WO2021201264A1

WO2021201264A1 - Pharmaceutical composition for parenteral administration for treating, preventing or alleviating osteoarthritis

Info

Publication number: WO2021201264A1
Application number: PCT/JP2021/014273
Authority: WO
Inventors: 裕士黒木; 晶博中畑; 泰彰中川; 厚今泉
Original assignee: 株式会社セラバイオファーマ; 国立大学法人京都大学; 独立行政法人国立病院機構
Priority date: 2020-04-02
Filing date: 2021-04-02
Publication date: 2021-10-07
Also published as: JPWO2021201264A1

Abstract

The present invention addresses the problem of providing a pharmaceutical composition for parenteral administration for treating, preventing or alleviating osteoarthritis.　A pharmaceutical composition for parenteral administration for treating, preventing or alleviating osteoarthritis, said pharmaceutical composition comprising, as an active ingredient, a conjugate of curcumin with a water-soluble substance.

Description

Parenteral pharmaceutical composition for the treatment, prevention or alleviation of osteoarthritis

The present invention relates to a pharmaceutical composition for parenteral administration for the treatment, prevention or alleviation of osteoarthritis.

The number of patients with arthritis tends to increase due to the aging of society and changes in lifestyle. Among arthritis, osteoarthritis (OA) is the most common degenerative joint disease with about 250 million patients worldwide (Non-Patent Document 1).
The number of patients with knee osteoarthritis in Japan is estimated to be about 10 million with subjective symptoms and about 30 million with potential patients (number of patients by X-ray diagnosis). The incidence increases with age, and the proportion of males and females aged 50 and over is 1.5 to 2 times higher in females than in males.
As a treatment method for knee osteoarthritis, an extremely large number are known, including exercise therapy, hyaluronic acid injection, steroid injection, surgery, and prevention by supplements.
However, the effect is not sufficient, and it is desired to provide an effective medical therapy, and further, an effective medical therapy that does not adversely affect articular cartilage and subchondral bone. Furthermore, preventive supplementation is desired.

In recent years, curcumin has been clarified to have pharmacological actions such as tumorigenesis inhibitory action, antioxidant action, anti-inflammatory action, cholesterol lowering action, antiallergic action, brain disease preventive action, and heart disease preventive and therapeutic action, and foods (for example, , Functional foods), pharmaceuticals, cosmetics, etc. are being considered for use.
Regarding arthritis, curcumin is exemplified as one of the other active ingredients in the preventive agent for arthritis containing lutein and glucosamine as active ingredients, but the effect thereof is not specifically described (Patent Document). 1).
On the other hand, a pharmaceutical composition for parenteral administration containing curcumin as a conjugate with a water-soluble substance (for example, glucuronic acid, sulfuric acid, glutathione, an amino acid, etc.) and containing this conjugate as an active ingredient can absorb curcumin which is poorly soluble in water. As a result of the improvement, it has been reported that sufficient pharmacological actions such as antitumor action of curcumin can be obtained (Patent Document 2).

Japanese Unexamined Patent Publication No. 2013-95723 International Publication No. 2018/003857

An object of the present invention is to provide a pharmaceutical composition for parenteral administration for treating, preventing or alleviating osteoarthritis with reduced side effects.

Therefore, the present inventor used a model animal of osteoarthritis to administer various components parenterally to the degree of damage to articular cartilage, type II collagen in articular cartilage and other articular disease markers, and subchondral bone. Evaluation of cysts and osteospores revealed that the water-soluble substance conjugate of curcumins improved or suppressed these symptoms peculiar to osteoarthritis in both articular cartilage and subchondral bone, and further, curcumins The present invention has been completed by finding that systemic symptoms are not observed by parenteral administration of a water-soluble substance conjugate.

That is, the present invention provides the following inventions [1] to [11].
[1] A pharmaceutical composition for parenteral administration for treating, preventing or alleviating osteoarthritis, which comprises a water-soluble substance conjugate of curcumin as an active ingredient.
[2] The pharmaceutical composition for parenteral administration according to the above [1], wherein the curcumins are one or more selected from curcumin and its derivatives.
[3] The pharmaceutical composition for parenteral administration according to the above [2], wherein the curcumin derivative is one or more selected from bisdemethoxycurcumin, demethoxycurcumin and tetrahydrocurcumin.
[4] The pharmaceutical composition for parenteral administration according to any one of the above [1] to [3], wherein the water-soluble substance is one or more selected from glucuronic acid, sulfuric acid, glutathione and amino acids.
[5] The pharmaceutical composition for parenteral administration according to any one of the above [1] to [3], wherein the water-soluble substance is one or more selected from glucuronic acid and sulfuric acid.
[6] The pharmaceutical composition for parenteral administration according to the above [1], wherein the water-soluble substance conjugate of the curcumins is curcumin monoglucuronide.
[7] The pharmaceutical composition for parenteral administration according to any one of the above [1] to [6], wherein the composition is a therapeutic agent for treating, preventing or alleviating osteoarthritis.
[8] The pharmaceutical composition for parenteral administration according to any one of the above [1] to [6], wherein the osteoarthritis is osteoarthritis of the knee, osteoarthritis of the hip, or osteoarthritis of the elbow. ..
[9] Use of a water-soluble substance conjugate of curcumin for the production of a pharmaceutical composition for parenteral administration for the treatment, prevention or alleviation of osteoarthritis.
[10] A water-soluble substance conjugate of curcumins for parenteral administration for the treatment, prevention or alleviation of osteoarthritis.
[11] A method for treating, preventing or alleviating osteoarthritis, which comprises parenterally administering an effective amount of a water-soluble substance conjugate of curcumins.

The pharmaceutical composition for parenteral administration for the treatment, prevention or alleviation of osteoarthritis, which comprises a water-soluble substance conjugate of curcumins of the present invention as an active ingredient, comprises articular cartilage and subchondral bone in osteoarthritis. Various symptoms can be improved or suppressed.

FIG. 1 shows the results of Safranin O and Fast-green staining of the rat tibia of Example 1. The upper two rows are drawings substitute photographs, the first row is the CMG administration group, and the second row is the control group, all of which are the staining results at 7 days, 14 days, 6 weeks, and 10 weeks. The lower two rows of the substitute photograph show the results of the OA score quantified based on the results of Safranin O and Fast-green staining. The first row shows the OARSI score, and the second row shows the modified Mankin histological score. FIG. 2 shows the results of immunohistological evaluation of the rat tibia of Example 1. The upper two rows are drawings substitute photographs, and the first row (upper and lower rows) shows the localization status of type I collagen, and the second row (upper and lower rows) shows the localization status of type II collagen. In the lower two rows of the substitute photograph, the first row shows the minimum brightness of type I collagen, and the second row shows the average brightness of type II collagen. FIG. 3 shows the immunohistological evaluation results of IL1-β and IL6 for the rat tibia of Example 1. The upper two rows show the expression status of inflammatory cytokines IL1-β and IL6 in the drawing substitute photograph, and the lower two rows show the percentage of positive cells of IL1-β and IL6 (%). FIG. 4 shows the immunohistological evaluation results of the inflammatory cytokine TNF-α related to the rat tibia of Example 1. The upper part shows the expression status of TNF-α in the drawing substitute photograph, and the lower part shows the ratio (%) of TNF-α positive cells. FIG. 5 shows the number of subchondral bone (SB) cysts, maximum cyst diameter and osteophyte mass in the rat tibia of Example 1. The upper part shows a drawing-substituting photograph showing the micro-CT imaging result of the subchondral bone, and the lower part shows the diameter (mm) of the number of cysts (Cysts) of the subchondral bone and the amount of osteophytes (mm ³ ). FIG. 6 shows the results of Safranin O and Fast-green staining of rat patella cartilage of Example 1 and the OA score. The upper two rows are drawings substitute photographs, the first row is the CMG administration group, and the second row is the control group, all of which are the staining results at 7 days, 14 days, 6 weeks, and 10 weeks. The lower two rows of the substitute photograph show the results of the OA score quantified based on the results of Safranin O and Fast-green staining. The first row shows the OARSI score, and the second row shows the modified Mankin histological score. FIG. 7 shows the results of immunohistological evaluation of rat patella cartilage of Example 1. The upper two rows are drawings substitute photographs showing the localization of type I and type II collagen. The lower two rows of the substitute photograph show the minimum brightness of type I collagen and the average brightness of type II collagen.

The active ingredient of the pharmaceutical composition for parenteral administration for the treatment, prevention or alleviation of osteoarthritis of the present invention is a water-soluble substance conjugate of curcumins.
Curcumin is the main component of curcuminoids contained in turmeric pigment, and is a compound represented by the following structural formula (1).

The curcumins of the present invention are one or more selected from curcumin and its derivatives. Derivatives of curcumin include bisdemethoxycurcumin, demethoxycurcumin and tetrahydrocurcumin.
In the present invention, as the curcumin, chemically synthesized curcumin may be used, or those commercially available as a turmeric pigment may be used. As the turmeric pigment, turmeric powder obtained by powdering the dried rhizome of Curcuma longa LINE, and the turmeric powder using an appropriate solvent (for example, ethanol, fat, propylene glycol, hexane, acetone, etc.) are used. Examples thereof include crude curcumin or oleoresin (turmeric oleoresin) obtained by extraction, and purified curcumin.
In addition, curcumin includes both keto type and enol type which are tautomers.

Examples of the water-soluble substance of the water-soluble substance conjugate of curcumins of the present invention include one or more selected from glucuronic acid, sulfuric acid, glutathione and amino acids. Examples of amino acids include amino acids existing in the living body, for example, essential amino acids.
The water-soluble substance conjugate of curcumins can maintain a high blood concentration of free curcumin. As a result, the pharmacological action of curcumin can be sufficiently obtained, and since the curcumin conjugate is a metabolite of curcumin in vivo, it is preferable because it is very safe.
The binding molar ratio of curcumin to the water-soluble substance in the conjugate of the water-soluble substance of curcumins is preferably curcumin: water-soluble substance = 1: 1 to 1: 3, more preferably 1: 1 to 1: 2. Preferably, 1: 1 is even more preferred.

The conjugation form (binding form) of curcumin and the water-soluble substance is, for example, the form of the formula (2).

(In the formula, ^{at least one of R 1} and R ² is a residue of the water-soluble substance, and the remainder is a hydrogen atom.)

In the formula (2), ^{one or both of R 1} and R ² are preferably glucuronic acid residues or sulfuric acid residues, and the remainder is preferably a hydrogen atom. Of these, ^{it is more preferable that one or both of R 1} and R ² are glucuronic acid residues and the remainder is a hydrogen atom. Of these, curcumin monoglucuronide, in which R ¹ is a glucuronic acid residue and R ² is a hydrogen atom, is more preferable because it is excellent in therapeutic, preventive and alleviating effects on osteoarthritis.

A water-soluble substance conjugate of curcumins can be produced by the method described in Patent Document 2.

Curcumin's water-soluble substance conjugate improves or suppresses various symptoms peculiar to osteoarthritis in both articular cartilage and subchondral bone by parenteral administration, as shown in Examples below, and further curcumin. Parenteral administration of a class of water-soluble substance conjugates eliminates systemic symptoms.
Here, as symptoms peculiar to osteoarthritis, damage to articular cartilage, type II collagen in articular cartilage, arthropathy markers such as inflammation markers, formation of subchondral bone cysts and osteophytes, and the like can be mentioned. The water-soluble substance conjugate of curcumins significantly suppresses damage to these articular cartilage, arthropathy markers such as type II collagen in articular cartilage and inflammation markers, and formation of subchondral bone cysts and osteophytes.
Examples of osteoarthritis targeted by the pharmaceutical composition for parenteral administration of the present invention include osteoarthritis of the knee, osteoarthritis of the hip, osteoarthritis of the elbow, and the like. Osteoarthritis can be mentioned as preferred.

The content of the water-soluble substance conjugate of curcumin in the pharmaceutical composition for parenteral administration of the present invention is appropriately determined according to the type of osteoarthritis, the sex, age, symptoms, etc. of the patient. However, for example, 1 to 100% by mass is preferable, 5 to 100% by mass is more preferable, and 10 to 100% by mass is further preferable.
The composition of the present invention is not particularly limited as a dosage form when used in the form of a therapeutic agent. For example, it can be used as a liquid preparation or a lyophilized preparation prepared at the time of use. It may also be a sustained release dosage form or a sustained release dosage form. It can also be prepared using additives such as carriers and excipients that are usually used in pharmaceutical preparations.

The route of administration of the pharmaceutical composition of the present invention may be parenteral administration, and examples thereof include systemic administration and topical administration depending on the disease, symptom, and the like. Specific examples thereof include injections (for example, local injection, intravenous injection, intramuscular injection, etc.). In the case of an injection, it can be administered by injection into joints, subcutaneously, intradermally, intramuscularly, etc., in addition to normal intravenous administration and intraarterial administration.

The aqueous solvent for the injection can be, for example, distilled water or saline. The non-aqueous solvent for injections can be, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, or polysorbate 80 (Public name). Such formulations also include tonicity agents (eg, sodium chloride, glucose, etc.), preservatives, wetting agents, emulsifiers, dispersants, stabilizers, pH adjusters (eg, sodium citrate, sodium acetate, phosphorus). It may contain a buffer (eg, sodium acetate, etc.), a local anesthetic (eg, prokine hydrochloride, lidocaine hydrochloride, etc.) or a solubilizing agent.
These formulations can be sterilized, for example, by filtration through a bacterial retention filter, formulation of a fungicide, or irradiation. Moreover, the composition obtained by dissolving or suspending a sterile solid composition in sterile water or a solvent for injection before use can also be used as these preparations. These preparations can be produced by a known method usually used in the preparation process.

When the pharmaceutical composition of the present invention is a therapeutic agent, preventive or palliative agent for osteoarthritis of the knee, hip osteoarthritis, elbow arthropathy, etc., it is administered as an injection, especially as a joint lumen injection. It is preferable to do so. In this case, the form of the composition of the present invention may be a powder filler in which it is dissolved at the time of use, or it may be an aqueous solution. Compositions for parenteral administration include water-soluble substance conjugates of curcumins, water, physiological saline, pH regulators, sugars, acids, alkalis, buffers, isotonic agents, stabilizers, and pain-relieving agents. , Preservatives and the like can be blended.
Examples of saccharides include monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols and the like. Examples of the acid and alkali include water-soluble inorganic acids, water-soluble inorganic acid salts, water-soluble organic acids, water-soluble organic acid salts, amino acids, amino acid salts and the like.

The pharmaceutical composition of the present invention can be further combined with a polymer base material such as hyaluronic acid.

For osteoarthritis of the knee, hip osteoarthritis or elbow arthritis, it is used not only for treatment but also for prevention or alleviation of pathological conditions.

Next, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to the specific aspects described below.

Manufacturing example 1
(Preparation method of curcumin water-soluble substance conjugate (curcumin monoglucuronide))
Curcumin monoglucuronide was synthesized according to the method described in WO2018 / 03857. That is, compound (1) (β-D-glucopyranoside uronic acid, 4) was reacted by reacting 1.0 g (2.52 mmol) of acetbromo-α-D-glucuronic acid methyl ester with 326.0 mg (2.15 mmol) of vanillin as raw materials. -Formyl-2-methoxyphenyl, methyl ester, triacetate) 491.6 mg (yield 49%) was obtained. On the other hand, compound (2) (5-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1, 4-Hexadien-3-one) was obtained. Compound (1) and compound (2) were reacted to obtain compound (3) (curcumin β-D-glucopyranoside

uronic acid

2,3,4-tri-O-acetyl, methyl ester). Next, curcumin monoglucuronide (253.1 mg) was obtained by deacetylation and purification of compound (3).

Example 1
(Effect of curcumin monoglucuronide (CMG) on in vivo rat OA model)
(I) Creation of In vivo rat OA model and administration of CMG Medial meniscus destabilization surgery (destabilization of the medical meniscus) to dissect the tibial attachment part of the anterior segment of the medial meniscus of the right knee of a rat (Wistar 12-week-old male) A rat OA model was created that induces OA by DMM surgery (DMM surgery) (Glasson, S. S. et al, The surgical restoration of the mental meniscus (DMM) model of osteoarthritis. 1061-1069, (2007)). Only the skin and joint capsule were incised on the left knee of the same rat, and the sham operation side was used.
The 64 OA model rats prepared were divided into an administration group in which CMG was administered into the knee joint and a control group in which physiological saline was administered into the knee joint. The observation period was 7 days (7D), 14 days (14D), 6 weeks (6W), and 10 weeks (10W) after the operation (n = 8 animals, respectively). Intra-articular administration (CMG 30 mg / ml or saline, 50 μl) was administered to rats with an observation period of 7D after surgery 3 days, 7 days, and 10 days after surgery, and for 14D rats after surgery, 3 days, 7 days, and 10 days after surgery. It was administered intra-articularly on the 14th. In rats with observation periods of 6 W and 10 W after surgery, they were administered intra-articularly weekly from 1 week after surgery to just before sacrifice.

At the end of the observation period, each patient was sacrificed 1 hour after intra-articular administration, and the knee joint was removed. After micro-CT imaging, the knee joint was fixed with PFA, decalcified, and dehydrated for tissue observation, and then embedded in paraffin to prepare a sliced specimen, which was used for the following evaluation.
(1) Histological evaluation SafraninO and Fast green staining (evaluation by staining a section prepared by sagittal section passing through the medial part of the tibial plateau and the center of the patellar cartilage)
(2) OARSI score ((Stateal articular cartilage tissue evaluation by histopathological scoring system by Osteoarthritis Research International): Glasson, S. S. et al, The OARSI histopathological tissue tissue Scoring (0-24) from the Grade and Stage below based on the Safranin On and Fast green stain according to the mouse, J. Osteoarthritis and Tissue, 18, 2010, S17-S23.
<Grade>
0: Normal, no articular cartilage damage 1: No cartilage damage, but fibrosis and cell death of articular cartilage surface 2: Articular cartilage surface damage and irregularity accompanied by deterioration of substrate staining 3: Articular cartilage There is a vertical crack in the cartilage, and the stainability of the substrate is deeply reduced. 4: The superficial articular cartilage is exfoliated. It is depressed to the middle layer of articular cartilage 5: The articular cartilage is deeply damaged and the subchondral bone is exposed 6: The articular cartilage is deeply damaged and the subchondral bone is exposed and bone spines are formed <Stage>
0: No OA findings 1: <10%
2: 10-25%
3: 25-50%
4:> 50%
(3) Modified Mankin (MM) score (evaluation of the severity of articular cartilage injury): The score is calculated by the total value of each of the following items.
<Pericellar Matrix Staining>
0: Normal 1: Partial brightness increases 2: Brightness increases with 40% or more cartilage <Spatial Arrangement of Chondrocytes>
0: Normal 1: Increase in chondrocytes in a wide range 2: Decrease in some chondrocytes 3: Decrease in chondrocytes in a wide range <Interterial Trix Staining>
0: Normal 1: Decreased staining 2: Partial disappearance of staining 3: Disappearance of staining in 40% or more of articular cartilage

(4) Immunohistological evaluation (evaluation of type I and type II collagen, IL1-β, IL6, TNF-α)
Type I collagen: After staining, measure the brightness of the articular cartilage of the medial tibial plateau and patellar cartilage using Image-J. Measure the average brightness of the articular cartilage using Image-J (NIH) IL1-β, IL6, TNF-α: Percentage of positive cells among the cartilage cells in the medial part of the tibial plateau after staining, respectively. (5) Evaluation of subchondral bone cysts and osteophyte by CT imaging Subchondral bone and osteoblast were evaluated from micro-CT images. The number of bone cysts and the diameter of the maximum cyst were measured. For osteophytes, the amount of osteophytes in the medial part of the tibial plateau was calculated using Amira (Thermo Fisher Scientific).

(II) Evaluation Results First, at 7D, 14D, and 6W after surgery, a fluorescent substance presumed to be curcumin could be detected from the articular cartilage.

(Histological evaluation of the medial part of the tibial plateau)
The Safranin O and Fast-green stained image and the OA score are shown in FIG.
Among the stained images, in the CMG administration group (upper row), the OA suppression effect by CMG administration was small in the early postoperative period when inflammation was strong, but the OA score at 10 W was the OARSI score (upper row) and the MMH score (lower row). In any of the scores, the CMG-administered group was significantly smaller, indicating that CMG administration was able to suppress the progression of OA in the chronic phase.
(Immunohistochemical evaluation of the medial part of the tibial plateau)
In addition, from the immunohistological evaluation results shown in FIGS. 2, 3 and 4, the average brightness of type II collagen at 10 W was significantly (p <0.05) in the CMG-administered group and was stained deeply. It can be seen that the degradation of type II collagen by OA is suppressed (Fig. 2). The positive cell expression rate (%) of the inflammatory cytokines IL1-β and IL6 (Fig. 3) and TNF-α (Fig. 4) decreased significantly (p <0.05) at 6 W in the CMG-administered group. , It can be seen that the expression of inflammatory cytokines is suppressed.

(Evaluation of subchondral bone on the medial side of the tibial plateau)
From the micro-CT imaging results (Fig. 5), the number of cysts and the maximum cyst diameter at 10 W were significantly smaller in the CMG-administered group (p <0.01), and the amount of osteophytes at 10 W was also significantly smaller in the CMG-administered group (Fig. 5). It can be seen that the progress of OA can be suppressed at p <0.01).
(Evaluation of patellar cartilage)
The patellar cartilage was evaluated in the same way as the articular cartilage in the medial part of the tibial plateau. The Safranin Oand Fast-green stained image and OA score are shown in FIG. 6, and the immunohistological evaluation results (type I and type II collagen) are shown in FIG. 7. The OARSI score at 10 W (upper part of FIG. 6) was significantly smaller in the CMG-administered group (p <0.01), and the average brightness of type II collagen at 10 W was significantly smaller in the CMG-administered group (p <0.05). , It can be seen that OA can be suppressed.

From the above, it was confirmed that the OA symptoms of both articular cartilage and subchondral bone were improved from 6W to 10W. No significant systemic toxic symptoms were observed in any of the CMG-administered groups, and it was confirmed that the cartilage and subchondral bone were not adversely affected.

Claims

A pharmaceutical composition for parenteral administration for the treatment, prevention or alleviation of osteoarthritis, which comprises a water-soluble substance conjugate of curcumin as an active ingredient.
The pharmaceutical composition for parenteral administration according to claim 1, wherein the curcumins are one or more selected from curcumin and its derivatives.
The pharmaceutical composition for parenteral administration according to claim 2, wherein the curcumin derivative is at least one selected from bisdemethoxycurcumin, demethoxycurcumin and tetrahydrocurcumin.
The pharmaceutical composition for parenteral administration according to any one of claims 1 to 3, wherein the water-soluble substance is at least one selected from glucuronic acid, sulfuric acid, glutathione and amino acids.
The pharmaceutical composition for parenteral administration according to any one of claims 1 to 3, wherein the water-soluble substance is at least one selected from glucuronic acid and sulfuric acid.
The pharmaceutical composition for parenteral administration according to claim 1, wherein the water-soluble substance conjugate of the curcumins is curcumin monoglucuronide.
The pharmaceutical composition for parenteral administration according to any one of claims 1 to 6, wherein the composition is a pharmaceutical composition for parenteral administration for treating or alleviating osteoarthritis.
The pharmaceutical composition for parenteral administration according to any one of claims 1 to 7, wherein the osteoarthritis is osteoarthritis of the knee, osteoarthritis of the hip, or osteoarthritis of the elbow.
Use of a water-soluble substance conjugate of curcumin for the production of a pharmaceutical composition for parenteral administration for the treatment, prevention or alleviation of osteoarthritis.
A water-soluble substance conjugate of curcumins for parenteral administration for the treatment, prevention or alleviation of osteoarthritis.
A method for treating, preventing or alleviating osteoarthritis, which comprises parentally administering an effective amount of a conjugate of a water-soluble substance of curcumins.