CS176291A3 - Pharmaceutical - Google Patents

Description

Pharmaceutical composition

Technology '- -

This invention relates to pharmaceutical compositions for the treatment of polycythemia vera tumors.

Background Art

The present invention relates to the treatment of polycythenia vera, a known disease in which myeloid cells are produced, produced by the bone marrow, in particular red blood cells and hemoglobin. In advanced disease, the spleen increases, removing the mature red blood cells from circulation. The disease usually starts in the middle years or a little later. Untreated patients survive approximately 2 years. 10 to 12 years of survival can be obtained from vein blood. Other treatments have been used to suppress bone marrow function by irradiation or by the action of chemical substances of an alkylating nature, such as melphalan, buffan and chlorambucil, but these may result in leukemia or other tumors. In addition, hydroxyurea was also used to inhibit DNA synthesis.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides pharmaceutical compositions for treating said disease, the active ingredient of which is an inhibitor of the enzymatic conversion of protoporphyrinogen to heme in cells. Thus, it has been shown that certain substances, for example certain types of herbicidal compounds, capable of causing the enzymatic conversion of protoporphyrinogen to chlorophyll in plant cells are also capable of causing the aforementioned conversion of this substance to heme. This inhibition is likely to affect the degree, in which the protoporphyrinogen is not converted to protoporphyrin by the IXenzyme, the protoporphyrinogenase, so that the anti-phyrinogen can not be converted to proto-porphyrin IX by a normal enzymatic process and instead is oxidized in the cell, e.g. outside the normal enzymatic conversion, 2, for example, outside the organelles, so that the proto-porphyrin IX accumulates at sites where it is unavailable for normal heme exchange. The effect of these substances on the enzymatic conversion can be demonstrated in experiments in which human tumor cells are grown, for example Hela cells in the dark in the presence of 1 or 10 to 100 µM of said substance, thereby forming an excess of protoporphyrin IX in both cells and culture medium. . For example, treatment can be performed orally or parenterally, such as intravenously or intraperitoneally. For this purpose, the active ingredient may be formulated into conventional pharmaceutical formulations such as tablets, for example compressed tablets, which may be coated, for example sugar, suppositories, capsules, for example hard or soft gelatin, suspensions, solutions, powders. or ampoule. In these compositions, the active ingredient may be present in admixture with a solid and / or liquid, pharmaceutically acceptable carrier, which may be a nutrient, the solid carrier may be corn starch, the liquid diluent may be water, an edible or mineral mineral or solvent For example, dimethylsulfoxide. For injection purposes, the active ingredient may be dissolved or dispersed in a sterile, pharmaceutically acceptable carrier, for example 0.9% aqueous sodium chloride solution or this phosphate buffered solution (Dulbecco), PBS, at a concentration of e.g. 5 mg / ml, or the active ingredient may be formulated into a liposomal system, for example using a phospho-lipid as the vehicle, as described in Remington's Pharmaceutical Sciences, 1985, 17th edition, pp. 1659-1660, Mack Publishing Company. For example, Oe can be performed according to Jori et al., 3r. 0. Cancer (1983), 48, p. 307 dissolves 51.4 mg of dipalmitoyldiphosphatidylcholine in 10 ml of a 1M solution of the active ingredient in a 9: 1 mixture of chloroform and methanol and after mixing, the solvent can be evaporated in vacuo at 30 ° C. to give a suspension which can be resuspended in 10 ml of 3 0.01 M phosphate buffer pH 7.4 containing 150 mM NaCl, and the supernatant solution is sonicated at 50 ° C for 30 minutes.

Especially in the case of oral administration, it may be desirable to be able to induce enzyme inhibition, in the form of water-soluble salts or, in the case of acidic substances, the sodium and potassium salts soluble in water, as is the case with the acid sulfonamide of PCT WO. No. 03732 or WO 85/001939 and WO 87/03873, for example 6c and 8h, further include water-soluble salts thereof, carboxylic acids or their water-soluble salts, e.g. sodium salt, known as acifluorfen.

The most convenient route of administration and dose at the most preferred therapeutic index may be determined by conventional assays commonly practiced in the art. The inhibitor-acting agent can be administered to laboratory animals, such as rats or mice, to determine the effect of different doses on the number of red blood cells and on the hemoglobin content of the blood. In vitro experiments can also be carried out using bone marrow cells collected from patients with the disease, such as the erythroleukemic A.T.C.C. K562, which is described, for example, in Bridges, J. Biol. Chem. 260, no. 11, 6811-6815, June 10, 1985. Depending on the data obtained in in vitro experiments and in animal experiments including toxicity studies in larger animals such as dogs or pigs, human clinical trials can be performed to ensure safety and at the same time good information on the effects of the substances under investigation, in which case the Human Experimentation Review Boards are followed. In rat and mouse experiments, 8h, which will be described below, is added to the feed at 3000 ppm and administered for 13 weeks, resulting in a statistically significant reduction in red blood cell counts, hemoglobin concentration, hematocrit, mean blood cell volume and mean content. hemoglobin in them without simultaneously inhibiting the production of white blood cells. After 28 days after omission of the test feed, the values returned to normal.

The invention also relates to the use of said compounds for the treatment of neonate or hyperbilirubinemia. As disclosed in U.S. Pat. No. 4,831,024 and in the publications cited therein, heme oxigenase inhibitors such as zinc and tin have been used for this purpose, presumably because they inhibit inhibition of heme to bilirubin. Compounds of the invention that cause protoporphyrin accumulation at sites where it is unavailable for normal heme conversion will also reduce the production of abnormal amounts of bilirubin. This will probably treat small children with too high carbon dioxide production, which is the cause of the risk of severe jaundice. The tests have been described in Smith and others as disclosed in U.S. Patent No. 4,831,024. For example, a mutant strain of Wistar, the so-called Gunn strain, which has hereditary acholuric jaundice as described in the literature cited therein, may be used in animals to determine the dose and route of administration.

The compounds of the invention are specific inhibitors of protoporphyrinogen conversion to heme but do not act as enzymatic poisons and do not denature the molecule as jetom in denaturing or crosslinking agents with sulfhydryl groups, preferably not materials that affect oxidation, such as acceptors electrons. Preferred compounds of the invention are compounds whose redox potential is more negative than -500 mV, such as less than -300 mV, as measured, for example, in a conventional aprotic solvent by cyclic voltmeter or polarographically. Preference is also given to substances of the tetrapyrrole ring with a protoporphyrin oxidase of less than 5 µM, ΐ r ΐ θ greater than 5, preferably less than 1 µM and a PI 5Q of greater than 6, for example a value of less than 0.3 µM is 0, 1 or 0.03 µM or 0.01 µM, respectively. The enzyme inhibiting agent preferably has a high ability to disrupt plasmalem of plant material. This ability test has been described in Halling and Peters,

Plant Physiology, 84, 1114-5 (1987). In this test, which will be described in more detail in the annex, the preferred materials have a total value of the exiting materials of at least 50% at a concentration of 100, preferably 1 µM or less, for example 100 nM. Highly active substances such as 1- (4-chloro-2-fluoro-5-propargyloxyphenyl) -9 -3-methyl-4-difluoromethyl-delta-1,2,4-triazolin-5-one quililactophene which will be further as described in more detail, causing the material to fully exit, the values being above 90% at a concentration of 100 nM.

Another test that can be used to verify the material's capacity to disrupt the plant material plasmolium is to lighten the greening, which will be described in more detail below in Appendix B. This assay measures the ability to inhibit lightning of Chlamydomonas reinhardi, a γ-1 mutant. It is an algae that does not form chlorophyll when grown in the dark, so white algae is formed, but the algae begin to produce chlorophyll when exposed to light. A number of preferred compounds of the invention have the ability to cause light greening inhibition to at least 50% when used at a concentration of 10 µm, preferably 10 µM, or even lower, for example, 10 µM. the negative charge does not give good results in this test, probably because algae cells do not allow acid group entry In addition, a number of preferred agents provide supernatant at the concentrations used, wherein the light absorption at 405 nm is higher than that for chlorophyll, -6 peak at 400 nm is compared to the peak for chlorophyll, which is twice, three or four times higher in this system at 668 nm, and herbicidal compounds of the following groups A are particularly suitable from the inhibitory agents useful in the practice of the invention. to GA Arylheterocyclic herbicides of the formula

Ph-NHet wherein Ph is substituted phenyl, preferably 2,4-disubstituted and more preferably 2,4,5-trisubstituted phenyl and NHet is a heterocyclic radical of 5 or 6 ring atoms, with 1 to 4 ring nitrogen atoms, in general of formula - N - C = O or - N- C - OH or -N - C - Cl

Q is a radical of the heterocyclic ring and R is a hydrogen atom or a substituent.

This class of materials includes, for example, materials referred to as "cyclic imide herbicides" as described by Wakabayashi et al., J. Pesticide Sci., 11, 635-606 (1986) with the following general formulas. : 7

O 8 (V)

The compound is aryloxadiazole, namely 2-tert-butyl-4- (2,4-o-dichloro-5-isapropoxyphenyl) -eca-1,3,4-oxadiazolin-5-one.

Other 2-alkyl-4- (2,4,5-trisubstituted phenyl) -delta [3,4-oxadiazolin-5-ones which can be used according to the invention have been described, for example, in U.S. Pat. 385,862, 3,836,539 and 3,876,413.

Compound II is aryltetrahydroindazole, 3-chloro-2- (4-chloro-2-fluoro-5-isopropoxyphenyl) -4,5,6,7-tetrahydro-2H-indazole. Other 3-substituted-2- (2,4,5-trisubstituted phenyl) -tetrahydroindazoles which can be used according to the invention have been described, for example, in U.S. Patent No. 4,670,043.

Compounds III, IV and V are aryltetrahydrophthalimides.

Other substances of this type which can be used according to the invention have been described, for example, in U.S. Pat. Nos. 4,431,322, 4,670,046, 4,670,042 and 4,439,229 and in International Publication No. WO 87/07602. In the last two citations, other applicable NHet circles are also listed, for example, Column 4, line 25 up to Column 5, line 20 of U.S. Patent 4,439,229 and pages 12-14 of WO 87/07602. .

Other suitable Ph-MHet herbicides are the following: aryltriazolinones described, for example, in U.S. Pat. 4,318,731, 4,398,943, 4,404,019, 4,702,945, 4,705,557, 4,702,763, 4,761,174, and in International Patent Applications WO 85/01637, WO 85/04307, WO 37/00730, U0 87/03782 U0 86/04481 and WO 33/01133, 9-aryl tetrazolinones disclosed in, for example, U.S. Pat. Nos. 4,734,124 and WO 85/01939 and WO 87/03873, aryl aryltriazinediones, for example those disclosed in U.S. Patent Nos. 4,755,217 and 4,766,233 and in the International Patent Application. WO / 86/00072, - arylhydantoins, described, for example, in U.S. Patent No. 4,427,438,

-arylimidazole pyrimidines, described, for example, in DE 2 604 989 (Derwent Abstract No. 65326X), in Japanese Kokai J60-158147A (Derwent Anstracts No. 85-240363) and in European Patent Application EP 230 874 (Derwent Abstracts no. Arylpyridyldiazines, for example compounds 8x of Example 8, - arylldiazinediones, for example compounds 8e and 8v of the following Example 8, - arylpyradiazinones, for example compounds 8f of the following Example 8, - aryloxadiazolinones, described, for example, in Japanese application Kokok359-148 769 (Derwent Abstracts No. 84-246947) or Kokai362-161772 (Derwent Abstract No. 87-238787), aryloxadiazinanes, for example compounds 8k and 8m of the following Example 8, - arylbenzamidazoles, e.g. Example 8, - aryliminotriazolpyridazines, for example those described in U.S. Pat. Nos. 4,913,723, 4,906,281 and 4,906,279, - arylthiazoles, e.g. of type 3 (5, 8r and 8ah of the following Example 8, - arylpyrroles, for example those disclosed in published European applications EP 255601 (Derwent No. 88-037583) and EP No. 260 288 (Derwent No. 83) Arylurazoles as described, for example, in U.S. Patent No. 4,452,981 and 10-arylhexahydropyridazines, such as described in U.S. Patent No. 4,619,687. 8. Heterocyclic arylurethanes, e.g., U.S. Patent No. 4,521,242 C. Heterocyclic aryl ureas, for example, according to Japanese application Kakai 358-225081 (Oerwent No. 84-034261). D. Arylamides, for example compounds of type 8a and 8 and following example 8. E. Phenyl ether herbicides, for example those having a β-halogen or p-nitrophenoxyphenyl structure, may be, for example, the following commercially available materials. 5- (2-chloro-4- (trifluoromethyl) phenoxy) -2-nitro-N-methanesulfonylbenzamide (fomesafen) methyl 5- (2,4-dichlorophenoxy) -2-nitrobenzoate (bifenox)

11 5- (2-Chloro-4- (frifluoromethyl) phenoxy) -2-nitrobenzoate sodium (acifluorfen) 2-chloro-1- (3-ethoxy-42-nitrophenoxy) -4-trifluoromethylbenzene (oxyfluorfen)

Other suitable diphenyl ether herbicides that are commonly supplied are: lactophene: 1- (carbethoxy) ethyl-5- (2-chloro-4-trifluoromethyl-phenoxy (-2-nitrobenzoate, fluorglycophene: (carbethoxy) methyl-5- (2 -chloro-4-trifluoromethyl-phenoxy) -2-nitrobenzoate, chlornitrofen: 2,4,6-trichloro- (4-nitrophenoxy) benzene, fluordifene: 2-nitro-1- (4-nitrophenoxy) -4-trifluoromethylbenzene, nitrofen 2,4-dichloro-1- (4-nitrophenoxy) benzene, chloromethoxyphene: 4- (2,4-dichlorophenoxy) -2-methoxy-1-nitrobenzene.

Other suitable diphenyl ether herbicides are, for example, methyl 5- (2-chloro-4-trifluoromethylphenoxy) -2-nitroacetophenone-dxim-O-acetate herbicides. F. F. Arylpyrazole herbicides, e.g.

Nos. 4,563,210, 4,496,390 and 4,459,150 and in accordance with DOS No. 3,520,327 A1. G. Compounds of general formulas

where

X b represents an oxygen or sulfur atom and

Oh has the above meaning, compounds of this type are described in European Patent Application No. 273 417, Derwent No. 37-040749. Some enzyme-inhibiting herbicides have been described by Matringe et al., FE3S Letters, Vol.245, No. 1, 2, pp. 35-33, 1939 and Yanase et al., Pesticide Biochemistry and Physiology, Vol. 35, pp. 70-30, 1989. Some specific substances useful for the inhibition of the invention will be described below. 13

O

14

15

O '.ό

17

19

C = CH 20

21

CH 22

23

24

25 81

CH-CH-3m 8n

26

27 8s 8t

F

28

Ί 29

Hi h3c ο CH. - 30 -

31

F

O 32

55 o

34 3ao

0 35

Ο

Cl 36

Determination a) When using enzyme and intact chloroplasts:

Protoporphyrinogen IX, further Protogen IX

Therefore, IX was obtained from Porfyrin Products, Logan, UT to be purified according to TP Fuesler et al., PlantPhysiol., 67, 246-249, 1981. Protogen IX was freshly prepared by reducing the proto IX amalgam of sodium in mercury by N. 3. Jacobs and 3. M. Jacobs, Enzyme, 28, 206-219,1982, using purified Proto IX at a concentration of 300 µM.

Plant material

Cucumis sativus L., cultivar "Wisconsin SMR18", was grown in a dark vermiculite chamber and watered with a commercially available 9-45-15 fertilizer. Seedlings were grown at 25 ° C and 80-90 relative humidity Intermittent illumination was used, 1 minute light at 60-2 -1 minute cycle, 25 / uE ms (PAR) intensity was used with a "light stick" (General Electric) , the control was coupled with an electronic timer. In this way, tissue was obtained, capable of rapidly synthesizing chlorophyll with simultaneous minimal starch stocks and a low initial chlorophyll content.

Chloroplast isolation

The developing chloroplasts were isolated according to T. P. Fuesler et al., Plant Physiol., 75, 662-664, 1984, with the difference that at the final purification step the plastides were centrifuged over 40% Percoll and not 45% vol. The chloroplasts were then resuspended in assay buffer, buffer containing 0.5 Mmannitol, 20 mL TES, 10 mM HEPES pH 7.7, 1 mM EDTA, 1 mM magnesium chloride, 1 ¾ serum bovine serum albumin. and 1mM di-37 thioerythritol, the final protein concentration in the buffer was 2mg protein / ml.

Protoporphyrinogen oxidase assay

The assays were carried out according to 3. M. Jacobs et al. 3. Jacobs, Arch. Biochem. Biophys, 229, 312-319, 1984. Samples of 0.2 ml chloroplast suspension were pre-incubated for 15 minutes in the dark at various concentrations of acifluorophenomethyl APM or with 0.2% acetone as a control.50 microliters of freshly prepared Protogen IX, i. about 15 nM is added to the suspension to initiate the reaction. The assay is terminated by the addition of 2.75 ml of fluorometric medium as described in M. 3. 3acobs and 3. M. 3acobs, Enzyme, 28, 206-219, 1982, this medium consisting of 1 ovým volume polyoxyethylene sorbitan monolaurate, i.e. Tween-20, 50 mM tris-HCl, pH 8.5, 1 mM EOTA, 1 mM dithioerythritol DTE was used instead of 5 mM glutathione. The suspensions were then read directly from a SPEX fluorolog-2 spectrofluorometer equipped with a fluorosensor screen for turbid biological samples. The amount of produced Proto IX was quantitatively subtracted from the standard curve obtained from purified Proto IX in a fluorometric environment, the amount of Proto IX compared to the emission at 640 nm at P5I excitation of 400 nm.

In order to determine the amount of non-enzymatic oxidation to Proto IX in this experiment, the experiment was performed using a suspension in which the chloroplasts were inactivated by heating at 85 ° C for 15 minutes in place of the active chloroplast suspension. By subtracting the amount of ProtoIX produced under these conditions from the amount of the same substance obtained in the presence of active chloroplasts, the amount of enzymatically produced Proto IX is obtained. The results are shown graphically in Fig. 1, in which the LSO is the smallest significant difference as commonly reported. Fig. 1 shows that the Ι ^ θ, ie the concentration at which 50¾ inhibition occurs, is less than 0.1 micromol, ie. about 0.03 micromol. In the tests for the effect of 10 micromoles of AFM on the enzymatic conversion of Proto IX to the magnesium salt of Proto IX in a chloroplast suspension in the presence of ATP, it can be shown that AFM does not inhibit the conversion. b) When using an isolated enzyme

Plant material and homogenization

Peas (Pisum sativum var. Little Marvel) were plated for 10 days in a dark chamber at 20 ° C. Rost-lines were illuminated for 1 hour per day, allowing developers to keep chlorophyll to a minimum. The particles were pale green in color. The leaves were homogenized in a 500 ml homogenization medium containing 0.5 M mannitol, 10 mM HEPES, 20 mM TES, 1 mM EOTA, 1 mM magnesium chloride, 5 mM cysteine 0.2 8SA at pH 7.7. The resulting slurry was filtered using four layers of gauze and then through a nylon mesh. with mesh size 43 microns. Thereafter, the monogenate was centrifuged at 4000 g for 3 minutes. The resulting pellet was used to isolate plasmas.

Etioplasts and their cleaning

The pellet, i.e. the plastid fraction, was re-dosed in 40 ml of the assay medium, with the same composition as the homogenization medium but without cysteine and containing 1 mM OTE 81¾ ESA. Then the material was centrifuged at 150 g to remove cell debris. The supernatant supernatant was centrifuged at 4000 g and the pellet resuspended and overlaid at 40 µl Percoll. An additional centrifugation of 3 m., Inute at 6500 g, fixed the etio-plastics at the bottom of the tube.

Protein content was determined using a method recommended by BioRad.

Enzyme solubilization

The enzyme was solubilized by the procedure that Jacobs Jacobs did not cast. The plastids were centrifuged and their pellet resuspended in 2.5 ml buffer containing 20 mM tris-HCl, 30 h glycerol and 1 mM OTE pH 7.6. The mixture was sonicated at 60 mHz and then the same volume of extraction medium containing 10 µl of Triton 10XX wetting agent, 8 µL of KCl, and 10 mM PMFS was added to a final wet to protein ratio of 0.7. After 3 hours at 48 ° C, the solution was centrifuged for 1 hour at 100,000 g and ultracentrifuged using a Beckman L2-65B centrifuge.

Gel filtration

The solubilized enzyme supernatant was then removed by filtration on a Sephadex G-25M column (Pharmacia PD-10). The columns were washed with 25 mL of elution buffer containing 20 mM Bistris-HCl, 20 µL glycerol and 0.1 µL Triton X opH 6.8. Samples were loaded onto columns and eluted with 3.5 ml buffer. The first 1 mL fraction was discarded, the remaining 2.5 mL fractions were collected and then lyophilized under nitrogen at -77 ° C. Purification by DEAE chromatography 0.75 ml of solubilized enzyme was applied to an EEA-5PW column of 8 mm x 7.5 cm (Waters Protein-Pak) 40 at steady state in elution buffer. After 60 minutes with wash buffer at a rate of 1 mL per minute, the enzyme was eluted using a linear 0.1 M sodium chloride gradient over 100 minutes. The gradient was then changed from 0.1 M to 0.8 M NaCl over 30 minutes. 4 ml fractions are collected and monitored for efficacy. The peaks are detected using a UV light detector (Waters 494) at 260 nm, sensitivity is 1UU. Efficacy of protoporphyrinogen oxidase

The enzymatic conversion of protoporphyrinogen IX to protoporphyrin IX is followed as in (a) above. In this assay, compound 6c had Ι ^ θ in the range of 0.8 to 0.3 µM, corresponding to ρΐ ≥ θ 6.1 to 6.5, with pI5Q being the negative logarithm in molar concentration. ProAFM is 0.8 µM, ρΙ5θ = 7.1, and for 8v, it is within the range of 0.08 to 0.03 µM, pI 50 is 7.1 to 7.5.

Supplement A

Percent Output Material Cotyledons from ethiolated cucumber seedlings were used. In the initial step, cotyledons are treated in the dark with an aqueous buffer solution containing radiolabelled sugar. The amount of sugar that is received is measured using a computer. The amount of cotyledons is then divided into two parts. One part of the cotyledons is treated with a buffer solution containing the test substance, the other is treated in the same manner with a buffer solution without the test substance, a control group. The cotyledons are then exposed to light for 16 hours in contact with aqueous solutions and then removed from the aqueous solutions. The solutions are measured by a computer to determine the content of the radiolabelled material. The results are expressed as 41% material output, which can be calculated as follows: material output in ¾ = -

S where S is the number of radiolabelled material, one cotyledon is received at initial exposure,

Sy is the number of pulses of radiolabelled material per cotyledon in aqueous solution containing the test compound after exposure to light and

Sg is the number of pulses of radiolabeled material per cotyledon in the control aqueous solution after exposure to light. The following assay conditions were used in the assay.

Plant material:

Cucumber seeds (Cucumis sativus L. cultivar WisconsinSMR 18) are allowed to germinate and grow on a vemiculite which is irrigated by conventional fertilizers 9-45-15. The seedlings are grown at 25 ° C and 80-90% relative humidity in a dark incubation chamber. The cotyledons are harvested from the etiolated seedlings after 5 days and rinsed in 1.0 mM calcium chloride, all done in a bright light.

Buffer solution:

From a solution containing 1 mM KCl, 1 mM calcium chloride and 2.0 mM potassium phosphate, the pH was adjusted to 6.5 with, for example, sodium hydroxide. 42

Radiolabelled sugar:

3-O-Methyl-3- (U-14C) glucose was used with a specific potency of 10.9 GBq / mmol (Amersham Corp., Arlington Heights IL)

Initial Processing:

The washed cotyledons at 180-230 were added to a 250 ml wide-necked Erlenmeyer flask with a stopper plug containing 50 ml of buffer, and the labeled sugar was added to a concentration of 600 nM. Shake the flask on a shaker in the dark for 24 hours at 125 rpm. Pakse cotyledons are separated using a nylon mesh and washed three times with 20 ml of 1 mM calcium chloride. The incorporation of radioactive sugar is measured by destroying three samples of five cotyledons in an NCS homogenizer (Amersham Corp.) and measuring the resulting macerate on a liquid scintillation spectrometer. The results of these tests are equivalent to the same assay that is performed by burning the cotyledons in an auto-oxidation apparatus. Treatment of the test compound and control experiment: Five cotyledons are placed with abaxial side up to 3 ml of buffer solution in a closed plastic mass 35 mm diameter petri dish. The test substance is then added in acetone solution in such a quantity as to obtain a predetermined concentration in the buffer. Finally, the acetone concentration is 0.1% by volume in both the test and control samples. The floating cotyledons are then shaken at 90 rpm for one hour on a shaker. Light Effect:

Cotyledon-containing dishes are exposed to light from four GE F2CT12-CU fluorescent lamps at 150 [mu] m m < 2 > s < 1 > Q for a symmetrically effective region of the spectra (PAR), measured on the surface of the cotyledons by shaking. 43 Measurement of Radiolabelled Material in Liquid: All liquid is separated from the cotyledon, using a liquid scintillation spectrometer for counting.

Processing in the dark: All pre-illumination treatments are performed in the dark or with green fluorescent light, which filters through a green plastic filter that transmits light through 450 to 600 nm.

Appendix B Environment

Medium M salt molarity stock ml of stock solution / 1 sodium citrate. 6H20 1,7 x 10 '10 ¾ 5 trace elements values below 10 FeCl3,6H20 0,37 x 10'3M 1 h 1 CaCl2.2H20 0,36 x 10 "3M 5,3 x 1Q'3M 10% 3 NH.NO, 4 i »3.7 x 10'3M 10 h 3 kh2po4 2.2 x 10'3M 10 ¾ 3 k2hpo4 1.7 x 10'3M 10 h 3 - 44 - Stock Mixture of trace element mixtures H38Q3 100 mg / 1 ZnS04.7H20 100 mg / 1 MnS04.4H20 40 mg / 1 C0Cl2.6H20 20 mg / 1 NaMo04.2H20 20 mg / 1 CuS04 4 mg / 1 Nutrient A is prepared by 10 ml / l of an aqueous 10¾ solution of sodium acetate is added with a molar concentration of 7.5 x M -M to the M medium.

The ingredients are added to the distilled water in the above order and then the mixture is heated for 15 minutes in an autoclave at 0.15 MPa. Stock cultures in the presence of light: The stock cultures of y-1 cells are maintained in liquid culture in A or M medium, preferably in M medium for 14 hours light and 10 hours dark at 25 ° C in an Erlenmeyer flask, sealed with a polyurethane stopper. The stock cultures are incubated without additional aeration on a rotary shaker at 125 rpm. Light intensity in culture is 12Q, uE-2-1m s (PAR). Under these conditions, the cultures grow in a semi-synchronous growth mode until a steady phase of 2-4 x 10 6 cells / ml is reached. 45

Non-dark cultures in the dark: 7.5 ml of cells from the stationary phase of the stock culture grown in the presence of light are transferred to 750 ml of medium A in an Erlenmeyer flask. The flasks are incubated in the dark air chamber with a submerged tube for 25 to 25 days air intake. During this period, 7 or 8 cells are separated by y-1 cells by cell division while losing all visible chlorophyll, the cell concentration of sepac is adjusted to 2-3 x 10 6 cells / ml. Preparation of cells for use in the experiment:

Cells are separated from 750 ml of freshly prepared culture grown in the dark by centrifugation at low speed, approximately 2000 rpm for approximately 5 min at 20 ° C. Then, the cells were resuspended carefully in 50 ml of A. The suspension sample of approximately 0.25 ml was subjected to a mobility test and the cell number in ml was determined after fixation with 1 l of an aqueous solution of glutaraldehyde. The normal number is 5 x 10? ml ml. Shares of 1 ml, with 10? the cells are placed in test wells, for example 24-well plates (Falcon). At this point, the cells are very mobile and have a yellow color.

The test substance is dissolved in a solvent such as acetone, ethanol, dimethylsulfoxide or water, preferably in acetone to a concentration that is 100x higher than the final concentration. Thereafter, 1 µl of this test solution is added to each of the two wells using a 5 or 10 microliter syringe. Four control wells are prepared per plate with the method described without the test substance. The plates are then covered with a transparent plastic film and placed in a growth chamber at 25 ° C for 13-16 hours at a intensity of -2-1 light 70-90 µmE. If the contents of the test wells are yellow, the wells are extracted in the manner described below. When the content is transparent or light green, it is placed on a rotary shaker at approximately 125 rpm and irradiated for 2 hours at a intensity of -2-1 light / 600 µE m sec prior to extraction.

Evaluation of results:

A 10 µl aqueous solution of sodium dodecyl sulfate in 250 µl and 1 ml of methanol is added to each well and mixed. The mixture was then allowed to stand for 5 to 4 hours at room temperature, and the plates were centrifuged at room temperature for 5 minutes at a rate of about 2000 rpm. Supernatants are collected and analyzed in a spectrophotometer (Beckman Model 35) at 350 to 500 nm for protoporphirin IX at 668 nm, the peak for chlorophyll absorption in this solvent system and at 720 nm to read the solution turbidity.

Value Analysis:

For each well, a green color value is obtained by subtracting the value obtained at 720 nm from that obtained at 668 nm. Calculate the mean for each test concentration and control. Percentage inhibition is calculated from the mean value for green coloring for 100 x (1 - Obedient concentration.

Claims (32)

PATENT N CLAIMS 1. A pharmaceutical composition for the treatment of a disease of polycythemia vera in mammals comprising as its active ingredient a specific inhibitor of protoporphyrinogen to heme enzymatic conversion in mammalian cells in an amount sufficient to inhibit this conversion and, in addition, a carrier acceptable from the pharmaceuticals. viewpoint. 2. A pharmaceutical composition according to claim 1 wherein the active ingredient is a different tetrapyrrole type and its redox potential is more negative than -500 mV. 3. A pharmaceutical composition according to claim 1, wherein the active ingredient for the protoporphyrinogen oxidase is less than 10 .mu.M. 4. A pharmaceutical composition according to claim 3 wherein the active ingredient is less than 1 micromole. 5. A composition according to claim 1, wherein the active ingredient is a nitrodiphenyl ether type herbicide. 6. A pharmaceutical composition according to claim 5 wherein the active nitrodiphenyl ether comprises a carboxylic acid ester substituent. 7. A pharmaceutical composition according to claim 6 wherein said substituent is a more alkoxycarbonyl-lower alkoxycarbonyl. 3. A pharmaceutical composition according to claim 5 wherein the nitrodiphenyl ether substituent is a lower alkoxy group. 48 9. A pharmaceutical composition according to claim 1 comprising the active ingredient of the formula Ph-NHet wherein Ph is substituted phenyl and NHet is a heterocyclic radical of 5 or 6 ring atoms containing 1 to 4 nitrogen atoms, of the formula -N-C = O or -N-C-OH or -N-C-Cl wherein Q is a heterocyclic ring radical and R is a hydrogen atom or a substituent. 10. A pharmaceutical composition according to claim 1, wherein the active ingredient is a general formula wherein Ph is substituted phenyl. 11. A pharmaceutical composition according to claim 1, wherein the active ingredient is represented by the general formula (49). wherein Ph is substituted phenyl. 12. A pharmaceutical composition according to claim 9 wherein Ph is phenyl with a substituent of formula N-SO2-R wherein R is lower alkyl. 13. A pharmaceutical composition according to claim 9 wherein Ph is phenyl with a substituent containing a terminal 1- (lower alkoxycarbonyl) ethoxy group. 14. A pharmaceutical composition according to claim 9 wherein said substituent is 1-9 lower alkoxycarbonyl) ethoxy / phenoxy. 15. A pharmaceutical composition for use in the treatment of elevated bilirubin in neonates, comprising, as an active ingredient, a compound which is a specific inhibitor of the enzymatic conversion of proto-porphyrinogen to heme in mammalian cells in an amount sufficient to promote and, in addition, a carrier acceptable from a pharmaceutical standpoint. 16. A pharmaceutical composition according to claim 15, wherein the active ingredient is 50 different from tetrapyrrole and its redox potential is less than -500 millivolts. 17. A composition according to claim 15, wherein the proprotoporphyrinogen oxidase active ingredient is less than 10 micromolar. 18. A pharmaceutical composition according to claim 17 wherein the Σ50 of the active ingredient is less than 1 micromole. 19. A pharmaceutical composition according to claim 15, wherein the active ingredient is a nitrodiphenyl ether herbicide. 20. A pharmaceutical composition according to claim 19 wherein the nitrodiphenyl ether active ingredient carries a carboxylic acid ester substituent. 21. A pharmaceutical composition according to claim 20 wherein said substituent is lower alkoxycarbonyl-lower alkoxycarbonyl. 22. The pharmaceutical composition of claim 19 wherein the nitro-diphenyl ether substituent is a lower alkoxy group. A pharmaceutical composition according to claim 15, wherein the active ingredient is a compound of the formula Ph-NHet wherein Ph is substituted phenyl and NHet is a heterocyclic radical of 5 to 6 atoms in the ring containing 1 to 4 nitrogen atoms of general formula 51 - N - C = O or -N - C - OH or - N - C - Cl wherein Q is a heterocyclic ring radical and R is a hydrogen atom or a substituent. 24. A pharmaceutical composition according to claim 15, wherein the active ingredient is represented by the general formula wherein Ph is substituted phenyl. 25. A pharmaceutical composition according to claim 15 wherein the active ingredient is represented by the general formula Wherein Ph is substituted phenyl. 26. A pharmaceutical composition according to claim 23, wherein Ph is phenyl with a substituent of formula HH-SO2-R wherein R is lower alkyl. 27. A pharmaceutical composition according to claim 23, wherein Ph is a phenyl substituent containing a terminal 1- (lower alkoxycarbonyl) ethoxy group. 23. A pharmaceutical composition according to claim 23 wherein said substituent is 1- (lower alkoxycarbonyl) ethoxy / phenoxy. 29. A pharmaceutical composition according to claim 1 wherein the active ingredient is an aryltriazolinone. 30. A pharmaceutical composition according to claim 29, which comprises 1- (2,4-dichloro-5- (N-methylsulfonylamino) -2-phenyl) -3-methyl-4-difluoromethyl-delta as active ingredient. -1,2,4-triazolin-5-one. 31. The pharmaceutical composition of claim 12, wherein the active ingredient comprises a compound in which NHet is a triazolinone ring moiety. 53 32. A pharmaceutical composition according to claim 15, wherein the active ingredient comprises an aryltriazolinone. 33. A pharmaceutical composition according to claim 32, comprising 1- (2,4-dichloro-5- (N-methylsulfonylamino) phenyl) -3-methyl-4-difluoromethyl-delta- 1,2,4-triazolin-5-one. 34. A pharmaceutical composition according to claim 32, wherein the active ingredient comprises a compound in which NHet is a triazolinone ring.