This application claims priority to JP Patent Application No. 2016-148854, filed on Jul. 28, 2016, which claims priority to JP Patent Application No. 2015-203282, filed on Oct. 14, 2015.
The present invention relates to a pharmaceutical composition for the prevention or treatment of a disease that can be ameliorated via immunomodulation, comprising, as an active ingredient, (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide (hereinafter, this compound is also referred to as “Compound (I)”) or a salt thereof.
The immune system has an important self-defense mechanism against various diseases caused by various factors inside and outside an organism. An impaired immune system adversely affects diseases, such as infections with bacteria or viruses, tumor incidence, and delayed recovery from injury or illness. Accordingly, modulation of the immune system is very critical for the prevention or treatment of various diseases. In the past, vaccination by means of administration of killed bacteria or antigens had been known as an immunomodulation method, and examples of other known techniques include methods involving the use of peptidoglycan, lipopolysaccharide, chitin, lactoferrin, or cyclophosphamide. In recent years, for example, cytokine therapy aimed at modulation of the immune system via administration of a protein such as IL-6, TNF, or IFN and immune cell therapy comprising sampling immune cells, exciting the activity thereof, and putting the cells back into the organism, have been possible. Such treatment techniques have exerted effects on the prevention or treatment of particular types of infections or tumors.
Epidermal growth factor receptor (EGFR) plays a very critical role in tumor growth. At present, various EGFR tyrosine kinase inhibitors have been developed and put into use in clinical settings. Specific examples of known EGFR tyrosine kinase inhibitors include: gefitinib (trade name: Iressa); erlotinib (trade name: Tarceva); and afatinib (trade name: Gilotrif). Such inhibitors are considered to have highly selective inhibitory activity against EGFR tyrosine kinase. In particular, such inhibitors are considered to exert anti-tumor effects on a subject having a mutation in the EGFR gene in a tumor-selective manner and contribute to improvement of the prognosis of the subject. In recent years, in addition, third-generation EGFR tyrosine kinase inhibitors exerting effects on T790M mutation, which is a resistance mechanism, and exhibiting enhanced selectivity to mutant EGFR (a representative example being AZ9291) have been developed. It is not too exaggeration to say that the anti-tumor effects of such compounds are achieved solely by highly selective inhibitory activity against EGFR tyrosine kinase, since such activity is capable of directly affecting tumor cells (Nature Rev. Cancer, vol. 6, pp. 803-811, 2006; and Journal of Thoracic Oncology, Vol. 3, No. 6, Supplement 2, June 2008).
Compound (I) and a salt thereof are known to inhibit EGFR tyrosine kinase with high selectivity and suppress tumor growth (WO 2013/125709). However, neither the compound of the present invention nor any of the known EGFR tyrosine kinase inhibitors described above are known to have immunomodulation activities.
The object of the present invention is to provide an immunomodulator and a pharmaceutical composition for the prevention or treatment of a disease that can be ameliorated via immunomodulation.
The object of the present invention is also to provide a method for the prevention or treatment of a disease that can be ameliorated via immunomodulation with the immunomodulator and the pharmaceutical composition.
The present inventors have conducted concentrated studies concerning pharmacological activities of Compound (I). As a result, they discovered that such compound had activities of modulating the immune system, thereby completing the present invention. The present invention is summarized as follows.
(1) An immunomodulator comprising (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof.
(2) The immunomodulator according to (1), which activates T cells.
(3) The immunomodulator according to (1), which induces IL-2 production.
(4) The immunomodulator according to (1), which induces IFN production.
(5) The immunomodulator according to (1), which induces immunocyte migration.
(6) The immunomodulator according to (1), which induces exudation and accumulation of immunocytes in an affected area.
(7) A method for modulating the immune system of a subject, comprising administering a pharmaceutical composition comprising (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof to the subject.
(8) A pharmaceutical composition for the prevention or treatment of an infection via immunomodulation, which comprises (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof.
(9) The pharmaceutical composition according to (8), wherein the infection is an infection with a parasite.
(10) The pharmaceutical composition according to (9), wherein the parasite is selected from the group consisting of trypanosomatid protozoa, malarial parasites, and Toxoplasma.
(11) The pharmaceutical composition according to (8), wherein the infection is an infection with a bacterium.
(12) The pharmaceutical composition according to (11), wherein the bacterium is selected from the group consisting of Streptococcus pneumoniae, Mycobacterium tuberculosis, Staphylococcus aureus, Bacillus anthracis, Vibrio cholerae, and Helicobacter pylori.
(13) The pharmaceutical composition according to (8), wherein the infection is an infection with a virus.
(14) The pharmaceutical composition according to (13), wherein the virus is selected from the group consisting of human T cell leukemia virus, papilloma virus, Epstein-Barr virus, cytomegalovirus, influenza virus, hepatitis B virus, and hepatitis C virus.
(15) A method for the prevention or treatment of an infection of a subject via immunomodulation, comprising administering a pharmaceutical composition comprising (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof to the subject.
(16) A pharmaceutical composition for the treatment of an immunodeficiency via immunomodulation, which comprises (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof.
(17) The pharmaceutical composition according to (16), wherein the immunodeficiency is caused by an infection with HIV.
(18) A method for the treatment of an immunodeficiency of a subject via immunomodulation, comprising administering a pharmaceutical composition comprising (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof to the subject.
(19) A pharmaceutical composition for the prevention or treatment of a disease caused by an immune system weakened with age via immunomodulation, comprising (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof.
(20) The pharmaceutical composition according to (19), wherein the disease caused by the weakened immune system is pneumonia.
(21) A method for the prevention or treatment of a disease caused by an immune system weakened with age of a subject via immunomodulation, comprising administering a pharmaceutical composition comprising (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof to the subject.
(22) A pharmaceutical composition for the prevention or treatment of a virus-associated tumor via immunomodulation, comprising (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof.
(23) The pharmaceutical composition according to (22), wherein the virus-associated tumor is Burkitt's lymphoma, hepatic carcinoma, uterine cervix cancer, adult T cell leukemia, Kaposi's sarcoma, or head and neck cancer.
(24) A method for the prevention or treatment of a virus-associated tumor of a subject via immunomodulation, comprising administering a pharmaceutical composition comprising (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof to the subject.
(25) A pharmaceutical composition for the potentiation of the activity of a medicine used for preventing or treating a disease by acting on an immune system, comprising (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof.
(26) The pharmaceutical composition according to (25), which is used for the potentiation of the activity of a vaccine to prevent an infection.
(27) The pharmaceutical composition according to (25), which is used for the potentiation of the activity of an antiviral agent.
(28) The pharmaceutical composition according to (25), which is used for the potentiation of the activity of an anti-PD-1 antibody or an anti-PD-L1 antibody.
(29) The pharmaceutical composition according to (25), which is used for the potentiation of the activity of a cancer vaccine.
(30) The pharmaceutical composition according to (25), which is used for the potentiation of the activity of an agent for inducing an antitumor immune response.
(31) The pharmaceutical composition according to (30), wherein the agent for inducing an antitumor immune response is an anti-PD-1 antibody or an anti-PD-L1 antibody.
(32) The pharmaceutical composition according to (31), wherein the agent for inducing an antitumor immune response is an anti-PD-1 antibody.
(33) The pharmaceutical composition according to (31), wherein the agent for inducing an antitumor immune response is an anti-PD-L1 antibody.
(34) A method for the potentiation of the activity of a medicine used for preventing or treating a disease of a subject by acting on an immune system, comprising administering a pharmaceutical composition comprising (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof to the subject.
The present invention provides an immunomodulator comprising, as an active ingredient, (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof and a novel pharmaceutical composition for the prevention or treatment of a disease that can be ameliorated via immunomodulation. The present invention also provides a novel method for the treatment of, for example, various infections, immunodeficiency, and tumors.
This description includes the disclosures of Japanese Patent Application Nos. 2015-203282 and 2016-148854, which are priority documents of the present application.
Compound (I), i.e., (S)—N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide, is represented by Structural Formula (I) below. Compound (I) is a known compound, and a method for producing the same is disclosed in WO 2013/125709.
Compound (I) may be in a free form or a salt form. When it is in a salt form, it may be in the form of a crystal. In such a case, a crystalline form may be a single crystal or a polymorphic mixture. Further, it may be a solvate (e.g., a hydrate) or a non-solvate. An example of a salt form is an acid addition salt, and specific examples thereof include: inorganic acid salts, such as salts of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and perchloric acid; sulfonic acid salts, such as salts of methanesulfonic acid, isethionic acid, benzenesulfonic acid, and p-toluenesulfonic acid; and other organic acid salts, such as salts of formic acid, maleic acid, fumaric acid, tartaric acid, citric acid, ascorbic acid, and trifluoroacetic acid.
Compound (I) and a salt thereof exert immunomodulation activity on subjects; that is, humans and other mammalians, such as monkeys, mice, rats, rabbits, dogs, cats, cows, horses, pigs, and sheep. Preferably, Compound (I) and a salt thereof exert immunomodulation activity on humans. The term “immunomodulation activity” used herein refers to activation of immunocytes. Specifically, the term refers to the activity of inducing division and differentiation of immunocytes, inducing the production of various cytokines, allowing immunocytes to migrate, and allowing the immunocytes to undergo exudation and/or accumulation in an affected area (i.e., a region in which a pathological change has occurred, such as a tumor tissue, infectious tissue, or inflammatory tissue); or enhancing the functions of immunocytes to eliminate foreign-matter-like components of endogenous origin or foreign matter of exogenous origin. Compound (I) and a salt thereof have activity of activating T cells, in particular, among immunocytes. Examples of induced cytokines include IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-17, IL-23, GM-CSF, IFN-γ, MCFA, MIP-1α, MIP-1β, and TNF-α, and a specific example is IL-2. In addition, Compound (I) and a salt thereof have activity of inducing cytokine production, particularly on peripheral blood mononuclear cells. Among various types of cytokines, in particular, production of IL-2 and/or IFN is induced. Also, Compound (I) and a salt thereof allow immunocytes to migrate. Further, Compound (I) and a salt thereof induce migration, exudation, and/or accumulation of immunocytes. Migration, exudation and/or accumulation of immunocytes in an affected area can be examined on the basis of tissue staining of the affected area and changes in expression levels of the gene characteristic to the immunocyte in the tissues of the affected area (examples of such genes include CD3, CD4, CD8, NK1.1, IL-2, IFN-γ, Perforin, Granzyme B, and CD69 genes). The present invention relates to: an immunomodulator containing Compound (I) or a salt thereof; Compound (I) or a salt thereof as an immunomodulator; and a method for immunomodulation of a subject comprising administering an effective amount of Compound (I) or a salt thereof to the subject in need of prevention or treatment.
The immunomodulation activity of Compound (I) and a salt thereof enables the prevention or treatment of various infections, immunodeficiency, diseases caused by an immune system weakened with age, and virus-associated tumors.
Specific examples of infections Compound (I) and a salt thereof can prevent or treat include infections with parasites (e.g., infections with parasites selected from the group consisting of trypanosomatid protozoa, malarial parasites, and Toxoplasma), infections with bacteria (e.g., infections with bacteria selected from the group consisting of Streptococcus pneumoniae, Mycobacterium tuberculosis, Staphylococcus aureus, Bacillus anthracis, Vibrio cholerae, Mycoplasmata, and Helicobacter pylori), and infections with viruses (e.g., infections with viruses selected from the group consisting of human T cell leukemia viruses (HTLV-1), papilloma viruses (HPV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV), influenza viruses (FLU), hepatitis B viruses (HBV), herpes virus, and hepatitis C viruses (HCV)). Other embodiments of the present invention relate to: a pharmaceutical composition for the prevention or treatment of an infection via immunomodulation, which contains Compound (I) or a salt thereof; Compound (I) or a salt thereof for the prevention or treatment of an infection via immunomodulation; and a method for the prevention or treatment of an infection of a subject via immunomodulation comprising administering an effective amount of Compound (I) or a salt thereof to the subject in need of prevention or treatment.
Specific examples of immunodeficiency Compound (I) and a salt thereof can treat include congenital immunodeficiency and acquired immunodeficiency, and a more specific example is acquired immunodeficiency caused by infection with human immunodeficiency virus (HIV). Accordingly, other embodiments of the present invention relate to: a pharmaceutical composition for the treatment of an immunodeficiency via immunomodulation, which contains Compound (I) or a salt thereof; Compound (I) or a salt thereof for the treatment of an immunodeficiency via immunomodulation; and a method for the treatment of an immunodeficiency of a subject via immunomodulation comprising administering an effective amount of Compound (I) or a salt thereof to the subject in need of treatment.
A specific example of a disease caused by an immune system weakened with age Compound (I) and a salt thereof can prevent or treat is pneumonia. Accordingly, other embodiments of the present invention relate to: a pharmaceutical composition for the prevention or treatment of a disease caused by an immune system weakened with age via immunomodulation, which contains Compound (I) or a salt thereof; Compound (I) or a salt thereof for the prevention or treatment of a disease caused by an immune system weakened with age via immunomodulation; and a method for the prevention or treatment of a disease caused by an immune system weakened with age of a subject via immunomodulation comprising administering an effective amount of Compound (I) or a salt thereof to the subject in need of prevention or treatment.
Specific examples of virus-associated tumors (i.e. tumors developed by infections with viruses) Compound (I) and a salt thereof can prevent or treat include Burkitt's lymphoma, hepatic carcinoma, uterine cervix cancer, adult T cell leukemia, Kaposi's sarcoma, and head and neck cancer. Accordingly, other embodiments of the present invention relate to: a pharmaceutical composition for the prevention or treatment of a virus-associated tumor via immunomodulation, which contains Compound (I) or a salt thereof; Compound (I) or a salt thereof for the prevention or treatment of a virus-associated tumor via immunomodulation; and a method for the prevention or treatment of a virus-associated tumor of a subject via immunomodulation comprising administering an effective amount of Compound (I) or a salt thereof to the subject in need of prevention or treatment.
The activity of Compound (I) and a salt thereof for immunomodulation can potentiate the effects of a medicine used for the prevention or treatment of a disease by acting on an immune system. Specific examples of medicines used for the prevention or treatment of a disease by acting on an immune system include: vaccines for the prevention of infections (e.g., vaccines for the prevention of infections such as diphtheria, tetanus, and pertussis), antiviral agents (e.g., influenza vaccines, hepatitis B vaccines, interferon α preparations, interferon β preparations, Telaprevir, Ribavirin, Simeprevir, Vidarabine, acyclovir, Ganciclovir, Valganciclovir, nucleoside analog reverse transcriptase inhibitors (NRTI) (e.g., AZT (zidovudine), ddI (Didanosine), ddC (Zalcitabine), d4T (Stavudine), and 3TC (lamivudine)), non-nucleoside reverse transcriptase inhibitors (NNRTI) (e.g., Nevirapine or Delavirdine), and protease inhibitors (e.g., Saquinavir, Ritonavir, Indinavir, or Nelfinavir)); agents for inducing antitumor immune responses (e.g., modulators having immunomodulation activity, such as CD28-like family members or CD28-like family ligand members: specific examples thereof include programmed death-1 (PD-1) inhibitors, programmed death-Ligand 1 (PD-L1) inhibitors, programmed death-Ligand 2 (PD-L2) inhibitors, anti-CTLA-4 inhibitors, anti-BTLA inhibitors, anti-CD28 modulators, anti-ICOS modulators, anti-ICOS-L modulators, anti-B7-1 modulators, anti-B7-2 modulators, anti-B7-H3 modulators, and anti-B7-H4 modulators; more specific examples thereof include anti-PD-1 antibodies, PD-1 peptide inhibitors, anti-PD-1 RNAi, anti-PD-1 antisense RNA, anti-PD-L1 antibodies, PD-L1 peptide inhibitors, anti-PD-L1 RNAi, anti-PD-L1 antisense RNA, anti-PD-L2 antibodies, PD-L2 peptide inhibitors, anti-PD-L2 RNAi, anti-PD-L2 antisense RNA, and anti-CTLA4 antibodies; and particularly specific examples thereof include anti-PD-1 antibodies and anti-PD-L1 antibodies), and cancer vaccines (e.g., Sipuleucel-T). Accordingly, other embodiments of the present invention relate to: a pharmaceutical composition for the potentiation of the activity of a medicine used for the prevention or treatment of a disease by acting on an immune system, which contains Compound (I) or a salt thereof; Compound (I) or a salt thereof for the potentiation of the activity of a medicine used for the prevention or treatment of a disease by acting on an immune system; and a method for the potentiation of the activity of a medicine used for the prevention or treatment of a disease by acting on an immune system comprising administering an effective amount of Compound (I) or a salt thereof in combination with such medicine to a subject.
The present invention also relates to use of Compound (I) or a salt thereof for the production of the immunomodulator and the pharmaceutical composition described above.
The immunomodulator and the pharmaceutical composition according to the present invention may comprise a pharmaceutically acceptable diluent, excipient, or adjuvant, according to need, and it may be prepared in a form adequate for an administration route. Specific examples of dosage forms include oral preparations (e.g., tablets, pills, capsules, granules, powders, and liquid preparations), injection preparations, suppositories, ointments, and adhesive skin patches. Such dosage forms can be prepared in accordance with conventional techniques. The immunomodulator and the pharmaceutical composition according to the present invention are preferably in the form of oral preparations because of the ease of administration. Specific examples of adjuvants include binders, disintegrators, lubricants, colorants, solubilizers, flavoring agents, suspending agents, isotonizing agents, buffers, and soothing agents. According to need, the immunomodulator and the pharmaceutical composition may contain additives, such as preservatives, antioxidants, colorants, sweeteners, and stabilizers.
The amount of the immunomodulator and the pharmaceutical composition according to the present invention to be administered varies depending on the purpose of administration, the age, gender, and body weight of the subject to which the immunomodulator and the pharmaceutical composition are to be administered, as well as the route of administration. In the case of an adult whose body weight is 50 kg, for example, Compound (I) or a salt thereof is administered in an amount of preferably 0.05 to 5,000 mg, and more preferably 0.1 to 1,000 mg per day. Administration can be performed, for example, once per two days, once per day, or two or three times per day.
Compound (I) and a salt thereof according to the present invention have the immunomodulation activity as described in the examples below, and Compound (I) and a salt thereof make contributions to the regulation of vital functions, health enhancement, and enhanced functions of eliminating foreign-matter-like components of endogenous origin or foreign matter of exogenous origin of humans and other mammalian animals.
Hereafter, the present invention is described in greater detail with reference to the examples, although the technical scope of the present invention is not limited to these examples.
The mouse spleen was extracted, grinded with glass slides with frosted areas, and then subjected to hemolysis, so as to obtain spleen cells. The resulting spleen cells were introduced into a complete medium (i.e., RPMI-1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 55 μM 2-mercaptoethanol) to a cell density of 2×106 cells/ml, the anti-CD3 antibody and the anti-CD28 antibody were added thereto to the final concentrations of 3 μg/ml and 0.5 μg/ml, respectively, and Compound (I) was further added thereto to each final concentration. The culture solution was seeded in a 96-well plate at 200 μl/well and then cultured in an incubator at 37° C. in the presence of 5% CO2 for 2 days. The culture supernatant was recovered, and the concentration of IL-2 contained therein was measured via ELISA using anti-mIL-2 antibody.
Also, culture was conducted in the same manner with the addition of the anti-CD3 antibody at a variable final concentration, the anti-CD28 antibody at the final concentration of 0.5 μg/ml, and Compound (I) at the final concentration of 0.0 μM or 0.1 μM, and the IL-2 concentration in the culture supernatant was measured.
These results demonstrate that Compound (I) could potentiate induction of IL-2 production of mouse spleen cells and that Compound (I) exerts immunomodulation activity.
Human peripheral blood mononuclear cells were introduced into a human complete medium (i.e., RPMI-1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin) to prepare a cell suspension (1×106 cells/ml). Phytohemagglutinin M (PHA-M) was added thereto to the final concentration of 5 μg/ml, and Compound (I) or an EGFR tyrosine kinase inhibitor (AZD9291) was further added thereto to each final concentration. This culture solution was seeded in a 96-well plate at 200 μl/well and then cultured in an incubator at 37° C. in the presence of 5% CO2 for 3 days. The culture supernatant was recovered, and the concentration of IL-2 therein was measured via ELISA using the anti-hIL-2 antibody.
A mixed lymphocyte reaction (MLR) is a representative experimentation technique for T cell modulation described in, for example, J. Exp. Med. 127 (5): 879-90, 1968. The influence of Compound (I) imposed on T cell modulation through this reaction was examined.
Spleens were extracted from C57BL/6N mice and BALB/c mice, grinded with glass slides with frosted areas, and then subjected to hemolysis, so as to obtain spleen cells. The C57BL/6N mouse spleen cells and the BALB/c mouse spleen cells were introduced into a complete medium (i.e., RPMI-1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 55 μM 2-mercaptoethanol). The BALB/c mouse spleen cells were irradiated with 30 Gy X-ray to destroy the growth activity. The spleen cells of these allogeneic mice were each added to the final concentration of 1×105 cells/well and mixed (Alo), and Compound (I) or the EGFR tyrosine kinase inhibitor (i.e., AZD09291 or Erlotinib) adjusted to relevant concentrations was added thereto. As a control sample, a mixture of syngeneic C57BL/6N mouse spleen cells (Syn) was also prepared. The culture solution was seeded in a 96-well plate at 200 μl/well and then cultured in an incubator at 37° C. in the presence of 5% CO2 for 3 days. Tritium-labeled thymidine (3H-Thd) was added thereto 2 days after the initiation of culture. The amount of 3H-Thd uptake was measured using a liquid scintillation counter.
The mouse spleen was extracted, grinded with glass slides with frosted areas, and then subjected to hemolysis, so as to obtain spleen cells. The spleen cells were suspended in 5 ml of a staining buffer (0.5% BSA, 2 mM EDTA, PBS(-)), and the cells were stained with 5 μM CFSE (5-carboxyfluorescein succinimidyl ester). Thereafter, the stained cells were washed with an ice-cooled complete medium (RPMI-1640). The CFSE-stained spleen cells were introduced into a complete medium (i.e., RPMI-1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 55 μM 2-mercaptoethanol) to prepare a cell suspension (1×106 cells/ml), the anti-CD3 antibody and the anti-CD28 antibody were added at 1 μg/ml and 1 μg/ml, respectively, and Compound (I) adjusted to various concentrations or the EGFR tyrosine kinase inhibitor (i.e., AZD09291, Erlotinib, Co1686 (Rociletinib), Ibrutinib, Sunitinib, or Dasatinib) was added at 0.1 μM. A control sample that does not contain the EGFR tyrosine kinase inhibitor was also prepared. These culture solutions were seeded in a 96-well plate at 200 μl/well and then cultured in an incubator at 37° C. in the presence of 5% CO2 for 3 days. The cells were recovered and stained with the anti-CD4 antibody and the anti-CD8 antibody. The amounts of CFSE in CD4- and CD8-positive cells were analyzed via flow cytometry.
Once CFSE is incorporated into a cell, the amount thereof is kept constant in the cell. Since the amount of CFSE per cell decreases by half upon cell division, according to flow cytometric analysis, the intensity of CFSE staining is attenuated and is shifted toward left (i.e., lower level) in the chart, as cell division proceeds. In the case of the sample to which Compound (I) had been added, as shown in
A cell suspension of the OVA-expressing mouse thymoma cell line (EG.7-OVA) was prepared with the use of PBS(-) and 50% Matrigel, and the cell suspension (1×104 cells/mouse) was implanted into the syngeneic C57BL/6n mice via subcutaneous injection. The mice were divided into groups based on their body weights 1 day after implantation, and Compound (I) was administered thereto at 50 mg/kg or the anti-PD-1 antibody was administered thereto at 100 μg/mouse. A control group to which neither Compound (I) nor the anti-PD-1 antibody had been administered was also prepared. The peripheral blood was sampled 14 days after implantation and analyzed via flow cytometry with the use of antibodies each reacting with a relevant immunocyte surface marker, so as to determine the relative number of CD4-positive cells, CD8-positive cells, and CD4-negative, CD8-negative, and NK1.1-positive cells.
A cell suspension of the mouse colon cancer cell line (colon26) was prepared with the use of PBS(-) and 50% Matrigel, and the cell suspension was implanted into the syngeneic BALB/c mice via subcutaneous injection at 2×103 cells/mouse. The mice were divided into groups based on their body weights 1 day after implantation, and Compound (I) was administered thereto at 50 mg/kg and/or the anti-PD-1 antibody was administered thereto at 100 μg/mouse. A control group to which neither Compound (I) nor the anti-PD-1 antibody had been administered was also prepared. The spleen cells were sampled 21 days after implantation and analyzed via flow cytometry with the use of antibodies each reacting with a relevant immunocyte surface marker.
The results demonstrate that Compound (I) induces the activation of immunocyte in vivo and has immunomodulation activity. [Example 7] Induction of cytokine production by Compound (I) in human peripheral blood mononuclear cell
Human peripheral blood mononuclear cells were introduced into a human complete medium (i.e., RPMI-1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin) to prepare a cell suspension (1×105 cells/ml). Compound (I), the EGFR tyrosine kinase inhibitor (i.e., AZD9291 or Erlotinib), or a cytokine-inducing positive control (i.e., Imiquimod) was added to the cell suspension. A control group to which the EGFR tyrosine kinase inhibitor and others were not added was also prepared (i.e., the non-treatment group). The culture solution was cultured in an incubator at 37° C. in the presence of 5% CO2 for 2 days. The culture supernatant was recovered, and various types of cytokines were inspected with the use of the Bio-Plex Pro human cytokine assay kit.
The mouse melanoma cell lines B16F10 and K1735M2 and the mouse colon cancer cell line MC38 were seeded in a 96-well plate at 3×103 cells/well. Compound (I) or the EGFR tyrosine kinase inhibitor (i.e., Erlotinib, Afatinib, AZD9291, or Co1686 (Rociletinib)) that had been diluted to the given concentration was added 24 hours later. Thereafter, culture was conducted for 3 days, and the number of cells was determined using CellTiter-Glo2.0 (Promega, G9243). Compound (I) did not suppress the growth of any cells. Also, the other EGFR tyrosine kinase inhibitors did not suppress the growth of any cells.
A cell suspension of the mouse melanoma cell line B16F10 was prepared with the use of PBS(-), and the cell suspension was injected intravenously into the mouse caudal portion at 5×105 cells/mouse. Compound (I) was administered orally to the mouse model at 12.5 mg/kg or 50 mg/kg on the previous day of B16F10 implantation. The number of pulmonary metastatic nodules was evaluated 14 days after implantation (i.e., 15 days after drug administration).
A cell suspension of the mouse colon cancer cell line MC38 was prepared with the use of PBS(-) and 50% Matrigel, and the cell suspension was injected subcutaneously into mice at 1×106 cells/mouse. The mice were divided into groups when the average subcutaneous tumor volume reached approximately 50 mm3, Compound (I) was administered at 50 mg/kg or the anti-PD-1 antibody was administered at 100 μg/mouse, and the tumor volume was measured with the elapse of time. The tumor volume was determined on the basis of the longer diameter and the shorter diameter of the tumor measured percutaneously in accordance with Equation A shown below.
Tumor volume (mm3)=longer diameter (mm)×shorter diameter (mm)2/2 (Equation A)
A cell suspension of the mouse melanoma cell line K1735M2 was prepared with the use of PBS(-) and 50% Matrigel, and the cell suspension was injected subcutaneously into mice at 1×106 cells/mouse. The mice were divided into groups based on their body weights 1 day after implantation, Compound (I) (50 mg/kg) or the anti-PD-1 antibody or anti-PD-L1 antibody (100 μg/mouse) was administered thereto alone or either the anti-PD-1 antibody or the anti-PD-L1 antibody was administered in combination with Compound (I), and the tumor volume was measured with the elapse of time. The tumor volume was determined on the basis of the longer diameter and the shorter diameter of the tumor measured percutaneously in accordance with Equation A shown above.
A cell suspension of the mouse colon cancer cell line MC38 was prepared with the use of PBS(-) and 50% Matrigel, and the cell suspension was injected subcutaneously into mice at 1×106 cells/mouse. The mice were divided into groups when the average subcutaneous tumor volume reached approximately 50 mm3, Compound (I) (50 mg/kg) or the anti-PD-1 antibody (50 μg/mouse) was administered thereto alone or Compound (I) (50 mg/kg) was administered in combination with the anti-PD-1 antibody (50 μg/mouse), and the tumor volume was measured with the elapse of time. The tumor volume was determined on the basis of the longer diameter and the shorter diameter of the tumor measured percutaneously in accordance with Equation A shown below.
Tumor volume (mm3)=longer diameter (mm)×shorter diameter (mm)2/2 (Equation A)
The present invention enables the prevention or treatment of a disease that can be ameliorated via immunomodulation. The present invention also provides a novel method for the treatment of various infections, immunodeficiency, and tumors.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
Number | Date | Country | Kind |
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2015-203282 | Oct 2015 | JP | national |
2016-148854 | Jul 2016 | JP | national |