JPH0717646B2 - Novel pheophorbide derivative and tumor diagnostic agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel pheophorbide derivative and a tumor diagnostic agent containing the same as an active ingredient.

[Conventional technology]

It is generally known that porphyrin-based compounds have an affinity for tumor tissues. Utilizing this property, a porphyrin compound, particularly a hematoporphyrin derivative, is administered to a patient, and a diagnostic method for confirming a tumor-occurring site by detecting fluorescence generated by irradiation with laser light after a certain period of time has been put into practical use. ing. However, it has been difficult to detect deep-seated tumors that are difficult to reach the irradiation light.

[Problems to be solved by the invention]

An object of the present invention is to synthesize a novel pheophorbide derivative in order to improve the drawback of the prior art as a tumor diagnostic agent, that is, the difficulty of detecting deep-seated tumors, and to provide this as a tumor diagnostic agent.

[Means for solving problems]

The following formula [II] which is a hydrolysis product of chlorophyll-a Pheophorbide-a is known to be effective as a medium for photochemotherapy of tumors [Photomedicin
e and Photobiology, 6 (2) 7-11 (1984)].

Moreover, as already revealed by the present inventors,
Pheophorbide-a is recognized to accumulate in tumors [Abstracts of the 43rd Annual Meeting of the Japanese Cancer Society (Showa 59) 446
page〕.

On the other hand, in the field of nuclear medicine in recent years, with the development of a positron-mography apparatus, a drug labeled with a radionuclide that releases a positron is administered in vivo, and the accumulation of tumors based on its metabolism in the body is measured from outside the body. Accurate tumor diagnosis is now possible [Eur J Nucl Med
7 294-297 (1982)].

In order to solve the above-mentioned problems, the present inventor has studied the combination of the compound (pheophorbide-a) represented by the formula [II] and various radioactive metal ions, and as a result, represented by the formula [I]. The present inventors have found that the novel pheophorbide derivative described above is suitable for this purpose, and completed the present invention.

Further, the drug according to the present invention is also administered as a therapeutic drug for inhibiting the growth and killing of the tumor by the radiation effect of β ⁺ and γ rays released from the drug by accumulating the drug in the tumor. It is useful.

Hereinafter, a general method for producing the compound of the formula [I] will be described.

The compound of the formula [II] is dissolved in an equal amount of acetic acid, an appropriate amount of ⁴⁸ V chloride or sulfate is added thereto, and this solution is heated at 110 ° C to 120 ° C.
When heated at reflux for 1 to 3 hours, metal ions are introduced into pheophorbide, and the reaction liquid changes from dark brown to dark green. After the reaction, the solvent was distilled off under reduced pressure, and the pheophorbide metal chelate compound was extracted from the residue with a mixed solvent of methanol 2% phosphoric acid (9: 1 V / V) to obtain unreacted metal chloride or metal sulfate. Be more separated.

When this is distilled under reduced pressure, a green to dark green compound of the formula [I] is obtained.

Although the compound of formula [I] is insoluble in water, methanol,
It is soluble in organic solvents such as acetone, benzene and chloroform.

Further, it is shown that a solution of the compound of the formula [I] in methanol or acetic acid has an absorption maximum in the visible region of 660 to 670 nm and 400 to 410 nm and retains the chlorin ring which is the basic skeleton of the chlorophyll derivative.

Next, the tumor diagnostic effect using the compound of the present invention will be described.

[Experimental Example 1] Mouse breast cancer (FM3A) was subcutaneously transplanted on the back (C3H /
He), ⁴⁸ VOSO ₄ and ⁴⁸ VO-pheophorbide-
2 aCi was intravenously injected through the tail vein.

Killed at 2 hours, 12 hours, 24 hours, 48 hours after administration,
The radioactivity distribution to each organ was measured using a γ-ray counter. The results are shown in Tables 1 and 2.

The highest uptake of ⁴⁸ VOSO ₄ was found in the lungs, followed by liver, kidney and bone uptake, but low uptake in tumors. On the other hand, unlike the distribution of ⁴⁸ VO-Pheophorbide-a in the body, uptake into the lung was low, and uptake into the kidney, liver and spleen was high. Uptake into blood was high and decreased significantly over time. Tumor uptake was high and increased over time. That is, the in vivo kinetics of ⁴⁸ VO-pheophorbide a reached a peak in uptake after 2 hours in normal tissue and was highest in the kidney. Excretion from normal tissues was very smooth, but there was a tendency to gradually accumulate in the tumor, with the highest level after 24 to 48 hours.

[Experimental Example 2] Whole-body autoradiogram of ⁴⁸ VO-administered rat Using the mouse to which ⁴⁸ VOSO ₄ , ⁴⁸ VO-pheophorbide-a was administered by the same method as described in Experimental Example 1, 2 hours and 24 hours after administration, A whole body autoradiogram was prepared by a conventional method. The degree of incorporation of ⁴⁸ VO compounds was quantified by computer image processing of this autoradiogram. The results are shown in FIGS.

As can be seen in Figure 4, ⁴⁸ VO-Pheophorbide-
Highest uptake of a into tumor (5.0%) dose / g tis
On the contrary, ⁴⁸ VOSO ₄ had a low 1.0% dose / gt issue. Furthermore, the tumor-muscle ratios are 4.17 and 2.08, respectively, and it is clear that ⁴⁸ VO-Pheophorbide-a can draw a tumor with good contrast.

As shown in the above experimental examples, the compound represented by the formula [I], ⁴⁸ VO-Pheophorbide-a, has a tumor tissue affinity and radioactivity, so that it is possible to detect a tumor development site in any part of the body. It is possible.

As nuclides other than ⁴⁸ V, Zn, Cu, Ni, Co, Fe, Mn, Ag, I
n, Hg, Tl, Sn, Pt, Rh, Ir, Cd, Si, Ge, Pb, Ga, Cr, Mo, Zr, Hf, Eu,
Pheophorbide complexes of radioisotopes of elements such as Pr, Yb, Y, Th, Ta, W, re, Os, Nb, Sb, Bi, Re, Ru and Ti also satisfy the object of the present invention. , ⁴⁸ V, ⁴⁵ Ti are the most effective.

Especially, ⁵⁵ Co, ⁵² Fe, ⁵⁴ Fe, ⁴⁴ Sc, ⁵² Mn, which are frequently used in nuclear medicine
⁵¹ Cr, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁷¹ Ge, ²⁰¹ Tl etc. are valid, Is a promising therapeutic agent.

Next, the results of testing in rats for acute toxicity of the compound represented by the formula [I] are shown by LD ₅₀ (mg / kg).
It is as shown in the table.

Next, the compound represented by the formula [I] can be made into an injection by using a suitable solution material, auxiliary agent, filler, carrier and the like used in the preparation, according to a conventional method for preparation of the preparation. It can be administered intravenously, intramuscularly, intradermally, subcutaneously or intraperitoneally.

The effective dose of the compound represented by the formula [I] is appropriately selected depending on the purpose of use, but usually 10 μg to 50
Although it is in the range of about 0 μg / kg, it is considered appropriate.

〔Example〕

Next, examples of the present invention will be described. Naturally, the following merely exemplifies the preferred embodiments of the present invention, and the present invention is not limited to these embodiments.

Example 1 13.7 mg ( ₄ mCi) of vanadyl sulfate ( ⁴⁸ VOSO ₄ ) is dissolved in 5 ml of acetic acid, to which 5.0 mg of pheophorbide-a is added. The mixture is heated to reflux at 100-120 ° C for about 2 hours. After the reaction, the solvent is distilled off under reduced pressure, and the residue is extracted with 10 ml of a mixed solvent of methanol-2% phosphoric acid (9: 1 V / V). The extract was distilled under reduced pressure, and the residue was crystallized from chloroform-hexane (1: 2 V / V) to obtain 3.5 mg of [I] dark green crystals.
The yield was 70%.

The physicochemical properties of this product are as follows.

・ Visible spectrum (in acetic acid solvent): Fig. 1 ・ Infrared spectrum (in chloroform solvent): Fig. 2 ・ Thin layer chromatogram That is, the ⁴⁸ V-labeled phophorbide-a produced in this example was used as a developing solvent (benzene: hexane: acetic acid = 10:
When analyzed by cellulose thin layer chromatography at 5: 1), the product gave a spot just below pheophorbide-a as shown in FIG. On the other hand, vanadyl sulfate that did not react remained at the origin.

In addition, the UV absorption spectrum of the acetic acid solution of vanadyl pheophorbide-a thus produced is shown in FIG. 1, and the absorption of the soret band characteristic of porphyrin is observed at 407 nm.
Four absorptions in the Q band (500 to 600 nm) observed with free pheophorbide-a have disappeared, and the spectrum has shifted to the short wavelength side. Furthermore, it does not fluoresce when illuminated with an ultraviolet lamp. The other facts indicate that vanadyl sulfate reacts with pheophorbide-a to form vanadyl pheophorbide-a complex. Formula [I] coordinated by porphyrin
It is recognized as the street.

Example 2 0.35 mg of ⁴⁸ VO-pheophorbide-a (100-200
was dissolved in dimethyl sulfoxide of mu), an emulsion 2.4ml and phosphate buffer 0.8ml of egg yolk lecithin was added to this solution, subjected to about 4 minutes emulsification operation in an ultrasonic emulsifier, ⁴⁸ VO
-Pheophorbide-a was incorporated into liposomes, and distilled water was added to bring the total volume to 4 ml.
It was filtered with a membrane filter having a hole diameter of 45 μm to obtain an injection solution.

By this method, liposomes having a diameter of 0.1 to 0.2 μm were obtained. The emulsification yield was 80-90%.

〔The invention's effect〕

According to the present invention, the present invention is extremely useful as a diagnostic agent for tumors because it detects the site of a tumor generated deep inside the body using a γ-ray camera or a positron camera with γ-rays by administration of a low concentration.

[Brief description of drawings]

FIG. 1 is the visible spectrum of ⁴⁸ VO-pheophoride-a obtained in the example, FIG. 2 is the infrared spectrum of the compound, and FIG. 3 is the compound and the pheophor of the formula [II]. Thin-layer chromatogram of Bide-a, FIG. 4 shows administration
Comparison of distribution of ⁴⁸ VOSO ₄ and ⁴⁸ VO-Pheophorbide-a after 24 hours, and FIG. 5 is a diagram showing distribution comparison of 2 hours and 24 hours after administration of ⁴⁸ VO-Pheophorbide-a.

 ─────────────────────────────────────────────────── --- Continuation of the front page (56) Reference JP-A-57-185220 (JP, A) JP-A-60-81128 (JP, A) JP-A-61-83185 (JP, A) JP-A-60- 23451 (JP, A)

Claims (2)

[Claims] 1. A novel pheophorbide derivative represented by the formula [I]. (In the formula, V is ⁴⁸ V.) 2. A tumor diagnostic agent comprising a compound represented by the formula [I] as an active ingredient. (In the formula, V is ⁴⁸ V.)