LT3997B - Process for preparing protoporphyrins and use thereof - Google Patents

Description

The present invention relates to a specific enzyme-mediated conversion of protoporphyrinogen to protoporphyrinogen oxidase, an inhibitor of cells and to its use in pharmaceutical compositions for the destruction, diagnosis and localization of mammary gland tumor cells and to the production of protoporphyrin IX.

A well-known method of introducing a hematoporphyrin derivative or a mixture of porphyrins, which are also hematoporphyrin derivatives, such as Photofrin II, into a mammary gland tumor is then exposed to light waves of a certain wavelength. This method of photodynamic therapy is the subject of a series of articles forming a special issue of the Journal of Photochemistry and Photobiology, 1987, Volume 46, No. 5 (hereafter P&P 46-5). The articles indicate that photodynamic therapy is widely used in the treatment of various cancers (including bronchial, bladder, esophagus, lung, skin, head organs, neck, brain, colon, etc. eye and gynecological cancers). It is claimed that said porphyrinic photosensitization is accompanied by biochemical effects such as formation of transverse protein bonds in membranes, lipid peroxidation, degradation of lysosomes and mitochondrial sheaths, decreased enzyme activity, and an increase in cellular uptake of porphyrin (P&P 46-5,695). The porphyrin preparation is usually administered by injection (e.g., intravenously or intraperitoneally); the typical dose is 2 mg Photofrin per kilogram body weight.

In addition, the porphyrin preparation can be incorporated into a liposome and administered by injection (e.g., into the abdominal cavity) as described in Spikes et al. Photodynamic Behavior of Porphyrins in Model Tissue Cell and Tumor Systems (Tissue and Tumor Systems) in Photodynamic Therapy of Tumor and Other Diseases, edited by Jori and Perria, Padua, eds. Libreria Pogretto, 1985, p. 45-53, and m.p. and in the special edition mentioned above.

Special literature reports that the administration of protoporphyrin to cells such as human erythrocytes or leukemia mouse cells results in a marked photosensitization effect; see: Dabbleman et al. Biochimica et Biophysica Act, 1978, vol. 511, pages 141-151; Kessel, Cancer Research, 1982, Vol. 42, No. 5, pp. 1703-1706. According to Kessel, protoporphyrin is the most active of all photosensitizing agents tested. However, when porphyrin was administered to animals by Kessel and other researchers, such as Berenbaum and coworkers (see Br.C. Cancer, 1982, 45 vol., Pp. 571-581), no detectable levels of protoporphyrin were detected in tumors, suggesting that when administered in the bloodstream, protoporphyrin may be rapidly transported in a manner similar to that of the body.

The active compound for use in photodynamic therapy or in known methods of tumor diagnosis and localization according to one aspect of the present invention is non-porphyrin or not only porphyrin.

Unlike plain porphyrin, the compound in question stimulates the enzymatic hematogenesis of protoporphyrinogen in said cells while inducing the formation of endogenous protoporphyrin IX in the cells. The authors found that reagents such as some herbicidal compounds that inhibit the enzymatic transfer of porphyrinogens to chlorophyll in plant cells also inhibit the enzymatic alteration of porphyrinogen in the mammary gland cell structure. The authors believe: The above inhibitory effects of these reagents probably also influence the conversion of protoporphyrinogen to protoporphyrinogen IX by the action of an enzyme (protoporphyrinogenoxidase, EC 1.2.3.4.) Such that protoporphyrinogen is no longer converted to protoporphyrin IX by the normal pathway, ( eg in plasma) by an unusual enzymatic process (eg, in the organelle envelope) which results in protoporphyrin IX accumulating in areas where it is not available for normal heme conversion; by exposing the cells to light, protoporphyrin IX accumulated in this way produces a cell disruption effect similar to that observed with the photodynamic therapy described above.

Corresponding enzyme inhibiting active compounds of the present invention are specific protoporphyrinogen oxidase inhibitors in the sense that they do not function as simple enzyme poisons, much like reagents that induce denaturation or inter-chain linkage (e.g., sulfihydryl-based reagents); they are not primarily oxidizing agents such as electron acceptors and thus the active compounds of the present invention have a more negative redox potential than -500 mV and even more negative, namely -800 mV, e.g. , suitable for the given compound in a solvent by a conventional method: cyclic voltammetry or polarography). It is valuable that the active compound is not tetrapyrol and that it has an index for protoporphyrinogen oxidase of less than 1 μΜ, e.g. less than 0.3 μΜ; namely, I ₅₀ approximately equal to 0.1, 0.03, or 0.01 μΜ or less.

An enzyme-inhibiting active compound having an increased capacity to degrade the plasma product of herbal preparations (plasmalemma) is suitable. One of the methods for investigating such capacity is described by Holing and Piters in the Leaching Experiment Plant Physiology, 1987, 84 vols., 1114-5 psi. According to this assay (described in more detail in Annex A), the most suitable reagents show at least 50% total leaching at a treatment level of 100 μΜ, preferably at a treatment level of 1 μΜ and less, e.g., 100 nM.

Total active substances such as 1- (4-chloro-2-fluoro-5-pro-2-pargyloxyphenyl) -3-methyl-4-difluoromethyl-Δ -1,2,4-triazoline-5 or lactofen (see below) with a degree of leaching above 90% at a concentration of 100 nM.

Another technique for determining the ability of a preparation to cleave the plasmolem of a plant material is the illumination inhibition assay, described in detail in Appendix B. According to that technique, the ability of a preparation to suppress graying in a dark culture of the microorganism Clamydomonas rein hardi, grows in the dark without forming chlorophyll). It is meant that the mass of algae is depleted because it contains fresh, uninjured cells but again forms chlorophyll in the light. Many suitable active compounds used in the present invention are capable of inhibiting irradiation-induced leaching by at least 50% at a concentration of 10 ⁵ M, preferably 10 ⁶ M and less, e.g. 10 ⁷ M. The compound may also be present. one which, during the course of a given study, is a rising product to the surface of a product with a maximum absorption at 405 nm, which exceeds the maximum for chlorophyll at a given system of 669 nm, e.g. 4x maximum at 668 nm.

Among the enzyme inhibiting active compounds that can be used in the practice of the present invention are several types of (A-D) herbicides.

A. Aryl-heterocyclic herbicides having the general formula:

Ph - NHet, wherein Ph - substituted phenyl, preferably 2,4-disubstituted phenyl, more preferably 2,4,5-triubstituted phenyl, and NHet a 5 or 6 membered heterocyclic ring (with 1-4 nitrogen atoms in the ring) of formula :

- N - C = O or - N - C - OH or - N - C - Cl

I / I II I II

Q Q - C - R Q - C - R where Q represents a heterocyclic ring closure group, o

R is hydrogen or a substituent group.

This class of compounds also includes such preparations as Wakabayashi et al. (see J. Pesticide Sci., 1986, vol. 11, pp. 635-460) as a class of herbicides based on cyclic imides, and p. the following compounds are given:

I II III

Compound I is a herbicide based on aryloxydiazole, namely 2-tert-butyl-4- (2,4-dichloro-5-isopropoxyphenyl) - Δ ² -, 3,4-oxydiazolin-5-one. Other 2-alkyl-4- (2,4,5-substituted-phenyl) - Δ -1,3,4-oxydiazolin-5LT 3997 B's which can be used in the present invention are characterized, for example, in U.S. Pat. 3,385,862,

836,539, 3,876,413.

Compound II is a herbicide based on aryl-tetrahydro-indazole, 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 which may be used in the present invention are characterized, for example, in U.S. Pat. 4,439,229 (page 4 of line 25 to page 5 of line 20).

Other suitable herbicides of the Ph - NHet type are:

aryl-trialinones disclosed in U.S. Pat.

318 731, 4 398 943, 4 404 019, 4 702 945, 4 705 557, 4 702 763, 4 761 174, as well as in International Application Nos. WO 85/01637, WO 85/04307, WO 87/00730, WO 87/03782, WO 86/04481 and WO 88/01133;

aryl-tetrazolinones described in U.S. Pat. 4,734,124, and m.p. International Applications No. WO 85/01939 and WO 87/03873;

aryl-triazindiones disclosed in U.S. Pat. 4,755,217 and 4,766,233, and m.p. International Application WO 86/00072;

arylhydantoin, described in U.S. Pat. 4,427,438;

aryl-urazoles described in U.S. Pat. 4,452,981;

aryl-hexahydropyridazines described in U.S. Pat

No. 4,619,687.

B. Ari1-heterocyclic urethanes described in U.S. Pat. 4,521,242.

C. Herbicides based on plain phenyl ether from those having a para-halogen or para-nitro-phenoxyphenyl structure, such as the following commercial preparations:

5- (2-Chloro-4- (trifluoromethyl) phenoxy) -2-nitro-Nmethanesulfonylbenzamide (fomesafen) sodium 5- (2-chloro-4- (trifluoromethyl) phenoxy) -2-nitrobenzoate (acifluorophen) methyl 5- (2,4) -dichlorophenoxy) -2-nitrobenzoate (bifenox)

2-Chloro-1- (3-ethoxy-4-nitrophenoxy) -4-trifluoromethyl-benzene (oxyfluorfen)

Other suitable and commercial herbicides based on simple diphenyl ethers are the following:

Lactofen: 1- (carboethoxy) -ethyl-5- (2-chloro-4-trifluoromethyl-phenoxy) -2-nitrobenzoate;

Fluoroglycofen: (Carboethoxy) -methyl-5- (2-chloro-4-trifluoromethyl-phenoxy) -2-nitrobenzoate;

Fromesofen: 5- [2-Chloro-4- (trifluoromethyl) -phenoxy] -N-methylsulfonyl-2-nitrobenzamide;

Chloronitrophen: 2,4,6-trichloro- (4-nitrophenoxy) benzene;

Chlorodiphen: 2-nitro-1- (4-nitrophenoxy) -4-trifluoromethyl-benzene;

Nitrofen: 2,4-Dichloro-1- (4-nitrophenoxy) -benzene;

Chlomethoxyphen: 4- (2,4-dichlorophenoxy) -2-methoxy-nitrobenzene.

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

D. Herbicides based on aryl-pyrazoles disclosed in U.S. Pat. 4,563,210; 4,496,390; and 4,459,150; 3,530,327 AI.

E. Compound of Formula:

Ii "b

Pin v

II o i | ii o

wherein x is oxygen or sulfur and Ph is as defined above; these compounds are described in European patent application no. 273417, and m.p. in the Dervent Abstracts directory (see # 87-040449).

The active compound can be used as a sterile formulation, which is a reagent, dissolved or dispersed in a pharmaceutical medium, such as an aqueous isotonic solution such as brine (0.9% NaCl) or Dulbecco's saline at a concentration of 2. , 5 mg / ml in a row. Alternatively, the active compound can be incorporated into a liposomal system obtainable in phospholipid medium as described on pages 1659-1660. see Remington's Pharmacentical Science, 17th issue of Mack Publishing Co. For example, in an analogous recipe described by Jori with co. (cf. Br.J. Cancer, 1983, 48 vol., 307), 51.6 mg of dipalmitoyl diphosphatidylcholine can be dissolved in 10 ml of a 1 mM reagent solution in a mixture of chloroform and methyl alcohol (9: 1 by volume). removal of the solvent (after thorough mixing) at 30 ° C under vacuum. The result is a solid which can be resuspended in 10 ml of 0.01 M phosphate buffer, 150 mM NaCl, pH 7.4 with further sonication at 50 ° C for 30 min.

Enzyme-inhibiting reagents can be coupled or bound to appropriate tumor-specific monoclonal antibodies (MABs) using a binding technique well known to those skilled in the art, and the enzyme-inhibiting reagent delivered to a specific tumor-affected area.

It is desirable to use enzyme inhibitors such as soluble aqueous salts or acidic compounds which form soluble aqueous sodium or potassium salts, e.g. such as sulfamide described in International Application WO 87/037873 (e.g. Reagent 6c in Example 6 below). WO 85/001939, WO 87/037873, or its water soluble salt, or the corresponding carboxylic acid or its water soluble salt, e.g., sodium salt known as Acifluorphene.

The enzyme inhibiting active compounds of the present invention may be incorporated into conventional pharmaceutical preparations such as tablets (e.g., compressed tablets coated with sugar paste and / or syrup), suppositories, capsules (e.g., hard gelatine capsules), suspensions. , solutions, powders or ampoules. In such formulations, the active ingredient may be in admixture with pharmaceutical solid or liquid carriers which, if desired, may be foods. For example, it may be a solid product such as corn starch or liquid, such as water, or an edible or mineral oil, or a solvent such as dimethylsulfoxide and the like. Mixtures of active ingredient inhibiting enzymes, such as a mixture of active ingredient 6c of Example 6 with Acifluorphene, may be used in approximately equal amounts. The dose administered may be determined by the usual experimental route well known to those skilled in the art, according to the method described in Photofrin, a photodynamic therapy (FDT) for malignant tumors, described by CRC in Critical Reviews, Geneology Hematology, 1984, Vol. , 2 editions,

Pp. 83-116.

The following examples illustrate the invention:

example

HeLa cells (a line of human tumor cells commonly used for tumor studies derived from the American Type Culture Collection) in logarithmic growth phase (grown at 37 ° C for 5 days in 1-liter Falcon flasks for tissue cultures) were lysed in phosphate buffered saline. in solution (BFDT). Subsequently, 0.25% trypsin solution in 2 ml BFDT was added. After a few minutes, the product was carefully removed from the flask and placed in a beaker with

100 ml of 25 mM HEPES solution in minimal stock medium (MEM), added to MEM 10% (w / w) inactivated calf plasma, 1.1 ml 200 mM glutamine in 0.85% saline, and so on. 11,000 units penicillin / 11,000 kg streptomycin.

The resulting cell culture, which was resuspended, was aliquoted into 5 ml aliquots of tissue culture flasks in 5 ml portions and (with the exception of the control) mixed with the active compound. The indicated portions were kept in the dark at 37 ° C for 3 days. The tissue cultures were then extracted and extracted under green light as follows:

Cells were lifted from the bottom of the flask by the addition of 0.8 mL of 0.25% trypsin solution in BFDT. After 1 or. after incubation in the dark, cells and media were removed from the flask, placed in a round bottom centrifuge tube, and subjected to two freeze-thaw cycles to disrupt the cells and allow extraction. Cell suspension assays after this disruptive operation did not reveal whole cells.

Subsequently, 10 mL of alkaline acetone solution (90% acetone: 10% 0.1 N NH ₄ OH) was added to each tube and centrifuged to remove protein and cellular fragments. 50 ml of water was added to the raised surface, followed by 0.5 ml of saturated aqueous NaCl solution. The pH was adjusted to 6.8 by dropwise addition of 2 M KH ₂ PO ₄ solution. It was then completed by C8 Baker Prop. type column with acetone - aqueous extract. The column was dried and washed with 2 ml of water each. Tetrapirolus was washed with 90:10 CH ₄ OH: H ₂ O; volume of the mixture 2x1.5 ml. The extracts were then quantitated by spectrofluorimeter.

The following active units were used in the experiment under consideration:

A. 5 - Aminolevulinic acid, known as an intermediate in tetrapyrene synthesis.

Active compound A was used as a 250mM aqueous solution (pH = 6.5) sterilized by filtration. The active compounds B, C, D and E (see formulas below) were used as 50 mM solutions in acetone. 0.2% (v / v) acetone was also added to the control. The following were introduced into tissue cultures

B. Herbicide with formula:

F α

o

C. Herbicide with formula:

Cl

CHg

D. Herbicide,

E. Herbicide with formula:

the amounts of the indicated active components to give the concentrations indicated in Table 1 below. The same table shows the quantities of protoporphyrin IX tetrapyrrole (Proto IX) obtained.

table

Tetrapyrol accumulation in treated HeLa cells

Active Active Agent Fluorescent IX of the mind component concentration, emission quantity, μΜ 400/630 s picomoles

A 5,000 41 842 4.2 B 100 12th 921 1.3 C 100 33 477 3.5 D 100 10th 551 1.1 E 100 8th 967 0.9 Control sample 2 795 0.3

example

HeLa cells were cultured in logarithmic growth phase for 6 days in six 1L Falcon flasks for tissue culture at 37 ° C, followed by washing with Buffered Phosphate Saline (BFDT). Subsequently, 0.25% trypsin in buffer (BFDT) was added and, after a few minutes, the cells were carefully removed and placed in a beaker with 110 ml of 25 mM HEPES in minimal medium (MEM) plus 10% (v / v) unactivated calves. plasma, 1.1 ml of 200 mM glutamine in 0.85% saline, and 11,000 units penicillin / 11,000 units streptomycin.

The resulting cell culture, which was resuspended, was placed in 5 ml aliquots in 5 ml Falcon flasks for tissue culture with or without herbicide; incubation treatment was carried out in the dark at 37 ° C for 4 days, in addition to the addition of the herbicide in dilute acetone solution for example 1. Acetone was also added to the controls so that the concentration of acetone was 0.2% (v / v). The amount of herbicide was such that the concentrations given in Table 2 below were obtained.

The following active components were used for processing:

B. As in Example 1.

C. As in Example 1.

F. Herbicide with formula:

The extraction was carried out as follows. The medium from each flask was placed in glass round bottom tubes and the cells adhered to the bottom of the falcon flasks were liberated by the addition of 0.5 ml of 0.25% trypsin solution in BFDT; after a few minutes at 37 ° C, the indicated cell culture was transferred from the flask and pooled with the cell medium.

Subsequently, equal portions of 100 μΐ were separated from each cell suspension to determine cell culture density. The remaining 5.4 ml of cell culture suspension for 30 min. was sonicated to disrupt the cells.

Subsequently, 10 mL of alkaline acetone solution (90% acetone: 10% NH ₄ OH) was added to each tube and centrifuged to remove protein and cellular fragments. 50 ml of water was added to the raised surface, followed by 0.5 ml of saturated aqueous NaCl solution. The pH was adjusted to 6.8 by dropwise addition of 2 M KH ₂ PO ₄ solution.

The product was extracted from water / acetone by loading onto C8 Baker Prop. column. The column was initially dried and then washed with 3 ml of a mixture of methyl alcohol and water (90:10). Analogously to Example 1, all extraction operations were carried out under conditions such that protoporphyrin was completely protected from its excitatory light action, i.e. in green light or dark, such as in white-covered dishes. Subsequently, the fluorescence parameters of the extracts were determined with a CPEX spectrofluorimeter.

Quantified protoporphyrin (Proto IX) was quantified using pre-determined quench rates. The results are shown in Table 2.

table

Mean growth retardation and Protein IX concentration

Active compound Active component concentration Growth retardation 0 IX of the mind quantity picomoles IX of the mind relative quantity B 100 99 5.20 2,364 F 100 106 2.71 1232 C 100 52 4.41 2005 C 10th -15 ⁺ 0.57 259 C 1 -26 ⁺ 0.19 86

Notes:

+ Negative growth retard value indicates that growth is still occurring ++ Mean value for 10 cells +++% compared to control

Another aspect of the present invention is a process for the preparation of protoporphyrin. Accordingly, it cultivated algae microorganisms belonging to the eukaryotic class in the dark (or in the light that does not induce Proto IX) by selecting a medium containing the active compounds described above that inhibit the enzymatic transfer of protoporphyrinogen to Proto IX. The medium and algae microorganisms, or both, were further treated to extract Proto IX and separate it from chlorophyll, carotenoids, disrupting cellular tissues, and so on. Separation can be accomplished by any conventional method, such as liquid phase chromatography, including reverse-phase liquid chromatography, or by solvent dissolution and precipitation of Proto IX by chelation with a metal such as iron, zinc. or magnesium followed, if necessary, by regeneration of Proto IX as known in the art with dilute acid chelation.

Operations at all stages of separation are performed under illumination that does not induce Proto IX, for example, in green light, as described in Appendix A, entitled Dark Processing, or in Full Dark. Iron chelate is insensitive to light, so this compound can work in light.

Algae microorganisms are preferably cultured in cell culture suspension at 15-30 ° C. The concentration of the active compound inhibitor can be, for example, from 10 ⁵ to 10 ⁷ M. Examples of suitable algae microorganisms: Scenedesmus sp., Chlamydomonas reinhardi, Englena sp., Bumilleriopsis filiformis.

example

Cultures of the microorganism Clamydomonas reinhardi (wild strain) were grown in a stainless steel reservoir, e.g., 300 L, using the culture medium described below. A solution of 1- (carboethoxy) -ethyl-5- (2-chloro-4-trifluoromethylphenoxy) -2-nitrobenzoate (so-called lactofen, technical phenylether herbicide) in acetone was added to the culture to provide a concentration of the active component of 10 ⁵ M and a final concentration of acetone 0.1%. For cultivation of microorganisms, the mixture was left at rest for 4-7 days at 25 ° C with careful mechanical and / or aerodynamic mixing. As with other operations, the contents of the reservoir were filtered in the dark: the filtrate was analyzed for protoporphyrin IX.

One of the analytical methods for the determination of protoporphyrin IX consists of filtration of the contents of the reaction tank (in the dark at all times) and addition of acetone to the filtrate. This mixture was subsequently extracted with petroleum ether.

The water-acetone medium was then extracted with diethyl ether, which was removed from the residue by evaporation. The residue was dissolved in methyl alcohol and reverse phase chromatography was used to determine protoporphyrin IX. Alternatively, the procedure for the determination of a given compound described in Examples 1 and 2 can be used.

Composition of the medium

Salts Molar concentration of Con x 10M centrate 0. o Concentrate ml / l (Na citrate) 6H ₂ O 1.7 10th 5 Traces of metals as below 10th FeCl ₂ .6H ₂ O 0, 37 1 1 CaCl ₂ .2H ₂ O 0.36 5.3 1 MgSO ₄ .7H ₂ O 1.2 10th 3 NH ₄ NO ₃ 3.7 10th 3 kh ₂ after ₄ 2.2 10th 3 k ₂ hpo ₄ 1.7 10th 3 CH ₃ CO ₂ Well 7.5 10th 10th

Mixture concentrate containing traces of metals:

H ₃ BO ₃ mg / l 100 ZnSO ₄ .7H ₂ O 100 MnSO ₄ * 4H ₂ O 40 CoCl ₂ .6H ₂ 0 20th NaMo0 ₄ -2H ₂ 0 20th CuSO ₄ 4

As an alternative to the application of the microorganism Clamydomonas reinhardi, the application of a culture of Sendesmus sp. grown in the above medium where sodium acetate is replaced by glucose.

example

This example is analogous to Example 3 except that 1- (4-chloro-2-fluoro-55-propargyloxyphenyl) -3-methyl-4-difluoromethyl-Δ ² -1,1,4-triazolin-5-one is used in place of lactofen.

example

Determination of I ₅₀

Protoporphyrinogen IX (Protogen IX)

Proto IX was supplied by Porphyrin Products of Logen, Utah, USA. This preparation was isolated in pure form and described by Fuesler and co-workers (T. P. Fuesler et al., Plant Physiol. 1981, 67, pp. 246-249 psi). Fresh Protogen IX was obtained by reduction of Proto IX with Na / H amalgams as described by Jacobs and Jacobs (Enzyme, 1982, 28 vols., 206-219 psi) working with 300 μΜ of purified Proto. IX.

Plant material

Cucumber sprouts (Cacumis sativus L., Wiskonsin SMR 18) were grown in a darkened germination chamber in vermiculite dampened with liquid branded fertilizer (9-45-15). Seedlings were grown at 25 ° C and 80-90% relative humidity. After seeding, the illumination was illuminated at a dose intensity of 25 μΕ / m with (PAR), using a Bright Stiek light source controlled by General Electric for 60 minutes. cycle by illuminating the sprouts for 1 min. In this way, plant tissue was obtained capable of accelerated synthesis of chlorophyll, minimizing the amount of reserve starch and the initial chlorophyll level.

Isolation of chloroplasts

Isolated chloroplasts were isolated according to the method described by T.P. Fuesler and coworkers (see Plant Physiol., 1984, 75 vol., Pp. 662-664) except that in the final purification step, the plastids were centrifuged at more than 40% (v / v) rather than at 45% by volume from Perco II. materials. The chloroplasts were then transferred again to the colloidal state in buffer containing 0.5 M mannitol, 20 μΜ TĘS ′, 10 μΜ HEPES, 1 μΜ ethylenediaminetetraacetic acid, 1 μΜ MgCl, 1% (w / v) bovine serum albumin, and 1 μΜ dithioerythritol at pH = 7.7 and a final protein content of 2 mg / L.

Protoporphyrinogen oxidase samples

Samples were prepared as described by Jacobs and Jacobs (J.M. Jacobs and N.J. Jacobs: Arch. Biochem. Biophys. 1984, 229 vol., Pp. 312-319). Chloroplast suspension samples (0.2 mL each) were tentatively 15 min. treated in the dark at different concentrations of acifluorophen-methyl (AFM) or, in the case of the control, at 0.2% (v / v) acetone. Then 50 μϊ of freshly prepared approximately 15 nM almost. Protogen was added to the suspension to initiate the reaction. The preparation of the samples resulted in the introduction of 2.75 ml of fluorometric material proposed by Jacobs and Jacobs (J.M. Jacobs and N.J. Jacobs: Enzyme 1982, Vol. 28, pp. 206-219, consisting of 1% v / v) 20 (polyoxyethylene sorbitan monolaurate), mM tris-HCl and 1 mM ethylenediaminetetraacetic acid pH = 8.5, and 1 mM dithioeri triol was replaced with 5 mM glutathione, and the suspension was then analyzed by direct reading on a SPEX Fluorog-2 spectrofluorometer. The amount of Proto IX formed was plotted against a standard curve of the quantitative relationship between Proto IX emission at 630 nm and excitation at 400 nm, incidentally obtained in pure Proto IX fluorometric medium.

To quantify the degree of non-enzymatic oxidation to Proto IX in the sample described above, the same sample was prepared except that a suspension of chloroplasts was used in place of the active chloroplast suspension for 15 min. by heating at 85 ° C. Subtracting the amount of Proto IX obtained in this way from the amount obtained in the presence of active chloroplasts gives the amount of Proto IX produced by the enzymatic pathway. The results are plotted in Figs. 1, where LSD is the smallest difference value (commonly accepted notation).

FIG. In Fig. 1, we can see that the index I ₅₀ (concentration providing 50% inhibition) is less than 0.1 μΜ, e.g., about 0.03 M.

Testing for the effect of 10 μ į AFM on the enzymatic conversion of Proto IX to magnesium in Proto IX chloroplast suspension (in the presence of ATP) did not show any inhibitory effect of AFM on this conversion.

example

In this example, enzyme inhibitory active components were added to the mice that are tumor carriers, while no preparations were added to the food of several control mice. The mice were then sacrificed, and the kidneys, intestines, adrenals, liver, tumors removed and analyzed for Proto IX. The following enzyme inhibiting active compounds have been used:

see Page 23

DBA / 2 Ha line mice were injected with SMT-F subcutaneous injection on the right shoulder on day zero. On the first day, the mice received a special diet for rodents: Purina Rodent Chow 5001 Mash without additives. Next, mice for 2 to 10 days received the same food with 2 per well of the enzyme inhibitory active compound under study (control mice received the same food but no additives). The additive was introduced into the food by adding a small amount of a solution of the test compound in acetone. On day 10, mice were sacrificed except for the mouse exposed to the active compound designated 6j: this mouse was sacrificed on day 7. The animal tissues were left overnight at 4 ° C to cool, then dampened with absorbent paper and the weight of each tissue sample determined. Subsequently, a homogeneous suspension of the tissues was prepared in 5 ml (10 ml for intestinal and liver tissues) of a mixture of acetone and 0.1 N NH ₄ OH, 9: 1 by volume. The homogeneous preparations were then centrifuged at 1500 g and the leached surface was analyzed with a SPEX FA112 spectrofluorometer. The optimum for the Proto IX is excitation at 400 nm and emission at 630 nm. Protein IX concentration was determined by the number of fluorescent flashes per gram of tissue per second. The results are shown in Table 3 below (mean values for each formulation are for a pair of mice except for the active compounds 6f, 6h, 6i and 6j, where only one mouse is used for the experiments).

table

Number of fluorescent flashes (x10 ³ s ³ ) per gram of tissue preparation Proto IX

Active compound Navi- who Kepe- nys Inks- this Antinks- chicks Gut- nas Tumor mass response to mg 6a 1203 4291 10817 6572 5870 * * ** 143 6b 1520 1261 2441 2529 5577 ** 90 6c 14336 2739 16186 13790 9937 ** 157 6d 2006 1581 12865 443 5565 ** 294 6e 836 876 915 1331 2069 ** 165 6f 449 1170 661 5611 1157 69 6g 166 803 606 1984 466 250 6h 298 914 585 2259 584 192 6i 932 1510 4014 36940 3454 20th 6j 1251 538 315 491 572 11th Controlled 239 536 494 1504 233 200

2 4 * *

Notes:

* The data in the table should be read with a factor x 10.

** Measurements were made on samples diluted with alkaline acetone (3 parts acetone for 1 part sample).

The data in Table 4 correspond to the concentrations of Proto IX in the respective tissues. Unit: Micro grams (µg) of Proto IX per g tissue.

table

Active power unit Tumor Liver The kidneys The adrenal glands Intestine 6a 11th 39 99 60 54 6b 14th 12th 22nd 23rd 51 6c 131 25th 148 126 91 6d 18th 14th 117 4 51 6e 8th 8th 8th 12th 19th 6f 4 11th 6th 51 11th 6g 2 7th 6th 18th 4 6h 3 8th 5 21st 5 6i 9th 14th 37 337 32 6j 11th 5 3 4 5 Controlled 2 5 5 14th 2 4

The Dougherty article quoted above indicates that the concentration of porphyrin in the same type (SMT-F) tumor after injection of 10 mg / kg hematoporphyrin in the same line of DBA / 2 mice is 3.6 pg / g. Another scientific publication (Moan et al: PP 46-5, page 713721, see Example 2, page 716) indicates that for CH / Tif tumors, 12 pg / g of porphyrin at a concentration of DBA / H ₂ line mice provide mg / kg intraperitoneal injection of Photofrin II (Photofrin II), widely used to provide photosensitivity in the photodynamic treatment and diagnosis of cancers.

The mice were fed the following amounts of food containing the active compound: 6a) 22 and 35 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) 10g; 6j) 20 g.

In the experiments described in Example 6, the non-tumor bearing animals, unless otherwise stated, used the active compound designated 6c in this example, and the sequence of operations was as follows:

(a) the LD ₅₀ for the active compound was found to be above 2600 mg / kg (ie mg active compound / kg body weight) in rats at 14 days after a single dose of corn oil with 15% active compound and above 700 mg / kg in mice at 14 days after oral administration of the same oil with 5% active compound.

(b) In the absence of active compound formulations for intraperitoneal injection, mice were able to withstand 0.5 ml dose of a mixture of equal parts dimethylsulfoxide and water.

c) Testing of the active compound by intraperitoneal injection in a mixture of 60% corn oil and 40% dimethylsulfoxide has shown that mice can withstand a dose of 100 mg / kg.

(d) The following experiments were carried out on mice:

1) 50 mg / kg dose daily (using a 1% solution of the active compound in acetone) for 8 days,

2) a daily dose of 50 mg / kg of a 1% dispersion of the active compound in mineral oil, prepared by dissolving the active compound in a drop of dimethylsulfoxide and mixing the resulting solution with mineral oil, for 8 days;

3) daily application of 50 mg / kg of 1% dimethylsulfoxide solution daily for 8 days,

4) fed standard food with 8 per cent by weight of the active compound within 8 days,

5) a daily intravenous dose of 50 mg / kg 2% dimethylsulfoxide for 4 days.

Thereafter, the experimental mice were sacrificed to detect the accumulation of Proto IX in their tissues.

In another experiment, the Fisher 344 male rat was fed with 5 perm of active compound 6c for 24 days, after which the animal was sacrificed. Increased levels of Proto IX were observed in the tissues of that rat compared to control animals, with significant increases in renal, intestinal, gastric, and cerebral tissues, whereas only minor increases were observed in muscle tissue.

In all the experiments described above, rats and mice were kept in standard illumination mode for 12 h. light and 12 hours. in the dark.

example

In a series of experiments according to Examples 1 and 2, HeLa culture cells were incubated with various drug active compounds at the concentration of 100 μΜ below. The percentages given after the labeling of each drug active compound show that the amount of Proto IX produced in the presence of a given drug active compound is increased relative to that produced in the control sample of the same experiment.

The active compounds used in Example 6 have the following values: 6a) 337%; 6b) 336%; 6c) 297%; 6d) 1200%; 6e) 1210%; 6f) 280%; 6g) 326%; 6h) 431%; 6i) 544%; 6j) 469%; the formulas of the other active compounds and their respective percentages are given below, (see page 29).

In this example, the enzyme-inhibiting active compound 6c from Example 6 was fed to rats with grafted tumor, followed by irradiation of the tumor area, thereby causing tumor resorption.

Namely, three rats of the Sprague Dawley line, weighing an average of 120 g each, were given a ration of food for 2 days with the addition of 2 perm of the active compound. On the third day, a chondrosarcoma was implanted on the right hind paw of each rat with 0.3 ml of tumor cell suspension (1 million tumor cells). On the sixth day, the hair of each rat's right hind leg was shaved and the size of each tumor was measured. Further, the affected lesion and surrounding skin were irradiated with non-thermal doses of light at 630 nm with an energy density of 270 J / cm. The next day, 2 rats appeared to be significantly rescued from their tumors (i.e., the tumor mass is more intangible) and the third rat almost resorbed. In all rats, the skin around the tumors and muscle cells were slightly pale and appeared to be slightly swollen, but to a much lesser extent than with Photofrin II in photodynamic therapy.

Annex A

Attempt to determine leaching percentage

Seedlings from cucumber seedlings grown in the dark were used to determine the percentage of leaching during the test. In the first step, the mass of the seminal vesicles was treated in the dark with an aqueous solution (in the presence of a buffer) containing sugar with radiolabeled atoms. It then measured the amount of sugar absorbed by the seminal vesicles according to the calculation procedure described below. The whole mass of the semen was divided into two portions. One of these portions was treated in the dark with an aqueous solution (in the presence of a buffer) containing the test compound and the other was treated in the dark with the same solution only, without the test compound (control). Soaked in seed solutions for 16 hours. They were irradiated with light and then separated from the aqueous solutions in which they were quantitated by measurement and calculation. The results obtained were then reported as the leaching percentage (IPR) calculated using the formula:

IPR = (S _T -S _C ) / S, where S, S _T and S _c are the number of degradation (s) per radiolabeled substance, respectively:

(a) in the material taken up by the seedlings during their initial processing,

(b) in aqueous solution of the test compound after irradiation; and

(c) in a control aqueous solution after irradiation.

The materials used, and t.p. the test conditions for the determination of the IPR are detailed below.

Plant materials

Vermiculite, moistened with commercially available fertilizer 9-45-15, was seeded with cucumber seeds Cocumis Salivus L., a species of Wisconsin SMR, from which seedlings were grown in an incubation chamber at 25 ° C and 8090% relative humidity. After 5 days from the time of sowing, the seedlings were collected from the pale, dark-grown seedling shoots and washed with 1.0 mM CaCl ₂ . All those operations were done in green light.

Buffer solution

The solution contains 1 mM KCl, 1 mM CaCl ₂ , 2.0 mM potassium phosphate; its pH was adjusted to 6.5 (as with NaOH).

Sugar with radioactive labeled atoms

That type of sugar is 3-O-methyl-3 [11-14C] -glucose with a specific activity of 10.9 GBk / mM; supplier: Amersham Corp. City of Arlington, Illinois, USA.

Pretreatment

180-230 units of the washed cotyledons were transferred into a wide-mouthed 250 ml Erlenmeyer flask with a polyurethane foam stopper, to which 50 ml of the buffer solution was added and added to the solution at a concentration of 600 nM. In the dark on a rotating device for 24 hours. run the flask for 125 min. ' ¹ frequency. The seedlings were then unloaded onto a nylon mesh and washed 3 times with 1 mM CaCl ₂ in portions of 20 ml each. The enrichment of radioactive labeled atoms 3997 B was determined by dissolving 3 samples, each containing 5 seedlings, in tissue solvent (NCS grade, manufactured by Amersham Corp.) and calculating total consumption on a liquid scintillation spectrometer.

The consumption determination experiment was found to be equivalent to the analogous setting by burning selected testis samples in a self-reducing device.

Treatment with test compound as well as control treatment

Five seed beads were allowed to float on the surface of 3 ml of buffer placed in a 35 mm plastic petri dish with a lid. Subsequently, an amount of the test compound (its solution in acetone) was added to ensure the required concentration (as stated above) of the test compound in the buffer solution. The amount of acetone in the buffer was 0.1% (v / v) in the control as well as in the test compound. Floating seedlings 16 hrs. spinning in a circular spin formed by shaking the plates for 90 min. ^-1 on the surface of the rotating shaker.

Irradiation with light

Petri dishes with seed beads floating on the surface of the solutions, 16 h. GE F20T12-CW fluorescent four fluorescent light at a given intensity of 150 μΕ / m spectral photosynthetic area (SFSS) and measured the intensity on the surface of the seminal vesicles while the plates were shaken continuously.

Measurement of the amount of radioactive tracer in a liquid

The whole fluid was separated from the seminal vesicles; liquid scintillation spectrometer determined the number of cleavages.

It's dark

Prior to the light irradiation stage, all other treatments were performed under green light emitted by a fluorescent source, i.e. in the light passed through a green separating plastic light filter that transmits light at 450-600 nm.

Annex B Table

A medium for cultures

M medium

Salts Molecularity Concentrate ml of concentrate 1 liter MgSO ₄ .7H ₂ O 1.2xlO ~ ³ M 10% 3 NH ₄ NO ₃ 3.7xlO ' ³ M 10% 3 kh ₂ after ₄ 2.2xlO ^'3 M 10% 3 k ₂ hpo ₄ 1.7x10 ^'3 M 10% 3

Concentrate of a mixture containing traces of metals

H ₃ BO ₃ 100 mg / l ZnSO ₄ .7H ₂ O 100 mg / l MnSO ₄ «4H ₂ O 40 mg / l COC1 ₂ .6H ₂ O 20th mg / l NaMo0 ₄ »2H ₂ 0 20th mg / l CuSO ₄ 4 mg / l

Medium A was prepared by adding 10 ml / l of 10% aqueous sodium acetate solution (concentration 7.5x10 M) to M medium.

All components were poured into distilled water in the above order, followed by 15 minutes. processed in an autoclave at a pressure of 1.05 kg / cm.

Stock of preserved preparation for light cultivation

The preserved yI cultures were axenically transferred to liquid cultures in media A or M, preferably media A, in Erlenmeyer flasks closed with polyurethane stoppers at 25 ° C for the following cycle: 14 hrs. lighting and 10 hrs. darkness. Canned cultures were incubated (without additional aeration) on a rotating shaker operating at 125 min ¹ . The illumination intensity at the culture layer level is 120 μΕ / sq.ms (SOFS). Under these conditions, the cultures are in equilibrium growth mode until they pass into a stationary phase containing (2-4) x105 cells per ml.

Cultivation of crops without light (crops grown in the dark)

7.5 ml of light-grown cells in the stationary culture phase from the preserved culture were transferred to an Erlenmeyer flask containing 750 ml of A medium.

The cells were incubated for 3 to 4 days in the dark by aeration at 25 ° C. During this time 7-8 cell y-I culture cleavage should have occurred, losing all visible chlorophyll and at a cell concentration of (2-3) x 10 units / ml.

Cell preparation for test specimens

Cells were isolated from 750 ml of freshly prepared dark culture by centrifugation for about 5 min. 20 ° C at low speed (about 2000 min ^x ). These cells were carefully transferred back to the suspended state in 50 ml A medium. This suspension in the sample (0.25 ml) was used to determine cell motility and, when fixed in 1% aqueous glyceraldehyde solution, counted the cell volume per ml. The most common cell number is 5x10 ⁷ units / ml. Portions of cell cultures containing 1 ml each containing 10 units. cells are placed in dishes for assays such as tissue culture plates with 24 wells. At this stage, the cells are yellow and highly motile.

The test compound was dissolved in a suitable solvent such as acetone, ethyl alcohol, dimethylsulfoxide or water (preferably acetone) to give a concentration up to 1000 times the final concentration. Five or ten μΐ microsyringe injected 1 μΐ of the test solution into each pair of wells. Analogically, 4 control wells without test compound were prepared on each plate. Plates for tissue cultures were covered with transparent plastic covers and 13-16 hours. was cultivated in a chamber at 25 ° C under illumination of 7090 μΕ / sq.ms. If the contents of the wells were yellow, they were removed as described below under Evaluation of Results. If the contents of the test wells appeared translucent or pale green, they were placed on a rotating shaker operating at a frequency of 125 min ' ^{1 for} 2 hours before removing it. light of an intensity of about 600 μΕ / sq.ms

Evaluation of results

Each well was filled with 250 μΐ of 10% aqueous sodium dodecyl sulphate solution and 1 ml of methyl alcohol, followed by thorough mixing. The resulting mixtures were kept in the dark for 3-4 hours. Plates containing the plants were treated in a low speed centrifuge (about 2000 min ^x ) for 5 min at room temperature. The resulting material was removed and analyzed by a 35 Beckman (Beckman) spectrophotometer at a wavelength range of 350-800 nm (detecting protoporphyrin IX, which has a chlorophyll absorption peak at 668 nm in the system) and a background opacity of 720 nm. .

Data Analysis

The gain rate for each well was obtained by subtracting from the value obtained at 668 nm the value obtained at 720 nm. Corresponding values are averaged for each concentration of test compound, and so on. for the control test. The percentage inhibition is determined by the formula:

Q = 100x (l-k) where k is the ratio of the mean value of the decay rate for each specific concentration to the mean value of the decay rate of the control.

Claims (10)

DEFINITION OF INVENTION 1. A specific inhibitor of the enzymatic conversion of protoporphyrinogen IX to protoporphyrinogen oxidase in mammary gland tumor cells, thereby inducing the production of protoporphyrin IX in these cells, which is not a tetrapyrrole derivative but an aryl-heterocyclic compound Ph-NHet wherein Ph is substituted phenyl and N-Het is a 5- or 6-membered heterocyclic ring having the formula: - N - C = O, - N - C - OH or - N - C - Cl! / I II I II Q Q-C-R Q-C-R wherein Q is a heterocyclic ring-closing group and R is hydrogen or a substituent group; aryl-heterocyclic urethane; phenylether; arylpyrazole or a compound of the formula: or wherein X is oxygen or sulfur and Ph is as defined above. 2. An inhibitor according to claim 1, characterized in that said compound I protoporfirinogenoksidazei ₅₀ is less than 1 μΜ. 3. An inhibitor according to claim 1 or 2, characterized in that it has a redox potential less than -500 mV. An inhibitor according to any one of claims 1 to 3, characterized in that it is 1- (4-chloro-2-fluoro-5-propargyloxyphenyl) -3-methyl-4-difluorometh-Δ ² 1,2,4 -triazolin-5-a. An inhibitor according to claims 1-4 for destruction of mammary gland tumor cells by exposure to light and in the presence of tetrapyrrole. An inhibitor according to claims 1-4 for diagnosing and localizing mammary gland tumor cells by increasing the amount of porphyrin in such cells and examining the mammary gland cells after irradiation with light activating porphyrin and measuring the fluorescence source of the tumor. 7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 which is a specific inhibitor of the enzymatic conversion of protoporphyrinogen to protoporphyrin IX, thereby inducing the production of protoporphyrin IX in mammary gland tumor cells, and a pharmaceutically acceptable carrier. A pharmaceutical composition according to claim 7 for the destruction of mammary gland tumor cells by exposure of the cells to light and in the presence of tetrapyrol. A pharmaceutical composition according to claim 7 for diagnosing or localizing mammary gland tumor cells by increasing the amount of porphyrin in these cells and assaying the mammary gland cells after irradiation with light activating porphyrin without measuring the fluorescence source of the tumor. 10. A process for the preparation of protoporphyrin IX, characterized in that it is isolated from eukaryotic class algae microorganisms grown in the medium of claims 1-4.