JPH0463853B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an anticancer substance sustained release agent.

As a result of repeated studies to obtain an anti-cancer substance sustained-release agent that can effectively act on cancer or tumors for a long period of time, the applicant has determined that both an anti-cancer substance and a blood coagulant are immobilized in a structure. It was discovered that by doing so, it was possible to achieve both sustained release properties and local retention properties, and this was proposed earlier (Japanese Patent Application Laid-open No. 140011/1983). However, subsequent research has revealed the problem that aggregation may occur when the anticancer substance and blood coagulant are coexisting and immobilized depending on the combination used.

In view of this situation, the inventors of the present invention have continued to study in order to solve the above problems, and as a result,
An anticancer substance and a blood coagulant can be immobilized on separate structures, and the structures thus obtained can be stacked on top of each other to form a laminate, or mixed to form a mixture to prevent aggregation during manufacturing. It was discovered that the formulation as a whole maintains excellent sustained release properties and local retention without any problems, and the present invention was achieved based on this finding.

That is, the present invention provides a fiber aggregate, a sponge, characterized by comprising the following steps (a), (b) and (c).
This is a method for producing an anticancer substance sustained release agent comprising a structure (A) in the form of a powder, monofilament, film, microcapsule, etc., an anticancer substance, and a blood coagulant.

(a) A step of immobilizing an anticancer substance on the structure (A) to obtain a structure (B) on which the anticancer substance is immobilized.

(b) A step of immobilizing a blood coagulant on the structure (A) to obtain a structure (C) on which the blood coagulant is immobilized.

(c) A step of combining the obtained structure (B) and structure (C).

In the present invention, examples of materials constituting the structure include cellulose, cellulose derivatives, proteins,
Synthetic polyamino acids, polyesters, polyamides,
Polyolefins, diene polymers, chlorinated polyolefins, N-vinyl compound polymers, aromatic vinyl compound polymers, polypinyl alcohol and its derivatives, unsaturated aldehyde polymers, unsaturated carboxylic acid polymers, Polymers of saturated carboxylic acid esters, polymers of unsaturated carboxylic acid anhydrides,
A polymer of unsaturated nitrile, a polymer of unsaturated carboxylic acid amide, polyether, silicone resin, polyurethane, natural rubber, etc. can be used.
Among these, collagen, gelatin, oxidized cellulose, polyglycolic acid, polylactic acid, glycolic acid-lactic acid copolymer, polyglutamic acid, amylose, succinic acid amylose have the advantage that there is no need to remove them after adaptation. Bioabsorbable substances such as oxidized amylose, such as oxidized amylose, among others, gelatin, amylose, chitin, and oxidized cellulose are preferably used.

The anticancer substance as used in the present invention refers to a substance generally called an anticancer agent, an anticancer agent, or an antitumor agent, and a substance generally called an immunopreparative or an immunostimulant. Examples of the former substance include, for example. Nitrogen mustard, nitromine, chlorambucil,
cyclofusphamide, melphalan, uracil mustard, mannomustine, dopan,
BCNU, triethylenemelamine, thio-TEPA,
Aza-TEPA, Trenimon, Isoprocyone,
Alkylating agents such as busulfan, dimethylmyleran, piposulfan, ethoglucide, epoxypropidine, epoxypiperazine, hexamethylmelamine, dibromomannitol, pipobroman, folic acid, aminopterin, methotrexate,
Antimetabolites such as guanine, 8-azaganine, 6-mercaptopurine, azathioprine, uracil, 5-fluorouracil, cytarabine, azaserine, diazomycin, actinomycin D, sacromycin, mitomycin C, daunomycin, bleomycin, chromomycin, cardinomycin Antibiotics such as filinadreamycin, 5-
Synthetic agents such as HP and IQ-1, plant ingredients such as thiotebacyclophosphamide, doxorubicin, daunorubicin, and neocarcinostane, Hg-hematoporphyrin, Co-protoporphyrin, stilbestrol, hydroxyurea, procarbazine, Methylglyoxal-bis-guanylhydrazone, L-asparaginase, etc. may be used alone or in combination of two or more, but one type of alkylating agent and one type of antimetabolite are used. It is common to select one type of antibiotics and use them in combination, and a combination of endoxan, 5-fluorouracil, mitomycin, or bleomycin is particularly common. Examples of the latter substances include chimic hormone and its related substances, BCG, cell wall skeleton and its methanol-insoluble fraction, bacteria and bacterial components such as Corynebacterium parvum and OK-432, picibanil, reentinan,
SPG, polysaccharides such as mannan, levan, glucan, muramyldipept and its derivatives, levamisole, bestatin, isoprinosine,
Vaccines such as NPT15392, azimecline, transfer factor, lymphokine, immunoPNA, interferon and its inducer, and Maruyama vaccine are listed, and each of these may be used alone or in combination of two or more.
It is generally used in combination with the above-mentioned anticancer agents or substances called anticancer agents or antitumor agents.

The blood coagulant used in the present invention includes, for example, blood coagulation factor factor, factor factor, factor factor, factor factor, factor factor, factor factor, factor factor, factor factor, factor XI, factor XII, and Examples include factor factor, brekaliklene, high-molecular-weight kininogen, and thrombin. These can be used alone or in combination of two or more. In the present invention, blood coagulation factor (hereinafter abbreviated as F) and thrombin are preferably used, and a combination of F and thrombin is particularly preferably used. F is called a fibrin stabilizing factor, and is a factor that promotes the production of stabilized fibrin through in-peptide bonds between fibrin molecules. F is isolated from blood or placenta of humans, cows, etc., but when used in humans, it is preferable to use human-derived F. Thrombin is a proteolytic enzyme that can convert fibrinogen to fibrin. Thrombin is isolated from blood of humans, cows, pigs, etc., but when used in humans, it is preferable to use human thrombin.

The anticancer substance and blood coagulant used in the present invention are:
to be bonded to or adsorbed to the structure;
Alternatively, it can be immobilized by encapsulating it. To attach anti-cancer substances or blood clotting agents to structures, see for example O. Zaborsky, “Immobilized
Conventionally known covalent bonding methods and ionic bonding methods such as those described in "Enzymes" CRC Press. 1973 can be used, and for adsorption,
Similarly, a physical adsorption method or an encapsulation method can be adopted, and for encapsulation, a method of microencapsulation using a known microencapsulation method using the material of the structure as an outer wall can be adopted. can.

In the present invention, an anticancer substance or a blood coagulant can be bound to a structure, for example, in the following manner. In other words, anti-cancer substances and blood clotting agents,
When it has a functional group capable of forming a covalent bond or an ionic bond, such as an amino group or a carboxyl group,
By treating a structure having a functional group capable of covalently or ionically bonding with these functional groups with a solution containing this, it is possible to perform immobilization through the desired bond. In addition, in this case, if the structure has no or only a few functional groups that can covalently engage or ionicly bond with the functional groups of the anticancer substance or blood coagulant, the structure should be prepared in advance with those functional groups. After introducing the functional groups by chemical reaction, anti-cancer substances or blood clotting agents can be attached to the structure. If the anti-cancer substance or blood coagulant does not have a functional group, it is possible to use it after introducing a functional group through a chemical reaction as in the case described above, but in many cases this is not possible. This method is not preferably employed because such a chemical reaction would impair the properties of the anticancer substance or blood coagulant as a drug. In addition, when covalently bonding,
It is preferable to use a dehydration condensation agent such as dicyclohexylcarbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-meth-p-toluenesulfonate.

Further, in the present invention, an anticancer substance or a blood coagulant can be adsorbed onto a structure by a physical adsorption method, an entrapment method, or the like as follows. That is, the anticancer substance or blood coagulant is separately dissolved or suspended in a solvent that can wet the structure, and the structure is treated with this solution to physically adsorb the anticancer substance or blood coagulant. I can do it. The entrapment method is a method in which an anticancer substance or a blood coagulant is encapsulated in a fine gel lattice so that it cannot be released. This adsorption method and entrapment method are effective for any combination of anticancer substances, blood coagulants, and structures, and are convenient and less likely to impair the properties of drugs, so they are preferably adopted in the present invention. be done.

In the present invention, in addition to the method of binding or adsorbing an anticancer substance or a blood coagulant to a structure as described above, the anticancer substance or a blood coagulant is first added to the material itself before being processed into a structure. It is also possible to bind or adsorb the anti-cancer substance or the blood coagulant and then process the material into a structure.

In order to produce an anticancer substance sustained release agent by the method of the present invention, the anticancer substance immobilized structure obtained by any of the above methods and the blood coagulant immobilized structure are then combined. All you have to do is combine them. For example, if the shape of the structure is sponge, film, woven fabric, or knitted fabric,
In the case of fiber aggregates such as nonwoven fabrics and paper, it is preferable to laminate the two, and in the case of a monofilament structure, it is preferable to mix them. Further, when the structure is in the form of a flocculent fiber aggregate, microcapsule, or powder, either lamination or mixing is possible, but lamination is preferable.

When producing the sustained-release agent according to the present invention, in addition to anticancer substances and blood coagulation agents, protease inhibitors such as antiplasmin, albumin, and _α2 -macroglobulin, ceruloplasmin, haptoglobin, and cold insoluble globulin are used. Blood proteins, fibronectin, antibiotics, etc. can be immobilized on the structure. Antiplasmin is an inhibitor of plasmin, which is a fibrinolytic enzyme, and therefore exerts its effect by inhibiting plasmin. In the present invention, antiplasmin includes, for example, ε-
Aminocaproic acid, tranexamic acid, etc. are preferably used.

The sustained release agent produced by the method of the present invention is preferably used for the treatment of cancer or tumors, and is particularly preferably used for vascular occlusion methods, puncture methods, and the like.

The sustained-release agent produced by the method of the present invention can be applied to vascular occlusion therapy by, for example, passing the tip of a vascular catheter through a blood vessel to the feeding artery to the target cancer or tumor tissue, and applying the embolic agent from the other end with physiological saline. This is done by injecting a suspension in water or the like.
At this time, a portion of the sustained-release agent reaches the target tissue and its neighboring tissues, adheres to the target site, and remains there, and a portion remains inside the feeding blood vessel, quickly occluding the blood vessel. Also, occluded blood vessels do not recanalize. Furthermore, the anti-cancer substance is sustainedly released from the sustained-release agent that remains on the target tissue, its neighboring tissues, and the vascular occlusion, and acts locally on the cancer or tumor tissue for a long time, accelerating and ensuring necrosis. Make it. This means that
In other words, when using the sustained-release agent manufactured according to the present invention, it is possible to perform vasoocclusive therapy quickly and accurately, and chemotherapy, which could not be performed in parallel in the past, can be performed in parallel. It means that it is possible to do it.

The sustained-release agent produced by the method of the present invention can be applied to a puncture method by, for example, suspending the sustained-release agent in physiological saline or the like through a puncture needle that reaches the target cancer or tumor tissue. After injecting the required amount of something,
Further, the needle is removed while gradually injecting this suspension. This allows the sustained release agent to remain attached to the target tissue and to its site distributed over the tissue near the path of the puncture needle. The sustained-release agent that remains on the target tissue immediately stops bleeding, repairs damaged blood vessels, and releases anti-cancer substances in a sustained manner to effectively act on cancer or tumor tissue for a long period of time. On the other hand, the sustained-release agent that is injected at the time of needle removal, dispersed on the tissue near the puncture needle route, and remains unadhered will immediately suppress bleeding from the injured site of the nearby blood vessel and repair it, and It acts effectively for a long time on cancer or tumor tissue that has been dispersed.

As described above, according to the present invention, when an anticancer substance and a blood coagulant are immobilized, their aggregation does not occur, so a sustained release agent with high activity can be easily produced. . Therefore, the sustained-release agent obtained in this way is preferably used in procedures such as vascular occlusion therapy and puncture, and combines excellent performance as an embolic agent and performance as a sustained-release agent for anticancer substances. It is something that we have. This sustained release agent also has applications in the direct spray method. In other words, even if conventional sustained-release anti-cancer agents are administered locally by being sprayed onto cancerous tissues exposed through incisional surgery, the sustained-release agents are dispersed due to blood, body fluids, etc. It is difficult to expect an effect because it washes off from the local area immediately after administration, but if this sustained-release agent is administered in the same way, it will not stick to the local area immediately and will not wash off.

The present invention will be explained in more detail with reference to Examples below.

Example 1 10 mg of powdered Xelform (absorbable powdered gelatin, manufactured by Nippon Upjiyon) was dissolved in 80 ml of water with 1 bottle of fibrogamin aqueous solution [1 bottle of concentrated dry preparation of human F (manufactured by Hoechst). ] 1 ml and physiological saline solution of thrombin [1 bottle of concentrated dry preparation of human thrombin (Midori Juji) dissolved in 50 ml of physiological saline. ] After immersing in 1 ml of the mixed solution for 2 minutes at 0°C, freeze-dry at -60°C for 15 hours.
An immobilized powder Zelform was obtained in which thrombin and thrombin were immobilized.

On the other hand, 10 mg of the same powdered Zelform used earlier was immersed in 2 ml of Adriacin (adriamycin, manufactured by Kyowa Hakko Co., Ltd.) aqueous solution [10 ml (potency) dissolved in 2 ml of water] at 0°C for 2 minutes. Immobilized powder Zelform on which adriacin was immobilized was obtained in the same manner as in the above case.

The two types of immobilized powder Zelform obtained were superimposed to obtain a laminate.

Further, two types of immobilized powder Zelform were obtained in the same manner as above, and both were uniformly mixed to obtain a mixture.

On the other hand, a loop was made using a 34 cm Siriyun medical tube with an inner diameter of 4 mm, and in a room at 2°C, 2 ml of ACD-preserved blood was added to a 10 wt% aqueous solution of _CaCl2 .
ml and then add the laminate prepared earlier into the tube and rotate it at 16 revolutions/min on a rotary plate with an inclination of 23 degrees. One minute after the start of rotation, formation of a clot was observed in the blood.
At this point, the rotation is stopped, and one hour after stopping, a paper disk (manufactured by Toyo Seisakusho, antibiotic test paper disk, diameter 8 mm) is fully immersed in the blood or clot in the loop, and this is used as a sample for testing. Bacillus Subtilis as a bacterium
Using ATCC6633, the size of the inhibition circle generated by the cylindrical plate method was determined, and the blood concentration of adriacin at 1 hour was determined from the size of this inhibition circle, which was 20μ.
g/mg. In the same way, the blood concentrations at the 5th, 10th, 24th, 1.5th, 2nd, and 3rd days were determined to be 31μg/mg and 44μ, respectively.
g/mg, 82 μg/mg, 168 μg/mg, 231 μg/mg,
It was 401 μg/mg.

When similar tests were conducted on mixtures,
Blood clot formation was observed 1 minute after the start of rotation, and the blood concentration of adriacin was 19 μg/mg after 1 hour.
29 μg/mg, 39 μg/mg, and
76μg/mg, 154μg/mg, 211μg/mg, 386μg/mg
It was mg.

Comparative Example 1 When the same aqueous solution of F used in Example 1, physiological saline solution of thrombin, and aqueous solution of Adriacin were mixed, an aggregate was formed, so this was used to immobilize it on powdered Zelform. Although attempts were made, uniform immobilization was difficult.

Comparative Examples 2 and 3 Immobilized powder Zelform in which F and thrombin were immobilized was obtained in the same manner as in Example 1. When this product was immersed in the same adriacin aqueous solution as in Example 1, aggregates were formed, although the amount was milder than in Comparative Example 1. In addition, adriacin-immobilized powder Zelform obtained in the same manner as in Example 1 was The same result was obtained when the sample was immersed in the same mixed aqueous solution of F and thrombin as in Example 1.

Example 2 201 mg of Oxycell cotton (oxidized cellulose cotton type manufactured by Absorption Co., Ltd., Sankyo Co., Ltd.) was immersed in 4 ml of a 0.4N calcium acetate aqueous solution at room temperature for 30 minutes, then taken out, washed with water, and air-dried.

Oxycel cotton on which F was immobilized and Oxycel cotton on which adriacin was immobilized were obtained in exactly the same manner as in Example 2, except that this Oxycel cotton was used instead of powdered Zelfoam and thrombin was not used. . Both were mixed uniformly to obtain a mixture.

Using the obtained mixture, a blood coagulation test using a rotating loop was conducted in the same manner as in Example 1, and a clot was formed 1 minute and 20 seconds after the start of rotation, and the fluidity of blood in the tube was lost. Also, 1st hour, 5th hour, 10th hour
The concentration of adriacin in the blood at 24 hours, 1.5 days, 2 days and 30 days was
24μg/mg, 38μg/mg, 49μg/mg, 89μg/mg,
They were 162 μg/mg, 220 μg/mg, and 391 μg/mg.

Example 3 Three pieces of sponzel (gelatin sponge for hemostasis, manufactured by Yamanouchi Pharmaceutical Co., Ltd., 5 x 2.5 cm) were prepared, one of them was added to 4 ml of a fibrogamine aqueous solution (1 bottle dissolved in 16 ml of water), and the other Add one piece to 5 ml of thrombin physiological saline solution (1 bottle dissolved in 25 ml of physiological saline) and the remaining one to 15 ml of adriacin aqueous solution (400 mg (potency) dissolved in 15 ml of water) at room temperature. After immersing them for 3 minutes, semi-freeze each of the three pieces separately at -30°C, place the fibrogamin-immobilized sponzel on the top row, and the adriacin-immobilized sponzel on the middle row.
Sponzel with immobilized thrombin was stacked on the bottom layer, and this was freeze-dried at -60℃ for 15 minutes.
A laminate was obtained over time. A 0.5 cm square piece was cut perpendicular to the laminated direction from this laminate, and a blood clot test was performed using the same rotating loop as in Example 1. A blood clot was formed within 1 minute after the start of rotation. It was done. Also, the 1st hour, 5th hour,
At the 10th hour, 24th hour, 1.5th day, 2nd day, and 30th day, the concentration of adriacin in the blood was
20μg/mg, 27μg/mg, 40μg/mg, 78μg/mg,
They were 154 μg/mg, 218 μg/mg, and 389 μg/mg.

Claims (1)

[Scope of Claims] 1. A structure having the shape of a fiber aggregate, sponge, powder, monofilament, film, microcapsule, etc., characterized by comprising the following steps (a), (b), and (c): A method for producing an anticancer substance sustained release agent comprising (A), an anticancer substance, and a blood coagulant. (a) A step of immobilizing an anticancer substance on the structure (A) to obtain a structure (B) on which the anticancer substance is immobilized. (b) A step of immobilizing a blood coagulant on the structure (A) to obtain a structure (C) on which the blood coagulant is immobilized. (c) A step of combining the obtained structure (B) and structure (C).