RU2074719C1 - Method of mammalian tumor cell destruction and pharmaceutical composition for mammalian tumor cells destruction - Google Patents

Abstract

FIELD: medicine, oncology. SUBSTANCE: mammary tumor cells were treated by light and specific inhibitor of enzymatic conversion of protoporphyrinogen to protoporphyrin-IX catalyzed by protoporphyrin oxidase in the presence of porphyrin. Method involves the use of pharmaceutical composition containing specific inhibitor of enzymatic conversion of protoporphyrinogen to protoporphyrin-IX catalyzed by protoporphyrin oxidase. Index I₅₀ for protoporphyrin oxidase is less 1 mcM and negative reductive-oxidizing potential is above -500 mV. EFFECT: improved method of tumor cells destruction. 11 cl, 5 tbl, 1 dwg

Description

 The invention (one aspect) relates to the photodynamic treatment of a mammary gland with tumor cells, as is the case for the photodynamic treatment of cancer.

 A known method of introducing a hematoporphyrin derivative or a mixture of porphyrins that are derivatives of this compound, for example, the introduction of the drug "Photofrin II" (Photofrin II) in breast tumor cells with subsequent exposure to these cells of light radiation of a certain wavelength in order to cause destruction of cells. This method of photodynamic therapy is the subject of a series of articles composing a special issue of the journal Photochemistry and Photobiology, 1987, v. 46, No. 5 (the abbreviation P P 46 5 is adopted below). These articles show that photodynamic therapy has found widespread use in the treatment of various cancers (including cancer of the bronchi, bladder, esophagus, lungs, skin, organs of the head and neck, brain, colon, and intraocular and gynecological cancers diseases). The biochemical effects of this porphyrin photosensitization are said to include cross-linking by proteins and membranes, lipid peroxidation, inhibition of the transfer of certain important metabolites, lysosomal and lysosomal lysosomal membranes, inhibition of the activity of a number of enzymes, and increased uptake of porphyrins by cells (PP 46-5, p. 695). Porphyrin drug is usually administered by injection (for example, intravenously or in the abdominal cavity); a typical dose is 2 mg of Photofrin per 1 kg of body weight.

 In addition, the porphyrin preparation can be incorporated into the liposome and injected (for example, into the abdominal cavity) as indicated, in particular, in the article by Spikos et al. "Photodynamic behavior of porphyrins in model cellular, tissue and tumor systems" (Photodynamic Behavior of Porphyrins in Model Cell, Tissue and Tumor System) in Photodynamic Therapy of Tamor and other Diceases, edited by Jori and Perria; Padua, ed. Libreria Progretto 1985, p. 45 53, as well as in the special issue mentioned above.

 Special literature states that the introduction of photoporphyrin into cells such as, for example, human erythrocytes or murine cells in leukemia, gives a noticeable photosensitizing effect, see respectively: Dubbelman et al. Biochimica et Biophysica Acta, 1978, t 511, p. 141 151; Kessel (Kessel), (Cancer Research) 1982, v. 42, No. 5, p. 1703 - 1706. Kessel's article states that of all the photosensitizing reagents tested, photoporphyrin is the most active. However, when Kessel and other researchers, such as Berenbaum et al. (see Br. J. Cancer, 1982, v. 45, p. 571 581) injected animals with photoporphyrin, no significant photoporphyrin content was found in tumors, indicating a possible rapid ablation of photoporphyrin introduced into the body in a similar way due to blood circulation .

 The use of a hematoporphyrin derivative and similar preparations for the diagnosis and localization of tumors, such as bladder or lung cancer, is also well known (see, for example, P P 46 5, p. 759).

 The active compound used according to one aspect of the present invention for treating cells in the photodynamic therapy under consideration or in known methods for diagnosing and localizing tumors is not porphyrin or not only porphyrin.

 Unlike pure porphyrin, this compound inhibits the enzymatic termination of photoporphyrinogen in the heme structure of these cells, thereby causing the formation of endogenous photoporphyrin IX in these cells. The authors found that reagents such as certain types of herbicidal compounds that inhibit the enzymatic conversion of porphyrinogens to chlorophyll in plant cells also inhibit the enzymatic conversion of porphyrinogen in the heme structure of breast cells. The authors believe that the mentioned inhibition by these reagents probably affects the stage of conversion of photoporphyrinogen to photoporphyrin IX under the action of an enzyme (photoporphyrinogen oxidase, EC 1.2.3.4) so that photoporphyrinogen no longer undergoes enzymatic conversion to photoporphyrin IX in a normal way, but instead undergoes intracellular oxidation (for example, in plasma) outside the normal enzymatic process (for example, all organelle shells), as a result of which photoporphyrin IX accumulates in areas where it is not accessible to normal th conversion to heme, and as a result of the irradiation of the accumulated thus fotoporfirin IX cell light effect causes destruction of cells similar to that observed in the case of the above-described photodynamic therapy.

Enzyme inhibitory active compounds of the present invention are specific inhibitors of photoporphyrinogen oxidase in the sense that they do not function like ordinary enzyme poisons like reagents that cause denaturation or crosslinking (for example, like reagents based on sulfhydryl); they are also not primarily such agents that affect the oxidation conditions as electron acceptors, and therefore, the active compounds preferred according to the present invention have a more negative redox potential than 500 mV and even a negative redox potential than 500 mV and even still negative, namely equal to 800 mV (measured, for example, by the usual method in a solvent suitable for the given compound: by the method of cyclic voltametry or polarog aficheskim method). It is also preferred that the active compound is not tetrapyrrole and that its I ₅₀ for photoporphyrogen oxidase is less than 1 μM, for example, less than 0.3 μM; in particular, the I ₅₀ value is approximately 0.1, 0.03 or 0.01 μM even less.

 Preferably, such an enzyme inhibitory active compound that is characterized by an increased ability to destroy the plasmalemma (Plasmalemma) of herbal preparations. One method for testing this ability is described under the title “Flushing Experiment” in a paper by Halling and Peters; Plant Physiology, 1987, v. 84, pp. 1114-6.

According to this test (described in more detail below in Appendix A), the preferred reagents show at least 50% total leaching at a treatment level of 100 μM, preferably at a treatment level of 1 μM or less, for example, at 100 nM. Highly active drugs, such as 1- (4-chloro-2-fluoro-5-propargyloxyphenyl) -3-methyl-4-difluoromethyl-Δ ² -1,2,4-triazolin-5-one or lactophen (see below) show a total washout of over 90% at a concentration of 100 nM.

 Another method for determining the ability of a drug to destroy the plant plasmalemma is the so-called. "Testing the inhibition of greening caused by light irradiation", described in more detail below in Appendix B. This method is intended to determine the ability of the drug to suppress the greening of bleached in the dark cultures of microorganisms Clamydomonas reinhardi, y-1 (a type of aglae microorganisms that grow in the dark without forming chlorophyll). At the same time, it is understood that the algae mass is bleached due to the presence of fresh, green cells in it, but again forms chlorophyll in the light.

Many of the preferred active compounds used according to the present invention are able to suppress greening caused by light irradiation by at least 50% at a concentration of the compound used 10 ^-5 M, preferred at 10 ^-6 or less, for example, at a concentration of 10 ^-7 M In addition, this compound should be such that, during the test of greening inhibition, it forms a product that floats to the surface with a maximum light absorption at 405 nM, which exceeds the maximum for chlorophyll constituting d in this system is 669 nM; for example, a pop-up product shows at 405 nM a maximum that is 2.3 or 4 times higher than the maximum at 668 nM.

 Among the inhibitory enzymes of the active compounds that can be used in the practice of the present invention, there are the following types of herbicides (A-D).

где Q означает замыкающую группу гетероциклического кольца, а R водород или замещающую его группу. Данный класс соединений включает также препараты, которые в статье Вакабаяси (Wakabayshi) и др. (см. J. Pesticide, Sci. 1986, т. II, с. 635 460) определены как класс гербицидов на основе циклических имидов, причем на с. I этой статьи представлены следующие соединения:

Соединение I представляет собой гербицид на основе арил-оксидиазола, а именно 2-трет. -бутил-4-(2,4-дихлор-5-изопропоксифенил)-Δ²-1,3,4-оксидиазолин-5-он. Другие 2-алкил-4-(2,4,5-тризамещенный фенил)-Δ²-1,3,4-оксидиазолин-5-оны, которые можно использовать согласно настоящему изобретению, охарактеризованы, например, в патентах США N 3385862, 3836539, 3876413.A. Aryl-heterocyclic herbicides of the general formula:
Ph nhet
Ph substituted phenyl, preferably 2,4-disubstituted phenyl, even more preferably 2,4,5 trisubstituted phenyl, and NHet a 5- or 6-membered heterocyclic ring (with 1 to 4 nitrogen atoms in the ring) of the formula:

where Q means the closing group of the heterocyclic ring, and R is hydrogen or a substituent group thereof. This class of compounds also includes preparations that are defined in the article by Wakabayshi et al. (See J. Pesticide, Sci. 1986, vol. II, p. 635 460) as a class of herbicides based on cyclic imides, and on p. I of this article presents the following compounds:

Compound I is an aryl-oxydiazole-based herbicide, namely 2-tert. -butyl-4- (2,4-dichloro-5-isopropoxyphenyl) -Δ ² -1,3,4-oxydiazolin-5-one. Other 2-alkyl-4- (2,4,5-trisubstituted phenyl) -Δ ² -1,3,4-oxydiazolin-5-ones that can be used according to the present invention are described, for example, in US patent N 3385862, 3836539, 3876413.

 Compound II is an aryl-tetrahydroindazole herbicide, namely 3-chloro-2- (4-chloro-2-fluoro-5-isopropoxyphenyl) -4,5,6,7-tetrahydro-2H-indazole.

 Compounds III, IV and V are aryl-tetrahydrophthalimide-based herbicides. Other aryl tetrahydrophthalimides that can be used according to the present invention are described, for example, in US Pat. Nos. 4,431,822, 4,670,046, 4,670,042, 4,439,229, as well as in international patent application laid out WO 87/07062. The last two sources also describe other NHet rings, the use of which is also permissible. Examples of such rings are given in particular on p. 12 14 of international application N WO 87/07602, as well as in US patent N 4439229 (from line 25 on p. 4 on line 20 on p. 5).

Other suitable NHet type P herbicides are:
aryl-trialinones, such as those described in US patents nos. 4318731, 4398943, 4404019, 4702945, 4705557, 4702763, 4761174, as well as in international applications under the numbers WO 85/01637, WO 85/04307, WO 87/00730, WO 87/03782, and WO 86/04481 and WO 88/01133;
aryl-tetrazolinones, such as those described in US patent under number 4734124, as well as in international applications under the numbers NN WO 85/01939 and WO 87/03873;
aryl-triazinediones, such as those described in US patents Nos. 4,755,217 and 4,766,233, as well as in international application N WO 86/00072;
aryl hydantoins, such as those described in US Pat. No. 4,427,438;
aryl-urazoles, such as described in US Pat. No. 4,452,981; and aryl-hexahydropyridazines, such as described in US Pat. No. 4,619,687.

 B. Aryl-heterocyclic urethanes, such as those described in US patent N 4521242.

Метил-5-(2,4-дихлорфенокси)-2-нитробензоат ("Бифенокс")

5-(2-(2-хлор-4-(трифторметил)-фенокси)-2-нитробензоат натрия ("Ацифлуоросфен")

2-хлор-1-(3-этокси-4-нитрофенокси)-4-трифторметилбензол ("Оксифлуорфен")

Другими подходящими и имеющимися в продаже гербицидами на основе простых дифениловых эфиров являются следующие соединения.C. Phenyl ether-based herbicides that have a para-halo or para-nitro-phenoxyphenyl structure, namely, such as the following commercially available preparations:
5- [2-chloro-4- (trifluoromethyl) phenoxy] -2-nitro-N-methanesulfonylbenzamide ("fomesafen")

Methyl 5- (2,4-dichlorophenoxy) -2-nitrobenzoate (Bifenox)

5- (2- (2-chloro-4- (trifluoromethyl) -phenoxy) -2-sodium nitrobenzoate (“Acifluorosthen”)

2-chloro-1- (3-ethoxy-4-nitrophenoxy) -4-trifluoromethylbenzene (“Oxifluorfen”)

Other suitable and commercially available diphenyl ether herbicides are the following compounds.

"Lactofen": 1- (carboethoxy) ethyl 5% (2-chloro-4-trifluoromethyl-phenoxy) -2-nitrobenzoate;
Fluoroglucophene: (carboethoxy) methyl 5- (2-chloro-4-trifluoromethylphenoxy) -2-nitrobenzoate;
Fromesophen: 5- [2-chloro-4- (trifluoromethyl) phenoxy] -N-methyl-sulfonyl-2-nitrobenzamide;
Chloronitrophen: 2,4,6-trichloro- (4-nitrophenoxy) benzene;
Chlorodifene: 2-nitro-1- (4-nitrophenoxy) -4-trifluoromethyl-benzene;
Nitrofen: 2,4-dichloro-1- (4-nitrophenoxy) benzene;
Chlomethoxifene;
4- (2,4-dichlorophenoxy) -2-methoxy-1-nitrobenzene.

 In addition, another suitable diphenyl ether is 5- (2-chloro-4-trifluoromethylphenoxy) -2-nitroacetophenone oxime-O-acetate.

 1. Herbicides based on aryl-pyrazoles, such as those described in US patents NN 4563210, 4496390 and 4459150, as well as in German patent application laid out N 3530327 A1.

где х^b кислород или сера, а Рh имеет смысл, указанный выше; данные соединения описаны в Европейской патентной заявке под N 273417, а также в каталоге Derwtnt Abstracts (см. N 87-040749).

where x ^{b is} oxygen or sulfur, and Ph has the meaning indicated above; these compounds are described in European patent application N 273417, as well as in the Derwtnt Abstracts catalog (see N 87-040749).

Treatment with the active compounds of the present invention can be carried out by injection both intravenously and into the abdominal cavity. The active compound can be used in the form of a sterile composition, which is a reagent dissolved or dispersed in a pharmaceutically acceptable carrier, for example, in an aqueous isotonic solution, such as salt water (0.9% NaCl) or saline Dulbecco buffer solution (concentration about 2.5 mg / ml. Alternatively, the active compound may be incorporated into the liposome system, such that it can be prepared in a phospholipid medium in the same way as described on page 1659 - 1660 directory ^Kemington, s Pharmacentical ^Science, 17th Edition company "Mack Publishing K ^{o" (Mack Publishing C o)} . For example, similar to the recipe described by Yori et al. (see Br. J. Cancer, 1983, v. 48, p. 307), 51.6 mg of dipalmitoyl diphosphatidylcholine can be dissolved in 10 ml of a 1 mM reagent solution in a mixture of chloroform with methyl alcohol (volume ratio 9: 1), followed by removal solvent (after thorough thorough mixing) under vacuum at 30 ^o C, resulting in a solid that can be re-suspended in 10 ml of 0.01 M phosphate buffer solution with a pH value of 7.4, containing 150 mm NaCl, s followed by sonication turbid solution at 50 ^o C for 30 mi .

 Enzyme inhibitory reagents that can be used in the practice of the present invention are allowed to be coupled or bound to suitable monoclonal antibodies specifically targeting tumors (MABs) using a binding technique well known to those skilled in the art as a means of delivering an enzyme inhibiting reagent to specific area affected by the tumor.

 The enzyme-inhibiting active compounds used according to the present invention can also be administered by simple administration by swallowing, preferably in a diluted form, together with a pharmaceutically acceptable carrier, and also by adding them to food, as shown below in Example 6.

 It is advisable, especially when administering the drug by swallowing, to use enzyme inhibiting reagents, which are water-soluble salts or acidic compounds that form water-soluble sodium or potassium salts, i.e., such as sulfamide of the kind described in International applications N WO 87/037873 (e.g., BS reagent in Example 6 below), as well as N WO 85/001939, N WO 87/037873 or a water-soluble salt thereof, or a corresponding carboxylic acid or a water-soluble salt thereof, for example, a sodium salt known under called eat "acifluorfen."

 The excimes of the active compounds used according to the present invention may be included in the usual pharmaceutical preparations, such as tablets (for example, compressed tablets, on which sugar paste and / or syrup can be applied), suppositories, capsules (for example, hard gelatine capsules), suspensions solutions, powders or ampoules. In such preparations, the active component may be mixed with a pharmaceutically acceptable solid or liquid carrier, which may be a food product if desired. For example, it can be a solid product, be a food product. For example, it may be a solid product, such as corn starch, or a liquid diluent, such as water or edible or mineral oil, or a solvent, dimethyl sulfoxide and the like. Mixtures of inhibitory enzymes of the active components can be used, for example, a mixture of the active component 6c shown in Example 6 below with “Acifluorfen” in approximately equal amounts. The prescribed dosage can be determined by the usual experimental method, well known to specialists in this field, by the experimental procedure described for the Photophrin preparation in the Dafferty article “Photodynamic Therapy (PDT) of Malignant Tumors”, see CRC Critical Reirews in Oncology / Hemotology, 1984, vol. 2, no. 2, p. 83 116.

 The following examples are provided below to illustrate the present invention.

PRI me R 1. HeLa cells (a line of human tumor cells commonly used in the study of tumors, derived from the American Type Cneture Collection culture bank), located in the phase of logarithmic growth (grown at 37 ^o C for 5 days in one-liter Falcon tissue culture flasks) were washed in saline phosphate buffered saline (SBS). Next, a 0.25% solution of trypsin in 2 ml of SFBR was added to them. After a few minutes, the preparation was carefully removed from the flask and placed in a beaker containing 100 ml of a 25 mM HEPES solution in a “Minimum Essential Medium” supplemented with 10% (fractional fraction) of inactive calf serum to MEM. 1.1 ml of a 200 mM glutamine solution in 0.85% saline, as well as 11,000 units of penicillin per 11,000 μg of streptomycin.

The resulting cell culture, reconverted to suspension, was placed in 5.0 ml portions in 50 ml Falcon tissue culture flasks and (with the exception of the control sample) mixed with the drug active compound. These portions were further kept in the dark at 37 ^o C for 3 days. After that, the cell culture was extracted and subjected to extraction under green light as follows.

 Cells were raised from the bottom of the flasks by adding 0.8 ml of a 0.25% solution of trypsin in SFBR. After incubation in the dark for 1 h, the cells and medium were removed from the flasks, placed in round-bottom centrifugation tubes, and subjected to two thawing freezing cycles to destroy the cells and allow extraction.

The study of cell suspensions after this destructive operation did not reveal intact cells. Then, 10 ml of acetone with the properties of a base (90% acetone 10% 0.1N NH OH) was added to each tube, after which the tubes were centrifuged to remove protein and pieces of cells. 50 ml of water and then 0.5 ml of a saturated aqueous solution of NaCl were added to the substance that floated to the surface. The pH was then adjusted to a value of 6.8 by dropwise adding a 2 M KH ₂ PO ₄ solution. The BakerPrep type C8 column was then filled with acetone-water extract. The columns were dried and each washed with 2 ml of water. Tetranyrroles were washed with a mixture of CH ₄ OH: H ₂ O at a ratio of 90 10 taken in a volume of 2 x 1.5 ml. After that, the extracts were quantified on a spectrofluorometer.

 In the experiment under consideration, the following drug active compounds were used.

 A. 5-aminolevulinic acid, known as an intermediate in the synthesis of terapyrroles.

 Active compound A was supplied as sterilized by filtration. A was delivered as a sterilized by filtration of a 250 mM aqueous solution (pH 6.5). Active compounds B, C, I and E were delivered as a 50 mM solution in acetone. Acetone was also added to the control sample so that the concentration in it was 0.2% (v / v). In cell cultures were introduced such quantities of these active components that would provide the concentrations shown below in table. 1. The same table shows the amounts of protoporphyrin IX tetrapyrrole obtained (Proto IX).

П р и м е р 2. Клетки Нe La в логарифмической фазе роста выращивали в течение 6 сут. в шести однолитровых фальконовских колбах для тканевых культур при 37^oС, после чего промывали фосфатным буферным солевым раствором (ФБСР). Далее туда добавляли 0,25% -ный раствор трипсина в СФБР и через несколько минут клетки осторожно извлекали и помещали в стаканы, содержащие по 110 мл 25 мМ раствора HEPES в минимально-эфирной среде (МЕМ) с добавлением 10% (об. доли) неактивированной телячьей сыворотки, 1,1 мл 200 мМ раствора глутамина в 0,85% солевом растворе, а также 11 000 единиц пенициллина на 11 000 мкг стрептомицина.

EXAMPLE 2. He La cells in the logarithmic growth phase were grown for 6 days. in six one-liter Falcon flasks for tissue cultures at 37 ^o C, and then washed with phosphate buffered saline (PBS). Then, a 0.25% solution of trypsin in SFBR was added thereto and after a few minutes the cells were carefully removed and placed in beakers containing 110 ml of a 25 mM HEPES solution in minimally ether medium (MEM) with the addition of 10% (v / v) non-activated calf serum, 1.1 ml of a 200 mM solution of glutamine in 0.85% saline, and 11,000 units of penicillin per 11,000 μg of streptomycin.

The cell culture, reconverted to suspension, was placed in equal portions of 5.0 ml each in Falcon flasks for tissue cultures with a capacity of 50 ml containing or not containing a herbicide, and incubation was performed in the dark at 37 ^° C for 4 days; analogously to example 1, the herbicide was introduced into a dilute acetone solution. Acetone was also added to control samples to provide 0.2% acetone concentration (sb). The amount of herbicide was such as to provide the concentrations indicated below in table. 2.

Экстрагирование проводили следующим образом. Среду из каждой колбы помещали в стеклянные круглодонные пробирки, причем клетки, прилипшие к донцам фальконовских колб освобождали добавлением 0,5 мл 0,25% -ного раствора трипсина в СФБР и после выдерживания в течение нескольких минут при 37^oС указанную культуру клеток сливали из колб и воссоединяли с клеточной средой.The following active components were used for processing:
B. As in example 1
C. As in example 1
F. Herbicide of the formula:

Extraction was carried out as follows. The medium from each flask was placed in round-bottom glass tubes, and the cells adhering to the bottoms of the Falcon flasks were released by adding 0.5 ml of a 0.25% trypsin solution in SFBR and, after standing for several minutes at 37 ^° C, the indicated cell culture was poured from flasks and reunited with the cell medium.

 Then, equal portions of 100 μl were taken from each cell suspension in order to determine the density of the cell culture. The remaining 5.4 ml of cell culture suspension was then sonicated for 30 min to destroy the cells.

Then, 10 ml of acetone was introduced into each tube with the main reaction (90% acetone 10% NH ₄ OH), after which the tubes were centrifuged to remove protein and scraps of cell tissue. 5 ml of water and then 0.5 ml of a saturated aqueous solution of NaCl were added to the substance floating onto the surface. After this, the pH value was added to 6.8, adding dropwise a 2 M solution of KH ₂ PO ₄ .

 Next, the product was extracted from water-acetone medium, loading into a C8 column Wacker Rer. The column was first dried, and then 3 ml of a mixture of methyl alcohol with water (90 10). Analogously to example 1, all the extraction operations mentioned above were carried out under conditions essentially excluding the excitation of protoporphyrin IX under the influence of light, namely, in green light or in the dark, for example, in white coated vessels. Then, the fluorescence parameters of the extracts were determined on a CPEX spectrofluorometer. Quantitative indicators of the proportional porphyrin accumulation (Proto IX) were determined using the attenuation coefficients found in advance. The results are presented below in table. 2.

 Another aspect of the present invention relates to the preparation of protoporphyrin IX (below Proto IX). In accordance with this, heterotrophic cultivation of algae microorganisms belonging to the eucaryotic class was carried out in the dark (or under illumination that does not cause Proto IX excitation) in a medium containing the active compound described above that inhibits the enzymatic conversion of protoporphyrinogen into Proto IX. The algae medium or microorganisms, or both, were further processed to extract Proto IX and separate it from chlorophyll, carotenoids, scraps of cell tissue, etc. The separation can be carried out by any conventional method, for example, by means of liquid phase chromatography, including phase reversed liquid chromatography, or by dissolving with a solvent, and also precipitating Proto IX by converting it into a chelate compound with a metal, for example, iron, zinc or magnesium, followed by ( optionally by regeneration of Proto IX, as is done according to the known method of treating a chelate with dilute acid.

 Operations at all stages of separation are carried out under illumination that does not cause Proto IX excitation, for example, in green light, as described in Appendix A, entitled “Processing in the dark”, or in complete darkness. The iron chelate is insensitive to light, so working in the light when using this compound is more permissible.

Algae microorganisms are preferably grown as a cell culture suspension at temperatures of 15-30 ^° C. The concentration of the inhibitory active compound may, for example, be from 10 ^-5 to 10 ^-7 M. Examples of suitable algae microorganisms are: Scenedesmus sp. Chlamydomonas reinhardi, Englena sp. Bumilleriopsis diliformis
Example 3. Cultures of the microorganism Clamydomonas reinhardi (wild variety) were grown in a stainless steel tank containing, for example, 300 l using the cultivation medium described below (Table A). A solution of 1- (carboethoxy) ethyl 5- (2-chloro-4-trifluoromethylphenoxy) -2-nitrobenzoate (the so-called "Lactophan") technical phenylether herbicide in acetone was added to the culture to ensure the concentration of the active component 10 ^{- 5} M and a final concentration of acetone equal to 0.1%. The mixture was left alone to grow microorganisms in it at 25 ^° C for 4-7 days with careful mechanical stirring and / or aeration.

 Continuing to perform operations in the dark, the contents of the tank were filtered off, the filtrate was analyzed for the detection of protoporphyrin IX in it.

 One of the analysis methods for detecting photoporphyrin IX was to filter the contents of the reaction tank (which had been in the dark all this time) and to add acetone to the filtrate. Then this mixture was subjected to extraction with petroleum ether. After that, the aqueous acetone medium was subjected to extraction with diethyl ether, which was then removed from the precipitate by evaporation. This precipitate was dissolved in methyl alcohol and subjected to phase reversal chromatography to obtain photoporphyrin IX. In addition, it was possible to use and methods of detection of this compound described in examples 1 and 2.

 An alternative to the use of the microorganisms Clamydomonas reinhardi is the use of a Scendesmus sp. grown on the above medium in which sodium acetate is replaced by glucose.

PRI me R 4. This example is similar to example 3 except that instead of "Lactofen" is used 1- (4-chloro-2-fluoro-5-propargyloxyphenyl) -3-methyl-4-difluoromethyl-Δ ² - 1,2,4-triazolin-5-one.

PRI me R 5. The definition of indicator I ₅₀
Protoporphyrinogen IX (Protogen IX)
The supplier of the drug Proto IX was the company "Porphyrin Products" (Porphyrin Products), Loghen, pc. Utah, USA. This drug was isolated in pure form and described by Füsler et al. (T.P. Fuesler et al. Plant Physiol, 1981, v. 67, p. 246 249). Fresh protogen IX was obtained by reducing Proto IX using an Na / H amalgam, as described by Jacobs and Jacobs (NJ Jacobs and JM Jacobs: Enzyme, 1982, v. 28, p. 206 219), working with purified Proto IX at a concentration of 300 μM .

Plant material
Cucumber seedlings (Cacumis saturis L. cultivar Wisconsin SMR18) were grown in a darkened seedling chamber on vermiculite moistened with liquid branded fertilizer (9-45-15). Seedlings were grown at 25 ^° C and at a relative humidity of 80 to 90%. Seedlings were illuminated with intermittent light of a dosed intensity of 25 µU / m ² .c (RAR) from a General Electric Bright Stick light source controlled by an electronic timer based on the calculation of I min illumination in over a 60 minute cycle. Thus, plant tissue was obtained that has the ability to accelerate the synthesis of chlorophyll while minimizing the amount of reserve starch and the levels of the initial chlorophyll.

Chloroplast isolation
The released chloroplasts were isolated by the method described by Füsler (T. R. Fuesler) et al. (see Plant Physiol. 1984, v. 75, p. 662,664) except that at the final stage of purification, the plasticides were centrifuged through 40% (v / v), rather than through a 45% pad of Pe material r co II. Then, the chloroplasts were resuspended in the sample buffer containing 0.5 M mannitol, 20 μM TES, 10 μM HEPES, 1 μM ethylenediaminetetraacetic acid, 1 μM mg Cl ₂ , 1% (w / v) bovine serum albumin and 1 μM dithioerythritol at pH 7.7 and a total protein content of 2 mg / L.

Samples of protoporphyrinogen oxidase
Samples were drawn so that it was described by Jacobs and Jacobs (JM Jacobs and NJ Jacobs: Arch. Biochem. Biophys. 1984, v. 229, p. 312 319). Samples (0.2 ml each) of the suspension of chloroplasts were pre-incubated in the dark for 15 minutes at various concentrations of aciofluorfenmethyl (AFM) or at an acetone concentration of 0.2% (v / v) in the case of a control sample. Then, 50 μl of a freshly prepared Protogen preparation with a concentration of about 15 nM was introduced into the suspension in order to initiate the reaction. The sample preparation was completed by the introduction of 2.75 ml of a fluorometric agent, which was proposed by Jacobs and Jacobs (JM Jacobs and NJ Jacobs: Enzyme), 1982, v. 28, p. 206 219) containing 1% (sb) of tween-20 (polyoxyethylene sorbitan monolaurate), 50 mM Tris-HCl, and 1 mM ethylenediaminetetraacetic acid at pH 8.5, with 1 mM dithioerythritol being substituted with 5 mM glutathione. Suspensions were then analyzed by direct reading on a SPEX Fluorolog 2 spectrofluoroteter equipped with a facial fluorescence indicator for turbid biological samples. The amount of Proto IX formed was determined by the standard curve of the graph between the quantitative emission of Proto IX at 630 nm and excitation at 400 nm, and this curve was obtained for pure Proto IX in a fluorometric medium.

To quantify the degree of non-enzymatic oxidation to Proto IX in the sample described above, the exact same sample was prepared, except that instead of a suspension of active chloroplasts, a suspension was used in which the chloroplasts were turned into an inactive state by heating at 85 ^° C for 15 minutes. Subtracting the amount of Proto IX obtained in this way from its amount obtained in the presence of active chloroplasts gives the amount of Proto IX, which was formed enzymatically. The results are presented graphically in the drawing, where LSD is the smallest significant difference (common designation). The drawing shows that the indicator I ₅₀ (concentration that provides 50% inhibition) is less than 0.1 μm, for example, about 0.03 μm.

 Testing for the effect of 10 μM AFM on the enzymatic conversion of Proto IX to magnesian Proto IX in a chloroplast suspension (in the presence of ATP) showed no inhibitory effect of AFM on this conversion.

 PRI me R 6. According to this example, inhibitory enzymes active components were added to the food of mice that are carriers of tumors, while the food of several control mice were not added. After this, the mice were sacrificed and their kidneys, intestines, adrenal glands, liver and tumors were removed, and then subjected to analysis for the content of Proto IX. The following enzyme inhibitory active compounds were used.

In particular, experiments were performed on mice of the DBA / 2 Na line, which, on day zero, were injected with a subcutaneous tumor preparation SMT-F in the right shoulder. Then, on the first day, the mice received special food for rodents:
Purina Rodent Chow 5001 Mash without additives. Then, on the second tenth day, the mice received the same food with the addition of the test enzyme inhibitory active compound in the amount of 2 ppm (control animals received the same food, but without additives). The additive was introduced into food by mixing with a small amount of a solution of the test active compound in acetone. Then, the mice were killed on the tenth day with the exception of the mouse treated with the active compound designated as 6j: this mouse was killed on the seventh day. Animal tissue was allowed to cool overnight at 4 ^{° C.} , and then moisture was removed with blotting paper and the weight of each tissue sample was determined. Next, a homogeneous suspension of 5 ml (in the case of intestinal and liver tissues 10 ml) of a mixture of acetone and 0.1 n NH ₄ OH taken in a volume ratio of 9: 1 was prepared from tissues. Then, the homogeneous preparations were centrifuged at 1500 g and surfaced to the surface substances were analyzed on a SPEX FA112 spectrofluorometer. For Proto IX, excitation at 400 nm and emission at a wavelength of 630 nm are optimal. The accumulation of Proto IX was determined by the number of fluorescent flashes per second per 1 g of tissue. The results are presented below in table. 3 (for each preparation, the values are average for a pair of mice, with the exception of tests using the active compounds 6f, 6h, 6i, and 6j, when only one mouse was taken for testing).

Указанные данные в табл. 4 числа соответствуют концентрациям Proto IX в соответствующих тканях. Единица измерения: ультраграммы (уг) Proto IX на 1 г ткани.

The indicated data in table. 4 numbers correspond to the concentrations of Proto IX in the corresponding tissues. Unit of measurement: Ultramograms (ug) of Proto IX per 1 g of tissue.

 In the Doupherty article cited above, a value of 3.6 ug / g is given for the concentration of porphyrin in a tumor of the same type of MT-F after injection of 10 mg / kg of hematoporphyrin to mice of the same line of 1 VA / 2 Na. In another scientific publication by Moan et al. (Moan et al: PP 46 5, p. 713 721, see note in Fig. 2 on p. 716) it is indicated that in the case of CH / Tif type tumors (a porphyrin concentration of the order of 12 ug / g provided by injection into the abdominal the cavity of mice of the DBA / H2 line of Photofrin II (Photofrin II) in an amount of 25 mg / kg, is widely used to give sensitivity to light in photodynamic treatment and diagnosis of cancer.

 Mice ate the following total amounts of food containing the active compound: 6a) 22 and 36 g; 6b) 37 and 35 g; 6c) 25 and 22 g; 6d) 41 and 32 g; 6e) 40 and 44 g; 6f) 27 g; 6g) 33 and 40 g; 6h) 36 g; 6i) 10 g; 6j) 20 g.

 When conducting the experiments described in this example 6 on animals that are not carriers of tumors, the following sequence was performed using the operation, unless otherwise specified, of the active compound, which in this example is designated 6c.

a) As a result of determining the LD ₅₀ value of the active compound, it turned out to be over 2600 mg / kg (i.e. mg of active compound per 1 kg of body weight) for rats, when this indicator was determined 14 days after a single meal of animals containing 15% corn oil active compound, and more than 700 mg / kg for mice, when this indicator was determined 14 days after a single meal of animals eating a portion of the same oil containing 5% of the active compound.

 b) When testing compositions for injection into the abdominal cavity that do not contain an active compound, the ability of mice to tolerate injections at a dose of 0.5 ml of a mixture of equal proportions of dimethyl sulfoxide and water was established.

 c) When testing the active compound, injected into the abdominal cavity in a mixture containing 60% corn oil and 40% dimethyl sulfoxide, it was found that mice are able to tolerate a dose of 100 mg / kg.

d) The following experiments were performed on mice:
1) Daily feeding doses of 50 mg / kg (using a 1% solution of the active compound in acetone) for 8 days.

2) daily injection of a dose of 50 mg / kg into the abdominal cavity for 8 days using a 1% dispersion of the active compound in mineral oil prepared by dissolving the active compound in a drop of dimethyl sulfoxide and mixing the resulting solution with mineral oil;
3) daily topical application to the skin of a dose of 50 mg / kg for 8 days using a 1% solution in dimethyl sulfoxide,
4) feeding for 8 days with standard food with the addition of the active compound in the amount of 2 ppm of the mass of food;
5) daily intravenous injection of a dose of 50 mg / kg for 4 days using a 2% solution in dimethyl sulfoxide.

 Then, the mice taken for the experiments were euthanized and the accumulation of Proto IX in their tissues was determined.

 In another experiment, a male Fischer 344 rat was fed food containing 5 ppm of active compound 6c for 24 days, after which the animal was sacrificed. In the tissues of this rat, an increase in the levels of Proto IX was observed compared with the control animal, and a noticeable increase in these levels was observed in the tissues of the kidney, intestines, stomach and head of the brain, while in the muscle tissues there was only a slight increase in the level.

 When conducting the above experiments on rats and mice, animals were kept in a standard mode of meeting: 12 hours of light and 12 hours of darkness.

 PRI me R 7. When conducting a series of experiments according to examples 1 and 2, cultures of HeL a cells were incubated in the presence of the following various drug active compounds with a concentration of 100 uM. The indicators c after each designation of the drug active compound show an increase in the amount of Proto IX formed in the presence of the drug active compound relative to the amount that is formed in the control sample in the same experiment.

 The active compounds of Example 6 used the following values: 6a) 337% 6b) 366% 6c) 297% 6d) 1200% 6e) 1210% 6f) 280% 6g) 326% 6h) 431% 6i) 544% 6j) 469% the formulas of other active compounds and the corresponding percentage values for them are given below.

П р и м е р 8. В данном примере ингибирующее энзимы активное соединение 6с из примера 6 скармливали крысам с привитыми опухолями, после чего опухолевую зону облучали светом, вызывая тем самым рассасывание опухоли.

Example 8. In this example, the enzyme inhibitory active compound 6c of Example 6 was fed to rats with inoculated tumors, after which the tumor area was irradiated with light, thereby causing the tumor to resorb.

In particular, three Sprague-Dawley rats weighing an average of 120 g for a period of 6 days were assigned a diet with the addition of the active compound in an amount of 2 ppm. On the third day, chondrosarcoma was implanted on the right hind limb of each rat by injection of 0.3 ml of a suspension of tumor cells (1 million tumor cells). On the 6th day, the right hind limb of each rat was shaved to remove hair and the size of each tumor was measured. Further, the area affected by the tumor and the surrounding areas of the skin were irradiated with non-thermal doses of light with a wavelength of 630 nm at an energy flux density of 270 J / cm ² . The next day, it turned out that two rats to a noticeable degree got rid of their tumors (that is, the mass of the tumor was no longer felt), and in the third the tumor almost completely resolved. In all rats, the areas of the skin and muscle tissue surrounding the tumor were slightly whitened and seemed to be swollen, but to a much lesser extent than is the case in photodynamic therapy using Photofrin II.

 Appendix A.

Leach Percentage Test
In the test to determine the percentage of leaching, cotyledons collected from cucumber seedlings germinated in the dark were used. In the first stage, the mass of cotyledons was treated in the dark with an aqueous solution (in the presence of a buffer compound) containing sugar with radioactive labeled atoms. Next, the amount of sugar assimilated by the cotyledons was measured according to the calculation procedure described below. Then the whole mass of cotyledons was divided into two portions. One of these portions was treated in the dark with an aqueous solution (in the presence of a buffer compound) containing the test compound, and the other was similarly treated in the dark with the same solution, but without the test compound (control sample). The cotyledons soaked in the indicated aqueous solutions were then irradiated with light for 16 hours, after which they were separated from the aqueous solutions, in which the content of a substance with radioactive labeled atoms was determined by measurement and calculation. The results were then presented as a percentage leaching rate (PPV), calculated by the formula:
PPV (St Sc) / s
where S, S _t and S _c, respectively, the number of decays (per cotyledon) of a substance with radioactive labeled atoms: a) in this substance absorbed by the cotyledons during their initial treatment, b) in an aqueous solution containing the test compound, after exposure to light and c) in a control aqueous solution after exposure to light.

 The materials used in more detail, as well as the conditions of the test to determine the percentage of leaching, are described below.

Plant material
In vermiculite moistened with commercially available fertilizer 9-45-15, seeds of cucumber Cocumis sativus L. cultivar Wisconsin SMR 18 were germinated, of which seedlings were grown in the dark in an incubation chamber at 25 ^° C and a relative humidity of 80 ^{° C.} After 5 days from sowing, seedlings were collected from under pale sprouted seedlings grown in the dark and washed in a 1.0 mM CaCl ₂ solution. All these operations were carried out under green light.

Buffer solution
The solution contains 1 mm KCl, 1 mm CaCl ₂ , 2.0 mm potassium phosphate and adjusted to a pH of 6.5 (as with NaOH).

Labeled Atom Sugar
This type of sugar is 3-0-methyl-3- [u-14C] -glucose with a specific activity of 10.9 GBq / mm; supplier: Amersham Corp. (Amersham Corp.), Arlington Heights, pc. Illinois, USA

Initial processing
180,230 washed cotyledons were introduced into a 250 ml wide-necked Erlenmeyer flask with a polyurethane foam stopper containing 50 ml of buffer solution, to which sugar with radioactive labeled atoms was also added in an amount ensuring its concentration in the solution of 600 nM. The flask was shaken in the dark on a rotating device for 24 hours at 125 min ^-1 . Then the cotyledons were discharged onto a sieve from nylon and washed 3 times with portions of 1 mM CaCl ₂ solution of 20 ml each. The assimilation of sugar with radioactive labeled atoms was determined by dissolving 3 samples of 5 pcs. cotyledons each in a tissue solvent (NCS brand, manufactured by Amersham Corp.) and the resulting depletion was calculated on a liquid scintillation spectrometer.

 It was found that these depletion experiments are equivalent to a similar determination made by burning selected cotyledon samples in a self-oxidizing agent.

 Processing using the test compound, as well as control processing.

Five Pieces the cotyledons were left to float non-axis side up on the surface of the buffer solution, poured in an amount of 3 ml into a plastic Petri dish with a diameter of 35 mm with a lid. Then the test compound (in the form of its solution in acetone) was introduced therein in an amount providing a predetermined concentration (as mentioned above) of the test compound in the buffer solution. The acetone content in the buffer solution was 0.1% (v / v) both in the control sample and in the solution containing the test compound. Then, the floating cotyledons made a circular motion in a whirlpool for 16 hours, which was formed as a result of shaking the cups with a frequency of 90 min ^–1 on the surface of the rotating shaking device.

Light exposure
Petri dishes with cotyledons floating on the surface of the solutions were irradiated for 16 hours with the light of four GE F20T12-CW fluorescent lamps at a given intensity of 150 μE / m ² in the spectral region of photosynthesis (SOFS), and the mentioned intensity was measured on the surface of cotyledons, at that time how the cups were continuously shaken.

 Measurement of the amount of substance in a liquid with radioactive labeled atoms.

 All liquid was taken from the cotyledons and then the number of decays was determined on a liquid scintillation spectrometer.

 TEMNOTA.

 All types of processing before the stage of irradiation with light was performed in the dark or under green light of a fluorescent source, that is, when the light passed through a green cut-off plastic filter, transmitting light with wavelengths of 450 600 nm.

 Appendix B.

Concentrate of trace metal
H ₃ BO ₃ 100 mg / L
ZnSO ₄ • 7H ₂ O 100 mg / L
MnSO ₄ • 4H ₂ O 40 mg / L
COCl ₂ • 6H ₂ O 20 mg / L
NaMoO ₄ • 2H ₂ O 20 mg / L
CuSO ₄ 4 mg / L
Medium A for cultures was prepared by adding to the medium M 10% aqueous solution of sodium acetate with a concentration of 7.5 x 10 ^-3 M in an amount of 10 ml / l.

All components were introduced in distilled water in the order indicated above, followed by their treatment in the autoclave under a pressure of 15 lb / ⁱⁿ² for 15 minutes.

The stock of canned preparation for growing crops under the influence of light
Preserved U-1 cell cultures were axenically transferred to liquid cultures on medium A or M, preferably on medium A, in Erlenmeyer flasks closed with polyurethane plugs, at 25 ^{° C.} under a cycle of 14 hours of light and 10 hours of darkness. Canned cultures were incubated (without additional aeration) on rotating shaking devices operating at a frequency of 125 min ^-1 . The illumination intensity at the level of the culture layer was 120 μE / m ² .s (SRFS). Under these conditions, the cultures are in semi-synchronous growth mode until they go into the stationary phase containing (2 4) x 10 ⁶ cells in 1 ml.

Growing crops in the absence of light (crops grown in the dark)
Cells in the stationary phase of cultivation from a canned culture grown in the world in an amount of 7.5 ml were transferred to an Erlenmeyer flask containing 750 ml of medium A. Cells were incubated in the dark by aeration through a buried aeration tube at a temperature of 25 ^o C for 3 4 days. During this time, this culture of u-1 cells had to undergo 7 8 cell divisions, losing all visible chlorophyll, at a cell concentration of (2 3) x 10 ⁶ pcs / ml.

Preparation of cells for trial use
Cells were taken from a freshly prepared culture in an amount of 750 ml grown in the dark by centrifugation from low revolutions (about 2000 min ^-1 ) at 20 ^° C for about 5 minutes. These cells were carefully transferred again to a suspension state in 50 ml of medium A. Cell motility was determined in a sample of this suspension (about 0.25 ml), and after fixing with a 1% aqueous solution of glutaraldehyde, the number of cells in 1 ml was counted. Typically, the number of cells is 5 x 10 ⁷ units / ml. Portions of cell culture 1 ml each containing 10 ⁷ pcs. cells, were placed in test vessels, for example, in 24-well Falcon plates intended for cell cultures. At this stage, the cells are very mobile and stained yellow.

The test compound was dissolved in a suitable solvent, for example, acetone, ethyl alcohol, dimethyl sulfoxide or water (preferably acetone) to provide a concentration 1000 times the final. In each pair of holes, using a microsyringe with a capacity of 5 or 10 μl, 1 μl of the test solution was introduced. On each plate for cell cultures were prepared 4 control wells in the same way as described above, but in the absence of the test compound. Plates for cell cultures were covered with transparent plastic covers and placed for 13 16 hours in a growth chamber at a temperature of 25 ^° C and an illumination intensity of 70 90 μE / sq.m. If the contents of the test wells turned yellow, then they were removed as described below in p. "Evaluation of the results." If the contents of the test holes turned out to be transparent or pale green, then they were placed on a rotating shaking device operating at a frequency of 125 min ^-1 before being removed and irradiated with light at an intensity of about 600 μE / m ² for 2 hours.

Evaluation of the results
250 μl of a 10% aqueous solution of sodium dodecyl sulfate and 1 ml of methyl alcohol were injected into each test well, after which the contents of the well were mixed well. The resulting mixtures were kept in the dark for 3–4 h. Plates with cell cultures were centrifuged at a low speed (about 2000 min ^–1 ) at room temperature for 5 min. The substance floating onto the surface was removed and analyzed on a Beckman spectrophotometer, the 35th model in the wavelength range of 350–500 nm (for the presence of protoporphyrin IX, which is characterized in the system with the mentioned solvent as a peak of chlorophyll absorption at 668 nm), as well as at 720 nm, in order to use the background turbidity reading.

Data analysis
For each well, by subtracting the value at 720 nm from the value at 668 nm, a green indicator was obtained. The corresponding values are average for each concentration of the test compound, as well as for the control sample. The percentage inhibition is found by the formula: Q 100 x (1 k), where k is the ratio of the average value of the greening index for this particular concentration to the average value of the greening index of the control sample.

Claims (10)

 1. A method of destroying mammalian tumor cells by exposing them to light in the presence of porphyrin, characterized in that the cells are treated with a specific inhibitor of the enzymatic conversion of protoporphyrinogen to protoporphyrin IX under the action of protoporphyrin oxidase before light treatment. 2. A method according to claim 1, characterized in that the inhibitors are used or aryl triazolinone or ariltetrazolinon or ariltriazindion or ariloksidiazol or ariltetragidroindazol or ariltetragidroftalimid or arilgidantoin or arilurazol or arilgeksagidropiridazin or aryl heterocyclic urethane, or fenoksifenilovoe compound or arylpyrazole or a compound of the formulas

where X _b O or S;
Ph substituted phenyl.  3. The method according to claim 2, characterized in that the inhibitor is a water-soluble salt or an acidic compound, the sodium salt of which is soluble in water.  4. The method according to PP. 1 to 3, characterized in that the inhibitor contains an aryl group, the substituent of which is the acid group of the sulfonylamide or its salt soluble in water. 5. The method according to PP. 1 to 4, characterized in that as the inhibitor use a compound of the formula

6. The method according to claim 1, characterized in that the processing of cells is carried out by oral administration of an inhibitor. 7. A pharmaceutical composition for the destruction of mammalian tumor cells, containing a biologically active compound and a pharmaceutically acceptable carrier, characterized in that the composition contains a specific inhibitor of the enzymatic conversion of protoporphyrinogen to protoporphyrin IX with protoporphyrin oxidase having a rate of ₅₀ for protoporphyrin oxidase less than 1 μm or having a negative redox potential of more than 500 MW. 8. The composition according to claim 7, characterized in that the inhibitors use either aryl triazolinone, or aryl tetrazolinone, or aryl triazinedione, or aryloxy diazole or aryl tetrahydroindazole, or aryl tetrahydrophthalimide, or aryl hydrazole, or aryl azheryl idryl, phenyl or aryl iride aryl iride or aryl azide or aryl iride aryl or aryl iride aryl or aryl iride aryl or aryl iride aryl or aryl azide or aryl azide or aryl azide or aryl azide or aryl azide or aryl azide or aryl aryl azide or aryl azide or aryl aryl azide or aryl azide or aryl azide are or or arylpyrazole, or a compound of the formulas

where X _b O or S;
Ph substituted phenyl.  9. The composition of claim 8, wherein the inhibitor is a water-soluble salt or an acidic compound, the sodium salt of which is soluble in water.  10. The composition according to paragraphs. 7 to 9, characterized in that the inhibitor contains an aryl group, the substituent of which is the acid group of the sulfonamide or its water-soluble salt. 11. The composition of claim 10, wherein the inhibitor is a compound of the formula

I am

Images