KR20230111069A - Adjuvant for Radioimmunotherapy of Cancer Comprising Genipin - Google Patents

Abstract

The present invention relates to an adjuvant for radioimmunotherapy of cancer containing genipin as an active ingredient, and when administered in combination with a radioimmunotherapy agent and genipin, a radioimmunotherapy effect is significantly improved. Therefore, the present invention enables effective anti-cancer treatment while minimizing the effect on normal cells, and can be usefully used to treat non-solid cancers such as blood cancer or lymphoma.

Description

Adjuvant for radioimmunotherapy of cancer containing Genipin {Adjuvant for Radioimmunotherapy of Cancer Comprising Genipin}

The present invention relates to an adjuvant for cancer radioimmunotherapy and a composition for enhancing radioimmunotherapy containing Genipin as an active ingredient.

BACKGROUND OF THE INVENTION Cancer is a disease that causes malignant tumors by unlimited proliferation of cells in biological tissues, and eventually invades surrounding tissues or metastasizes to other organs, leading to death of the living body. In addition to hereditary causes, physical stimulation, chemical stimulation, and viral infection are the causes, and it is difficult to cure.

Cancer treatment methods include radiation therapy, chemotherapy using anticancer agents, and radioimmunotherapy in which tumor-specific antibodies and therapeutic radioactive isotopes are combined. Treatment using radiation has a high frequency of side effects, and there is a problem that its use is limited. In particular, when it is difficult to treat through surgery, the treatment is performed using radiation. In order to minimize damage to normal organs and increase the treatment effect, radiation should be adjusted according to the stage and location of cancer. Although there is a difference in sensitivity between individuals or tissues during radiation therapy for cancer cells, if the sensitivity to radiation can be increased, the same therapeutic effect can be obtained by irradiating a patient with a smaller amount of radiation.

Radioimmunotherapy (RIT) is a treatment that can maximize the therapeutic effect by labeling a tumor epitope-specific monoclonal antibody with a therapeutic radioisotope (I-131, Y-90, Lu-177, etc.) that emits beta rays, and using a combination of anti-cancer effects of monoclonal antibodies and cytotoxic radiation. Radioimmunotherapy can expect a synergistic effect of cancer treatment by targeted therapy of tumor antibodies and cancer treatment effect by radiation. Accordingly, studies on radioimmunotherapy are being actively conducted, but there are also cases in which the response of radioimmunotherapy is low depending on the type or progress of some patients or cancer.

Genipin is a type of non-saccharide glycoside derived from an iridoid glycoside known as geniposide present in Gardenia jasminoides, and is less toxic than commonly used synthetic crosslinkers, so it is widely used as an excellent natural protein crosslinking agent. Genipin is known to promote secretion of gastric acid and alleviate age-related insulin resistance. In addition, genipin is also known to help choleretic action in liver disease, and recent studies have reported that genipin has an anti-inflammatory and anti-angiogenic inhibitory role (Non-Patent Document 1 and Non-Patent Document 2). However, no literature has been reported yet showing that genipin can improve the therapeutic effect in anticancer treatment using radioimmunotherapy.

Koo et al., Eur J Pharmacol, 495, 201-208, 2004 Nam et al., Int Immunopharmacol, 10, 493-499, 2010

As a result of research efforts to develop a combination agent capable of increasing the cancer therapeutic effect of radioimmunotherapeutic agents, the inventors of the present invention completed the present invention by experimentally confirming that the therapeutic effect of radioimmunotherapeutic agents increases synergistically when Genipin is used in combination with radioimmunotherapeutic agents.

Accordingly, an object of the present invention is to provide an adjuvant for cancer radioimmunotherapy comprising genipin as an active ingredient.

In addition, an object of the present invention is to provide a composition for enhancing cancer radioimmunotherapy comprising genipin as an active ingredient.

In addition, an object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of cancer comprising genipin and a radioimmunotherapy agent as active ingredients.

One aspect of the present invention provides an adjuvant for cancer radioimmunotherapy comprising genipin as an active ingredient.

The genipin has a structure of Formula 1 below, and can be purified from nature, purchased commercially and used, or prepared by a chemical synthesis method known in the art.

[Formula 1]

Genipin is a non-sugar part of geniposide, a natural component of the iridoid glycoside system, and is a component that exhibits the main pharmacological efficacy of gardenia fruit (gardenia). Gardenia is widely used in food, such as producing a yellow color as a non-toxic, non-polluting natural dye or food coloring, and is also widely used for medicinal purposes. In addition, gardenia has long been used as an anti-inflammatory, antipyretic, diuretic, and hemostatic agent in oriental medicine. As such, Genipin is derived from gardenia, a natural product, and has the advantage of being proven safe.

In one embodiment, the 'adjuvant for cancer radioimmunotherapy' may be administered in combination with a radioimmunotherapy agent. The combined administration of the adjuvant may be concurrently administered with the radioimmunotherapy agent, separately or sequentially.

The adjuvant containing the genipin can synergistically improve the cancer treatment or prevention effect of the radioimmunotherapy agent by being co-administered with the radioimmunotherapy agent.

The adjuvant of the present invention may be one that exhibits a synergistic therapeutic effect with the radioimmunotherapy agent or enhances the anticancer effect of the radioimmunotherapy agent for cancer in anticancer therapy using the radioimmunotherapy agent. Enhancement of the anticancer effect of the radioimmunotherapy agent for cancer may be to improve the therapeutic sensitivity or radiosensitivity of the radioimmunotherapy agent. Therapeutic sensitivity or sensitivity of a radioimmunotherapy agent means the degree of therapeutic response to radiation therapy, and high sensitivity or sensitivity is described when a high therapeutic effect is exhibited for a small amount of radiation. In the present invention, when an adjuvant is used in combination with a radioimmunotherapeutic agent, even if a smaller dose of the radioimmunotherapeutic agent is treated than when the adjuvant is not used in combination, it may mean that an anticancer effect equivalent to the case of using a larger amount of the radioimmunotherapeutic agent appears. Alternatively, when the adjuvant is used in combination with a radioimmunotherapeutic agent, it may mean that a superior anticancer effect is exhibited as compared to the use of an equivalent dose of the radioimmunotherapeutic agent alone.

In the present invention, radioimmunotherapy (RIT) is a type of radiation therapy in which a radioactive material is combined with an immunotherapeutic agent and injected into the body, and is an immunotherapeutic agent that can specifically bind to a target antigen located on the surface of cancer cells. For example, it is a targeted radiotherapy method in which a radioactive isotope is bound to a monoclonal antibody. Radioimmunotherapy has the advantage of being able to specifically treat cancer cells with a target antigen, thereby minimizing the effect on normal cells and increasing the anticancer effect.

In the present invention, the radioimmunotherapy agent may be a drug used for radioimmunotherapy, specifically an antibody paired with a radioactive material called a radiotracer, and more specifically an antibody labeled with a radioactive isotope.

The antibody is a polypeptide comprising a framework region from an immunoglobulin gene or fragment thereof that specifically binds and recognizes an antigen, and includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies. The monoclonal antibody refers to an antibody derived from a single B cell clone, and the polyclonal antibody refers to an antibody produced and secreted from different B cell clones. The chimeric antibody refers to an antibody molecule artificially formed by processing two types of antibodies against different antigens so that one molecule binds to both antigens. In addition, the humanized antibody is an antibody that retains the reactivity of a non-human antibody while having low human immunogenicity. Such humanized antibodies can be obtained, for example, by retaining non-human CDR regions and substituting the remaining portions of the antibody with their human counterparts. The antibody includes not only a complete antibody form, but also an antigen-binding fragment (antibody fragment) of an antibody molecule. A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain linked to the heavy chain by disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and subclasses include gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types. An antigen-binding fragment of an antibody or antibody fragment refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2, Fv, and the like.

In one embodiment, the radioimmunotherapy agent may be a radioisotope-labeled anti-CD20 antibody. The anti-CD20 antibody may be an antibody that specifically binds to the CD20 antigen expressed on the surface of immune cells. The immune cells may be, for example, pre-mature or mature B cells (B lymphocytes).

The anti-CD20 antibody may be (Rituximab).

In one embodiment, the radioimmunotherapy agent may be radioisotope-labeled Rituximab. The Rituximab (trade names: Mabthera, Truxima) binds to the CD20 antigen expressed on malignant B cells as a target, and the body's immune system dissolves antibody-labeled B cells, thereby causing pharmacological effects on lymphoma, chronic lymphocytic leukemia, Wegener's granulomatosis, polyarteritis nodosa, and rheumatoid arthritis.

The radioactive isotope refers to an isotope that decays into a stable element by emitting radiation such as alpha particles, neutrons, beta rays, gamma rays, and X-rays. Among them, isotopes that emit alpha particles, beta rays, and gamma rays have low penetrating power and excellent cell ionization ability, so they can be used for medical purposes. The radioisotope may be an alpha particle emitting radioisotope, a beta ray emitting radioisotope or a gamma ray emitting radioisotope, for example, Cu-67, Y-90, Pb-103, Sn-117m, Ho-166, I-131, Lu-177, Re-188, At-211, Bi-213, Ra-223, Ac-225, Th-227, or In-1 11, specifically I-131 ( ¹³¹ I).

In one embodiment, the radiolabeled rituximab may be ¹³¹ I -rituximab.

In the present invention, cancer, which is a disease to be treated in the present invention, generally refers to a physiological state of a mammal having abnormal cell growth characteristics, and abnormal overproliferation due to a problem in the regulation function of normal division, differentiation, and death of cells. Infiltrate into surrounding tissues and organs to form lumps and destroy or transform existing structures. The cancer may be any one cancer selected from the group consisting of blood cancer, breast cancer, head or cervical cancer, uterine cancer, cervical cancer, ovarian cancer, lung cancer, bladder cancer, esophageal cancer, neoplastic tumor, neuroblastoma, testicular cancer, leukemia, stomach cancer, liver cancer, colon cancer, skin cancer, brain cancer, laryngeal cancer, prostate cancer, thyroid cancer, kidney cancer, and rectal cancer. Specifically, hematologic cancer, lymphoma, acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, myeloproliferative tumor, myelodysplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, Hodgkin lymphoma, primary central nervous system lymphoma, malignant lymphoma, lymphoid cancer, progenitor cell tumor, diffuse progenitor cell tumor, in which abnormal proliferation of lymphoid tissue occurs Sunken B-Cell Lymphoma, Mantle Cell Lymphoma, Follicular Lymphoma, Extranodular Marginal Cell Lymphoma, Burkitt Lymphoma, Extranodular NK/T-Cell Lymphoma, Peripheral T-Cell Lymphoma, Immunodeficiency-Related Lymphocytic Proliferative Disorders, Nodular Lymphocyte Predominantly Hodgkin's Disease, Classical Hodgkin's Disease with Hyperpoptosis, Lymphocytic Classic Hodgkin's Disease, Mixed Cellular Classical Hodgkin's Disease, Lymphocyte Depleted Classical Hodgkin's It can be kin disease or bucket lymphoma.

The 'anti-cancer effect' means prevention or treatment of the cancer. The prevention refers to any action that suppresses or delays the occurrence or progression of cancer, and the treatment means not only death of cancer cells, but also all effects in which cancer cells do not get worse, such as reduction in growth, reduction in proliferation, reduction in recurrence, alleviation of one or more symptoms associated with cancer to some extent, formation of immune system anticancer activity and immune memory formation in cancer cells, or reduction in invasion of cancer cells, but are not limited thereto.

The adjuvant of the present invention may be provided in the form of a pharmaceutical composition. In this case, the pharmaceutical composition may further include an active ingredient exhibiting anticancer activity in addition to genipin. The pharmaceutical composition may further include a pharmaceutically acceptable carrier or additive in addition to the active ingredient.

The meaning of 'pharmaceutically acceptable' means that it does not inhibit the activity of the active ingredient and does not have toxicity more than is adaptable to the subject of application (prescription). The carrier is defined as a compound that facilitates the addition of the compound into cells or tissues.

The pharmaceutical composition may be administered by mixing with any convenient carrier, etc., and such dosage form may be a single or repeated dosage form. The composition may be a solid formulation or a liquid formulation. Solid preparations include, but are not limited to, powders, granules, tablets, capsules, and suppositories. Solid formulations may include carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, and the like, but are not limited thereto. Liquid preparations include solutions, suspensions, and emulsions such as water and propylene glycol solutions, but are not limited thereto, and may be prepared by adding appropriate colorants, flavoring agents, stabilizers, viscosifiers, and the like. For example, a powder may be prepared by simply mixing an appropriate pharmaceutically acceptable carrier such as lactose, starch, or microcrystalline cellulose in addition to genipin, which is an active ingredient of the present invention. Granules can be prepared by mixing the active ingredient of the present invention with a suitable pharmaceutically acceptable carrier and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone or hydroxypropylcellulose, and then using a wet granulation method using a solvent such as water, ethanol, or isopropanol or a dry granulation method using compression force. In addition, tablets may be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate and then tableting them using a tableting machine. When formulating the pharmaceutical composition as an injection, it may be prepared according to a conventional method for preparing an injection known in the art. When formulated as an injection, it may be dispersed in a sterile medium so that it can be used as it is when administered to a patient, or may be administered after dispersing in an appropriate concentration by adding distilled water for injection.

The pharmaceutical composition may be administered as an injection (e.g., intravenous injection, intramuscular injection, intraperitoneal injection, infusion, subcutaneous injection, implant), inhalant, oral agent, nasal agent, vaginal agent, rectal agent, sublingual agent, transdermal agent, topical agent, etc., depending on the disease to be treated and the condition of the subject, but is not limited thereto. Depending on the route of administration, it can be formulated into an appropriate dosage unit formulation containing a pharmaceutically acceptable carrier, excipients, and vehicles that are commonly used and non-toxic. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences ( ^19th ed., 1995).

The pharmaceutical composition may be administered at about 0.0001 mg/kg (body weight) to about 10 g/kg (body weight) daily, and at a daily dosage of about 0.001 mg/kg (body weight) to about 1 g/kg (body weight). The therapeutically effective amount or effective dose of the pharmaceutical composition may vary depending on the formulation method, administration method, administration time and/or route of administration, etc., and may vary depending on the type and degree of response to be achieved by administration of the composition, the type of subject to be administered, age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, drugs used simultaneously or at different times in the subject, and other components of the composition, etc., and similar factors well known in the pharmaceutical field. A person of ordinary skill in the art can readily determine and prescribe an effective dosage for the desired treatment. In addition, if necessary, for convenience, the total daily dose may be divided and administered several times during the day. The term “treatment-effective amount” refers to an amount sufficient to produce a desired effect, including treatment or improvement of cancer in a patient with cancer, side effects of anti-cancer treatment or resistance to anti-cancer drugs, improvement of a condition (e.g., one or more symptoms), delay of progression of a disease, and the like, in a patient with cancer.

In addition, another aspect of the present invention provides a composition for enhancing cancer radioimmunotherapy comprising genipin as an active ingredient.

The composition may be administered in combination with a radioimmunotherapy agent.

The radioimmunotherapy agent may be an antibody labeled with a radioactive isotope.

In addition, another aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of cancer comprising genipin and a radioimmunotherapy as active ingredients.

The radioimmunotherapy agent may be an antibody labeled with a radioactive isotope.

In addition, another aspect of the present application provides a method for treating cancer by administering Genipin in combination with a radioimmunotherapy agent.

The radioimmunotherapy agent may be an antibody labeled with a radioactive isotope.

In the other aspect of the present invention, the composition for enhancing cancer radioimmunotherapy, the pharmaceutical composition for treating or preventing cancer, and the method for treating cancer, the contents related to co-administration, Genipin, radioimmunotherapy, radioimmunotherapy, pharmaceutical composition, cancer, etc. are the same as those described while describing the adjuvant for cancer radioimmunotherapy, which is one aspect of the present invention, and will not be duplicated and described in order to avoid excessive complexity in the specification.

The cancer radioimmunotherapy adjuvant or cancer radioimmunotherapy composition containing Genipin as an active ingredient of the present invention can enhance the anticancer effect of the radioimmunotherapy agent. According to the present invention, a desired therapeutic effect can be obtained even when a radioimmunotherapeutic agent is used at a reduced dose, thereby minimizing the radiation effect on normal cells and increasing the specific therapeutic effect on cancer cells. In particular, the adjuvant or composition of the present invention can be more usefully used to treat blood cancer or lymphoma in which a specific cancer site cannot be removed by surgical operation or external radiation therapy.

However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

Figure 1 shows the anticancer effect of Genipin on Raji cells, and shows Raji cell viability when Genipin is treated by dose.
Figure 2 shows Raji cell viability by dose of the treated drug when rituximab, ¹³¹ I, ¹³¹ I-rituximab, and ¹³¹ I-rituximab and Genipin (10 μM) were used in combination.

Hereinafter, the present invention will be described in detail by examples. However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[Example 1]

culture of cells

Raji cells were prepared by culturing in RPMI-1640 medium (Corning, Inc., NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Inc., MA, USA) and 1% penicillin/streptomycin antibiotics (Gibco, Thermo Fisher Scientific, Inc., MA, USA). The Raji cells were cultured at 37°C in a 5% CO ₂ incubator.

[Example 2]

Confirmation of anti-cancer effect of Genipin

Cell viability was measured using the EZ-Cytox cell viability, proliferation, and cytotoxicity assay kit (Daeil Lab, Seoul, Korea), and was performed according to the manufacturer's protocol. Specifically, Raji cells (2 x 10 ⁴ cells/0.1 ml/well) were plated in a 96-well plate and cultured for 24 hours at 37°C in a humidified incubator under a 5% CO ₂ atmosphere. After culturing, the Raji cells were treated with different concentrations of genipin (0.1-50 μM) and incubated again at 37°C for 24 hours. After incubation, 10 μL of Ez-Cytox solution was added to each well and further incubated for 0.5-4 hours. Absorbance was quantified by measuring with a 450 nm SpectraMax i3 microplate reader (Molecular Devices, Sunnyvale, CA). Cell viability was calculated as a percentage of the optical density (OD) ratio of treated and control samples. All assays were repeated three times to measure cell viability and to evaluate the degree of apoptosis in Genipin-treated Raji cells.

EZ-Cytox assay was performed for 24 hours by treating Raji cells with increasing concentration of genipin from 0.1 to 50 μM. The percentage of cell proliferation measured at 12 hours was plotted against the concentration of Genipin (FIG. 1). As a result, as shown in FIG. 1, the proliferation percentage of Raji cells decreased in a concentration-dependent manner in the range of 6.25-50 μM. When Raji cells were treated with a high concentration of genipin (50 μM), the cell proliferation rate decreased the most to 9%. However, when treated with low concentrations of genipin (0.1 - 3.13 μM), no difference in the proliferation rate of Raji cells was observed. It was confirmed that genipin has an anticancer effect on Raji cells.

[Example 3]

Confirmation of synergistic effect when using a combination of radioimmunotherapy and Genipin

¹³¹ An experiment was conducted to determine whether there was a synergistic effect of radioimmunotherapy when I-rituximab and genipin were used in combination.

Specifically, the same number of Raji cells (2 x 10 ⁴ /0.1 ml/well) were plated in a 96-well plate and cultured for 24 hours. The cultured cells were treated with various concentrations of rituximab (10, 50 and 100 μg), ¹³¹ I (10, 50 and 100 μCi), ¹³¹ I -rituximab (10, 50 and 100 μCi) or a combination of ¹³¹ I-rituximab (10, 50 and 100 μCi) and Genipin (10 μM). After culturing the cells for 24 hours, 10 μl of EZ-Cytox solution was added to each well. After further incubating the plate at 37°C for 3 hours in a CO ₂ incubator, cell viability was calculated by measuring the optical density in the same manner as in Example 2 above. After culturing for 12 hours, the degree of apoptosis was evaluated in Raji cells treated with ¹³¹ I-rituximab (10, 50, and 100 μCi) and 10 μM genipin in combination.

As a result, as shown in Figure 2, when treated with ¹³¹ I-rituximab and Genipin (64%, 51%, and 32%), when treated with ¹³¹ I (113%, 103%, 90%), when treated with rituximab (95%, 90%, 91%), or when treated with ¹³¹ I-rituximab (106%, 84%) %, 71%), increased apoptosis was confirmed in Raji cells. In addition, when 10 μM of Genipin was combined with ¹³¹ I-Rituximab (100 μCi), it was confirmed that there was a higher cell killing effect than the sum of the cases where Genipin (10 μM) and ¹³¹ I-Rituximab were treated alone. In particular, when Genipin (10 μM) and ¹³¹ I-Rituximab (100 μCi) were treated together, the cell viability was 32%, and the highest degree of apoptosis was confirmed, which was ¹³¹ I-Rituximab (100 μCi) alone. It was confirmed that the degree of cell death was more than twice that of the treatment group (71%). From the above experimental results, it was confirmed that genipin and ¹³¹ I-rituximab had a synergistic anticancer effect when used in combination.

Claims (12)

An adjuvant for cancer radioimmunotherapy comprising genipin as an active ingredient.
The method of claim 1,
The adjuvant is a radioimmunotherapy adjuvant for cancer, which is administered in combination with a radioimmunotherapy agent.
The method of claim 2,
The adjuvant is a radioimmunotherapy adjuvant for cancer, which is administered concurrently, separately, or sequentially with the radioimmunotherapy agent.
The method of claim 2,
The radioimmunotherapy agent is an antibody labeled with a radioisotope, an adjuvant for cancer radioimmunotherapy.
The method of claim 4,
The antibody is an anti-CD20 antibody, an adjuvant for cancer radioimmunotherapy.
The method of claim 4,
The radioactive isotope is Cu-67, Y-90, Pb-103, Sn-117m, Ho-166, I-131, Lu-177, Re-188, At-211, Bi-213, Ra-223, Ac-225, Th-227, and At least one selected from the group consisting of In-111, radioimmunotherapy adjuvant for cancer.
The method of claim 1,
Wherein the cancer is blood cancer or lymphoma, an adjuvant for cancer radioimmunotherapy.
A composition for promoting radioimmunotherapy of cancer comprising genipin as an active ingredient.
The method of claim 8,
The composition is a composition for promoting radioimmunotherapy of cancer, which is administered in combination with a radioimmunotherapy agent.
The method of claim 9,
The radioimmunotherapeutic agent is an antibody labeled with a radioisotope, a composition for promoting radioimmunotherapy of cancer.
A pharmaceutical composition for the treatment or prevention of cancer comprising genipin and a radioimmunotherapy agent as active ingredients.
The method of claim 11,
The radioimmunotherapy is an antibody labeled with a radioactive isotope, a pharmaceutical composition for the treatment or prevention of cancer.