CN107569686A

CN107569686A - Medical composition and its use comprising vital stain and metallic compound

Info

Publication number: CN107569686A
Application number: CN201610519691.5A
Authority: CN
Inventors: 邹方霖; 卢星; 邹礼常; 王建霞
Original assignee: Chengdu Kuachang Science and Technology Co Ltd
Current assignee: Chengdu Kuachang Science and Technology Co Ltd
Priority date: 2016-07-05
Filing date: 2016-07-05
Publication date: 2018-01-12

Abstract

The present invention relates to a kind of pharmaceutical composition and its application in mammal local patholoic change relevant disease is prevented and treated comprising pharmaceutically acceptable vital stain and metallic compound.

Description

Medical composition and its use comprising vital stain and metallic compound

Technical field

The present invention relates to a kind of pharmaceutical composition comprising pharmaceutically acceptable vital stain and metallic compound and its Application in the local administration of prevention and treatment mammal local patholoic change relevant disease.

Background technology

Vital stain is one kind in dyestuff.Studies have shown that vital stain has the work of different effectiveness to multiple biological targets Property.By taking methylenum careuleum as an example, its main activity is dyeability (being used as coloring agent), light sensitivity (being used as sensitising agent) and removing toxic substances Effect (treats exogenous methemoglobinemia and cyanide poisoning) as intravenous antidote.About next active report A lot, including for treating the activity of following disease:Anti parasitic, tumor suppression, resist non-struma big (hemangioma), anti-local inflammation, Anti-microbial infection, anti-function of organization abnormal (trigeminal neuralgia) etc..But methylenum careuleum is clinically still mainly used as dyeing Agent (main activity), the application of other activity (secondary activity) are very limited.

In its main active clinical practice, the usual validity of vital stain is higher and adverse reaction is less.And secondly Activity is not by a large amount of clinical practices, otherwise it is not high to be that validity carries at all, otherwise it is when improving dosage to improve validity Side effect also significantly increases.For example, when methylenum careuleum is injected intravenously dosage excessive (500mg), you can cause nausea, vomit, backache, Precordial pain, lip, nail are livid purple, shiver with cold, headache, dizziness, perspire and clouded in mind.In a word, it is existing with these applications The drug effect (validity and security) of clinical medicine, which is compared, does not show obvious competitive advantage.

In order to improve main active (such as photosensitive activity) curative effect of vital stain, or/and next activity is (such as antitumor Activity) also transition into the drug effect of clinical competitiveness, with through having carried out long-term research in global range.However, result of study Lose and be obviously improved its competitiveness.Thus, vital stain can only be used as a kind of sensitising agent or synergist to use.

Clinically, metallic compound is mainly used in extra-nutrition metal, such as is mainly used in treatment iron-deficient containing molysite Anaemia.Movable influence of the metal salt for local lesion's (such as tumour) is complex.By taking iron as an example, when asiderosis, Caspase signalling channels are activated.But when iron builds up excessive, ROS can be caused to increase again, the consequence of primary cellular defect may be caused. Iron is again relevant with the elimination of free radical simultaneously, helps to protect cells from peroxide damage.Numerous results of study show, machine The reserves of iron and the risk positive correlation of many tumours in body, such as the carcinoma of the rectum, liver cancer, kidney, lung cancer and stomach cancer.

Most medicines are developed in the form of systemic administration.When it is employed, still its be local administration Using when, it is not only possible to show entirely different drug effect, and the shared effect of metallic compound and vital stain also height It is uncertain, it may be possible to antagonism, addition, synergistic effect.

The treatment of local patholoic change relevant disease at present, mainly operation, radiation cure and chemotherapy.Operation causes to body Injury, it is especially frustrating on some injuries for influenceing gland (such as mammary gland and thyroid gland) attractive in appearance.Radiation cure Side effect is also clearly.Existing chemotherapy is substantially systemic administration, there is very high systemic side effects risk.It is for example, existing Anticarcinogen has also killed a large amount of normal cells while a wide range of interior killing cancer cell.Some are used for the chemotherapy of Local treatment Agent is just under development.Some curing agents (such as absolute ethyl alcohol, urea, etc.) use get up more.However, these curing agents It is not high with very high local irritation and curative effect.

The content of the invention

The goal of the invention of the present invention, which is that exploitation is a kind of, safer compared with prior art effectively contains vital stain and gold Belong to composition, the pharmaceutical preparation comprising said composition and its local patholoic change relevant disease for treating mammal of compound In application.

According to an aspect of the present invention, it provides one kind and includes pharmaceutically acceptable vital stain and metallic compound Pharmaceutical composition, wherein the metal salt and relative quantity (w/w) of the vital stain in the pharmaceutical composition are 1:2-1: 200th, preferably 1:5-1：100.

In one embodiment, the metallic compound is to be selected from one below kind or a variety of metal salts：Molysite, copper Salt, zinc salt, manganese salt, aluminium salt, preferably molysite and mantoquita, more preferably selected from one below kind or a variety of ferrous salts：Dichloro Change iron, ferrous sulfate, ferrous succinate, ferrous fumarate, ferrous gluconate, ferrous lactate.

In one embodiment, the vital stain is selected from the one or more of following organic dyestuff and its derivative： Methylenum careuleum, patent blue, isosulfan blue, toluidine blue, trypan blue, alkali blue, Yihong, basic fuchsin, crystal violet, gentian violet, neutrality Red, janus green B, sarranine；It is preferably selected from the one or more of following organic dyestuff and its derivative：Methylenum careuleum, patent blue, Isosulfan blue, crystal violet, dimethyl diaminophenazine chloride, the one or more more preferably selected from following organic dyestuff and its derivative：Methylenum careuleum, specially Sharp blue, isosulfan blue.

In one embodiment, described pharmaceutical composition also contributes to strengthen prevention and treatment comprising one or more The active material of drug effect.The active material can show effective antitumor activity when being included in systemic administration in safe dose Active material, it is preferably selected from following compound and its one or more of derivative：Adriamycin, cis-platinum, taxol, Changchun are new Alkali, endoxan, 5 FU 5 fluorouracil (hereinafter referred to as effective antitumor material).The active material is additionally included in safe dose Effective antitumor activity is not shown during interior systemic administration but still has the active material of certain antitumor activity (hereinafter referred to as Poorly efficient antitumorigenic substance), it is preferably selected from one below kind or a variety of：Nonsteroidal anti-inflammatory compound, quinolines, qinghaosu Derivative, phenolic compound.

In further embodiment, wherein the nonsteroidal anti-inflammatory compound is selected from one of the following group kind or a variety of： Salicylic acid compounds, phenoxy propionic acid anti-inflammatory compound, indoles anti-inflammatory compound, phenyl amines anti-inflammatory compound, fenamic acids anti-inflammatory Compound, pyrazolone anti-inflammatory compound, are preferably selected from salicylic acid compounds, more preferably selected from salicylic acid, acetyl water One or more in poplar acid, diflunisal, sasapyrin, bicoumarin.

In one embodiment, the quinolines are selected from one below kind or a variety of：Quinine, chloroquine, primaquine Quinoline and its isomers and derivative, it is preferably selected from quinine and quinine derivative, one kind or more of more preferably following compound Kind：Quinine, a quinin hydrochloride and quinine dihydrochloride.

In further embodiment, the artemisinin derivative includes the ester derivative of qinghaosu, ethers derives Thing, peptide derivative, dimer derivate and polymer derivative etc., preferably Artesunate (Artesunate, ATS), double hydrogen Qinghaosu (dihydroartemisinin, DHA), Artemether (artemether), arteether.

In further embodiment, the phenolic compound is selected from one below kind or a variety of：Nitrophenols chemical combination Thing, chlorinated phenols and aminophenol compound, Nitro-phenols and aminophenol compound are preferably selected from, are more preferably selected from The one or more of following compound：2,4- dinitrophenol, metanitrophenol, paracetamol and its derivative.

In further embodiment, described pharmaceutical composition can also include curing agent and/or anodyne.

In one embodiment, it is Topical dosage forms according to the pharmaceutical composition of the present invention, preferably following formulation： Target area is injected and/or perfusion formulation, respiratory tract administration formulation, surface administration formulation, mucosa delivery formulation and cavity/canal drug administration formulation.

In further embodiment, according to the present invention Topical dosage forms for liquid preparation, semisolid preparation or The liquid stock solution of person's gaseous formulation, wherein the concentration of the metallic compound (such as molysite or mantoquita, especially ferrous salt) is 0.02-2%w/v, it is preferably 0.05-1.5%w/v, more preferably 0.1-1.2%w/v or 0.1-1.6%w/v, the live body The concentration of dyestuff is 1-6%w/v, preferably 1-5%w/v, 1.5-5%w/v or 2-5%w/v, more preferably 2-4%w/v or 3-4%w/v.

In further embodiment, the curing agent is preferably liquid hardening agent, more preferably alcohol-based liquid curing agent, Such as ethanol, propane diols, liquid PEG, glycerine, isopropanol etc., it is preferably selected from one of the following group kind or a variety of：Ethanol, third Glycol, liquid PEG.Now, these alcohol compounds are both used as curing agent, may also function as the effect of solvent, and its concentration can be 55-95%v/v, preferably 60-80%v/v.When being shared such as ethanol, propane diols, liquid PEG, the concentration ratio (v/v between three:v/ v:V/v) it is preferably (0.5-1):(0.5-1):(0.5-1).

In further embodiment, in the pharmaceutical composition according to the present invention of Topical dosage forms, if deposited The concentration of the effective antitumor material can be 0.1-10%w/v, and the concentration of the poorly efficient antitumorigenic substance can be 0.1-15%w/v.

In further embodiment, in the pharmaceutical composition according to the present invention of Topical dosage forms, if deposited The concentration of the phenolic compound can be 0.1-10%w/v, preferably 0.1-8%w/v, wherein the Nitro-phenols Concentration can be 0.1-1%w/v, and the concentration of the aminophenol compound can be 2-10%w/v.

In further embodiment, in the pharmaceutical composition according to the present invention of Topical dosage forms, if deposited The concentration of the nonsteroidal anti-inflammatory compound is 1.5-15%w/v, preferably 3-15%w/v, more preferably 5-12%w/v.

In further embodiment, in the pharmaceutical composition according to the present invention of Topical dosage forms, the quinoline The concentration of quinoline class compound is 1.5-10%w/v, preferably 2-10%w/v, more preferably 3-8%w/v.

In further embodiment, in the pharmaceutical composition according to the present invention of Topical dosage forms, the green grass or young crops The concentration of artemisin derivative is 1-6%w/v, preferably 2-5%w/v, more preferably 3-5%w/v.

According to another aspect of the present invention, it is also provided is preparing medication treatment office according to the pharmaceutical composition of the present invention Application in the medicine of portion's focus lesion, wherein local lesion's lesion includes one or more of：Tumour, non-struma Greatly, local inflammation, microorganism infection and function of organization are abnormal.

According to a further aspect of the invention, it also provides a kind of method for treating local lesion's lesion, wherein the office Portion's focus lesion includes one or more of：Tumour, non-struma are big, local inflammation, microorganism infection and function of organization are abnormal, This method includes the pharmaceutical composition according to the present invention to the local administration of individual thus needed.

Advantages below is had according to embodiment of the present invention compared with prior art：Controlled with existing operation and radioactive ray Treatment is compared, and shows same efficient more friendly " treatment-patient body " relation (being disturbed to body smaller)；With it is existing Systemic chemotherapy medication is compared, and can improve curative effect to conspicuousness under the toxic side effect of minimum；Used with existing local chemotherapy Medicine, especially curing agent (such as absolute ethyl alcohol) treatment are compared, and the compatibility between perilesional tissue is higher, and curative effect is more It is good；Compared with the composition of the prior art containing salicylic acid compounds, it is free of the material for being pharmaceutically difficult to receive, and logical Cross maximize the drug effect of salicylic acid compounds using best of breed dosage, side effect minimize.In addition, pass through different pharmacology The introducing of medicine (such as alcohol-based liquid curing agent, antitumorigenic substance etc.) also generate extra synergy, so as to Further improve the security and validity for the treatment of.In addition, prepared by said composition, convenient, cost is cheap, helps to make to be difficult to hold Effective treatment is also enjoyed by many people of high cost.

The present invention is described in more detail below with reference to accompanying drawing.

Brief description of the drawings

Fig. 1 provides an Experiment on therapy result using pancreatic tumor borne cell nude mice as model, and wherein Figure 1A shows dichloride Concentration of iron is fixed on 0.4% and the tumour inhibiting rate curve when 0-4% changes methylenum careuleum concentration, and Figure 1B methylenum careuleum concentration is fixed on 1% and 0-1% change ferrous chloride concentration when tumour inhibiting rate curve.

Embodiment

In the present invention, unless otherwise indicated, liquid phase compound concentration at room temperature is with volume/volume percent concentration (v/v%) represent, solid-phase compound concentration is represented with weight/volume percent concentration (w/v%).

The composition of the present invention, is also abbreviated as the component 2 of component 1/ or the component 3 of 1/ component of component 2/ in the following description. The concentration of each component in composition, can also be preced with before component.Such as the methylenum careuleum of 0.4% ferrous chloride/5% refers to one The concentration of the composition of ferrous chloride and methylenum careuleum, wherein ferrous chloride is 0.4%, and the concentration of methylenum careuleum is 5%.It is other multigroup It is grouped the shorthand way of compound by that analogy.

According to an aspect of the present invention, it provides one kind and includes pharmaceutically acceptable vital stain and metallic compound Pharmaceutical composition, wherein the metal salt and relative quantity (w/w) of the vital stain in the pharmaceutical composition are 1:2-1: 200th, preferably 1:5-1：100.The present inventor conducts in-depth research for vital stain compound, is surprised to find The pharmaceutical composition of metallic compound and vital stain with aforementioned proportion can treat or suppress tumour, non-struma Greatly, the abnormal aspect of local inflammation, microorganism infection and function of organization has unexpected synergy.

Within the scope of this invention, term " metallic compound " includes metal salt and organometallic comprising respective metal Compound (it does not include the salt that metal and organic acid are formed), wherein the metal for example can be iron, copper, magnesium, zinc, manganese, aluminium and One or more in calcium.The metallic compound is preferably selected from one below kind or a variety of metal salts：Molysite, mantoquita, magnesium Salt, zinc salt, manganese salt, aluminium salt and calcium salt, preferably molysite and mantoquita.

The salt can be the salt that the metal is formed with inorganic acid or organic acid.The inorganic acid and organic acids are as wrapped Include sulfuric acid, nitric acid, phosphoric acid, boric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, acetic acid, benzoic acid, benzene sulfonic acid, camphorsulfonic acid, citric acid, Cyclamic acid, esilate, formic acid, butanedioic acid, albumen butanedioic acid, cholic acid, fumaric acid, glucoheptonic acid, gluconic acid, glucose Aldehydic acid, isethionic acid, lactic acid, fumaric acid, malic acid, maleic acid, malonic acid, methanesulfonic acid, methylsulfuric acid, nicotinic acid, breast Clear acid, oxalic acid, palmitic acid, pyroglutamic acid, saccharic acid, stearic acid, succinic acid, tannic acid, tartaric acid, toluenesulfonic acid and trifluoro second Acid etc..

For molysite, it includes iron (III) ion salt and ferrous (II) ion salt, and it is collectively referred to as molysite.Iron Salt includes any pharmaceutically acceptable molysite, can be iron ion or ferrous ion and above-mentioned inorganic acid or organic acid shape Into salt.For example, ferrous ion salt includes ferrous chloride, ferrous sulfate, ferrous succinate, ferrous fumarate, gluconic acid Asia Iron, ferrous lactate etc..Iron (III) ion salt includes such as Ferric Ammonium Citrate, iron-dextrin, iron protein succinylate.In basis In the pharmaceutical composition of the present invention, the metallic compound is preferably ferrous ion salt, more preferably selected from one below kind or A variety of ferrous salts：Ferrous chloride, ferrous sulfate, ferrous succinate, ferrous fumarate, ferrous gluconate, ferrous lactate.

The metallic compound also includes metallo-organic compound, such as transferrins, iron content ferroheme, amino-acid complex Iron, polyferose etc..

Within the scope of this invention, term " vital stain " refers to make in local lesion's lesion during local be administered Dyestuff within the scope of color but its side effect and pharmaceutically acceptable.The vital stain can be art technology Any appropriate person known to personnel, such as can include one below kind or a variety of organic dyestuff and its derivative：Methylenum careuleum (also including hydrate in the present invention), patent blue, isosulfan blue, toluidine blue, trypan blue, alkali blue, Yihong, basic fuchsin, knot Crystalviolet, gentian violet, dimethyl diaminophenazine chloride, janus green B, sarranine；It is preferably selected from one kind or more of following organic dyestuff and its derivative Kind：Methylenum careuleum, patent blue, isosulfan blue, crystal violet, dimethyl diaminophenazine chloride, more preferably selected from one of following organic dyestuff and its derivative Kind is a variety of：Methylenum careuleum, patent blue, isosulfan blue.By taking methylenum careuleum as an example, its derivative is generally also dyestuff, such as 1,9- diformazan Base methylenum careuleum, 1- methyl methylenum careuleum etc..

According in one embodiment of the invention, described pharmaceutical composition, which can also include one or more, to be helped to strengthen The active material of the drug effect of prevention and treatment.The active material can be shown effectively when being included in systemic administration in safe dose The active material of antitumor activity, it is preferably selected from following compound and its one or more of derivative：Adriamycin, cis-platinum, Taxol, vincristine, endoxan, 5 FU 5 fluorouracil (hereinafter referred to as effective antitumor material).The active material is also It may include not showing that effective antitumor is active but still has the activity of certain antitumor activity during the systemic administration in safe dose Material (hereinafter referred to as poorly efficient antitumorigenic substance), is preferably selected from one below kind or a variety of：Nonsteroidal anti-inflammatory compound, quinoline Class compound, artemisinin derivative, phenolic compound.

Within the scope of this invention, term " nonsteroidal anti-inflammatory compound " refers to not contain steroidal structure but has anti-inflammatory The compound and its derivative of effect.NSAIDs (Nonsteroidal Antiinflammatory Drugs, NSAIDs) it is the nonsteroidal anti-inflammatory compound with clinical competitiveness.The nonsteroidal anti-inflammatory compound can be this area skill Any appropriate person known to art personnel, it can include such as salicylic acid compounds, phenoxy propionic acid anti-inflammatory compound (such as cloth Ibuprofen, Fenbid, naproxen etc.), indoles anti-inflammatory compound (such as Indomethacin, sulindac etc.), phenyl amines anti-inflammatory chemical combination Thing (such as phenacetin, N- (4- hydroxy phenyls) antifebrin (paracetamol)), fenamic acids anti-inflammatory compound (such as first Fenamic acid, clofenamic acid, Diclofenac and flufenamic acid etc.), acetic acid class anti-inflammatory compound (such as C14H10Cl2NNaO2 etc.), happiness health class anti-inflammatory Compound (such as feldene etc.), pyrazolone anti-inflammatory compound (such as phenylbutazone, crovaril etc.).The present invention's In embodiment, the nonsteroidal anti-inflammatory compound is preferably salicylic acid compounds.

Within the scope of this invention, term " salicylic acid compounds " refers to salicylic acid and its derivative.The salicylic acid The chemical entitled 2 hydroxybenzoic acid of (Salicylic acid).The salicyclic acid derivatives can be those skilled in the art Any appropriate person known, its can be include such as metalline salicyclic acid derivatives and not metalline salicylic acid derive Thing.The former can include such as sodium salicylate, magnesium salicylate, zinc salicylate, metallic element complex compound (such as aspirin Copper) etc., the latter can include such as acetylsalicylic acid (Aspirin aspirin), di-lysine-aspirin, diflunisal, ammonia Base salicylic acid, PAS, N- phenylanthranilic acids, salicylanilide, O-ethoxyl acid amides, salicylic acid benzene Ester, gaultherolin, methyl p-hydroxybenzoate, ethyl-para-hydroxybenzoate, sasapyrin, bicoumarin and their medicine The upper acceptable derivates of thing.By taking aspirin therein as an example, salicyclic acid derivatives can be include for example its single spread out Derivative that biological (such as low-density lipoprotein, hydrogen sulfide aspirin etc.), two kinds of derivatives are formed (such as an oxidation Nitric oxide hydrogen sulfide (NOSH) aspirin that nitrogen aspirin and hydrogen sulfide aspirin are formed).According to the present invention's In pharmaceutical composition, the salicylic acid compounds are selected from salicylic acid and following compound and its one kind of derivative It is or a variety of：Acetylsalicylic acid, diflunisal, sasapyrin, bicoumarin.

Within the scope of this invention, term " phenolic compound " refers to the compound containing phenolic group group in its chemical constitution. The phenolic compound can be any appropriate person well known by persons skilled in the art, preferably uncoupling reactive phenolic compounds, It can include such as Nitro-phenols, chlorinated phenols and aminophenol compound.

The Nitro-phenols can include such as 2,4-DNP and (dinitrophenol, are abbreviated in of the invention Make DNP), metanitrophenol, o-nitrophenol etc. and their derivative.DNP derivatives can include for example 2,4- dinitros- 1- naphthols (Ma Diwusi is yellow), 4,6- dinitro-o-cresols (Victoria yellow), phenols fluorine curcumin derivative etc..It is described Chlorinated phenols can include such as 2,4,5- trichlorophenol, 2,4,6,-Ts, 3,3 ', 4 ', 5- tetrachlorosalicylanilides (TCS) etc. and they Derivative.The aminophenol compound can include such as paracetamol and its derivative.

In the pharmaceutical composition according to the present invention, the phenolic compound is preferably selected from Nitro-phenols and amino Phenolic compounds, the more preferably one or more selected from following compound：2,4- dinitrophenol, metanitrophenol, to acetyl Amino phenols and its derivative.

Within the scope of this invention, term " artemisinin derivative " refers to the derivative of qinghaosu.Qinghaosu (artemisinin, ART) is a kind of sesquiterpene lactone chemical combination of the group containing endoperoxides extracted from feverfew artemisia annua Thing, its molecular formula are C₁₅H₂₂O₅.The artemisinin derivative can be any appropriate person well known by persons skilled in the art, and it can To be ester derivative, ether derivative, peptide derivative, dimer derivate and the polymer derivative for including qinghaosu Deng preferably Artesunate (Artesunate, ATS), dihydroartemisinine (dihydroartemisinin, DHA), Artemether (artemether), arteether etc..

Within the scope of this invention, term " quinolines " refers to the quinoline that pharmaceutically acceptable contains quinoline ring Quinoline derivant, such as including chloroquine (chloroquine), HCQ quinoline, ammonia first quinoline, amodiaquine, quinine, nitroquine, PIPERAQUINE, primaquine Quinoline (primaquine), pamaguine, pentane quinoline and its isomers and pharmaceutically acceptable salt, wherein preferably quinine, chlorine Quinoline, primaquine and its isomers and pharmaceutically acceptable salt, especially quinine and its isomers and pharmaceutically it is subjected to Salt.

The pharmaceutically acceptable salt of quinolines includes acid-addition salts, as inorganic acid addition salt and organic acid add Into salt.The example of inorganic acid addition salt includes but is not limited to：Halogen acid salt (such as hydrochloride, such as quinin hydrochloride), hydrobromate, Hydriodate, dihydrochloride (such as quinine dihydrochloride), dihydrobromide, two hydriodates), disulfate, sulfate, phosphoric acid Salt (such as chloroquine diphosphate, piperaquine phosphate, primaquine phosphate) etc..The example of organic acid addition salt includes but is not limited to：Bicarbonate Salt or carbonate, ethyl carbonate salt, formates, acetate, Chinese holly edge hydrochlorate or tannate.

For quinine, its isomers for example can be quinindium, cinchonine and cinchonidine, and its salt for example can be Quinin hydrochloride, quinine dihydrochloride, quinine sulfate etc..

Above-mentioned quinolines can be used alone, and also can be combined and uses, such as quinin hydrochloride and other quinolines Combination, the composition of quinine dihydrochloride and other quinolines, the composition of primaquine and other quinolines, chloroquine With the composition of other quinolines.

According to the present invention, the quinolines are selected from one below kind or a variety of：Quinine, chloroquine, primaquine and its Isomers and derivative, it is preferably selected from quinine and quinine derivative, the one or more of more preferably following compound：Kui Rather, a quinin hydrochloride and quinine dihydrochloride.

In further embodiment, curing agent and/or anodyne can also be included according to the pharmaceutical composition of the present invention.

Within the scope of this invention, term " curing agent " refers to local organization can be caused to produce aseptic inflammation, causes scorching group Constantly it is fiberized during being woven in physiology reparation, finally makes the chemistry system of local-pathological-changed tissues necrosis, diminution and fibrosis Agent.It for example can be liquid hardening agent and solid hardener, and the latter is for example including one below kind or a variety of：Lauromacrogol, fish Liver enuatrol, ethanolamine oleate, polidocanol, urea, sodium sulphate, bleomycin A5, bleomycin etc..

Within the scope of the invention, term " alcohol-based liquid curing agent " refers to there is curing agent in liquid at normal temperatures The alcohol compound of property.It is well known that many alcohols materials all have the property of curing agent.Alcohol-based liquid curing agent can be Any appropriate person well known by persons skilled in the art, such as including one below kind or a variety of：It is ethanol, propane diols, glycerine, different Propyl alcohol, liquid macrogol (abbreviation PEG in the present invention), are preferably selected from lower Group one or more：Ethanol, propane diols, Liquid macrogol.Alcohol-based liquid curing agent can play a part of solvent under many circumstances, but in locally administration and highly concentrated Under conditions of degree, influence of its curing agent effect to pharmacology is bigger.For example, ethanol is turning into tumour, especially non-malignant swollen One of the most frequently used curing agent in knurl treatment.

In the pharmaceutical composition according to the present invention, the content of alcohol-based liquid curing agent for example can be 55-95%v/v, It is preferred that 60-80%v/v, and Lauromacrogol, sodium morrhuate, ethanolamine oleate, polidocanol, bleomycin A5 and bleomycin Concentration can be respectively 0.5-3.0%, and the concentration of sodium sulphate is 5-30%, and the concentration of urea is 10-40%.

The anodyne can be any appropriate person well known by persons skilled in the art, to mitigate the pain of patient, example Such as phenmethylol, procaine hydrochloride, anesin, hydrochloric acid benefit card.If it does, the concentration of anodyne for example can be 0.1-3% (weight).Such as the concentration of phenmethylol can be 0.1-3%, procaine hydrochloride, anesin, hydrochloric acid benefit The concentration of card can be respectively 0.1-2%.

In further embodiment, according to the present invention pharmaceutical composition can also include to increase pharmaceutical activity into Divide the immunologic adjuvant of drug effect.The immunologic adjuvant can be any appropriate person well known by persons skilled in the art, add it to promote Enter the related activity for being beneficial to treat the antigen of tumour in ill area, it for example can be inorganic adjuvant, such as aluminium hydroxide, bright Alum etc.；It is microorganism and its product such as mycobacteria (tubercle bacillus, BCG vaccine), bacillus pumilis, Bordetella pertussis, endotoxin, thin Fungus extract (muramyl dipeptide) etc.；Synthetic adjuvant, such as artificial synthesized double stranded polynucleotide (double-strand polyadenylic acid, urine Thuja acid), levamisol, isoprinosine etc.；Finish, such as Fei Shi adjuvants, adjuvant 65, mineral oil, vegetable oil；It is immune Stimulant (BCG vaccine, corynebacteria, endotoxin, trehalose, thymic peptide, OK432 etc.)；Cell factor, such as interferon, leucocyte Interleukin, TNF, TGF, colony stimulating factor, chemotactic factor (CF), thymosin extrasin etc.；Heterogenetic antigen, such as inactivate Streptococcus, human red blood cells membranous antigen, tumor infiltrating lymphocyte, tumor vaccine etc..If it does, the concentration of immunologic adjuvant is for example For 0.01-1% (weight).

The composition of the present invention, can also optionally include other treatment medicine.The other treatment medicine can be ability Any appropriate person known to field technique personnel, the treatment level that it is advantageous to further improve human or animal patient is added, such as Improve drug effect or improve resistance to the action of a drug of these other treatment medicines etc..

In the pharmaceutical composition according to the present invention, it includes pharmaceutically acceptable carrier or excipient.The load Body or excipient can be any appropriate persons well known by persons skilled in the art, and it can be for example including one below kind or a variety of： Decentralized medium, preservative, stabilizer, wetting agent and/or emulsifying agent, solubilizer, tackifier, ease up for adjusting the salt of osmotic pressure Electuary.The tackifier are, for example, that sodium carboxymethylcellulose, carboxymethyl cellulose, glucan, polyethylene adjoin pyrrolidone or gelatin. The solubilizer is, for example, meglumine, dimethyl sulfoxide (DMSO), formamide, Tween80.The preservative is such as antioxidant example (such as ascorbic acid) or microbicide (such as sorbic acid or benzoic acid).

Pharmaceutical composition according to the present invention can be solid pharmaceutical preparation, semisolid preparation, liquid preparation and gaseous state preparation Formulation.The preparation or formulation include but is not limited to powder agent, spray, cream, ointment, suppository, gel, paste, Lotion, ointment, supensoid agent, solution, elixir, syrup.It will be appreciated by those skilled in the art that according to required dosage and medicine generation Kinetic parameter, different preparations can be made in the compound of the present invention.More specifically, pharmaceutical composition of the invention can be with It is injection, spray, aerosol, powder spray, externally used solution agent, lotion, liniment, ointment, emplastrum, paste, patch, drop Eye agent, nasal drop, ophthalmic ointment, gargle, sublingual tablets, sticking tablet, patch, suppository, aerosol, effervescent tablet, drops, Pill.

According to the present invention pharmaceutical composition be preferably Topical dosage forms, injected preferably by target area, respiratory tract to Medicine, surface administration, the formulation of mucosa delivery and cavity/canal drug administration.

According to one embodiment of the invention, composition of the invention is solution.The solution can be pass through by It is that composition components are dissolved in decentralized medium and formed or mentioned component is individually dissolved in decentralized medium, so Mix and formed afterwards.The decentralized medium of the solution includes solvent, preferably pharmaceutically suitable hydrophilic solvent.Term " parent Water-soluble matchmaker " refers to there is hydrophilic solvent, and it includes such as miscible organic solvent of water, water or can comprising water and water The vehicle system of misci-ble organic solvents.

According to one embodiment of the invention, composition of the invention is supensoid agent.The supensoid agent can be pass through by Composition components are suspended in the supensoid agent formed in decentralized medium or mentioned component individually are suspended in into scattered Jie In matter then mixing and formed.The decentralized medium of the suspension can with any appropriate person well known by persons skilled in the art, Such as it is usually used in injecting purpose vegetable oil, artificial oil or semi synthetic base oils as oily group.Vegetable oil can be such as cottonseed oil, almond Oil, olive oil, castor oil, sesame oil, soybean oil and peanut oil.

By taking composition solution agent (such as injection) as an example, it can be prepared in accordance with the following methods.

Method one：If contained vital stain, metallic compound and the other compositions (example being optionally present in composition Such as effective or poorly efficient antitumorigenic substance, curing agent, other medicines, anodyne) required concentration in water-soluble concentration model When enclosing interior, then corresponding injection can be obtained by these raw materials being directly added into water for injection.

Method two：If the institute of contained vital stain, metallic compound and the other compositions being optionally present in composition Need concentration to have the not person in water-soluble concentration range, then these raw materials are dissolved in liquid alcohols curing agent, then be diluted with water to required Concentration can obtain corresponding injection.

Method three：The solution of (such as passing through the above method) containing whole components is prepared first, then prepared (such as pass through Lyophilized technique) it is dry powder, combined using preceding be mixed to form the dry powder with the solvent of the liquid alcohols curing agent containing required concentration Thing solution.

Method four：The solution of (such as passing through the above method) component containing part is prepared first, then prepared (such as pass through Lyophilized technique) it is dry powder, using preceding by the dry powder and solution (such as the liquid alcohol containing required concentration containing other part component The solution of class curing agent) it is mixed to form composition solution.

By taking respiratory tract administration formulation (such as spray) as an example, its can be added outside vital stain and metallic compound with Lower one or more excipient prepare its liquid stock solution：Tween-80, benzalkonium chloride, microcrystalline cellulose-carboxymethyl cellulose Plain sodium etc..Its preparation method refers to above-mentioned composition solution manufacturing method.

By taking cavity/canal drug administration formulation (such as suppository) as an example, it can be added outside salicylic acid compounds and metallic compound It is prepared by one or more of excipient：Cottonseed oil, apricot kernel oil, olive oil, blue or green punt-pole oil, menthol, borneol, solubility emulsifiability agent Deng.Its preparation method refers to the preparation method of above-mentioned composition solution.

By the principle of these above-mentioned methods, those skilled in the art can be prepared a variety of using any appropriate specific method Local administration preparation comprising the present composition.For example, the change in the medical preparation of the present invention includes：Containing variety classes and The vital stain of concentration, the active material containing variety classes and concentration, the metallic compound containing variety classes and concentration, containing difference The other compositions of species and concentration (such as other active drugs, anodyne, curing agent etc.).

Within the scope of the invention, it is effective acting time of the increase pharmaceutical composition of the present invention at administration, improves it Bioavilability, it for example can be with slow release formulation medication.Those skilled in the art can use any appropriate specific method Prepare a variety of slow release formulation preparations comprising the present composition.For example, the slow release formulation of pharmaceutical composition of the present invention, Ke Yitong Cross and added outside metallic compound, vital stain as slow-released carrier or the micro particles of matrix, nano-particle, micella and original It is prepared by position gel-type vehicle.

It is well known that the initial release amount of sustained release preparation is 20-50%.Thus, it will be appreciated by those skilled in the art that slow Release dosage form is generally administered with the 2-5 multiple doses or concentration of regular dosage form.For example, in the slow release formulation of the present composition, gold The concentration for belonging to compound can be 0.04-10%w/v, and it is preferably 0.1-7.5%w/v, more preferably 0.2-6%w/v, described The concentration of vital stain can be 2-30%w/v, more preferably preferably 3-20%w/v, 4-15%w/v.

Within the scope of the invention, term " local lesion " refers to the part that primary or secondary disease becomes.Term is " local Focus lesion " refers to all structures related to lesion pathology in local lesion, and it can be well known by persons skilled in the art Any appropriate person, such as can include one or more of：Tumour, non-struma are big, local inflammation, microorganism infection and group Knit dysfunction.Term " part " includes the part in following organ：Secretory, blood circulation where excretory system Cardiovascular organ and skin where system.Local lesion's lesion of these organ or tissues includes one or more of：It is swollen Knurl, non-struma are big, local inflammation, microorganism infection and function of organization are abnormal.One example of cardiovascular organ disease is hemangioma.

Within the scope of the invention, " local application " refers to that its medicine composition (active component and its concentration) can pass through part Means are given is also suitable for the requirement of the target area environment so as to produce the medicine of curative effect with to target area and medicine composition.Term " target " Refer to local patholoic change, " target area " refers to the region where local patholoic change.For example, when lesion is tumour, target is tumour, and target area is Knurl area (such as knurl body or/and medicine are up to periphery of knurl body)；When lesion is that non-struma is big, target is lump, and target area is enlargement Area (such as the lesion such as hyperplasia, tumour, tubercle or/and periphery)；When lesion is local inflammation, target area be inflamed area (such as The periphery of inflammation lump or/and medicine up to lump)；When lesion is diacrisis tissue, target is the gland of diacrisis, Target area is then the tissue comprising these abnormal secretion glands or organ and/or its periphery.For example, during insulopathic, target is For pancreas islet, target area is then pancreas islet or/and periphery.When disease is skin disease, target is skin or the lesion of skin accessory organ, Target area is then the diseased region or/and periphery.

Within the scope of the invention, term " secretory disease " refer to it is primary or after raw where gland or gland Tissue, the lesion of organ, it can be any appropriate person well known by persons skilled in the art, such as can include following one kind It is or a variety of：Tumour, non-struma are big, local inflammation, microorganism infection and function of organization are abnormal.The abnormal symptom of function of organization is for example Gland dysfunction.

Within the scope of the invention, term " gland " refer to it is being made up of gland cell or gland cell group, perform secretion work( The structure of energy (secretion), it includes exocrine gland and incretory.Exocrine gland is a kind of gland for having conduit, is wrapped Include pancreas, intestinal glands, sweat gland, sebaceous glands, glands of large intestine, apocrine sweat gland, the parotid gland, glandula submandibularis, mammary gland, gastric gland, liver etc..Incretory It is some glands without output duct in human body, including thyroid gland, parathyroid gland, adrenal gland, hypophysis, pineal body, pancreas islet, chest Gland and sexual gland etc..

Within the scope of the invention, term " skin disease " refer to it is primary or after sick in skin or skin accessory organ Become, it can be any appropriate person well known by persons skilled in the art, such as can include tumour, non-struma greatly, locally to send out Scorching, microorganism infection.

Within the scope of the invention, term " tumour " includes malignant tumour and non-malignant tumors.The malignant tumour includes Such as breast cancer, cancer of pancreas, thyroid cancer, nasopharyngeal carcinoma, prostate cancer, liver cancer, lung cancer, intestinal cancer, carcinoma of mouth, cancer of the esophagus, stomach cancer, Laryngocarcinoma, carcinoma of testis, carcinoma of vagina, uterine cancer, oophoroma, etc..The nonmalignant tumor includes such as mastadenoma, Vipoma, first shape Adenoma, prostate tumor, hepatoma, lung knurl, enteroncus, oral cancer, esophagus knurl, stomach knurl, rhinopharyngocele, laryngeal tumor, testicular tumor, elytroncus (elytrophyma), son Palace knurl, salpingioma, ovarioncus etc..The tumour is in addition to above solid tumor, in addition to hemangioma.

Within the scope of the invention, term " non-struma is big " includes such as non-inflammation and non-knurl enlargement, including for example Hyperplasia (such as hyperplasia of mammary gland, pancreas, thyroid gland, parathyroid gland, prostate etc.), tumour (such as mammary gland, thyroid gland, first shape The tumour of other gland etc.), tubercle (such as tubercle of mammary gland, thyroid gland, parathyroid gland etc.), abnormal veins group (such as hemorrhoid etc.). The hemorrhoid include internal piles, external piles, mixed hemorrhoid.

Within the scope of the invention, term " local inflammation " refers to the non-knurl inflammation of part, including for example rotten Property scorching (alterative inflammation), exudative inflammation (exudative inflammation) and productive inflammation.Secretion The inflammation of glandular organ includes such as mastitis, pancreatitis, thyroiditis, prostatitis, hepatitis, pneumonia, enteritis, stomatitis, pharynx Inflammation, esophagitis, gastritis, gastric ulcer, rhinitis, nasosinusitis, laryngitis, tracheitis, bronchitis, vaginitis, hysteritis, fallopian tubal Inflammation, oaritis etc..

Within the scope of the invention, skin disease is big etc. including cutaneum carcinoma, skin non-malignant tumors, inflammation, non-struma.Specifically For, the skin disease in addition to cutaneum carcinoma and skin non-malignant tumors includes:Virus dermatopathy (such as bleb, wart, rubella, Hand-foot-and-mouth disease), bacterial dermatosis (such as impetigo, furuncle, leprosy), fungal dermatopathy (such as various tinea), animal cause Skin disease (such as scabies, acarodermatitis, paederus dermatitis, pediculosis, worm sting or bite), sexually transmitted disease (such as syphilis, leaching Disease and condyloma acuminatum), anaphylaxis and autoimmune skin disease (such as contact dermatitis, eczema, nettle rash), physical skin Sick (such as daylight dermatoses, pernio, corn, rhagadia manus et pedis, pressure sore), connective tissue disease (such as lupus erythematosus), pigment Obstacle dermatoses (such as tattoo, freckle, mole, various spots), cutaneous appendages disease (such as acne, brandy nose, seborrhea Property dermatitis, alopecia areata, baldness, ephidrosis and bromhidrosis).

Within the scope of the invention, term " pathological change of function " refer to the part as caused by the cause of disease cell, The dysfunction of tissue or organ, including for example eccrine pathocrinia.The pathocrinia includes gland Hyperfunction disease and gland hypofunction.Term " gland hyperfunction disease " refers to that secretory cell function is too enlivened then Hormone sensitive lipase gene increase secretion also increases, such as hyperthyroidism." the gland hypofunction " refers to that secretion is thin Born of the same parents' function reduction or certain distinctive enzyme, which lack, to be reduced hormone sensitive lipase gene and secretes reduction, such as Jia shape gland Gong Neng Minus move back disease, pancreas Island work(energy Minus moves back disease (one kind of diabetes) etc..Term " hormone " refers to that endocrine cell synthesizes and is directly secreted into the change of body fluid Information substance is learned, it includes steroid hormone (such as cortex hormone of aadrenaline, sex hormone etc.), amino acid derivativges hormone (example Such as thyroxine, adrenal medullary hormone, epiphysin), (such as hypothalamic hormone, hypophysis swash for peptide and proteohormone Element, gastrointestinal hormone, insulin, calcitonin etc.), derivative of fatty acid hormone (such as prostaglandin).

The composition of the present invention and the medical preparation of said composition can also be with other interventional therapies, systemic chemotherapy, immune Therapy, photodynamic therapy, sound motivation therapy, the combined administration of the combination of surgical intervention or such therapy are treated with further improve Effect.

In the present invention, the shared composition-medicine efficacy relation of medicine is investigated with the effect method principle such as Loewe.It is with phase It is line of demarcation to add effect (additivism), and definition of all effects more than summation action is synergy (synergism), is acted on Definition less than summation action is antagonism (antagonism).Within the scope of the invention, the summation action of composition is determined Justice is as follows：The effect of it is active material (vital stain and metallic compound share) by synergy in said composition (such as press down Ratio of outflow) 115%.Inventor has found there is these in above-mentioned application and treatment method according to the pharmaceutical composition of the present invention Synergy.

Based on the research being more particularly described hereinafter, although specific mechanism waits further to study, combination of the invention Thing show promote mammal tumor in dependency structure (such as pathological tissues, sick cell and participate in form they appoint One structure) effective destruction (such as effectively facilitate sick cell apoptosis, necrosis, autophagy etc. be advantageous to its disappearance activity), from And reach the pharmaceutical properties for the treatment of disease.

Embodiment

By specific examples below, the present invention is further illustrated, but not as limitation of the present invention.With In lower embodiment, all experiments are carried out according to relevant regulations on cell or experimental animal.Unless otherwise specified, Suo Youshi Test and carry out according to a conventional method.

Material, reagent used in specific examples below etc., unless otherwise specified, commercially obtain.Below Portion of reagent used is listed in Table 1 below in embodiment.

Table 1

In the examples below, tested tumour cell includes：Human liver cancer cell (HepG2), human breast cancer cell (MDA- MB231), human lung carcinoma cell (A549), human thyroid cancer cell (SW579), Human Prostate Cancer Cells (LNCaP/AR), human pancreas Cancer cell (PANC-1), human colon cancer cell (COLO205), people's head ＆ neck cancer cell (Fuda), KB cell (CNE1), people Stomach cancer cell (BGC823), oophoroma (PA1), skin cancer cell (A341) etc..The research method and knot of embodiments of the invention Fruit is also applied for other tumour cells.

In the examples below, unless otherwise indicated, the experiment that subcutaneous transplantation knurl animal experiment is issued by pencil authorities refers to Southing row.Animal is 6~8 week old, body weight 17.5-20.5g Female nude mice (BALB/C-Nude).Above-mentioned tested tumour is thin Born of the same parents' subcutaneous vaccination treats tumour length to required volume (such as 70-100mm in nude mice³Left and right), using the software (Sichuan of PEMS 3.2 West China HSPH of university works out) random district's groups are negative control group, positive controls and several seminar, every group 8 Nude mice.

The packet same day starts to be administered.Experiment sets negative control group, positive controls and several seminar, is separately added into the moon Property tester, positive control and several research medicines.Negative control thing is composition solvent, and its administering mode and volume are equal It is identical with seminar.Positive control is selected from above-mentioned existing antitumorigenic substance, and its administering mode is oral gastric infusion, and it is administered Dosage carries out (such as adriamycin 25mg/kg, 5 FU 5 fluorouracil 8mg/kg) by the convention of selected medicine.Unless otherwise indicated, grind It is knurl area injection research medicine to study carefully group, and injection volume is determined by dosage and concentration.Experimental observation, measurement and the project of analysis, bag Include general state, body weight, food ration, gross tumor volume, knurl weight etc..

Gross tumor volume calculation formula is as follows：

TV=l/2 × a × b², a represents length of tumor in formula, and b represents tumor width.

Relative tumour volume calculation formula is as follows：

RTV=Vt/V₀, V in formula₀The gross tumor volume of the same day (i.e. first day) measurement gained is administered for packet, Vt is each time Gross tumor volume during measurement.

Relative tumor proliferation rate calculation formula is as follows：

T/C (%)=TRTV/CRTV × 100, TRTV is the RTV of positive controls or seminar in formula, and CRTV is feminine gender The RTV of control group.

Inhibition rate of tumor growth (being abbreviated as tumour inhibiting rate in the present invention) calculation formula is as follows：

Tumour inhibiting rate (%)=(TW-CW)/CW × 100%, wherein TW are positive controls or the average knurl weight of seminar；CW For the average knurl weight of negative control group.

The evaluating drug effect standard based on Relative tumor proliferation rate of seminar is：T/C (%)>40 be invalid activity, T/C (%)≤40 are (still>And Analysis of variance P compared with negative control group 15%)<0.05 is that effective active (is lived without preferred pharmacy Property), T/C (%)≤20 and Analysis of variance P compared with negative control group<0.05 is preferred activity.

The evaluating drug effect standard based on tumour inhibiting rate of seminar is：Tumour inhibiting rate<40% is invalid activity, tumour inhibiting rate >=40% (still<And Analysis of variance P compared with negative control group 75%)<0.05 is effective active, tumour inhibiting rate >=75% and through variance Analysis P compared with negative control group<0.05 is preferred activity.

In the examples below, experiment is using duplicate measurements variance analysis (Repeated Measures ANOVA) point The other group differences to index mean carry out statistical test.When group differences are statistically significant (P≤0.05), using minimum Significant difference method is to each group compared with negative control group difference.Quantitative target is retouched using mean ± standard error (X ± SEM) State.When heterogeneity of variance is prompted in LEVENE homogeneitys test of variance (P≤0.05), using Mann-Whitney U rank tests (M-W Method) compare group difference.All statistical analyses, completed under the softwares of SPSS for Windows 13.0.

Embodiment 1：The preparation of composition

Using aforementioned preparation process, fraction compositions solution manufactured in the present embodiment is listed in table 2.In the present invention, unless It is otherwise noted, each component in composition solution, liquid compound concentration at room temperature is with volume-concentration of volume percent (V/V%) represent, solid chemical compound concentration is represented with weigh-volume percent concentration (W/V%).

Table 2

^*:In all embodiments of the invention, unless otherwise indicated, alcohol-based liquid curing agent used be ethanol, PEG300 and Propane diols shares, their concentration ratio (% concentration:% concentration:% concentration) it is 1:1:1.For example, one contains 75% alcohols liquid In the solution of body curing agent, the concentration of ethanol, PEG300 and propane diols can be 25%, 25% and 25%, etc. respectively.

Specifically, for example, with final 10ml composition solutions stereometer, at room temperature, measured according to the amount shown in table 2 The dry powder of corresponding vital stain dry powder, metal salt and the other compositions (such as salicylic acid compounds) being optionally present adds The appropriate solvents of 8ml (solvent of such as curing agent containing alcohol-based liquid) dissolve, add other liquid additives (such as phenmethylol, 0.2ml) and solvent makes cumulative volume reach 10ml, and it is standby to be packed as 2ml/ bottles after well mixed, and composition solution is made.

In order to ensure the composition stability of composition, in addition to it can add relevant stable agent, vital stain can also be done Powder and the other compositions (such as salicylic acid compounds) being optionally present are divided with solid-state (such as freeze-dried powder) or/and liquid form Do not store, be well mixed use before application.

Embodiment 2：Application method comparative study

For pancreatic tumor borne cell nude mice (tumor volume 85-135mm³) zoopery.Positive controls are injected intraperitoneally The 5 FU 5 fluorouracil aqueous solution (25mg/kg/ times) (A groups), once a day, continuous 10 days is administered.2 gavage seminar and are administered respectively The methylenum careuleum composition solution (C groups) of 0.2% aqueous solution of methylene blue (B groups) and 0.08% ferrous chloride/0.2%, is gavaged every time 250 μ l, once every other day, it is administered 5 times.Negative control group intratumor injection physiological saline, injection seminar of 2 Ge Liu areas intratumor injection point The methylenum careuleum composition solution (E groups) of 1% aqueous solution of methylene blue (D groups) and 0.4 ferrous chloride/1%, per injection are not administered 100 μ l, once every other day, it is administered 5 times.Next day after drug withdrawal, animal is euthanized, tumour inhibiting rate is determined after dissection.As a result it is as follows： A, the tumour inhibiting rate of B, C, D, E group is respectively 61%, 8%, 10%, 27% and 61%.

Using the other compositions (such as composition 1-10 in table 2) prepared in embodiment 1, also there are similar results.This A little experiment displays, synergistic effect of the iron containing compoundses to vital stain, in being locally administered apparently higher than in systemic administration.

Embodiment 3:Vital stain/metal salt compositions research

For lotus liver cancer cells nude mice (knurl body 95-165mm³) experiment, positive controls (A groups) intraperitoneal injection 5- The fluorouracil aqueous solution (25mg/kg/ times), daily injection once, are administered 10 times.Negative control group intratumor injection physiological saline, Seminar intratumor injection pharmaceutical aqueous solution 75-100 μ l, are administered once.Animal was euthanized and dissected in 3 days after drug withdrawal, determined Tumour inhibiting rate.

In 3 are studied medicine series, First Series are 5 methylenum careuleum groups, concentration is respectively 0.5%, 1.0%, 1.5%th, 2.0%, 3.0%.Second series are 5 ferrous chloride/methylenum careuleum group, and ferrous chloride concentration is 0.4%, and methylene Blue concentration is respectively 0.5%, 1.0%, 1.5%, 2.0%, 3.0%.3rd series and 5 ferrous chloride/methylenum careuleum group, it is sub- First indigo plant concentration is 1%, and ferrous chloride concentration is respectively 0.02%, 0.05%, 0.1%, 1%, 2%.As a result such as Fig. 1 institutes Show, the tumour inhibiting rate curve for the first and second series that wherein Figure 1A is shown, and the tumour inhibiting rate that the 3rd series is shown in Figure 1B is bent Line.

In Fig. 1, the composition group of least concentration (the methylenum careuleum group of 0.4% ferrous chloride/0.5% and 0.02% ferrous chloride/ 1% methylenum careuleum group) tumour inhibiting rate be 28% and 41%, more than 115% be higher than 0.5% methylenum careuleum group and 1% methylenum careuleum group respectively The effect of (tumour inhibiting rate is respectively 15% and 27%), show synergistic function.The effect of the two Synergistic compositions But still it is significantly lower than positive control (tumour inhibiting rate 72%).However, when the ferrous chloride in composition and the knurl body of methylenum careuleum After injection concentration reaches a certain critical value (being respectively 0.1% and 1%), drug effect is shown to the extremely sensitive of concentration, it presses down Ratio of outflow is improved until being more than positive control rapidly, preferred curative effect occurs.

More experiment displays, when the local administration concentration of metal salt is 0.2-2%, methylenum careuleum, patent blue, isosulfan blue Safety and be 1-4%, preferably 1.5-3% by the concentration of preferred synergy.

In above-mentioned experiment, metal salt/vital stain composition middle and high concentration (such as methylenum careuleum of the present invention is also observed More than 1%) relation of vital stain and security.In safe-dosaging limits, local administration is important to pay close attention to local irritation. In Lump body injection, state high concentration composition group and occur obvious color and luster change and slight myodegeneration in injection site. In another identical confirmatory experiment, the injection site for cuing open the nude mice killed in 30 days after stopping to administration has carried out pathological section inspection Look into, as a result show negative control group and the injection site of seminar and the difference of normal non-injection site with through unobvious.

In addition, another battery of tests shows that when being administered by target area, pharmaceutical composition of the invention is in same combined concentration Under, a larger administered volume shows more preferable therapeutic effect., can to control dosage if Gross Target Volume is too big To carry out branch's processing to it.Each administered volume of injection is necessary for more than the 50% of part volume to be handled.Work as tumour When sufficiently small, part volume to be handled is single gross tumor volume.

In addition, we have also investigated the composition in the present invention to be used for the anti-different tumours developed.In the implementation of the present invention In example, unless otherwise indicated, the research method of the tumor experiment of anti-different developments is as follows.

Nude mice, which is divided into, is first inoculated with mouse group and rear inoculation mouse group.Mouse group is first inoculated with prior to 7 days inoculation human liver cancers of rear inoculation mouse group Cell.7 days after rear inoculation mouse group inoculation, nude mice packet (every group 8) is carried out.First it is inoculated with mouse group (tumor volume 100- 150mm³) it is divided into negative control group (A groups) and seminar's (B groups), it is rear to be inoculated with mouse group (tumor volume 35-65mm³) be divided into it is negative right According to group (C groups) and seminar's (D groups).A, B groups intratumor injection administration 70-100 μ l, C, D groups intratumor injection administration 30-70 μ l.

In the present embodiment, negative control thing is physiological saline, and research medicine is 0.7%/2% aqueous solution of methylene blue.Administration 1 It is secondary, after drug withdrawal next day dissect, the tumour inhibiting rate of D groups and B groups is respectively 95% and 71%.The composition of the present invention can be applied not only to Treating malignant tumor, removal completely even can be used for have the tumour of certain carcinogenic risk, to prevent it from developing into malignant tumour.

Other metal salts/vital stain the composition (such as composition 1-10 in table 2) prepared using embodiment one, It is observed that similar validity and safety results.

Embodiment 4：Vital stain/metal salt/other antitumorigenic substances composition research

In one experiment, 3 serial research drug solutions contain 75% alcohol-based liquid curing agent, therefore with 75% alcohols liquid The body curing agent aqueous solution is negative control thing.

When the ferrous chloride in fixing composition and acetyl salicylic acid concentration (being respectively 0.7% and 3%) in 0.5- 3% change methylenum careuleum concentration (0.5%, 1%, 1.5%, 2%, 3%) when, ferrous chloride/acetylsalicylic acid/methylenum careuleum with it is identical Ferrous chloride/methylenum careuleum of concentration of component, ferrous chloride/acetylsalicylic acid compare with methylenum careuleum/acetylsalicylic acid, tumour inhibiting rate It is higher by more than 115%.

When the ferrous chloride in fixing composition and methylenum careuleum concentration (being respectively 0.7% and 1%) become in 1.5-12% Change acetyl salicylic acid concentration (1.5%, 3%, 5%, 8%, 12%) when, ferrous chloride/acetylsalicylic acid/methylenum careuleum with identical group Ferrous chloride/methylenum careuleum of point concentration, ferrous chloride/acetylsalicylic acid compare with methylenum careuleum/acetylsalicylic acid, and tumour inhibiting rate is high Go out more than 115%.

When the methylenum careuleum in fixing composition and acetylsalicylic acid concentration (being respectively 1% and 3%) in 0.02- During 2% change ferrous chloride concentration (0.02%, 0.05%, 0.1%, 1%, 2%), ferrous chloride/acetylsalicylic acid/methylenum careuleum Compared with ferrous chloride/methylenum careuleum, ferrous chloride/quinine dihydrochloride and methylenum careuleum/quinine dihydrochloride with same composition concentration, Tumour inhibiting rate is higher by more than 115%.

By by taking the following group as an example, the methylenum careuleum of the acetylsalicylic acid of 0.7% ferrous chloride/3%/1%, 0.7% ferrous chloride/3% The tumour inhibiting rate of the methylenum careuleum of the acetylsalicylic acid of acetylsalicylic acid, 1% methylenum careuleum/3%, 0.7% ferrous chloride/1% is respectively 89%, 46%th, 67%, 73%.

In another experiment, negative control group injecting normal saline, 3 serial seminar's injection pharmaceutical aqueous solutions.

When the ferrous chloride in fixing composition and quinine dihydrochloride concentration (being respectively 0.7% and 3%) in 0.5- 3% change methylenum careuleum concentration (0.5%, 1%, 1.5%, 2%, 3%) when, ferrous chloride/quinine dihydrochloride/methylenum careuleum with it is identical Ferrous chloride/methylenum careuleum of concentration of component, ferrous chloride/quinine dihydrochloride compare with methylenum careuleum/quinine dihydrochloride, tumour inhibiting rate It is higher by more than 115%.

When the ferrous chloride in fixing composition and methylenum careuleum concentration (being respectively 0.7% and 1%) change in 1-8% During quinine dihydrochloride concentration (1%, 2%, 4%, 6%, 8%), ferrous chloride/quinine dihydrochloride/methylenum careuleum and same composition are dense Ferrous chloride/methylenum careuleum of degree, ferrous chloride/quinine dihydrochloride are compared with methylenum careuleum/quinine dihydrochloride, and tumour inhibiting rate is higher by More than 115%.

Become when the methylenum careuleum in fixing composition and quinine dihydrochloride concentration (being respectively 1% and 3%) and in 0.02-2% Change ferrous chloride concentration (0.02%, 0.05%, 0.1%, 1%, 2%), ferrous chloride/quinine dihydrochloride/methylenum careuleum with it is identical Ferrous chloride/methylenum careuleum of concentration of component, ferrous chloride/quinine dihydrochloride compare with methylenum careuleum/quinine dihydrochloride, tumour inhibiting rate It is higher by more than 115%.

By by taking the following group as an example, the methylenum careuleum of the quinine dihydrochloride of 0.7% ferrous chloride/3%/1%, 0.7% ferrous chloride/3% The tumour inhibiting rate of the methylenum careuleum of the quinine dihydrochloride of quinine dihydrochloride, 1% methylenum careuleum/3%, 0.7% ferrous chloride/1% is respectively 82%, 61%th, 62%, 68%.

In addition, we have also investigated metal salt/other synergist/vital stain composition to be used for the swollen of anti-different developments Knurl.Research method is as previously described.In the present embodiment, negative control thing is the aqueous solution containing 70% alcohol-based liquid curing agent, research Medicine is the aqueous solution of methylene blue of the acetylsalicylic acid of 0.7% ferrous chloride containing 70% alcohol-based liquid curing agent/5%/2%.Administration 1 It is secondary, dissect within 20 days after drug withdrawal, the tumour inhibiting rate of D groups and B groups is respectively 95% and 81%.

In other analogous compositions (such as group in table 2 that other tumor animals are tested and are prepared using embodiment 1 Compound 11-21) experiment when, can also be observed that similar long-term effect and safety results.

Embodiment 6：Tumor suppression research

Cancer cell as previously described, for a variety of lotus cancer cell nude mices (knurl body 85-165mm³) experiment, positive controls (A Group) the intraperitoneal injection 5 FU 5 fluorouracil aqueous solution (25mg/kg/ times), once a day, it is administered 10 times.Negative control thing is made a living Manage salt solution.It is respectively the medicine prepared by preceding method to study medicine：2% aqueous solution of methylene blue (B groups), 0.7% ferrous chloride/ The acetyl salicylic acid solution of the methylenum careuleum of 2% methylenum careuleum composition solution (C groups) and 0.7% ferrous chloride/2%/5% (contains 70% Liquid alcohols curing agent) (D groups).Negative control group and seminar intratumor injection 75-100 μ l, 1 time altogether.After drug withdrawal 20 days it is right Animal is euthanized and dissected, and determines tumour inhibiting rate.As a result it is as follows.

The experiment carried out for lotus thyroid carcinoma cell nude mice, from experiment the 5th day, compared with negative control group, The gross tumor volume of positive controls and seminar all reduces, the statistically significant (P of difference<0.05).Anatomical results show, A, B, C, the tumour inhibiting rate of D groups is respectively 71%, 63%, 76% and 93%.

The experiment carried out for lotus breast cancer cell nude mice, from experiment the 5th day, compared with negative control group, sun Property control group and the gross tumor volume of seminar all reduce, the statistically significant (P of difference<0.05).Anatomical results show, A, B, C, The tumour inhibiting rate of D groups is respectively 68%, 63%, 75% and 95%.

The experiment carried out for lotus nasopharyngeal carcinoma cell nude mice, from experiment the 5th day, compared with negative control group, sun Property control group and the gross tumor volume of seminar all reduce, the statistically significant (P of difference<0.05).Anatomical results show, A, B, C, The tumour inhibiting rate of D groups is respectively 73%, 70%, 82% and 96%.

The experiment carried out for lotus skin cancer cell nude mice, from experiment the 5th day, compared with negative control group, sun Property control group and the gross tumor volume of seminar all reduce, the statistically significant (P of difference<0.05).Anatomical results show, A, B, C, The tumour inhibiting rate of D groups is respectively 78%, 68%, 81% and 94%.

In another serial lotus cancer cell nude mice (knurl body 100-150mm³) in experiment, positive control, negative control Thing with it is upper identical.It is respectively the following medicine prepared by preceding method to study medicine：The 2% patent blue aqueous solution (B groups), 0.7% The quinine dihydrochloride water of the methylenum careuleum of the patent blue composition solution (C groups) of ferrous chloride/2% and 0.7% ferrous chloride/2%/3% Solution (D groups).Eligible result is similar with the above results.

In other tumor bearing nude mices, (inoculated tumour cell is respectively human lung carcinoma cell (A549), Human Prostate Cancer Cells (LNCaP/AR), ovarian cancer cell (PA1), head ＆ neck cancer cell (FADU)) experiment in, and using implement 1 prepare it is other During composition (such as table 2), similar result can also be observed that.

Embodiment 8：Resist the non-big research of struma

The anti-non-big effect of struma of the composition of the present invention is studied by model of the proliferation of mammary gland first, its test method It is as follows：

Animal used is body weight 150-180g unpregnancy female rats.Intramuscular injection oestradiol benzoate (0.5mg/kg, 1 time/ My god, continuous 20 days), then intramuscular injection progesterone (5mg/kg, 1 times/day, continuous 5 days) carry out animal modeling.Sampled biopsy checking After modeling success, it is grouped at random using the softwares of PEMS 3.2.Experimental animal be divided into blank control group (end modeling animal) and Following modeling group：Positive controls, negative control group, 3 seminar, every group of 6 animals.The packet same day starts to be administered.It is positive Tester is RUZENGNING PIAN (gavaging, once a day, each 1g/kg, be administered 27 times).Negative control thing is molten for research medicine Matchmaker.Seminar and negative control group inject 100 μ l in enlargement area.Administration frequency is 5 times altogether once every other day.

The project observed, measure and analyzed in experiment, in addition to the food ration, body weight, general state of routine, in addition to Change with respect to nipple Magnification (T/C%) and breast tissue pathology.

It is with respect to papilla diameter calculation formula：

RTD=Dt/D0, D0 is the packet gained papilla diameter of administration measurement for the first time in formula, and Dt is when measuring each time Papilla diameter.

It is with respect to nipple Magnification calculation formula：

T/C (%)=TRTD/CRTD × 100, TRTD is positive controls in formula or seminar RTD, CRTD are negative right According to a group RTD.

The relative nipple Magnification evaluating drug effect standard of seminar is：

T/C (%)>50 be inactive, and T/C (%)≤50 is (still>25%) and Analysis of variance is compared with negative control group P<0.05 is active, T/C (%)≤25 and Analysis of variance P compared with negative control group<0.05 is to have preferred synergy.

Breast tissue pathology is analyzed as follows：The 4th day euthanasia animal, wins rat second to mammary gland, stone after last dose Wax is cut into slices, in the change of light Microscopic observation breast tissue pathology form after HE dyeing.It is right by observing the form of leaflet and acinus Each group rat breast tissue pathology is integrated, wherein：Lobule of mammary gland not hyperplasia, body of gland quantity is few, and acinus is not expanded, note 0 Point；For lobule of mammary gland without obvious hyperplasia, indivedual acinuses have slight hyperplasia, but without expansion, remember 1 point；Lobule of mammary gland major part hyperplasia, portion Divide acinus substantially to expand, remember 2 points；The obvious hyperplasia of lobule of mammary gland, acinus are in extreme expansion state, and glandular epithelium is in flat Shape, there is substantial amounts of secretion in acinus and in conduit, remember 3 points；Mammary gland alveolus, conduit, the pathological proliferation of leaflet are obvious, note 4 Point.

The pathological analysis evaluating drug effect standard of seminar is：Pathology integrates >=3 to be inactive, and 1<Pathology integrates<3 simultaneously classical prescriptions Difference analysis P compared with negative control group<0.05 is active, pathology integration≤1 and Analysis of variance P compared with negative control group <0.05 is to have preferred synergy.

In an experiment, 3 seminar inject the following solution prepared by preceding method and (contain 65% liquid alcohols respectively Curing agent):The patent blue composition solution (C groups) of the 2% patent blue aqueous solution (B groups), 0.7% ferrous chloride/2% and 0.7% The acetyl salicylic acid solution (containing 70% liquid alcohols curing agent) (D groups) of the patent blue of ferrous chloride/2%/5%.

From after medication the 7th day, C, D group papilla diameter are obvious compared with negative control group to be reduced, and difference has statistics Meaning (P<0.05).The 10th day after medication, C, D group are less than 50% with respect to nipple Magnification (T/C)；The 25th day after medication, D, the relative nipple Magnification of C, B group and positive controls is respectively 8%, 11%, 23% and 39%.The 25th day after medication Pathology integrates, and D group≤1 approaches with negative control group, and C groups and positive controls are more than 2, B groups and are more than 3.Drug safety is seen It is essentially identical to examine result.

In another experiment, 3 seminar inject the following solution prepared by preceding method and (contain 65% liquid alcohol respectively Class curing agent)：The isosulfan blue composition (C groups) of 2% isosulfan blue (B groups), 0.7% ferrous chloride/2% and 0.7% ferrous chloride/ The isosulfan blue composition solution (D groups) of 3% quinine dihydrochloride/2%.Experimental result is similar with upper one experiment.

When implementing other compositions (such as composition in table 2) that 1 prepares using the present invention, it can also be observed that similar Result.

The anti-non-big effect of struma of the composition of the present invention is also that model is studied with non-inflammatory goitre.With non-inflammation Property goitre be model studied.Simple goiter (simple goiter) is also known as non-inflammatory goitre, is by non- Inflammatory or tumprigenicity reason cause thyroid gland compensatory enlargement caused by thyroxine dyssynthesis.The examination of goitre treatment Proved recipe method is as follows：

Experimental animal is body weight 150-180g adult rat.Rat is raised 3 months in the environment of iodine-deficient forage nursing More than.It is decreased obviously using urinating iodine, the obvious enlargement of thyroid gland is models successfully.Random area is carried out to animal using the softwares of PEMS 3.2 Group, it is divided into blank control group (end modeling animal) and following modeling group：Positive controls, negative control group (A groups), seminar, Every group 6.It is less than 100mm from lump volume³Nude mice, packet the same day start to be administered.Positive control is Potassiumiodate (KIO3, gavaging, administration number of times is 27 times, and administration frequency is once a day, dosage is 0.4 μ g/kg every time).Negative control thing is Study the solvent of medicine.Seminar and negative control group inject 100 μ l in enlargement area.Administration frequency is once every other day, and altogether 5 It is secondary.

The project observed, measure and analyzed in experiment, in addition to the food ration, body weight, general state of routine, in addition to Thyroid Gland Swell, 24h iodine discharge rate and thyroid pathology inspection.

From experiment the 10th day, C, D group Thyroid Gland Swell were obviously improved compared with negative control group, iodine discharge rate and blank Control group reaches unanimity (difference<25%), effect is suitable with positive controls.In pathological examination, negative control group observable To the obvious enlargement of thyroid gland, folliculus is intensive, and epithelial hyperplasia hypertrophy is in high column, it is seen that the cell mass rope of hyperplasia, between folliculus Blood vessel showed increased, lumen distention is congested, and fibr tissue increases between leaflet.

The 25th day after medication, D groups fibr tissue etc. between thyroid size, folliculus form size, epithelial cell, leaflet The difference of aspect and blank control group is less than 10%.C groups approach with positive controls, are less than with the difference of blank control group 20%.The difference of B groups and blank control group is 35%.The Drug safety observation result of each group is essentially identical.

In another experiment, 3 seminar inject the following solution prepared by preceding method and (contain 65% liquid alcohol respectively Class curing agent):The methylenum careuleum composition solution (C groups) of 2% aqueous solution of methylene blue (B groups), 0.7% ferrous chloride/2% and The methylene solution (containing 70% liquid alcohols curing agent) (D groups) of the acetylsalicylic acid of 0.7% ferrous chloride/5%/2%.Experimental result with A upper experiment is similar.

Embodiment 9：The research of anti-local inflammation

The anti-local inflammation effect of the composition of the present invention is studied by model of allergic rhinitis, its animal experiment Method is as follows.

Experimental animal is body weight about 150-180g Adult male rats, is randomly divided into blank control group and modeling group.Make Module is with oralbumin (OVA) sensitization liquid (every milliliter of (OH) containing 0.5mgOVA and 30mg Al₃) as allergen pass through abdominal cavity (once a day, totally 7 times) sensitization is injected, is then made with oralbumin modeling liquid (3%OVA) collunarium (once a day, totally 7 times) Mould.After modeling success, using the random district's groups of the softwares of PEMS 3.2, it is divided into following modeling group：Positive controls, negative control group (A Group), seminar, every group 6.The packet same day starts to be administered.Positive control is commercially available momestasone furoate nasal spray (Belgium Schering Plough).

The daily nasal cavity injection of each group is administered once, totally 7 times.With administration 30 minutes after in 3 minutes rat scratching, sneezing time Number is observation index.With the multilevel iudge drug effect with blank control group.Scratching and sneeze number such as modeling group respectively reach sky More than 7 times and more than 1 times of white control group, then modeling success.Scratching and sneeze number such as medicine group respectively reach and the positive Horizontal similar in control group, then medicine is effective.

In an experiment, negative control thing and research medicine are spray, former with aerosol fluid before the use Liquid is stored in the container of sprayer unit.The preparation method of spray is as described in Example 1.The liquid stock solution contains 75% alcohol Class I liquid I curing agent and following auxiliary material：Glycerine (2.5%), Tween-80 (1.5%), benzalkonium chloride (0.02%), crystallite are fine Tie up element-sodium carboxymethylcellulose (1.5%).The liquid stock solution of 3 research medicines also contains following components respectively：3% crystal violet water The acetyl water of the crystal violet composition solution (C groups) of solution (B groups), 0.7% ferrous chloride/3% and 0.7% ferrous chloride/10% Poplar acid/3% crystal violet solution (D groups).

The scratching of the 15th day and sneeze number, D, C, B group and the numeral of positive controls and blank control group after medication Difference is respectively 15%, 18%, 37% and 25%.In experimentation, each group is showed no obvious mucosal irritation effect, also not Cause allergic reaction.

In another experiment, 3 research aerosol fluid stostes contain following components respectively:3% aqueous solution of methylene blue (B Group), the methylenum careuleum composition solution (C groups) of 0.7% ferrous chloride/3% and 0.7% ferrous chloride/10% salicylic acid/3% it is sub- First indigo plant solution (D groups).Experimental result is similar with upper one experiment.

Similar results are also obtained using the spray of the same composition with implementing the 1 other compositions (such as table 2) prepared.

Embodiment 10：The research of anti-microbial infection composition

The anti-microbial infection effect of the composition of the present invention, it is that model is ground with fungal dermatopathy (ringworm of the foot) Study carefully, test method is as follows.

Positive control is miconazole (Xian-Janssen Pharmaceutical Ltd.), and negative control thing and research medicine are spray Mist agent, it is stored in before the use with aerosol fluid stoste in the container of sprayer unit.The preparation method of spray is as implemented Shown in example 1.The liquid stock solution contains 75% alcohol-based liquid curing agent and following auxiliary material：Glycerine (2.5%), Tween-80 (1.5%), benzalkonium chloride (0.02%), microcrystalline cellulose-sodium carboxymethylcellulose (1.5%).The liquid of 3 research sprays Stoste contains following components respectively：Methylenum careuleum composition solution (the C of 4% aqueous solution of methylene blue (B groups), 0.7% ferrous chloride/4% Group) methylene blue solution (D groups) of the salicylic acid of and 0.7% ferrous chloride/10%/3%.

The people of volunteer 50 with the ringworm of the foot for participating in experiment is divided into 5 groups, every group of 10 people.Positive controls are smeared in affected part to be reached Gram peaceful frost, once a day, continuous 7 times.Corresponding spray is sprayed in affected part by negative control group and 3 seminar, once a day, even It is continuous 7 times.

Curative effect judging standard is：Smelting is cured, and skin lesion is disappeared>90%；Effectively, skin lesion is disappeared>50%；Invalid, skin lesion is disappeared< 50%.

7 days after medication, compared with negative control group, there is significantly larger skin in positive controls and 4 seminar Damage disappears.The result of 10 days is as shown in the table after medication.

Table 3

Group	Case load	Smelting more counts	Significant figure	Invalid number	Inefficiency (%)
						Negative control group	10	0	0	10	100
Positive controls	10	0	4	6	60
						B groups	10	0	2	8	80
C groups	10	1	5	4	40
						D groups	10	3	7	0	0

In another experiment, the liquid stock solution of 3 research sprays contains following components respectively:The 3% crystal violet aqueous solution Salicylic acid/3% of the crystal violet composition solution (C groups) of (B groups), 0.7% ferrous chloride/3% and 0.7% ferrous chloride/10% Crystal violet solution (D groups).Experimental result is similar with upper one experiment.

Embodiment 11：The research of anti-gland diacrisis

The anti-gland diacrisis effect of the composition of the present invention is studied by model of hyperthyroidism, its test method It is as follows.

Experimental animal is body weight about 150-180g Adult male rats.Hyperthyroidism modeling medicine is levothyrocine (abdomen Chamber is injected, the μ g/100g body weight of dosage 50, continuous injection 10 days).After Serological testing and pathological examination confirmation model successfully, Using the random district's groups of the softwares of PEMS 3.2, it is divided into blank control group (end modeling animal) and following modeling group：Positive controls, the moon Property control group (A groups), 3 seminar, every group 6.The packet same day starts to be administered.Positive control is that methimazol (is gavaged, is administered Number is 27 times, and administration frequency is once a day, dosage is each 2mg/kg).Negative control thing is the solvent of research medicine.Grind Study carefully group and negative control group and inject 100 μ l in enlargement area.Administration frequency is 8 times altogether once every other day.

The project observed, measure and analyzed in experiment, in addition to the food ration, body weight, general state of routine, in addition to The Serological testing and thyroid pathology inspection of the 16th day after the administration same day and administration.Serological testing uses and puts the method for exempting from (RIA) its serum T 3, T4 and thyrotropic hormone (TSH) concentration value are determined.Pathological examination observation thyroid morphology, folliculus shape Fibr tissue form between state, epithelial cell form, leaflet.Result of the test is as follows.

In an experiment, the following aqueous solution of thyroid gland internal injection is distinguished by 3 seminar：3% aqueous solution of methylene blue (B Group), the quinine dihydrochloride of the methylenum careuleum composition solution (C groups) of 0.7% ferrous chloride/3% and 0.7% ferrous chloride/6%/ 3% methylene blue solution (D groups).Negative control group knurl area injecting normal saline.

Upon administration in the Serological testing of the 25th day, C, D group serum T 3, T4 T3, T4 compared with negative control group are raised Ease up, TSH declines and tends to improve.For Serological testing result, D, C, B group and positive controls and the blood for not modeling animal Clear value difference Fen Biewei 15%, 38%, 47% and 35%.

In pathological examination, negative control group can be observed thyroid gland and increase in diffusivity, and follicular epithelium hyperplasia is in Gao Zhu Shape simultaneously has small folliculus to be formed, and folliculus periphery many epithelial cells not of uniform size occurs and absorbs vacuole, interstitial rich blood vessel, fills Blood, lymphadenia.Compared with negative control group, the thyroid gland diffusivity of C, D group has declined, follicular epithelium hyperplasia and small The situation that folliculus is formed significantly reduces；Folliculus periphery occur epithelial cell absorb vacuole, interstitial blood vessel number and hyperemia and Lymphadenia has weakened.C, D groups approach with not modeling the pathological examination result of animal blank control group, hence it is evident that ratio Positive controls and other seminar are good.Each group does not observe irreversible local lesion and obvious weight loss.

In another experiment, the following aqueous solution prepared by preceding method is injected respectively by 3 seminar:3% patent blue The disalt of the patent blue composition solution (C groups) of the aqueous solution (B groups), 0.7% ferrous chloride/3% and 0.7% ferrous chloride/6% The patent blue solution (D groups) of sour quinine/3%.Experimental result is similar with upper one experiment.

Claims

1. a kind of pharmaceutical composition comprising pharmaceutically acceptable vital stain and metallic compound, wherein the metal salt with Relative quantity (w/w) of the vital stain in the pharmaceutical composition is 1:2-1:200th, preferably 1:5-1：100.

2. pharmaceutical composition according to claim 1, wherein the metallic compound is to be selected from one below kind or a variety of gold Belong to salt：Molysite, mantoquita, zinc salt, manganese salt, aluminium salt, preferably molysite and mantoquita, more preferably selected from one below kind or a variety of Ferrous salt：Ferrous chloride, ferrous sulfate, ferrous succinate, ferrous fumarate, ferrous gluconate, ferrous lactate.

3. pharmaceutical composition according to claim 1, wherein the vital stain be selected from following organic dyestuff and its derivative it It is one or more：Methylenum careuleum, patent blue, isosulfan blue, toluidine blue, trypan blue, alkali blue, Yihong, basic fuchsin, crystal violet, Gentian violet, dimethyl diaminophenazine chloride, janus green B, sarranine；It is preferably selected from the one or more of following organic dyestuff and its derivative：It is sub- First indigo plant, patent blue, isosulfan blue, crystal violet, dimethyl diaminophenazine chloride, one kind or more more preferably selected from following organic dyestuff and its derivative Kind：Methylenum careuleum, patent blue, isosulfan blue.

4. according to one of claim 1-3 pharmaceutical composition, it also contributes to enhancing to prevent and treat comprising one or more Drug effect antitumorigenic substance, the antitumorigenic substance includes effective antitumorigenic substance or/and poorly efficient antitumorigenic substance, wherein: The effective antitumor material preferably is selected from the one or more of following antineoplastic and its derivative：Adriamycin, cis-platinum, Japanese yew Alcohol, vincristine, endoxan, 5 FU 5 fluorouracil；One below kind or a variety of preferably is selected from the poorly efficient antitumorigenic substance：It is non- Steroidal anti-inflammatory compound, quinolines, artemisinin derivative, phenolic compound.

5. according to one of claim 1-4 pharmaceutical composition, wherein described pharmaceutical composition can also include curing agent and/or stop Pain agent.

6. according to one of claim 1-5 pharmaceutical composition, it is Topical dosage forms, preferably following formulation：Note target area Penetrate and/or irrigate formulation, respiratory tract administration formulation, surface administration formulation, mucosa delivery formulation and cavity/canal drug administration formulation.

7. pharmaceutical composition according to claim 6, wherein the Topical dosage forms be liquid preparation, semisolid preparation or The liquid stock solution of gaseous formulation, wherein the concentration of the metallic compound is 0.02-2%w/v, it is preferably 0.05-1.5%w/ V, more preferably 0.1-1.2%w/v or 0.1-1.6%w/v, the concentration of the vital stain is 1-6%w/v, preferably 1-5% W/v, 1.5-5%w/v or 2-5%w/v, more preferably 2-4%w/v or 3-4%w/v.

8. pharmaceutical composition according to claim 6, wherein the curing agent is preferably liquid hardening agent, more preferably alcohol-based liquid Curing agent, such as ethanol, propane diols, liquid PEG, glycerine, isopropanol, it is preferably selected from one of the following group kind or a variety of：Second Alcohol, propane diols, liquid PEG, its concentration can be 55-95%v/v, preferably 60-80%v/v.

9. pharmaceutical composition according to claim 6, wherein the concentration of the effective antitumor material can be 0.1-10%w/ V, and the concentration of the poorly efficient antitumorigenic substance can be 0.1-15%w/v.

10. pharmaceutical composition according to claim 6, wherein the concentration of the nonsteroidal anti-inflammatory compound can be 1.5-15% W/v, preferably 3-15%w/v, more preferably 5-12%w/v；And/or

The concentration of the phenolic compound can be 0.1-10%w/v, preferably 0.1-8%w/v, wherein the Nitro-phenols Concentration can be 0.1-1%w/v, and the concentration of the aminophenol compound can be 2-10%w/v；And/or

The concentration of the artemisinin derivative is 1-6%w/v, preferably 2-5%w/v, more preferably 3-5%w/v；And/or

The concentration of the quinolines is 1.5-10%w/v, preferably 2-10%w/v, more preferably 3-8%w/v.