JP4796758B2 - Composition and antitumor agent containing the same - Google Patents

Description

  The present invention relates to a composition having an ability to suppress the growth of tumor cells and an antitumor agent comprising the same.

  Tumor mortality accounts for the highest proportion of current Japanese causes of death. Therefore, studies on antitumor agents have been continued using various raw materials. However, since tumor cells are cells originally transformed by self cells, drugs having a strong medicinal effect on tumor cells usually have strong side effects on normal cells.

In recent years, by using animal-derived materials, development of drugs with few side effects and a high growth inhibitory effect on tumor cells has been promoted. For example, research on tumor cell growth inhibition by sulfated polysaccharides such as heparin has been conducted. According to this, it has become clear that heparin has the ability to suppress tumor cell growth (see, for example, Patent Document 1 and Non-Patent Document 1).
JP-A-63-88128 ANTACHOPOULOS CT, ILIOPOULOS DC, GAGOS S, AGAPITOS MV, KARAYANNACOS PE, ROBOLI SK, SKALKEAS GD, (Athens Univ.School of Medicine, Athens, GRC) "In vitro Effects of Heparin on SW480 Tumor Cell-Matrix Interaction." Anticancer Res ., VOL. 15 NO. 4 P1411-1416 (1995 / 07-1995 / 08)

  However, when a large amount of heparin is administered to the human body, there is a problem that it involves the risk of bleeding complications, which are serious complications. This is because heparin acts on both the intrinsic and extrinsic systems of the blood coagulation system.

  Heparin is extracted and purified from the lungs and intestines of cattle, pigs, lambs and the like. As the use of heparin derived from bovine organs has been prohibited since the epidemic of mad cow disease, heparin extracted from animals cannot basically completely eliminate any contamination of viruses or prions. Moreover, it is expensive to extract heparin from a living body, and the heparin itself becomes expensive.

  In view of such circumstances, an object of the present invention is to provide a safe and inexpensive composition having a tumor cell growth inhibitory ability and an antitumor agent comprising the same.

  The present invention has the following configuration.

[1] A composition having a tumor cell growth-inhibiting ability, comprising a sulfated polysaccharide comprising glucose, glucuronic acid, and rhamnose, wherein one or more hydroxyl groups are sulfated. .
[2] The composition according to [1], wherein the sulfated polysaccharide is constituted by repeating a structural unit consisting of two molecules of glucose, one molecule of glucuronic acid, and one molecule of rhamnose.
[3] The composition according to [2], wherein the structural unit is a unit represented by the following formula (1).
(In the formula, each R is independently selected OH or OSO ₃ H.)
[4] A composition having tumor cell growth-inhibiting ability, comprising gellan in which one or more hydroxyl groups are sulfated.
[5] An antitumor agent comprising the composition according to any one of [1] to [4].
[6] The antitumor agent according to [4], which is formulated for intravenous administration, parenteral administration or oral administration.

  According to the present invention, it has become possible to provide a safe and inexpensive composition having the ability to suppress the growth of tumor cells and an antitumor agent comprising the same.

  Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Sulphated polysaccharide and its production method ==
The composition according to the present invention contains a sulfated polysaccharide composed of glucose, glucuronic acid, and rhamnose and having one or more hydroxyl groups sulfated. The ratio and binding order of glucose, glucuronic acid, and rhamnose constituting the sulfated polysaccharide skeleton are not particularly limited, but 4 molecules of 2 molecules of glucose, 1 molecule of glucuronic acid, and 1 molecule of rhamnose are structural units. In this case, the ratio of the number of constituent molecules is 2: 1: 1.

In particular, the repeating unit is more preferably the following formula (1).

  The degree of sulfation of this polysaccharide, that is, the sulfate group substitution rate of the sulfatable hydroxyl groups is preferably 8 to 90%, more preferably 30 to 80%. Furthermore, the average molecular weight of the polysaccharide after sulfation is preferably in the range of 1 to 1000 KDa, more preferably 1 to 300 KDa.

  The sulfated polysaccharide contained in the composition of the present invention may be chemically synthesized, or may be a fermentation product of a microorganism present in nature or an extract from seaweed, and its origin is not particularly limited.

  In the case of chemical synthesis, the polysaccharide having a hydroxyl group as a raw material may be either chemically synthesized or an extract from microorganisms or seaweed that exist in nature. When the raw material is a polymer, it may be used for the reaction after its molecular weight has been lowered by hydrolysis with an acid such as hydrochloric acid, sulfuric acid or trifluoroacetic acid or an alkali such as sodium hydroxide.

  An example of a raw material existing in nature is gellan (gellan CAS 71010-52-1) obtained by purifying a polysaccharide produced by Pseudomonas elodea after deacylation. Gellan is a polysaccharide composed of glucose, rhamnose and uronic acid, and it can be obtained in large quantities at a low cost, so it can be preferably used as a raw material. Also, saccharides produced by microorganisms, such as welan produced by the genus Alcaligenes (US Pat. No. 4,342,866) or rhamsan (US Pat. No. 4,401,760). It is done.

As a method for sulfating these raw materials, a generally known method can be used. For example, chlorosulfonic acid is allowed to act on polysaccharides in dimethylformamide as in the method of Keiichi Miyamoto et al. (International Journal of Biological Macromolecules 28 (2001) p.381-385). It can be prepared by a method in which a DMF / -SO ₃ complex is allowed to act on a polysaccharide in dimethylformamide (DMF), such as a method or a method by Kenji Kamide. As another similar method, a method in which an anhydrous sulfuric acid complex such as a dioxane-SO ₃ complex, a trimethylamine-SO ₃ complex, or a pyridine-SO ₃ complex is allowed to act on a polysaccharide can be used.

  The average molecular weight of the sulfated polysaccharide used in the present invention is obtained by using gel filtration chromatography by HPLC, 0.2M-NaCl as an eluent, and pullulan (Shodex STANDARD P-82) having a known molecular weight as a molecular weight standard. It can be measured by a differential refractive index detector.

== Composition and antitumor agent having tumor cell growth inhibitory ability ==
The sulfated polysaccharide can constitute a composition having tumor cell growth inhibitory ability. The composition may contain a pharmaceutically acceptable carrier.

  This composition can be converted to a pharmaceutical dosage form in a manner similar to that commonly used for heparin. That is, the composition may be orally administered in the form of emulsions, tablets, capsules, granules, powders, syrups and the like according to a conventional method, or dissolved in a buffer or physiological saline, and injected. Inject subcutaneously, intramuscularly, intraperitoneally or intravenously into a formulation, or administer rectally as a suppository (suppository), or spray into the oral cavity or respiratory tract mucosa as a formulation such as a spray. Parenteral administration in such a manner that it is administered to the mucosa, or is transdermally or mucosally administered by applying or pasting it to the affected area (for example, skin or mucous membrane) as a preparation such as an ointment or a tape. You can also

  The composition thus configured has the ability to suppress the growth of tumor cells as shown in the following examples. Therefore, when this composition is formulated, it can be used as an antitumor agent.

  The type of tumor to be targeted is not particularly limited, but can be particularly preferably applied to lung cancer cells, colon cancer cells, osteosarcoma, melanoma, and the like.

  Hereinafter, the present invention will be described in detail with reference to examples.

Definitions of terms used in the examples and measurement methods are as follows.
1) Average molecular weight (KDa) measurement: The polysaccharide was dissolved in 0.2 M NaCl aqueous solution at a concentration of 1.0 mg / ml, and subjected to gel filtration by HPLC. As the HPLC column, Shodex Ionpak KS-804 and KS-G were used, and the eluate using 0.2M NaCl aqueous solution as an eluent was detected by a differential refractive index detector. A calibration curve of elution time and molecular weight was prepared in advance using a pullulan (Shodex STANDARD P-82) having a known molecular weight separately measured, and the average molecular weight of the polysaccharide was determined by applying the calibration curve to the calibration curve.
2) Sulfate group substitution rate (%): The percentage of hydroxyl groups that were sulfated out of the hydroxyl groups per polysaccharide constituting the polysaccharide was expressed as a percentage. In this measurement method, the total amount of S in the polysaccharide is measured by elemental analysis by ICP, and the amount of free S released from the polysaccharide is measured by ion chromatography. The sulfate group substitution rate was calculated from the combined S amount obtained by subtracting the free S amount from the total S amount.

Example 1 Synthesis of Sulfated Gellan 2.0 g of gellan (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 200 ml of a 0.5 M aqueous solution of trifluoroacetic acid and reacted at 80 ° C. for 30 minutes for hydrolysis to lower the molecular weight. 1.0 g of the resulting low molecular weight gellan was added to 100 ml of dehydrated pyridine in nitrogen gas, 6.4 g of pyridine sulfur trioxide complex was added, and the mixture was reacted at 115 ° C. for 5 hours. After completion of the reaction, pyridine was distilled off. After dissolving in 10 ml of water, 2 volumes of ethanol was added to precipitate the reaction, which was collected by filtration. The collected precipitate was dissolved in 10 ml of water, neutralized with 1N NaOH, precipitated again with 2 volumes of ethanol and collected. This precipitation / recovery was repeated three times and dried for one day by drying at 50 ° C. under reduced pressure to obtain a sulfated gellan powder.

<Example 2> Measurement of tumor cell growth inhibitory activity in vivo (using sulfated gellan GS46)
Sulphated gellan (average molecular weight 27.7 KDa, sulfate substitution rate 69.69) synthesized in Example 1 and dissolved in 100 μl of HBSS in each tail vein of 7 mice (C57BL / 6 ／, 6 weeks old, the same applies hereinafter). 1%) 1 mg was injected. As a negative control, another 7 mice were injected with 100 μl of HBSS, and as another positive mouse, another 7 mice were injected with 1 mg of heparin (molecular weight 10 KDa) dissolved in 100 μl of HBSS. Ten minutes later, 2 × 10 ⁵ Lewis lung cancer-derived highly metastatic H11 cells suspended in 100 μl of HBSS were injected into the tail vein of each mouse. Sixteen days later, each mouse was sacrificed and the lung was removed and fixed overnight with Buan Dubosk fixative.

  When each fixed lung was washed with 70% ethanol and wet weight was measured, all seven mice injected with sulfated gellan showed marked growth inhibition of tumor cells (see FIGS. 1 and 2). The appearance of the tumor was also confirmed to suppress the growth of tumor cells.

<Example 3> Measurement of tumor cell growth inhibitory activity in vivo (using sulfated gellan GS29)
In Example 2, the suppression of tumor cell growth was examined in the same manner except that sulfated gellan (molecular weight 9.6 KDa, sulfate group substitution rate 38.6%) having a lower sulfate group substitution rate was used. 2 out of 7 mice injected with chlorinated gellan showed significant tumor cell growth inhibition (see FIGS. 1 and 2), but the overall inhibitory effect was milder than GS46.

<Example 4> Measurement of tumor cell growth inhibitory activity in vivo (using sulfated gellan GS12)
In Example 2, when the sulfated gellan having the lowest sulfate group substitution rate (molecular weight 197.7 KDa, sulfate group substitution rate 17.5%) was used, inhibition of tumor cell proliferation was examined by the same method. The mice also showed milder inhibition of tumor cell growth (see FIGS. 1 and 2).

  Thus, since the composition of the present invention has the ability to suppress the growth of tumor cells, it can be used as an antitumor agent. Moreover, since the inhibitory ability depends on the sulfate group substitution rate, it is considered to be dose-dependent, and this composition is expected to be easy to use as a drug. In addition, since the suppression ability is independent of the molecular weight, it is considered that this composition can be easily formulated and can take various forms.

In the Example of this invention, it is the observation photograph of the lung which compared the tumor cell growth inhibitory ability. In the Example of this invention, it is the graph showing the measured value of the lung wet weight which compared the tumor cell growth inhibitory ability.

Claims (2)

An antitumor agent having an ability to suppress the growth of tumor cells, comprising gellan in which one or more hydroxyl groups are sulfated,
An antitumor agent, which is a dosage form administered to a living body. The antitumor agent according to claim 1, which is formulated for intravenous administration, intestinal administration or oral administration.