CN1875973B - A pharmaceutical composition containing N3-o-toluyl-fluorouracil and its liposome preparation - Google Patents
A pharmaceutical composition containing N3-o-toluyl-fluorouracil and its liposome preparation Download PDFInfo
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- CN1875973B CN1875973B CN2006100422460A CN200610042246A CN1875973B CN 1875973 B CN1875973 B CN 1875973B CN 2006100422460 A CN2006100422460 A CN 2006100422460A CN 200610042246 A CN200610042246 A CN 200610042246A CN 1875973 B CN1875973 B CN 1875973B
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Abstract
The invention relates a method for preparing a drug of 5-Fu precursor N3-o-toluyl-flulorouraci (TFu), a drug compound thereof and a liposome preparation and application. The invention includes the found of anti-cancer activity of the N3-o-toluyl-flulorouraci (TFu), the drug compound and liposome preparation, wherein the liposome contains any different neutral or charged liposome forming substance. The drug compound and the liposome preparation can be advantageously used with a second therapeutic agent different from the N3-o-toluyl-flulorouraci (TFu), and the second therapeutic agent comprise an antitumor drug, an antifungal drug, antibiotics and other active agents. The invention provides a method for supplying the effective dose of the preparation for treating mammalian such as human.
Description
Technical field
The present invention relates to a kind of N of containing
3The pharmaceutical composition of-adjacent toluyl fluorouracil (TFu) with and the preparation method and the application of Liposomal formulation, belong to medical technical field.
Background technology
The pyridine of atropic fluorine is a 5-Fu prodrug of researching and developing, and to late gastric cancer, colorectal cancer, esophageal carcinoma curative effect certainly, is fluorouracil series antineoplastic medicament of new generation.Clinical preceding drug effect, pharmacology and toxicological experiment according to this chemical compound show, the anti-tumor activity height of this chemical compound, and antitumor spectra is wide, toxicity is little, and is safe, and particularly outstanding is its no immunosuppressive action, be fluorouracil series antineoplastic medicament of new generation, Application Prospect is boundless.Yet, clinical before and clinical trial show that all this drug stabilisation is very poor, be very easy to decompose, in external exposure air, can partly decompose.According to Yang Yubin etc.; the pyridine of atropic fluorine is at the intravital pharmacokinetic of tumor patient, Chinese Journal of New Drugs 2001,10 (3): 196-198; this medicine main metabolites in vivo is the adjacent toluyl fluorouracil (TFu) of 3-, and it can further slowly be metabolized to 5-Fu in vivo.
1980, Tetsuji Kametani was at J.Med.Chem.1980, and 23, reported among the 1324-1329 that a series of fluorouracil derivants are (comprising N
3-adjacent toluyl fluorouracil) the activity of synthesizing and having tested these chemical compounds.According to Table IV in the document, N
3The anti-tumor activity of-adjacent toluyl fluorouracil is too low, thereby has negated N
3-adjacent toluyl fluorouracil is as the prospect of antitumor drug.Do not have in the prior art N yet
3-adjacent toluyl fluorouracil is as the research of medicinal application.The inventor has carried out a large amount of external and in vivo tests at TFu, determined that TFu is used for the treatment of the effectiveness of cancer as active constituents of medicine, and determined its appropriate dosage in pharmaceutical composition, and the characteristics poor at this compound water soluble, that bioavailability is low design and prepared its Liposomal formulation.
Summary of the invention
At the deficiencies in the prior art, the invention provides the dosage of active component in the pharmaceutical composition, said composition of the adjacent toluyl fluorouracil of N3-(TFu) and the preparation method and the application of Liposomal formulation thereof.
Technical scheme of the present invention is as follows:
A kind of pharmaceutical composition comprises adjacent toluyl fluorouracil (TFu) of N3-and pharmaceutically acceptable excipient.
The adjacent toluyl fluorouracil of above-mentioned N3-content is 1～150mg/g or 0.1～15mg/ml.
Above-mentioned excipient can be the excipient of any regular dosage form pharmaceutically, includes but not limited to the excipient of tablet, capsule, pill, granule, suppository, solution, suspensoid and Emulsion, paste, ointment, gel, cream, lotion, powder, spray, injection or transfusion.
Therapeutic N
3-adjacent toluyl fluorouracil (TFu) liposome composition comprises and contains adjacent toluyl fluorouracil (TFu) of N3-and lipid components.N wherein
3The mol ratio of-adjacent toluyl fluorouracil (TFu) and lipid components was 1: 40～1: 5 scope.
Above-mentioned N
3-adjacent toluyl fluorouracil (TFu) liposome composition, wherein bag carries N
3The liposome of-adjacent toluyl fluorouracil (TFu) comprises the vesicle with 5 μ m or the following size of 5 μ m.
Above-mentioned N
3-adjacent toluyl fluorouracil (TFu) liposome composition, wherein said bag carries N
3The liposome of-adjacent toluyl fluorouracil (TFu) comprises the vesicle with 0.1 μ m or the following size of 0.1 μ m.
Above-mentioned N
3-adjacent toluyl fluorouracil (TFu) liposome composition, wherein said lipid components comprise a kind of or combination in cholesterol, phosphatidylcholine or the Phosphatidylserine.Preferably, described lipid components also comprises vitamin E.
Adjacent toluyl fluorouracil (TFu) liposome composition of above-mentioned N3-; wherein said phosphatidylcholine is selected from distearoyl phosphatidylcholine; the hydrogenated soya phosphatide phatidylcholine; S-PC, lecithin phatidylcholine, hydrolecithin phatidylcholine; dipalmitoyl phosphatidyl choline; the dioleoyl phospholipid phatidylcholine, two anti-oleoyl phosphatidylcholines, a kind of or combination in the dimyristoyl phosphatidyl choline.
The purposes of the adjacent toluyl fluorouracil of N3-of the present invention (TFu) in the preparation antitumor drug.
Adjacent toluyl fluorouracil (TFu) the zoopery result of N3-shows that if dosage is higher than 130mg/kg, animal can not tolerate, and obvious poisoning symptom occurs, tests defective.Be lower than 50mg/kg, suppression ratio may be lower than 30%, does not meet the basic demand of antitumor drug (chemicals), so selecting dosage is 130～50mg/kg.
In general, human dosage is effectively to measure according to the animal of the best clinically, and come out in conjunction with the comprehensive reckoning of long term toxicity test.Our animal (mice) experiment effective dose is 130～50mg/kg, and according to these data, according to current people and mice body surface area conversion factor (70 kilograms is example), mice is standard with 20g, and people's body surface area is about 383.5 times of mice.So, 130～50 (mg/kg)/50=2.6～1mg/ mice, 2.6～1mg * 382 (doubly)=997～382mg/ people.50 the meaning is that the kg among 130～50mg/kg (1000g) is become 20g mice body weight, so, divided by 50.Last result of calculation, it is 997～382mg/ people or 1000～380mg/ people that the people intends with dosage, or is calculated as 14.24～5.46mg/kg according to kg body weight, promptly about 15～5mg/kg.
Pharmaceutical composition of the present invention can be any conventionally form pharmaceutically, such as tablet, capsule, pill, granule, suppository, solution, suspensoid and Emulsion, paste, ointment, gel, cream, lotion, powder, spray and injection and transfusion.
TFu is fat-soluble strong, and its suspension oral administration biaavailability only 40～45% is unsuitable for direct injection.TFu is a fat-soluble medicine, can be scattered in the interlayer of hydrophobic group of liposome bilayer.With lecithin and cholesterol is the feedstock production liposome.Liposome can obviously improve the medicine character, improves dissolubility, can be for oral or injection.This dosage form can increase the targeting and the histiocyte compatibility of medicine, reduces its toxicity, improves stability.The invention provides this based composition and method.In addition, compare with free drug, as anti-tumor agent, liposome has the pharmacokinetics and the enhanced effect of improvement.Liposome, its preparation method and treatment such as cancer, particularly mammal, especially human body in application in the such disease of cancer.This method comprises the adjacent toluyl groups fluorouracil medicine of the N3-compositions treatment effective dose, that medicine can be accepted in the excipient is comprised that liposome gives mammal.Liposome of the present invention comprises N
3The Liposomal formulation of-adjacent toluyl fluorouracil, wherein said liposome can contain any various neutrality or charged liposome forms material.It can be amphiphile, amphiphilic molecule that described liposome forms material, such as phospholipid, and as phosphatidylcholine, Phosphatidylserine, cholesterol etc., they form liposome in polar solvent.Phospholipid in this liposome can derive from natural origin or synthetic.With the difference that this liposome is formed, this liposome can carry negative charge or positive charge or can be neutral.Preferred liposome can also contain vitamin E.
" excipient " that uses in the liposome refers to the material of the stability that helps drug products, including, but not limited to the stability of pH, and the chemical stability of the stability of the colloidal nature of liposome and medicine and phospholipid.The example of excipient is including, but not limited to acid, the sodium of univalent anion or ammonium form such as chloride, acetate, Lactobionate and formates; Dianion such as aspartate, succinate and sulfate; With trivalent ion such as citrate and phosphate.
" phospholipid " refers to any phospholipid that can form liposome or the combination of phospholipid.Phosphatidylcholine (PC), comprise those available from ovum, the phosphatidylcholine of Semen sojae atricolor or other plant source, or those part or all of synthetic phosphatidylcholines, or have the variable lipoid chain length and the phosphatidylcholine of degree of unsaturation, be suitable for using in the present invention.Phosphatidylcholines synthetic, semisynthetic and natural prodcuts are suitable for using in the present invention; including, but not limited to distearoyl phosphatidylcholine (DSPC); hydrogenated soya phosphatide phatidylcholine (HSPC); S-PC (PC); lecithin phatidylcholine (ovum PC); dioleoyl phospholipid phatidylcholine (DOPC); hydrolecithin phatidylcholine (HEPC); two palmityl phosphatidylcholines, two anti-oleoyl phosphatidylcholines (DEPC); dipalmitoyl phosphatidyl choline (DPPC), and dimyristoyl phosphatidyl choline (DMPC).
The present invention has considered the adjacent toluyl fluorouracil of any effective phospholipid: N3-ratio.The adjacent toluyl fluorouracil of preferred N3-: the phospholipid mol ratio is 1: 40～1: 5, is more preferably 1: 20～1: 10.Preferred Liposomal formulation comprises phospholipid: the cholesterol mol ratio surpasses 18: 1～2: 1 scope.Most preferred Liposomal formulation is S-PC (PC): the cholesterol mol ratio is 15: 1.In preferred embodiments, liposome is the vesicle that has less than the 450nm median particle diameter, and wherein phospholipid is S-PC (PC), and comprises cholesterol with 15: 1 mol ratio.
Although in this preparation, can use the N of wide concentration range
3-adjacent toluyl fluorouracil, still useful concentrations is in the scope of 1～10mg/ml.N
3The mol ratio of-adjacent toluyl fluorouracil and lipid components also can extensively change, but the most useful scope is about N
3-adjacent toluyl fluorouracil (TFu): the scope of lipid components 1: 40～1: 5 (mol ratio).If desired, can make the filter membranes of this liposome by different sizes to control its size.Advantageously, can be with this liposome composition with the second kind of therapeutic agent coupling that is different from the adjacent toluyl fluorouracil of N3-, described second kind of therapeutic agent comprises antineoplastic agent, antifungal agent, antibiotic and other activating agent.If desired, liposome of the present invention can be multilamellar liposome, unilamellar liposome or its mixture.
Usually, the method for preparing preparation of the present invention is the solution that initial preparation forms liposome.For example by weighing up a certain amount of phosphatidylcholine and cholesterol, and they are dissolved in the mixture of the preferred chloroform of mixture of preferred ether of organic solvent or solvent and ether implement.For example evaporate this solution to form solid-state lipoid mutually as thin film or powder with rotary evaporator, spray dryer or other instrument.Preferred drying means is to use rotary evaporator.Then with the aqueous solution hydration thin film or the powder that contain active medicine, contain or do not contain excipient, to form the liposome dispersion.Preferably except acid or alkali, do not use excipient to adjust the pH of drug solution.According to employed phospholipid, the class membrane of lipoprotein or the powder that will be scattered in the drug solution are heated to about 25 ℃～70 ℃ temperature.
By stirring, preferably by shaking or hybrid dispersions, form multilamellar liposome.For example by supersound process or use the Micro Fluid device or extrusion device or homogenizer or French extrude instrument the aqueous dispersion of lipoid solid phase is imposed energy such as shearing force or hole to form multilamelar vesicles.Also can use injection, freezing and melt, detergent solution is fallen in dialysis from lipoid, or other known method that is used to prepare liposome prepares liposome.Use the size of various known technologies (comprise and continue to use energy) may command liposome.
Use dialysis, size exclusion chromatography (SEC) (Sephadex G-50 resin) or ultrafiltration (50,000～300,000 molecular weight),, from the liposome dispersion, remove the excipient and/or the medicine of not embedding by the exchange of buffer and aqueous solution.
The therapeutic use of liposome can comprise the common deleterious medicine of transmission free form.In liposome, can make toxic medicament away from can causing toxic sensitive organization, and guiding can be brought into play the selection area of their therapeutical effect.Liposome also can therapeutic modality uses slowly discharging medicine through an elongated segment phase, thereby by the enhanced pharmacokinetics reduction administration frequency that distributes.In addition, liposome can provide a kind of formation to be suitable for the method for the dewatering medicament aqueous dispersion of intravenous conveying.
The landline of liposome also can influence their distributions in vivo.The passive conveying of liposome relates to the various paths of administration such as the application of oral administration, although other effective form of medication such as injection, inhalant, preparation capable of permeating skin or suppository also are considered.Every kind of path is all variant on the liposome location.
Description of drawings
Fig. 1: morphological observation result behind the cancer of biliary duct FRH-0201 cell adding variable concentrations TFu.A, b be not for adding the matched group of TFu medicine, and cell attachment stretches good; C:TFu concentration is 0.016 μ mol/ml, d:TFu concentration is 0.063 μ mol/ml, e:TFu concentration is 0.25 μ mol/ml, f:TFu concentration is 1 μ mol/ml, g:TFu concentration is 2 μ mol/ml, h:TFu concentration is 4 μ mol/ml, and i, j:TFu concentration are greater than 4 μ mol/ml, and cell is all dead.As seen from the figure, stretch variation with TFu concentration rising cell, stand density reduces, and the gap increases, and occurs granule in the endochylema.The concentration cell rounding that raises again suspends, and is dead then.
Fig. 2: Giemsa staining result behind the cancer of biliary duct FRH-0201 cell adding variable concentrations TFu, matched group a, shrinkage appears in experimental group b-g cell, and dense dying sprouted, and forms apoptotic body.
Fig. 3: acridine orange coloration result behind the cancer of biliary duct FRH-0201 cell adding variable concentrations TFu.Matched group a, the dosing cell does not stretch well, and most of cell is fusiformis, and endochylema dyeing is light, and nuclei dyeing is a yellow green, kernel is clear; The cell rounding shrinkage of experimental group b.TFu effect back, endochylema is unclear, and nucleus is dyed darker yellow green, karyopycnosis, kernel is unclear.
Fig. 4: the FRH-0201 cell growth curve after the variable concentrations TFu effect.
Fig. 5: TFu suppresses the FRH-0201 cell of In vitro culture, and this inhibitory action has time dependence (a) and dose dependent (b).
Fig. 6: different pharmaceutical to the FRH-0201 cell inhibitory rate relatively.Medicine DDP among the figure is a cisplatin, and ADM is an amycin, and VCR is a vincristine, and MMC is a mitomycin, and TAXOL is a taxol.
Fig. 7: colony forms the ability testing result.A is a matched group, and cloning efficiency is (35.33 ± 4.16) %, and b is 0.0156 a μ mol/mlTFu group, and cloning efficiency is (16.67 ± 2.31) %.
Fig. 8: DNALadder detects the apoptosis result.M:Marker, C1～C5:4,1,0.25,0.063,0.016 μ mol/mlTFu effect 72h, matched group is the auxocyte same period of not dosing.
Fig. 9: mice plasma TFu content HPLC bioassay standard curve chart.
Figure 10: normal mouse is irritated stomach TFu suspendible aqueous solution group blood drug level-time plot (n=3).
Figure 11: normal mouse is irritated stomach TFu liposome group blood drug level-time plot (n=3).
Figure 12: normal mouse intravenous injection TFu solution group blood drug level-time plot (n=3).
Figure 13: normal mouse intravenous injection TFu liposome group blood drug level-time plot (n=3).
Detailed Description Of The Invention
The invention provides with the invention provides and comprise N
3The dosage of active component and the preparation method and the application of Liposomal formulation thereof in the pharmaceutical composition of-adjacent toluyl fluorouracil (TFu), the said composition.Pharmaceutical composition of the present invention can be any pharmaceutically conventionally form, and especially its Liposomal formulation has following advantage: avoided the adjacent toluyl fluorouracil of problems of dissolution, N3-of the adjacent toluyl fluorouracil of N3-and liposome stability, can the adjacent toluyl fluorouracil of N3-, the adjacent toluyl fluorouracil of N3-toxicity reduce as giving with high concentration bolus injection or short-term infusion mode, the therapeutic efficiency of the adjacent toluyl fluorouracil of N3-improves.
Preferably, compositions of the present invention is to contain N
3The Liposomal formulation of-adjacent toluyl fluorouracil.In general, can prepare Liposomal formulation by known technology.For example, in a kind of preferred technology, all lipophilic component are dissolved in suitable solvent, can evaporate this system then and form the lipophilic film.To join on the described lipid film and such as the such polar solvent of aqueous solvent subsequently and become liposome of the present invention the violent homogenize of gained mixture.
Ideal situation is, in case liposome form, then with its by suitable membrane filtration to control its size.Suitable filter membrane comprises that those can be used for obtaining from filtrate the filter membrane of the liposome of required range size.For example, can form liposome and after this obtain having the liposome of about diameter below 5 microns or 5 microns by 5 microns membrane filtrations.Perhaps, can be by the liposome that uses 1 μ m, 500nm, 200m, 100nm or other filter membrane to obtain to have corresponding size.
In order to prepare N
3-adjacent toluyl groups-fluorouracil preparation is with N
3-adjacent toluyl fluorouracil is dissolved in suitable solvent.Suitable solvent is those N
3-adjacent toluyl groups-fluorouracil can dissolve therein and can evaporate and can not leave over the solvent of unacceptable residue on the medicine of unacceptable amount on the medicine.For example, can use non-polar solven such as ether, chloroform etc.
The phospholipid of any appropriate can be used for the present invention.For example, phospholipid can purification from natural origin or can be synthetic with chemical mode, phospholipid can be dissolved in suitable solvent, described solvent comprises that phospholipid can dissolve therein and can evaporate and can not leave over the solvent of unacceptable residue on the medicine of unacceptable amount on the medicine.Can be with phospholipid solution and N
3-adjacent toluyl fluorouracil mixes.Perhaps, phospholipid and the adjacent toluyl fluorouracil of N3-directly can be dissolved.Phospholipid derivative can also be used for Liposomal formulation of the present invention, enough stablize and to N as long as the Liposomal formulation of gained is used treatment
3-adjacent toluyl fluorouracil has suitable capacity.
The liposome of any appropriate can be formed material and be used for Liposomal formulation of the present invention.Suitable liposome forms the chemical compound with hydrophilic segment and hydrophobic part that material comprises synthetic, semisynthetic (natural modified) or natural appearance.This compounds is amphiphile, amphiphilic molecule and can has clean positive and negative or be neutral charge.The hydrophobic part that forms the chemical compound of liposome can comprise one or more nonpolar aliphatic chains, for example palmityl.The examples for compounds of suitable formation liposome comprises phospholipid, sterols, fatty acid etc.The preferred chemical compound that forms liposome comprises phosphatidylcholine, cholesterol, two palmityl phosphatidylcholines, Phosphatidylserine and vitamin E.
Liposome can be formed material be dissolved in it therein can dissolved suitable solvent, this solvent can be such as the such low polar solvent of chloroform or such as the such non-polar solven of ether.Suitable solvent only comprises that liposome forms material and can dissolve therein and can evaporate and the solvent that can not leave over unacceptable residue on the medicine of unacceptable amount on the medicine.In this solution, can be mixed with and comprise N
3To form solution, wherein all components all is soluble and this solvent evaporation can be obtained a kind of uniform lipid film subsequently at this solution-adjacent toluyl fluorouracil at other interior composition.Can be by keeping N
3Any suitable mode of-adjacent toluyl fluorouracil and other lipophilic component stability is carried out solvent evaporation.
Suitable liposome can be neutral, electronegative or positively charged, and this electric charge is the function of liposome component electric charge and liposome solutions pH.For example, under neutral pH, positively charged liposome can be formed by the mixture of phosphatidylcholine, cholesterol and stearylamine.Electronegative liposome for example can be formed by phosphatidylcholine, cholesterol and Phosphatidylserine.In a preferred embodiment, N
3The Liposomal formulation of-adjacent toluyl fluorouracil contains injection S-PC and cholesterol.
More generally situation is, can use the proper method of any formation liposome, as long as the method causes N
3-adjacent toluyl groups-fluorouracil liposome produces.Therefore, can use and do not relate to the film formed solvent evaporated method of dried lipid.For example, can prepare liposome by in water and organic facies, forming Emulsion and evaporating organic solvent.The invention is intended to comprise the N of which kind of method preparation of don't work
3The Liposomal formulation of-adjacent toluyl groups-fluorouracil.
The adjacent toluyl groups of preferred N3--fluorouracil Liposomal formulation contains the N of suitable relative molecular weight
3-adjacent toluyl fluorouracil and lipid.Wherein the mol ratio of the adjacent toluyl groups-fluorouracil of N3-(TFu) and lipid components is in about 1: 1～100 scope, more preferably from about 1: 2～80, more preferably from about 1: 5～50, more preferably from about 1: 15～30, and most preferably from about 1: 28.
This Liposomal formulation also contains the phosphatidylcholine and the cholesterol of suitable relative molecular weight.Suitable relative molecular weight comprises about 1～99: 0.1～50 phosphatidylcholine: cholesterol.More preferably the relative molecular weight scope is 2～50: 1～25, more preferably 4～25: 2～15 and preferred amount ranges 5～15: 4～10, most preferred ratio is 15: 1.Preferred Liposomal formulation also contains an amount of such as vitamin E such antioxidant or other suitable antioxidant.An amount of scope is 5wt.% or below the 5wt.%.
Can disperse this film to form liposome by polar solvent, preferred aqueous solutions such as saline solution are joined on the lipid film and by violent mixing.This solution can be that pure water or its can contain salt, buffer or other soluble active agent.Can use any mixed method, condition is that method selected is induced the enough shearing forces of generation between lipid film and the polar solvent so that the violent described mixture of homogenize and form liposome.For example, can mix by vortex, magnetic stirring and/or ultrasonic Treatment.Can form multilamellar liposome by the described solution of vortex merely.Unilamellar liposome can comprise ultrasonic Treatment and/or filtration step so in the method if desired.
Can by in aqueous solution with the aliquot dialysed overnight of described Liposomal formulation and subsequently this liposome is dissolved in methanol and analyzes this sample by the standard method of use high performance liquid chroma-tography (HPLC) and measure N
3The bag of-adjacent toluyl groups-fluorouracil carries efficient.Perhaps, after centrifugal 1 hour, collect liposome, then it is dissolved in methanol and is used for the HPLC analysis with 50,000 * g.Usually, N
3The encapsulation efficient of-adjacent toluyl fluorouracil in liposome is higher than 80% of dispensing dosage.
The present invention includes pharmaceutical preparation, also contain N the excipient that suits on avirulent except containing, the inert medicine of this preparation
3The Liposomal formulation of-adjacent toluyl groups-fluorouracil, the present invention also comprises the production method of these preparations.Be understandable that the proper excipient on the so-called medicine refers to all kinds of solids, semisolid or liquid diluent, filler and formulation aid.The present invention also comprises the pharmaceutical preparation of unit dosage forms.This means that described preparation is the form of individual part, for example bottle, syringe, capsule, pill, suppository or ampoule, the N of the liposome entrapment in them
3-adjacent toluyl fluorouracil content is equivalent to a part of unit dose or a plurality of unit dose.For example, this unit dosage forms can contain 1,2,3 or 4 unit dose or 1/2,1/3 or 1/4 unit dose.Unit dose preferably contains the N that gives in single administration
3-adjacent toluyl fluorouracil consumption, and its be equivalent to usually whole every day of dosage, half dosage every day or every day dosage 1/3rd or 1/4th N
3-adjacent toluyl fluorouracil consumption.
Tablet, capsule, pill, granule, suppository, solution, suspensoid and Emulsion, paste, ointment, gel, cream, lotion, powder, spray and injection and transfusion can be suitable pharmaceutical preparation.Suppository removes and contains N
3-adjacent toluyl fluorouracil liposome also contains suitable water solublity or water-insoluble excipient outward.Suitable excipient is those liposome N of the present invention
3It is stable to be used for the treatment of the excipient of application that-adjacent toluyl fluorouracil fully keeps therein, for example the mixture of Polyethylene Glycol, some fats and esters or these materials.Ointment, paste, cream and gel also can contain N
3-adjacent toluyl fluorouracil liposome keeps stable suitable excipient therein.
According to known method, for example by with liposome N
3-adjacent toluyl fluorouracil and excipient or multiple mixed with excipients and prepare the said medicine preparation in a usual manner.
Can be through the part, intravenous, non-intestinal, intraperitoneal and/or rectum, preferably give described reactive compound or its pharmaceutical preparation through non-intestinal, but, the preferred oral administration.
Embodiment shows N
3The administration of-adjacent toluyl fluorouracil is not to having been produced pharmacology's effect by the mammal tumor that inclusion slackened in the Liposomal formulation.In addition, when as the Liposomal formulation administration, the N that animal can tolerate higher doses
3-adjacent toluyl fluorouracil, and they have the better effects of measuring as half survival time or have than giving conventional N
3The littler gross tumor volume of animal of-adjacent toluyl fluorouracil.In the mice body, show higher chemical compound plasma concentration and in the mice body, shown the long chemical compound elimination half-life.Under dosage very nearly the same, the intravital peak plasma concentrations of mice is more than 1.5 times.In animal, the adjacent toluyl fluorouracil of the N3-of higher dosage liposome more has tolerance preferably.But, toxicity profile seems similar.
Invention has been described, is described referring now to some embodiment, and these embodiment only are used for task of explanation rather than are used for limiting the present invention.Embodiment 1 describes the synthetic route and the structural identification of the adjacent toluyl fluorouracil of N3-.Embodiment 2 describes the discovery of the active anticancer of the adjacent toluyl fluorouracil of N3-.Embodiment 3 describes determining of dosage.Embodiment 4 describes the external tumor killing effect of the adjacent toluyl fluorouracil of N3-.Embodiment 5 describes the conventional capsule preparation of the adjacent toluyl fluorouracil of N3-.Embodiment 6 describes the pellet capsule preparation of the adjacent toluyl fluorouracil of N3-.Embodiment 7 describes the conventional tablet of the adjacent toluyl fluorouracil of N3-.Embodiment 8 describes the Liposomal formulation of the adjacent toluyl fluorouracil of N3-.Embodiment 9 describes the another kind of Liposomal formulation of the adjacent toluyl fluorouracil of N3-.Embodiment 10 describes the tumor killing effect of the adjacent toluyl fluorouracil of N3-Liposomal formulation.Embodiment 11 describes mice LD50 test.Embodiment 12 describes the pharmacokinetic study behind the adjacent toluyl fluorouracil of free N3-and oral back of the adjacent toluyl fluorouracil of N3-Liposomal formulation and the quiet notes.
The specific embodiment
Embodiment 1: present embodiment is represented the synthetic route and the structural identification of the adjacent toluyl fluorouracil of a kind of N3-.
1.1 pyridine: hydro-oxidation sodium is dry more than 24 hours, and 115～116 ℃ fraction is collected in air-distillation then.
1.2 triethylamine: air-distillation before using, collect 88～89 ℃ fraction.
1.3 benzene: add calcium chloride after dry 24 hours, 80～81 ℃ fraction is collected in air-distillation.
1.4 ether: add sodium metal backflow air-distillation after 2 hours, collect 35～36 ℃ fraction.
1.5 dioxane: air-distillation, collect 101.32 ℃ fraction.
1.6 the preparation of adjacent toluyl chlorine
Take by weighing ortho-toluic acid 176.8g (1.3mol), add thionyl chloride 200ml (excessive), stir, reflux 5 hours is to the white cigarette of no longer emerging.Excessive thionyl chloride is removed in distilling under reduced pressure, gets the adjacent toluyl chlorine (1.27mol) of 195.5g yellow oil.Yield 97.7%.
1.7N
3-adjacent toluyl groups-5-Fu's (TFu) is synthetic
Take by weighing 5-Fu 2.6g (0.2mol) and be dissolved in the 48ml anhydrous pyridine, slowly be added drop-wise in the adjacent toluyl chlorine (about 0.06mol) of 12ml anhydrous pyridine and 7.8ml, low temperature (10～15 ℃) stirred after 3 hours, poured in the frozen water jolting into.Add the benzene shake well, divide three extractions, collect three times extract, wash back sodium sulfate dried overnight with water, filter.Benzene is removed in the filtrate decompression distillation, after residue oiliness residue is used the normal hexane thorough washing, uses the ether crystallization, uses the chloroform recrystallization, gets white plates crystallization TFu, and yield is 76%.Fusing point is: 156.5～157.5 ℃.
1.8 the structural confirmation of target compound
Synthetic target compound is carried out structural identification with FT-IR infrared spectrum, proton nmr spectra and single crystal diffraction collection of illustrative plates, and result and peak position ownership see Table 1.
Table 1 target compound FT-IR infrared spectrum and proton nmr spectra
Embodiment 2: present embodiment has proved the active anticancer of the adjacent toluyl fluorouracil of N3-.
2.1.1 morphological observation:
To be in exponential phase, cancer of biliary duct FRH-0201 cell inoculation in good condition in 24 orifice plates, every hole 1 * 10
4Individual cell, cell attachment behind the 24h, discard original culture fluid, matched group adds culture fluid to be continued to cultivate, experimental group adds the culture fluid that contains variable concentrations TFu (8,4,1,0.25,0.063,0.016 μ mol/ml) respectively, continue to cultivate, under inverted microscope, dynamic observe the cellular morphology variation and take the photograph sheet.Row Giemsa staining behind the 72h.
Autoclaved coverslip is put into 6 orifice plate cell culture, take out the cell film flying of making behind the drug effect 3d, PBS washes 2 times, and 95% ethanol is 5min fixedly, and with 0.01% acridine orange dyeing 5min, sheet observed and take the photograph by the interim mounting of PBS under the fluorescence microscope.
The result: 1. the morphological observation inverted microscope is observed down, and the cellular control unit growth is vigorous, and adherent stretching, extension is good, cell good transmittance, several no suspension cells; 0.016-4umol/mlTFu group attached cell density reduces gradually, cell stretches variation gradually, and the endochylema endoparticle increases, and intercellular substance increases, and suspension cell increases, and sees cell debris; TFu concentration is during greater than 4umol/ml, and cell rounding no longer stretches, and the part cell is suspended in the culture fluid, cell disruption death then, and platform is expected the no living cells of blue dyeing.See accompanying drawing 1.
2.1.2 Giemsa staining: the experimental group cell shrinkage, dense dying sprouted, and forms apoptotic body, the results are shown in accompanying drawing 2.
2.1.3 acridine orange fluorescent staining microscopic examination: visible cell stretches well behind the matched group FRH-0201 cell dyeing, and nucleus is dyed yellow green, visible kernel, and the endochylema homogenizing, third dimension is strong; After variable concentrations TFu effect, the visible cell circle that diminishes, cell shrinkage, the kytoplasm densification, nucleus is dyed yellow green, nuclear chromatin limit collection, no kernel presents the feature of apoptotic cell.The results are shown in accompanying drawing 3.
2.2 MTT experiment
2.2.1 variable concentrations TFu influences the cholangiocarcinoma cell multiplication capacity
With every hole 2 * 10
3Individual cell (200ul) is inoculated in 5 96 well culture plates; The careful suction removed culture fluid after cultivation 24h made cell attachment, and matched group adds culture fluid and continues cultivation.Experimental group adds the culture fluid that contains variable concentrations TFu (make final concentration is 4,1,0.25,0.0625,0.015625umol/ml) respectively, and every block of plate of every kind of concentration of matched group and experimental group is provided with 3 multiple holes, continues incubator and cultivates.The 1st～5d, the cell that detected in the plate every 24 hours, every hole adds people MTT (5g/L) 20ul respectively, incubator continues to cultivate 4h, stops cultivating, and careful the suction removed culture fluid in the hole, every hole adds the dimethyl sulfoxide low-speed oscillation 10min of 150ul, and crystal is fully dissolved.Measure the light absorption value (OD value) in each hole at enzyme-linked immunosorbent assay instrument 540nm place; With time is transverse axis, and the OD value is the longitudinal axis, draws cell growth curve.Calculate suppression ratio: suppression ratio=(1-experimental group OD value/matched group OD value) * 100%, and be transverse axis with time and concentration, suppression ratio is the longitudinal axis, curve plotting.
Result: TFu suppresses the FRH-0201 cell of In vitro culture, and this inhibitory action has time dependence and dose dependent.See accompanying drawing 4, accompanying drawing 5.
2.2.2 drug susceptibility detects
Selecting kind of a chemotherapeutics, is respectively TFu, 5-Fu, cisplatin (DDP), amycin (ADM), vincristine (VCR), mitomycin (MMC), VP-16, taxol (TAXOL) according to document (contemporary Internal Medicine-Oncology therapeutic scheme is estimated) and clinical dosage commonly used, calculates experimental concentration according to formula.Formula: drug level (ug/ml)=drug dose (mg) * average body surface area * 100 ÷ average weight ÷ 60.The results are shown in accompanying drawing 6.
2.3 colony formation ability detects
50 cell inoculations in every hole are in 24 orifice plates; Matched group is changed culture fluid and is continued to cultivate behind the cell attachment, and test group adds the TFu of variable concentrations, makes that final concentration is 4,1,0.25,0.0625,0.0156,0.004,0.001umol/ml; Leave standstill in the incubator and cultivated 7-12 days, during take the circumstances into consideration to change culture fluid according to medium pH value; The PBS washed twice; Methanol is fixed 10 minutes; Rui Shi-Jim Sa liquid dyeed 3 minutes; Microscopically observation of cell clone's formation situation is also taken the photograph sheet, and counting calculates cloning efficiency, triplicate greater than clone's number of 50 cells.
The result: the matched group cloning efficiency is (35.33 ± 4.16) %, and 0.0156umol/ml group cloning efficiency is (16.67 ± 2.31) %, and 0.004umol/ml group cloning efficiency does not form for (4.67 ± 4.16) % has the clone greater than this concentration group.
See Table 2, accompanying drawing 7.
Matched group and experimental group clone formation situation
2.4DNALadder detection apoptosis
Collect the cell of variable concentrations drug effect 72h, experimental procedure cell lysis extraction DNA carries out electrophoresis routinely.The results are shown in accompanying drawing 8.After the variable concentrations TFu effect, raise with concentration, the DNA Ladder band of appearance is obvious more.Typical apoptosis ladder band all appears in each concentration group.
2.5 flow cytometer detects cell cycle distribution and apoptosis
Collect＞1 * 10
6Individual cell, test group are the cell of 0.0625umol/mlTFu and 5-Fu effect 24,48h, and matched group is the auxocyte same period, is divided into two groups, apoptosis group and periodic groups; The apoptosis group gets about 1 * 10
5Individual cell is pressed the operation of test kit step, makes apoptosis and detect on flow cytometer; The centrifugal back of remaining cell is fixed with 80% ethanol of-20 ℃ of pre-coolings; The 1mlPI 30min that dyes; On flow cytometer, make cell cycle analysis, measure cell proliferation index (PI=(S+G2/M) % * 100), G0/G1 phase cell proportion, the variation of S phase cell proportion detects not the vegetative state and the cytoactive of cell on the same group.(Backman?counter?cytomics?FC?500)
The result: as seen cell cycle detects, and along with prolong action time, G1 phase cell increases, and S phase cell reduces, and the stunt of experimental group cell stagnated in the G1 phase, and phase TFu group cell cycle arrest situation is higher than the 5-Fu group simultaneously.As seen apoptosis respectively organizes the apoptosis rate rising with prolonging action time, compares simultaneously, and TFu group apoptosis rate is higher than the 5-Fu group.
Apoptosis detect to find that obvious apoptosis does not appear in 24h, and 48h begins to occur apoptosis, obviously raises to the 72h apoptosis rate.Compare simultaneously, TFu group apoptosis rate is higher than 5-Fu.See Table 3.
Each organizes cell not phase cell cycle and apoptosis rate simultaneously
2.6CA19-9, the detection of CA125
Collect cell culture fluid that TFu concentration is 0.0625umol/ml effect 72h and the same period each 1ml of normal cell culture fluid, the centrifuging and taking supernatant; Experimentize according to the test kit step; Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument 420nm place.The drawing standard curve is also obtained test group and the value of matched group, sees Table 4.
Matched group and different experiments group tumor markers expression are relatively
Conclusion:
TFu can effectively suppress the FRH-0201 cell proliferation, and this inhibitory action has time, dose dependent;
TFu may be by the retardance cell cycle, and further cell death inducing is had an effect;
TFu apparently higher than 5-Fu, can be used as the new drug reference of clinical cancer of biliary duct chemotherapy to the FRH-0201 cell inhibitory rate.
Embodiment 3: describe determining of dosage.
Definite result of zoopery dosage shows that if dosage is higher than 130mg/kg, animal can not tolerate, and obvious poisoning symptom occurs, tests defective.Be lower than 50mg/kg, suppression ratio may be lower than 30%, does not meet the basic demand of antitumor drug (chemicals), so selecting dosage is 130～50mg/kg.Experimental result sees Table 5,6,7 and table 8.
Table 5 TFu is to the growth inhibited effect of S180 murine sarcoma
Compare with matched group,
*P＜0.01,
*P＜0.05n=3.
Table 6 TFu is to the growth inhibited effect of H22 rat liver cancer
Matched group compares,
*P＜0.05,
*P＜0.01, n=3
Table 7 TFu is to nude mice transplantability people SGC-7901 vitro growth of gastric cancer cell inhibitory action
Compare with matched group,
*P＜0.05,
*P＜0.01, n=3
Table 8 TFu is to the growth inhibited effect of nude mice transplantability people NKM-45 cell
Compare with matched group,
*P＜0.05,
*P＜0.01, n=3
In general, human dosage is to come out according to comprehensive reckoning of definite toxicity test of the animal effective dose of the best clinically.Our animal (mice) experiment effective dose is 130～50mg/kg, according to these data, and according to current people and mice body surface area conversion factor, (70 kilograms is example), and mice is standard with 20g, people's body surface area is about 383.5 times of mice.So, 130～50 (mg/kg)/50=2.6～1mg/ mice, 2.6～1mg * 382 (doubly)=997～382mg/ people.50 the meaning be with the kg among 130～50mg/kg (1000g becomes 20g mice body weight, so, divided by 50.Last result of calculation, it is 997～382mg/ people or 1000～380mg/ people that the people intends with dosage, or calculates 14.24～5.46mg/kg, promptly about 15～5mg/kg according to kg body weight.
Embodiment 4: present embodiment proof TFu inside and outside is to the inhibition effect of growth of tumour cell.
Dissolve with DMSO during N3-neighbour toluyl fluorouracil (TFu) experiment in vitro, culture medium is diluted to needs concentration.During zoopery, make the TFu suspension with 5% starch.Capecitabine (Capecitabine, Cap), Shanghai Co., Ltd., Factory of Roche Group product, lot number SH0040.Hyclone and RPMI1640 culture medium are purchased the company in Gibco BRL; Tetramethyl azo azoles orchid (MTT), Sigma company product; 96 well culture plates, U.S. Costar company product, production number 3599.
4.1 rat liver microsomes mixed-function oxidase (S-9): with Polychlorinated biphenyls male Wister rat is carried out enzyme induction, get liver, homogenate, 9000r/min is centrifugal, and getting supernatant is S-9.10ml S-9 mixed liquor contains 1ml S-9,0.2ml 0.4mmol/L magnesium chloride, 0.2ml 1.65mmol/L potassium chloride, 4ml 0.2mmol/L phosphate buffer (pH7.4), 0.014g 6-glucose 1-phosphate1-, 0.0314g coenzyme II (NADP), 4.6ml distilled water.
4.2 tumor cell line and cultivation: low differentiation of human adenocarcinoma of stomach cell SGC-7901, people's epithelium sample adenocarcinoma of stomach cell NKM-45, cell grows in RPMI1640 culture medium+10% hyclone, go down to posterity with the 0.02%EDTA+0.25% trypsinization, provide by Shanghai Inst. of Cytobiology, Chinese Academy of Sciences.Murine sarcoma cell strain S180, rat liver cancer cell strain H22 with the preservation of going down to posterity of Kunming mouse ascites, is provided by the Pharmaceutical Research Inst. of Shandong Prov. Medical Science Academy.
4.3 animal: Kunming mouse, body weight 20 ± 2g, the male and female dual-purpose, Shandong University's Experimental Animal Center provides.The Balb/c nude mice, body weight 19-21g, male, Institute of Experimental Animals, Chinese Academy of Medical Sciences's animal breeding field provides.
4.4 experimental technique
4.4.1 cell growth inhibition test: get SGC-7901 and NKM-45 cell exponential phase of growth respectively, after the washing of Hank ' s liquid, be dispersed into individual cells with the 0.02%EDTA+0.25% trypsinization, the blue dyeing counting living cells of platform dish is more than 95%.Get cell inoculation in 96 well culture plates, cell number 5000/well/0.2ml puts 37 ℃, cultivates 12 hours in the 5%CO2 incubator.Behind the cell attachment, add variable concentrations TFu, final concentration is respectively 0.01,0.1,1,10,100,1000 μ g/ml, and each concentration was established three multiple holes, continues to cultivate, and got culture plate every 24 hours, detected inhibitory rate of cell growth, to 120 hours.
4.4.2 hepatomicrosome enzyme mixed liquor (S-9) suppresses the influence of growth of tumour cell effect to TFu: the same method, inoculating cell added chemical compound, adds S-9 mixed liquor 2 μ l/ holes again and cultivates, and got culture plate every 24 hours, measured inhibitory rate of cell growth.
4.4.3MTT method is measured inhibitory rate of cell growth: with the cell of 96 well culture plates growth, after the PBS washing, every hole adds serum-free medium (containing MTT 5mg/ml) 200 μ l, continue to cultivate after 4 hours and abandon supernatant, the PBS washing, add 200 μ l DMSO joltings 5 minutes again, microplate reader 570nm place surveys the OD value.
4.4.4 animal transplanting tumor growth inhibited experiment: extract inoculation 5 days S180 and H22 mouse tumor ascites respectively, make the tumor cell suspension after the normal saline dilution, adjust cell number to 1 * 10
7/ ml, every right side of mice armpit subcutaneous vaccination 0.2ml.Random packet, 10 every group.Next day gastric infusion, 0.8ml/ only, dosage is respectively 50,100,200mg/kg, successive administration 12-15 days.After the last administration 24 hours, put to death animal, to get tumor and weigh, the computerized compound is to inhibition rate of tumor growth.Tumour inhibiting rate=(matched group tumor weight-experimental group tumor is heavy)/matched group tumor is heavy.Each tumor line experiment repeats calculating mean value 3 times.
4.4.5 gastric carcinoma cells nude mice transplantation experiments: get SGC-7901 and NKM-45 cell 107 respectively and be suspended in 200 μ lmatrigel (MA), it is subcutaneous to be inoculated in nude mice right side armpit for Collaborative Biomedical, Bedford.Random packet, 8 every group.The 7th day, when gross tumor volume grows to 0.2-0.3cm
3The time, gastric infusion, dosage is respectively 0.20,0.40, and 0.80mmol/kg sets up matched group, and administration is 5-6 time weekly, continuous 4 weeks.Get tumor and weigh, the same calculating inhibition rate of tumor growth.
4.4.6 statistics: data represent with x ± s, adopt SPSS10.0 software to carry out statistical analysis, not on the same group between parameter relatively adopt non-paired t test, there is statistical significance p＜0.05 for difference.
4.5 experimental result
4.5.1SGC-7901 and the normal growth curve of NKM-45 cell: inoculate 96 orifice plates with 5000/well/0.2ml, cell growth curve when observing no drug treating.When cell grows to 120hrs (5 days), cell growth curve is platform status, the 6th day, growth curve begins to descend, illustrate under this experiment condition that cell still shows as the exponential growth state before growing to 120 hours, the last minute point of growth curve should be at 120 hours.
4.5.2TFu to SGC-7901 and NKM-45 cell growth inhibition: (1) variable concentrations TFu and SGC-7901 cells contacting different time, cells survival curve performance dose dependent and incubation time dependency inhibitory action relation.With the 72h time point is example, and relatively each dosage group cells survival rate compares P＜0.05 between group; 10 μ g/ml dosage groups record TFu to each time point cell inhibiting rate, 24h, 13.09%; 48h, 15.69%; 72h, 22.24%; 96h, 28.93%; 120h, 36.86%, compare P＜0.05 between group.The highest suppression ratio occurred in 120 hours, 1000 μ g/ml, suppression ratio are 48.68%.Hatch with cell simultaneously as TFu and S-9, each dosage group and time point all obviously increase the effect of SGC7901 growth inhibited, each test point tumor cell survival rate decline 29.24-42.57%.(2) TFu and NKM-45 cell are hatched different time, the cells survival curve also display density and time of contact the dependency interactively.Handle cell with 10 μ g/ml dosage, each time point of 24-120h, the growth inhibition ratio of TFu pair cell are 10.28-34.99%.The 72h time point, 0.1-1000 μ g/ml dosage, the suppression ratio 10.01-32.97% of pair cell.1000 μ g/ml and cells contacting 120 hours, suppression ratio are 42.11%.When TFu, S-9 and cell are hatched jointly, significantly improve growth inhibited effect to NKM-45.At the 72h time point, 0.1-1000 μ g/ml dosage reaches 65.11-93.03% to the NKM-45 growth inhibition ratio.Calculate 10 μ g/ml dose points, incubation time 24-120h, suppression ratio are 31.28-94.11%.1000 μ g/ml, 120 hours, suppression ratio reached 96.09%.With do not add S-9 relatively, increase suppression ratio 53.98-66.19%, table 9.
Table 9 promotes that in the presence of hepatomicrosome enzyme (S-9) (be 72 hours detection time to TFu, n=3) to SGC7901 and NKM-45 cell growth inhibition (%)
4.5.3TFu suppress mice S180 sarcoma and the effect of mice H22 liver cancer cell growth:
With 0.20,0.40,0.80mmol/kg all has stronger inhibitory action to S180 sarcoma and H22 liver cancer cell growth.Between 41.81-59.55%, is 39.77-87.65% to the suppression ratio of H22 hepatocarcinoma to the suppression ratio of S180 sarcoma.In the experimentation, TFu increases the weight of animals inhibitory action.Wherein, obvious with the 0.80mmol/kg successive administration to the body weight gain inhibitory action, p＜0.05, table 10,11.
Table 10 TFu is to the inhibitory action of S180 murine sarcoma
Compare with matched group,
*P＜0.01,
*P＜0.05n=3,
Table 11TFu is to the inhibitory action of H22 rat liver cancer
Matched group compares,
*P＜0.05,
*P＜0.01, n=3
4.5.4TFu nude mice is transplanted SGC-7901 and the effect of NKM-45 gastric carcinoma cells growth inhibited: with 0.20,0.40,0.80mmol/kg, the continuous oral administration, to the SGC7901 inhibitory rate of cell growth is 43.55%, 60.30% and 71.07%, be 33.21%, 47.96% and 60.44% to the NKM-45 inhibitory rate of cell growth.In the therapeutic process, 0.80mmol/kg dosage has obvious inhibitory action to the growth of nude mice body weight, p＜0.05, table 12,13.
Table 12 TFu is to the effect of nude mice transplantability people SGC-7901 cell inhibiting
Compare with matched group,
*P＜0.05,
*P＜0.01, n=3
Table 13 TFu is to the effect of nude mice transplantability people NKM-45 cell inhibiting
Compare with matched group,
*P＜0.05,
*P＜0.01, n=3
With N3-neighbour toluyl fluorouracil (TFu), starch uniform mixing, be 100mg by the adjacent toluyl fluorouracil of N3-content, starch 200mg, the adding starch slurry is an amount of, constantly stirs as making wet granular by granulator.After 50 ℃ of oven dry, capsule machine is encapsulated to get final product.
Embodiment 6: present embodiment prepares the adjacent toluyl fluorouracil of a kind of N3-pellt capsule.
With adjacent toluyl fluorouracil (TFu) 100mg of N3-, microcrystalline Cellulose 400mg uniform mixing adds an amount of adhesive, gets soft material, can make micropill by the micropill machine.After 50 ℃ of oven dry, capsule machine is encapsulated to get final product.
Embodiment 7: present embodiment prepares the adjacent toluyl groups of a kind of N3--fluorouracil conventional tablet.
With adjacent toluyl groups-fluorouracil (TFu) 100mg of N3-, starch 200mg and disintegrating agent (5% weight) uniform mixing adds an amount of starch slurry, constantly stirs as making wet granular by granulator.After 50 ℃ of oven dry, add the lubricant tabletting and get final product.
Embodiment 8: present embodiment has prepared the adjacent toluyl groups of a kind of N3--fluorouracil Liposomal formulation.
Adjacent toluyl groups-the fluorouracil of N3-(TFu) is dissolved in the chloroform; S-PC (PC): the cholesterol mol ratio is to be dissolved in chloroform at 15: 1; the adjacent toluyl fluorouracil of N3-: the S-PC mol ratio is 1: 15, then with mixed solution evaporating solvent and the lipid that obtains approaching and dried lipid film of medicine in about vacuum below 30 ℃ or 30 ℃.Add the PBS buffer again, constantly stir as promptly getting liposome by violent mixing of vortex.The liposome that makes like this is a multilamellar liposome, after ultrasonic Treatment, can obtain the littler unilamellar liposome of particle diameter.
Embodiment 9: present embodiment has prepared the adjacent toluyl groups of a kind of N3--fluorouracil Liposomal formulation.
With S-PC (PC): the cholesterol mol ratio is to be dissolved in chloroform or ether at 15: 1, then with mixed solution evaporating solvent and dried lipid film of obtaining approaching in about vacuum below 30 ℃ or 30 ℃.Add the diethyl ether solution of the adjacent toluyl fluorouracil (TFu) of N3-(TFu: the PC mol ratio is 1: 15) again, add an amount of PBS buffer, constantly stir as promptly getting liposome by violent mixing of vortex.The liposome that makes like this is a multilamellar liposome, after the high pressure dispersing emulsification machine is handled, can obtain the littler unilamellar liposome of particle diameter.
Embodiment 10: present embodiment has proved the tumor killing effect of TFu liposome.
S
180Tumor inoculation: the selection tumor growth is vigorous and do not have diabrosis, the tumor-bearing mice that health condition is good, and dislocation is put to death, and is fixed on the operation plate, disinfects mouse skin in alcohol, strips tumor under the aseptic condition, and used apparatus is through scalding.Added physiological saline solution by 1: 2, make cell suspension with Potter-Elvehjem Tissue Grinders, in tucking in nest subcutaneous vaccination 0.2ml.Disinfect the skin of inoculation position before the inoculated tumour in alcohol.
Animal transplanting tumor growth inhibited experiment: extract 5 days S180 mouse tumor ascites of inoculation, make the tumor cell suspension after the normal saline dilution, adjust cell number to 1 * 107/ml, every right side of mice armpit subcutaneous vaccination 0.2ml.Random packet, 10 every group.Next day gastric infusion, 0.8ml/ only, dosage is respectively 50,100,200mg/kg, successive administration 12-15 days.After the last administration 24 hours, put to death animal, to get tumor and weigh, the computerized compound is to inhibition rate of tumor growth.Tumour inhibiting rate=(matched group tumor weight-experimental group tumor is heavy)/matched group tumor is heavy.Result of the test is as shown in table 14.
The tumor killing effect of table 14 TFu liposome
| The TFu liposome | 200mg/kg | 100mg/kg | 50mg/kg | The Cap positive control | Negative control group |
| Number of mice (beginning/end) | 10/4 | 10/9 | 10/9 | 10/10 | 13/13 |
| Body weight (g) | 19.28±2.78 | 22.8±3.29 | 23.8±2.45 | 22.02±3.55 | 22.2±3.56 |
| Tumor heavy (g) | 0.129±0.151 | 0.512±0.510 | 0.544±0.809 | 0.636±0.594 | 1.193±0.589 |
| Tumour inhibiting rate % | 89.2 | 57.1 | 54.4 | 46.7 |
Annotate: 1, the dosage group molal quantity of the dosage of Cap positive controls and TFu 200mg/kg is suitable;
2, in this body weight, the heavy statistical data of tumor, mice dead before the off-test is not counted
3, the mice body weight 12.9～16.9g of each dosage group death of TFu, the tumor weight average is: 0
Embodiment 11: present embodiment is described adjacent toluyl fluorouracil (TFu) mice of N3-LD
50Test.
11.1 animal is selected 44 of kunming mouses, body weight 18～22g, and male and female half and half: Shandong University experimental animal center provides.
11.2 the medicine preparation takes by weighing sample 3340mg, arabic gum 0.4g is ground to fine powder in mortar, adds a little carboxymethylcellulose sodium solution of 0.5% and continues to be ground to the evenly follow-up 20ml of adding to, and stirs evenly, and promptly gets the suspension that concentration is 167mg/ml.
11.3 44 of mice group mices, male and female are respectively done, and are divided into 4 groups at random, 11 of every group of mices, and the dosage spacing between each group is 0.7.
11.4 medication gives oral administration gavage 0.6ml, 0.42ml, 0.29ml, 0.21ml respectively by the difference of each group dosage, promptly observes the dead mouse situation after the administration, observes continuously 9 days.
11.5 test results test the results are shown in Table 15, the Bliss method is calculated.
Table 15 TFu mice LD
50Result of the test
Median lethal dose(LD 50) LD
50=3165.77mg/kg, 95% fiducial limit=2543.69～3939.98mg/kg
11.6 under experimental condition, given liposome Chinese medicine concentration increases the administration volume to maximum, does not observe the death condition of liposome administration group.
Embodiment 12: present embodiment is described the pharmacokinetic study behind the adjacent toluyl fluorouracil of free N3-and oral back of the adjacent toluyl fluorouracil of N3-Liposomal formulation and the quiet notes.
12.1. animal experiment method
12.1.1 the foundation of body inner analysis method
12.1.1.1 the selection of ultraviolet detection wavelength
The preparation of standard reserving solution: precision takes by weighing TFu standard substance an amount of (about 10mg) and puts in the 100ml volumetric flask, adds the mobile phase dissolving and is diluted to scale, shakes up, and is made into the standard reserving solution that concentration is 100 μ g/ml, and is standby.In the scanning of 200～400nm wavelength place, sample has absorption maximum at the 258nm place, and blank adjuvant is noiseless.
12.1.1.2 chromatographic condition
Chromatographic column: venusi lXBP C-18 post (4.6mm * 250mm, 5 μ m); Mobile phase: acetonitrile-distilled water (50: 50, V/V); Flow velocity: 1.0ml/min; Detect wavelength: 258nm; Column temperature: room temperature; Sample size: 20 μ l.
12.1.1.3 the processing of plasma sample
The accurate plasma sample 1mL that draws puts in the centrifuge tube, adds 0.5molL
-1NaH
2PO
4Solution 0.2ml, ethyl acetate 0.3ml, suspendible concussion 3min, centrifugal 5min (3000rmin
-1), get supernatant 0.1ml and be transferred in the 5ml centrifuge tube, nitrogen dries up in 50 ℃ of water-baths, and residue accurately with the dissolving of 0.5ml mobile phase, is got 20 μ l sample introductions.
12.1.1.4 the foundation of standard curve
12.1.1.5 the foundation of mice plasma standard curve
Precision is measured standard reserving solution in right amount to centrifuge tube, N
2Dry up, add mice blank plasma 0.5ml, the vortex mixing, being made into concentration is 0.5,1,4,6,20,40 μ gml
-1Serial pastille plasma sample, after carrying out pretreatment by aforementioned plasma sample processing method, sample introduction 20 μ l, the record chromatogram, try to achieve peak area A, with peak area A concentration C is carried out linear regression processing, get regression equation: A=12271C+5444.4 (r=0.9995), canonical plotting is seen accompanying drawing 9.
By accompanying drawing 9 as can be known, the TFu plasma concentration is at 0.5～40 μ gml
-1Scope internal linear relation good (r=0.9995), lower limit of quantitation is 0.5 μ g/ml.
12.1.1.6 mice plasma method recovery test
Precision is measured the TFu standard solution and is put in the centrifuge tube N in right amount
2Dry up, add mice blank plasma 0.5ml, be made into basic, normal, high (1,4,40 μ gml
-1) plasma sample of three kinds of concentration, pressing under the 1.1.3 item after the blood sample disposal methods, sample introduction 20 μ l try to achieve peak area A, try to achieve the method response rate.The results are shown in Table 16.
Table 16 mice plasma method recovery test result
12.2. mice interior medicine dynamics test
12.2.1 normal mouse gastric infusion
Get 66 of normal mouses, male and female half and half, body weight 27 ± 1g is divided into 2 groups at random, 33 every group.Gastric infusion, every mice is irritated stomach 0.5ml respectively, dosage 100mg/kg (pressing TFu calculates), fasting 12h before the administration freely drinks water.The 1st group is TFu suspendible aqueous solution group, and the 2nd group is TFu liposome group, gets blood respectively at oral preceding, oral back 5min, 15min, 30min, 45min, 1,2,4,6,12,24,48h in the eyeground vein clump, adds in the heparinization centrifuge tube 3500rmin
-1Centrifugal 15min gets upper plasma, and-20 ℃ of refrigerators are preserved to be measured.Handle and measure by plasma sample preprocess method and assay method; The determination of plasma concentration result is handled with DAS ver1.0 (Drug And Statistics for Windows) pharmacokinetics program, try to achieve pharmacokinetic parameter.Available pair of chamber model comes match, weight coefficient w=1/c
2Determination of plasma concentration the results are shown in Table 17; Draw matched group and the average blood drug level-time graph of liposome group, the results are shown in accompanying drawing 10, accompanying drawing 11.
Table 17 normal mouse gastric infusion determination of plasma concentration result (n=3)
Come match with two chambers model, weight coefficient w=1/c
2, crude drug group t
1/2Be 17h, area under the drug-time curve AUC is 18.4862 μ g/ml*h, and Cmax is 15.6900 (mg/L); Liposome group t
1/2Be 20h, area under the drug-time curve AUC is 26.6145 μ g/ml*h, and Cmax is 24.0000 (mg/L);
12.2.2 normal mouse intravenous administration
Get 66 of normal mouses, male and female half and half, body weight 27 ± 1g is divided into 2 groups at random, 33 every group.The tail vein injection administration, every mice is injected 0.1ml respectively, dosage 100mg/kg (pressing TFu calculates), fasting 12h before the administration freely drinks water.The 1st group is TFu solution (50% ethanol) group, the 2nd group is TFu liposome group, get blood respectively at 5min, 15min, 30min, 45min, 1,2,4,6,12,24,48h before the injection, after the injection in the eyeground vein clump, add in the heparinization centrifuge tube 3500rmin
-1Centrifugal 15min gets upper plasma, and-20 ℃ of refrigerators are preserved to be measured.Handle and measure by plasma sample preprocess method and assay method; The determination of plasma concentration result is handled with DASver1.0 (Drug And Statistics for Windows) pharmacokinetics program, try to achieve pharmacokinetic parameter.Available pair of chamber model comes match, weight coefficient w=1/c
2. determination of plasma concentration the results are shown in Table 18; Draw matched group and the average blood drug level-time graph of liposome group, the results are shown in accompanying drawing 12, accompanying drawing 12.
Table 18 normal mouse intravenous administration determination of plasma concentration result (n=3)
Come match with two chambers model, weight coefficient w=1/c
2, crude drug group t
1/2Be 18h, area under the drug-time curve AUC is 40.5624 μ g/ml*h, and Cmax is 19.0000 (mg/L); Liposome group t
1/2Be 103h, area under the drug-time curve AUC is 36.3720 μ g/ml*h, and Cmax is 8.10000 (mg/L);
As from the foregoing, oral crude drug, oral liposome, the absolute bioavailability of injecting lipid body is respectively 45.6%, 65.61%, 89.67%.
Claims (3)
1. therapeutic N
3-adjacent toluyl fluorouracil liposome composition is characterized in that, contains N
3-adjacent toluyl fluorouracil and lipid components, N
3The mol ratio of-adjacent toluyl fluorouracil and lipid components is 1: 15; liposome is the vesicle that has less than the 450nm median particle diameter; lipid components comprises the compositions of S-PC and cholesterol, and S-PC comprises cholesterol with 15: 1 mol ratio.
2. therapeutic N as claimed in claim 1
3-adjacent toluyl fluorouracil liposome composition is characterized in that described bag carries N
3The liposome of-adjacent toluyl fluorouracil comprises the vesicle with the following size of 0.1 μ m.
3. therapeutic N as claimed in claim 1 or 2
3-adjacent toluyl fluorouracil liposome composition is characterized in that described lipid components also has vitamin E.
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| CN2006100422460A CN1875973B (en) | 2006-02-10 | 2006-02-10 | A pharmaceutical composition containing N3-o-toluyl-fluorouracil and its liposome preparation |
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| CN101485887B (en) * | 2008-01-17 | 2011-06-29 | 中国人民解放军第二军医大学 | 5-fluorouracil-sn2-phosphatidyl choline copolymer as well as preparation method and application thereof |
| CN102488655B (en) * | 2011-12-01 | 2013-07-24 | 山东大学 | N3-ortho toluene formoxyl uracil nanosuspension and lyophilized preparation thereof |
| CN107496357A (en) * | 2017-08-29 | 2017-12-22 | 平顶山学院 | A kind of N3-O-toluyl-flulorouracil liposome and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4196202A (en) * | 1977-12-20 | 1980-04-01 | Taiji Okada | Carcinostatic composition containing 3-N-o-toluyl-5-fluorouracil producing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4196202A (en) * | 1977-12-20 | 1980-04-01 | Taiji Okada | Carcinostatic composition containing 3-N-o-toluyl-5-fluorouracil producing the same |
Non-Patent Citations (1)
| Title |
|---|
| 平其能.现代药剂学 1998年 1.中国医药科技出版社,1998,588-593、618. * |
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