CN1171552A - Use of porphyrins - Google Patents

Abstract

Disclosed is a method and a pharmaceutical composition for killing mammalian tumer cells by subjecting said cells to light in the presence of a light-activatable tetrapyrrole, in which the improvement comprises treating said cells with a compound which inhibits the enzymatic conversion of protoporphyrinogen to protoporphyrin IX by protoporphyrinogen oxidase in said cells thereby causing a buildup of protoporphyrin IX in said cells. Also disclosed is a method for the production of protoporphyrin IX which comprises growing eukaryotic microalgae in the presence of a protoporphyrinogen oxidase inhibitor.

Description

The application of porphyrin

The present patent application is that application number is dividing an application of 90104921.2 patented claims.

One aspect of the present invention relates to mammalian tumor cell is carried out fluorescigenic processing in light, as cancer being carried out fluorescigenic processing in light.

As everyone knows, be used for mammalian tumor cell, then these cells carried out the illumination of suitable wavelength, make cytoclasis with hematoporphyrin derivative or by its porphyrin potpourri of deriving (as Photofrin II ).Have the theme of a series of papers relate to this in light fluorescigenic methods of treatment, these papers are published in photochemistry and photobiology (Photochemistry andphotobiogy) the 46th volume, 5 phases, in November, 1987 is (hereinafter with " P﹠amp; P46-5 " expression) special issue in.In these papers, think, this in light fluorescigenic cure be used to handle various cancers (cancer that comprises bronchus, bladder, esophagus, lung, skin, head and neck, brain, colon and intraocular and gynaecology).Show, the biochemical effect of this porphyrin photosensitization comprises the dissolving, the deactivation of several enzymes of inhibiting effect, lysozyme and mitochondrial membrane of conveying of peroxidation, some analytic metabolism thing of crosslinked, the lipid of memebrane protein, and the cell that increases porphyrin absorbs (P﹠amp; The 695th page of P46-5).Generally be to use injection to introduce porphyrin material (as vein or intraperitoneal injection).Common dose is the about 2 milligrams of Photofrin II of per kilogram of body weight.

Also the porphyrin material can be introduced liposome, and inject (for example intraperitoneal injection), for example in people's such as Spikes " in the fluorescigenic behavior in light of model cell, tissue and tumour system mesoporphyrin " (at " Photodynamic Therapy of Tumors and otherDiseases ", edit by G.Jori and C.A.Perria, the 45-53 page or leaf, Libreria Progetto, Padua (1985)) in, and wherein introduced list of references.

Technical literature is pointed out, introduce protoporphyrin in cell, as human erythrocyte (people such as Dubbel-man, Biochimica et Biophysica Acta, 511 (1978) 141～151) or murine leukemia cell (Kessel, Cancer Research 42,1703～1706, May 1982), can produce tangible photosensitizing effect.The article of Kessel points out that protoporphyrin is the most effective photosensitizer of being tested.But as Kessel and other people are (as people such as Berenb-aum, Br.J.Cancer (1982) 45, when 571～581) being injected into protoporphyrin in the animal body, but discovery does not have the protoporphyrin of detected level in tumour, and they think that such introducing protoporphyrin may lose easily in the circulation system in body.

Hematoporphyrin derivative and similar substance are used for the detection and the location of tumour, as bladder or lung's cancer (P ﹠amp; P 46-5,759 pages) in, also know.

Another aspect of the present invention, relate to a kind of in this light, fluoresce to be used in handling handle cell or be used for detecting or the reagent of positioning tumor by known method, this reagent is not porphyrin, perhaps singly is not porphyrin.It comprises a kind of enzyme inhibitor, and this inhibitor suppresses former porphyrinogen (protoporphyrinogen) in said cell enzymatic is converted into protoheme, thus the accumulation of the protoporphyrin IX of giving birth in described cell, producing.We find out, this reagent, and certain herbicides compounds class for example, it can suppress former porphyrinogen in vegetable cell enzymatic is converted into chlorophyll, and the enzymatic that also can suppress former porphyrinogen in mammalian cell is converted into protoheme.We believe, inhibiting effect by this reagent, can influence by enzyme (former porphyrinogen oxidase, EC1,2,3, the enzymatic conversion of the former porphyrinogen that 4) causes is the step of protoporphyrin IX, so just can make former porphyrinogen not be transformed into protoporphyrin IX by normal enzymatic pathway, but in cell (as in blood plasma) oxidized, but, consequently accumulated protoporphyrin IX being difficult for the position that acquisition normally changes into protoheme not according to normal enzymatic approach (as outside quasi-lipid film).Therefore, when cell was subjected to illumination, the protoporphyrin IX of this accumulation had the effect of destruction cell shown in the processing that fluoresces mentioned above in light.

Enzyme inhibitor of the present invention, in some sense, it is the special inhibitor that the enzymatic of former porphyrinogen is converted into protoheme, they do not produce common poisoning by enzyme, for example modifier or crosslinking chemical (as sulfydryl reagent), preferably they are not those materials that influence oxidizing condition (as electron accepter).Therefore the redox potential of the preferred reagent of the present invention will more be defeated by pact-500mV, as more being defeated by-800mV (for example by classic method, adopting cyclic voltammetry measurement method or polarography to measure in the aprotic solvent of reagent).Preferably this reagent is not tetrapyrrole, and to the oxidasic I of former porphyrinogen ₅₀Be lower than 10 μ m (and pI ₅₀Greater than about 5), be more preferably and be lower than about 1 μ m (and pI ₅₀Be greater than about 6), for example be lower than about 0.3 μ m, as I ₅₀Be about 0.1 or 0.03 or 0.01 μ m or lower.

Preferred enzyme inhibitor has the ability of high destruction plasmalemma of plant, and a kind of method of testing this ability is that loss experiment (Efflux Experiment) is described in the article (Plant Physiology, 84,1114-5 (1987)) of Halling and Peters.In this test (detailed description see below appendix A), preferred reagent, in handling rate is 100 μ m, be preferably in handling rate 1 μ m or still less, during as 100nM, its total number of dropouts is at least 50%, and high active substance is as 1-(4-chloro-2-fluoro-5-propargyl oxygen phenyl)-3-methyl-4-difluoromethyl-Δ ²-1,2,4-triazoline-5-ketone or newborn fen (lactophen) (description is hereinafter arranged), under 100nM concentration, its percentage that always runs off surpasses 90%.

The method that another kind of test substances is destroyed the plasmalemma of plant ability is the green inhibition test of photo induced strain (Light-Induced Greening Inhibition Test), and it is specified among the following appendix B.This method of testing is to measure horse tongue butterfly chlamydomonas (Chlamydomonas reinbardi) mutant strain Y-1 (a kind of algae that suppresses dark decolouring, do not produce chlorophyll during growth in darkness, so this algae is owing to existing new non-green cell to become decolouring, but after illumination, produce chlorophyll again) the green ability of light-turn.Many preferred compounds of the present invention (reagent) that are used for are when compound used therefor concentration is 10 ^-5M, preferred 10 ^-6M or lower (as 10 ^-7) time, the green ability at least 50% of the inhibition light that has-turn.But, the compound of ionization in water, as compound 6C (embodiment 6 sees below), wherein Yun Hang ion band negative charge does not just have fine reaction in this test, and obviously frustule stops acidic-group to enter.In addition, many preferred compounds are arranged, when light-turn uses described concentration in the green inhibition test, its supernatant at the optical absorption peak at about 405nm place greater than chlorophyll peak (at about 668nm place), for example, the peak height of supernatant at the 405nm place is 2,3 or 4 times of 668nm peak heights.

Can be used for having in the enzyme inhibitor of the invention process following A to G class herbicides compounds:

The A general formula is

The aryl-heterocyclic herbicide of Ph-NHet, wherein " Ph " is the phenyl that replaces, preferably 2, the dibasic phenyl of 4-, more preferably 2,4,5-trisubstd phenyl, NHet are one 5 or 6 joint heterocycles, and have 1～4 theheterocyclic nitrogen atom, its general formula is

Or

Or

Wherein Q is the other parts of heterocycle and R is a hydrogen or a substituting group, this compounds is included in people's such as Wakabayashi article (J.Pesticide Sci.11,635-460 (1986)) be called in " cyclic imides class herbicide " those materials, following compound is arranged in its Fig. 1:

Compound I is an aryl oxadiazoles herbicide, i.e. the 2-tert-butyl group-4-(2,4-two chloro-5-isopropyl oxygen phenyl)-Δ ²-1,3,4-oxadiazoline-5-ketone.Other can be used for 2-alkyl-4-of the present invention (2,4, the 5-tri-substituted phenyl)-Δ ²-1,3,4-oxadiazoline-5-ketone is disclosed in, as United States Patent (USP) 3385862; 3836539; In 3876413.

Compound I I is an aryl tetrahydrochysene indazole herbicide, i.e. 3-chloro-2-(4-chloro-2-fluoro-5-isopropyl oxygen phenyl)-4,5,6,7-tetrahydrochysene-2H-indazole.Other can be used for 3-replacement-2-of the present invention (2,4, the 5-trisubstituted benzene) tetrahydrochysene indazole class and is disclosed in, in United States Patent (USP) 4670043.

Compound III, IV and V are aryl tetrahydrochysene phthalimide herbicides.Other can be used for aryl tetrahydrochysene phthalimide class of the present invention and is disclosed in, as United States Patent (USP) 4431822; 4670046; 4670042 and 4439229 and disclosed international application (PCT) WO 87/07602 in.The two pieces of documents in back also disclose other and can be used for NHet ring of the present invention, as walking to the 5th hurdle the 20th row on US 4439229 the 4th hurdle the 25th, and among the WO 87/07602 in the 12nd to 14 page, this ring are illustrated.

Other suitable PH-NHet type herbicide has:

Aryl triazolineone is as at United States Patent (USP) 4318731; 4398943; 4404019; 4702945; 4705557; 4702763; 4761174; And international application (PCT) WO85/01637, WO85/04307, WO87/00730, WO87/03782, disclosed compound among WO86/04481 and the WO88/01133;

The aryl Tetrazoline series is as disclosed compound in United States Patent (USP) 47734124 and international application (PCT) WO85/01939 and WO87/03873;

Aryl-triazine two ketones, as United States Patent (USP) 4755217 and 4766233 and international application (PCT) WO86/00072 in disclosed compound;

The aryl hydantoins is as disclosed compound in United States Patent (USP) 4427438;

The Aryimidazole miazines is as speciallyying permit disclosed compound among J 60-158147A (Derwent digest registration number 85-240363) and the European publication application Ep230874 (Derwent digest registration number 87-215141) Deutsches Reichs-Patent DT 2604989 (Derwent digest registration number 65326X) day disclosure;

The aryl-pyridine diazines is as the compound of the compound 8X type of following embodiment 8;

The compound 8e of following embodiment 8 of aryl diazine two ketones and the compound of 8v type;

Aryl draws thiadiazinthion (aryl pyradiazinones) class, as the compound of the compound 8f of following embodiment 8;

Aryl oxadiazoline ketone is as disclosed compound in day disclosure special permission J59-148769 (Derwent digest registration number 84-246947) or day disclosure special permission J62-161772 (Derwent digest registration number 87-238787);

Aryl oxadiazines ketone is as the compound of the compounds of the 8k of following embodiment 8 and 8m;

Aryl benzamides azoles (aryl benzamidazolos) class of rattling away is as the compound of the 8ad compounds of following embodiment 8;

Aryl imido Triazolopyridazine is as at United States Patent (USP) 4913723; Disclosed compound in-4906281 and 4906279;

Aryl thiazole is as compound 8L, the 8r of following embodiment 8 and the compound of 8ah class;

Aryl pyrrolines is as disclosed compound in European publication application EP255601 (Derwent digest registration number 88-037583) and EP260288 (Derwent digest registration number 88-127433);

Aryl urazole class is as disclosed compound in United States Patent (USP) 4452981;

The aryl six hydrogen azole of rattling away is as disclosed compound in United States Patent (USP) 4619687;

B. aryl-heterocyclic urethanes is as disclosed compound in United States Patent (USP) 4521242.

C. aryl-heterocyclic ureas is as disclosed compound in day disclosure special permission J58-225081 (the Derwent digest number of stepping on 84-034261).

D. aryl amides is as the compound 8aj of following embodiment 8 and the compound of 8ak class.

E. phenylate classes of herbicides, as have right-halogen in pairs-those compounds of the phenoxy group benzene structure of nitro, (2-chloro-4-(fluoroform methyl 5-(2, the 4-dichlorophenoxy) phenoxy group)-2-nitro-N ' yl)-2-nitrobenzoic acid methyl esters-methylsulfonyl benzamide (fomasafen) (bifenox) as following commercially available material: 5- 5-(2-chloro-4-(fluoroform 2-chloro-1-(3-ethoxy-yl) phenoxy group)-2-nitrobenzoyl 4-nitro-phenoxy group)-4-three sour sodium (acifluorfen) toluene fluorides (oxyfluorfen)

Other suitable commercially available diphenyl ether herbicide is:

Breast fen (lactophen): 1-(carbethoxyl group) ethyl 5-(2-chloro-4-4-trifluoromethylphenopendant)-2-nitrobenzoyl acid esters,

Fluoroglycofen (fluoroglycofen): (carbethoxyl group) methyl 5-(2-chloro-4-4-trifluoromethylphenopendant)-2-nitrobenzoyl acid esters,

DCNP (chloronitrofen): 2,4,6-three chloro-(4-nitrophenoxy)-benzene,

Preforan (fluorodifen): 2-nitro-1-(4-nitrophenoxy)-4-trifluoromethylbenzene,

Nitre fen (nitrofen): 2,4-two chloro-1-(4-nitrophenoxy) benzene,

Pl 3468 (chlomethoxyfen): 4-(2, the 4-dichlorophenoxy)-2-methoxyl-1-nitrobenzene.

Suitable in addition diphenyl ether herbicide also has methyl 5-2-chloro-4-4-trifluoromethylphenopendant)-2-nitro acetophenone ketoxime-O-acetic acid esters.

F. arylpyrazole classes of herbicides is as at United States Patent (USP) 4563210; 4496390 and 4459150 and German publication 3520327A1 in disclosed compound.

G. the compound of following general formula:

Or

X wherein ^bBe O or S, " Ph " has above-mentioned implication, as publishing excerpts of disclosed compound among the mark 87-040749 at European publication application EP273417 or moral temperature German.

Some enzyme suppresses herbicide at people such as Matringe (FEBS Letters, Vol245, No1,2,35～38 pages (1989)) and people such as Yonase (Pesticide Biochemistry andPhysiology, existing open in the article of Vol 35,70～80 pages (1989).

With the processing of reagent of the present invention, can implement by vein or intraperitoneal injection.This reagent can be used as sanitizing composition, comprises dissolving or is dispersed in the carrier that allows on the medicine, and as the isotonic solution of water, the phosphate buffered saline (PBS) (PBS) as salt solution (as 0.9%NaCl) or Dulbecco (is about 2.5mg ml as concentration ^-1) in reagent, perhaps be included in the reagent in liposome (liposomal) system, as according to Remington ' s Pharmaceutical Sciences, method described in 1985 (17 editions) 1659-1660 page or leaf (Mack PublishingCompany publication) prepares with Phospholipids bubble (vesicle).For example; with people (Br.J.Cancer (1983) .48 such as Jori; 307 pages) the similar method of described preparation; 51.4mg two palmityls two phosphatidyl choline are dissolved in the chloroform-methanol (9: in solution 1v/v) of 10ml 1mM reagent; after mixing fully; under 30 ℃ of vacuum, remove and desolvate; obtain a kind of solid; solid can be suspended in 10ml PH7.4 again; contain in the 0.01M phosphate buffer of 150mM NaCl, carried out sonicated (sonicate) 30 minutes at 50 ℃ of solution then muddiness.

Can be used for the connexon technology that enzyme inhibitor of the invention process could mutually combine or use prior art and be attached on the suitable tomour specific monoclonal antibody (MABs), so that the guiding enzyme inhibitor is to concrete knub position.

Be used for enzyme inhibitor of the present invention and also can use, preferably,, they are added in the food as hereinafter embodiment 6 is described with the form after the carrier dilution that allows on the medicine by simple oral introducing.

Especially for oral, require preferably water soluble salt or acid and form water-soluble sodium or sylvite of employed enzyme inhibitor, acid sulfamido or its water soluble salt described in international patent application (PCT) WO 87/03782 (as the reagent 6c of embodiment 6 hereinafter) or WO 85/001939 and WO 87/037873, perhaps carboxylic acid or its water soluble salt, as be called the sodium salt of sour fluorine fen (acifluorfen).

Enzyme inhibitor used in the present invention can join in the pharmaceutical preparation commonly used, as tablet (as, the compressing tablet of available massecuite and/or syrup coat), suppository, capsule (as hard gelatin capsule), suspending liquid, solution, powder or ampoule.In these preparations, reagent can be with medicine on the solid that allows and/or the liquid-carrier form of mixing, if desired, these carriers can be nutrients, for example it can be solid or the liquid diluent as cornstarch, as water or edible oil or mineral oil or as the solvent of dimethyl sulfoxide (DMSO).Also can use the potpourri of enzyme inhibitor, for example, the reagent 6c of following embodiment 6 and sour fluorine fen, as potpourri by about equal proportion, used dosage can be measured by the normal experiment of knowing in the prior art, as test to Photofrin II class, be described in the article of " Photody-namic Theropy (PDT) of Malignant Tumors " (CRC Critical Reviews in Oncology/Hematology vol2, (1984) 83～116 pages of issue2) of Dougherty.

Reagent can combine with some known medicines (as verapamil, cyclosporin or quinine) that can improve the anticancer cellular sensitivity of cancer therapy drug and use.

List following embodiment to further specify the present invention.

Embodiment 1

Will be (in 1 liter of Falcon tissue culture flasks at exponential phase, 37 ℃ of insulations 5 days) the sea draw (Hela) cell (a kind of human tumour cell line who is usually used in tumor research, and obtain from American type culture collection) phosphate buffered saline (PBS) (PBS), to wash.Add 0.25% tryptic PBS solution 2ml, after a few minutes, lightly cell is shifted out from bottle, and put into the solution of 110ml at the 25mM Hepes of minimum minimal medium (MEM), be added with the nonactive calf serum of 10%v/v, 0.85% brine solution of 1.1ml 200mM glutamine in this minimum minimal medium, and 11000 units penicillin/11000mcg streptomysin.

The branch solution such as 5ml of the cell culture of suspension again of gained are respectively charged in the 50mlFalcon tissue culture flasks, and (except being used for contrast) mixes with treating agent.To wait separatory in the dark then, 37 ℃ are incubated three days down.Subsequently cell is shifted out, and under green glow, extracts as follows:

By adding 0.8ml 0.25% tryptic PBS solution, cell is shifted out at the bottom of bottle.After cultivating 1 hour in the dark, cell and medium are shifted out from bottle, centrifuge tube at the bottom of the garden of packing into is then through twice freezing-thaw cycles condition, with ruptured cell and extract.

After this breaks operation, detect cell suspending liquid, there is not to find to have complete cell.In each test tube, add 10ml alkalescence acetone (90% acetone: 10%1N NH then respectively ₄OH), centrifuge tube is to remove deproteinize and cell fragment.After in supernatant, adding the saturated NaCl aqueous solution of 0.5ml, add 5ml water again.Drip 2MKH then ₂PO ₄So that pH value is transferred to 6.8.The aqueous acetone extract is installed to C8 Baker Prep post.After draining off, post uses the 2ml water washing.CH with 2 * 1.5ml volume ₃OH/H ₂O (90/10) wash-out tetrapyrrole.With spectrofluorimeter quantitative measurement extract.

Used in this experiment treating agent is:

A. 5-amino-laevulic acid, known it be the precursor of tetrapyrrole.

B. the sour fluorine fen-methyl that has following structural formula:

C. the herbicide that has following structural formula:

D. the herbicide that has following structural formula:

E. the herbicide that has following structural formula:

Reagent A is mixed with the bacterium filtering solution of 250mM.pH6.5.Reagent B, C, D and E are mixed with the acetone soln of 50mM.Also add acetone in reference examples, making its concentration is 0.2%v/v.Be added to amount of reagent in the cell culture by the listed concentration of table 1.The amount of the tetrapyrrole protoporphyrin IX that is produced (" Proto IX ") is shown in table 1.

Table 1

Tetrapyrrole accumulation reagent reagent concentration fluorescent emission Proto IX content in the HeLa cell of handling

(μ m) CPS400/630 (Pmoles) A 5,000 41,842 4.2 B 100 12,921 1.3 C 100 33,477 3.5 D 100 10,551 1.1 E 100 8,967 0.9 contrasts-2,795 0.3

Embodiment 2

To in phosphate buffered saline (PBS) (PBS), wash at the HeLa cell of exponential phase (in six 1 liter of Falcon tissue culture flasks) 37 ℃ of insulations 6 days.Add 0.25% tryptic PBS solution, after a few minutes, at leisure cell is shifted out, and in the 25mM Hepes solution of 110ml in minimum minimal medium (MEM) of packing into, be added with 0.85% brine solution of the nonactive calf serum of 10%v/v, 1.1ml 200mM glutamine in this minimum minimal medium, 11000 units penicillin/11000mcg streptomysin.

The separatory (each 5ml) that waits of the cell culture that suspends again is respectively charged in the 50mlFalcon tissue culture flasks, adds in the bottle or do not add herbicide, and in the dark, in the time of 37 ℃, cultivated 4 days, herbicide is pressed the acetone diluted solution form adding among the embodiment 1.Also add acetone in reference examples, making its acetone concentration is 0.2%v/v.The herbicide addition is by the specified concentration of following table 2.

Treating agent is:

B. as example 1

C. as example 1

F. the following herbicide of structural formula:

Extraction: the nutrient culture media in each bottle is packed in the glass round bottom pipe, by adding the tryptic PBS solution of 0.5ml0.25%, the cell at the bottom of sticking to bottle is beaten pine, then 37 ℃ keep a few minutes after, it washed from bottle and merge with its nutrient culture media.From each cell suspending liquid, take out separatory such as 100 μ l then to measure cell density.Then remaining 5.4ml cell suspending liquid is carried out sonicated with ruptured cell.

In each pipe, add 10ml alkalescence acetone (90% acetone: 10%1N NH then respectively ₄OH), centrifuge tube is to remove deproteinize and cell fragment.After in supernatant, adding the saturated NaCl aqueous solution of 0.5ml, add 5ml water again.Drip 2MKH ₂PO ₄, make PH be transferred to 6.8.The aqueous acetone extract is added on the C8Backer Prep post.After post drains off, use the 2ml water wash.CH with 3ml90/10 ₃OH/H ₂O wash-out tetrapyrrole.Identical with embodiment 1, all these extraction steps carry out down or under dark (as in lighttight container with lid) condition at the light (as green glow) that non-protoporphyrin IX activates basically fully.On the SPEX spectrofluorimeter, measure the fluorescence of extract.Use the semi-invariant of the predetermined quantitative protoporphyrin IX of extinction coefficient (" Proto IX ").The results are shown in table 2.

Table 2

The accumulation reagent reagent concentration growth inhibited Proto IX amount Proto IX amount of average growth inhibited and Proto IX

(μ m) be (Pmoles) (%) ^*Relative % ^{* *}B 100 99 5.20 2364 F 100 106 2.71 1232 C 100 52 4.41 2005 C 10-15 ^*0.57 259 C 1-26 ^*0.19 86 * " growth inhibited " grow for negative value is meant.* per 10 ⁵The average magnitude of cell.The percentage of * * contrast.

Another aspect of the present invention relates to the preparation of protoporphyrin IX (" Proto IX ").In this respect, in containing the mentioned reagent medium of (this reagent can suppress former porphyrinogen enzymatic and be converted into protoporphyrin IX), in dark (or under light that non-protoporphyrin IX excites), the little algae heterotrophism of eucaryote ground is cultivated.Treatment media or algae or medium and algae with the extraction protoporphyrin IX, and separate from chlorophyll, carotenoid and cell fragment etc. then.Separation can be by any method commonly used, as adopt liquid phase chromatography, comprise reversed phase liquid chromatography, or solvent extraction, or precipitate protoporphyrin IX by chelation with metal such as Fe, Zn or Mg, shift out the chelate that is produced, if desired, by known method regeneration protoporphyrin IX with diluted acid processing chelate.

Separating step is to carry out down or in the dark at the light that non-protoporphyrin IX excites (described green glow under as appendix A, title " dark ").The photosensitivity of the chelate of iron is relatively poor, so when handling this material, more can illumination.

Preferred little algae is cultivated in 15～30 ℃ temperature range preferably with cell suspending liquid.The concentration of inhibitor for example can be about 10 ^-5～10 ^-7M.Used little algae example is Scenedesmus kind (Scenedesmus sp), horse tongue butterfly chlamydomonas (Chlamydomonas reinhardi), Euglena kind (Euglenasp) and linear Bumilleriopsis (Bumilleriopsisfiliformis).

Embodiment 3

Logarithmic phase culture with horse tongue butterfly chlamydomonas (wild type) uses following nutrient culture media, and (as 300 liters) are cultivated again in a stainless steel cask.Add the acetone soln of 1-(ethoxycarbonyl)-ethyl-5-(2-chloro-4-4-trifluoromethylphenopendant)-2-nitrobenzoyl acid esters (newborn fen, a kind of phenylate herbicide that is purchased), its concentration is 10 ^-5M, the acetone ultimate density is 0.1%.Make this potpourri in the dark under 25 ℃, stir gently and/or ventilation condition under cultivating 4～7 days.When keeping dark, the interior contained thing of filter vat is also handled its filtrate to reclaim protoporphyrin IX wherein.

A kind of disposal route that reclaims protoporphyrin IX is the contained thing (will keep dark) in the filtering reaction bucket and adds acetone in filtrate.Use the petroleum ether extraction potpourri.Use extracted with diethyl ether aqueous acetone potpourri then, remove ether, stay residue by evaporation.This residue is dissolved in methyl alcohol and carries out reverse-phase chromatography to obtain protoporphyrin IX.Also can use at the recovery method described in embodiment 1 and 2.The composition of nutrient culture media:

ml

Former storage

The former liquid storage liquid of salt volumetric molar concentration/L sodium citrate 6H ₂O 1.7 * 10 ^-3The M 10% 5 following 10FeCl of trace meter ₃6H ₂O 0.37 * 10 ^-3M 1% 1CaCl ₂2H ₂O 0.36 * 10 ^-3M 5.3% 1MgSO ₄7H ₂O 1.2 * 10 ^-3M 10% 3NH ₄NO ₃3.7 * 10 ^-3M 10% 3KH ₂PO ₄2.2 * 10 ^-3M 10% 3K ₂HPO ₄1.7 * 10 ^-3M 10% 3CH ₃CO ₂Na 7.5 * 10 ^-3M 10% 10

The former liquid storage of trace meter potpourri

H ₃BO ₃ 100mg/L?ZnSO ₄·7H ₂O 100mg/L?MnSO ₄·4H ₂O 40mg/L?COCl ₂·6H ₂O 20mg/L?NaMoO ₄·2H ₂O 20mg/L?CuSO 44mg/L

Use the scenedemine kind without horse tongue butterfly chlamydomonas, on above-mentioned nutrient culture media, cultivate, but replace sodium acetate with glucose.

Embodiment 4

Except using herbicides compounds 1-(4-chloro-2-fluoro-5-propargyl oxygen phenyl)-3-methyl-4-difluoromethyl-Δ ²-1,2,4-triazoline-5-ketone replaces outside the newborn fen, and present embodiment is identical with embodiment 3.

Embodiment 5

I ₅₀Mensuration

(a) in complete chloroplast, use enzyme:

Former porphyrinogen 1X (" Protogen 1X ").Protoporphyrin 1X is by Porphgrin Products, and Logan, UT buy and by the described method purifying of people such as T.P.Fuesler (Plant Physiol, 67,246～249 (1981)).Press the described method of N.J.Jacobs and J.M.Jacobs (Enzyme, 28,206～219 (1982)), use Na/Hg amalgam reduction protoporphyrin 1X and the former porphyrinogen 1X of prepared fresh, used purifying protoporphyrin 1X concentration is 300 μ M.

Plant material in the dark culturing chamber, on vermiculite, is irrigated and cultivation cucumber (cucumber of ' Wisconsin SMR18 ' kind (Cucumis sativusL)) with commercially available (9-45-15) chemical fertilizer.Rice shoot is grown under 25 ℃, the condition of relative humidity 80～90%.Optical density is 25 μ E/m ^-2Sec ^-1(PAR) per 60 minutes cycle of light shone 1 minute, and intermittent irradiation, light source are provided by the general electronic corporation " bright rod " (General Electric " BrightStick ") that electronic timer is controlled.Can make tissue quicken chlorophyll like this and synthesize, make starch storage and initial chlorophyll content reduce to minimum simultaneously.

Chloroplast separates people (Plant Physiol75 such as pressing T.P.Fuesler, 662～664 (1984)) described method is separated the chloroplast that is generated, except final purification step, plastid is centrifugal by the Percoll cushion of 40% rather than 45% (v/v) (Percoll cushion).Chloroplast is suspended in a kind of calibrating damping fluid again, to its ultimate density be 2ng protein/ml, this damping fluid contains 0.5M mannitol, 20mM TES, 10mMHEPES, PH7.7,1mM EDTA, 1mM MgCl ₂, 1% (w/v) calf serum albumin and 1mM dithioerythritol.

The oxidasic detection of former porphyrinogen. the described method of J.M.Jacobs and N.J.Jacobs (Arch. Biochem. Biophys., 229,312～319 (1984)) of pressing detects.(v/v acetone (in contrast) gives 0.2ml Chloroplast Suspension sample in the dark and cultivating 15 minutes with the sour fluorine fen-methyl (acifluorfen-mefhyl) (" AFM ") or 0.2% of various concentration.Then, the former porphyrinogen 1X (about 15nM) with the new preparation of 50 μ l joins suspending liquid to begin reaction.By adding the fluorescent media (Enzyme of 2.75ml N.J.Jacobs and J.M.Jacobs, 28,209～219 (1982)) end to analyze, this medium contains Tris-HCl, the 1mM EDTA of 1% (v/v) polysorbas20 (polyoxyethylene sorbitol acid anhydride-laurate), 50mM PH8.5; Replace the 5mM glutathione with 1mM dithioerythritol (" DTE ").On the SPEX Fluorolog-2 type spectrofluorimeter that is equipped with the front fluorescence that is used for muddy biological sample to select to the direct reading of suspending liquid, amount by the quantitative protoporphyrin 1X that produces in the typical curve of the purifying protoporphyrin 1X gained in fluorescent media (exciting at 400nm) in the amount of the corresponding protoporphyrin 1X of emission measure of 630nm.

For in above-mentioned detection, measuring the amount of the protoporphyrin 1X that non-enzymatic catalysis is oxidized to, can carry out same analysis, but except adopting 85 ℃ of heating 15 minutes Chloroplast Suspension of inactivation replace active Chloroplast Suspension.The protoporphyrin IX amount that deducts such generation when active chloroplast is arranged in the protoporphyrin IX amount that is generated just obtains the amount of the formed protoporphyrin IX of enzymatic.The result is to be illustrated in Fig. 1 (by convention, wherein LSD represents minimum effectively deviation).Fig. 1 shows, I ₅₀Value (50% concentration that suppresses is arranged) is lower than 0.1 μ M, the i.e. about 0.03 μ M of Q.

In Chloroplast Suspension (having in the presence of the ATP) 10 μ MAFM to protoporphyrin IX enzymatic change into protoporphyrin IX magnesium effect the analysis showed that AFM does not have inhibiting effect to this conversion.

(b) use the enzyme that separates

Plant material and homogeneization: pea (Peas mutation (Pism sativumvar.) Little Marvel) was germinateed 10 days in 20 ℃ of dark culturing chambers.1 hour every day of illumination plant, make leaf obviously launch the synthetic minimum of reducing to of chlorophyll simultaneously.It is yellowish green that leaf is.

Then leaf is put into the 500ml grinding and carry out homogenizing with medium, this medium contains 0.5M mannitol, 10mM HEPES, 20mM TES, 1mMEDTA, 1mM MgCl ₂, 5mM halfcystine and 0.2BSA, PH7.7.Then this slurry is passed through 4 layers of coarse sheeting, pass through the nylon mesh of 43-micron again.Then with homogenate under 4000g centrifugal 3 minutes.The gained precipitation is used for plastid and separates.

The separation of leucoplastid and purifying: will precipitate (plastid part) and be suspended in again in the 40ml tested media (grind deduct halfcystine but contain 1mMDTE and 1%ESA) with medium, and under 150g, carry out centrifugal, to remove cell fragment.The gained supernatant is centrifugal at 4000g, and the precipitation that will suspend again is added on the 40%Percoll.At 6500g centrifugal 3 minutes, whole leucoplastid was at the pipe bottom sediments.

Measure protein content with the BioRad method.

The enzyme solubilization is according to the method solubilising enzyme of Jacobs and Jacobs.The plastid precipitation also is resuspended in the 2.5ml damping fluid, and this damping fluid contains 20mM Tris-MCl, 30% glycerine and 1mMDTE PH7.6.After using the sonicated of 60mHz, add the washing spe medium, 10%Triton 100X, 8%KCl and 10mMPMSF make finally to obtain: washing agent: albumen is that 0.7 (w/w) is 48 ℃ of insulations after 3 hours, in BeckmanL2-65B, under the 100000g film preparation is carried out ultracentrifugation.

Gel filtration, the supernatant that will contain the solubilising enzyme then carries out desalination on a Pharmacia PD-10G-25 Sephadex post.(20mM Bistris-HCl, 20% glycerine, 0.1%Triton PH6.8) wash with the 25ml elution buffer with post.Sample is added on the post and with 3.5ml slow-towards the liquid wash-out.Discard the fraction of 1ml and collect remaining 2.5ml.Then, at-77 ℃, N ₂Freezing these fractions under the condition.

Use the DEAE chromatography purification, with the plastid-solubilising of 0.75ml volume-enzyme preparation is added to a Waters Protein-Pak DEAE-5pw post, and (on the 8mm * 7.5cm), this post has been used elution buffer balance mistake.Wash (1ml/ branch) after 60 minutes, with elution buffer with linear 0.1M NaCl gradient liquid wash-out enzyme in 100 minutes.Change to 0.8M NaCl at 30 minutes inside gradient liquid by 0.1M NaCl.Collect the 4ml fraction, and analyze its activity.With Water494 UV-detector (UV detector) at the 260nm place, sensitivity: during 1AU, detect its peak value.

Former porphyrinogen oxidase active: press the method described in the present embodiment 5 (a), detect the enzymatic transformation of former porphyrinogen IX to protoporphyrin IX.

In this test, the I of compound 6c (among the embodiment 6) ₅₀Value is that 0.8～0.3 μ M (is equivalent to pI ₅₀6.1～6.5, pI wherein ₅₀Be I ₅₀The negative logarithm of volumetric molar concentration); The I of AFM ₅₀Value is 0.08 μ M (pI ₅₀Be 7.1), the I of compound 8v (among the embodiment 8) ₅₀Value is 0.08～0.03 μ M (pI ₅₀7.1～7.5).

Embodiment 6

In the present embodiment, enzyme inhibitor is joined in the mouse feed of suffering from tumour; Some mouse with comparing, are not added inhibitor in its feed.Then mouse is killed, take out its kidney, intestines, adrenal gland lymph node, liver and tumour and analyze the content of their protoporphyrin IX.Used following enzyme inhibitor:

More particularly, mouse is the DBA/2Ha mouse, takes on the hypodermic tumour to injected in mice SMT-F the same day (0 day) on the right side.The mouse that locellus is placed, fed in first day with untreated Purina Rodent Chow 5001 mixed fodders, at the 2nd to 10 day, mouse fed with the same mixed fodder of handling with the 2000ppm inhibitor (perhaps, the contrast mouse then being fed with the same mixed fodder that is untreated) subsequently.Mixed fodder is mixed with the acetone soln of the reagent that will test on a small quantity and carry out the processing of mixed fodder.At the 10th day mouse is killed then, except the mouse of handling with reagent 6j, this mouse just killed at the 7th day.To be organized in 4 ℃ of refrigerations and spend the night, blot then, and write down the fresh weight of each tissue sample.To be organized in 5ml (then being 10ml) acetone-0.1NNH for intestines and liver ₄OH (9: 1, v/v) middle homogenizing.Homogenate is centrifugal at 1500g, and on SPEX FA 112 type spectrofluorimeters, analyze its supernatant, to protoporphyrin 1X, select the 400nm wavelength to excite the 630nm wavelength emission.Measure the accumulation of protoporphyrin 1X, with CPS (the institute's emitted fluorescence counting p.s.) expression of every gram tissue.The results are shown in following table (result to each processing is the mean value of two mouse, except handling with reagent 6f, 6h, 6i and 6j, has only handled a mouse with them).Average cps * 10 of the protoporphyrin 1X of every gram ^3*

Weight (mg) 6a 1,203 4,291 10,817 6572 5870** 1436b 1,520 1,261 2,441 2529 5577** 906c 14,336 2,739 16,186 13790 9937** 1576d 2,006 1,581 12,865 443 5565** 2946e 837 876 915 1331 2069** 1656f 449 1,170 661 5,611 1157 696g 166 803 606 1,984 466 2506h 298 914 585 2,259 584 1926i 932 1,510 4,014 36,940 3454 206j 1,251 538 315 491 572 11 contrasts 239 536 494 1,504 233 200 of tumor resection reagent tumour liver kidney adrenal gland intestines

424**

* numerical value equals former data * 10 in the table ^-3

The * test is to carry out on the sample of 3 parts of alkaline acetone diluted.

These data are equivalent to the concentration of the following protoporphyrin 1X in each self-organizing, and it is that protoporphyrin ng with every gram tissue measures to represent: reagent tumour liver kidney adrenal gland intestines 6a 11 39 99 60 546b 14 12 22 23 516c 131 25 148 126 916d 18 14 117 4 516e 888 12 196f 4 11 6 51 116g 276 18 46h 385 21 56i 9 14 37 337 326j 11 5345 contrasts 255 14 2

4

In the Dougherty article of quoting in the above, to behind the hematoporphyrin derivative of same-type (DBA/2Ha) injected in mice 10mg/kg, the porphyrin concentration that draws in same-type (SMT-F) tumour is 3.6 μ g/g.At other technical literature (people such as Moan, P ﹠amp; P46-5,713～721 pages; Fig. 2 note at 716 pages) proposes in, giving DBA/H ₂After injecting 25mg/kg Photofrin II (widely used sensitizer during fluorescigenic processing and knurl disease are detected in light) in the mouse peritoneum, at tumour (C ₃H/Tif mammal cancer) the protoporphyrin concentration in reaches about 12 μ g/g.

The general assembly (TW) that contains the reagent feed that mouse consumes is as follows: 6a, 22 and 35g; 6b, 37 and 35g; 6c, 25 and 22g; 6d, 41 and 32g; 6e, 40 and 44g; 6f, 27g; 6g, 33 and 40g; 6h, 36g; 6i, 10g; 6j, 20g.

In the preparation of present embodiment 6 described experiments, carry out following conventional steps, use the reagent of pointing out at this embodiment 6c (unless otherwise noted), and with the animal of non-trouble tumour:

(a) to the LD of mouse ₅₀Surpass 2600mg/kg (being reagent mg/ body weight kg) (behind the dosage of the once oral corn oil that contains 15% reagent, build-in test during 14 days) and to mouse LD ₅₀The reagent that surpasses 700mg/kg (behind the once oral dosage that contains 5% reagent and corn oil, build-in test during 14 days) is measured.

(b) in the excipient test of intraperitoneal injection, do not have reagent, learn after the test, mouse can be stood the equivalent DMSO (dimethyl sulfoxide (DMSO)) of injection 0.5ml dosage and the potpourri of water.

(c) in the reagent test of intraperitoneal injection, reagent is in the potpourri of 60% corn oil and 40%DMSO, records the result and shows, mouse can be stood 100mg/kg dosage.

(d) carry out following test with mouse:

(i) the oral 50mg/kg of tube feed every day (with the acetone soln of 1% reagent) is 8 days;

(ii) intraperitoneal injection 50mg/kg every day (with the mineral oil dispersion liquid of 1% reagent, be dissolved in 1 droplet DMSO and with mineral oil make reagent) is totally 8 days;

(iii) on skin, use 50mg/kg (DMSO solution) every day, totally 8 days with 1%;

(iv) feed, be mixed with the standard feed of 2000ppm reagent (based on feed weight) with 8 days;

(v) intravenous injection every day 50mg/kg (using 2%DMSO solution), totally four days.

The mouse that these are used to test kills then, and measures the accumulation of the protoporphyrin IX of its tissue.

In another experiment, male Fisher 344 rats of forage feed 27 days with containing 5000ppm reagent 6c kill then, the tissue of this mouse shows, compared with the control, and at kidney, intestines, the protoporphyrin amount that is increased in stomach and the brain increases obviously, increases and have only in musculature seldom.

In superincumbent rat and the mouse test, animal is 12 hours dark of the standard of remaining on---in the bright circulation in 12 hours.

Embodiment 7

In series a series of experiments together of embodiment 1 and 2, the HeLa cell culture is cultivated in the presence of the following various treating agents that 100 μ M are arranged.Almost the percentage that provides of each treating agent represents that all the protoporphyrin IX amount that is produced in the presence of the treating agent is being arranged, and compares with the amount that reference examples produced of identical experiment, and increase is arranged.

The reagent that embodiment 6 is used: 6a, 337%; 6b, 366%; 6c, 297%; 6d, 1200%; 6e, 1210%; 6f, 280%; 6g, 326%; 6h, 431%; 6i, 544%; 6j, 469%.Other reagent:

With the same in embodiment 1 and 2, the data of present embodiment 7 are based on fluorescence method and measure (for example pressing per second counting " cps ").It is (different with embodiment 1 and 2 that but a pair cell extract is measured in this example, in embodiment 1 and 2, broken cell and nutrient culture media are extracted together), fluorescence matrix is that the sort of described in embodiment 5 (contains 1% Tween-20,50mM Tris-Hcl, 1mMEDTA and 1mM dithioerythritol, PH8.5), and fluorescence matrix itself has also been carried out the cps measurement.When meter sample example earlier data, available following formula calculates the percentage increased numbers: $\[\frac{\mathrm{Ca}-\mathrm{Cm}}{\mathrm{Cc}-\mathrm{Cm}}\×100\]-100$ Wherein Ca is the per second counting with gained sample after the agent treated, and Cc is the per second counting of control sample, and Cm is the per second counting (being background fluorescence) of fluorescence matrix itself.From these data as can be seen, the Cc-Cm value is compared quite little with the Cm value in this cover experiment; For example the independent measurement value to Cc is 49025 and 52069, and is 4295 to Cm.So the formula above using can not illustrate the effectiveness of compound well.Therefore, use following formula to recomputate data: $\frac{\mathrm{Ca}}{\mathrm{Cc}}\×100\%$ The result is as follows: 6a 336%; 6b 148%; 6c 154%; 6d 510%; 6e 320%; 6f 280%; 6g 329%; 6h 178%; 6i 171%; 6j 185%; 7a 118%; 7b 142%; 7c 103%; 7d 128%; 7e 173%; 7f 118%; 7g 155%; 7h 118%.

Embodiment 8

In the present embodiment, respectively the HeLa cell handled and the protoporphyrin 1X content of nutrient culture media thereof are analyzed, proved that a large amount of protoporphyrin 1X that generated in the cell secrete in nutrient culture media in processing procedure.

Press the same quadrat method of embodiment 1 and 2, to cultivate under the HeLa cell culture condition that listed various treating agents exist below 100 μ M are arranged, treating agent is joined in the dilute solution of dimethyl sulfoxide (DMSO) (DMSO), and only contain 0.2%DMSOv/v in the reference examples.After cultivating like this, nutrient culture media is poured out telling from cell, by adding the 0.25%w/v tryptose in 37 ℃ of dipping cells 20 minutes, cell is unclamped from bottle after, in bottle, add fluorescent media again.Fluorescent media is with identical described in the embodiment 7, except its pH value is 6.5.This medium also can be used as extractant, and the gained extract is carried out the protoporphyrin IX content analysis.Isolated nutrient culture media is also carried out the fluorescence analysis of protoporphyrin IX content.Almost the given percentage of every kind for the treatment of agent is right, all illustrates, the protoporphyrin IX amount that has treating agent to exist to be produced down is corresponding to the protoporphyrin IX amount that is produced in the reference examples in same experiment; First value of every pair of data is the concentration of protoporphyrin IX in the nutrient culture media and second value is the concentration in cell. 8e.107％，98％

8f.207％，108％

8g.149％，105％

8h.776％，252％ 8l.101％，99％ 8j.153％，105％ 8h.124％，102％ 8l.129％，100％ 8m.289％，134％ 8n.297％，135％

8o.112％，103％ 8p.110％，96％

8q.120％，101％ 8r.140％，102％

8s.115％，106％ 8t.150％，105％

8u.122％，100％ 8v.172％，111％

8w.121％，102 8x.192％，118％ 8g. 126％，101％?8x. 183％，112％

8aa.?101％，100％?8ab.?122％，101％

8ac.?123％，101％?8ad.?104％，96％ 8ae.?126％，101％?8af.?115％，97％

8ag.110％，99％ 8ah.109％，97％

8ai.200％，118％ 8aj.104％，100％

8al.102％，98％

8ak.110％，99％

8am，434％，191％ 8an.712％291％

8ao.?587％，214％ 8ap. 304％，185％

8ag.?210％，126％ 8ar. 122％，106％

6b. 271％，142％

6c. 772％，280％

6e. 690％，256％

For compound 8t-8ar, to compare with reference examples, the rough observation of cell growth shows that some reagent cell growth has following inhibiting effect:

8aj, ag ar:25% suppresses

T, ac, ai, ak, an, ap:50% suppresses

U:60% suppresses

V:90% suppresses

Ab, ad, ae, af, ah: deadly

Embodiment 9

In the present embodiment,, tumour is implanted in the rat, illumination is carried out in the zone that tumour is arranged, make tumor regression with enzyme inhibitor 6c hello the rat of embodiment 6.

Particularly, the Spraque-Dawley rat of 3 average weights 120 gram is fed with the food that contains 2000ppm reagent 6 days totally.At the 3rd day, chondrosarcoma is implanted the right hind of every rat by the suspending liquid (about 1,000,000 tumour cells) of injection 0.3ml tumour cell.。At the 6th day, with left hind shaving and the unhairing of every rat, measure the size of each tumour, use the light of the 630nm of unlikely thermal dose that tumor region and surrounding skin thereof are carried out total amount and be 270J/cm ²Illumination.Found that two rats did not obviously have tumour (promptly not having palp tumor mass) in second day, born of the same parents' knurl of the 3rd rat almost completely disappears, skin on every side and musculature have slightly and bleach and some swelling, but with compare with the treatment that fluorescence takes place in the indirect light of Photofrin II, swelling is obviously little.

In another set of experiment, give 20 rats preceding 10 days in photo-irradiation treatment, at random feed, and after 7 days, inject tumor cell suspension at feeding animal with above-mentioned identical food (feed that promptly contains 2000ppm reagent).Be injected at like this and can cause to generate to have the tumour of fully determining rete vasculosum in 1-2 days.When using optical processing, diameter of tumor is 5～8mm, and significantly not downright bad.In next day of optical processing, observing 75% tumour that is subject to processing has obviously and disappears, and this disappearing is significantly can touch tumor mass and have some black dull in tumour formation place because of not having.But, at the time past tense, there is 95% tumour to regrow, illustrate that this specific light processing has also stayed the tumour cell of some survivals, is desirable by following one or more methods to change this processing: long optical processing, one or more other optical processing, higher reagent dosage, make tumour cell carry out handling, change application method, for example injection than the enzyme inhibitor of 3 days longer times.

Used optical processing can not cause that tangible blood flow stops up to processing region, and this is by after illumination, injects Sodium fluoroscin immediately and confirms.

Repeat giving of rat and be equipped with experiment, use the compound 8c (in the test of embodiment 8 than active much lower a kind of reagent of the compound 6c) of oral same dose to handle, do not produce any tumor regression.

Can screen enzyme inhibitor (for example known " snperoxiaized herbicide ") by compound easily to the test (as above-mentioned embodiment 1,2,7 and 8) of the effect of the protoporphyrin IX that in the HeLa cell culture, produced.Can replace HeLa cell with other clone, for example undifferentiated mammalian cell is as lymphocyte, mouse leukemia cell, human fetal lung fibroblast, Chinese hamster gonad cell or any other mammal cell line renewable or the generation protoheme.

Appendix A

The test of loss percentage

The run off test of percentage of the cotyledon that use is collected from yellow cucumber seedling.Initial step is to handle blades in the dark with the aqueous buffer solution that contains radiolabeled sugar.Measure the sugar amount (counting as follows) that is absorbed by cotyledon then.With the blades separated into two parts, a part of cotyledon wants the buffering liquid of test compounds to handle with containing in the dark then, and another part is not then handled with not containing the other identical solution that will survey compound in the dark in the same way, in contrast.The cotyledon illumination that contacts with aqueous solution was told from aqueous solution after 16 hours, measured (by counting) aqueous solution then to measure its radioactive marker substance's content.The result represents with loss percentage, it can be marked with following method meter, the radioactive marker substance's that cotyledon absorbed when wherein S was initial treatment counting (each cotyledon), ST is the counting (each cotyledon) that contains radioactive marker substance in the aqueous solution of wanting test compounds after the illumination, and Sc is the counting (each cotyledon) that contrasts radioactive marker substance in the aqueous solution after the illumination:

More particularly, following substances and condition are employed in the test of loss percentage.

Plant material: in vermiculite, make it to germinate and growth with commercially available (9-45-15) chemical fertilizer pouring cucumber (" the Wisconsin SMR 18 " cucumber of kind).Rice shoot is grown in 25 ℃ and 80～90RH.Collect cotyledon in the back 5 days yellow rice shoot and use 1.0mM CaCl by planting ₂Flushing, all operations all is to carry out under green glow.

Buffer solution: 1mM KCl, 1mM CaCl ₂With the 2.0mM potassium phosphate, pH value transferred to for 6.5 (as using NaOH).

Radiolabeled sugar: than 3-0-methyl-3-(u-14c) glucose (Amersham Corp., Arlington Heights IL) of putting to 10.9GBq/mmol.

Initial treatment: washed cotyledon (180～230) is added in the Erlenmeyer bottle of the 250ml wide-mouth foam plugs good (foam-stoppered) that contains 50ml buffer solution, and adds radiolabeled sugar, making its concentration in solution is 600nM.In the dark, on gyratory shaker with bottle with the rotating speed jolting of 125rpm 24 hours.Then cotyledon is covered the 1nMCaCl that on nylon mesh, also uses the 20ml volume ₂Wash 3 times.Measure the radiolabeled sugar amount of being absorbed by the sample of organizing three parts of 5 cotyledons of digestion in the solubilizer (Amersham Corp) at NCS, and in liquid scintillation spectrometer, the gained macerate is counted.(found by the result who draws in these digestive juices with by the coming to the same thing of burning sample cotyledon in the autoxidation device to the same mensuration of gained.)

Handle (and control treatment) with wanting test compounds:

Have in the plastics pendant Ti Shi double dish of lid at a diameter 35mm, 5 cotyledons up are suspended on the 3ml damping fluid from axle, add the compound (with its acetone soln form) that to test then, the compound that will test is reached in buffer solution give and decide concentration (below will discuss), the same with the solution that contains test compounds, in reference examples, the concentration of acetone in buffer solution is 0.1% (v/v).On the gyratory shaker face, make the cotyledon turn 16 hours of suspension with 90rpm rotating speed jolting double dish.

Illumination: in the photosynthetic activity district of form (PAR) with four GE F20T12-CW fluorescent lights with 150 μ Em ^-2Sec ^-1Measured intensity carry out illumination 16 hours (in the surface measurement of cotyledon) to filling the double dish that is suspended in cotyledon on the solution, jolting double dish simultaneously.

Radioactive marker substance's Determination on content in the liquid: from cotyledon, separate whole liquid, in liquid scintillation spectrometer, count then.

Dark: the whole processing before illumination step all are to carry out (i.e. the green plastic valve optical filter (plastic cut-off filter) that passes through by the light that can make 450～600nm filter light) in the dark or under green fluorescence.

Appendix B

Nutrient culture media:

Former liquid storage ml is former for nutrient culture media M salt volumetric molar concentration

Liquid storage/L sodium citrate 6H ₂O 1.7 * 10 ^-3The M 10% 5 following 10FeCl of trace meter ₃6H ₂O 0.37 * 10 ^-3M 1% 1CaCl ₂2H ₂O 0.36 * 10 ^-3M 5.3% 1MgSO ₄7H ₂O 1.2 * 10 ^-3M 10% 3NH ₄NO ₃3.7 * 10 ^-3M 10% 3KH ₂PO ₄2.2 * 10 ^-3M 10% 3K ₂HPO ₄1.7 * 10 ^-3M 10% 3

The former liquid storage of trace meter potpourri

H ₃BO ₃ 100mg/L

ZnSO ₄·7H ₂O 100mg/L

MnSO ₄·4H ₂O 40mg/L

COCl ₂·6H ₂O 20mg/IL

NaMoO ₄·2H ₂O?20mg/L

CuSO ₄ 4mg/L

Culture medium A is by add 10ml/l10% aqueous sodium acetate solution (7.5 * 10 in nutrient culture media M ^-3M) prepare.

By top listed order each composition is added in the distilled water, and with the 15psi pressure in (pound/time 2), autoclaving 15 minutes.

The cultivation of stock culture under illumination:

At 25 ℃, in the good Erlenmeyer bottle of poly-urethane stopper plug, the stock culture of Y-1 cell is contamination-freely remained among fluid nutrient medium A or the M, be preferably among the nutrient culture media M.Stock culture is cultivated under the condition of not replenishing ventilation on the gyratory shaker of 125rpm rotating speed.The intensity of illumination of cultivating on the object plane is 120 μ Em ^-2Sec ^-1(PAR).Culture is in half synchronous growth pattern under these conditions, reaches 2-4 * 10 until them ⁶The stationary phase of cell/ml.

The cultivation of culture under the unglazed photograph (culture of dark culturing)

To cultivate down by illumination stationary phase stock culture cell (7.5ml) change in the 750ml culture medium A in the Erlenmeyer bottle.By a gas-filled tube inflation under water, with cell in the dark, cultivated 3-4 days for 25 ℃.During this period, the Y-1 cell culture will carry out 7-8 cell division, lose all visible chlorophyll, and concentration be 2-3 * 10 ⁶Cell/ml.

The cell preparation that is used to test:

About 5 minutes of about 20 ℃ of low-speed centrifugals (promptly about 2000rpm), collecting cell from the culture (750ml) of the dark growth of prepared fresh.Cell is suspended in the 50ml culture medium A lightly again.Measure the activity of this suspended sample (about 0.25ml), and with 1% glutaraldehyde water solution fixing after, carry out cell count to measure cell/ml value.Normal cell count is about 5 * 10 ⁷Cell/ml.With 1ml aliquot sample (10 ⁷Cell/ml) is added to test container (for example, Falcon ^24 1 hole tissue culture wares) in.This moment, cell was very movable and be yellow.

Test compounds is dissolved in the solvent (acetone, ethanol, dimethyl sulfoxide (DMSO) or water, preferably acetone), and making its concentration is 1000 times of ultimate density.With 5 or 10 μ l microsyringes this test fluid of 1ul is added in per two holes.

In a manner described, four of preparations do not have the contrast of test compounds according to the hole in each tissue culture ware.The tissue culture ware is covered with transparent plastic cover, and put into 25 ℃, have 70～90 μ Em ^-2Sec ^-1The incubator of light intensity in kept 13～16 hours.If the inclusions in the instrument connection presents yellow, then identify method extraction described in the joint by following result.If the inclusions in the instrument connection presents transparent or light green, then should before extraction, be placed on the rotation bed of 125rpm rotating speed, and with about 600 μ Em ^-2Sec ^-1Light intensity irradiation 2 hours.

The result identifies

The aqueous solution (250 μ l) and the methyl alcohol (1ml) of 10% lauryl sodium sulfate are added in each instrument connection, and thoroughly mix.Potpourri was placed 3-4 hour in the dark.With the tissue culture ware centrifugal 5 minutes of room temperature low speed (about 2000rpm).Shifting out supernatant, and on Beckman35 type spectrophotometer, carry out the former analysis that quinoline IX exists that frightens between 350nm and 500nm wavelength, is the chlorophyll absorption peak of this solvent system at the 668nm place, is the reading that is used for the turbidity background at the 720nm place.

Data analysis

To each hole, from the 668nm reading, deduct 720 readings and just obtain green value.These values are each mean value of wanting test compounds concentration and reference examples.Percent inhibition is:

Claims (3)

1. one kind is used for cancer effectively at the fluoresce screening technique of non-porphyrin compound of treatment of light, it is characterized in that, method comprises with described compound treatment mammalian tumor cell, perhaps handle mammal, and measure the protoporphyrin IX that in described cell or described tumour, is produced with the cell that can detect the tumour form.

2. according to the method for claim 1, it is characterized in that described measurement is to be undertaken by observing the fluorescence that is sent by described processing cell or tumour under the light that excites at porphyrin.

3. according to the method for claim 1, it is characterized in that described cell is in in-vitro culture medium, and the about 1-100 μ of the concentration of the wanting test compounds M in described nutrient culture media.

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