JP7549287B2 - Pharmaceutical compositions containing plant-derived alkaloids - Google Patents

Description

The present invention relates to a pharmaceutical composition containing a plant-derived alkaloid.

Traditionally, corticosteroids, which have strong anti-inflammatory and immune system suppressive effects, have been widely used to treat various immune system diseases. Diseases, disorders, and conditions for which corticosteroids are suitable include many autoimmune diseases and organ transplant rejection.

Corticosteroids are used to treat many diseases, disorders and conditions, but there are quite a few patients who are resistant to the therapeutic effects, and the efficacy of the drug may not be fully demonstrated. In addition, side effects such as increased susceptibility to infection, peptic ulcers (steroid ulcers), osteoporosis (steroid osteoporosis), diabetes (steroid diabetes), glaucoma, cataracts, arteriosclerosis, hypertension and thrombosis may hinder treatment.

In order to reduce the side effects of these corticosteroids, it is generally necessary to reduce the dosage and the administration period. The combined use of corticosteroids with various immunosuppressants is one way to compensate for the above-mentioned shortcomings of corticosteroids, but immunosuppressants such as cyclosporine and tacrolimus can also cause a variety of serious side effects that cannot be ignored. Therefore, there has been a demand for alternative treatment methods.

The present inventors have been searching for novel immunosuppressants using natural products as seeds, while also focusing on the combined effects of the novel immunosuppressants and corticosteroids, and have conducted research from both basic and clinical perspectives. As background, the present inventors have reported in international journals that the in vitro immunosuppressant responsiveness of patients' peripheral blood mononuclear cells (PBMCs) correlates with the therapeutic effects of drugs (Non-Patent Documents 1, 2, and 3). The present inventors believe that this research system is not only useful for promoting personalized medicine with existing immunosuppressants, but can also be applied to discover compounds whose immunosuppressive effects have not been known until now, and have used this research system to find alkaloids derived from plants of the genus Stephania as candidate compounds from herbal medicines and seeds derived from nature (e.g., flavonoids, lignans, peptides, and alkaloids), as well as existing pharmaceuticals (e.g., antibacterial drugs, fat-soluble vitamins, etc.).

Plant-derived alkaloids contained in plants of the genus Stephania, such as Stephania gracilis and Stephania japonica, of the family Menispermaceae, include, for example, cepharanthine, isotetrandrine, berbamine, cyclaine, homoaromorine, and cepharanolin. Cepharanthine (registered trademark) is an example of a pharmaceutical preparation that contains an alkaloid extracted from plants of the genus Stephania, particularly Stephania gracilis, as an active ingredient. Cepharanthine (registered trademark) is suitable for leukopenia and viper bites, and is commercially available in the form of tablets, powder, and intravenous injection. However, there are many unknowns regarding its mechanism of action.

On the other hand, Yamazaki et al. conducted a multi-center clinical study in which Cepharanthin (registered trademark) was used off-label in patients with thrombocytopenic purpura to examine the recovery of platelet counts (Non-Patent Document 4). As a result of this study, it was reported that Cepharanthin (registered trademark) was effective in patients who were taking corticosteroids but for whom steroids were not effective, and that it was possible to reduce the amount of steroids. However, there have been no reports on the mechanism of action of Cepharanthin (registered trademark) in improving platelet counts or reducing the amount of steroids in patients with thrombocytopenic purpura. Furthermore, there have been no reports of improved efficacy in patients with other immune system diseases or organ transplant patients by combining Cepharanthin (registered trademark) with corticosteroids.

Hirano T et al., Clin Pharmacol Ther 74: 581-590; 2003 Muhetaer et al., Cell Medicine 3:75-80; 2012 Sugiyama K et al., Cell Medicine 6: 47-55; 2014 Yamazaki T et al., Pediatric Int 59:303-308; 2017

The present invention aims to provide a pharmaceutical composition containing a plant-derived alkaloid.

As a result of intensive research conducted by the inventors to solve the above problems, they have discovered that alkaloids derived from plants of the genus Stephania in the family Menispermaceae, particularly Stephania nigra, have the effect of suppressing pathological activation of lymphocytes in patients with rheumatoid arthritis (see Figure 1). This suggests that Cepharanthine (registered trademark) can be used for rheumatoid arthritis and other diseases for which steroids have traditionally been used. Based on the above findings, the inventors have arrived at the present invention by applying Cepharanthine (registered trademark) to diseases for which steroids are used.

Furthermore, it has been found that the use of Cepharanthin (registered trademark) in combination with a corticosteroid drug significantly enhances the immunosuppressive effect of the corticosteroid drug. Specifically, using peripheral blood lymphocytes from healthy individuals and patients with rheumatoid arthritis, preliminary experiments have confirmed that the combined use of cepharanthin with a very small amount (pg/mL level) of a corticosteroid strongly and significantly suppresses the pathological activation of T lymphocytes (see Figure 2). Based on these findings, the present inventors aim to enhance the efficacy of steroid drugs and reduce side effects by co-administering cepharanthin or a preparation containing cepharanthin when administering corticosteroid drugs to patients with immune system diseases such as rheumatoid arthritis and nephrotic syndrome, or to patients with organ transplants.

That is, the present invention provides:
[1] A pharmaceutical composition comprising a plant-derived alkaloid or a pharma- ceutical acceptable salt or solvate thereof for the treatment or prevention of a disease, disorder, or condition involving pathological activation of lymphocytes;
[2] The pharmaceutical composition according to [1], wherein the plant-derived alkaloid is an alkaloid derived from a plant of the genus Stephania in the family Menispermaceae;
[3] The pharmaceutical composition according to [1] or [2], wherein the plant-derived alkaloid is an alkaloid derived from Stephania gracilis plant of the family Menispermaceae;
[4] The pharmaceutical composition according to any one of [1] to [3], wherein the plant-derived alkaloid is at least one selected from the group consisting of cepharanthine, isotetrandrine, berbamine, cycleanine, homoaromorine, cephalanoline, aromaline, obamedine, norcycleanine, 2-norcepharanthine, 2-norcephalanoline, 2-norberbamine, secocepharanthine, ovaberine, 2-norisotetrandrine, oxyacanthine, stephibaberine, tarrugosine, coclaurine, reticulin, rhodanidine, protosinomenine, N-methylcoclaurine, FK-3000, sinomenine, cefamonine, tannadine, cephamulin, lastawavirin, isocoridine, collidine, stephalin, cephalamin, acunadinine, and acunadiractam;
[5] The pharmaceutical composition according to any one of [1] to [4], wherein the plant-derived alkaloid is at least one selected from the group consisting of cepharanthine, isotetrandrine, berbamine, cycleanine, homoaromorine, cephalanoline, aromaline, obamedine, norcycleanine, 2-norcepharanthine, 2-norcephalanoline, 2-norberbamine, secocepharanthine, ovaberine, 2-norisotetrandrine, oxyacanthine, stefibaberine, and tarrugosine;
[6] The pharmaceutical composition according to any one of [1] to [5], wherein the plant-derived alkaloid is at least one selected from the group consisting of cepharanthine, isotetrandrine, berbamine, cycleanine, homoaromorine, and cepharanolin;
[7] The pharmaceutical composition according to any one of [1] to [6], wherein the plant-derived alkaloid is cepharanthine;
[8] The pharmaceutical composition according to any one of [1] to [7], further comprising a corticosteroid or a pharma- ceutical acceptable salt or solvate thereof;
[9] The pharmaceutical composition according to [8], wherein the corticosteroid is selected from the group consisting of methylprednisolone, prednisolone, hydrocortisone, dexamethasone, betamethasone and triamcinolone;
[10] The pharmaceutical composition according to [8] or [9], wherein the corticosteroid is selected from the group consisting of methylprednisolone and prednisolone;
[11] The pharmaceutical composition according to any one of [1] to [10], wherein the disease, disorder or pathology associated with pathological activation of lymphocytes is at least one selected from the group consisting of rheumatoid arthritis, nephrotic syndrome, autoimmune disease, inflammatory disease, neurodegenerative disease, allergic disease, autoimmune hepatitis, pancreatitis, bronchial asthma, systemic lupus erythematosus, purpura, glomerulonephritis, myasthenia gravis, ulcerative colitis, Crohn's disease, multiple sclerosis, dermatomyositis, polymyositis, atopic dermatitis, transplant rejection, graft rejection and graft-versus-host disease;
[12] The pharmaceutical composition according to any one of [1] to [11], wherein the disease, disorder, or pathology associated with pathological activation of lymphocytes is selected from the group consisting of rheumatoid arthritis, nephrotic syndrome, and organ transplant rejection;
[13] For simultaneous, separate, sequential or consecutive use for the treatment or prevention of diseases, disorders or pathologies involving pathological activation of lymphocytes;
(i) a plant-derived alkaloid or a pharma- ceutically acceptable salt or solvate thereof;
(ii) in combination with a corticosteroid or a pharma- ceutical acceptable salt or solvate thereof;
[14] For the treatment or prevention of a disease, disorder or pathology involving pathological activation of lymphocytes,
(i) a plant-derived alkaloid or a pharma- ceutically acceptable salt or solvate thereof;
(ii) The present invention relates to a combination comprising a corticosteroid or a pharma- ceutically acceptable salt or solvate thereof.

The present invention provides a pharmaceutical composition containing a plant-derived alkaloid.

Fig. 1 shows the effect of cepharanthine (CEP) on the proliferation of peripheral blood mononuclear cells (PBMCs) stimulated with concanavalin A in healthy subjects and rheumatoid arthritis (RA) patients. PBMCs were suspended in RPMI1640 medium and cultured in the presence of concanavalin A and various drugs for 4 days, after which cell proliferation was examined by the WST method. Fig. 1(A) shows the dose-dependent inhibitory effect on the proliferation of PBMCs from healthy subjects and PBMCs from RA patients, and Fig. 1(B) shows a comparison of the _IC50 value of CEP on the proliferation of PBMCs from healthy subjects (○) and PBMCs from RA patients (●). FIG. 2 shows the effect of cepharanthine alone and in combination with methylprednisolone on the proliferation of PBMC from healthy subjects stimulated with T cell mitogens.

The present invention will be described in more detail below.

The first aspect of the present invention is a pharmaceutical composition containing a plant-derived alkaloid as an active ingredient. The plant-derived alkaloid as an active ingredient also includes a pharma- ceutical acceptable salt or solvate of the plant-derived alkaloid. The pharmaceutical composition according to the present invention contains a plant-derived alkaloid and thus has the effect of suppressing pathological activation of lymphocytes.

Plant-derived alkaloids can be extracted from plants of the genus Stephania in the family Menispermaceae, such as Stephania gracilis and Stephania quinata, in accordance with conventional methods. The plant of the genus Stephania is preferably Stephania gracilis.

The active ingredient of the present invention may be an extract obtained by concentrating an extract from a plant of the genus Stephania, a precipitate formed when an acidic solution of the extract is made alkaline, an alkaloid-containing fraction separated from the precipitate, or crystals obtained by separating and purifying the extract by a conventional method. For example, an alkaloid fraction can be separated by extracting a plant of the genus Stephania (including, but not limited to, rhizomes, stems, seeds, leaves, etc.) with a solvent such as methanol, ethanol, acetone, ethyl acetate, or benzene, concentrating the extract, dissolving the concentrate in an acidic aqueous solution such as dilute hydrochloric acid, dilute sulfuric acid, an aqueous citric acid solution, or an aqueous oxalic acid solution, making the solution alkaline, and collecting the resulting precipitate. The fraction obtained may be further purified by known means such as various types of chromatography or recrystallization.

Plant-derived alkaloids include the following:
(a) Bisbenzylisoquinoline alkaloids: for example, cepharanthine, isotetrandrine, berbamine, cycleanine, homoaromorine, cephalanoline, aromaline, obamedine, norcycleanine, 2-norcepharanthine, 2-norcephalanoline, 2-norberbamine, secocepharanthine, obaverine, 2-norisotetrandrine, oxyacanthine, stephibaberine, and tarrugosine;
(b) Benzylisoquinoline alkaloids: for example, coclaurine, reticulin, rhodanidine, protosinomenine, N-methylcoclaurine, etc.;
(c) Morphinan alkaloids: for example, FK-3000, sinomenine, cefamonine, tannadine, cefamulin, etc.;
(d) Aporphine alkaloids: for example, lasta avalanche, isocoridine, collidine, etc.;
(e) Proaporphine alkaloids: for example, stephaline;
(f) Lotus flower alkaloids: Examples include cephalamin, acunadinine, and acunadirachtam. The pharmaceutical composition of the present invention contains one or more plant-derived alkaloids selected from the group consisting of these alkaloids.

The plant-derived alkaloid contained in the pharmaceutical composition of the present invention is preferably at least one selected from the group consisting of the bisbenzylisoquinoline alkaloids of group (a) above, i.e., cepharanthine, isotetrandrine, berbamine, cycleanine, homoaromorine, cephalanoline, aromaline, obamedine, norcycleanine, 2-norcepharanthine, 2-norcephalanoline, 2-norberbamine, secocepharanthine, ovaberine, 2-norisotetrandrine, oxyacanthine, stephibaberine, and tarrugosine, and more preferably at least one selected from the group consisting of cepharanthine, isotetrandrine, berbamine, cycleanine, homoaromorine, and cepharanoline.

Bisbenzylisoquinoline alkaloids are represented by the following formulas (I), (II), (III) and (IV).

The plant-derived alkaloid may be a pharma- ceutically acceptable salt or solvate. For example, pharma-ceutically acceptable salts include addition salts of inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid, and addition salts of organic acids such as acetic acid, succinic acid, malic acid, tartaric acid, citric acid, maleic acid, fumaric acid, methanesulfonic acid, and p-toluenesulfonic acid.

As a pharmaceutical preparation containing alkaloids derived from plants of the genus Stephania, Cepharanthine (registered trademark, Kaken Seiyaku Co., Ltd.), an alkaloid preparation extracted from Stephania oleracea, is commercially available. Cepharanthine (registered trademark) preparations contain as alkaloids one or more selected from the group consisting of cepharanthine, isotetrandrine, berbamine, cycleanine, homoaromorine, cephalanolin, aromarine, obamedine, norcycleanine, 2-norcepharanthine, 2-norcephalanolin, 2-norberbamine, secocepharanthine, ovaberine, 2-norisotetrandrine, oxyacanthine, stephibaberin, and tarrugosine, with the main components being cepharanthine, isotetrandrine, berbamine, cycleanine, homoaromorine, and cephalanolin. In another embodiment, the main components may be cepharanthine, isotetrandrine, berbamine and cyclaine.

In this specification, the term "Cepharanthine (registered trademark)" refers to a commercially available alkaloid preparation extracted from Cepharanthus nigra (sold by Kaken Seiyaku Co., Ltd.), and the term "Cepharanthine" refers to the compound represented by the above chemical formula (I) (wherein _R1 is _CH3 , _R2 and _R3 form _-CH2- , and _R4 is _CH3 ).

As described above, the pharmaceutical preparation containing alkaloids derived from Stephania plants may contain multiple plant-derived alkaloids as active ingredients, but in another embodiment, the pharmaceutical preparation containing alkaloids derived from Stephania plants may contain only cepharanthine as the active ingredient.

The pharmaceutical composition according to the present invention may typically contain pharma- ceutical acceptable additives, such as excipients, binders, lubricants, solvents, diluents, stabilizers, isotonicity agents, etc., known in the art. Examples of excipients include starch, lactose, methylcellulose, crystalline cellulose, synthetic aluminum silicate, etc.; examples of binders include hydroxypropylcellulose, polyvinylpyrrolidone, etc.; examples of lubricants include talc, magnesium stearate, calcium stearate, etc.; and examples of isotonicity agents include sodium chloride, glucose, glycerol, mannitol, etc.

The pharmaceutical composition of the present invention is formulated into, for example, tablets, powders, granules, capsules, injections, liquids, suppositories, etc.

The pharmaceutical composition is used for treating or preventing a disease, disorder, or condition involving pathological activation of lymphocytes. Here, "pathological activation of lymphocytes" refers to activation of lymphocytes involved in immune responses that are detrimental to the patient, such as the expression of reactivity against self-antigens or transplanted organs.

The disease, disorder or condition involving pathological activation of lymphocytes is, for example, selected from the group consisting of rheumatoid arthritis, nephrotic syndrome, autoimmune disease, inflammatory disease, neurodegenerative disease, allergic disease, autoimmune hepatitis, pancreatitis, bronchial asthma, systemic lupus erythematosus, purpura, glomerulonephritis, myasthenia gravis, ulcerative colitis, Crohn's disease, multiple sclerosis, dermatomyositis, polymyositis, atopic dermatitis, transplant rejection, graft rejection and graft versus host disease.

The disease, disorder or condition involving pathological activation of lymphocytes is preferably selected from the group consisting of rheumatoid arthritis, nephrotic syndrome and transplant rejection. Here, transplant rejection includes organ transplant rejection.

The pharmaceutical composition of the present invention can be administered, for example, orally, intravenously, subcutaneously, or intraperitoneally. The dosage of the pharmaceutical composition of the present invention varies depending on the age, weight, and condition of the patient.

The second aspect of the present invention relates to a pharmaceutical composition that further comprises, in addition to a plant-derived alkaloid, a corticosteroid or a pharma- ceutical acceptable salt or solvate thereof. By using a combination of a plant-derived alkaloid and a corticosteroid, a synergistic inhibitory effect on pathological activation of lymphocytes can be obtained.

The corticosteroid is, for example, selected from the group consisting of methylprednisolone, prednisolone, hydrocortisone, dexamethasone, betamethasone and triamcinolone, and is preferably selected from the group consisting of methylprednisolone and prednisolone.

Another aspect of the present invention relates to a combination of (i) a plant-derived alkaloid or a pharma- ceutically acceptable salt or solvate thereof and (ii) a corticosteroid or a pharma- ceutically acceptable salt or solvate thereof for simultaneous, separate, sequential or consecutive use for the treatment or prevention of a disease, disorder or condition involving pathological activation of lymphocytes.

Yet another aspect of the present invention relates to a combination comprising (i) a plant-derived alkaloid or a pharma- ceutical acceptable salt or solvate thereof, and (ii) a corticosteroid or a pharma-ceutical acceptable salt or solvate thereof, for the treatment or prevention of a disease, disorder, or condition involving pathological activation of lymphocytes.

With regard to the combination, component (i) (first active ingredient) and component (ii) (second active ingredient) may be administered together, one after the other or separately in one combined unit dosage form or in two separate unit dosage forms. A therapeutically effective amount of each active ingredient of the combination of the present invention may be administered simultaneously, separately, or in any order, sequentially or consecutively. In the combination, the first active ingredient and the second active ingredient may be present in multiple units. The combination also includes a kit comprising, for example, components (i), (ii) and instructions for use.

By applying the above-mentioned components (i) and (ii) in combination, a synergistic inhibitory effect on pathological activation or proliferation of lymphocytes is achieved compared to monotherapy using each of these components, and for example, a synergistic effect is achieved in alleviating symptoms and delaying or inhibiting progression in diseases, disorders, or pathological conditions involving pathological activation of lymphocytes. In addition, by applying these in combination, it is possible to reduce the dosage of each active ingredient or reduce the frequency of administration. This is expected to reduce side effects and improve quality of life.

The dosage of each active ingredient used in the combination of the present invention may vary depending on the compound or pharmaceutical composition used, the method of administration, and the severity of the disease, disorder, or condition.

Cepharanthin (registered trademark) preparations have been used clinically for a long time, and are considered to have few side effects, and even if they do occur, they are minor, making them far superior to immunosuppressants in terms of safety. If a synergistic effect is observed in clinical trials when used in combination with corticosteroids, it is expected that the dosage and administration period of corticosteroids will be reduced, leading to a significant reduction in drug resistance and side effects of the drugs.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

<1> Separation and culture of human peripheral blood mononuclear cells (PBMC) Lymphocyte separation solution (Nakarai, Japan) was dispensed into a centrifuge tube, and 20 mL of heparinized peripheral venous blood from a healthy subject was gently layered on top, followed by centrifugation at 2300 rpm and 23°C for 20 minutes. After separation, the mononuclear cell (PBMC) layer was separated, RPMI1640 liquid medium (Gibco BRL, Grand Island, NY, USA) was added thereto, and centrifugation was further performed twice, and the PBMC was washed and then resuspended in RPMI1640 medium. This was separated onto a hemocytometer, the number of cells was counted using an inverted optical microscope, and the PBMC suspension was obtained by diluting with the medium to 1.0 x 10 ⁶ cells/mL. The PBMC includes all T lymphocytes in peripheral blood.

200 μL of the PBMC suspension was transferred to each well of a 96-well sterile flat-bottom microplate (Cosmo Bio, Japan), and then concanavalin A (Seikagaku Corporation, Japan) was added as a T cell proliferation stimulating substance (mitogen) to a final concentration of 5 μg/mL, followed by the addition of cepharanthine (Cayman Chemical, USA) and methylprednisolone (Sigma Aldrich, St. Louis. Mo., USA), and the plate was cultured at 37° C. in the presence of 5% CO ₂ for 4 days. All of the above operations were performed aseptically in a clean bench.

<2> Method for evaluating the effect of cepharanthine and methylprednisolone on the proliferation of human peripheral blood mononuclear cells (PBMCs) After culturing PBMCs in the presence of each drug as described in <1> above, the proliferation rate of the cells was examined by the MTT method. Specifically, MTT solution was first added to the cell suspension after the end of the culture, and the cells were further cultured at 37°C and in the presence of 5% _CO2 for 4 hours. Since MTT changes into a pigment (formazan crystal) in the proliferating cells, the proliferation rate of the cells can be determined by measuring the amount of this pigment by colorimetric quantification. The absorbance of each well of the plate was measured at a wavelength of 550 nm using a multispectro plate reader. From the absorbance value thus obtained, the proliferation rate of PBMCs in the presence of each concentration of drug was calculated. For each concentration of cepharanthine, the proliferation rate of PBMCs was plotted against the concentration of methylprednisolone. The results are shown in Figure 2.

Figure 2 shows that cepharanthine inhibited the proliferation of peripheral blood lymphocytes from healthy subjects stimulated with T cell mitogen (concanavalin A). Furthermore, it shows that the use of cepharanthine at a concentration of 1 μM or more in combination with methylprednisolone synergistically and significantly inhibited the proliferation of peripheral blood lymphocytes from healthy subjects stimulated with T cell mitogen in a methylprednisolone concentration-dependent manner.

Claims (5)

A pharmaceutical composition for treating or preventing a disease, disorder, or condition involving pathological activation of lymphocytes, comprising a plant-derived alkaloid, or a pharma- ceutical acceptable salt or solvate thereof, and a corticosteroid, or a pharma-ceutical acceptable salt or solvate thereof,
The plant-derived alkaloid is cepharanthine ,
A pharmaceutical composition, wherein the corticosteroid is methylprednisolone . The pharmaceutical composition according to claim 1, wherein the disease, disorder or condition associated with pathological activation of lymphocytes is at least one selected from the group consisting of rheumatoid arthritis, nephrotic syndrome, autoimmune diseases, inflammatory diseases, neurodegenerative diseases, allergic diseases, autoimmune hepatitis, pancreatitis, bronchial asthma, systemic lupus erythematosus, purpura, glomerulonephritis, myasthenia gravis, ulcerative colitis, Crohn's disease, multiple sclerosis, dermatomyositis, polymyositis, atopic dermatitis, transplant rejection, graft rejection and graft-versus - host disease. 3. The pharmaceutical composition according to claim 1 or 2 , wherein the disease, disorder or condition associated with pathological activation of lymphocytes is selected from the group consisting of rheumatoid arthritis, nephrotic syndrome and transplant rejection. For simultaneous, separate, sequential or consecutive use for the treatment or prevention of diseases, disorders or conditions involving pathological activation of lymphocytes;
(i) a plant-derived alkaloid or a pharma- ceutically acceptable salt or solvate thereof;
(ii) A combination with a corticosteroid or a pharma- ceutical acceptable salt or solvate thereof,
The plant-derived alkaloid is cepharanthine ,
A combination wherein the corticosteroid is methylprednisolone . For the treatment or prevention of a disease, disorder or condition involving pathological activation of lymphocytes,
(i) a plant-derived alkaloid or a pharma- ceutically acceptable salt or solvate thereof;
(ii) a combination comprising a corticosteroid or a pharma- ceutically acceptable salt or solvate thereof,
The plant-derived alkaloid is cepharanthine ,
A combination wherein the corticosteroid is methylprednisolone .

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