subjects who have received or have been administered BRAF inhibitors. To suppress (reduce or prevent) the appearance of cells. That is, the “preventive agent for BRAF inhibitor-resistant melanoma” of the present invention shows the suppression of the appearance of BRAF inhibitor-resistant melanoma cells. The preventive agent for BRAF inhibitor-resistant melanoma of the present invention contains interferon-β as an active ingredient. In the context of the present invention, “showing suppression of appearance of BRAF inhibitor-resistant melanoma cells” means having an action of suppressing the appearance of melanoma cells that have acquired resistance to BRAF inhibitors. Therefore, the present invention also relates to a BRAF inhibitor-resistant melanoma appearance inhibitor containing interferon-β as an active ingredient. The preventive agent for BRAF inhibitor-resistant melanoma or the suppressor for appearance of BRAF inhibitor-resistant melanoma may be for use in combination with a BRAF inhibitor. Alternatively, the BRAF inhibitor-resistant melanoma preventive agent or BRAF inhibitor-resistant melanoma appearance inhibitor of the present invention may optionally further contain a BRAF inhibitor in addition to interferon-β.

  The therapeutic effect or the preventive effect on the BRAF inhibitor-resistant melanoma and the BRAF inhibitor-resistant melanoma appearance suppressing effect can be evaluated by a model using a BRAF inhibitor-resistant melanoma or a melanoma having a V600 mutation in the BRAF gene. Such models include, for example, an in vitro model of BRAF inhibitor-resistant melanoma (Molecular cancer therapeutics, 2012, Vol. 11, p. 909-920), BRAF inhibitor-resistant melanoma transfer model mice (Nature, 2013). Year, Vol. 494, p.251-255). Moreover, an evaluation test can be implemented according to the below-mentioned Example.

  The therapeutic or prophylactic agent for BRAF inhibitor-resistant melanoma containing the above-mentioned interferon-β as an active ingredient has an antitumor action against BRAF inhibitor-resistant melanoma, and melanoma cells that have acquired resistance to BRAF inhibitor Therefore, it can be used for treatment or prevention (suppression of appearance) of BRAF inhibitor-resistant melanoma.

  The therapeutic or prophylactic agent for BRAF inhibitor-resistant melanoma containing the above-mentioned interferon-β as an active ingredient is used in combination with a BRAF inhibitor in the treatment of melanoma, particularly in the treatment of melanoma in which the BRAF inhibitor is highly effective. Are preferably used. Thereby, the onset of BRAF inhibitor resistant melanoma by administration of the BRAF inhibitor can be prevented. The above-mentioned therapeutic or prophylactic agent for BRAF inhibitor-resistant melanoma used in combination with a BRAF inhibitor can be co-administered with a BRAF inhibitor. In other words, the therapeutic or preventive agent for BRAF inhibitor-resistant melanoma and the BRAF inhibitor are each prepared separately, that is, the therapeutic or preventive agent for BRAF inhibitor-resistant melanoma and the BRAF inhibitor are separately prepared as single agents, Can be administered to a subject simultaneously or sequentially, as they are or in combination with a pharmaceutically acceptable carrier. Alternatively, for the purpose of enhancing the therapeutic effect or preventive effect on BRAF inhibitor-resistant melanoma, the above-mentioned BRAF inhibitor-resistant melanoma is treated or prevented at an appropriate time interval before or after BRAF inhibitor administration, It can also be administered to a subject separately from the BRAF inhibitor. In this case, the dosage form and administration route of the BRAF inhibitor-resistant melanoma treatment or prevention agent and the BRAF inhibitor need not be the same, and may be different from each other.

  The therapeutic or prophylactic agent for BRAF inhibitor-resistant melanoma and the BRAF inhibitor can be prepared as a mixture, that is, a combined preparation, and can be administered as it is or after further mixing with a pharmaceutically acceptable carrier. The present invention also provides such a medicament for treating melanoma comprising interferon-β and a BRAF inhibitor as active ingredients. This pharmaceutical for treating melanoma has a therapeutic effect on melanoma in which a BRAF inhibitor is highly effective, that is, melanoma that has not acquired resistance to a BRAF inhibitor. Since this melanoma therapeutic drug shows the suppression of the appearance of BRAF inhibitor-resistant melanoma cells, not only for the treatment of melanoma, but also for suppressing the appearance of BRAF inhibitor-resistant melanoma cells accompanying melanoma treatment with BRAF inhibitors It can be advantageously used as a medicine.

  The present invention also provides a combination drug for treating melanoma, comprising interferon-β and a BRAF inhibitor, wherein the interferon-β and the BRAF inhibitor are administered to a subject simultaneously or separately. Such a combination drug usually includes a drug containing interferon-β as an active ingredient and a drug containing a BRAF inhibitor as an active ingredient, and these drugs are administered to a subject simultaneously or separately. It is a combination medicine for treating melanoma. Interferon-β and BRAF inhibitor are each prepared separately, that is, interferon-β and BRAF inhibitor are separately prepared as a single agent (medicine), and these are further added to each as it is or a pharmaceutically acceptable carrier. Can be administered to a subject simultaneously or sequentially. Alternatively, interferon-β can be administered to the subject separately from the BRAF inhibitor at an appropriate time interval before or after administration of the BRAF inhibitor. In this case, the dosage form and administration route of each of interferon-β and BRAF inhibitor need not be the same, and may be different from each other. This combination drug for the treatment of melanoma is not only a pharmaceutical kit containing a plurality of drugs, that is, not only interferon-β (a drug containing an active ingredient) and BRAF inhibitor (a drug containing an active ingredient), but also other drugs It may be a pharmaceutical kit containing This combination drug for melanoma treatment can be advantageously used not only for melanoma treatment, but also as a medicine for suppressing the emergence of BRAF inhibitor-resistant melanoma accompanying melanoma treatment with BRAF inhibitors.

  Since the administration of interferon-β, which exhibits the suppressive effect of BRAF inhibitor-resistant melanoma, and BRAF inhibitor can be administered together with the therapeutic effect on melanoma, the appearance of BRAF inhibitor-resistant melanoma cells can be suppressed. A melanoma therapeutic drug for use in suppressing the appearance of inhibitor-resistant melanoma cells and the above-described combination melanoma therapeutic drug can be used as an excellent therapeutic agent for melanoma.

  The BRAF inhibitor-resistant melanoma treatment or prevention agent, the BRAF inhibitor-resistant melanoma cell appearance inhibitor, the melanoma treatment medicament, or the melanoma treatment combination medicament is any administration subject, preferably a mammal (for example, , Humans, hamsters, mice, rats, hamsters, rabbits, dogs, cats, cows, horses, pigs, sheep, monkeys, etc.), particularly preferably humans. The administration subject of the therapeutic agent for BRAF inhibitor-resistant melanoma of the present invention is preferably a subject (patient) having BRAF inhibitor-resistant melanoma. The subject of administration of the preventive agent for BRAF inhibitor-resistant melanoma or the inhibitor of the appearance of BRAF inhibitor-resistant melanoma cells of the present invention is preferably a subject (patient) having a melanoma that has not acquired BRAF inhibitor resistance, Particularly preferred are those subjects who are or will receive administration of a BRAF inhibitor. It is preferable that the administration target of the melanoma therapeutic drug or the melanoma therapeutic combination drug is a subject having melanoma that has not acquired BRAF inhibitor resistance.

  The BRAF inhibitor-resistant melanoma treatment or prevention agent, BRAF inhibitor-resistant melanoma cell appearance inhibitor, melanoma treatment drug, or melanoma treatment combination drug can be administered orally or parenterally. . The administration route of the therapeutic or preventive agent for BRAF inhibitor-resistant melanoma, the inhibitor for the appearance of BRAF inhibitor-resistant melanoma cells, the therapeutic agent for melanoma, or the combined pharmaceutical agent for melanoma treatment of the present invention is not particularly limited, and is subcutaneously or intradermally. , Transdermal, transmucosal, pulmonary, nasal, enteral, intravenous, intraarterial, intramuscular, intraperitoneal, rectal, and intravaginal administration, or oral administration There may be.

  The therapeutic or preventive agent for BRAF inhibitor-resistant melanoma, the inhibitor for the emergence of BRAF inhibitor-resistant melanoma cells, or the therapeutic drug for melanoma according to the present invention is used as an active ingredient of interferon-β or interferon-β and a BRAF inhibitor. In addition, it may be a pharmaceutical composition containing any pharmaceutically acceptable additive. The combination drug for the treatment of melanoma of the present invention comprises a pharmaceutical composition containing any pharmaceutically acceptable additive in addition to the active ingredient interferon-β, and any pharmaceutically acceptable drug in addition to the BRAF inhibitor. Combinations with pharmaceutical compositions containing additives may also be included. Examples of additives include carriers, excipients, stabilizers, suspending agents, emulsifiers, thickeners, dispersants, absorption enhancers, binders, lubricants, disintegrants, wetting agents, buffering agents, Examples include, but are not limited to, flavoring agents, preservatives, and coloring agents.

  Examples of the dosage form for oral administration in the present invention include tablets, pills, capsules, granules, syrups, emulsions and suspensions, and these can be produced by known methods. . These dosage forms contain additives such as carriers or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, maltose, saccharose, starch, and magnesium stearate.

  Examples of the dosage form for parenteral administration in the present invention include injections, eye drops, ointments, poultices, suppositories, nasal absorption agents, pulmonary absorption agents, transdermal absorption agents, and topical sustained release agents. These can be produced by known methods. The solution preparation can be prepared, for example, in a state where components such as interferon-β are dissolved in a sterile aqueous solution used for injection or suspended and emulsified in an extract and embedded in liposomes. The solid preparation can be prepared, for example, as a lyophilized product by adding mannitol, trehalose, sorbitol, lactose, glucose or the like as an excipient to interferon-β. Further, it can be used in the form of powder. Further, these powders can be mixed with polylactic acid, glycolic acid or the like to be solidified. The gelling agent can be prepared, for example, by dissolving interferon-β in a thickener or polysaccharide such as glycerin, PEG, methylcellulose, carboxymethylcellulose, hyaluronic acid or chondroitin sulfate.

  The preparation of any dosage form may contain human serum albumin, human immunoglobulin, α2 macroglobulin, amino acid or the like as a stabilizer, and alcohol as a dispersant or absorption enhancer as long as it does not impair interferon activity. , Sugar alcohol, ionic surfactant or nonionic surfactant may be included. Moreover, you may contain trace metal or organic acid salt as needed.

  The BRAF inhibitor-resistant melanoma therapeutic or preventive agent, the BRAF inhibitor-resistant melanoma cell appearance inhibitor, the melanoma therapeutic drug, or the melanoma therapeutic combination drug of the present invention may further contain other pharmacological components. .

  The dose ratio or combination ratio of interferon-β and BRAF inhibitor (eg, vemurafenib, dabrafenib, encolafenib, PLX-8394, EBI-945, BGB-283, etc.) is the subject of administration, administration route, target disease, symptom, Or it can select suitably by the combination etc. of the component to mix | blend.

  Interferon which is an active ingredient in the administration of the preparation of a BRAF inhibitor-resistant melanoma of the present invention, such as a BRAF inhibitor-resistant melanoma cell appearance inhibitor, a melanoma therapeutic drug, or a combination drug for melanoma treatment -Β can be administered daily at a dose of 10,000 U to 18 million U / dose once or twice daily. These may also be administered at a single administration interval with a period of two days or more, and may be administered intermittently at an administration interval of once every two days to one month. In addition, a preparation containing a chemically modified interferon-β (for example, a covalent conjugate of interferon-β and polyalkylene glycol, preferably a covalent conjugate of interferon-β and polyethylene glycol) is 1 It is preferable to intermittently administer once or twice a week to once a month, and particularly preferably intermittently administrate once or twice a week.

  In addition, in the administration of the BRAF inhibitor in combination with interferon-β, the daily dose of the BRAF inhibitor varies depending on the condition and body weight of the subject, the type of BRAF inhibitor, the administration route, etc., for example, BRAF When the inhibitor is administered orally, if it is an adult (body weight of about 60 kg), the BRAF inhibitor is 0.1 μg / kg to 100 mg / kg, preferably 1 μg / kg to 10 mg / kg 1-3 times a day. It is preferable to administer them separately.

  The present invention administers interferon-β, the above-mentioned BRAF inhibitor-resistant melanoma treatment or prevention agent, BRAF inhibitor-resistant melanoma cell appearance inhibitor, melanoma treatment drug, or combination drug for melanoma treatment to the above subject A method for treating or preventing BRAF inhibitor-resistant melanoma is also provided.

  The following examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof.

Example 1 Preparation of Covalent Conjugate of Interferon-β and PEG Recombinant human interferon-β (SEQ ID NO: 1) was prepared according to Goeddel et. al. Nucleic Acid Research, (1980), Vol. 8, p. It was prepared according to the method described in 4057-4074.

  Subsequently, according to the method described in Example 6 of WO 2005/018260, PEG having a molecular weight of 42,000 was added to the amino group of the 134th lysine of the amino acid sequence of recombinant human interferon-β (SEQ ID NO: 1). Was prepared as a “covalent conjugate of interferon-β and PEG”. Specifically, first, ethylene glycol was added to recombinant human interferon-β (final concentration 200 μg / mL) dissolved in 100 mmol / L acetate buffer (pH 5.0) containing 0.5 mol / L sodium chloride. After addition (at a final concentration of 20%), the pH was adjusted to 7.6 using a 1 mol / L disodium hydrogen phosphate solution. To this, hydroxysuccinimide ester activated PEG (molecular weight 42,000, NOF Corporation; product number 61G99122B01) was added and mixed, and a binding reaction was carried out at 4 ° C. overnight. A cation exchange column (TOYOPEARL CM 650 (S)) was added to this binding reaction solution by adding 5 volumes of 10 mmol / L acetate buffer (pH 4.5) and equilibrated with 10 mmol / L acetate buffer (pH 4.5). ; Tosoh Corporation). The protein was eluted and fractionated with the following developing solvent.

<Developing solvent>
Solvent A: 10 mmol / L acetate buffer (pH 4.5)
Solvent B: 10 mmol / L acetate buffer (pH 4.5) containing 1 mol / L sodium chloride

  The mixed solution of the solvents A and B is passed through 40 times the resin amount of the cation exchange column, and the solvent B feed rate is continuously increased from 0% to 65% during the passage. Elution. The eluted fraction was further applied to a cation exchange column SP-5PW (Tosoh Corporation), and the molecular weight of the amino group of the 134th lysine of the amino acid sequence of recombinant human interferon-β (SEQ ID NO: 1) was determined. A “covalent conjugate of human interferon-β and PEG” (hereinafter “PEG-IFN-β”) in which one molecule of 42,000 PEG was covalently bonded was isolated.

(Example 2) Cell growth inhibitory action of interferon-β on BRAF inhibitor-resistant melanoma In this example, BRAF inhibitor-resistant melanoma cells were treated by the method of Greger et al. (Molecular cancer therapeutics, 2012, Vol. 11, p. 909). -920), and the cell growth inhibitory effect of interferon-β on BRAF inhibitor-resistant melanoma was evaluated. As interferon-β, natural human interferon-β (Feron (registered trademark); Toray Industries, Inc.) and PEG-IFN-β prepared in Example 1 were used.

Human melanoma cell line A375 (CRL-1619; American type culture collection) having a V600E mutation in the BRAF gene (600th valine is changed to glutamic acid) at 37 ° C. using DMEM medium containing 10% fetal bovine serum, The cells were cultured and maintained in a 5% CO ₂ incubator. Note that a DMEM medium containing 10% fetal bovine serum was also used as the culture medium at the time of evaluation by the test substance treatment.

  A DMEM medium containing 10% fetal bovine serum supplemented with human melanoma cell line A375 (CRL-1619; American type culture collection) and vemurafenib (VMF-150; LC Laboratories) with a final concentration of 1.5 μmol / L was used. Then, BRAF inhibitor resistant A375 cells were established by subculture for 4 weeks. In addition, at the time of evaluation by the test substance treatment, a DMEM medium containing 10% fetal calf serum was also used as a culture medium for these cells.

A375 cells or BRAF inhibitor-resistant A375 cells prepared above were seeded in a 96-well plate at 5 × 10 ³ cells / well, a test substance was added, and the cells were cultured at 37 ° C. and 5% CO ₂ . As test substances, Vemurafenib (final concentration: 1.5 μmol / L; VMF treatment group), natural human interferon-β (Fueron (registered trademark); Toray Industries, Inc.) (final concentration: 1,000 or 10,000 U) / ML; IFN-β treatment group) and PEG-IFN-β (final concentration: 1,000 or 10,000 U / mL; PEG-IFN-β treatment group) were used for treatment. The number of living cells (relative level) was measured 48 hours after the start of the test substance treatment.

Viable cell count was performed as follows. 10 μL of CellTiter 96 (registered trademark) AQueous One Solution Cell Proliferation Assay (G3581; Promega) reagent was added to each well. The 96-well plate was allowed to stand for 60 minutes in a 37 ° C., 5% CO ₂ incubator, and then the absorbance (490 nm) was measured with a plate reader. As a control, 4 wells (control well; untreated group) to which a test substance was not added were provided.

After obtaining the absorbance correction value from the following formula (1) from each measured value, the cell viability (% relative to the control) 48 hours after the test substance treatment was calculated from the following formula (2). The absorbance correction value of the control well was the average value of 4 wells.
Formula (1):
Absorbance correction value = absorbance measurement value of each well−absorbance measurement value of a well not seeded with cells Formula (2):
Cell viability (% of control) = (absorbance correction value of each well) / (absorbance correction value of control well) × 100

  It shows that the cell growth inhibitory effect by test substance treatment is so high that the value of cell viability (% with respect to control) is small.

  The results are shown in FIG. The vertical axis | shaft of FIG. 1 shows cell viability (% with respect to control) (N = 4, average value +/- standard error). In the figure, “control” is an untreated group, “VMF” is a VMF (vemurafenib) treated group, “IFN-β” is an IFN-β treated group, and “PEG-IFN-β” is a PEG-IFN-β treated group. Show. “A375” indicates A375 cells, and “BRAF inhibitor resistant A375” indicates BRAF inhibitor resistant A375 cells.

  Vemurafenib treatment resulted in a cell survival rate of A375 cells of about 30% compared to the number of living cells in the untreated group, but the cell survival rate of BRAF inhibitor-resistant A375 cells was the same as the number of living cells in the untreated group. It was about 70% in comparison, and resistance to the cell growth inhibitory action by vemurafenib was recognized (FIG. 1, VMF treatment group). On the other hand, natural human interferon-β (FIG. 1, IFN-β treatment group) and PEG-IFN-β (PEG-IFN-β treatment group) have a cell growth inhibitory effect on BRAF inhibitor-resistant A375 cells. The intensity of the cell growth inhibitory action was the same as that of the parent strain A375 cells.

  From this result, it was shown that interferon-β has a cell growth inhibitory action, that is, an antitumor action, against BRAF inhibitor-resistant melanoma.

Example 3 Comparison of Cell Growth Inhibitory Action of Interferon-α and Interferon-β against BRAF Inhibitor-Resistant Melanoma Using the BRAF inhibitor-resistant A375 cells prepared in Example 2, interferon-α against BRAF inhibitor-resistant melanoma And interferon-β were compared and evaluated for their cell growth inhibitory effects. PEG-IFN-α2a (trade name: Pegasys, Chugai Pharmaceutical) was used as interferon-α, and PEG-IFN-β prepared in Example 1 was used as interferon-β. BRAF inhibitor resistant A375 cells were cultured and maintained in a DMEM medium containing 10% fetal bovine serum supplemented with vemurafenib (final concentration: 1.5 μmol / L) at 37 ° C. in a 5% CO ₂ incubator. . At the time of evaluation by the test substance treatment, a DMEM medium containing 10% fetal bovine serum (no vemurafenib added) was used as a culture medium.

BRAF inhibitor-resistant A375 cells were seeded in a 96-well plate at 5 × 10 ³ cells / well, a test substance was added, and the cells were cultured at 37 ° C. and 5% CO ₂ . As test substances, PEG-IFN-α2a and PEG-IFN-β were used for treatment (final concentrations: 100 U / mL, 1,000 U / mL or 10,000 U / mL, respectively). The number of living cells (relative level) was measured 48 hours after the start of the test substance treatment.
Measurement of the number of viable cells and calculation of cell viability were carried out in the same manner as in Example 2.

  The results are shown in FIG. The vertical axis | shaft of FIG. 2 shows the cell viability (% with respect to control) of BRAF inhibitor resistant A375 cell 48 hours after the test substance treatment start (N = 4, average value +/- standard error). “PEG-IFN-α2a” indicates a PEG-IFN-α2a treatment group, and “PEG-IFN-β” indicates a PEG-IFN-β treatment group.

  Both PEG-IFN-α2a and PEG-IFN-β more strongly suppressed the growth of BRAF inhibitor-resistant A375 cells with increasing concentrations. However, it was shown that PEG-IFN-β has a much stronger cell growth inhibitory effect than PEG-IFN-α2a.

  From this result, it was shown that interferon-β has a superior cell growth inhibitory effect on BRAF inhibitor-resistant melanoma than interferon-α, that is, an excellent antitumor effect.

(Example 4) Effect of combined treatment of interferon-β and vemurafenib In this example, the influence of the combined treatment of interferon-β and vemurafenib on the cell growth inhibitory effect was evaluated using A375 cells. As interferon-β, natural human interferon-β (Feron (registered trademark); Toray Industries, Inc.) and PEG-IFN-β prepared in Example 1 were used.

A375 cells were cultured and maintained in a DMEM medium containing 10% fetal calf serum at 37 ° C. in a 5% CO ₂ incubator. Also at the time of evaluation by the test substance treatment, a DMEM medium containing 10% fetal calf serum was used as a culture medium.

A375 cells were seeded in a 96-well plate at 2 × 10 ³ cells / well, a test substance was added, and the cells were cultured at 37 ° C. and 5% CO ₂ . Test substances include vemurafenib (final concentration: 0.5 or 1.5 μmol / L), natural human interferon-β (Feron (registered trademark); Toray Industries, Inc.) (final concentration: 1,000 U / mL) and PEG-IFN-β (final concentration: 1,000 or 10,000 U / mL) was used. Vemurafenib and natural human interferon-β or PEG-IFN-β were added alone (single treatment) or combined and added simultaneously (combination treatment). The number of living cells (relative level) was measured 96 hours after the start of treatment with the test substance.
Measurement of the number of viable cells and calculation of cell viability were carried out in the same manner as in Example 2.

  The results are shown in FIG. The vertical axis of the figure shows the cell survival rate (% with respect to control) of A375 cells after 96 hours from the start of the test substance treatment (N = 4, mean value ± standard error). In the figure, “VMF (μmol / L)” indicates vemurafenib treatment concentration, “−” indicates no vemurafenib treatment, “0.5” and “1.5” indicate final concentrations of 0.5 μmol / L, 1. Treatment with 5 μmol / L vemurafenib is shown. “No interferon-β” indicates no interferon-β treatment, “PEG-IFN-β” indicates PEG-IFN-β treatment, and “IFN-β” indicates natural human interferon-β treatment. For example, “no interferon-β” and “VMF (−)” in the leftmost bar indicate the test substance-untreated group (ie, “control”). Also, “interferon-β-free” and “VMF 0.5 μmol / L” in the second bar from the left indicate the vemurafenib single treatment group at the final concentration of 0.5 μmol / L, and “PEG” in the fourth bar from the left. “IFN-β 1,000 U / mL” and “VMF (−)” indicate the PEG-IFN-β alone treatment group at a final concentration of 1,000 U / mL, and “PEG-IFN-” in the fifth bar from the left “β 1,000 U / mL” and “VMF 0.5 μmol / L” indicate a combination treatment group of PEG-IFN-β having a final concentration of 1,000 U / mL and vemurafenib having a final concentration of 0.5 μmol / L.

  As shown in FIG. 3, in the PEG-IFN-β (1,000 or 10,000 U / mL) or natural human interferon β (1,000 U / mL) alone treatment group, the cell viability of A375 cells is 50-70% compared with the number of living cells in the test substance-untreated group, and the number of living cells in the test substance-untreated group in the vemurafenib (final concentration 0.5 or 1.5 μmol / L) alone-treated group In comparison, they were 13% and 6%, respectively, and it was confirmed that the growth of A375 cells was suppressed by treatment with each test substance alone. On the other hand, by the combined treatment of PEG-IFN-β or natural human interferon-β and vemurafenib, the cell survival rate of A375 cells was 4-10% compared with the number of living cells in the test substance-untreated group However, an additive or synergistic cytostatic effect by the combined treatment was not observed.

  From this result, the combined treatment of interferon-β and BRAF inhibitor has no additive or synergistic cytostatic effect, ie, antitumor effect, on melanoma that has not acquired BRAF inhibitor resistance. It was shown that.

(Example 5) Inhibitory effect of interferon-β on the appearance of BRAF inhibitor-resistant melanoma In this example, the inhibitory effect of interferon-β on the appearance of BRAF inhibitor-resistant melanoma cells was evaluated using A375 cells. As interferon-β, natural human interferon-β (Feron (registered trademark); Toray Industries, Inc.) and PEG-IFN-β prepared in Example 1 were used.

A375 cells were cultured and maintained in a DMEM medium containing 10% fetal calf serum at 37 ° C. in a 5% CO ₂ incubator. Also at the time of evaluation by the test substance treatment, a DMEM medium containing 10% fetal calf serum was used as a culture medium.

A375 cells were seeded in a 12-well plate at 2 × 10 ³ cells / well, a test substance was added, and the cells were cultured at 37 ° C. and 5% CO ₂ . Test substances include vemurafenib (final concentration: 0.5 or 1.5 μmol / L), natural human interferon-β (Feron (registered trademark); Toray Industries, Inc.) (final concentration: 1,000 U / mL) and PEG-IFN-β prepared in Example 1 (final concentration: 1,000 or 10,000 U / mL) was used. Vemurafenib and natural human interferon-β or PEG-IFN-β were added alone (single treatment) or combined and added simultaneously (combination treatment).

  Twelve days after the start of treatment with the test substance, the morphology of each group of cells was observed using a phase contrast microscope, and the state was obtained as an image. The result is shown in FIG. “Control” in FIG. 4 indicates the test substance-untreated group. In FIG. 4, “VMF” indicates vemurafenib treatment, “IFN-β” indicates natural human interferon-β treatment, and “PEG-IFN-β” indicates PEG-IFN-β treatment. For example, “VMF 0.5 μmol / L” indicates a vemurafenib alone treatment group at a final concentration of 0.5 μmol / L, and “PEG-IFN-β 1,000 U / mL” indicates PEG at a final concentration of 1,000 U / mL. -IFN-β alone treatment group, “VMF 0.5 μmol / L + PEG-IFN-β 1,000 U / mL” is PEG-IFN-β and final concentration 0.5 μmol at a final concentration of 1,000 U / mL The combination treatment group with vemurafenib at / L is shown.

  As shown in FIG. 4, in the test substance-untreated group (ie, “control”), 12 days after the start of the test substance treatment, small cells are densely formed to form a cell mass, and the cells actively proliferate. The situation was recognized. PEG-IFN-β 10,000 U / mL single treatment group (“PEG-IFN-β 10,000 U / mL”) and natural human interferon β 1,000 U / mL single treatment group (“IFN-β 1,000 U / mL "), the cell density was greatly reduced as compared with the test substance-untreated group, and the cell morphology was also enlarged. Moreover, in the single treatment group of PEG-IFN-β 1,000 U / mL (“PEG-IFN-β 1,000 U / mL”), epithelial cells arranged in a paving stone form a monolayer and divide. There were hardly any cells inside. On the other hand, in the vemurafenib (0.5 or 1.5 μmol / L) single treatment group, a cell population that formed colonies and proliferated while maintaining the density was observed. Furthermore, combined treatment of PEG-IFN-β (1,000 or 10,000 U / mL) or natural human interferon-β (1,000 U / mL) and vemurafenib (0.5 or 1.5 μmol / L) Compared to the single treatment group of each test substance, the group has clearly fewer cells and is enlarged, it seems to be rounded and peeled off from the adhesive surface, and an air bubble-like organelle is formed inside the cell It was also recognized that.

In addition, the number of viable cells was measured 14 days after the start of the test substance treatment.
Viable cell count was performed as follows. After 14 days from the start of treatment with the test substance, the cells were washed with PBS and then detached from the plate with a trypsin-EDTA solution. The detached cells were washed, suspended in a culture medium, stained with trypan blue solution, and the number of viable cells was counted using a hemocytometer. The cell viability of the test substance-treated group is the percentage (% of control) of the viable cell count relative to the viable cell count (100%) of the test substance-untreated group (ie, “control” not treated with any test substance). Represent.

  Table 1 shows the viable cell count (per well) and cell viability (% of control) of A375 cells 14 days after the start of test substance treatment. In Table 1, “No interferon-β” indicates no interferon-β treatment, “PEG-IFN-β” indicates PEG-IFN-β treatment, and “IFN-β” indicates natural human interferon-β treatment. “Vemurafenib (μmol / L)” indicates vemurafenib treatment, “−” indicates no vemurafenib treatment, “0.5” and “1.5” indicate a final vemurafenib concentration of 0.5 μmol / L, 1.5 μmol / L, respectively. L treatment is indicated. For example, “no interferon-β” and “vemurafenib (−)” indicate the test substance-untreated group (ie, “control”), and “no interferon-β” and “vemurafenib 0.5 μmol / L” indicate vemurafenib 0.5 μmol. / PEG single treatment group, “PEG-IFN-β 1,000 U / mL” and “vemurafenib (−)” indicate PEG-IFN-β 1,000 U / mL single treatment group, “PEG-IFN-β “1,000 U / mL” and “Vemurafenib 0.5 μmol / L” indicate a combination treatment group of PEG-IFN-β 1,000 U / mL and Vemurafenib 0.5 μmol / L.

As shown in Table 1, after 14 days from the start of the test substance treatment, the cell viability of A375 cells in the vemurafenib (0.5 or 1.5 μmol / L) alone treatment group was not treated with the test substance untreated group (ie, “control”). ) Was about 2 to 6% compared with the number of living cells, but the number of living cells was increased more than the number of cells at the time of seeding (2 × 10 ³ ) (0.5 μmol / L treatment; 167 × 10 ³ and 1.5 μmol / L treatment; 51 × 10 ³ ). From this result, it was shown that the cell which acquired the vemurafenib tolerance appeared by the vemurafenib single treatment for 14 days. This can also be confirmed from the observation results of the cells 12 days after the start of treatment with the test substance (FIGS. 4B and 4C). A cell population that forms colonies and proliferates while maintaining the density was observed, and the cells that acquired vemurafenib resistance were observed. Existence was shown. On the other hand, combined use of PEG-IFN-β (1,000 or 10,000 U / mL) or natural human interferon-β (1,000 U / mL) and vemurafenib (0.5 or 1.5 μmol / L) In the treatment group, the cell viability of A375 cells is 0.3% or less compared to the number of living cells in the test substance-untreated group, and the number of living cells is the number of cells at the time of seeding (2 × 10 ³ cells). Was less than. Therefore, it is shown that treatment with PEG-IFN-β or natural human interferon-β significantly suppresses the growth of tumor cells in the vemurafenib treatment group, that is, the appearance of cells that have acquired vemurafenib resistance is suppressed. It was done.

  From these results, it was shown that interferon-β has an action of suppressing the appearance of melanoma cells that have acquired resistance to BRAF inhibitors.

  From the results of Example 4 and Example 5, interferon-β does not show an additive or synergistic antitumor effect on melanoma more than BRAF inhibitor alone treatment in combination treatment with BRAF inhibitor. Was shown to have an inhibitory effect on the appearance of BRAF inhibitor-resistant melanoma cells. That is, interferon-β was shown to have a preventive effect against the development of BRAF inhibitor-resistant melanoma.

  From the above examples, interferon-β has an anti-tumor effect on BRAF inhibitor-resistant melanoma (ie, has a therapeutic effect on BRAF inhibitor-resistant melanoma), and BRAF inhibitor-resistant melanoma cells. It has been shown to have an excellent inhibitory effect on appearance (ie, has a preventive effect on BRAF inhibitor-resistant melanoma).

  The agent of the present invention can be used in the field of medicine as a therapeutic agent for BRAF inhibitor-resistant melanoma or a prophylactic agent for BRAF inhibitor-resistant melanoma. In addition, the agent of the present invention can be used for suppressing the appearance of BRAF inhibitor-resistant melanoma cells as a therapeutic agent for melanoma exhibiting the suppression of the appearance of BRAF inhibitor-resistant melanoma cells.

  SEQ ID NO: 1: amino acid sequence of human interferon-β

Claims (9)

  A therapeutic or prophylactic agent for BRAF inhibitor-resistant melanoma, comprising interferon-β as an active ingredient.   The therapeutic or prophylactic agent for BRAF inhibitor-resistant melanoma according to claim 1, wherein the interferon-β is covalently bonded to polyalkylene glycol. The B according to claim 2, wherein the polyalkylene glycol is polyethylene glycol.
A therapeutic or prophylactic agent for RAF inhibitor-resistant melanoma.   A pharmaceutical for treating melanoma for use in suppressing the emergence of BRAF inhibitor-resistant melanoma cells, comprising interferon-β and a BRAF inhibitor as active ingredients.   Melanoma treatment for use in suppressing the emergence of BRAF inhibitor-resistant melanoma, comprising interferon-β and BRAF inhibitor, wherein interferon-β and BRAF inhibitor are administered simultaneously or separately Combination medicine.   The medicament for treating melanoma according to claim 4, wherein the interferon-β is covalently bonded to a polyalkylene glycol.   The medicament for treating melanoma according to claim 6, wherein the polyalkylene glycol is polyethylene glycol.   The combination drug for melanoma treatment according to claim 5, wherein the interferon-β is covalently bonded to a polyalkylene glycol.   The combination drug for melanoma treatment according to claim 8, wherein the polyalkylene glycol is polyethylene glycol.

Images