CN1074219A

CN1074219A - New 10-hydroxyl pheophytin a

Info

Publication number: CN1074219A
Application number: CN92115272A
Authority: CN
Inventors: 宋佖淳; 韩宝燮; 李元荣
Original assignee: First Sugar Refining Corp; Samsung Electronics Co Ltd
Current assignee: First Sugar Refining Corp; Samsung Electronics Co Ltd
Priority date: 1991-12-17
Filing date: 1992-12-17
Publication date: 1993-07-14
Also published as: WO1993012114A1; MX9207313A; AU2956992A; KR950013563B1; KR930012781A

Abstract

The present invention provides 10-hydroxy pheophytin a of formula (1) or a pharmaceutically acceptable salt thereof. These compounds are used as photosensitizers in photodynamic cancer therapy.

Description

New 10-hydroxyl pheophytin a

The invention relates to new 10-hydroxyl pheophytin a (following be referred to as sometimes " CpD-I ") and its pharmacy acceptable salt, and the preparation method of this compound.The invention still further relates to the photosensitiser composition that contains as the above-claimed cpd of active ingredient, and utilize above-mentioned optical dynamic therapy method for cancer.

In recent years, " photodynamic therapy (PDT) " caused the interest and the concern of medical circle, and so-called " photodynamic therapy (PDT) " is meant the therapy of utilizing actinic rays kill tumor cell.T.J.Dougherty has reported representative this photodynamic therapy.This photodynamic therapy comprises the use photosensitizers, this photosensitizers by preferential absorption in solid tumor growth tissue part rapidly.In a single day photosensitizers is injected into, and just is enriched in around the tumor locus.Then to the light of the inherent maximum absorption wavelength of tumour photosensitizer administration own, so that kill tumor cell and do not damage healthy cell around the tumour cell optionally [referring to people such as T.J.Dougherty, Cancer Res.38, PP.2628-2635(1978)].

When utilizing photodynamic therapy treatment cancer, key is to select used photosensitizers.Photosensitizers should have the preferential also specificity of emitting fluorescence that combines with tumour cell.By utilizing these character can the localization of cancer cell.In photosensitizers injects checked health body, in health, behind the placement endoscope, be easy to observe the fluorescence position, i.e. the growth of tumour cell position then.

During irradiation, be in the sensitiser absorption light of ground state.The photoexcited state of photosensitizers shifts energy to intravital molecular oxygen, produces singlet oxygen.The various biological compounds of singlet oxygen oxidation comprise cellularstructure component such as steroide and the phosphatide with unsaturated acyl group chain, amino acid such as tryptophane, Histidine, light Gelucystine and methionine(Met); And nucleic acid component such as guanosine and cytidine or the like.Therefore, in case singlet oxygen and tumour cell effect, it will be by the cellular component kill tumor cell of oxidation tumor tissues.

As mentioned above, photodynamic therapy can be used for the diagnosis and the treatment field of cancer.Photodynamic therapy is better than the methods of treatment of other type.This methods of treatment has good therapeutic action and very wide range of application, even steal mirror in also can passing by light and treat with succeeding because be in the tumour (no matter be what tumour) at any remote position.This methods of treatment also has another advantage, and promptly it only produces less side effect for normal cell, but thereby repetitive administration.

Up to now, a large amount of photosensitizerss have been developed, the wherein existing natural synthetic that also has.These photosensitizerss comprise psoraline, flavine, porphyrin, acridine, thiodiphenylamine, xanthene, quinone, polyenoid, halogenated aromatic compound, and mineral ion is [referring to J.D.Spikes and R.Livingston, Advances in Radiation Biology, Vol, II, PP.29-121(1969)].Many this photosensitizerss now have been used as light power antineoplastic agent and have been used for clinical trial.A small amount of photosensitizers can have been bought from the market and be used for PDT.Its representative example comprises that hematoporphyrin derivative (HpD) is (by The Quadra Logic Technology Inc(Vancouver, Canada) sell, trade name is " a Photofrin-I ", the mixture of the ether of polyhematoporphyrin ether and ester in one, " Photofrin-II ", a kind of purer product.

Inventor of the present invention concentrates and has carried out a series of tests, so that seek a kind of effective photosensitizers with good result of treatment, found that isolated new phoeophytin derivative can greatly improve photosensitivity from the silkworm extract, thereby finished the present invention.

Therefore, the purpose of this invention is to provide new phoeophytin derivative, this derivative can be used as photosensitizers and is used for photodynamic therapy treatment cancer.

Another object of the present invention provides the method for the new phoeophytin derivative of preparation the present invention.

Another object of the present invention provides photosensitiser composition, and it comprises as the new phoeophytin derivative of the present invention of activeconstituents with it and mixing and the bonded usual component, as carrier, and vehicle, swelling agent or the like.

Still a further object of the present invention provides the new phoeophytin derivative optical dynamic therapy method for cancer with the present invention.

The rest part of other purpose of the present invention by reading specification sheets is with fully aware of.

Accompanying drawing 1A and 1B represent various silkworm extracts, the thin-layer chromatography of spinach and Sang Zi (TLC);

Accompanying drawing 2A, 2B and 2C represent to be described among accompanying drawing 1A and the 1B with 2 reversed phase high efficiency liquid chromatography (HPLC); Wherein accompanying drawing 2A be illustrated under the fluorescence compartment lamp exposure after several hours with 2 reversed phase high efficiency liquid chromatography, its expression is converted to CpD-2 by CpD-1 light; Accompanying drawing 2B be illustrated under the lamp exposure after 5 minutes with 2 reversed phase high efficiency liquid chromatography; Accompanying drawing 2C represent without exposure with 2 reversed phase high efficiency liquid chromatography;

Accompanying drawing 3A represents CpD-1 and chlorophyll a separately ¹H-NMR spectrum, wherein arrow indication C ₁₀-OH and C ₁₀The position of-H;

Accompanying drawing 3B represents CpD-1(CpD-1 and heavy water (D ₂O) mix) and chlorophyll a ¹H-NMR spectrum, with CpD-1 blended heavy water be to be used to measure C ₁₀-OH's ¹H resonance;

Accompanying drawing 3C represents CpD-1's ¹³C-NMR spectrum;

Accompanying drawing 3D represents CpD-1's ¹H two dimension COZY NMR spectrum;

The Fourier Tranform infrared spectra of accompanying drawing 4 expression CpD-1;

Accompanying drawing 5 is illustrated under 295 ° of K 1, the chart of the creating singlet oxygen by using quantity that produces during the maximum initial light rate of oxidation of 3-phenyl isobenzofuran (DPBF);

The absorbability of CpD-1 and HpD in accompanying drawing 6 expression tumours and the normal cell; And

Accompanying drawing 7A and 7B represent to use CpD-1 and HpD difference human body T-4 lymphoma cell line (MOLT-4) and human body end blood lymphocytes (PBL) slightly, are exposed to the percentage survival of the above-mentioned cell in ruddiness (accompanying drawing 7A) and gold-tinted (accompanying drawing 7B) back then.

The invention provides new 10-hydroxyl pheophytin or its pharmaceutically acceptable salt with following formula:

Pheophytin derivative of the present invention can high yield produce the light activation creating singlet oxygen by using, compares with other sensitising agent commonly used, demonstrates better selection and absorb in tumor tissues. In addition, aspect the selection destruction of cancer cells and treatment tumour efficient of experimental animal, sensitising agent of the present invention is more superior than other sensitising agent commonly used.

The The compounds of this invention structure is similar to chlorophyll a, but still has architectural feature, i.e. Mg in the chlorophyll a⁺⁺And C₁₀Hydrogen atom is replaced by two hydrogen atoms and hydroxyl respectively on the position.

Known to the inventor, 10-hydroxyl pheophytin of the present invention is the noval chemical compound that structure also was not determined, and also is not used as sensitising agent in vivo with in the in vitro test.

The formula I compound is convertible into its non-toxic salt, i.e. its pharmacy acceptable salt that obtains according to any ordinary method.Described non-toxic salt comprises the inorganic salt of formula I compound, as its metal-salt, and inorganic acid salt and organic salt.Metal-salt comprises an alkali metal salt such as sodium salt, sylvite etc., and alkaline earth salt, and as calcium salt, magnesium salts etc.Inorganic acid salt can comprise hydrogen chlorate, hydrobromate, vitriol, phosphoric acid salt or the like.Organic salt can comprise organic amine salt, as front three amine salt, triethylamine salt, pyridinium salt, procaine salt, picoline salt, dicyclohexyl amine salt, N, N-dibenzyl-1, the 2-ethylenediamine salt, N-methylglucosamine salt, diethanolamine salt, triethanolamine salt, three-(methylol amino) methane salt, phenylethyl benzylamine salt, dibenzyl ethylenediamine salt or the like.Non-toxic salt also comprises organic carboxyl acid or sulfonate, as formate, acetate, maleate, tartrate, mesylate, benzene sulfonate, tosylate etc., and with alkalescence or acidic amino acid such as arginine, aspartic acid, the salt that L-glutamic acid, Methionin etc. form.

The present invention also provides the method for preparing new phoeophytin derivative of the present invention, comprises step:

(a) from the silkworm extract, extract the chlorophyll metabolism thing with suitable solvent;

(b) substance dissolves that extraction is obtained is in suitable solution, to supersaturation;

(c), from gained solution, separate the photosensitizers component through chromatography; And

(d) collection shows the quick agency part of high light, uses the ordinary method purifying subsequently.

The extraction of silkworm extract (step (a)) can be carried out according to conventional solvent-extraction process, for solvent, can use known any solvent of this specialty such as ketone, alcohol, and composition thereof.The material that extracts is dissolved in the suitable solvent then, obtains a supersaturated solution (step (b)).Any solvent all can be used for the dissolved solids extract.Yet, be effective dissolving, preferred tertiary butanols, the mixture of acetone and pentane.Gained solution is then through chromatography, the solid ingredient (step (c)) of separate dissolved in extracting solution.The known any preparative chromatography of this specialty, as liquid chromatography (LC), thin-layer chromatography (TLC), and other class shape and color spectrum all can be used.After having separated, collect part, use conventional purification process purifying (step (d)) then with strong photosensitivity.The present invention preferably uses high pressure liquid chromatography (HPLC).

Analyze products therefrom, determine its chemical structure.Can use any analytical procedure known in the art, as infrared spectra (IR), Fu Liye changes infrared spectra (FT-IR), UV spectrum (UV), rotatory dispersion and circular dichroism, fluorometry, flash spectrum, mass spectrum, NMR (Nuclear Magnetic Resonance) spectrum etc.

In addition, the formula I compound also can be made raw material by utilizing chlorophyll a, and is synthetic with chemical process.The synthetic method of preparation formula I compound comprises the steps:

(a) with chlorophyll a under room temperature, in ethanol or chloroform,, slough Mg ion wherein with HCl reaction;

(b) under the safety lamp or dark situations of room temperature and dimness, in gained solution, slowly blasted air one day or the longer time, replace C with oh group ₁₀Hydrogen atom on the position; With

(c) with products therefrom purifying according to a conventional method.

The raw material chlorophyll a can obtain from any suitable raw material with currently known methods, but preferred the use is rich in chlorophyllous marine alga, and Spirulina platensis is as the raw material of chlorophyll a.This synthetic method advantage is to be fit to produce by batch on a large scale CpD.

Although the above-mentioned part of the present invention has only been described single formula I compound, but should know, for similar compound, can carry out with the wherein substituent improvement foot of other substituting group replacement, referring to G.N.La Mar, " Model Compounds As Aids In Interpreting NMR Spectra of Hemoproteins " in Biological Applications of Magnetic Resonance, R.G.Shulman Ed., pp.305-343(1979), Academic Press, New York; And J.D.Satterlee, " NMR Spectroscopy of Paramagnetic Haem Proteins " in Annual Reports On NMR Spectroscopy, G.A.Webb Ed., pp.84-85(1986), Academic Press, New York.According to these prior arts, utilize ordinary method known in the art to be easy to design and have the substituent analogue different in a large number with the formula I compound with synthesizing, therefore, the present invention also comprises the 10-hydroxyl-pheophytin a analogue with following formula structure:

Wherein, R ₁, R ₃, R ₄, R ₅, R ₆And R ₈Represent C ₁-C ₃Alkyl aldehydes or C ₃-C ₅Cycloalkyl;

R ₂Represent C ₁-C ₃Alkenyl, and

R ₇Representative

Wherein 1, m and n represent 1 to 3 integer separately.

On the other hand, provide the photosensitiser composition that is used for the treatment of cancer cells, comprised active ingredient formula I compound or its pharmacy acceptable salt and mix or the bonded usual component with it, as carrier, vehicle, swelling agent, and other additive.

On the other hand, the invention provides the optical dynamic therapy method for cancer, comprise to health and use new formula I phoeophytin derivative or its pharmacy acceptable salt.

Formula I phoeophytin derivative can pass through various injecting methods such as intravenously, intramuscular, and subcutaneous and peritoneal injection feeds in the human body, and per daily dose is 5 milligrams to 10 milligrams of every kg body weight.

The present invention is described in more detail by following embodiment.Described embodiment only is used to illustrate the object of the invention, and should not be considered to be limited in strict the present invention who describes in claims.

Embodiment 1: prepared CpD-1 and determined its structure by the silkworm extract

According to conventional solvent-extraction process, from the silkworm extract, extract the chlorophyll metabolism thing with acetone.As a comparison, independently repeat identical extracting method with spinach with Sang Zi.

After the drying, the 0.1g crude extract is dissolved in the 1ml trimethyl carbinol, in the mixed solvent of the pentane of acetone (5: 60: 235V/V/V), obtain a supersaturated solution.Gained solution is put (20 * 20cm) (TLC plastic plate, Art, 5735, EM Science) on thin-layer chromatography (TLC) the plastics silica-gel plate according to a conventional method.After having put, the sample band is dry in nitrogen gas stream.Filter paper (25cm height) is placed in the TLC separation chamber (30 * 28 * 10cm), before launching, will pour in the separation chamber with the same solvent of above-mentioned dissolving usefulness in 20 minutes, be stamped the separation chamber of cover glass with the vacuum grease sealing.Developing time is about 40 minutes.All operations all carries out under dark or dim green glow.

The results are shown in shown in accompanying drawing 1A and the 1B.Referring to accompanying drawing 1A and 1B, for spinach and Sang Zi, in launching chromatogram second band (" being with 2 ") does not appear.In vivo test shows with 2 to have fabulous photosensitivity.

To be with 2 from the TLC plate, carefully to scrape, and acetone extraction.Elutriant evaporates in nitrogen gas stream then, preserves residue, so that be further purified.

Subsequently, according to the ordinary method reversed-phase HPLC, used instrument is ISCO type 2350 HPLC, 2360 type gradient sequencers, UV-5 extinction/fluorescence monitor (ISCO, Inc), and C-18 Dynamax-300

(1 * 25cm) post (Rainin Instrument Company, Inc.).The HPLC operational condition is as follows:

Flow velocity: 1.7ml/min,

Moving phase: 35% methyl alcohol and 65% acetonitrile,

Gradient: last 25 minutes to 100% moving phase from 80% moving phase and 20% water.

Use has absorbancy and is about 7.0(600nm) 200 μ l volume injected samples (about 25 μ g).Each operating time is set to about 80 minutes, and monitoring 660nm place's absorbancy also is stored in the microcomputer.Automatically collect each peak.Obtain P1, P2, P3 and four groups of peaks of R4 as describing among the accompanying drawing 2A-2C.

With reference to accompanying drawing 2A-2C, accompanying drawing 2A represents with 2 at the reversed-phase HPLC curve of fluorescence compartment lamp exposure after several hours; Accompanying drawing 2B represents with 2 at the reversed-phase HPLC curve of exposure after 5 minutes under the lamp; Accompanying drawing 2C represents unexposed with 2 reversed-phase HPLC curve, is CpD-1 corresponding to the main ingredient at the 3rd peak (P3), and after exposure under lamp, CpD-1 is oxidized to the isomer C pD-2 of CpD-1, corresponding to second peak (P2).

The high-purity C pD-1 that obtains thus is by utilizing FAB/MS, ¹H-peacekeeping bidimensional COZY NMRS, ¹³C NMR and FT-IR determine its structure.

Accompanying drawing 3A represents CpD-1 and chlorophyll a ¹H-NMR spectrum, wherein arrow is represented C in CpD-1 and the chlorophyll a respectively ₁₀-OH and C ₁₀The position of-H.The chemical shift of CpD-1 and chlorophyll a is listed in the following table 1.Accompanying drawing 3B represents CpD-1, with heavy water (D ₂O) blended CpD-1 and chlorophyll a ¹H-NMR spectrum.Accompanying drawing 3C and accompanying drawing 3D represent CpD-1's respectively ¹³C-NMR spectrum and CpD-1's ¹H bidimensional COZY NMR spectrum.The FT-IR spectrum of accompanying drawing 4 expression CpD-1.

According to above-mentioned chart, can determine the structure of CpD-1, wherein the Mg in the chlorophyll a ⁺⁺Replaced C by two hydrogen atoms ₁₀Hydrogen atom is replaced by OH on the position.

Table 1

（δ，ppm）＊

¹H** chlorophyll a CpD-1

α 9.61 9.75

β 9.47 9.59

δ 8.63 8.75

2a 8.00 8.03

2b _t6.29 6.31

2b _*6.20 6.22

10-H/OH 6.27 5.57

P2 5.14 5.22

P1 4.49 4.56

8 4.44 4.53

7 4.24 4.20

4a 3.87 3.77

10b 3.87 3.76

3a 3.70 3.60

1a 3.41 3.44

5a 3.26 3.29

7a ₁2.64 2.95

7b ₁2.48 2.56

7a ₂2.34 2.26

7b ₂2.34 2.26

P4 1.88 1.91

4b 1.81 1.70

P3a 1.70 1.61

8a 1.57 1.10

P15 1.49 1.49

P(CH ₂) _*→1.24 1.24

P15a?&?P16 0.84 0.84

P7?&?P11 0.78 0.79

Note: * solvent for use: CDCl ₃

The * position is represented by the Fischer coding scheme.

Embodiment 2: synthetic CpD-1

The dry marine alga of about 40g (Spirulina platensis is available from Mexico City) is immersed in the 1L flask that contains 500ml methyl alcohol, to wherein adding 5ml0.01MKOH.Under dark situations, slowly blasted air 6 days to gained solution, refluxed 15 minutes at about 50 ℃ then.Gained solution is then poured 100ml methanol rinse residual substance into by filter paper filtering in the funnel.Filtrate was handled 3 minutes with the dense HCl of 1ml then.(Buchi, Switzerland) evaporation gained solution to volume is less than 30ml with the optically-active vaporizer.In the material that produces, add 200ml ddH then ₂O and 200mlCCl ₄After the separation, organic phase 200ml ddH ₂The O washed twice is abandoned water.Organic solvent is further removed by evaporation, and the gained resistates is dissolved in about 20mlCCl ₄In, obtain crude extract.

About 60g230 to 400 order silica gel (Sigma Chemical Co.) is immersed in CCl ₄In, sonic treatment is 5 minutes then.Pillar is filled with 2.5 internal diameters (cm) Glass tubing, and uses CCl ₄Pre-equilibration.Press similarity condition and fill second and the 3rd silicagel column.Be packed into after the crude extract, inject about 50ml CCl ₄Pink and the yellow substance of drip washing.Inject the CCl that contains 20% acetone then ₄Solution, wash-out 10-OH pheophytin a/the contain pheophytin a component of CpD.Abandon the residual substance in the post, evaporating solvent, residue are dissolved in 15ml CCl again ₄In be used for silica gel chromatography for the second time.After in sample is filled into new silicagel column, with about 50ml CCl ₄Wash-out is pink/yellow substance.Use the CCl of 10% acetone then ₄Wash-out target CpD part.Equally can not be by the CCl of 10% acetone ₄The surplus materials of wash-out abandons.Repeat solvent is steamed from the part of collecting, resistates is dissolved in 15ml CCl then ₄After will being filled in the 3rd silicagel column from the sample that second silica gel obtains; Inject earlier about 150ml(enough till) contain the CCl of 2% acetone ₄Solution, the non-target CpD(of wash-out utilize absorption spectrum to detect).Then will be up to the CCl that contains 5% acetone ₄Drips of solution is added to moving phase, and wash-out contains 10-OH pheophytin a part.Dry collected part and dark preservation under nitrogen atmosphere are so that utilize reversed-phase column chromatography to be further purified.Aforesaid operations carries out in Fume Hoods.

Preparation type C-18 reversed-phase column (Chromosorb LC-1, Sigma Chemical Co.) is filled in long (cm) adjustable Glass tubings in 2.2 internal diameters * 12 (Amicon).Elution program is as follows:

Moving phase: 50% ethanol, 40% acetonitrile, and 10%CCl ₄

Inject the about 250 μ g CpD of sample: 1ml

Flow velocity: 2ml/min

Detect: 660nm absorbs

Elution program: (1) from 99%M.P. and 1% water gradient elution 15 minutes to 100%M.P.

(2) the 100%M.P. wash-out is 13 minutes

(3) 20%CCl ₄Aqueous isopropanol wash-out 7 minutes

(4) 99%M.P. and 1% water elution are 20 minutes.

Inject back 28 minutes wash-outs and go out 10-OH pheophytin a part.Through the TLC assay determination, products therefrom purity is greater than 95%.For TLC, this product unfolded Rf value is identical with crude extract second band of silkworm metabolite.The TLC analysis condition is described identical with embodiment 1.

The contriver has measured TLC stratographic band 1 by determining NMR proton/carbon spectrum, through comparing with 10-OH pheophytin a and chlorophyll a, is defined as pheophytin a.The retention time of pheophytin a is longer than 10-OH pheophytin a on the C-18 post, and they can not be separated under above-mentioned elution requirement.The 10-OH pheophytin a product of dry gained is also stored in the dark under nitrogen atmosphere.Productive rate: more than or equal to 0.4%, calculate, suppose that chlorophyll a 100% is extracted with the dry weight seaweed powder, (in fact the percentage extraction yield of every batch of chlorophyll a is all changing), this productivity ratio is high 10 times from the CpD productive rate (0.04) that the crude extract of silkworm extract obtains.

Embodiment 3: the singlet oxygen productive rate

One of most important index of measuring PDT photosensitizers maximum efficiency is exactly that it is by shifting the ability that produces singlet oxygen from the triplet state photosensitizers to the molecular oxygen energy.There are two kinds of independent solutions can measure the quantum yield of singlet oxygen, see described method I of present embodiment and method II.Photosensitized oxidation method (method I) uses 1, and 3-phenylbenzene isobenzofuran (DPBF) is as the singlet oxygen trapping agent.According to the minimizing of DPBF fluorescence or absorbancy, measure photosensitized oxidation speed, thus the initial photosensitized oxidation speed of estimation DPBF in the presence of various photosensitizerss.Initial photosensitized oxidation speed is inversely proportional to the initial rate that produces singlet oxygen.

Method I: the photosensitized oxidation of relative DPBF

Use 632.8nm he-Ne laser (maximum output 20mw, Metrologic Instruments, Inc.) as source of radiation, excited sample solution.Use freshly prepd DPBF(recrystallization under nitrogen atmosphere in the dark) ethanolic soln, concentration is utilized 420nm, ε=22,500cm ^-1M ^-1Detect.In containing the sealing cuvette that total amount is a 2ml solution, various photosensitizerss almost have identical absorbancy at 632nm.To slowly blast in the solution through the pre-saturated oxygen of solvent, and last 20 minutes, the DPBF with a small amount of five equilibrium different concns mixes with solution then.Measure DPBF at 455nm(ethanol) and 465nm(CCl ₄) locate the relation with irradiation time of weakening of fluorescence intensity, determine the initial photosensitized oxidation speed of DPBF.

Method II: direct luminescent method

In order directly to detect singlet oxygen, measure near infrared phosphorescence.(Quantel YG-571-C), excites at the 355nm place to use Q-switch Nd:YAG laser apparatus.Silkworm CpD and chlorophyll a sample dissolution by being adjusted in 355nm place absorbancy to about 0.55, make sample concentration basic identical in the ethanol-OD of air saturation.

Intersect according to inverse, by 1/ speed to 1/[DPBF] coordinate diagram two reciprocal obtain maximum initial rate (accompanying drawing 5).The quantum yield of the singlet oxygen of chlorophyll a in ethanol (Φ △, X, ethanol) is by maximum initial rate, with respect to quantum yield (Φ △, ch1=0.57, the CCl of the singlet oxygen of known chlorophyll a in tetracol phenixin ₄), calculate by following formula (I):

φΔ,x=φΔ,chl (Achl·[RATEmax]x)/(Ax·[RATEmax]chl) (1)

Formula (1) also can be used for calculating the quantum yield of various photosensitizerss in ethanol.The results are shown in following table 2 and the accompanying drawing 5.According to these results, can determine that the singlet productive rate of CpD-1 is higher than chlorophyll a.CpD-1 demonstrates high 20% productive rate than HpD.

Table 2

Singlet oxygen productive rate (Φ △) is measured by relative DPBF photosensitized oxidation (method I) and straight line luminescent method (method II)

Method parameter chlorophyll a CpD-1 CpD-2

I φΔof 0.44 0.50 0.52

(DPBF) _ox

The relative I Δ 1.00 1.01 ± 0.05 1.09 ± 0.04 of II

τΔ(μsec) 30.5±0.8 31.6±0.4 28.9±1.1

Relative φ Δ 1.00 1.05 ± 0.06 1.16 ± 0.05

φΔ 0.44 0.46±0.03 0.51±0.02

The extinction ability of embodiment 4:CpD-1 in tumour and normal cell

Utilize human body T-4 lymphocyte series (MOLT-4) standard tumour cell as a comparison, freshly prepd human body peripheral blood lymphocyte (PBL) is the standard healthy cell as a comparison, the cell sorption of assessment CpD-1 and HpD.

To 1 * 10 ⁶Add the 1ml photosensitizers solution that contains 10 μ g/ml CpD-1 and HpD in the cell respectively, and in drum, cultivated 30 to 210 minutes.Culture is at 0 ℃, and 2, under the 000rpm centrifugal 5 minutes.The cell sheet that is settled out washs with cool physiological saline, and then centrifugal.In throw out, add 400 μ l DMF.Room temperature vibrate formed mixture and 4 ℃ centrifugal, get 300 μ l supernatant liquids and as cell extract.

In 300 μ l cell extracts, add 200 μ l distilled water and 500 μ l 1N hydrochloric acid, make it reach 1ml.Be determined at 660 and the CpD-1 at 550nm place and the absorbancy of HpD respectively.And the absorbancy comparison under presetted wavelength with measuring result and standardized solution, obtain the absorbed dose of test photosensitizers in cell.

The results are shown in shown in the accompanying drawing 6.From accompanying drawing 6, absorbed dose in cell increases with incubation time as can be seen, irrelevant with porphyrin and test cell category, absorbed dose is greater than the absorbed dose in the human body peripheral blood lymphocyte (PBL) of standard healthy cell as a comparison in MOLT-4.Using under the identical test cell, CpD shows bigger absorbancy than HpD.In sum, can determine preferentially to be absorbed at tumour cell position CpD-1, its locating effect is more than the high twice of HpD.

Embodiment 5: select to destroy tumour cell (in vitro tests)

Utilize with the foregoing description 2 in identical tumour and healthy cell, assess the selection destructiveness of CpD-1.

CpD-1 is injected into respectively contains 1 * 10 ⁶In the tumour cell and healthy cell of cell.The cell that to handle thus shone 10 minutes under ruddiness then.Photosensitizers as a comparison injects the repetition same steps as with HpD replaced C pD-1.Calculate the cell survival percentage of every interval in the time of 1 hour in 8 hours after the illumination immediately.The results are shown in shown in the accompanying drawing 7A.

Found after illumination, can be killed fully in 2 hours through the MOLT-4 that CpD-1 handles and crossed by red light irradiation, and the survival rate time of the PBL that handles and handled by CpD-1 and shone through CpD-1 increases and reduces, and 8 hours survival rates reach 69.4% after the illumination.Handled by HpD and MOLT-4 that red light irradiation is crossed after illumination 8 hours the time survival rate be 83.4%, and the PBL that is crossed by HpD processing and red light irradiation after illumination 8 hours the time survival rate be 91.4%.

Repeat above-mentioned same steps as, but replace red light irradiation MOLT-4 and PBL with yellow.The results are shown in shown in the accompanying drawing 7B.

The survival rate of MOLT-4 after being handled by CpD-1 increases in time and reduces, and survival rate was 2.7% when gold-tinted shone back 8 hours, and the PBL survival rate when gold-tinted shines back 8 hours after being handled by CpD-1 is 81.5%.MOLT-4 survival rate when gold-tinted shines back 8 hours after being handled by HpD is 58.2%, and the survival rate of PBL when gold-tinted shines back 8 hours after being handled by HpD is 83.3%.

These presentation of results CpD-1 demonstrates higher cytotoxicity at long wave (ruddiness) than shortwave (gold-tinted).With skin-deep anti-, HpD shows than high cell toxicity under gold-tinted than under ruddiness.Nonetheless, the cytotoxicity of HpD is still much smaller than CpD-1.In a word, with respect to CpD-1, HpD is very ineffective.

In sum, can determine that CpD-1 is better than HpD with regard to the selectivity tumor destruction.

Embodiment 6: to the result of treatment (in vitro tests) of mouse skin tumour

Female C3H/HeJ mouse (the about 20g of body weight) and BA mastocarcinoma are used for this experiment.By injecting 1 μ l BA mastocarcinoma sheet in the back flank of acceptor mouse, import subcutaneous tumors.When tumor growth was transplanted the back about 10 days to diameter 6-7mm(), use PDT and treat.

In this PDT therapeutic process, the 1st group that includes 12 mouse is injected into two kinds of various dose CpD, injects back 24 hours, with the rayed of two kinds of different light dosages.

From 12 mouse of the 1st group, take out 6, handle (5mg/Kg), used the 670nm rayed then 24 hours with 0.2ml liquid storage 2.In 6 mouse, wherein 3 total light dosages of acceptance are 100J/cm ², accept 300J/cm for other 3 ²Other 6 mouse are injected into more heavy dose of liquid storage 2(0.4ml) (10mg/Kg), and inject back 24 hours rayed at CpD.Equally, wherein 3 mouse are accepted 100J/cm ²Light dosage, other 3 mouse are accepted 300J/cm ²Like this, under the same conditions, per 3 mouse have constituted the PDT treatment time group that accepts.

In the tripping device of the 2nd group that forms by 12 mouse PDT treatment, use same PDT condition, but just inject beginning optical processing in 4 hours at CpD.The above results is listed in table 3 and the table 4 respectively.

Table 3

PDT treatment result when injecting behind the CpD-1 24 hours

The regrowth of CpD dosage light dosage mouse quantity toxicity is cured

(mg/Kg) (J/cm ²)

5 100 3 - 3 -

5 300 31 dead-2

10 100 3 - - 3

10 300 32 dead-1

Table 4

PDT treatment result when injecting behind the CpD-1 4 hours

The regrowth of CpD dosage light dosage mouse quantity toxicity is cured

(mg/Kg) (J/cm ²)

5 100 3 - 3 3

5 300 31 is dead, and 2-2

1 loses pin

10 100 33 dead--

10 300 33 dead--

Embodiment 7: to the result of treatment (in vitro tests) of malignant tumour

Inject by subcutaneous abdomen, the ICR mouse is used the S-180 cell, thereby induce the malignant tumour that produces the 5-10mm size.The S-180 cell obtains by the long-time sarcoma inducing cell system-Sarcoma 180 that cultivates in ICR mouse body.

To test mouse and be divided into two groups, each group is handled with CpD-1 and HpD respectively.Two groups after handling like this are divided into two, obtain total 4 groups.Two groups of rayed twice that have the wavelength of maximum absorbance with porphyrin in four groups wherein, each 10 minutes.Illumination is to carry out in the time of 24 hours then when treating back 1 hour with photosensitizers earlier.Two groups of rayed twice with identical wavelength of in four groups another, each 10 minutes, illumination was to carry out after 48 hours then when treating back 24 hours with photosensitizers earlier.

Earlier after injecting CpD-1 1 hour the time, the group of twice of illumination shows 100% curative ratio (cure quantity/always treat quantity=20/20) in the time of 24 hours then, and the group of handling with HpD shows 80% curative ratio (16/20).These presentation of results: compare with HpD, CpD-1 has high light sensitization ability (P＜0.025).

Equally, earlier after injecting CpD-1 24 hours the time, the group of twice of illumination shows 60% curative ratio (12/20) in the time of 48 hours then.And the group of handling with HpD shows 80% curative ratio (16/20).According to these results (P＞0.1), can infer that CpD-1 retention time in tumour cell is very short, therefore, patient's rayed once more arranged to what handle through CpD-1 even in the relatively short time, the effect of illumination for the second time is also very little.As for HpD, because HpD for the second time cytotoxic effect such as flush, edema and skin pain, the patient who stands optical dynamic therapy has to and light was isolated 24 hours at least.[referring to Photobiology of Porphyrins, In Doiron DR, Gomer CJ eds., Porphyrin Localization and treatment of tumors, New York, Alan R Liss Inc., 1984, pp.19-39].

Above-mentioned experimental result discloses CpD-1 and all is better than HpD in every respect, can be effective as the photosensitizers of treatment tumour cell.

Embodiment 8: to the swollen result of treatment (in vitro tests) of abdomen

Inject by subcutaneous abdomen, the ICR mouse is used the S-180 cell, induce abdomen swollen, and mouse is divided into five groups.Every group comprises 10 mouse.According to the mouse body weight with the growth of handling the time increase situation, assessment CpD-1 is to the swollen result of treatment of abdomen.

Inject S-180 after 6th day (first group), with CpD-1 handle respectively by the 13rd day (second group) and the 20th day (the 3rd group) for each group, and respectively after injecting CpD-1 24 hours then, rayed was three times when 48 hours and 72 hours, each 10 minutes.Handle but not illumination with CpD-1 for the 4th group.Handle without CpD-1 for the 5th group.

More than in 50 days time limit, if the weight increase of processed mouse and the 5th group is basic identical and still live, then mouse is considered to cure fully after above-mentioned processing.For first group, 8 mouse are cured fully.For second group, only there are 3 mouse to be cured fully, and remain 7 continuation growth death in 35 days owing to body weight.For the 3rd group, all mouse are dead because the continuation of body weight increases in 35 days.Equally, for the 4th group, all mouse are the death owing to increasing rapidly of body weight in 35 days, and its initial weight of weight ratio when mouse is dead nearly weighs twice.

Above-mentionedly work out explanation for less tumor locus, the clearance rate of tumour is higher.Can conclude that utilizing PDT method, CpD-1 to can be used for treating abdomen swells.

Embodiment 9: to the inhibition effect of the reversed transcriptive enzyme of retroviral

Five times of diluting solns that at first prepare infectivity Gross leukosis virus (GLV).Wherein GLV has leukemic C by cultivating from the patient in substratum ₃The TGV clone that obtains in the HeB/Fe mouse and obtaining (referring to Machala O, Bodyd R, Youn JK, and Barski G:Long-term in Vitro culture of pathogenic Gross Leukemia Virus in mouse thymusCells, Eur.J.Cancer 10,467-472,1974).Gained solution is handled with CpD-1, then rayed.Reverse transcriptase products is examined and determine by the toxicity of assessing virus.

GLV is inoculated into 1 * 10 ⁵On the normal mice fetus cells, and containing 10% penicillin (100 units per ml), cultivating 24 hours in the essential substratum of the Eagle bottom line of Streptomycin sulphate (100 μ g/ml) and foetal calf serum.Follow rayed after adding CpD-1, culture is with trypsin treatment and use trypan blue staining so that calculating survivaling cell.To grind with stain remover by the centrifugal resulting virion of ultra-high speed, and react with active mixing solutions, this solution has measurement ³H dTTP combines the ability of level with reversed transcriptive enzyme.The mixture that is produced, is washed to glass filter paper by point sample, and drying utilizes scintillometer to measure intrinsic specific activity.

Virus after CpD-1 processing and rayed is showed 5000cpm or littler radioactivity, the enzyme product is had remarkable effect (promptly reducing) or enzyme (reversed transcriptive enzyme) is had significant photodynamic action, and do not have CpD-1 to handle but demonstrate greater than 40 through the comparative group that rayed is crossed, the 000cpm radioactivity, equally, through CpD-1 handle and the identical virus of illumination to the infectivity attenuating of mouse fetus cells.

According to these results, can infer that CpD-1 has antiviral activity.

As mentioned above, 10-hydroxyl pheophytin a of the present invention and pharmacy acceptable salt demonstrate the photosensitivity of improvement and good tumour cell absorbability.In addition, The compounds of this invention is being better than other conventional photosensitizers, especially HpD aspect the throughput of selecting destruction of cancer cells and singlet oxygen.Therefore The compounds of this invention is adapted in the photodynamic therapy application as photosensitizers.

Claims

1, formula I 10-hydroxyl pheophytin a or its pharmacy acceptable salt.

2, the pheophytin a described in the preparation claim 1 or the method for its pharmacy acceptable salt comprise the steps:

(a) from the silkworm extract, extract the chlorophyll metabolism thing with appropriate solvent;

(b) substance dissolves that extraction is obtained is in appropriate solvent, to supersaturation;

(c) gained solution is gone out photosensitive component through chromatographic separation; And

(d) collect to show the quick agency part of high light, subsequently purifying according to a conventional method.

3, the pheophytin a described in the preparation claim 1 or the method for its pharmacy acceptable salt comprise the steps:

(a) under the room temperature, chlorophyll a and HCl are reacted in ethanol or chloroform, slough Mg ion wherein;

(b) under the room temperature, under the safety lamp of dimness or in the dark, in the solution of gained, slowly blasted air one day or the longer time, replace C with hydroxyl ₁₀Hydrogen atom on the position; And

(c) will generate product purifying according to a conventional method.

4, a kind of photosensitiser composition that is used for the optical dynamic therapy cancer, comprise as activeconstituents at the 10-hydroxyl pheophytin a described in the claim 1 or its pharmacy acceptable salt, with mix with it or the conventional component of bonded such as carrier, vehicle, swelling agent and other additive.

5, a kind of by giving in the body with the 10-carbonyl pheophytin a in the claim 1 or the optical dynamic therapy method for cancer of pharmacy acceptable salt.