WO1991019418A1

WO1991019418A1 - Treatment of disease

Info

Publication number: WO1991019418A1
Application number: PCT/US1991/004087
Authority: WO
Inventors: James Warren Winkelman; Blaik Phillip Halling; Debra Ann Witkowski
Original assignee: Fmc Corporation
Priority date: 1990-06-11
Filing date: 1991-06-10
Publication date: 1991-12-26
Also published as: AU8185491A; IE911971A1; IL98405A0; CS176291A3; ZA914435B; PT97937A

Abstract

Treatment of polycythemia vera or hyperbilirubinaemia by administering a compound which is a specific inhibitor of the enzymatic conversion of protoporphyrinogen to heme in mammalian cells.

Description

TREATMENT OF DISEASE

One aspect of this invention relates to the treatment of polycythemia vera, a disease in which there is an increased production of myeloid elements (that is materials produced in the bone marrow), particularly hemoglobin and red blood cells. Late in the disease the spleen (which functions to remove matured red cells from the blood) becomes greatly enlarged. The disease generally begins in late middle life. Patients

receiving no therapy survive only about two years.

Treatment by repeated phlebotomy (bleeding) has

extended survival time to 10 to 12 years. Other treatments have included suppression of marrow function by radiation or chemotherapy with alkylating agents such as melphalan, busulfan and chlorambucil (which may lead to leukemia or other neoplasms) or with

hydroxyurea which inhibits DNA synthesis.

In this invention patients with polycythemia vera are treated with an enzyme-inhibitor agent which inhibits the enzymatic conversion of protoporphyrinogen to heme in the cells of the body. We have ascertained that agents, such as certain types of herbicidal compounds, which inhibit the enzymatic conversion of protoporphyrinogen to chlorophyl in plant cells also inhibit the enzymatic conversion of protoporphyrinogen to heme in mammalian cells. We believe that the inhibition by such agents probably affects the step of conversion of protoporphyrinogen to protoporphyrin IX by an enzyme (protoporphyrinogen oxidase) so that the protoporphyrinogen cannot follow the normal enzymatic pathway to protoporphyrin IX but instead becomes oxidized in the cell (for example in the cytosol) but outside the normal enzymatic pathway (for example outside the organelle membrane) and that the result is an accumulation of protoporphyrin IX in locations where it is unavailable for the normal conversion to heme.

The effect of the agent on the enzymatic pathway is shown, for instance, in experiments in which cultures of human tumor cells (for example HeLa cells) are grown in the dark in the presence of, say, a concentration of about 1 or 10 to 100 μM of the agent, producing excess protoporphyrin IX in both the cells and the culture medium.

Treatment with the enzyme-inhibiting agent may be effected by ingestion (for example orally) or injection (for example intravenous or intraperitoneal injection). For this purpose the agent may be incorporated into conventional pharmaceutical preparations such as tablets, (for example compressed tablets which may be coated, as with sugar paste and/or syrup),

suppositories, capsules (for example hard or soft gelatine capsules), suspensions, solutions, powders or ampules. In such preparations the agent may be present in admixture with a pharmacologically acceptable solid and/or liquid carrier which may, if desired, be a nutrient; for example it may be a solid such as corn starch or a liquid diluent such as water or an edible oil or mineral oil or a solvent, for example dimethyl sulfoxide. For injection the enzyme-inhibiting agent may be used as a sterile composition comprising the agent dissolved or dispersed in a pharmaceutically acceptable carrier, for example as an aqueous isotonic solution such as aqueous saline (for example 0.9% NaCl) or Dulbecco's phosphate saline (PBS) at a concentration of, say, 2.5 mg mL-¹, or comprising the agent in a liposomal system such as one prepared with a

phospholipid vesicle in a manner such as described at pages 1659-1660 of Remington's Pharmaceutical Sciences, 1985, (17th edition), published by Mack Publishing Company. For instance, analogously to the formulation described by Jori et al., Br. J. Cancer (1983), 48 at page 307, 51.4 mg. of dipalmitoyl-diphosphatidylcholine may be dissolved in 10 mL of a 1 mM solution of the agent in chloroform-methanol (9:1, v/v), and after thorough mixing the solvent may be removed under vacuum at 30°C, giving a solid which may be resuspended in 10 mL of 0.01M phosphate buffer at pH 7.4 containing 150 mM NaCl, and the cloudy solution then sonicated for 30 minutes at 50°C.

It may be desirable, especially for oral

administration, to employ enzyme-inhibiting agents which are water-soluble salts or which are acidic and form water-soluble sodium or potassium salts, such as an acidic sulfonamide of the type disclosed in International applications (PCT) WO 87/03782 or WO 85/001939 and WO 87/037873 (for example agents 6c and 8h below) or a water-soluble salt thereof, or a carboxylie acid or water-soluble salt thereof such as the sodium salt known as acifluorfen.

The best route of administration and the dosage with the best therapeutic ratio may be determined by routine experimentation, well known in the art. Thus, the enzyme-inhibiting agent may be administered to laboratory animals such as rats or mice to determine the effect of various doses on red blood cell count and blood hemoglobin content. It may be tested in vitro, using bone marrow cells from polycythemia vera

patients, such as the erythroleukemic cell line known as A.T.C.C. K562 (which cell line is referred to in, for instance, an article by Bridges in J. Biol. Chem., 260 (11), 6811-6815 of June 10, 1985). Based on data from such in vitro and animal experiments, plus further animal experimentation (including conventional short and long term toxicity studies with larger mammals such as dogs or pigs), dosage trials for humans would be initiated in accordance with protocols that insure safety and full disclosure (according to institutional Human Experimentation Review Boards as required by law and the policies of the medical centers in which the trials are conducted).

In tests on rats and mice, agent 8h (described below) was added to the diet of the animals in concentration of 3000 ppm, for 13 weeks, resulting in a significant decrease in erythrocyte counts, hemoglobin levels, hematocrit levels, mean corpuscular volume and mean corpuscular hemoglobin content, without inhibiting the production of white blood cells. After a 28 day recovery period in which the agent was omitted from the diet, hemoglobin and hematocrit levels returned to normal.

Another aspect of the invention relates to the use of the same enzyme-inhibiting agents to treat neonatal jaundice or hyperbilirubinaemia. As discussed in U.S. patent 4,831,024 and references cited therein,

inhibitors of heme oxygenase such as zinc and tin protoporphyrin have been used for this purpose,

presumably because they inhibit the conversion of heme to bilirubin. We expect that our enzyme-inhibiting agents which cause an accumulation of protoporphyrin in locations where it is unavailable for the normal conversion to heme will also reduce the production of abnormal amounts of bilirubin. The treatment may be employed for treatment of infants who exhibit above-normal carbon monoxide production, as determined by the known screening test for that purpose and who thus are considered to be of increased risk of serious jaund ce; see the test described in the articles by Smith et al discussed in U.S. patent 4,831,024, for example. For preliminary tests on animals to help establish the dosage and mode of administration in conventional manner, rats of the mutant strain of Wistar rats (so- called Gunn rats which have hereditary acholuric jaundice) may be used, as described in the literature cited in that U.S. patent.

The enzyme inhibiting agents of this invention are specific inhibitors of the enzymatic conversion of protoporphyrinogen to heme in the sense that they do not operate as general enzyme poisons such as

denaturing or cross-linking agents (for example

sulfhydryl reagents), preferably they are not materials which affect the oxidation conditions such as electron acceptors; thus, preferred agents for this invention have redox potentials more negative than about -500 mV, such as more negative than -800 mV (measured, for instance in the conventional manner in an aprotic solvent for the agent, as by cyclic voltametry or polarographically). It is also preferred that the agent not be a tetrapyrrole and that its I50 for protoporphyrinogen oxidase be less than about 10 μM (a PI50 greater than about 5), more preferably less than about 1 μm (a PI₅₀ greater than about 6) such as less than about 0.3 μM, for example an I₅₀ of about 0.1 or 0.03 or 0.01 μM or less.

The enzyme-inhibiting agent is preferably one which has a high capacity for disrupting the plasmalemma of plant material. One test for that capacity is the Efflux Experiment described in the article by Hailing and Peters in Plant Physiology, 84, 1114-5 (1987). In such a test (described in more detail in Appendix A below), preferred agents show a total efflux of at least 50% at a treatment rate of 100 μM, preferably at a treatment rate of 1 μM or less, such as 100 nM;

highly active materials, such as 1-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3-methy1-4-difluoromethyl-Δ²- 1,2,4-triazolin-5-one or lactophen (described below,), give total efflux percentages of over 90% at 100 nM concentration.

Another test of the capacity of a material for disrupting the plasmalemma of plant material is the Light-Induced Greening Inhibition Test described more particularly in Appendix B below. This test measures the capacity to inhibit the light-greening of dark-bleached Chlamydomonas reinhardi mutant y-1 (a type of algae which when grown in the dark does not make chlorophyll, so that the mass of algae becomes bleached owing to the presence of new non-green cells, and which produces chlorophyll again when it is exposed to light). Many of the preferred compounds (agents) used in this invention have the ability to inhibit the light-greening by at least 50% when the compound is used at a concentration of 10^-5M, more preferably at a concentration of 10^-6M or less, for example 10^-7M.

However, compounds which are ionized in water, such as compounds like 6c in which -the operative ion is

negatively charged do not respond well in this test; apparently the algal cells resist entry of the acidic group. In addition, many of the preferred compounds, when used at said concentration in the Light-Greening Inhibition Test, give a supernatant which shows a light absorption peak at about 405 nm which is higher than the chlorophyll peak (the peak at about 668 nm in this system), for example the supernatant shows a 405 nm peak whose height is 2, 3 or 4 times the height of the 668 nm peak.

Among the enzyme-inhibiting agents which may be employed in the practice of this invention are herbicidal compounds of the following classes (A to G) :

A. Aryl heterocyclic herbicides of the general formula Ph-NHet where "Ph" is a substituted phenyl, preferably 2,4-disubstituted phenyl, more preferably a 2,4,5-trisubstituted phenyl, and NHet is a 5- or 6-membered heterocyclic ring having one to four ring-nitrogen atoms and having the formula

where Q represents the balance of the heterocyclic ring and R represents hydrogen or a substituent group. This class of compounds includes such materials as those designated as "the cyclic imide class of herbicides" in an article by Wakabayashi et al., J. Pesticide Sci. 11, 635-460 (1986) which shows the following compounds in its Fig. 1:

Compound I is an aryl oxadiazole herbicide, namely 2-tert-butyl-4-(2,4-dichloro-5-isopropoxyphenyl)-Δ²- 1,3,4-oxadiazolin-5-one. Other 2-alkyl-4-(2,4,5-trisubstituted phenyl)-Δ²-1,3,4-oxadiazolin-5-ones which may be used in the present invention are

disclosed, for instance, in U.S. patents 3,385,862;

3,836,539; and 3,876,413. Compound II is an aryl tetrahydroindazole

herbicide, namely 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)tetrahydro indazoles which may be used in the present invention are disclosed, for instance, in U.S. patent 4,670,043.

Compounds III, IV and V are aryl tetrahydrophthalimide herbicides. Other aryl tetrahydrophthalimides which may be employed in the present invention are disclosed, for instance, in U.S. patents 4,431,822;

4,670,046; 4,670,042; and 4,439,229 and published

International application (PCT) WO 87/07602. The latter two citations above also disclose other NHet rings which may be used, such rings being illustrated, for example, at column 4 line 25 to column 5 line 20 of U.S. 4,439,229 and at pages 12 to 14 of WO 87/07602.

Other suitable herbicides of the PH-NHet type are: aryl triazolinones, such as those disclosed in U.S. patents 4,318,731; 4,398,943; 4,404,019; 4,702,945;

4,705,557; 4,702,763; 4,761,174 and International applications (PCT) WO 85/01637, WO 85/04307, WO

87/00730, WO 87/03782, WO 86/04481, and WO 88/01133; aryl tetrazolinones, such as those disclosed in U.S. patents 4,734,124 and International applications (PCT) WO 85/01939 and WO 87/03873;

aryl triazinedioneε, such as those disclosed in U.S. patents 4,755,217 and 4,766,233 International application (PCT) WO 86/00072;

aryl hydantoins, such as those disclosed in U.S. patent 4,427,438;

aryl imidazolopyrimidines, such as those disclosed in West German patent DT 2604989 (Derwent Abstracts accession No. 65326X), Japanese Kokai J60-158147A

(Derwent Abstracts accession No. 85-240363), and European published patent application EP 230874

(Derwent Abstracts accession No. 87-215141);

aryl pyridyldiazines, such as compounds of the type of compound 8x of Example 8 below;

aryl diazinediones, such as compounds of the type of compounds 8e and 8v of Example 8 below;

aryl pyradiazinones, such as compounds of the type of compound 8f of Example 8 below;

aryl oxadiazolinones, such as those disclosed in Japanese Kokai J59-148769 (Derwent Abstracts accession No. 84-246947) or Japanese Kokai J62-161772 (Derwent Abstracts accession No. 87-238787);

aryl oxadiazinones, such as compounds of the type of compounds 8k and 8m of Example 8 below;

aryl benzamidazoles, such as compounds of the type of compound 8ad of Example 8 below;

aryl iminotriazolopyridazines, such as those disclosed in U.S. patents 4,913,723; 4,906,281; and 4,906,279;

aryl thiazoles, such as compounds of the type of compounds 8L, 8r and 8ah of Example 8 below;

aryl pyrroles, such as those disclosed in European published patent applications EP 255601 (Derwent

accession No. 88-037583) and EP 260288 (Derwent

accession No. 88-127433);

aryl urazoles, such as those disclosed in U.S patent 4,452,981; and

aryl hexahydropyridazines, such as those disclosed in U.S. patent 4,619,687.

B. Aryl heterocyclic urethanes, such as those disclosed in U.S. patent 4,521,242.

C. Aryl heterocyclic ureas, such as those disclosed in Japanese Kokai J58-225081 (Derwent Abstracts accession No. 84-034261).

D. Aryl amides, such as compounds of the type of compounds 8aj and 8ak of Example 8 below.

E. Phenyl ether herbicides, such as those having a p-halo or p-nitro phenoxyphenyl structure, such as the following commercially available materials:

5-(2-chToro-4-(trifluoromethyl) methyl 5-(2,4-dichlorophenoxy)phenoxy)-2-nitro-N-methanesul2-nitrobenzoate (bifenox) fonylbenzamide (fomesafen)

sodium 5-(2-chloro-4-(trifluoro2-chloro-1-(3-ethoxy-4-nitromethyl)phenoxy)-2-nitrobenzoate phenoxy)-4-trifluoromethylbenzene

Other suitable commercially available diphenyl ether herbicides are

lactophen: 1-(carboethoxy)ethyl 5-(2-chloro-4-trifluoromethylphenoxy)-2-nitrobenzoate,

fluoroglycofen: (carboethoxy)methyl 5-(2-chloro-4-trifluoromethylphenoxy)-2-nitrobenzoate,

chloronitrofen: 2,4,6-trichloro-(4-nitrophenoxy)-benzene, fluorodifen: 2-nitro-1-(4-nitrophenoxy)-4-trifluoromethylbenzene,

nitrofen: 2,4-dichloror-1-(4-nitrophenoxy)benzene, chlomethoxyfen: 4-(2,4-dichlorophenoxy)-2-methoxy- 1-nitrobenzen.

Still another suitable diphenyl ether herbicide is methyl 5-(2-chloro-4-trifluoromethylphenoxy)-2-nitroacetophenone oxime-O-acetate.

F. Aryl pyrazole herbicides such as those disclosed in U.S. patents 4,563,210; 4,496,390; and

4,459,150 and German Offenlegungsschrift 3520327 Al.

G. Compounds of the formula

where X^b is O or S and "Ph" has the meaning described above, such as compounds disclosed in published

European patent application 273417 or Derwent Abstracts accession no. 87-040749.

Some enzyme-inhibiting herbicides are disclosed in Matringe et al., FEBS Letters, Vol. 245, no. 1, 2, pages 35-38 (1989) and Yanase et al., Pesticide

Biochemistry and Physiology, Vol. 35, pages 70-80

(1989).

Some specific treating agents are:

Determination of I₅₀

(a) Using enzyme in intact chloroplasts:

Protoporphyrinogen IX ("Protoαen IX"). Proto IX was purchased from Porphyrin Products, Logan, UT and purified as outlined by T. P. Fuesler et al., Plant Physiol., 67, 246-249 (1981). Protogen IX was freshly prepared by reduction of Proto IX with a Na/Hg amalgam as outlined by N. J. Jacobs and J. M. Jacobs, Enzyme, 28, 206-219 (1982), utilizing the purified Proto IX at a concentration of 300 μM.

Plant Material. Cucumber (Cucumis sativus L.

cultivar 'Wisconsin SMR18') was raised in a dark growth chamber on vermiculite irrigated with a commercial (9- 45-15) fertilizer. The seedlings were grown at 25°C and a relative humidity of 80 to 90%. Intermittent illumination, one minute of light per 60 minute cycle with a measured intensity of 25 μE/m^-2 sec^-1 (PAR), was supplied by a General Electric 'Bright Stick'

controlled by an electronic timer. This provided tissue capable of rapid chlorophyll synthesis while minimizing starch reserves and initial chlorophyll levels.

Chloroplast Isolation. Developing chloroplasts were isolated as described by T. P. Fuesler et al.,

Plant Physiol., 75, 662-664 (1984), except that in the final purification step, plastids were centrifuged through a 40% rather than 45% (v/v) Percoll cushion. The chloroplasts were resuspended in an assay buffer containing 0.5 M mannitol, 20 mM TES, 10 mM HEPES, pH 7.7, 1 mM EDTA, 1 mM MgCl₂ , 1% (w/v) bovine serum albumin and 1 mM dithioerythritol to a final concentration of 2 mg protein/mL.

Assays of Protoporphyrinoqen Oxidase. Assays were conducted as outlined by J. M. Jacobs and N. J. Jacobs, Arch. Biochem. Biophys., 229, 312-319 (1984). Samples (0.2 mL) of the chloroplasts suspension were pre-incubated in the dark for 15 minutes with various.

concentrations of acifluorfen-methyl ("AFM") or with 0.2% (v/v) acetone (as a "control"). Then fifty μL of freshly prepared Protogen IX (about 15 nM), was added to the suspension to initiate the reaction. Assays were terminated by the addition of 2.75 mL of the fluorometric media of N. J. Jacobs and J. M. Jacobs, Enzyme, 28, 206-219 (1982) consisting of 1% (v/v)

Tween-20 (polyoxyethylene sorbitan monolaurate), 50 mM Tris-HCl, pH 8.5, 1 mM EDTA; 1 mM dithioerythritol ("DTE") was substituted for 5 mM gluthathione. The suspensions were then read directly on a SPEX

Fluorolog-2 spectroflourometer equipped with a

frontface fluorescence option for turbid biological samples. The amount of Proto IX produced was

quantified from a standard curve (of quantity of Proto IX versus emission at 630 nm, on excitation at 400 nm) generated from purified Proto IX in fluorometric media.

To determine the amount of nonenzymatic oxidation to Proto IX in the above assay, the same assay was carried out except that in place of the suspension of active chloroplasts, there was used a suspension in which the chloroplasts had been inactivated by heating for 15 minutes at 85°C. Subtracting the quantity of Proto IX thus produced from the quantity produced in the presence of the active chloroplasts gave the amount of Proto IX formed enzymatically. The results are shown graphically in Figure I (in which LSD indicates least significant difference, as is conventional).

Figure I shows that the I50 value (the concentration which provides 50% inhibition) is less than 0.1 μM, i. e., about 0.03 μM.

Assays of the effect of 10 μm AFM on the enzymatic conversion of Proto IX to magnesium Proto IX in the chloroplast suspension (in the presence of ATP) showed that AFM had no inhibiting effect on that conversion. (b) Using isolated enzyme:

Plant Material and Homoqenization: Peas (Pisum sativum var. Little Marvel) germinated at 20°C in a dark growth chamber ten days. Plants were illuminated for one hour per day, which allowed for significant leaf expansion while minimizing chlorophyll synthesis. Leaves were pale yellow-green in color.

The leaves were then homogenized in 500 mL of grinding media consisting of 0.5 M mannitol, 10 mM HEPES, 20 mM TES, l mM EDTA, ImM MgCl₂, 5mM cysteine, and 0.2 BSA, pH 7.7. The brei was then passed through four layers of cheesecloth, then through a 43-micron nylon mesh. The hemogenate was then centrifuged at 4000 g for 3 minutes. The resultant pellet was

utilized for plastid isolation.

Etioplast Isolation and Purification: The pellet (plastid fraction) was resuspended in 40 mL of assay media (grinding media minus cysteine and containing ImM DTE and 1% ESA) and centrifuged at 150 g to remove cellular debris. The resultant supernatant was

centrifuged at 4000 g, and the resuspended pellets were overlaid on 40% Percoll. After centrifugation for 3 minutes at 6500 g, intact etioplasts pelleted at the bottom of the tube.

Protein content was determined utilizing the BioRad method.

Enzyme Solubilization: The enzyme was solubilized following the procedure of Jacobs and Jacobs. The plastids were pelleted, and resuspended in 2.5 mL of buffer consisting of 20 mM Tris-MCl, 30% glycerol, and 1 mM DTE, pH 7.6. After sonification at 60mHz, a volume of detergent extraction media, 10% Triton 100X, 8% KCl, and 10 mM PMSF, was added to give a final detergent:protein ratio of 0.7 (W/W). After three hours at 48°C, the membrane preparation were ultra-centrifuged at 100,000 g for 1 hr. on a Beckman L2-65B.

Gel Filtration: The supernatants containing the solubilized enzyme were then desalted on a Pharmacia PD-10 G-25M Sephadex column. Columns were washed with 25 mL of Elution Buffer (20 mM Bistris-HCl, 20%

glycerol, 0.1% Triton X, pH 6.8). Samples were loaded onto the column and eluted with 3.5 mL of buffer. The first mL fraction was discarded while the remaining 2.5 mL was collected. These fractions were then frozen under N₂ at -77°C.

Purification Utilizing DEAE Chromatography: A 0.75 mL volume of plastid-solubilized-enzyme preparation was loaded onto a Waters Protein-Pak DEAE-5PW column (8mm × 7.5cm) equilibrated with Elution Buffer. After 60 minutes of washing with Elution Buffer (lmL/minute), the enzyme was eluted with a linear 0.1M NaCl gradient over 100 minutes. The gradient was then changed from 0.1M NaCl to 0.8 M NaCl in 30 minutes. Four-mL

fractions were collected and assayed for activity.

Peaks were detected using a Waters 494 UV detector at 260 nm, sensitivity: 1AU.

Protoporphyrinogen Oxidase Activity: Enzymatic conversion of protoporphyrinogen IX to protoporphyrin IX was assayed as described in section (a) above.

In this test, compound 6c showed I₅₀ values of 0.8 to 0.3 μM (corresponding to a PI₅₀ of 6.1 to 6.5, where pI₅₀ is negative log of the I₅₀ Molarity); for AFM the value was 0.08 μM (PI50 of 7.1) and for compound 8v the value was 0.08 to 0.03 μM (pI₅₀ of 7.1 to 7.5). Appendix A

Percent Efflux Test

The Percent Efflux test employs cotyledons

harvested from etiolated cucumber seedlings. In an initial step a mass of cotyledons is treated in the dark with an aqueous buffered solution containing a radiolabelled sugar. The amount of the sugar taken up by the cotyledons is then measured (by counting, as described below) . The mass of cotyledons is then divided into two portions; one portion of the

cotyledons is treated in the dark with an aqueous buffered solution containing the compound to be tested, while the other portion is treated in the same way, in the dark, with an otherwise identical solution without the test compound, as a control. The cotyledons, in contact with the aqueous solutions, are then exposed to light for 16 hours, and then separated from the aqueous solutions; the latter are then measured (by counting) to determine their contents of radiolabelled material. The results are expressed as Percent Efflux which is calculated as follows, where S is the count of

radiolabelled material (per cotyledon) taken up by the cotyledons in their initial treatment, S_T is the count of radiolabelled material (per cotyledon) in the aqueous solution containing the compound to be tested, after the exposure to light and S_c is the count of radiolabelled material (per cotyledon) in the aqueous solution of the control, after exposure to light:

More specifically, the following materials and conditions are employed in the Percent Efflux test. Plant material: Cucumber seed (Cucumis sativus L. cultivar 'Wisconsin SMR 18') was germinated and grown in vermiculite irrigated with a commercial (9-45-15) fertilizer. Seedlings were grown at 25°C and 80-90% RH in a dark incubation chamber. Cotyledons were

harvested from the etiolated seedlings five days after planting and were rinsed in 1.0 mM CaCl₂, all under green light.

Buffered solution: 1 mM KCl, ImM CaCl₂ and 2.0mM potassium phosphate, adjusted to pH 6.5 (as with NaOH).

Radiolabelled sugar: 3-O-methyl-3-[U-14C]glucose of specific activity 10.9 GBq/mmol (Amersham Corp., Arlington Heights IL).

Initial treatment: Washed cotyledons (180 to 230) are added to a 250 mL widemouth foam-stoppered

Erlenmeyer flask containing 50 mL of the buffered solution, and the radiolabelled sugar is added in an amount to give a concentration of 600 nM thereof in the solution. The flask is shaken for 24 hours at 125 rpm on a gyratory shaker in the dark. The cotyledons are then recovered on a nylon mesh and rinsed three times in 20 mL volumes of ImM CaCl₂. The uptake of the radiolabelled sugar is measured by digesting three samples of five cotyledons each in NCS tissue

solubilizer (Amersham Corp.) and counting the resulting macerate in a liquid scintillation spectrometer.

(Results from these digestions were found to be

equivalent to the same determination made by combusting sampled cotyledons in an autooxidizer.)

Treatment with compound to be tested (and control treatment):

Five of the cotyledons are floated, abaxial side up, on 3 mL of the buffered solution in a 35 mm

diameter covered plastic petri dish. The test compound is then added (as a solution thereof in acetone) in such amount as to give a predetermined concentration (discussed below) of the test compound in the buffered solution; the acetone concentration in the buffered solution is then 0.1% (v/v) in the control as well as in the solution containing the test compound. The floating cotyledons are then swirled by shaking the dishes at 90 rpm on the surface of a gyratory shaker for 16 hours.

Exposure to light: The dishes containing the cotyledons floating on the solutions are exposed for 16 hours to illumination provided by four GE F20T12-CW fluorescent lamps at a measured intensity of 150μE m^-2 sec^-1 in the photosynthetically active region of the spectrum (PAR) (measured at the surface of the

cotyledons) while the dishes are being shaken.

Measurement of amount of radiolabelled material in the liquid: All the liquid is separated from the cotyledons and then counted in a liquid scintillation spectrometer.

Darkness: All treatments prior to the illumination step are carried out in the dark or under green fluorescent light (that is light filtered through a green plastic cut-off filter which passes light of 450-600 nm).

Appendix B

Culture Media

Medium M

Salts Molarity Stock mL Stock/L

Na Citrate·6H₂O 1.7 × 10^-3M 10% 5

Trace Metals as below as below 10

FeCl₃·6H₂O 0.37 × 10^-3M 1% 1

CaCl₂·2H₂O 0.36 × 10^-3M 5.3% 1 MgSO₄·7H₂O 1.2 × 10^-3M 10% 3

NH₄NO₃ 3.7 × 10^-3M 10% 3

KH₂PO₄ 2.2 × 10^-3M 10% 3

K₂HPO₄ 1.7 × 10^-3M 10% 3

Stock of Trace Metals Mixture

H₃BO₃ 100 mg/L

ZnSO₄·7H₂O 100 mg/L

MnSO₄ · 4H₂O 40 mg/L

COCl₂·6H₂O 20 mg/lL

NaMoO₄ ·2H₂O 20 mg/L

CuSO₄ 4 mg/L

Culture Medium A is prepared by adding 10 mL/L of an aqueous, 10% sodium acetate solution (7.5 × 10^-3M) to Medium M.

The components are added to distilled water in the order listed above and are autoclaved at 15 psi for 15 minutes.

Stock Cultures Grown in the Presence of Light

Stock Cultures of y-1 cells are maintained

axenically in liquid culture on Medium A or M,

preferably on Medium M, in a 14/10 hour light/dark cycle at 25°C in Erlenmeyer flasks which are stoppered with polyurethane plugs. The stock cultures are incubated without supplemental aeration on rotary shakers at 125 rpm. The light intensity at the culture level is 120 μE m^-2 sec^-1 (PAR). Under these conditions the cultures are in a semi-synchronous mode of growth until they reach a stationary phase of 2-4 × 10⁶ cells/mL.

Cultures Grown Without Light (Dark-Grown Culture)

Cells from the stationary phase of stock cultures grown in the presence of light (7.5 mL) are transferred to 750 mL of Medium A in an Erlenmeyer flask. The cells are incubated in the dark, with aeration through a submersed aeration tube, at 25°C for three to four days. During this period this culture of y-1 cells will undergo seven to eight cell divisions, losing all visible chlorophyll, and the concentration should be 2- 3 × 10⁶ cells/mL.

Preparation of Cells for Use in Assay

Cells are harvested from a freshly prepared dark- grown culture (750 mL) by low speed centrifugation, that is, about 2000 rpm, for approximately five minutes at about 20°C. The cells are gently resuspended in 50 mL of Medium A. A sample of this suspension (approximately 0.25 mL) should be examined for motility and, after fixation with an aqueous, 1% gluteraldehyde solution, a cell count should be made to determine the number of cells/mL. A normal cell count is about 5 x 10⁷ cells/mL. Aliquots of 1 mL (10⁷ cells/mL) are placed into the testing vessels (for example, FalconΨ 24-well tissue culture plates). At this point the cells are very motile and are yellow in color.

The test compound is dissolved in a solvent

(acetone, ethanol, dimethylsulfoxide, or water,

preferably acetone) to provide a concentration which is 1000 times the final concentration. One μL of this test solution is added to each of two wells with a 5 or 10 μL microsyringe.

Four control wells per tissue culture plate are prepared in the manner described above without the test compound present. The tissue culture plates are

covered with a transparent plastic cover and are placed in a growth chamber with a light intensity of 70-90 μE m^-2 sec^-1 for 13-16 hours at 25°C. If the contents of the test wells appear yellow, they are extracted as described in the Evaluation of Results section below. If the contents of the test wells appear transparent or are a pale-green color, they are placed on a rotary shaker at approximately 125 rpm and are irradiated for two hours with a light intensity of about 600 μE m^-2 sec^-1 before being extracted.

Evaluation of Results

An aqueous, 10% sodium dodecyl sulfate solution (250 μL) and methanol (1 mL) is added to each test well and is mixed thoroughly. The mixtures are allowed to stand in the dark for three to four hours. The tissue culture plates are centrifuged at room temperature at low speed (ca. 2000 rpm) for five minutes. The

supernatants are removed and analyzed in a Beckman

Model 35 spectrophotometer between 350 nm and 500 nm for the presence of protophorphrin IX, at 668 nm which is the absorption peak of chlorophyll in this solvent system, and at 720 nm to use as a turbidity background reading.

Analysis of Data

For each well a greenness value is obtained by subtracting the 720 nm reading from the 668 nm reading. These values are averaged for each concentration of the compound to be tested and for the control. The percent inhibition is

Claims

CLAIMS :

1. A method of treating polycythemia vera characterized by administering a compound which is a specific inhibitor of the enzymatic conversion of protoporphyrinogen to heme in mammalian cells, in an amount effective to inhibit such conversion.

2. A method as in claim 1 characterized in that said compound is not a tetrapyrrole and said compound has a redox potential more negative than -500 mV.

3. Method as in claim 1 characterized in that said compound has an I₅₀ for protoporphyrinogen oxidase of less than 10 μM.

4. Method as in claim 3 characterized in that said I₅₀ is less than 1μM.

5. Method as in claim 1 characterized in that said compound is a nitrodiphenyl ether herbicide.

6. Method as in claim 5 characterized in that said nitrodiphenyl ether has a carboxylic acid ester substituent.

7. Method as in claim 6 characterized in that said substituent is a lower alkoxycarbonyl-lower alkoxycarbonyl substituent.

8. Method as in claim 5 characterized in that said nitrodiphenyl ether has a lower alkoxy

substituent.

9. Method as in claim 1 characterized in that said compound has the formula Ph-NHet where "Ph" is a substituted phenyl and NHet is a 5- or 6-membered heterocyclic ring having one to four ring-nitrogen atoms and having the formula

where Q represents the balance of the heterocyclic ring and R represents hydrogen or a substituent group.

10. Method as in claim 1 characterized in that said compound has the formula

where Ph is substituted phenyl.

11. Method as in claim 1 characterized in that said compound has the formula

where Ph is substituted phenyl.

12. Method as in claim 9 characterized in that Ph is phenyl having a substituent of the formula

/

where R is lower alkyl.

13. Method as in claim 9 characterized in that Ph is phenyl having a substituent which includes a

terminal 1-(lower alkoxycarbonyl)ethoxy group.

14. Method as in claim 9 characterized in that said substituent is a [1-(lower alkoxycarbonyl)ethoxy]-phenoxy substituent.

15. A method for treating neonatal hyperbilirubinaemia, or preventing its occurrence in infants susceptible thereto, characterized by administering a compound which is a specific inhibitor of the enzymatic conversion of protoporphyrinogen to heme in mammalian cells, in an amount effective to inhibit such

conversion.

16. A method as in claim 15 characterized in that said compound is not a tetrapyrrole and said compound has a redox potential more negative than -500 mV.

17. Method as in claim 15 characterized in that said compound has an I₅₀ for protoporphyrinogen oxidase of less than 10 μM.

18. Method as in claim 17 characterized in that said I₅₀ is less than 1μM.

19. Method as in in claim 15 characterized in that said compound is a nitrodiphenyl ether herbicide.

20. Method as in claim 19 characterized in that said nitrodiphenyl ether has a carboxylic acid ester substituent.

21. Method as in claim 20 characterized in that said substituent is a lower alkoxycarbonyl-lower alkoxycarbonyl substituent.

22. Method as in claim 19 characterized in that said nitrodiphenyl ether has a lower alkoxy

substituent.

23. Method as in claim 15 characterized in that said compound has the formula Ph-NHet where "Ph" is a substituted phenyl and NHet is a 5- or 6-membered heterocyclic ring having one to four ring-nitrogen atoms and having the formula

24. Method as in claim 15 characterized in that said compound has the formula

where Ph is substituted phenyl.

25. Method as in claim 15 characterized in that said compound has the formula

where Ph is substituted phenyl.

26. Method as in claim 23 characterized in that Ph is phenyl having a substituent of the formula

/

where R is lower alkyl.

27. Method as in claim 23 characterized in that Ph is phenyl having a substituent which includes a

terminal 1-(lower alkoxycarbonyl) ethoxy group.

28. Method as in claim 23 characterized in that said substituent is a [1-(lower alkoxycarbonyl)ethoxy]-phenoxy substituent.

29. Method as in claim 1 characterized in that said compound is an aryl triazolinone.

30. Method as in claim 29 characterized in that said compound is 1-[2,4-dichloro-5-(N-methylsulfonylamino)phenyl]-3-methyl-4-difluoromethyl-Δ²-1,2,4-triazolin-5-one.

31. Method as in claim 12 characterized in that NHet is a triazolinone ring.

32. Method as in claim 15 characterized in that said compound is an aryl triazolinone.

33. Method as in claim 32 characterized in that said compound is 1-[2,4-dichloro-5-(N-methylsulfonylamino)phenyl]-3-methyl-4-difluoromethyl-Δ²-1,2,4- triazolin-5-one.

34. Method as in claim 32 characterized in that NHet is a triazolinone ring.

35. Process for preparing pharmaceutical

compositions containing a compound which is a specific inhibitor of the enzymatic conversion of

protoporphyrinogen to heme in mammalian cells

characterized in that one or more of said compounds, having been prepared by methods known in the art are formulated into pharmaceutical composition for

treatment of polycythemia vera by using

pharmaceutically acceptable carriers and/or other auxiliaries.

36. Process for preparing pharmaceutical

protoporphyrinogen to heme in mammalian cells

treatment of neonatal hyperbilirubinaemia by using pharmaceutically acceptable carriers and/or other auxiliaries.