JPH0554489B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing a porphyrin polymer, and more particularly to a method for producing a porphyrin polymer useful for diagnosis and treatment of cancer.

[Prior art and its problems] One of the methods that has recently attracted attention as a means of diagnosing and treating cancer is a method using a photosensitizer and a laser beam. This method involves intravenously administering a photosensitizing substance that has novel affinity to cancer cells, allowing it to accumulate in the cancer cells, and then irradiating it with a laser beam to induce a physicochemical reaction, thereby diagnosing and diagnosing cancer tissue.
(Harufumi Kato et al.; Clinical Surgery, 37
(4), 517 (1982)). Hematoporphyrin has long been known as the photosensitizer used here, but later, hematoporphyrin derivatives (hereinafter referred to as "HpD") with improved tumor localization were developed ( RLLipson, et al.; Arch.
Dermatol., 82 , 508 (1960): TJ Dougherty, et.
al., Cancer Research, 38 , 2628 (1978)).

However, HpD has been confirmed by electrophoresis to be a mixture of several compounds, and is known to be taken up by normal cells. Also,
There are various problems regarding the intratumoral binding site and distribution state of HpD, and it is known that the occurrence of photochemical reactions is uneven and there is a possibility that the tumor may persist even with sufficient light irradiation (Haruo Kusumi et al.; History of Medicine , 132 , 107
(1985)). Furthermore, HpD must be used in large amounts to provide sufficient sensitivity, such as fluorescence intensity and electron spin resonance absorption (hereinafter referred to as "ESR"), and it is difficult for HpD to be taken up in large amounts not only by tumor cells but also by normal cells. It has side effects such as sun sensitivity.

On the other hand, when HpD is used for tumor diagnosis,
Because the autofluorescence of normal mucosa, which has a peak at 580 nm, overlaps with the specific fluorescence of HpD at 630 nm, the boundaries between fine lesions and normal areas become unclear, leading to problems such as inability to make accurate diagnoses. Yes (Harufumi Kato et al., Clinical Surgery, 37 , 517, (1982)). Furthermore, when used in vitro for tumor diagnosis, there are problems with reproducibility.

Therefore, the present inventors have conducted intensive research to find a highly safe photosensitizer that provides sufficient sensitivity at a low dose and has a specific affinity for tumor cells, and as a result, they have completed the present invention. I've reached it.

[Structure of the Invention] The present invention has the following formula (): (In the formula, Y and Z may be the same or different, and each represents -CH( _CH3 )OH or -CH=
Represents _CH2 . ) is characterized by heating and reacting one compound or a mixture of two or more compounds selected from the group consisting of the compounds shown in an organic solvent at 40 to 200°C while blocking light and oxygen. This invention relates to a method for producing porphyrin polymers.

Specific examples of the compound represented by the formula () include hematoporphyrin (hereinafter referred to as "Hp"),
Examples include protoporphyrin (hereinafter referred to as "Pp"), and 7(12)-(1-hydroxyethyl)-12(7)-vinyldeuteroporphyrin isomers.
Further, examples of the organic solvent used include acetic acid, dimethylformamide, dimethyl sulfoxide, and the like.

The production method of the present invention can be carried out either in the presence or absence of an acid. When the reaction is carried out in the absence of an acid, it is preferable to heat the reaction at 100 to 150°C. The reaction time in this case is usually
30 minutes to 78 hours. When the reaction is carried out in the presence of an acid, examples of the acid include concentrated sulfuric acid or hydrogen chloride. The reaction time in this case is usually 5 minutes to 78
It's time.

If the reaction temperature is below the lower limit, the degree of polymerization of the product will be insufficient and it will not be able to exhibit sufficient photosensitivity when bound to cancer cells.

After the reaction is complete, add a large amount of water to the reaction solution to adjust the pH.
If the concentration is adjusted to 2.5 to 4.0, a precipitate will form, which is removed and thoroughly washed with water to remove the organic solvent. The obtained powder was dissolved in a 0.1N aqueous sodium hydroxide solution, left at room temperature for 5 to 60 minutes, the pH was adjusted to 7.0 to 7.5, and the low molecular weight portion was removed using an ultrafiltration membrane with a molecular weight cutoff of approximately 5000. Remove. Next, the pH of the solution containing the high molecular weight portion is adjusted to 2.5 to 4.0, and the resulting precipitate is collected and dried while shielding from light.

Before use, dissolve the dry powder in a 0.1N aqueous sodium hydroxide solution, neutralize it, and pass it through a 0.22μ filter to sterilize it before use.

The porphyrin polymer obtained in the above manner has the following formula (): (In the formula, X represents a bonding group derived from the above-mentioned Y and/or Z.)
2000 or more (corresponds to a polymer with a degree of polymerization of 3 or more).

[Effects of the Invention] According to the present invention, it is possible to provide a highly safe photosensitizer that exhibits sufficient sensitivity at a lower dose than conventional products and has specific affinity for tumor cells.

[Examples of the Invention] Hereinafter, the present invention will be explained in more detail with reference to Examples, Comparative Examples, and Test Examples, but these are not intended to limit the scope of the present invention in any way.

Example 1 A solution of 1 g of Hp in 100 ml of dimethylformamide,
Heat at 130 to 135℃ while replacing with nitrogen gas under light shielding.
Heated for 17 hours. After the reaction is complete, add 500ml of water to the reaction solution.
was added and the pH was adjusted to 3.5 with hydrochloric acid. The resulting precipitate was collected, thoroughly washed with water, dissolved in 50 ml of 0.1N aqueous sodium hydroxide solution, and allowed to stand at room temperature for 10 minutes. After adjusting the pH to 7.3 with hydrochloric acid and removing the low molecular weight portion using an ultrafiltration membrane with a molecular weight cutoff of 5000, the solution containing the high molecular weight portion was adjusted to pH 3.5 with hydrochloric acid and the precipitate was removed. Porphyrin polymer (hereinafter referred to as “N-
570 mg of HpD-A was obtained.

Example 2 1 g of Hp was dissolved in 100 ml of dimethylformamide in the same manner as in Example 1 and incubated at 130-135°C for 8 hours and 10 hours.
Each porphyrin polymer is reacted for an hour.
850 mg and 700 mg were obtained (hereinafter, the porphyrin polymer reacted for 10 hours is referred to as "N-HpD-B",
The time-reacted porphyrin polymer was
HpD-C”).

Example 3 In the same manner as in Example 1, 1 g of Pp was dissolved in 10 ml of dimethylformamide and reacted at 130 to 135°C for 17 hours to form a porphyrin polymer (hereinafter referred to as "N-HpD-D").
) 600 mg.

Example 4 100 mg of Hp was dissolved in a mixed solution of 90 ml of acetic acid and 10 ml of concentrated sulfuric acid, and the mixture was replaced with nitrogen gas while shielding from light.
Heated at 100°C for 1 hour. After the reaction was completed, the reaction solution was treated according to Example 1 to obtain 70 mg of porphyrin polymer (hereinafter referred to as "N-HpD-E").

Example 5 100 mg of Pp was dissolved in a mixed solution of 90 ml of acetic acid and 10 ml of concentrated sulfuric acid, and the mixture was heated for 50 ml while replacing the water with nitrogen gas in the dark.
Heated at ℃ for 20 hours. After the reaction was completed, the reaction solution was treated according to Example 1 to obtain 50 mg of porphyrin polymer (hereinafter referred to as "N-HpD-F").

Example 6 1 ml of concentrated sulfuric acid was added to a solution of 1 g of Hp in 10 ml of dimethylformamide, and heated at 100° C. for 60 minutes while purging with nitrogen gas while shielding from light. After the reaction was completed, the reaction solution was treated according to Example 1 to obtain 700 mg of porphyrin polymer (hereinafter referred to as "N-HpD-G").

Example 7 4g of Hp (dihydrochloride) was mixed with 76ml of glacial acetic acid and 4ml of concentrated sulfuric acid.
It was dissolved in a mixed solution of and left at room temperature for 5 hours. Add this to 1.2L of 3% sodium acetate aqueous solution, collect the resulting precipitate by filtration, thoroughly wash and dry, and powder
3.4g was obtained. 100 mg of the obtained powder was dissolved in 5 ml of 0.1N aqueous sodium hydroxide solution and treated at room temperature for 1 hour. Hydrochloric acid was added to this solution to adjust the pH to 4.0, and the solution was washed with 20 ml of tetrahydrofuran and 200 ml of saturated saline. Next, the mixture was extracted with 300 ml of ethyl acetate, washed with water, and concentrated to dryness to obtain a powder. This powder was subjected to high performance liquid chromatography (Zorbax column; elution solvent, tetrahydrofuran:water = 1:1 (V/
V)+10% acetate buffer), the composition was as follows.

Hp: 40% 7(12)-(1-hydroxyethyl)-12(7)-vinyldeuteroporphyrin isomer: 45% Pp: 5% Add 50 mg of the above powder to 5 ml of dimethylformamide in the same manner as in Example 1. The mixture was dissolved, reacted at 130 to 135°C for 6 hours, and purified to obtain 40 mg of porphyrin polymer (hereinafter referred to as "N-HpD-H").

When the mass spectrum of N-HpD-H was measured, it was confirmed that it was a polymer containing three or more porphyrin rings.

Comparative Example The method described in the literature (Cancer Research, 38 , 2628 (1978)), namely, ``Hp was reacted with acetic acid and sulfuric acid, left overnight, filtered using a Whatman No. 1 filter, and neutralized by adding sodium acetate to this solution. and dry the resulting precipitate under reduced pressure.
Got HpD. Furthermore, 1 g of this HpD was dissolved in a 0.1N aqueous sodium hydroxide solution, and this solution was adjusted to pH 9.5, and the low molecular weight portion was removed using an ultrafiltration membrane with a molecular weight cutoff of 10,000 (manufactured by Amicon, PM10). high molecular weight fraction (hereinafter referred to as “IN-HpD”)
Got 600mg.

Test Example 1 Inventive product N-HpD prepared in Examples 1 and 2
-A(-〇-, N-HpD-B(-×-), N-HpD-
C

Claims (1)

[Claims] Primary formula (): (In the formula, Y and Z may be the same or different, and each represents -CH( _CH3 )OH or -CH=
Represents _CH2 . ) is characterized by heating and reacting one compound or a mixture of two or more compounds selected from the group consisting of the compounds shown in an organic solvent at 40 to 200°C while blocking light and oxygen. , the following formula (): (In the formula, X represents a bonding group derived from Y and/or Z in the formula ().) A method for producing a porphyrin polymer comprising repeating units represented by the following and having a degree of polymerization of 3 or more. 2. Claim 1, in which one type of compound or a mixture of two or more types of compounds selected from the group consisting of compounds represented by the formula () is heated and reacted at 100 to 150°C in the absence of an acid. Manufacturing method described in section. 3. Claim 1, wherein one type of compound or a mixture of two or more types of compounds selected from the group consisting of compounds represented by the formula () is heated and reacted at 40 to 200°C in the presence of an acid. Manufacturing method described.