JPH05286868A - Carcinostatic agent complex and its screening method - Google Patents

Abstract

PURPOSE:To obtain a carcinostatic agent complex for treating multiple drug- cancers, based on inhabitation of pump function of p-glucoprotein, high in safety and reduced in side effects. CONSTITUTION:The objective carcinostatic agent-carrier complex consists of a carcinostatic agent-pharmacologically inert carrier complex having <=1/1.5 IC50 ratio of the complex to carcinostatic agent itself in sensitivity test using AH66DR cell and is effective in cancer cell having multidrug resistance based on p-glucoprotein(Pgp).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anticancer agent-carrier complex which is effective for drug-resistant cancer cells, and more specifically, p-glycoprotein (hereinafter referred to as "Pgp").
The present invention relates to an anticancer agent-carrier complex effective for cancer cells having multidrug resistance.

[0002]

2. Description of the Related Art Drug resistance is a phenomenon in which a used anticancer drug loses its efficacy, which is the greatest problem facing cancer chemotherapy. Therefore, this problem of drug resistance must be solved by all means in order to improve the treatment results using anticancer agents.

In this drug resistance phenomenon, there is a more troublesome problem, that is, the problem of multidrug resistance. This multidrug resistance is a phenomenon in which when a cancer cell becomes resistant to adriamycin, which is one of the anticancer agents, the cell also becomes resistant to many other anticancer agents. This phenomenon of multidrug resistance is believed to be due to the action of cancer cells to keep the intracellular anticancer drug concentration low. Furthermore, a protein having a molecular weight of 170 to 180 kDa (P-glycoprotein, hereinafter referred to as "Pgp") is overexpressed on the cell membrane of multidrug-resistant cancer cells, and this P-glycoprotein causes the carcinostatic agent to be extracellular. It has been found to have a function as a pump for discharging (eg, BIO medica, 6 (5), 4
42-445 (1991)).

In order to overcome such multidrug resistance, various attempts have been made to increase the retention time (half-life) of cancer drug in cancer cells. For example, anti-Pg
A method of blocking the function itself of Pgp using a p monoclonal antibody (Jpn. J. Cancer Res., 80 , 627-6.
31 (1989); BIO medica 6 (5), 442.
445 (1991)) or other small molecule (specifically, verapamil, which is a calcium antagonist), is administered in large amounts at the same time as a carcinostatic agent to preferentially administer verapamil to P.
The method of prolonging the intracellular half-life of an anticancer agent by excreting it by gp has been carried out.

[0005]

However, in the method using the above monoclonal antibody, since such an antibody is derived from another animal (for example, mouse),
Since it is highly antigenic in itself, it is not always easy to use it in clinical practice. On the other hand, in the method of administering a large amount of another low molecular weight calcium antagonist together with the carcinostatic agent, the “side effect” due to the calcium antagonist itself cannot be ignored. Therefore, an object of the present invention is to solve the above-mentioned problems, and to provide a carcinostatic agent complex for treating multidrug-resistant cancer, which is highly safe and has few side effects.

[0006]

Means for Solving the Problems As a result of earnest research, the inventors of the present invention utilized P activity by utilizing the pharmacological activity as in the past.
By not increasing the so-called pump function of gp (P-glycoprotein), but rather increasing the molecular size of the carcinostatic agent or its bulkiness (bulkyness), the pump function of Pgp works with high safety and few side effects. I found that I could not get it, and further, such Pg
It was found that the effect of the anticancer drug complex on p can be extremely effectively screened by using specific cells.

The carcinostatic agent complex for treating multidrug-resistant cancer of the present invention is based on the above-mentioned findings. More specifically, from the complex of a carcinostatic agent and a pharmacologically inactive carrier. It is characterized by becoming.

According to the present invention, there is further provided a carcinostatic agent composite comprising a carcinostatic agent and a carrier having a molecular weight of less than 10,000. According to the present invention, there is further provided a complex consisting of a carcinostatic agent and a carrier, which comprises AH66DR
There is provided a carcinostatic agent complex characterized in that the IC ₅₀ ratio of the complex to the carcinostatic agent itself in a susceptibility test using cells is 1 / 1.5 or less.

According to the present invention, AH66DR cells are further incubated in the presence of an antitumor agent complex,
There is provided a method for screening an anticancer agent complex, which comprises measuring inhibition of cell growth.

The present invention will be described below in detail with reference to the drawings as necessary. (Anticancer agent) In the present invention, an anticancer agent refers to an agent that suppresses the growth / proliferation of cancer cells. In the present invention,
As the carcinostatic agent, known substances such as metabolic inhibitors, hormones, cell division inhibitors, nucleic acid synthesis inhibitors and polysaccharides can be used without any particular limitation, but especially Pgp
An anticancer drug that is easily transported by (P-glycoprotein) is effectively used. The term "anticancer drug easily transported by Pgp" as used herein means its IC ₅₀ (50% in a free state (a state in which other carriers are not bound) in a sensitivity test using AH66DR cells described later. Growth inhibitory concentration)
, In the susceptibility test using the parent strain AH66 cells
Compared with C ₅₀ , it means 5 times or more (more preferably 10 times or more, particularly 50 times or more).

Susceptibility test using AH66DR cells Live cells (2 × 10 ⁴ ) of AH66DR were cultured in a 24-well culture plate (Corning, NY, USA) in a predetermined medium (for example, 10% fetal bovine serum ( RPMI 1640) containing FBS) and various concentrations (eg, 0.
Incubate at 37 ° C. for 96 hours with an anticancer drug to be tested (about 0001 to 500 μmol / L).
Then, the number of viable cells was counted by the trypan blue exclusion method, and the degree of cell proliferation was determined by measuring the degree of cell proliferation into the acid-insoluble fraction [ ³
H] -thymidine uptake is used for calculation ("Technology of tissue culture", 2nd edition, edited by the Japan Tissue Culture Society,
1988). The susceptibility test is used only for evaluating the "ease of transport by Pgp" of the anticancer drug itself to be used in the present invention. It does not limit the range of.

The anticancer agent preferably used in the present invention is more specifically, for example, vincristine,
Vin alkaloid anticancer agents such as vinblastine and vindesine; anthracycline anticancer agents such as adriamycin, THP-adriamycin, daunomycin, aclacinomycin A; etoposite, maytansine, taxol, mitomycin C, actinomycin D, mitoxantrone, Menogalol and the like can be mentioned.

(Carrier) In the present invention, the carrier means a substance which does not have a carcinostatic action and is itself pharmacologically inactive. The carrier used in the present invention has an ED ₅₀ (50% effective amount) as an index of pharmacological action of 1 μmol /
The substance is preferably L or more (more preferably 10 μmol / L or more). The carrier of the present invention does not have a specific antigenic determinant (epitope) recognition action as in an antibody against a specific antigen.

The carrier used in the present invention may be a natural product, synthetic product, semi-synthetic product, or in vivo degradable / non-degradable carrier, and its molecular weight ( In the case of polymers, etc., it refers to the average molecular weight, the same applies hereinafter)
Is preferably 200 or more, more preferably 500 or more (particularly 1,000 or more). This carrier has a high molecular weight (molecular weight 1
However, the molecular weight is 2,000 or more, more preferably 10, or more from the viewpoint of easiness of binding with a carcinostatic agent or the half-life in blood in vivo. 0
It is preferably 00 or more (particularly 30,000 or more). The upper limit of the molecular weight of the carrier is such that it can be reached to cancer cells after administration to a living body, and to the extent that the uptake of the antitumor agent complex into the cancer cells is not significantly inhibited, more specifically, usually, The molecular weight is preferably 8,000,000 or less, more preferably 5,000,000 or less (particularly 3,000,000 or less).

In the present invention, as the carrier,
Binders, disintegrating agents, thickeners (viscosi) commonly used as formulation additives
ty-increasing agent) or the like can be used. More specifically, natural products such as albumin (eg, human serum albumin (HSA), globulin, immunoglobulin, hemocyanin, fibrinogen, dextran, polyamino acid, starch, gelatin; maleic anhydride-
Examples thereof include synthetic products such as divinyl ether copolymer, styrene-maleic anhydride copolymer, polylysine and polyaspartic acid.

(Complex) In the present invention, the carcinostatic agent and the carrier can be bound to each other by a known binding method to form a complex, and the binding method is not particularly limited, but in the present invention, The method generally used for binding the hapten and the carrier can be used. A known spacer may be inserted between the carcinostatic agent and the carrier, but in this case, it is preferable that the molecular weight of (spacer + carrier) satisfies the above-mentioned molecular weight condition regarding the carrier. In addition, the above-mentioned complex may be G
It can be used after being purified by a PC or the like.

More specifically, in the present invention, an acylating agent (N-hydroxysuccinimide ester (NH
S), S-acetylmercaptosuccinic anhydride, etc.),
Maleimides (N-ethylmaleimide etc.), allyl halides (dihalonitrobenzene of 2,4-dinitro-1-fluorobenzene etc.), triisocyanate compounds (tolylene-2,4-diisocyanate etc.), carbodiimides (EDC, DCC, CMC, etc.), alkyl chloroformates, isoxazolium salts, glutaraldehyde, diazonium salts and the like.

The molar ratio of the carcinostatic agent and the carrier constituting the complex of the present invention is not particularly limited, but (the carcinostatic agent) /
(Carrier) = 1 / 1-100 / 1 (further 3/1
Is preferably about 50/1). In the present invention, the formation of the complex (which varies depending on the drug) is performed by, for example, GPC (gel permeation chromatography) under the following conditions to obtain a complex having the above-mentioned quantitative ratio, It can be confirmed by being eluted.

GPC condition column: Sephadex G-100 (Pharmacia, Sweden) Mobile phase: 10 mM phosphate buffered saline (pH 7.
0) Flow rate: 15 mL / hr Temperature: 4 ° C

As described above, the carcinostatic agent composite of the present invention comprising a carcinostatic agent and a carrier has the following AH6
In a sensitivity test (growth inhibition inhibition test) using 6DR cells, (IC _{50 of} complex) / (I of carcinostatic agent itself)
_C50 ) is 1 / 1.5 or less, further 1/10 or less (especially 1
/ 50 or less) is preferable.

(AH66DR cells) AH66DR cells which are preferably used for screening the complex of the present invention are D
Rat ascites hepatoma cells AH66 (Gann, 47 , 612-61), which are NR (daunorubicin (daunomycin chemical name)) resistant cells and established by azo dye.
5,1956) as follows. A above
DNR of H66 cells (10 ⁵ cells) continuously for 2 months or more
(2 μg / ml), and obtained 2 to 3 times D
NR-resistant AH66 cells are further continued in vitro with gradually increasing concentrations of DNR (from 2 μg / ml to increasing every 3 weeks) until the final DNR concentration is 75 μg / ml. The obtained cells are further treated for 3 months at 60 μg /
AH66DR cells are obtained by culturing in the presence of ml of DNR. The AH66 cells and the AH66DR cells can be maintained in, for example, RPMI1640 medium (Nissui Pharmaceutical Co., Ltd.).

(Composite screening using AH66DR cells) The anticancer agent composite screening using AH66DR can be suitably carried out, for example, as follows. 24 live cells of AH66DR (2 × 10 ⁴ cells)
Predefined medium (eg, RP containing 10% FBS) in a well culture plate (Corning, NY, USA)
MI1640) and various concentrations (for example, 0.000
Incubation is carried out at 37 ° C. for 96 hours each with an anticancer agent or an anticancer agent complex to be tested (about 1 to 100 μmol / L). After that, the number of viable cells was counted by the trypan blue dye exclusion method, and the degree of cell proliferation was measured by [ ³ H] -thymidine in the acid-insoluble fraction of the cells.
Calculated by incorporation.

The carcinostatic agent complex of the present invention may, if necessary,
It may be used in the same manner as the administration method and dose of the above-described anticancer agent except that the amount used is converted into the used amount (dose) of the anticancer agent itself constituting the complex. It is possible. Therefore, when the anticancer drug complex of the present invention is administered, it is basically administered to humans or animals according to the administration method and dose of the underlying anticancer drug.
Although the dose varies depending on the administration subject, symptoms, administration method, etc., it is generally 0.2 to
The dose may be administered within the range of 0.6 mg / kg / day (converted to a carcinostatic agent).

According to the findings of the present inventors, the complex of the present invention is considered to be free because of the reduced toxicity (LD ₅₀ ) of the carcinostatic agent due to its sustained release property and the like. In comparison, large-scale administration of at least 2 times or more and 10 times or less (preferably about 5 times or less) is possible.

[0025]

The present invention will be described in more detail with reference to the following examples. In the following, all statistical processing was performed using Student's t test.

Example 1 (Preparation of DXR-protein complex) DXR (doxoru)
bicin (chemical name of adriamycin), Tokyo Farmitalia-C
arlo Erba) and the carrier protein are bound by H
Method of urwitz et al. (Cancer Res., 35 , 1175-1181 (1975))
It was carried out according to.

Specifically, 3 mg of commercially available BSA (bovine serum albumin, Sigma, USA) or commercially available goat immunoglobulin G (IgG, US Sigma) and 0.5 mg of DXR (unlabeled / and ¹⁴ C-labeled). To 1
Cross-linked with glutaraldehyde solution (concentration 0.1%) in mL of phosphate buffered saline. Free and bound DXR were separated by gel filtration using Sephadex G-100 (Pharmacia, Sweden).

The degree of substitution depends on the absorption of DXR (280 nm
And 495 nm). In addition, the protein concentration is based on the commercially available protein assay kit (US Bio
-Rad). For comparison, a BSA-glutaraldehyde conjugate was also prepared in the same manner as above except that DXR was not used.

Example 2 (Drug Sensitivity) Each of AH66 cells and AH66DR cells (2 × 10 ⁴ cells) at various concentrations (0.01 to
200 μmol / L) in the presence of DXR (10% F
It was cultured in RPMI 1640) containing BS at 37 ° C. for 96 hours. After the culture was completed, the number of viable cells was measured by the trypan blue dye exclusion method in which only dead cells were stained without being incorporated into viable cells.

Example 3 (Cell Proliferation) Live cells (2 × 10 ⁴ ) of AH66DR were placed in a 24-well culture plate (Corning, NY, USA) in a predetermined medium (for example, R containing 10% FBS).
PMI1640) with various concentrations (eg 0.0
It was incubated at 37 ° C. for 96 hours with an anticancer drug to be tested (about 1-2 μmol / L). Then, the number of viable cells was counted by the trypan blue dye exclusion method, and the degree of cell proliferation was determined by the acid-insoluble fraction (DNA fraction) of the cells.
[ ³ H] -thymidine (specific activity 20 Ci)
/ Mol, Amersham Japan KK).

Example 4 (Incorporation of DXR and DXR complex) AH66DR cells (1 × 10 ⁵ cells) were plated on a 24-well microplate.
1 μmol / L of [ ¹⁴ C] -DXR (specific activity 35 mC
i / mmol, Amersham Japan KK) or [ ¹⁴ C] -DXR-BSA complex obtained in Example 1 together with 3
Incubated at 7 ° C. After incubation for various times, the microplate was ice-cold 0.1.
After washing well with 5 mol / L physiological saline and terminating the incubation, the amount of drug incorporated into cells (radioactivity) was measured by a liquid scintillation counter (LS6).
000 IC, manufactured by Beckman, USA).

Example 5 (SDS-PAGE and Western blotting) The cell extract was diluted with a buffer for SDS-PAGE (polyacrylamide gel electrophoresis) sample, and then SDS.
-Fractionation using PAGE (7.5% separation gel), Towb
in et al. (Proc. Natl. Acad. Sci. USA, 76 , 4350-
4354 (1979)) electro on nitrocellulose paper.
transferred. The obtained nitrocellulose was treated with anti-Pgp
(P-glycoprotein) monoclonal antibody (C219, USA
Centocor) at 4 ° C for 17 hours and then horseradish peroxidase-labeled anti-mouse IgG
(Bio-Rad). By incubating with 3,3′-diaminobenzidine (Bio-Rad), it was colored and the enzyme activity was detected.

Results (DXR sensitivity of AH66DR) In the culture system in which DXR was continuously contacted for 96 hours, the IC ₅₀ (50% inhibitory concentration) of DXR to AH66DR cells was 16 μmol /
It was L. On the other hand, in the parental AH66 cells, the IC ₅₀ was 0.08 μmol / L (see FIG. 1).

Also, after SDS-PAGE, Western
In blotting (Fig. 2), Pgp showed AH66.
It was detected only in extracts from DR cells.

(Complex of protein and carcinostatic agent) As a result of various concentration changes, 3 to 5 mol of the carcinostatic agent was bound to 1 mol of BSA or IgG.
This composite was used in the above examples.

(Cytotoxic effect of DXR-protein complex on AH66DR cells) BSA or Ig described above
The anticancer cytotoxic effect of the complex of G and DXR is shown in FIG.

In FIG. 3, DXR itself or BSA is used.
-The apparent and potential growth inhibitory effect of the BSA-DXR and IgG-DXR conjugates of the invention is shown against the dose (horizontal axis) in comparison with the glutaraldehyde conjugate. As is clear from FIG. 3, AH66DR
BSA-DXR or IgG-DX of the invention for cells
The IC _{50 of the} R complex is 0.05 μmol / L and 0.07 μmol / L (in terms of DXR concentration), respectively.
Met.

The growth inhibitory effect (IC ₅₀ ) of these conjugates on multidrug resistant AH66DR was determined by the IC ₅₀ (0.08μ) of DXR itself against AH66 (which does not have multidrug resistance).
mol / L). Further, the growth inhibitory effect calculated by thymidine incorporation almost corresponded to the result of the above-mentioned assay based on the viable cell count.

(Uptake and accumulation of drug into cells)
The drug concentration as a function of time in AH66 and AH66DR was compared with 1 μmol / L [ ¹⁴ C] -DXR,
1 μmol / L BSA [ ¹⁴ C] converted to DXR concentration
-The DXR complex was evaluated. The results are shown in Fig. 4.
With DXR treatment within 1 hour, intracellular drug concentration was
In 66 (sensitive cells), it is almost twice as much as in AH66DR (resistant cells) (significant difference P <0.05),
This significant difference continued thereafter.

On the other hand, the resistant AH66DR was administered at a dose equivalent to that of the above DXR in the BSA-of the present invention.
Treatment with DXR conjugate resulted in relatively low drug accumulation after 1 hour of treatment. However,
After this, as shown in FIG. 4, BSA-D to AH66DR
The XR uptake gradually increased, reaching a level almost equivalent to DXR (equivalent concentration) for the sensitive AH66 after 24 hours of treatment.

As described above, the antitumor agent complex (BSA-DXR or IgG-DXR) of the present invention acts extremely effectively on AH66DR cells resistant to DXR, and for a long time (after 24 hours). ), It was found that it is taken up almost in the same manner as DXR itself for sensitive AH66 cells.

That is, the carcinostatic agent-carrier complex of the present invention is the same as the carcinostatic agent itself for cancer cells (susceptible cells) substantially not having Pgp (P-glycoprotein). It was confirmed that it exerts a carcinostatic effect on multidrug-resistant cancer cells having Pgp.

[0043]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, a carcinostatic agent comprising a carrier having substantially no pharmacological activity and a carcinostatic agent and effectively acting on multidrug resistant cancer cells. An agent complex is provided.

[Brief description of drawings]

FIG. 1 is a graph showing the number of viable cells in a sensitive AH66 cell (parent cell line) and a resistant cell line AH66DR cell after continuous contact with DXR for 96 hours.

FIG. 2 is a photograph showing the result of Western blotting of Pgp after SDS-PAGE.

FIG. 3 is a graph showing a comparison of carcinostatic effect between free DXR and DXR-BSA or DXR-IgG complex in resistant strain AH66DR cells.

FIG. 4 Comparison of uptake of free DXR with time in AH66 cells (parent line) and AH66DR cells (resistant line), and BSA-DX in AH66DR cells.
It is a graph which shows the time-dependent change of the amount of R taken in.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Nozomu Hibi 51 Komiyacho, Hachioji City, Tokyo SRL EL Hachioji Laboratory

Claims (5)

[Claims] 1. A carcinostatic agent complex for the treatment of multidrug-resistant cancer, which comprises a carcinostatic agent and a pharmacologically inactive carrier complex. 2. A carcinostatic agent complex comprising a carcinostatic agent and a carrier having a molecular weight of less than 10,000. 3. A complex comprising a carcinostatic agent and a carrier, wherein the IC ₅₀ ratio of the complex to the carcinostatic agent itself in a sensitivity test using AH66DR cells is 1/1.
An anticancer drug complex, which is 5 or less. 4. The carrier is human serum albumin (H
SA), The composite according to claim 1 or 3. 5. A method for screening an anticancer agent complex, which comprises incubating AH66DR cells in the presence of the anticancer agent complex to measure inhibition of cell growth.