JP5182858B2 - Sensitizer for X-ray therapy - Google Patents

Description

  The present invention relates to a sensitizer for X-ray treatment that exhibits an effect of damaging or killing malignant neoplasms such as cancer cells, cancer precursor cells, cells infected with viruses and bacteria by X-ray irradiation.

  In conventional radiotherapy, when high-energy X-rays or gamma rays are intensively irradiated to a tumor, intracellular water is decomposed into hydroxy radicals and hydrogen atoms, and reacts with intracellular oxygen to produce superoxide and hydroperoxy Radicals are generated. These active oxygens and free radicals cause direct damage to DNA in the cell, and indirectly, by extracting hydrogen from lipids in cell membranes and transforming them into lipid peroxides through a chain of peroxide radicals, causing cell membrane damage. There are two systems of damage pathways. However, since it is not possible to selectively irradiate only the target cells with radiation, these reactions also proceed in normal cells around the affected area, and there are tumor cells and normal cells in the path and around the affected area. The result is damaging without discrimination.

In order to perform radiation therapy more effectively, sensitizers that suppress normal tissue damage and specifically increase the radiosensitivity of tumors are being developed, and metalloporphyrins are used as such sensitizers. It has been proposed. (For example, see Patent Documents 1 and 2)
Japanese Unexamined Patent Publication No. 2006-289897 JP-T-2005-504012

  The metalloporphyrins described in these patent documents contain a metal ion such as divalent or trivalent iron with a porphyrin derivative as a skeleton, and have a structure having a plurality of benzene rings in the side chain. Compared to protoporphyrin, hematoporphyrin, porphyrin salt and the like, which are derived from the living body, the structure is large and complicated. Therefore, there is a possibility that the uptake into tumor cells may be reduced, or the catalytic action of radicals by iron proceeds to damage the cells.

On the other hand, a hematoporphyrin derivative, which is a kind of porphyrin compound, is used for photodynamic therapy (PDT) in which cancer is treated by combining a laser and a photosensitive substance as a photosensitive substance. The mechanism of action is due to singlet oxygen ¹ O ₂ and when a photosensitive substance is irradiated with light of a specific wavelength, the molecule absorbs light and the energy of the light raises the molecule to an excited state, and then the excitation energy Is passed to the oxygen molecule, and at the same time it converts the oxygen molecule to ¹ O ₂ , the dye molecule returns to the ground state. ¹ O ₂ oxidatively damages the residues of protein methionine, tryptophan, histidine, and cysteine, and reacts with unsaturated fatty acids to produce lipid peroxides. PDT uses a red laser as an excitation light source, and thus has low permeability to living tissue, and is limited to the affected area about 1 cm deep from the surface, and is not suitable for large tumors or deep tumors.

  Therefore, the present invention can be applied to a deeply affected area when the target cells of the affected area such as a tumor are treated by irradiating X-rays by solving the problems of the prior art. To provide a low-exposure and minimally invasive sensitizer for X-ray treatment that can efficiently transfer the energy of the target to the target cell to damage the target cell and reduce damage to surrounding normal cells And

As a result of intensive studies, the present inventors have found that certain porphyrins such as protoporphyrin derived from a living body having no benzene ring in its side chain and its sodium salt have low cytotoxicity when X-ray irradiation is not performed, and X-ray irradiation The present invention has been completed by finding that the sensitizing action can be efficiently expressed only at times.
That is, the present invention employs the following configuration.
1. A sensitizer for X-ray therapy containing a compound selected from the group consisting of protoporphyrin, protoporphyrin sodium, hematoporphyrin and 5-aminolevulinic acid as an active ingredient.
5-Aminolevulinic acid (ALA) itself does not have photosensitivity, but protoporphyrin IX (PpIX) is selectively produced in the tumor after in vivo administration, and is similarly sensitized for X-ray therapy at the absorption wavelength of PpIX. It has an action as an agent. Rezaphyrin and photophyllin currently applied clinically in PDT have a porphyrin skeleton as a basic structure but do not have a benzene ring around it, and therefore have low cytotoxicity and exert a sensitizing effect by X-rays. It falls within the scope of the present invention.
2. 2. The X-ray therapeutic sensitizer according to 1, wherein the sensitizer for X-ray therapy exhibits a sensitizing effect with an absorbed dose of 10 Gy or less at a low energy X-ray of 200 keV or less, desirably about 1 to 100 keV. Sensitizer for radiation therapy.

The sensitizer for X-ray treatment of the present invention has the following remarkable effects.
(1) Since no transition metal ion is contained in the porphyrin ring, cell damage under conditions where X-ray irradiation is not performed can be suppressed. In addition, since it is a porphyrin derivative derived from animals and plants that do not contain an organic compound such as a benzene ring in its side chain, it can be easily taken into cells.
(2) Low X-ray energy in the region of diagnostic X-ray equipment (200 keV or less), lower X-ray energy than therapeutic X-ray generator (200 keV or more), and lower than conventional therapeutic dose (50-70 Gy) Sensitization effect is expressed in the range (10 Gy or less).
(3) By converting the absorbed X-ray energy into a redox reaction and generating reactive oxygen species or free radicals, the intensity of treatment can be controlled according to the amount of X-ray irradiation. It is.
(4) By using porphyrin that is biocompatible and is selectively taken into the tumor, it is possible to treat large tumors and deep tumors by long-distance X-ray irradiation.

In the present invention, a compound selected from the group consisting of protoporphyrin, sodium protoporphyrin, hematoporphyrin and 5-aminolevulinic acid is used as a sensitizer for X-ray treatment.
As shown in the following chemical formulas (1) to (3), protoporphyrin (1), hematoporphyrin (2) and protoporphyrin sodium (3) are living animals and plants that do not contain an organic group such as a benzene ring in the side chain. Since it is a derived porphyrin derivative, it can be easily incorporated into a living body as compared with a porphyrin derivative having a large and complicated structure having a plurality of benzene rings in the side chain described in Patent Document 1.

In addition, 5-aminolevulinic acid (ALA) itself is a compound represented by the following formula (4), which itself does not have photosensitivity, but selectively receives protoporphyrin IX ( PpIX) is produced and has the same action as a sensitizer for X-ray therapy at the absorption wavelength of PpIX.
H ₂ NCH ₂ COCH ₂ CH ₂ COOH (4)
Since these X-ray therapeutic sensitizers do not contain a transition metal ion in the porphyrin ring, they have low cytotoxicity when not irradiated with X-rays, and efficiently develop a sensitizing action only when irradiated with X-rays. be able to. Rezaphyrin and photophyllin currently applied clinically in PDT have a porphyrin skeleton as a basic structure but do not have a benzene ring around it. Therefore, cytotoxicity is low, and X-rays exert a sensitizing effect. This is within the scope of the present invention.

  The sensitizer for X-ray treatment of the present invention is an ordinary method such as injection, suppository, tablet, powder, granule, capsule, pill, ointment, liquid, patch, buccal, aerosol, etc. Various dosage forms. When manufacturing an injection, add an appropriate solvent, if necessary, pH adjuster, buffer, stabilizer, suspension, solubilizer, carrier, etc. Can do.

  As the stabilizer, sodium sulfite, sodium metasulfite and the like are used. As the suspending agent, for example, methylcellulose, polysorbate 80, gum arabic and the like are used. As the solubilizer, polyoxyethylene hydrogenated castor oil, nicotinamide, polyoxyethylene sorbitan monolaurate, or the like is used.

For injection, for example, an X-ray therapeutic sensitizer is dissolved, dispersed, emulsified or the like in advance in an aqueous carrier such as physiological saline for injection, or dissolved, dispersed or emulsified at the time of use as a powder for injection. It can manufacture by doing.
Examples of the administration method of the injection include intravenous administration, intraarterial administration, intraportal administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, and intralesional direct administration.

Solid preparations such as tablets, powders, granules, capsules, pills, suppositories, or liquids such as syrups are sensitizers for X-ray treatment, excipients, binders, disintegrants, lubricants, A coloring agent, a flavoring agent, a flavoring agent, a bulking agent, a coating agent, and the like can be added to produce the composition according to a conventional method. For example, a sensitizer for X-ray treatment may be formulated into tablets, powders, granules, capsules, pills, and syrups according to a conventional method and administered orally.
Ointments, liquids, patches, patches, aerosols can be manufactured from sensitizers for X-ray treatment and appropriate carriers by conventional methods, and may be applied directly to the lesion as an external preparation. . The sensitizers for X-ray treatment of the present invention may be used alone or in combination of two or more.

  The dose of the sensitizer for X-ray treatment of the present invention varies depending on the age, weight, sex, administration method, and symptoms of the patient, but is usually selected within a range of about 1-1500 mg per day per adult. . In general, irradiation is performed within 12 hours after administration of the sensitizer for X-ray therapy of the present invention, but it is more desirable to receive irradiation within 1 hour.

Next, the sensitizer for X-ray treatment of the present invention will be further described with reference to examples, but the following specific examples do not limit the present invention. In the following examples, X-ray irradiation and measurement of the fluorescence intensity of the sample were performed as follows.
(X-ray irradiation)
The X-ray source is Toshiba KXO-15E for X-ray diagnosis, and the prepared sample is placed at a position 18 cm away from the X-ray irradiation port. The X-ray is irradiated under the constant conditions of tube voltage 100 kV and tube current 4 mA. The absorbed dose was changed by changing the time.
(Measurement of fluorescence intensity)
Using a microplate spectrofluorometer (TECAN, infinite M200), the fluorescence intensity at 585 nm was measured when excited at a wavelength of 456 nm.

Example 1
Protoporphyrin (Protoporphyrin IX C ₃₄ H ₃₄ N ₄ O ₄ molecular weight 562.66) is suspended in a phosphate buffer, and its concentration is 0.3 μg / ml, 1.0 μg / ml, 3.0 μg / ml, 10.0 μg / ml, A sample solution of 30.0 ug / ml was prepared, and 100 μl of each was dispensed into each 96-well microplate.
Next, dihydroethidium, so that the final concentration of each sample is 25 μM.
Invitrogen-Molecular Probes) was added, followed by irradiation with X-rays of 0 Gy, 1 Gy, 2 Gy, 3 Gy, 5 Gy, and 10 Gy. FIG. 1 shows the result of measuring the fluorescence intensity (Ex 456 nm, Em 585 nm) of the sample after irradiation. The numerical value is a relative fluorescence intensity, and the higher the numerical value, the more active oxygen species or free radicals are produced.

(Example 2)
In the first embodiment, protoporphyrin sodium instead of protoporphyrin _{(Protoporphyrin Disodium Salt, C 34 H} 32 N 4 Na 2 O 4, 606.6, Tokyo Kasei) was used instead of the, X-rays in the same manner as in Example 1 The result of measuring the fluorescence intensity of the irradiated sample is shown in FIG.

(Example 3)
In Example 1 above, X-ray irradiation was performed in the same manner as in Example 1 except that hematoporphyrin (Hematoporphyrin IX base, C ₃₄ H ₃₈ N ₄ O ₆ , 598.71, Funakoshi) was used instead of protoporphyrin. The result of measuring the fluorescence intensity of the sample is shown in FIG.

(Comparative Example 1)
X in the same manner as in Example 1 except that iron porphyrin (Fe (III) Mesoporphyrin IX chloride, C ₃₄ H ₃₆ FeN ₄ O ₄ Cl, 656.0, Funakoshi) was used instead of protoporphyrin. FIG. 4 shows the result of measuring the fluorescence intensity of the sample irradiated with the beam.

Dihydroethidium reagents are derived from dihydroethidium (excitation wavelength: 385 nm, fluorescence wavelength: 480 nm) in the presence of active oxygen such as O _2- (superoxide anion), HO ₂ (peroxy radical), and HO (hydroxy radical). Since ethidium (excitation wavelength: 465 nm, fluorescence wavelength: 585 nm) is generated, it is used as one method for measuring active oxygen. (Reference: Nethery D, Stofan D, Callahan L, DiMarco A, Supinski G .: Formation of reactive oxygen species by the contracting diaphragm is PLA (2) dependent. J Appl Physiol. 1999 Aug; 87 (2): 792- 800.).

From these results, it was confirmed that in the specific porphyrin derivative used in the present invention, a large amount of active oxygen was generated by X-ray irradiation as compared with the conventional iron porphyrin.
Fe (III) Mesoporphyrin IX chloride, one of the iron porphyrins, showed almost no generation of active oxygen in the low-dose region of the diagnostic X-ray set in the above example, as shown in FIG. . This indicates that the porphyrin iron complex containing iron and the porphyrin iron complex inclusion albumin compound described in Patent Document 1 are not necessarily effective in a low dose region of 100 keV or less and 10 Gy or less.

Radiation therapy is thought to act by damaging the DNA of cells by cleaving DNA strands through free radicals generated in the vicinity of DNA by radiation. From this, protoporphyrin, protoporphyrin sodium and hematoporphyrin with a large amount of active oxygen generation such as O _2- (superoxide anion), HO ₂ (peroxy radical), and HO (hydroxy radical) In the set low dose range, it is more effective as an X-ray sensitizer than iron-containing porphyrin.

Example 4
HELA cells were seeded on a 96-well microplate, and cultured in a CO ₂ incubator using medium (MEM, 10% FCS) so as to be subconfluent. Separately, a sample was prepared by adding the protoporphyrin used in Example 1 to the medium (MEM, 10% FCS) so that the final concentrations were 0.3 μg / ml and 1.0 μg / ml.
The medium was sucked out from the microplate, and 30 μl of each sample was added to each 96-well microplate and incubated for 1 hour, followed by irradiation with 0 Gy, 1 Gy, 2 Gy, and 3 Gy X-rays. In this process, the influence of cell damage due to radiation itself increases as the X-ray dose increases, so the process was performed with a lower X-ray dose than in Example 1.
After X-irradiation, the sample was sucked out, the cells were detached by trypsin treatment, and seeded in a 25 cm ² flask. The medium is changed the next day, and after culturing for about 9 days so that the number of cells in the control group is 50 colonies or more, the cells are fixed with 4% formalin buffer, and the number of colonies of 50 colonies or more is image-processed. I counted. The results are shown in FIGS. Table 1 shows the inhibition of colony forming ability of porphyrins by 1 Gy X-ray irradiation, with the colony forming ability of the control group having a dose of 0 Gy as 1.

(Comparative Example 2)
In Example 4, except that the iron porphyrin used in Comparative Example 1 was used instead of protoporphyrin, the results of counting the number of colonies for the samples treated in the same manner as in Example 4 are shown in FIGS. Shown in

(Example 5)
In Example 4, instead of protoporphyrin, the protoporphyrin sodium used in Example 2 was used, and the X-ray irradiation dose was set to 1 Gy. Table 1 shows the results of counting the number and evaluating the inhibition of colony forming ability.

(Example 6)
In Example 4, instead of protoporphyrin, hematoporphyrin used in Example 3 was used, and the X-ray irradiation dose was set to 1 Gy. The number of colonies for each sample treated in the same manner as in Example 4 Table 1 shows the results of the evaluation of colony forming ability inhibition.

As described above, in the diagnostic X-ray region where the X-ray energy is 100 keV or less, which is lower than that for conventional treatment, and the absorbed dose is 3 Gy or less, which is lower than that for conventional radiation therapy, the protoporphyrin is added to the X-ray alone than X-ray alone It was confirmed that the colony formation rate was low even at the same X-ray dose.
This indicates that what enhances the effect of tumor cell damage by X-ray irradiation due to the presence of the sensitizer of the present invention is effective. Furthermore, under the irradiation conditions described above, it was confirmed that the porphyrin containing no iron had a lower colony formation rate than the porphyrin containing iron.

The damage to higher animals caused by radiation stems from the effects on this cell that make it up, particularly its cell death. Cell death includes proliferative death and interphase death. When a high dose of radiation is irradiated, damage to a target other than a DNA molecule directly results in a state called interphase death in which the cell is killed. In radiotherapy, proliferation death due to damage to DNA in the cell nucleus is important. Proliferative death means that cells that originally have proliferative ability lose their ability to divide. Therefore, even if the cells continue to metabolize normally, if one cell does not grow into a cell population that is visible as a colony by culturing, that is, if it loses the ability to grow, it is regarded as proliferative death. When considering a cultured cell population, the number of dead cells increases and the number of colonies decreases with increasing radiation dose.
For this reason, the radiation effect on cells has been established by a quantitative index called colony formation rate, and has been used for a long time to predict the results of animal experiments (TT Puck and PI Marcus, J Exp. Med. 103, 653-666 (1956)).
Therefore, as seen in each of the above examples, the effect of animal experiments can be accurately predicted based on the two data of the amount of reactive oxygen species or free radicals generated and the colony formation rate indicating cell damage.

The present invention provides a sensitizer for X-ray therapy that is useful in treating or treating malignant neoplasms such as cancer cells, cancer precursor cells, cells infected with viruses and bacteria in radiation therapy. By using this linear sensitizer, it is possible to perform a minimally invasive treatment using radiation at a low exposure dose in which X-ray irradiation is limited to the affected area.
Sterilization and sterilization by sterilization of bacteria (Escherichia coli, endotoxin, O157, verotoxin, MRSA (methicillin-resistant Staphylococcus aureus), enterotoxin, chlorophyll), toxic degradation, and oxidative degradation, damage, and death of bacteria and viruses It is also possible to perform.

In Example 1, it is a figure which shows the result of having measured the fluorescence intensity (Ex 456nm, Em 585nm) about the sample after X-ray irradiation. In Example 2, it is a figure which shows the result of having measured the fluorescence intensity (Ex 456nm, Em 585nm) about the sample after X-ray irradiation. In Example 3, it is a figure which shows the result of having measured the fluorescence intensity (Ex 456nm, Em 585nm) about the sample after X-ray irradiation. In Comparative example 1, it is a figure which shows the result of having measured the fluorescence intensity (Ex 456nm, Em 585nm) about the sample after X-ray irradiation. In Example 4 and Comparative Example 2, it is a figure which shows colony formation ability inhibition about the sample after X-ray irradiation. In Example 4 and Comparative Example 2, it is a figure which shows colony formation ability inhibition about the sample after X-ray irradiation.

Claims (1)

Protoporphyrin, protoporphyrin sodium c arm及 beauty X-ray therapy sensitizer containing a compound selected from the group as an active ingredient consisting of 5-Aminorevurin acid.