JPH01211530A - Antitumor agent - Google Patents

Abstract

PURPOSE:To obtain an antitumor agent having high affinity to cancer cell and remaining in cancer tissue for a long period to keep the antitumor activity, by bonding an antitumor substance to a human-originated (modified) albumin and using the obtained compound as an active component. CONSTITUTION:A human-originated albumin or a modified albumin produced by the partial acetylation or succinylation of albumin is bonded with an antitumor substance directly or through a spacer such as dextran or polyglutamic acid. When the antitumor substance has e.g., a free amino group, said amino group is condensed with the amino group of albumin using succinimidyl pyridyl dithiopropionate, etc., or with the carboxyl group of albumin using carbodiimide.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to an antitumor agent, and more particularly, to an antitumor agent that has high affinity for cancer cells, remains in cancer tissues for a long period of time, and has an antitumor effect. The present invention relates to a novel antitumor agent with sustained activity.

[Conventional technology and its problems]

Today, various anti-cancer substances are used for the treatment of cancer. For example, antibiotics include neocarzinostatin (NC8), pleomycin, adriamycin,
Mitomycin, etc. are widely used in clinical practice, anti-metabolites such as sFU, ftorafur, cytosine arabinoside, 6-mercazotoprine, etc., and alcohol-alcoholizing agents, such as ACNU, cyclophosphamide, melphalan, etc., are widely used clinically.

However, all of these antitumor substances have the drawback of high toxicity, and in recent years many studies have been conducted to improve this drawback.

For example, so-called missile therapy, in which these anti-tumor substances are conjugated to monoclonal antibodies against cancer-related antigens and then administered, has been actively attempted in order to concentrate drugs only at cancer sites. However, these monoclonal antibodies have only been obtained against specific cancer-specific antigens, and none that can be used against all cancers have yet been obtained.

Furthermore, since most of the drugs are obtained by immunizing foreign animals, they have not been put to practical use because they may cause side effects such as anaphylaxis when administered to humans. For this reason, attempts have been made to use human-derived tanno-Q substance as a drug carrier. A known method for this is to use albumin as microspheres and wrap the drug in them, but since albumin has been denatured by Calo's fever, etc., it is quickly captured by the reticuloendothelial system. In reality, it has been ineffective because it has the disadvantage of causing problems.

[Means to solve the problem]

The present inventors conducted intensive research to improve the drawbacks of these antitumor substances and monoclonal antibody-bound antitumor agents, and found that albumin, which is contained in large amounts in human serum, has an anti-malignant effect.
A! Similar to immunoglobulins such as monoclonal antibodies, compounds chemically bound to I substances are more abundant at tumor sites than antitumor substances alone.

The present invention was completed based on the discovery that high antitumor activity can be obtained by remaining for a long period of time.

That is, the present invention provides anti-IId! containing as an active ingredient a compound obtained by binding an antitumor substance to human-derived albumin or a modified product thereof. 4J4 agent.

Human albumin used as a raw material in the present invention is a protein with a molecular weight of 69,000, and can be easily isolated and purified in large quantities from human serum by conventional methods. There is no need to worry about anaphylaxis, etc.

Furthermore, modified albumin that retains the properties of albumin, such as albumin that is partially acetylated or succinylated, can also be used as a raw material in the present invention.

Anti-cancer substances that bind include antibiotics such as neocarzinostatin, adriamycin, aclacinomycin, chromomycin A3, pleomycin, and pezoreomycin, and metabolizers such as 6-mercazotoprine, cytosine arabinoside, cyclocytidine, and ftorafur. Antagonist, Nisugyunon, Melphalan, Chlorambutyl, ACNU
Examples include alkali-forming agents such as In particular, neocarzinostatin has a chemical structure in which it has a tangy moiety and a low-molecular chromophore of the active substance in its molecule, so even when it binds to a polymeric substance such as albumin, it easily binds only the chromophore of the active substance. It is most preferable for binding to albumin because it can easily enter cells, the reaction is easy, and the activity does not decrease during the reaction.

The bond between albumin and the antitumor substance is achieved by chemically bonding the amino group or carboxyl group of albumin and the amino group, carboxyl group, or aldehyde group of the antitumor substance directly or via a spacer such as dextran or -riglutamic acid. It is done by doing. Specifically, for example, there are the following methods. That is, for example, when the antitumor substance has a free amino group, the amino group and the amino group of albumin are condensed with succinimisolbilizorzothiolobionate (5PDP) or the chalcomyl group of albumin is condensed with the amino group of albumin. can be condensed with carbodiimide. Furthermore, when the antitumor substance has a carboxyl group, it can be condensed with the amide 7 group of albumin. Examples of antitumor substances that can be bound to albumin by the above method include neocarzinostatin, adriamycin, ACNC, melphalan, and chlorambutyl. In addition, when the antitumor substance has sugar as a constituent component, the adjoining hydroxyl groups in the sugar moiety are cleaved using sodium metaperiodate to generate an aldehyde group, and then the amino group of albumin is cleaved to form an aldehyde group. A Schiff pace can be formed in between and combined. Antitumor substances that can be bound to albumin in this way include, for example, pmomycin Ah, aclacinomycin,
Examples include adriamycin, cytarabine, and ftorafur.

The molar ratio of albumin and anti-111 tumor substance for binding is not limited to % and can be adjusted as appropriate by considering the type of anti-tumor substance used, condensing agent, etc., reaction-1 reaction time, etc. .

After the reaction, a compound obtained by binding albumin and an antitumor substance (hereinafter referred to as "albumin-binding antitumor agent") is further purified by known means such as chromatography.

The alpine-conjugated anti-vaginal ulcer agent obtained by pasting is made into a paper and stiffened by a known method. For example, physiological saline, buffer solution,
Dilute with a suitable solvent such as a stabilizer, suspending agent, or non-aqueous solvent,
Can be used.

The anti-ms agent of the present invention can be administered intravenously, kinetically, or peripatrically. Although the dosage varies depending on the type and degree of cancer and the bound antitumor substance, a range of 219-s (1*/Kp) per day for adults is appropriate.

[Action and effect of the invention]

The alpine-binding antitumor agent, which is the active ingredient of the antitumor agent of the present invention, has lower toxicity and higher antitumor action than the original antitumor substance. Therefore, the antitumor agent of the present invention can be administered frequently and is an effective drug for treating various types of human cancer, cancer, leukemia, etc.

〔Example〕

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

Example 1 Production of albumin-bound NC8: 100 μl of albumin was dissolved in 10 μg of phosphate buffer (pH 7.5) containing 0.1 M NaCl, and N-succinimisol-3-(2-birisoldithio)propionate ( A solution of 5Pi)P)1,8IIg in ethanol is added, and the mixture is stirred at 25°C for 30 minutes. After the reaction, Sephadex G equilibrated with the same buffer solution.
-25 (250 m) column and eluted with the same buffer.
I got it. In addition, NC8400 was reacted with 5PDP2&4 in the same manner as with albumin described above to obtain NC8-bilisorzothiothioturopionate 40-. Further, this solution was adjusted to -45 using lN-I (Ci, and then 10
Add 0 mM zothiothreitol aqueous solution 4-, stir at room temperature for 30 minutes in the dark, and then add 0.1M acetate buffer (pH 45).
) through a Sephadex G-25 column equilibrated with
NC8-mercaptopropionate 70WIl was obtained.

Next, alpmin-birisorzothioglopionate 3
Add NC8-mercaptopropionate 15- to 0-, add 1N-NaOH to 7.2, and heat at 25°C after i14 period.
Stir under protection from light for 20 hours. After stirring, the reaction solution was concentrated to 7 by ultrafiltration, and Sephacryl S was equilibrated with 0.01 M IJ acid buffered saline (m7.2) in advance.
-200 column (500 m) and eluted with the same buffer to obtain a solution 8-(albumin-NC8 conjugate amount: 96 mg) of aldimine-NC8 conjugate.

Example 2 Preparation of albumin-bound cytosine arabinolad: 40 kg of cytosine arabinolad was added to 5 ml of 0.01 M
7
decompose the um. This solution is 0.01 M containing albumin 50m+9! In addition to J acid buffered saline 5-, adjust the - to 9.5 with 1N-NaOH, and then stir at room temperature for 1 hour. Add sodium borohydride to this reaction solution! 5
19 was added, and after reacting at room temperature for 2 hours, 0.01
M IJ was added to a Sephacryl S-200 column (s 00tRt) equilibrated with phosphate buffered saline to remove unreacted cytosine arabinoside, and a bath containing 48 μm of albumin-cytosine arabinoside complex was added. I got fi4go.

Example 3 Preparation of albumin-bound chlorambutyl: 2089 chlorambutil was dissolved in DMF 0.5-, water-soluble carbodiimide 10M4I was added, and after stirring for 10 minutes, 0,0I M phosphate buffered saline (m7) containing 501f albumin was dissolved. Add .2) and react at room temperature for 24 hours. Immediately after the reaction, a Sephacryl S-200 column (s
o oIIIt), unreacted chlorambutyl was removed, and alpumi/-chlorambutyl complex 36m19
A bath liquid 4- containing the following was obtained.

Example 4 Anti-inflammatory effect on cultured cancer cells: Each cultured cancer cell was placed in one well of a 96-well grate at I x 10
4 cells/100 μj of medium were added, and at the same time, 100 μl of albumin-bound NC8 (obtained in Example 1) and NC8 diluted medium were added. After culturing at 37° C. for 48 hours, a portion of the cells was collected, and their viability was determined by staining with IJ and Q Nplu to determine s ”CI!@. The results are shown in the table below.

Table 1 *Displayed as NC8 activity.

Example 5 Antitumor effect on mouse transplanted cancer: The effect of albumin-bound NC8 (obtained in Example 1) on Ehrlichi ascites cancer and Meth-Abkifi cancer was tested using mice. In addition, below, the life extension rate was calculated from the following formula.

(1) Effect on Ehrlich ascites cancer: Inject Ehrlich cancer cells 2X into the cavity of ddY male 6-month-old mice.
After the 10-part individual contact P1, the drug dissolved in physiological saline was intraperitoneally administered five times on days 2, 4, 6, 8, and 10. Efficacy was determined by observing whether the animals were alive or dead for 60 days after inoculation. The results are shown in the table below.

(2) Meth-A effect on solid cancer: B
AL B/c male 6 week old trout back ironically Me
For 3 days from 1 day after inoculating 1 x 10 th-A cancer cells,
Drugs dissolved in saline were administered intravenously. Efficacy was determined by observing whether the animals were alive or dead for 60 days after inoculation. The results are shown in the table below.

Margins below Example 6 Acute Toxicity: Albumin NC8 and NC8 were administered via the tail vein to 1 #10 male ICR mice (8 withdrawals) and observed for 2 weeks after administration to determine LDllo.

LDs.

* Albumin NC83042U/l’# NC82895U/hour *Displayed as NC8 activity.

More applicants? -La Kasei Kogyo Co., Ltd.- 1',! Agent: Patent attorney Miyuki Ariga1:' □Patent attorney: Nobuo Ono:...

Claims (1)

[Scope of Claims] 1. An antitumor agent containing as an active ingredient a compound obtained by binding an antitumor substance to human-derived albumin or a modification thereof. 2. The antitumor agent according to item 1, wherein the antitumor substance is neocarzinostatin. 3. The anti-tumor substance is selected from the group consisting of adriamycin, aclacinomycin, chromomycin A_3, bleomycin, pepleomycin, 6-mercaptopurine, cytosine arabinoside, cyclocytidine, futrafur, aesquinone, melphalan, chlorambutyl and ACNU. The antitumor agent according to item 1, which is an antitumor agent. 4. The antitumor agent according to any one of items 1 to 3, wherein the administration form is an injection.