CN105288646A - Photosensitizer phospholipid compound as well as pharmaceutical composition and application of photosensitizer phospholipid compound - Google Patents
Photosensitizer phospholipid compound as well as pharmaceutical composition and application of photosensitizer phospholipid compound Download PDFInfo
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- CN105288646A CN105288646A CN201510650463.7A CN201510650463A CN105288646A CN 105288646 A CN105288646 A CN 105288646A CN 201510650463 A CN201510650463 A CN 201510650463A CN 105288646 A CN105288646 A CN 105288646A
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Abstract
The invention discloses a photosensitizer phospholipid compound, a preparation method of the photosensitizer phospholipid compound, a pharmaceutical composition and application of the photosensitizer phospholipid compound. The pharmaceutical composition is the photosensitizer phospholipid or a combined pharmaceutical composition of the photosensitizer phospholipid and a pharmacologically acceptable carrier, and the pharmaceutical composition is in the form of a liquid preparation, a solid preparation, a semisolid preparation, a capsule, granules, a gel or an injection. The photosensitizer phospholipid compound or a liposome of the photosensitizer phospholipid is applicable to the photodynamic therapy of tumors; the photosensitizer phospholipid compound or the liposome of the photosensitizer phospholipid compound can be rapidly taken by cells and can release a photosensitizer crude drug, and under laser action, the photosensitizer phospholipid compound or the liposome of the photosensitizer phospholipid compound can rapidly release active oxygen to take a strong anti-tumor effect. The photosensitizer phospholipid compound and a liposome nanoparticle thereof can serve as a liquid preparation, a solid preparation, a semisolid preparation, a sterilized preparation and a sterile preparation; and the photosensitizer phospholipid compound and the liposome nanoparticle are low in toxicity and are applicable to the efficient treatment of various tumors.
Description
Technical field
The present invention be a kind of there is antitumor action the photosensitizer phosphatide cpd for optical dynamic therapy and pharmaceutical composition and purposes, relate to medical art.
Background technology
Photodynamic therapy is the novel tumor Therapeutic Method grown up after operation, radiotherapy, chemotherapy and immunization therapy.Its principle is, by injecting photosensitizer in tumor patient body, utilize tumor cell to the absorption of photosensitizer and delay effect, tumor locus is irradiated with the laser of specific wavelength, under the participation of molecular oxygen in biological tissues, bring out strong photochemical reaction, produce very active singlet oxygen and free radical isoreactivity oxygen composition, amino-oxide group acid, unsaturated fatty acid, the multiple biomacromolecule such as adenosine, generate and there is chemically active photooxidation secondary intermediate product in a large number, thus destruction albumen, lipid, the cellular component that nucleic acid etc. are important, cause major injury and the dysfunction of many organelles, finally cause tumor cell dead because of irreversible damage, to reach therapeutic purposes.Photosensitizer is the key element of photodynamic therapy, relevant with its stability, tissue selectivity, absorption spectrum and activity etc.
Mixing Porphyrin-Based Sensitizer is first generation photosensitizer, although determined curative effect in oncotherapy, but still there is many deficiencies, as poor in complicated component, tissue selectivity, lucifuge time length, HONGGUANG (630nm) absorption difference, the treatment degree of depth cause skin allergy etc. not, easily, become the chief culprit causing normal structure generation photosensitivity reaction.Second filial generation photosensitizer is mostly monomeric compound, at present for clinical treatment or carrying out having of clinical trial: Verteporfm, Temoporfin, Photrex, Motexafin, 5-ALA, Hexvix, hematoporphyrin monomethyl ether and hypocrellin etc.Their chemical constitution is clear and definite, and singlet oxygen productive rate is high, and the photosensitive phase is short, and maximum absorption wavelength red shift, photolytic activity, absorption spectrum and tissue selectivity are improved than first generation photosensitizer, but distribution is wide in body tissue, and dark toxicity is obvious.
Dark toxicity is very important index for optical dynamic therapy, and it can not have any impact to human body with laser pre-irradiation after ensureing that medicine injection itself enters human body.The dark toxicity of photosensitizer is lower, and its safety is better.Therefore, by structural modification, reducing dark toxicity is important problem clinically.By the combination such as antibody or part corresponding to the antigen of photosensitizer and aminoacid, polymer, protein, saccharide, expression of tumor tissue, receptor, build a kind of " the biological missile type photosensitive drug " that not only there is tumor targeting location but also optical dynamic therapy effect can be played, curative effect can be improved and reduce side effect, strengthen tissue selectivity and the targeting of medicine, reduce dark toxicity, improve phototoxicity, strengthen therapeutic effect.But, the product with premium properties of clinical practice so far or rare.
Liposome is a kind of new drug carrier with release function, hydrophobic drug usually in two lipid layers of liposome, in the aqueous phase of hydrophilic medicament in liposome.Hydrophobic photosensitizer also can load in liposome.The liposomal form medicament that namely fast Da Er is Verteporfin tieed up by photosensitizer as conventional clinically, and expensive.But, due to the mobility of liposome membrane, cause medicine to be easy to leak out, make the medicine of parcel be difficult to play good drug effect, and tissue distribution be extensive, cause certain dark toxicity.Therefore, be necessary the defect overcoming existing photosensitizer, the photosensitizer of development of new, improve optical dynamic therapy effect.
The present invention utilizes the photosensitizer of 2 molecules to be connected with the spacerarm of easily degrade respectively to form pair hydrophobic tail, and is connected by covalent bond with phospholipid hydrophilic head, prepares phosphatide cpd, can improve the dissolubility of photosensitizer; This photosensitizer phosphatide cpd separately or can be assembled into elaioplast nanometer particle together with auxiliary agent, is easy to by cellular uptake, reduces dark toxicity, for oncotherapy; Photosensitizer phosphatide cpd of the present invention and liposome thereof, through the degraded such as enzyme, glutathion, discharge high concentration photosensitizer fast, have strong phototoxicity under light illumination, be beneficial to photodynamic tumor-treatment.
Summary of the invention
Technical problem: the invention provides and a kind of there is lower cytotoxicity, dark toxicity and stronger phototoxicity, improve medicine parcel efficiency, photodynamic tumor-treatment better effects if, there is the photosensitizer phosphatide cpd of target function, based on the pharmaceutical composition of this photosensitizer phosphatide cpd, and this photosensitizer phosphatide cpd is preparing the application in anti-tumor photosensitizer medicine.
Technical scheme: photosensitizer phosphatide cpd of the present invention is the pharmaceutically acceptable salt that the compound of following structural (1) or the compound of described structural formula (1) and counter ion counterionsl gegenions are formed:
In formula (1), X is spacerarm, to be carbon number be 2 ~ 30 the alkylene alkyl containing ehter bond, the carbon number oxygen base alkylene alkyl that is 1 ~ 30, the carbon number alkylene alkyl containing cystine linkage-S-S-that is 2 ~ 30, the carbon number alkylene alkyl containing peptide bond that is 2 ~ 80, the carbon number alkylene alkyl containing ester bond that is 2 ~ 30, carbon number be 2 ~ 30 the alkylene alkyl containing hydrazone key or carbon number be 2 ~ 30 not containing heteroatomic alkylene alkyl/sub-alkylene; Y is the photosensitizer be connected with X by ester bond, amido link, amino-formate bond or hydrazone key; L is 2-amino-ethyl, 2-trimethyl amido ethyl cation, 2, the end group of 3-dihydroxypropyl, molecular weight to be the Polyethylene Glycol-amino-ethyl of the N-without targeting end group of 200-4000 or molecular weight be 200-4000 is the N-Polyethylene Glycol-amino-ethyl of targeting group.
Further, in photosensitizer phosphatide cpd of the present invention, photosensitizer Y be following any one: Verteporfin, benzene derivatives of porphyrin monocyclic acids A, amino-laevulic acid methyl ester, photofrin, hematoporphyrin derivative, biporphin ether, phytochrome II, the non-nurse sodium of porphin, phototherapy element, light spirit element, compound numbers is the photosensitizer of haematodrex, ProtoporphyrinIX, cancer light quinoline, amino-laevulic acid, the own ester of amino-laevulic acid, amino-laevulic acid phenyl ester, m-tetrahydroxy chlorin, chlorin e 6, photosensitizer chlorin e 6-C15 mono-methyl, single asparagine chlorin e 6, first alizarinopurpurin, octaethyl alizarinopurpurin, the former alizarinopurpurin of stannum, stannum octaethyl alizarinopurpurin, alizarinopurpurin 18, 1,2,4-trihydroxyanthraquinone, alizarinopurpurin, sun-proof purple LB, hematoporphyrin monomethyl ether, hypocrellin, diphenyl porphin, compound numbers is the photosensitizer of BCPD-17, happiness pool point, m-THPC, hemporfin, compound numbers is the photosensitizer of WST09, compound numbers is the photosensitizer of CGP55847, talaporfin, good fortune contest because of, Sorafenib, light clo, phthalein blue or green-4, motexafin lutecium, hypericin, deuteroporphyrin, thiophene furan quinoline, different porphyrin, compound numbers is the photosensitizer of LuTex, compound numbers is the photosensitizer of Sapphyrin, compound numbers is the photosensitizer of Pentaphyrin, compound numbers is the photosensitizer of Platyrin, compound numbers is the photosensitizer of Porphycene, compound numbers is the photosensitizer of Conphycene, compound numbers is the photosensitizer of Hemiporphycene, compound numbers is the photosensitizer of Isoporphycene, compound numbers is the photosensitizer of N-confusedporphyrin, compound numbers is the photosensitizer of DoublyN-confusedporphyrin, compound numbers is the photosensitizer of 21-core-modifiedporphyrin, compound numbers is the photosensitizer of 21,23-core-modifiedporphyrin, 3
1, 3
2the many chlorins-13 of-two dehydrogenation-15-carbonyl sieve
1, 15
1-anhydride.
In the present invention, photosensitizer can also be phthalocyanines, fluorine boron two pyroles, porphyrin alkene class, phorbin, Bacteriochlorin class, get Ke Sa porphyrin, phthalocyanines, porphyrin, porphin class, Psoralens resistance, chalcogen pyrans drone salt form II type, rhodamine type type III, methylene flange-type IV type, Buddhist nun rowland type V-type.
Further, in photosensitizer phosphatide cpd of the present invention, end group is in the N-Polyethylene Glycol-amino-ethyl of targeting group, and targeting group is any one in folic acid, galactose, polypeptide, antibody, antibody fragment, hyaluronic acid, asialoglycoprotein, polysaccharide, nucleic acid and plan peptide.
Further, when in photosensitizer phosphatide cpd structural formula (1) of the present invention, L is uncharged group, described counter ion counterionsl gegenions are combinations of a kind of cation and a kind of anion, described cation be hydrion, sodium ion, potassium ion, calcium ion, iron ion, magnesium ion, ammonium ion, zinc ion any one; When in structural formula (1), L is positively charged group, described counter ion counterionsl gegenions are any one in hydrion, sodium ion, potassium ion, calcium ion, iron ion, magnesium ion, ammonium ion, zinc ion, and described anion is any one in chloride ion, sulfate ion, sulfate ion, nitrate ion, carboxylic acid ion, carbanion, bromide ion, phosphate anion, formate, acetate, citrate, lactate, fumaric acid radical, tartrate anion, gluconic acid radical ion.
The compounds of this invention can the form of isomer exist, and usually described " the compounds of this invention " comprises the isomer of this compound.Can be there is asymmetric center and have S configuration or R configuration in the compounds of this invention, the present invention includes the mixture of all possible stereoisomer and two or more isomers.
Pharmaceutical composition of the present invention comprises above-mentioned photosensitizer phosphatide cpd, or comprises acceptable carrier on above-mentioned photosensitizer phosphatide cpd and pharmacodynamics.
Pharmaceutical composition of the present invention can be liquid preparation, solid preparation, semi-solid preparation, capsule, granule, gel or injection.
Usual pharmaceutical composition of the present invention contains the compounds of this invention of 0.1-100 % by weight.
The compounds of this invention or the route of administration containing its pharmaceutical composition can be drug administration by injection.The compounds of this invention can be made ordinary preparation, also can be slow releasing preparation, controlled release preparation, targeting preparation and various particulate delivery system.
Further, pharmaceutical composition of the present invention is elaioplast nanometer particle, and grain diameter 10-1000 nanometer, also comprises auxiliary agent.
Further, pharmaceutical composition of the present invention, the auxiliary agent of use be phospholipid, Polyethylene Glycol phospholipid (molecular weight polyethylene glycol 400-3000), containing one or more in the Polyethylene Glycol phospholipid of targeting group or cholesterol.Targeting group is folic acid, galactose, polypeptide, antibody, antibody fragment, hyaluronic acid, asialoglycoprotein, polysaccharide, nucleic acid and plan peptide.
Pharmaceutical composition of the present invention is liquid preparation, solid preparation, semi-solid preparation, capsule, granule, gel, injection, slow releasing preparation or controlled release preparation.
The preparation method of photosensitizer phosphatide cpd elaioplast nanometer particle of the present invention, the mixture by the compounds of this invention photosensitizer phosphatide cpd or photosensitizer phosphatide cpd of the present invention and auxiliary agent, by method preparations such as film dispersion method, reverse phase evaporation, freeze-drying, ultrasonic dispersion, spray drying method, film squeezing and pressing method or high pressure homogenize.
The present invention utilizes the photosensitizer of 2 molecules to be connected by covalent bond with phospholipid hydrophilic head as hydrophobic tail with spacerarm, prepares photosensitizer phosphatide cpd, and dissolubility is significantly better than the former medicine of photosensitizer;
The present invention utilizes the photosensitizer of 2 molecules to be connected by spacerarm covalency with phospholipid hydrophilic head as hydrophobic tail with spacerarm, prepares photosensitizer phosphatide cpd, and under the effect such as polypeptidase, glutathion, fast degradation discharges the former medicine of high concentration photosensitizer; Photosensitizer phosphatide cpd is prepared into elaioplast nanometer particle by the present invention, has low dark toxicity, high light toxicity.
Beneficial effect: the present invention compared with prior art, has the following advantages:
Photosensitizer phosphatide cpd of the present invention is the compound of following structural (1), or the pharmaceutically acceptable salt that the compound of described structural formula (1) and counter ion counterionsl gegenions are formed:
Photosensitizer phosphatide cpd of the present invention, in structural formula (1), photosensitizer Y is connected with phospholipid hydrophilic head by spacerarm X, and improve the water solublity of photosensitizer, photosensitizer phosphatide cpd and liposome thereof have lower cytotoxicity;
Photosensitizer phosphatide cpd of the present invention is assembled into liposome, there is passive target effect or because containing targeting group, there is active targeting effect, be easy to by cellular uptake, cause photosensitizer distribution in the tissue more concentrated, overcome the extensively distribution and cause the defect of significantly dark toxicity in body of common photosensitizer, make photosensitizer phosphatide cpd and liposome thereof have lower dark toxicity when light is irradiated, there is better safety;
Photosensitizer phosphatide cpd of the present invention is assembled into liposome, there is passive target effect or because containing targeting group, there is active targeting effect, be easy to by cellular uptake, the distribution of photosensitizer in body is caused more to concentrate on target tissue, overcome common photosensitizer extensively distribution and defect of causing phototoxicity to reduce in body, make photosensitizer phosphatide cpd and liposome thereof have stronger phototoxicity when light is irradiated, there is better optical dynamic therapy effect;
Photosensitizer phosphatide cpd of the present invention, in structural formula (1), photosensitizer Y is connected with phospholipid hydrophilic head by spacerarm X, improve the water solublity of photosensitizer, this spacerarm X is fast fracture under glutathion or polypeptidase etc. exist, a part photosensitizer phosphatide cpd can discharge two molecular photoactive agent fast, in target cell, obtain high concentration photosensitizer, there is stronger phototoxicity under light illumination, cause more efficient optical dynamic therapy effect;
In photosensitizer phosphatide cpd of the present invention, photosensitizer Y and spacerarm X has collaborative hydrophobic interaction, assembling forms stable liposome, it is a part for liposome structure, when overcoming general liposome dewatering medicament, medicine is easy to the shortcoming of leaking, improve the efficiency of medicine parcel simultaneously, there is low cytotoxicity and dark toxicity;
Photosensitizer phosphatide cpd of the present invention is assembled into liposome, due to the protective effect of liposome, the photosensitizer being positioned at lipid layer is not subject to destruction, and photosensitizer active structure is kept very well before by cellular uptake, makes the photodynamic tumor-treatment better effects if of photosensitizer phosphatide cpd;
Photosensitizer phosphatide cpd of the present invention is assembled into elaioplast nanometer particle, is a kind of brand-new photosensitizer, is also a kind of carrier of photosensitizer;
Photosensitizer phosphatide cpd of the present invention is assembled into stable elaioplast nanometer particle, there is the membrane structure similar with cell, be easy to by cellular uptake, photosensitizer phosphatide cpd is made to enter in cell by liposomal form, and photosensitizer is discharged in cell, there is lower dark toxicity, stronger phototoxicity and more efficient optical dynamic therapy effect;
The compound system that photosensitizer phosphatide cpd of the present invention or itself and phospholipid auxiliary agent etc. form can adopt simple process, as membrane process etc. is assembled into elaioplast nanometer particle easily, and particle diameter 10-1000 nanometer;
The spacerarm X of photosensitizer phosphatide cpd of the present invention, by high concentration esterase, glutathion, polypeptidase or low ph value microenvironment fast degradation in tumor cell, discharges the active photosensitizer molecule of high concentration fast;
In photosensitizer phosphatide cpd structural formula (1) of the present invention, the end group of L to be molecular weight be 200-4000 is the N-Polyethylene Glycol-amino-ethyl of targeting group, the surface of liposome that this photosensitizer phosphatide cpd is assembled into has targeting group, has active targeting effect; The surface of liposome that photosensitizer phosphatide cpd of the present invention is assembled into altogether with the phospholipid containing targeting group has targeting group, has active targeting effect, has stronger phototoxicity and more excellent antineoplaston effect;
The pharmaceutical composition of compound of the present invention or compound of the present invention and Conventional pharmaceutical carriers, the compounds of this invention containing 0.1-100 % by weight, has excellent phototoxicity, has strong killing action to human tumor cells.
Accompanying drawing explanation
Fig. 1 is the synthetic route chart of two (methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol-succinate) phosphatidyl choline compounds
Fig. 2 is the synthetic route chart of two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine
Fig. 3 is the synthetic route chart of two (HPPH-GFLG) phosphatidylcholine
Fig. 4 is the synthetic route chart of two (Verteporfin-diethylene glycol-succinate) phosphatidylcholine
Fig. 5 is the synthetic route chart of two (Verteporfin-butyryl) phosphatidylcholine
Fig. 6 is the synthetic route chart of two (Verteporfin-diethylene glycol-succinyl) phosphatidyl glycerol
Fig. 7 is the synthetic route chart of two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE
Fig. 8 is the synthetic route chart of two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE-N-Polyethylene Glycol
Fig. 9 is the synthetic route chart of two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE-N-Polyethylene Glycol-folic acid compound
Figure 10 is the synthetic route chart of two (HPPH-dithio diethylene glycol-succinate) phosphatidyl choline compounds
Figure 11 is the particle size distribution schematic diagram of two (methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol-succinate) phosphatidylcholine liposome
The form (transmission electron microscope picture) of two (Verteporfin-dithio diethylene glycol-succinate) the phosphatidylcholine elaioplast nanometer particle of Figure 12
Figure 13. photosensitizer is to (a) of human breast cancer cell dark toxicity and (b) phototoxicity, vertical coordinate is cell survival rate (unit: %), and abscissa is concentration (unit: μM) " V " group: Visudyne Verteporfin liposome; " Lipo " two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome; " B ": blank group.
Figure 14. photosensitizer is to tumor-bearing mice tumor suppression experimental result: tumor-bearing mice divides 5 groups, often organize 6, random packet, be respectively administration light group " Lipo-1 " (2mg/kg), administration light group " Lipo-2 " (1mg/kg), contrast administration illumination " V " group (2mg/kg), blank " B " group (normal saline), a not administration irradiation " Light " group, administration Lipo not light group " Lipo-N " (2mg/kg).Abscissa is natural law (unit: day), and vertical coordinate is gross tumor volume (unit: mm
3).
Detailed description of the invention
Below in conjunction with Figure of description and embodiment, technical scheme of the present invention is described in further details.
Photosensitizer phosphatide cpd of the present invention is the compound of following structural (1), or the pharmaceutically acceptable salt that the compound of described structural formula (1) is formed with counter ion counterionsl gegenions respectively:
Photosensitizer Y is shown in technical scheme.Photosensitizer Y can also be phthalocyanines, fluorine boron two pyroles, porphyrin alkene class, phorbin, Bacteriochlorin class, get Ke Sa porphyrin, phthalocyanines, porphyrin, porphin class, Psoralens resistance, chalcogen pyrans drone salt form II type, rhodamine type type III, methylene flange-type IV type, Buddhist nun rowland type V-type.Spacerarm X is shown in technical scheme.The chemical constitution of group L is shown in technical scheme.
In the present invention, described counter ion counterionsl gegenions are a kind of cationes, or the combination of a kind of cation and a kind of anion.Cation is hydrion, sodium ion, potassium ion, calcium ion, iron ion, magnesium ion, ammonium ion, zinc ion, and anion is chloride ion, sulfate ion, sulfate ion, nitrate ion, carboxylic acid ion, carbanion, bromide ion, phosphate anion, formate, acetate, citrate, lactate, fumaric acid radical, tartrate anion, gluconic acid radical ion.
By the elaioplast nanometer particle having the photosensitizer phosphatide cpd of structural formula (1) structure and phospholipid or targeting phospholipid co-assemble and form particle diameter 10-1000 nanometer, targeting group is one or more in folic acid, galactose, polypeptide, antibody, antibody fragment, hyaluronic acid, asialoglycoprotein, polysaccharide, nucleic acid and plan peptide.
Photosensitizer phosphatide cpd of the present invention, preparing the application in antitumor drug by described photosensitizer phosphatide cpd or its pharmaceutically acceptable salt, is prepared into medicament with acceptable carrier on pharmacodynamics.The compounds of this invention or the compounds of this invention and one or more solids or liquid pharmaceutical excipients and/or adjuvant can be combined, make and can be used as the suitable administration form or dosage form that people's medicine uses.Usual pharmaceutical composition of the present invention contains the compounds of this invention of 0.1-100 % by weight.
The compounds of this invention or the pharmaceutical composition containing it can be liquid dosage form, solid dosage forms, semi-solid preparation, capsule, granule, gel, injection, slow releasing preparation or controlled release preparation.The compounds of this invention can be made ordinary preparation, also can be slow releasing preparation, controlled release preparation, targeting preparation and various particulate delivery system.
Pharmaceutical composition of the present invention is the elaioplast nanometer particle of particle diameter 10-1000 nanometer, is made up of the photosensitizer phosphatide cpd self assembly with general formula (1) structure.Elaioplast nanometer particle is made, particle diameter 10-1000 nanometer by the compounds of this invention and auxiliary agent.Auxiliary agent is one or more in phospholipid, pegylated phospholipids (molecular weight polyethylene glycol 200-4000), targeting pegylated phospholipids (molecular weight polyethylene glycol 200-4000) cholesterol, and targeting group is one or more of folic acid, galactose, polypeptide, antibody, antibody fragment, hyaluronic acid, asialoglycoprotein, polysaccharide, nucleic acid and plan peptide.
The present invention utilizes the photosensitizer of 2 molecules to be connected with spacerarm to be connected by covalent bond with phospholipid hydrophilic head as hydrophobic tail, and prepare photosensitizer phosphatide cpd, dissolubility is significantly better than photosensitizer.
Photosensitizer phosphatide cpd is prepared into nano-particle by the present invention, has the characteristic of liposome, can form liquid preparation, solid preparation, for photodynamic therapy.
Photosensitizer phosphatide cpd of the present invention, its spacerarm contains disulfide bond, hydrazone key, ehter bond, peptide bond, ester bond, under glutathion or polypeptidase etc. exist, fast fracture, discharge active medicine hydroxy camptothecin or its analog fast, a part photosensitizer phosphatide cpd discharges the photosensitizer of two molecules fast, forms high concentration photosensitizer in cell, has high phototoxicity.
In photosensitizer phosphatide cpd formula (1) of the present invention, photosensitizer and the combination of hydrophobic spacerarm realize excellent collaborative hydrophobic interaction, form stable liposome, play synergistic stability effect, when overcoming general liposome dewatering medicament, medicine is easy to the shortcoming of leaking, improve the efficiency of medicine parcel, make photosensitizer phosphatide cpd take in cell easily through liposomal form simultaneously.
Photosensitizer phosphatide cpd and auxiliary agent are prepared into nano-particle by the present invention, have the characteristic of liposome, have the characteristic that can form liquid preparation, solid preparation, semi-solid preparation, sterilization preparation and sterile preparation, for photodynamic therapy.
The preparation method of photosensitizer phosphatide cpd elaioplast nanometer particle of the present invention, the mixture by the compounds of this invention photosensitizer phosphatide cpd or the compounds of this invention and auxiliary agent, by method preparations such as film dispersion method, reverse phase evaporation, freeze-drying, ultrasonic dispersion, spray drying method, film squeezing and pressing method or high pressure homogenization methods.
From toxicity screening, compared with photosensitizer, the phototoxicity that the performance of the compounds of this invention photosensitizer phosphatide cpd is more excellent, extremely low dark toxicity.From toxicity screening, compared with photosensitizer, the phototoxicity that the performance of the compounds of this invention photosensitizer phosphatide cpd liposome is excellent, extremely low dark toxicity.
Portion of reagent code name below for using in preparation process:
DMAP4-dimethylamino naphthyridine
CDIN, N '-carbonyl dimidazoles
DMSO dimethyl sulfoxine
GPC Phosphorylcholine glycerol
DBU1,5-diazabicylo [5.4.0] 11-5-alkene
EDCIN, N '-thio-carbonyldiimidazole
EDC1-ethyl-(3-dimethylaminopropyl) carbodiimide
NHSN-N-Hydroxysuccinimide
(BOC)
2o Bis(tert-butoxycarbonyl)oxide
TFA trifluoroacetic acid
DMF dimethyl formamide
DMSO dimethyl sulfoxine
BOC tertbutyloxycarbonyl
TEA triethylamine
GFLG glycyl-phenylalanyl-leucyl-glycine
Followingly further illustrate the present invention by embodiment, but the invention is not restricted to following examples.
Embodiment 1
The synthesis (synthetic route is shown in Fig. 1) of two (methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol-succinate) phosphatidyl choline compounds
Amino-laevulic acid methyl ester 1g is dissolved in 20mL chloroform; add triethylamine 0.6g; triphosgene 0.3g, reacts 6h under room temperature, revolves steaming and desolventizes; solid is dissolved in 10mLDMSO; add triethylamine 0.3g, add dithio diethylene glycol 0.6g, room temperature reaction 24h; gained reactant liquor, by column chromatography purification, obtains methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol 0.31g.Methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol, succinic anhydrides 2g is dissolved in 30mL pyridine, 40 DEG C of reaction 48h; Revolve steaming to desolventize, separate out precipitation in cold diethyl ether, dilute hydrochloric acid washs, and obtains intermediate product methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol monomester succinate 0.81g.Intermediate product methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol monomester succinate 0.8g is dissolved in 20mLDMSO; add CDI0.6g; activation 1h; add GPC0.4g and DBU0.6g; room temperature reaction 24h; gained reactant liquor, by column chromatography purification, obtains two (methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol-succinate) the phosphatidylcholine 0.56g of product.
1HNMR(500MHz，CD
3OD∶CDCl
31∶1)：δ4.64-4.12(15H，m)，3.97-3.43(12H，m)，3.30(9H，s)，2.84-2.53(24H，m)。[M+H]
+m/z，1073.13。
Two (methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol-succinate) phosphatidylcholine is dissolved in the sodium-chloride water solution of 0.01M, and lyophilizing obtains two (methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol-succinate) the phosphatidylcholine white solid powder containing sodium ion and chloride ion.
Embodiment 2
The synthesis (synthetic route is shown in Fig. 2) of two (Verteporfin-dithio diethylene glycol-succinate) phosphatidyl choline compounds
Verteporfin (Verteporfin) 1g is dissolved in 20mL chloroform, adds CDI0.5g, activation 1h, add dithio diethylene glycol 0.6g, DBU0.5g, room temperature reaction 24h, gained reactant liquor, by column chromatography purification, obtains Verteporfin-dithio diethylene glycol monoesters 0.31g.Verteporfin-dithio diethylene glycol monoesters, succinic anhydrides 2g is dissolved in 30mL pyridine, 40 DEG C of reaction 48h; Revolve steaming to desolventize, separate out precipitation in cold diethyl ether, dilute hydrochloric acid washs, and obtains intermediate product Verteporfin-dithio diethylene glycol monomester succinate 0.81g.Intermediate product Verteporfin-dithio diethylene glycol monomester succinate 0.8g, be dissolved in 20mLDMSO, add CDI0.8g, activation 1h, add GPC0.4g and DBU0.8g, room temperature reaction 24h, gained reactant liquor, by column chromatography purification, obtains two (Verteporfin-dithio diethylene glycol-succinate) the phosphatidylcholine 0.51g of product.
1HNMR(500MHz，CD
3OD∶CDCl
31∶1)：δ7.0-6.49(8H，m)，5.9-5.2(8H，m)，4.64-4.31(15H，m)，3.97-3.43(24H，m)，3.30(9H，s)，2.84-2.10(38H，m)，1.71-1.65(12H，d)。[M+H]
+m/z，2132.39。
Two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine is dissolved in the potassium chloride solution of 0.01M, and lyophilizing obtains two (Verteporfin-dithio diethylene glycol-succinate) the phosphatidylcholine white solid powder containing potassium ion and chloride ion.
Embodiment 3:
The synthesis (synthetic route is shown in Fig. 3) of two (HPPH-GFLG) phosphatidyl choline compounds
Light clo (photochlor, HPPH) 1g is dissolved in 20mLDMF, add 0.5gCDI and 0.5gTEA, activation 1h, then N-glycyl-phenylalanyl-leucyl-glycine (GFLG) 0.5g is added, room temperature reaction 24h, reactant liquor pillar layer separation, obtains intermediate product HPPH-GFLG0.65g.Product is dissolved in 15mL dimethyl sulfoxine, adds CDI0.6g, adds GPC0.3g and DBU0.6g, room temperature reaction 24h, separates out precipitation in cold diethyl ether, and column chromatography for separation obtains two (HPPH-GFLG) phosphatidylcholine 0.45g.
1HNMR(500MHz，CD
3OD∶CDCl
31∶1)：δ8.01，7.21，7.12，7.08，4.92，4.64，4.53，4.32，4.16，4.09，3.97，3.77，3.70，3.43，3.37，3.30，3.07，3.05，2.24，2.22，2.05，1.83，1.75，1.71，1.46，1.33，1.31，1.29，1.01，0.96。[M+H]
+m/z，2244.71。
Two (HPPH-GFLG) phosphatidylcholine is dissolved in the dibastic sodium phosphate aqueous solution of 0.01M, and lyophilizing obtains two (HPPH-GFLG) phosphatidylcholine white solid powder containing sodium ion and phosphate anion.
Embodiment 4
The synthesis (synthetic route is shown in Fig. 4) of two (Verteporfin-diethylene glycol-succinate) phosphatidylcholine
Verteporfin 1g is dissolved in 15mLDMSO, adds CDI0.8g, adds diethylene glycol 0.4g and DBU0.8g, room temperature reaction 24h, and gained reactant liquor obtains Verteporfin-diethylene glycol monoesters 0.65g by column chromatography purification.Verteporfin-diethylene glycol monoesters 0.4g is dissolved in 20mL pyridine, succinic anhydrides 2g, 40 DEG C of reaction 48h; Revolve steaming to desolventize, separate out precipitation in cold diethyl ether, dilute hydrochloric acid washs, and obtains intermediate product Verteporfin-diethylene glycol-monomester succinate 0.81g.Intermediate product 0.8g is dissolved in 20mLDMSO, adds CDI0.6g, activation 1h, add GPC0.4g and DBU0.6g, room temperature reaction 24h, gained reactant liquor, by column chromatography purification, obtains two (Verteporfin-diethylene glycol-succinate) the phosphatidylcholine 0.56g of product.
1HNMR(500MHz，CD
3OD∶CDCl
31∶1)：δ6.9，6.7，6.49，6.3，5.9，5.3，5.27，5.20，4.64，4.32，4.25，3.97，3.77，3.67，3.65，3.43，3.30，2.82，2.64，2.52，2.35，2.29，2.05，1.71，1.36。[M+H]
+m/z，2036.13。
Two (Verteporfin-diethylene glycol-succinate) phosphatidylcholine is dissolved in the aqueous sodium persulfate solution of 0.02M, and lyophilizing obtains two (Verteporfin-diethylene glycol-succinate) the phosphatidylcholine white solid powder containing sodium ion and sulfate ion.
Embodiment 5:
The synthesis (route is shown in Fig. 5) of two (Verteporfin-butyryl) phosphatidylcholine
Hydroxybutyric acid 1g is dissolved in 15mL chloroform, adds triethylamine 0.5g, drips tertiary butyl dimethylchlorosilane 0.6g, reaction 2h, revolves steaming and desolventizes, add the anhydrous DMSO of 20mL, CDI0.6g, activation 1h, adds GPC0.4g and DBU0.6g, room temperature reaction 24h, precipitation is separated out in cold diethyl ether, process 2h at adding acetic acid 50 DEG C, gained reactant liquor, by column chromatography purification, obtains two (4-maloyl group) phosphatidylcholine 0.6g.
Verteporfin 1g is dissolved in 15mLDMSO, add CDI0.8g, add two (4-maloyl group) phosphatidylcholine 0.4g and DBU0.8g, room temperature reaction 24h, gained reactant liquor obtains two (Verteporfin-butyryl) phosphatidylcholine 0.56g by column chromatography purification.
1HNMR(500MHz，CD
3OD∶CDCl
31∶1)：δ6.9，6.7，6.49，6.3，5.9，5.3，5.27，5.20，4.64，4.32，4.25，4.08，3.97，3.77，3.67，3.65，3.43，3.30，2.82，2.64，2.52，2.35，2.29，2.05，1.96，1.71，1.36。[M+H]
+m/z，1831.95。
Embodiment 6:
The synthesis (synthetic route is shown in Fig. 6) of two (Verteporfin-diethylene glycol-succinyl) phosphatidyl glycerol
Prepared by the method for embodiment 4 intermediate product Verteporfin-diethylene glycol-monomester succinate 1g, CDI0.5g are dissolved in 20mLDMSO, room temperature reaction 1h, adds 3-(4-methoxyl group benzyloxy) propane-1,2-glycol 0.5g, DBU0.5g, room temperature reaction 12h, revolves solvent evaporated; Add spirit of vinegar, logical hydrogen under Pd/C catalyst exists, room temperature reaction 12h, except desolventizing, through pillar layer separation, obtain two (Verteporfin-diethylene glycol-succinyl) the glyceride 0.78g of intermediate product.
Get two (Verteporfin-diethylene glycol-succinyl) the glyceride 0.4g of intermediate product to be dissolved in 20mL chloroform, add phosphorus oxychloride 0.6g, triethylamine 0.8g, 10 DEG C of reaction 4h, revolve solvent evaporated; Add anhydrous glycerol contracting acetone 0.6g, triethylamine 0.3g, chloroform is reaction dissolvent, and 10 DEG C of reaction 24h, revolve solvent evaporated; Add isopropyl alcohol, aqueous acetic acid, 50 DEG C of reaction 24h, reactant liquor, by column chromatography purification, obtains two (Verteporfin-diethylene glycol-succinyl) the phosphatidyl glycerol compound 0.31g of product.
1HNMR(500MHz，CD
3OD∶CDCl
31∶1)：δ6.9，6.7，6.49，6.3，5.9，5.3，5.27，5.20，4.64，4.32，4.25，4.08，3.97，3.77，3.68，3.65，3.39，2.82，2.64，2.52，2.35，2.29，2.05，1.71，1.36。[M+H]
+m/z，2025.06。
Two (Verteporfin-diethylene glycol-succinyl) phosphatidyl glycerol is dissolved in the sodium hydrate aqueous solution of 0.01M, and lyophilizing obtains the phosphatidyl glycerol pressed powder containing counter ion counterionsl gegenions sodium ion.
Embodiment 7:
The synthesis (synthetic route is shown in Fig. 7) of two (Verteporfin-diethylene glycol-succinyl) phosphatidyl ethanol amines
Producing two (Verteporfin-diglycol-succinyl) glyceride 0.3g in the middle of in embodiment 6 is dissolved in 10mLDMSO, adds phosphorus oxychloride 0.5g, triethylamine 1.0g, and 10 DEG C of reaction 24h, revolve solvent evaporated; Adding 10mL chloroform is reaction dissolvent, adds N-t-butoxycarbonyl-amino ethanol 0.6g, triethylamine 0.6g, 10 DEG C of reaction 24h, and revolve solvent evaporated, pillar layer separation obtains crude product; Drip trifluoroacetic acid TFA5mL, room temperature reaction 3h.Gained reactant liquor, by column chromatography purification, obtains two (Verteporfin-diglycol-succinyl) the phosphatidyl ethanol amines 0.21g of product.
1HNMR(500MHz，CD
3OD∶CDCl
31∶1)：δ6.9，6.7，6.49，6.3，5.9，5.3，5.27，5.20，4.64，4.32，4.27，4.25，4.08，3.97，3.77，3.68，3.65，3.52，2.82，2.64，2.52，2.35，2.29，2.05，1.71，1.36。[M+H]
+m/z，1994.05。
Embodiment 8:
The synthesis (synthetic route is shown in Fig. 8) of two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE N-Polyethylene Glycol
Get mPEG (terminal hydroxy group poly glycol monomethyl ether, mean molecule quantity 2000) 0.5g is dissolved in 20mL chloroform, add CDI0.2g, activation 1h, add two (Verteporfin-diethylene glycol-succinyl) phosphatidyl ethanol amines 0.1g, DBU0.2g, the room temperature reaction 24h in embodiment 7, reactant liquor, by column chromatography purification, obtains two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE-N-Polyethylene Glycol 0.08g of product.
1HNMR(500MHz，CD
3OD∶CDCl
31∶1)：δ6.9，6.7，6.49，6.3，5.9，5.3，5.27，5.20，4.64，4.32，4.27，4.25，4.08，3.79，3.77，3.68，3.65，3.52，3.24，3.15，2.82，2.64，2.52，2.35，2.29，2.05，1.71，1.36。
Embodiment 9:
The synthesis (synthetic route is shown in Fig. 9) of two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE-N-Polyethylene Glycol-folic acid compound
Terminal hydroxy group-N-BOC-amino-polyethyleneglycols (mean molecule quantity 2000) 0.5g is dissolved in 20mL chloroform, add CDI0.2g, activate after 1 hour, revolve and steam except desolventizing, add DMSO10mL, DBU0.2g, add two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE 0.1g of embodiment 8, room temperature reaction 12 hours, pillar layer separation; Then, remove polyoxyethylene end group protecting group BOC with TFA process, carry out purification by column chromatography process, obtaining end group is amino two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE-N-Polyethylene Glycol.Product joins in 5mLDMSO, adds 0.2gCDI and 0.2gTEA, activation 1h, add folic acid 0.3g, room temperature reaction 24 hours, reactant liquor, through column chromatography, obtains two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE-N-Polyethylene Glycol-folic acid 0.06g of product.
1HNMR(500MHz，CD
3OD∶CDCl
31∶1)：δ8.57，8.02，7.73，6.9，6.7，6.61，6.49，6.3，5.9，5.3，5.27，5.20，4.64，4.46，4.32，4.27，4.25，4.08，3.79，3.77，3.68，3.65，3.52，3.24，3.15，2.82，2.64，2.52，2.35，2.29，2.18，2.10，2.05，1.71，1.36。
Embodiment 10:
The synthesis (synthetic route is shown in Figure 10) of two (HPPH-dithio diethylene glycol-succinate) phosphatidyl choline compounds
Light clo (photochlor, HPPH) 1g is dissolved in 20mL chloroform, adds CDI0.5g, activation 1h, adds dithio diethylene glycol 0.6g, DBU0.5g, room temperature reaction 24h, gained reactant liquor, by column chromatography purification, obtains HPPH-dithio diethylene glycol monoesters 0.31g.HPPH-dithio diethylene glycol monoesters, succinic anhydrides 2g is dissolved in 30mL pyridine, 40 DEG C of reaction 48h; Revolve steaming to desolventize, separate out precipitation in cold diethyl ether, dilute hydrochloric acid washs, and obtains intermediate product HPPH-dithio diethylene glycol monomester succinate 0.81g.Intermediate product HPPH-dithio diethylene glycol monomester succinate 0.8g, be dissolved in 20mLDMSO, add CDI0.8g, activation 1h, add GPC0.4g and DBU0.8g, room temperature reaction 24h, gained reactant liquor, by column chromatography purification, obtains two (HPPH-dithio diethylene glycol-succinate) the phosphatidylcholine 0.51g of product.
1HNMR(500MHz，CD
3OD∶CDCl
31∶1)：δ6.4，4.92，4.64，5.01，4.53，4.38，4.32，4.16，4.09，3.97，3.77，3.70，3.43，3.37，3.30，3.07，2.84，2.64，2.35，2.29，2.05，1.83，1.75，1.71，1.46，1.33，1.31，1.29，1.01，0.96。[M+H]
+m/z，1984.49。
Embodiment 11:
The preparation of two (methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol-succinate) phosphatidylcholine liposome
Two (methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol-succinate) phosphatidylcholine 1mmol of Example 1, add chloroform 20mL, 60 DEG C revolve solvent evaporated; Add 20mLPBS (pH=7.4) 60 DEG C of skinnings, 200nm membrane filtration, obtain two (methoxycarbonyl propiono methylene carbamate-dithio diethylene glycol-succinate) phosphatidylcholine elaioplast nanometer particle solution.The following Figure 11 of granularmetric analysis result, mean diameter 330nm.
Embodiment 12:
The preparation of two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome
Two (Verteporfin-dithio diethylene glycol-succinate) the phosphatidylcholine 1mmol obtained by embodiment 2, add chloroform 20mL, 60 DEG C revolve solvent evaporated; Add 20mLPBS (pH=7.4) 60 DEG C of skinnings, 200nm membrane filtration, obtain two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine elaioplast nanometer particle solution.Granularmetric analysis result display mean diameter 106nm.By two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine elaioplast nanometer particle solution lyophilizing, obtain powdered nanoparticles granule.Transmission electron microscope measures the form of nano-particle as Figure 12.
Embodiment 13:
The preparation of two (HPPH-GFLG) phosphatidyl choline compounds liposome
Two (HPPH-GFLG) phosphatidyl choline compounds 0.3mmol obtained by embodiment 3, add chloroform 10ml, 60 DEG C revolve solvent evaporated; Add 10mlPBS (pH=7.4) 60 DEG C of skinnings, obtain two (HPPH-GFLG) phosphatidyl choline compounds elaioplast nanometer particle solution.Granularmetric analysis result shows, mean diameter 154nm.
Embodiment 14:
The preparation of two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE-N-Polyethylene Glycol-folic acid compound liposome
Two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE-N-Polyethylene Glycol-folic acid compound 0.3mmol obtained by embodiment 9, add chloroform 10ml, 60 DEG C revolve solvent evaporated; Add 10mlPBS (pH=7.4) 60 DEG C of skinnings, obtain two (Verteporfin-diethylene glycol-succinyl) PHOSPHATIDYL ETHANOLAMINE-N-Polyethylene Glycol-folic acid compound elaioplast nanometer particle solution.Granularmetric analysis result display mean diameter 170nm.
Embodiment 15:
The preparation of two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome B
Two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine 0.1mmol, distearoyl phosphatidylcholine DSPC0.4mmol of embodiment 2 are dissolved in 10mL chloroform, and 60 DEG C revolve solvent evaporated; Add 10mlPBS (pH=7.4) 60 DEG C of skinnings, obtain two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome B nanoparticles solution.Granularmetric analysis display mean diameter 180nm.
Embodiment 16:
The preparation of two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome C
Two (Verteporfin-dithio diethylene glycol-succinate) phosphatidyl choline compounds 0.1mmol of Example 2, distearoyl phosphatidylcholine DSPC0.3mmol, DSPE-PEG DSPE-PEG-folic acid (molecular weight polyethylene glycol 2000) 0.1mmol, add chloroform 10ml, 60 DEG C revolve solvent evaporated; Add 10mlPBS (pH7.4) 60 DEG C of skinnings, obtain two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome C nano particle solution.Granularmetric analysis shows, mean diameter 520nm.Lyophilization, obtains two (Verteporfin-dithio diethylene glycol-succinate) Powdered nano-particle of phosphatidylcholine liposome C.
Embodiment 17:
The preparation of two (HPPH-dithio diethylene glycol-succinate) phosphatidylcholine liposome
Two (HPPH-dithio diethylene glycol-succinate) phosphatidylcholine 1mmol of Example 10, add chloroform 20mL, 60 DEG C revolve solvent evaporated; Add 20mLPBS (pH=7.4) 60 DEG C of skinnings, 200nm membrane filtration, obtain two (HPPH-dithio diethylene glycol-succinate) phosphatidylcholine elaioplast nanometer particle solution, mean diameter 330nm.
Embodiment 18:
The external degradation test of two (HPPH-GFLG) phosphatidyl choline compounds liposome
In two (HPPH-GFLG) phosphatidyl choline compounds elaioplast nanometer particle 10mLPBS solution of 0.1mmol prepared by embodiment 13, be divided into 2 parts, a copy of it adds the PBS buffer 0.5mL (enzyme concentration 50mmol) of papain (papain), another part adds PBS buffer, place and hatch at 37 DEG C in incubator, content (the Agilent1100LC of HPPH is detected by high performance liquid chromatography, the anti-phase C18 post of Zorbax, 150 × 4.6mm, 5 μm, sample size 20 μ L, column temperature 25 DEG C, determined wavelength λ=254nm; Gradient elution: 2-90% buffer B/A, flow velocity 1.0mL/min, the deionized water of buffer A: 0.1%TFA, the acetonitrile of buffer B: 0.1%TFA).
Result shows, the HPPH discharged after the HPPH that the liposome solutions of Papain ferment treatment discharges after 6 hours reaches 45%, 15 hours of total amount reaches 75%; Liposome solutions without ferment treatment does not detect HPPH.Visible, two (HPPH-GFLG) phosphatidylcholine liposome has enzymatic degradation, discharges HPPH fast.
Embodiment 19:
The external degradation test of two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome
In two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine elaioplast nanometer particle 10mLPBS solution of 0.1mmol prepared by embodiment 12, be divided into 2 parts, a copy of it adds the PBS buffer 0.5mL (GSH concentration 50mmol) of glutathion (GSH), another part adds PBS buffer, place and hatch at 37 DEG C in incubator, content (the Agilent1100LC of Verteporfin is detected by high performance liquid chromatography, the anti-phase C18 post of Zorbax, 150 × 4.6mm, 5 μm, sample size 20 μ L, column temperature 25 DEG C, determined wavelength λ=254nm; Gradient elution: 2-90% buffer B/A, flow velocity 1.0mL/min, the deionized water of buffer A: 0.1%TFA, the acetonitrile of buffer B: 0.1%TFA).
Result shows, the liposome solutions of GSH process discharges Verteporfin reaches total amount 90% after 3 hours; Liposome solutions without GSH process did not detect Verteporfin after 3 hours.Visible, two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome has GSH sensitivity, can by GSH Co ntrolled release Verteporfin.
Embodiment 20:
The external degradation test of two (HPPH-dithio diethylene glycol-succinate) phosphatidyl choline compounds liposome
In two (HPPH-dithio diethylene glycol-succinate) the phosphatidylcholine elaioplast nanometer particle 10mLPBS solution of 0.1mmol prepared by embodiment 17, be divided into 2 parts, a copy of it adds the PBS buffer 0.5mL (GSH concentration 50mmol) of glutathion (GSH), another part adds PBS buffer, place and hatch at 37 DEG C in incubator, content (the Agilent1100LC of HPPH is detected by high performance liquid chromatography, the anti-phase C18 post of Zorbax, 150 × 4.6mm, 5 μm, sample size 20 μ L, column temperature 25 DEG C, determined wavelength λ=254nm; Gradient elution: 2-90% buffer B/A, flow velocity 1.0mL/min, the deionized water of buffer A: 0.1%TFA, the acetonitrile of buffer B: 0.1%TFA).
Result shows, two (HPPH-dithio diethylene glycol-succinate) phosphatidylcholine liposome solutions of GSH process discharge HPPH reaches total amount 93% after 3 hours; Liposome solutions without GSH process did not detect HPPH after 3 hours.Visible, two (HPPH-dithio diethylene glycol-succinate) phosphatidylcholine liposome has GSH sensitivity, discharges HPPH fast.
Experimental example 21:
The anti-tumor activity of two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome
Photodynamics is tested: the dark toxicity of MCF-7 Human Breast Cancer Cells and Photodynamic therapy effect assessment
Sample: two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome (Lipo-Verteporfin is labeled as " Lipo " group), is prepared by embodiment 12.
Contrast: injection Verteporfin (Verteporfin liposome, Visudyne are designated as " V " group): Novartis Co., Ltd of Switzerland MCF-7 Breast cancer lines: purchased from Chinese Academy of Sciences's Shanghai cell bank
Semiconductor laser, American I ntense company, 7404 types.
Microplate reader, BioRad company 680 of U.S. type.
To take the logarithm the cell of phase, with 025% trypsin cell MCF-7 digestion dispelled, count, bed board 96 orifice plate 5000, every hole cell, overnight incubation is supernatant discarded after it is adherent.Every hole adds pastille (" Lipo " group, " V " group) the culture medium 200 μ L of variable concentrations (0-16 μM), separately establishes blank group (be labeled as " B " group) to add the culture fluid of not drug containing.Lucifuge adopts mtt assay to measure the survival rate of cell after cultivating 24 hours.
To take the logarithm the cell of phase, with 025% trypsin cell MCF-7 digestion dispelled, count, bed board 96 orifice plate 5000, every hole cell, overnight incubation is supernatant discarded after it is adherent.Every hole adds pastille (" Lipo ", " V ") the culture medium 200 μ L of variable concentrations (0-16 μM), separately establishes the blank group of culture fluid adding not drug containing.Lucifuge adopts mtt assay to measure the survival rate of cell after cultivating 24 hours.Then the laser irradiating cell 20min (laser energy density 6J/cm2) of different light dosage is used.Irradiation terminates to continue to hatch 24h in rear incubator, adopts mtt assay to measure the survival rate of cell.
The operation of mtt assay is as follows: every hole adds MTT solution (5mg/mL) 20 μ L, after continuing to hatch 4h in incubator, take out supernatant discarded, every hole adds 150 μ LDMSO, plate shaker shakes up, microplate reader 490nm reads plate, calculates cell inhibitory rate, calculate cell survival rate as follows: survival rate %=A490 (administration group)/A490 (blank) × 100% according to the absorbance recorded.Data are expressed as average ± SD (n=6).And by middle efficacious prescriptions journey calculation of half inhibitory concentration IC
50).Data SPSS10.0forWindows statistical package analyzing and processing, carry out t inspection and variance analysis (One-wayANOVA), P < 0.05 has statistical significance for difference.
Result: the dark toxicity of cell in vitro and phototoxicly the results are shown in Figure 13 (a), (b).As seen from the figure; " Lipo " IC50 that organizes dark toxicity is approximately 16 μMs; and the IC50 of the dark toxicity that " V " organizes is less than 12 μMs, result shows under the same conditions, and the dark toxicity that " Lipo " organizes is less than " V " group (P < 0.05).On the basis of dark toxicity test result, investigate medicine to the Photodynamic therapy of cell, from the test of the experimental data of dark toxicity known phototoxicity, drug level used does not occur that cells survival suppresses phenomenon.Phototoxic experimental result shows, along with the survival rate of the lifting cell of drug level presents downward trend, and in the same circumstances, the cell survival rate that " Lipo " organizes lower than " V " group (P < 0.05), i.e. " Lipo " phototoxicity organized is greater than under the same conditions " V " phototoxicity organized.
Cause the reason of the above results to be that in photosensitizer liposome, the active structure of photosensitizer is positioned at lipid layer, before by cellular uptake, keep stable, dark toxicity is reduced; Photosensitizer liposome is by after cellular uptake, and liposome disintegrates, and 1 molecular photoactive agent phosphatide cpd discharges the former medicine of 2 molecular photoactive agent, forms high local concentrations photosensitizer, under illumination condition, generates a large amount of active oxygen, improve phototoxicity.
Experimental example 27:
Tumor suppression experiment semiconductor laser in two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome, American I ntense company, 7404 types.
MCF-7 Breast cancer lines: Chinese Academy of Sciences's Shanghai cell bank
Sample: two (Verteporfin-dithio diethylene glycol-succinate) phosphatidylcholine liposome (Lipo-Verteporfin is below designated as " Lipo "), is prepared by embodiment 12.
Contrast: injection Verteporfin (Verteporfin liposome, Visudyne are below designated as " V "): Novartis Co., Ltd of Switzerland
The foundation of bearing mouse model: the MCF-7 breast cancer cell of phase of taking the logarithm, with the trypsinization of 0.25%, dispels, counting, select weight at the C57BL/6 male mice of about 16-18g, every mice, in dorsal sc inoculation MCF-7 breast cancer cell suspension 0.2mL, about contains cell 1x10
6individual, observe tumor growth situation.After inoculation about two weeks, tumor growth was to suitable volumes (about 75mm
3).By animal random packet.The computing formula of gross tumor volume (TV) is: TV=1/2 × a × b
2(formula 3-2), wherein a, b represent length and width respectively.Each group of nude mice starts administration, and dosage regimen is shown in grouping and administration, uses the method measuring tumor footpath, dynamically observes the Graft Versus Tumor of given the test agent.
Experiment grouping and administration: get tumor-bearing mice 36, random packet, often organize 6, be respectively administration " Lipo-1 " light group (2mg/kg), administration " Lipo-2 " light group (1mg/kg), contrast administration illumination " V " group (2mg/kg), blank " B " group (normal saline), a not administration irradiation " Light " group, administration Lipo not light group " Lipo-N " (2mg/kg).
By fresh photosensitizer liposome solutions of preparing by tail vein injection administration mice (dosage is equivalent to Verteporfin 2mg/kg).Once, volume is 0.2ml, continuous 3 weeks in injection in every three days.Administration adopts laser to irradiate mouse tumor tissue (150J/cm after 3 hours
2, 500s).
Experimental result:
Assessed the suppression of drug on tumor by the change of the mouse tumor volume measuring after irradiation 21 days, the results are shown in Figure 14.As seen from the figure, the mouse tumor of negative control group constantly grows, and does not have significant difference between these three groups of data.Compared with negative control group, the mouse tumor volume of photosensitizer phosphatide cpd liposome group (" Lipo-1 " " Lipo-2 ") of the present invention all obtains suppression in various degree, and the inhibition that " Lipo-1 " organizes is better than " Lipo-2 " group, and all there is significant difference (P < 0.05orP < 0.01) compared with the gross tumor volume of negative control group mice.Result shows, photosensitizer phospholipid liposome of the present invention has good Tumor suppression growth, is better than contrast injection Verteporfin medicine (Novartis Co., Ltd of Switzerland).The function of tumor inhibition of " Lipo " demonstrates certain dose-effect relationship.In addition, the weight of animals detection display, the Mouse Weight of administration " Lipo " light group (2mg/kg), without significant change, shows that photosensitizer phosphatide cpd liposome of the present invention is without overt toxicity.But the Mouse Weight that contrast administration injection Verteporfin illumination " V " is organized slightly declines, and after 21 days, is 80% of original body mass, shows that matched group photosensitizer has certain toxicity.Result shows, photosensitizer phospholipid liposome of the present invention has optical dynamic therapy effect more better than injection Verteporfin marketed products.
Above-described embodiment is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention; some improvement and equivalent replacement can also be made; these improve the claims in the present invention and are equal to the technical scheme after replacing, and all fall into protection scope of the present invention.
Claims (9)
1. a photosensitizer phosphatide cpd, is characterized in that, this phosphatide cpd is the compound of following structural (1), or the pharmaceutically acceptable salt that the compound of described structural formula (1) and counter ion counterionsl gegenions are formed:
In formula (1), X is spacerarm, to be carbon number be 2 ~ 30 the alkylene alkyl containing ehter bond, the carbon number oxygen base alkylene alkyl that is 1 ~ 30, the carbon number alkylene alkyl containing cystine linkage-S-S-that is 2 ~ 30, the carbon number alkylene alkyl containing peptide bond that is 2 ~ 80, the carbon number alkylene alkyl containing ester bond that is 2 ~ 30, carbon number be 2 ~ 30 the alkylene alkyl containing hydrazone key or carbon number be 2 ~ 30 not containing heteroatomic alkylene alkyl/sub-alkylene; Y is the photosensitizer be connected with X by ester bond, amido link, amino-formate bond or hydrazone key; L is 2-amino-ethyl, 2-trimethyl amido ethyl cation, 2, the end group of 3-dihydroxypropyl, molecular weight to be the Polyethylene Glycol-amino-ethyl of the N-without targeting end group of 200-4000 or molecular weight be 200-4000 is the N-Polyethylene Glycol-amino-ethyl of targeting group.
2. photosensitizer phosphatide cpd according to claim 1, is characterized in that, described photosensitizer Y be following any one: Verteporfin, benzene derivatives of porphyrin monocyclic acids A, amino-laevulic acid methyl ester, photofrin, hematoporphyrin derivative, biporphin ether, phytochrome II, the non-nurse sodium of porphin, phototherapy element, light spirit element, compound numbers is the photosensitizer of haematodrex, ProtoporphyrinIX, cancer light quinoline, amino-laevulic acid, the own ester of amino-laevulic acid, amino-laevulic acid phenyl ester, m-tetrahydroxy chlorin, chlorin e 6, photosensitizer chlorin e 6-C15 mono-methyl, single asparagine chlorin e 6, first alizarinopurpurin, octaethyl alizarinopurpurin, the former alizarinopurpurin of stannum, stannum octaethyl alizarinopurpurin, alizarinopurpurin 18, 1,2,4-trihydroxyanthraquinone, alizarinopurpurin, sun-proof purple LB, hematoporphyrin monomethyl ether, hypocrellin, diphenyl porphin, compound numbers is the photosensitizer of BCPD-17, happiness pool point, m-THPC, hemporfin, compound numbers is the photosensitizer of WST09, compound numbers is the photosensitizer of CGP55847, talaporfin, good fortune contest because of, Sorafenib, light clo, phthalein blue or green-4, motexafin lutecium, hypericin, deuteroporphyrin, thiophene furan quinoline, different porphyrin, compound numbers is the photosensitizer of LuTex, compound numbers is the photosensitizer of Sapphyrin, compound numbers is the photosensitizer of Pentaphyrin, compound numbers is the photosensitizer of Platyrin, compound numbers is the photosensitizer of Porphycene, compound numbers is the photosensitizer of Conphycene, compound numbers is the photosensitizer of Hemiporphycene, compound numbers is the photosensitizer of Isoporphycene, compound numbers is the photosensitizer of N-confusedporphyrin, compound numbers is the photosensitizer of DoublyN-confusedporphyrin, compound numbers is the photosensitizer of 21-core-modifiedporphyrin, compound numbers is the photosensitizer of 21,23-core-modifiedporphyrin, 3
1, 3
2the many chlorins-13 of-two dehydrogenation-15-carbonyl sieve
1, 15
1-anhydride.
3. photosensitizer phosphatide cpd according to claim 1 and 2, it is characterized in that, described end group is in the N-Polyethylene Glycol-amino-ethyl of targeting group, and targeting group is any one in folic acid, galactose, polypeptide, antibody, antibody fragment, hyaluronic acid, asialoglycoprotein, polysaccharide, nucleic acid and plan peptide.
4. the photosensitizer phosphatide cpd according to claim 1,2 or 3, it is characterized in that, when L is uncharged group in described structural formula (1), described counter ion counterionsl gegenions be hydrion, sodium ion, potassium ion, calcium ion, iron ion, magnesium ion, ammonium ion, zinc ion any one; When in structural formula (1), L is positively charged group, described counter ion counterionsl gegenions are combinations of a kind of cation and a kind of anion, described cation is any one in hydrion, sodium ion, potassium ion, calcium ion, iron ion, magnesium ion, ammonium ion, zinc ion, and described anion is any one in chloride ion, sulfate ion, sulfate ion, nitrate ion, carboxylic acid ion, carbanion, bromide ion, phosphate anion, formate, acetate, citrate, lactate, fumaric acid radical, tartrate anion, gluconic acid radical ion.
5. a pharmaceutical composition, is characterized in that, said composition comprises the photosensitizer phosphatide cpd described in claim 1,2,3 or 4, or comprises acceptable carrier on described photosensitizer phosphatide cpd and pharmacodynamics.
6. pharmaceutical composition according to claim 5, is characterized in that, said composition is liquid preparation, solid preparation, semi-solid preparation, granule, gel or injection.
7. pharmaceutical composition according to claim 5, is characterized in that, said composition is the elaioplast nanometer particle of particle diameter 10-1000 nanometer, also comprises auxiliary agent in this pharmaceutical composition.
8. pharmaceutical composition according to claim 7, is characterized in that, described auxiliary agent is phospholipid or cholesterol.
9. the application of photosensitizer phosphatide cpd in photodynamics oncotherapy described in claim 1,2,3 or 4, it is characterized in that, this application, by described photosensitizer phosphatide cpd or its pharmaceutically acceptable salt, is combined with acceptable carrier on pharmacodynamics and is prepared into medicament.
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