JPS6156128A - Novel blend of doxorubicin and analogue and anionic polymer,manufacture and anticancer medicinal composition - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel combination of Dokinrubishi/and its analogs and anionic & IJ mom, a method for producing the same, and a pharmaceutical composition containing the combination having anticancer activity. .

BACKGROUND OF THE INVENTION The conjugation of anticancer drugs to suitable water-soluble polymeric carrier compounds has been proposed as a new approach to improve the therapeutic efficacy of drugs in cancer chemotherapy. Indeed, the curative index of most anticancer drugs (including anthracycline antibiotics) is small, and host toxicity continues to primarily limit the use of applicable medicines.

Means for Solving the Problems According to the invention, a 1:2 regularly alternating cyclopolymer of divinyl ether and maleic anhydride is added to doxorubicin or an analog thereof in the form of polycarboxylic V-) to an anthracycline. an anthracycline bonded by an ester bond to 140 atoms of
An anionic polymer blend is obtained.

A 1:2 regularly alternating cyclopolymer of divinyl ether and maleic anhydride is also known as a D- or pyran copolymer with the formula: [divinyl ether-maleic anhydride copolymer (IWA-MA)].

This uses a strong base, preferably 12 or more -,
For example, it can be converted into the polycarboxylate form by hydrolysis with an aqueous solution of IJ (0.2N-hydroxide). The sodium salt in the form of polycarboxy V-) has the formula: [polycarboxylate form].

The anthracycline-anionic polymer blend of the present invention can gradually release the drug in free form in vivo by hydrolyzing the ester bond, thus altering the pharmacological behavior of the drug. Ru. Side chain (C-13,
0-14) avoids denaturation of the pharmaceutical amino shield, which appears to be essential for optical pharmaceutical activity.

Anticancer activity in in vivo experimental tumor models appears to be reflected by reduced drug toxicity and increased efficacy in all animal tumors used when using anthracyclines conjugated to polyanionic polymers. shows clear therapeutic benefits. Improving the healing index of polymer-conjugated drugs can yield promising improvements in pharmacological distribution and selective delivery of drugs to cancer cells with large intracellular volumes.

The present invention further provides that in preparing the anthracycline-anionic polymer blend according to the present invention, a 1:2 regularly alternating copolymer of divinyl ether and maninic anhydride is hydrolyzed with a strong base and obtained. A process is obtained which consists of reacting a polymer in the form of a polycarboxylate with a 14-bromo derivative of 14-deromdoquinrubicin or a doquinrubicin analogue.

This method can be applied simply to doxorubicin, 4-demethoxy-
Doxorubicin and 4'-deoxide quinolubicin are shown in the following reaction diagram.

Meng ↑ Example The present invention will be explained in detail below with reference to Examples.

Example 1 Pyran copolymer [average molecular weight 32000 (100m9
Dissolve K in 5 ml of distilled water) and 0.2N hydroxide)
It was converted to the polycarboxylate form by hydrolysis in aqueous IJum solution (pH>12) for a minimum of 15 minutes. Complete hydrolysis of maleic anhydride was confirmed by titration curve. After the water decomposition, the OPH value of the bathtub was adjusted to 8.5. 14-bromo-daunorubicin (20-5'0+n
9 freshly dissolved in a small volume of methanol),
It was added to the polymer bath under stirring. The reaction was carried out at room temperature. The acid produced during the nucleophilic substitution reaction was neutralized by resuscitation with dilute aqueous sodium hydroxide solution. The reaction mixture was stirred for 2 hours and then adjusted to pH 7.0. All free antibiotics were removed by Dowex.
x) was removed by ion crossing chromatography on 5Q W-x2. Generally, daunorubicin-polymer blends were diluted with water or an aqueous solvent to reduce polymer concentration. Thin layer chromatography [Chloroform:methanol:acetic acid 80:20:4 (unit:
volume)] and high pressure liquid chromatography on a C-18 reversed-phase column [water-niacetonitrile 66:34 (
(unit: volume), adjusted to pH 3.8 with phosphoric acid, flow rate 1.511 tg/min as mobile phase; photometric detection, excitation wavelength: 500 nm; emission wavelength: 590 nm)
was used to detect free anthracycline derivatives in samples of polymer derivatives. Additionally, samples showing remaining free drug were collected using Dowex 50W-
Rechromatographed on X2. The concentration of bound drug in the polymer derivative was estimated by absorbance at 48 Dnm.

Polymer content in the formulation was determined by dry weight after the formulation was drain dialysed against distilled water to remove all salts. Titration data were also used to calculate the applicable carboxyl groups and thus evaluate the degree of drug substitution. This method gave the same results. Also, since the anhydride functional group could react with the pharmaceutical amino group to form an ami-P bond, this type of reaction was ruled out by the following observations: a) Complete hydrolysis of the anhydride under the coupling reaction conditions; titration curve.

b) The presence of residual anhydride was also ruled out by the fact that no mKN-acyl derivative was formed when the free aminoglycoside was admixed and the alkaline hydrolyzed polymer was seeded under standard reaction conditions.

Example 2 14-deromudeoxide quine rubicin was used in place of 14-bromudeoxide quine rubicin in the method of Example 1 to give the title compound.

Example 6 Ichinoseki compound (sulfur) 14-bromo-4'-deoxy-daunorubicin was used in place of 14-bromdoquine lupicin in Example 1, and the indicated compound was converted to ? It was 8.

Dokin rubicin - bilan cobolimer\mNk compound (I) (
P-DX (hereinafter abbreviated as P-DX) was subsequently tested for cytotoxic activity and anticancer activity against tumors in laboratory mice, and the results were used to evaluate the effectiveness of this type of anticancer drug as a standard drug.
daunorubicin (DR) and doxorubicin (DX)
) (7) compared with the results. Inhibition of the colony forming ability of HeLa cells was used to quantify the cytotoxic activity of the drug conjugated to the polymer. Even though some reduction in cell S inactivation of the bound drug when compared to the free drug was consistently observed (Table 1), the conjugated drug required for a 50% inhibition of the colony forming ability of HeLa cells. concentration (15fig/ml
) was only slightly larger than once required by free drug (10n&/d).

Comparative studies of anthracyclines were performed on tumor tissue implanted with both i,p, (238 day leukemia, J-744 lymphoma) and 1°v, (gross leukemia). In these studies, the route of drug administration was similar to that of tumor cell inoculation.

The results of a number of experiments on P388 leukemia are summarized in Table 2. Free daunorubicin is available for its optimal use f4ff
'I'/C209% was obtained with 19//Cg. Doxorubicin was tested at -ffi 10mp/9 for its optimal use and was more active than daunorubicin ('I'/c - 274%
)Met. P-DX tested at various doses (expressed as quinrubicin in the formulation) showed a dose-dependent 7ff
t's activity. Small doses produced anticancer effects similar to the free drug. However, the low toxicity of the Doquinrubicin-polymer combination allowed for increased doses, thus improving the therapeutic efficacy of the anticancer drug. Actually large t(>
40 η/g) was easily tolerated, thus indicating a significant reduction in the pharmacotoxicity of P-DX. Using the same treatment schedule, simultaneous administration of a mixture consisting of free drug and polymer is only marginally superior to free drug alone within optimal (i.e., best tolerated) doses. Anticancer effect was obtained. However, pyran copolymers had no detectable effect on drug toxicity. In addition, the great activity of the polymeric derivative of doxorubicin was obtained by the optimal dose for a long period of time (Tumor Funk: t
umourfrec) reflected in the relative proportion of the number of survivors.

This therapeutic effect was not observed with daunorubicin and was only slightly observed with doxorubicin. The polymer itself had only marginal activity in this tumor tissue (maximum T/C-165%).

To highlight the differences between free anthracyclines and macromolecular derivatives, other studies were performed on T-744 lymphoma and gross leukemia (Table 6). The results of a comparative study using all mice bearing J-744 lymphoma showed that P
Results were similar to those using mice with 388 leukemia. P-DX increased lifespan more than free daunorubicin or free doxorubicin.

Only P-DX produced long-term survivors.

These drugs were evaluated against a gross leukemia model using multiple injection sites (Table 4).

Even though doxorubicin's binding to polymers reduces the drug's efficacy, P-DX at its optimal dose is significantly more active than daunorubicin or l-"quinrubicin, resulting in significantly increased lifespan and significant long-term There were no survivors.

4-demethoxygen rubicin-pyran copolymer blend (P-DmX) is used in multiple treatment schedules (q3dX3,i) for gross leukemia.

When tested using 4-demethoxydaunorubicin (DmDR) and slightly better anticancer effects than 4-demethoxidequinrubicin (Drr, DX) (Table 5).

However, as evidenced by P-DX, only the formulation produced significant numbers of long-term survivors.

The reduction in tissue toxicity allowed the administration of large amounts of drug in the form of formulations: free drug (LDlo"0.6 m9/kg) and polymer-bound drug (LD10=1.7Tn97).
The ratio between the isotoxic doses of 9′) was approximately 3.

Table 1 Cytotoxin activity of daunorubicin, doxorubicin and P-quinrubicin-pyran copolymer blend against HeLa cells Cells were exposed to the drug for 24 hours. Viability was determined by colony forming ability of r1 treated cells.

After treatment, the medium was removed, cells were washed and suspended and spread in Eagle's minimum essential medium containing 10% fetal calf serum. Colonies were counted 8 days after inoculation.

Table 2 Comparison of therapeutic effects of 1, p and treatment on P388 leukemia 1 Note 1 Experiments were conducted with 1 xio' cells/mouse. p, performed in inoculated BDF I mice. The mean survival time (TST) of control mice ranged from 9 to 9 in 6 experiments.
It was the 10th.

Note 2 DR+P is a mixture of daunorubicin and pyran copolymer; P is pyran copolymer O Note 3 1. p6 treated.

For P-DX, it was expressed as dose Radkin rubicin content. For DR+P, doses were expressed as daunorubicin content.

Note 4: MAT of dead mice in treatment group only divided by MAT of control group, 100 times (X100)t,
Ta. Data or ranges from single experiments are in parentheses.

Note 5 LTS is the long-term survival number (>60 days).

Note 6 Dose expressed as polymer glue; new drug/
The molar ratio of the polymers ranged from 19 to 29° in the various formulations. p.

The test was carried out in inoculated BALB/c mice.

The MST of control mice was torn for 19 days.

Note 2 One day after cancer cell transplantation, one target was covered.

Note 3: LTS is the long-term survival number (>90 days).

Table 4 Effect of multiple administrations on gross leukemia 1 Note 1 Experimental gold, I X 16' cells/mouse 1. v.

It was performed in inoculated C3H/He mice.

The MST of control mice was 5.5 days in two experiments.

Note 2 i, v at 1, 4 and 7 days after cancer cell transplantation.

Treated.

Note 3: LTS is the long-term survival number (>60 days).

Table 5 4-demethoxy-daunorubicin (DmDR), 4-demethoxydoquin lupicin (DmDX) and 4-demethoxydoquin lupicin-Bilanco sumer combination (P
-DmDX) effect on gross leukemia 1 (DaA9) (chi) DmDRO, 751431/10 0/10DmDX
0.50 288 (285-291) O
/20 1/200.75 229 (200-25
8) IZ/20 1/20p-DmX 1.13
350 1/10 0/101
．． 70 286 (272-300) 3/20
3/20 Note 1 Experiments were conducted with 1x106 cells/mouse.
．． v.

It was performed in inoculated C3H/He mice.

Note 2 1, 4 and 7 days after cancer cell transplantation. v.

Treated.

Note 3 The range is in parentheses.

Note 4 LT8 is the long-term survival number (>60 days).

Claims (1)

[Claims] 1. Doxorubicin and its analogs and pyran copolymer in the form of its polycarboxylate with 0.2-NaO
A novel blend consisting of an anionic polymer obtained by mild hydrolysis with H. 2. Doxorubicin and its analogs and pyran copolymer in the form of its polycarboxylate with 0.2N-Na
In producing a compound consisting of an anionic polymer obtained by mild hydrolysis with OH, 14-
bromodaunorubicin or its 14-bromo analog in aqueous methanol at room temperature and from 120 to 180
reacting with an anionic polymer in the form of said polycarboxylate for a period of minutes and maintaining the solution neutral by adding dilute aqueous sodium hydroxide solution;
Novel formulations of doxorubicin and its analogues with anionic polymers, characterized in that all free antibiotics are subsequently removed by chromatography on ion exchange resins to obtain pure solutions of the desired new formulations. manufacturing method. 3. Doxorubicin and its analogs and anionic polymer in the form of its polycarboxylate 0.2N-N
Anticancer activity containing a novel combination of doxorubicin and its analogs and an anionic polymer, characterized in that it contains a combination consisting of an anionic polymer obtained by mild hydrolysis with aOH A pharmaceutical composition comprising: