CN106860457A

CN106860457A - A kind of pharmaceutical composition, preparation method and applications for treating secretion gland disease

Info

Publication number: CN106860457A
Application number: CN201510920676.7A
Authority: CN
Inventors: 邹方霖; 卢星; 邹礼常; 王建霞
Original assignee: Sichuan Nbond Medical Technology Co Ltd
Current assignee: Sichuan Nbond Medical Technology Co Ltd
Priority date: 2015-12-11
Filing date: 2015-12-11
Publication date: 2017-06-20

Abstract

The application in treatment secretion gland disease and method the present invention relates to a quinin hydrochloride, and the pharmaceutical composition comprising a quinin hydrochloride.

Description

A kind of pharmaceutical composition, preparation method and applications for treating secretion gland disease

Technical field

The present invention relates to a kind of pharmaceutical composition, its preparation method and its application in treatment secretion gland disease for including a quinin hydrochloride and the miscible organic solvent of water.

Background technology

One quinin hydrochloride is well-known anti-malarial agents.Its application outside antimalarial, is much carried out with composition forms.This based composition is once applied to treat various diseases, such as hemorrhoid, rhinitis, hemangioma, facial paralysis etc..It is generally believed that other components (such as urethane) in composition not only also can in the treatment play some drug effects with hydrotropy.Thus, for specified disease the pharmaceutical composition comprising a quinin hydrochloride always among research.For example, Chinese patent 87104056 discloses a kind of six components compositions " injection for whole prolapse haemorrhoids " for containing a quinin hydrochloride and quinine dihydrochloride, for the anal fissure, rectal polyp and the papilla fiber histioma that remove hemorrhoid and associate with hemorrhoid.But in the composition, a quinin hydrochloride concentration very little (is less than 0.01M), and contains urethane.Chinese patent 200910103187.7 is used to treat hemorrhoid and tumour there is provided a kind of nine components composition.The concentration of quinine also very little (be less than 0.01M) in said composition, and contain urethane.Because the side effect of urethane is stronger, Chinese Government has limited or even has forbidden this kind of application.Chinese patent 96110932 discloses a kind of composition comprising a quinin hydrochloride and Anodynine, and it is used for Treatment of Hyperthyroidism.However, Anodynine side effect is very strong, in developed country, oneself is eliminated.

Possible application of the quinine derivative in terms of gland disease treatment, for example anti-knurl, once by excessively very many concerns.In fact, in the in vitro test with tumour cell as model, the cell 503nhibiting concentration (IC of some quinine derivatives and conventional antitumor medicine (such as 5-FU, adriamycin, taxol etc.)₅₀) all in every liter of level of (μM ol/L) of micromole.According to common understanding, a concentration dependent for the Antineoplastic effect of medicine is in IC₅₀Increase with concentration afterwards and reduce.Conventional antitumor medicine is generally with than IC₅₀When being higher by the concentration of several times (still micromole every liter in the range of) and being injected in knurl body, anti-knurl validity is just shown.Even if however, the injection concentration of a quinin hydrochloride is improved about 500 times (0.025M) by we, effective anti-knurl effect (tumour inhibiting rate is less than 40%) is also not observed, the drug effect more considerably higher than existing antitumor medicine is not shown.In the prior art, the tumour inhibiting rate of other quinine class medicines is not also high.In the last few years, they were just no longer regarded as a kind of competitive antitumor medicine by people, and were treated as anti-knurl activity increase agent and are studied.For example, quinine and antitumor medicine (vincristine class medicine) are exactly used to reduce the multidrug resistance of cell so that antitumor medicine reaches preferable drug effect by No. 201310459568.5 Chinese patent application.Have not yet to see and be prohibited because side effect is relatively strong not comprising urethane and antipyrine etc. or limit a quinin hydrochloride pharmaceutical composition of the drug ingedient for using for other gland disease treatments.

The treatment of secretion gland disease, the mainly chemotherapy of operation, radiation cure and other medicines at present.Operation is damaged to body, and the injury of the gland (such as mammary gland and thyroid gland) attractive in appearance on some influences is frustrating, it is become a kind of painful choice.The side effect of radiation cure is also clearly.Existing chemotherapy is substantially systemic administration, there is systemic side effects risk very high.For example, existing antitumor medicine has also killed a large amount of normal cells while interior killing cancer cell on a large scale, immune system is seriously damaged, and curative effect is also and unsatisfactory.

The content of the invention

Goal of the invention of the invention be to provide it is a kind of compared with prior art more effectively smelting treat secretion gland disease pharmaceutical composition and treatment method.

According to an aspect of the present invention, it provides a kind of pharmaceutical composition for treating secretion gland disease, it includes a quinin hydrochloride and the pharmaceutically miscible organic solvent of acceptable water, and be the formulation for being suitable for being administered through target area, wherein when being administered through target area, the concentration of a quinin hydrochloride is 0.15-0.4M (Mol/L), more preferably preferably 0.15-0.35M, 0.2-0.3M in described pharmaceutical composition；The concentration of the miscible organic solvent of water is 3-20%, more preferably preferably 5-12%, 5-9%.

According to another aspect of the present invention, it provides application of the quinin hydrochloride in treatment secretion gland disease, a wherein described quinin hydrochloride is included in pharmaceutical composition, described pharmaceutical composition includes the miscible organic solvent of pharmaceutically acceptable water and is the formulation for being suitable for being administered through target area, wherein when target area is administered, the concentration of a quinin hydrochloride is 0.15-0.4M, more preferably preferably 0.15-0.35M, 0.2-0.3M in described pharmaceutical composition；The concentration of the miscible organic solvent of water is 3-20%, more preferably preferably 5-12%, 5-9%.

According to a further aspect of the invention, it provides a kind of method for treating secretion gland disease, it is included to individuals in need knurl area administration comprising a quinin hydrochloride and pharmaceutically pharmaceutical composition of the miscible organic solvent of acceptable water, wherein described pharmaceutical composition is the formulation for being suitable for being administered through target area, and when the target area is administered, the concentration of a quinin hydrochloride is 0.15-0.4M, more preferably preferably 0.15-0.35M, 0.2-0.3M in described pharmaceutical composition；The concentration of the miscible organic solvent of water is 3-20%, more preferably preferably 5-12%, 5-9%.

Within the scope of the invention, when target area is administered, the administered volume of described pharmaceutical composition is not less than the 70% of the target block product for being intended to process, is preferably not less than 110%.

Within the scope of the invention, the pharmaceutically acceptable miscible organic solvent of water includes one below kind or various：Ethanol, propane diols, PEG, isopropanol.Pharmaceutical composition of the invention is prohibited because side effect is relatively strong not comprising urethane and antipyrine etc. or limits the drug ingedient for using.

Within the scope of the invention, in addition to a quinin hydrochloride and the pharmaceutically miscible organic solvent of acceptable water, the composition can also further include the medicine and/or synergist that anodyne, other treatment secrete gland disease.

Within the scope of the invention, wherein the gland disease includes form disease and functional disease.Form disease for example can be enlargement, and enlargement includes big malignant tumour, non-malignant tumors, non-struma or non-knurl hyperplasia, tumour.Functional disease for example can be hyperthyroidism.The malignant tumour for example includes breast cancer, liver cancer, cancer of pancreas, thyroid cancer, prostate cancer, lung cancer, head and neck cancer, colon cancer, nasopharyngeal carcinoma.

Embodiment of the invention has advantages below compared with prior art：Compared with existing operation and radiation cure, same efficient more friendly " treatment-patient body " relation (being disturbed to body smaller) is shown；Compared with existing systemic administration is treated, can be under the toxic and side effect for minimizing, conspicuousness ground improves curative effect；It is higher with compatibility between perilesional tissue compared with existing local application, especially curing agent (such as absolute ethyl alcohol) are treated, and better efficacy.

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

Brief description of the drawings

Fig. 1 is the injection concentration-tumour inhibiting rate relation curve of a quinin hydrochloride injection, and wherein abscissa X is knurl area injection concentration (4 × 10^XμM) index (X), and ordinate Y is the tumour inhibiting rate (Y%) measured in zoopery.

Specific embodiment

Within the scope of this invention, pharmaceutically the miscible organic solvent of acceptable water includes one below kind or various：Ethanol, propane diols, polyethylene glycol (PEG), isopropanol.Wherein PEG can optionally employ the molecular weight PEG less than 600, such as PEG200, PEG300 and PEG400 etc..

Within the scope of the invention, except a quinin hydrochloride, water and pharmaceutically in addition to the miscible organic solvent of acceptable water, the composition can also further include anodyne, other smeltings and treat the medicine and/or synergist of secretion gland disease.

The anodyne can be any appropriate person well known by persons skilled in the art, to mitigate the pain of patient, such as phenmethylol, procaine hydrochloride, anesin, hydrochloric acid benefit card etc..

The medicine that secretion gland disease is treated in described other smeltings for example can be antineoplastic.Antineoplastic can be any appropriate person well known by persons skilled in the art, to further enhance antitumor activity, such as including adriamycin, 5-FU, gemcitabine, cis-platinum, chloroquine, primaquine, qinghaosu and its derivative etc..

The synergist can be any appropriate person well known by persons skilled in the art, to further enhance treatment tumor effect, such as including curing agent and activity increase agent.

The curing agent can be any appropriate person well known by persons skilled in the art, add it to promote the necrosis of enlargement in ill area, reduce and fibrosis, and it for example can be Lauromacrogol, sodium morrhuate, EO, polidocanol and bleomycin A5 etc..

In the present invention, term " activity increase agent " refers to that activity is compared smaller or even there is no (such as the tumour inhibiting rate in zoopery is less than 40%) but can be used to increase the material of other medicines anti-tumor activity, including immunologic adjuvant.The immunologic adjuvant can be any appropriate person well known by persons skilled in the art, add it to promote the activity of the antigen for being beneficial to treat tumour related in ill area, and it for example can be inorganic adjuvant, such as aluminium hydroxide, alum；Microorganism and its product such as mycobacteria (tubercle bacillus, BCG vaccine), bacillus pumilis, Bordetella pertussis, endotoxin, bacterial extract (muramyl dipeptide) etc.；Synthetic adjuvant, such as artificial synthesized double stranded polynucleotide (double-strand polyadenylic acid, uridylic acid), levamisol, isoprinosine；Finish, such as Fei Shi adjuvants, adjuvant 65, mineral oil, vegetable oil；Immunostimulant (BCG vaccine, corynebacteria, endotoxin, trehalose, thymic peptide, OK432 etc.)；Cell factor, such as interferon, interleukins, TNF, TGF, colony stimulating factor, chemotactic factor (CF), thymosin extrasin；Heterogenetic antigen, such as inactivates streptococcus, HRBC's membranous antigen, tumor infiltrating lymphocyte, tumor vaccine.

Within the scope of the invention, term " gland " refers to being made up of gland cell or gland cell group, structure performing secreting function (secretion), including exocrine gland and incretory.Exocrine gland is the gland that a class has conduit, including pancreas (not including pancreas islet), 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 are some glands without output duct in human body, including thyroid gland, parathyroid gland, adrenal gland, hypophysis, pineal body, pancreas islet, thymus gland and sexual gland etc..Secretion gland disease refer to gland or comprising it is eccrine tissue or organ in function or/and paramophia.Wherein described gland disease includes form disease and functional disease.Form disease for example can be enlargement.Term " enlargement " refers to a kind of block-shape morphology exception enlargement occurred in structure, tissue or organ, including malignant tumour, non-malignant tumors, non-struma be big or non-knurl hyperplasia, tumour.Functional disease for example can be hyperthyroidism.The malignant tumour for example includes breast cancer, liver cancer, cancer of pancreas, thyroid cancer, prostate cancer, lung cancer, head and neck cancer, colon cancer, nasopharyngeal carcinoma.

Term " target area administration " refers to that medicine is added directly into the local interval interior of drug target Relatively centralized.For example, when disease is enlargement, target area is the enlargement area including lump and block week.When lump is tumour, target area is including knurl body and Liu Zhouliu areas；When lump is non-struma block (or accretion prism), target area is the enlargement area including lump (or accretion prism) and block (or accretion prism) week；When lump is tumour, target area is capsule.When disease is hyperthyroidism, target area is thyroid gland.When target area is administered, pharmaceutical composition of the invention is the formulation for being suitable for target area administration, for example, can be injection etc., and in the pharmaceutical composition of target area administration, the concentration of one quinin hydrochloride is 0.15-0.4M, more preferably preferably 0.15-0.35M, 0.2-0.3M；The concentration of the miscible organic solvent of water is 3-20%, more preferably preferably 5-12%, 5-9%.In addition, when target area is administered, the administered volume of described pharmaceutical composition is not less than the 70% of the volume for being intended to process, is preferably not less than 110%, so that the pharmaceutical composition comprising a quinin hydrochloride can infiltrate target spot and its peripheral region, realizes therapeutic action higher.When disease is enlargement, if the volume of its lump (such as tumour, non-struma block, capsule) is sufficiently small, the volume is the above-mentioned volume for being intended to process.The enlargement larger for lump and hyperthyroidism, then need to be allocated as the medication in batches of multiple parts, then the part volume is the above-mentioned volume for being intended to process.

The pharmaceutical composition being administered through target area in for the application of the present invention and method, by taking injection as an example, can prepare in accordance with the following methods.

Method one：The miscible immiscible organic solvent of water is first prepared into required vehicle system in water, add the quinin hydrochloride of solid phase one and the anodyne, the medicine and/or synergist of other smeltings treatment secretion gland disease that are optionally present, you can obtain a quinin hydrochloride and the injection of the miscible organic solvent of water of the concentration comprising needed for.

Method two：First the quinin hydrochloride of solid phase one is dissolved in the miscible organic solvent of water, add anodyne, the medicine and/or synergist of other smeltings treatment secretion gland disease being optionally present, it is subsequently adding appropriate water, you can obtain a quinin hydrochloride and the injection of the miscible organic solvent of water of the concentration comprising needed for.

Method three：Medicine and/or synergist that the quinin hydrochloride of solid phase one and the anodyne being optionally present, other smeltings treat secretion gland disease are dissolved in the miscible organic solvent of water respectively first, two solution are mixed again, it is subsequently adding appropriate water, you can obtain a quinin hydrochloride and the injection of the miscible organic solvent of water of the concentration comprising needed for.

By above-mentioned these methods, the various injections for including a quinin hydrochloride can be prepared.For example, the change in different injections includes：A quinin hydrochloride containing various concentrations, containing the different miscible organic solvents of water, the miscible organic solvent of water containing various concentrations, the anodyne containing variety classes and concentration, other smeltings treat medicine and/or synergist of secretion gland disease, etc..But change anyway, concentration of the quinin hydrochloride in the pharmaceutical composition that knurl area is administered should remain 0.15-0.4M, more preferably preferably 0.15-0.35M, 0.2-0.3M；The concentration of the miscible organic solvent of water should remain 3-20%, more preferably preferably 5-12%, 5-9%.

If it does, the concentration of anodyne is 0-3% (weight)；The concentration that the medicine of secretion gland disease is treated in other smeltings is 0-0.15M；The concentration of synergist is 0-3% (weight).

Within the scope of the invention, the pharmaceutical composition comprising a quinin hydrochloride can also be the form of solid powder before knurl area is administered, and be restored with corresponding solvent before administration.

Within the scope of the invention, it is to increase effective acting time of the quinin hydrochloride at administration, improve its bioavilability, it can be for example mixed in micro particles as slow-released carrier or matrix, nano-particle, micella and situ-gel matrix.

Embodiment

By specific examples below, the present invention is further illustrated, but not as limitation of the present invention.

In the examples below, unless otherwise indicated, subcutaneous transplantation knurl animal experiment is carried out as follows.

Animal：SPF grades of nude mice (BALB/C-Nude), all females of sex, the week old of animal about 6~8 during packet, body weight is 17.5-20.5g.

Animal inoculation pvaccination and packet：The tested tumour cell of in vitro culture, collects the tumour cell of exponential phase, by the tumor cell inoculation of required number in the subcutaneous of nude mice.Tested tumour cell includes：Human liver cancer cell (HepG2), human breast cancer cell (MDA-MB231), human thyroid cancer cell (SW579), Human Prostate Cancer Cells (LNCaP/AR), human pancreatic cancer cell (PANC-1), etc..Research method and result in embodiments of the invention are also applied for a quinin hydrochloride other tumour cells of anti-tumor activity to it.Treat that tumour is long to 50-100mm³When, random district's groups, respectively negative control group, positive controls and several drug research groups, every group 8 are carried out to animal subject using the softwares of PEMS 3.2 (establishment of West China HSPH of Sichuan University).

Administration：The packet same day starts administration, and injection volume is determined by dosage and concentration.Administration number of times is 6 times, and administration frequency is per once two days.The injected material of negative control group is corresponding solvent.The positive control of positive controls injection is selected from above-mentioned existing antitumor medicine, and administering mode and dosage are carried out (such as tail vein injection) by the convention of selected medicine.Drug research group is needed to inject the research medicine of variety classes and concentration according to experiment, and the injection of knurl area is unless otherwise indicated.

Observation, measurement and analysis：

1) general state：Observation 1 time daily, self administration of medication starts to experiment the 28th day, and observation index or content include but is not limited to situations such as animal administration part, appearance sign, general behavior activity, the state of mind, death and other Novel presentations.

2) body weight：Quarantine determines 1 time, is determined 1 time before administration and administration phase is determined weekly 2 times.

3) food ration：Administration phase is determined weekly 1 time.

4) relative tumor growth inhibiting rate：In the 1st, 3,5,7,11,16,21,27 days measurement transplantable tumor sizes after administration.

Gross tumor volume computing formula is as follows：TV=l/2 × a × b², a represents length of tumor in formula, and b represents tumor width.

Relative tumour volume computing formula is as follows：RTV=V_t/V₀, V in formula₀Gross tumor volume obtained by the packet administration same day (i.e. first day) measurement, V_tGross tumor volume during to measure each time.

Relative tumor proliferation rate computing formula is as follows：T/C (%)=T_RTV/C_RTV× 100, T in formula_RTVIt is positive controls or the RTV of drug research group, C_RTVIt is the RTV of negative control group.

The evaluating drug effect standard of drug research group is：T/C (%)>40 for inactive, T/C (%)≤40 (but>And Analysis of variance P compared with negative control group 15%)<0.05 is active (without preferred agents activity), T/C (%)≤15 and Analysis of variance P compared with negative control group<0.05 is have preferred agents activity.

5) inhibition rate of tumor growth (tumour inhibiting rate) is determined：System is carried out after being administered in first time within the 28th day to become celestial, carry out knurl body and weigh.Tumour inhibiting rate computing formula is as follows：Tumour inhibiting rate %=(TW-CW)/CW × 100%, TW is the average knurl weight of positive controls or drug research group in formula；CW is the average knurl weight of negative control group.

The evaluating drug effect standard of drug research group is：Tumour inhibiting rate<40% for inactive, tumour inhibiting rate >=40% (but<And Analysis of variance P compared with negative control group 85%)<0.05 for active, tumour inhibiting rate >=85% and Analysis of variance P compared with negative control group<0.05 is have preferred agents activity.

In the examples below, zoopery carries out statistical test using the group differences of duplicate measurements variance analysis (Repeated Measures ANOVA) respectively to index mean.When group differences are statistically significant (P≤0.05), each group is compared with negative control group difference using least significant difference method.Quantitative target is described using mean ± standard error (X ± SEM).When heterogeneity of variance is pointed out in LEVENE homogeneitys test of variance (P≤0.05), group difference is compared using Mann-Whitney U rank tests (M-W methods).All of statistical analysis, completes under the softwares of SPSS for Windows 13.0.

Embodiment 1：The preparation of one quinin hydrochloride injection

Table 1

Remarks:+ to refer in addition to the composition in table remaining be full water for injection

- refer to being free of.

In table 1, the preparation method of A2 is as follows：At room temperature, mixed during 0.9ml absolute ethyl alcohols are added into 7.5ml waters for injection, 0.2ml phenmethylols are added thereto dissolving again, then dissolved in the quinin hydrochloride dry powder of 595mg mono- being added into the solution, adding water for injection makes cumulative volume reach 10ml, 2ml/ bottle standby, just prepared injection A2 is packed as after well mixed.

The preparation method of A3 is as follows：At room temperature, mixed during 0.9ml absolute ethyl alcohols are added into 7.5ml waters for injection, 0.2ml phenmethylols are added thereto dissolving again, then dissolved in the quinin hydrochloride dry powder of 595mg mono- being added into the solution, add the dissolving of 1g polidocanols, being eventually adding water for injection makes cumulative volume reach 10ml, it is well mixed after be packed as 2ml/ bottle standby, just prepared injection A3.

NM A1 and A4 are prepared according to method similar to the above.

Embodiment 2：The optimum condition research of application

The optimum condition of application of the quinin hydrochloride of the invention in gland disease treatment, is studied in anti-knurl experiment (external inhibiting tumor cell experiment and transplantable tumor animal model experiment) first.

In the experiment of anti-knurl, positive control is usually existing clinical medicine (such as cis-platinum, adriamycin, mitomycin, gemcitabine, 5-FU etc.).Their clinical meaning is hypotoxicity high activity, for example, hypotoxicity and the tumour inhibiting rate up to 75% are shown in zoopery.Term " anti-tumor activity ", it is sometimes again referred to as active, refer to that there is certain anti-homophylic property of knurl compared with existing clinical medicine.A kind of material in such as testing in vitro there is relatively low 503nhibiting concentration (to be abbreviated as IC₅₀50 μM are less than in generally suppressing reaction at 72 hours) or inhibition rate of tumor growth (abbreviation tumour inhibiting rate higher is shown in zoopery, typically larger than 40% but less than 75%), just it is considered as having similarity with existing clinical medicine, so as to show anti-tumor activity, the material is also just referred to as active material.Term " reactive conditions " refers to that the something that makes that researcher provides shows the condition of its anti-tumor activity.For example, make a quinin hydrochloride tumour inhibiting rate brought up to from 35% or so 75% or so condition (application method, dosage etc.).

Term " preferably anti-tumor activity ", it is sometimes again referred to as preferred active, refer to the property compared with existing clinical medicine with more hypotoxicity and considerably higher anti-tumor activity.For example, it is preferable to activity can refer to show significantly greater tumour inhibiting rate (such as more than 85%) with less side reaction cost (non-toxic only slight side effect) in zoopery.Term " optimum condition " refers to that the something that can make that researcher provides shows the condition of its preferred anti-tumor activity.Term " preferred substance " refers to the material for not only giving anti-tumor activity condition and giving optimum condition.

The optimum condition of application of the quinin hydrochloride of the invention in oncotherapy, is studied on anti-tumor activity experiment and transplantable tumor animal model in vitro first.

1) anti-tumor activities

In the present invention, medium effective concentration (is abbreviated as IC₅₀) it is drug concentration when cell survival rate reduces 50% in experiment in vitro.11 plants of man―machine systems are selected in this experiment, and the antitumor action of medicine is determined using tetrazolium MTT reducing process.Experiment sets positive and negative control group and seminar.Negative control group adds isometric physiological saline, and positive controls are the conventional chemotherapeutic drugs 5-FU of various concentrations (in μM ol/l levels).Seminar is a quinin hydrochloride solution (in μM ol/l levels) of the various concentrations that the A2 prepared with embodiment 1 dilutes as mother liquor.

In experiment, from the attached tumor cells of exponential phase, after being digested with pancreatin, the suspension of required cell concentration is made into the RPMI l640 culture mediums containing 10% calf serum, be seeded in 96 well culture plates, 200 μ l are inoculated with per hole, then at 37 DEG C and 5%CO₂Middle culture 24h.The culture medium containing various concentrations sample that seminar and positive controls renew, negative control group then changes the culture medium containing isometric physiological saline, and every group sets 5 parallel holes, makes final volume for 200ul/ holes, cultivates 48 hours.Then abandoning supernatant, adds .37 DEG C of the serum free medium containing 0.2mg/ml MTT of 200 μ l Fresh to continue to cultivate 4h per hole.It is careful to abandon supernatant, and 200 μ l DMSO are added, after being mixed with miniature ultrasonic oscillator, with tested wavelength as 570nm on ELIASA, reference wavelength is that 450nm determines OD value.It is calculated as follows the inhibiting rate of drug on tumor cell growth：Growth of tumour cell inhibiting rate %=(1-OD experiment/OD controls) × 100%

Dose-effect curve can obtain to inhibition rate of tumor cell mapping with same sample various concentrations, medium effective concentration (IC is then calculated using Logit methods₅₀Value).Usual IC₅₀Concentration be less than 100 μM when, then judgement sample has lethal effect to tumour cell in vitro.Table 2 is test composition to testing the IC of tumour cell₅₀, concentration value is with the quinin hydrochloride densimeter in composition.Generally speaking, they all μM concentration level, significant difference is not seen statistically.And the IC of positive control₅₀Then between 0.01-0.5 μM.

Table 2

By Try tumour cells
		Human breast carcinoma MDA-MB231	62.2
Human liver cancer HepG2	31.9
		Human prostata cancer LNCaP/AR	32.9
Human thyroid cancer cell SW579	35.6
		Human pancreatic cancer cell PANC-1	45.7

Further experiment also demonstrates that the miscible organic solvent of water in concentration range introduced in the present composition has no negative effect to the anti-tumor activity of a quinin hydrochloride.

2) application method

Experimental animal is lotus HCC nude mice, is divided into negative control group, positive controls and drug research group.Negative control group injecting normal saline, the conventional antitumor medicine 5-FU of positive controls injection, drug research group injects a quinin hydrochloride solution.When tail vein injection is small, in, heavy dose of concentration for 0.025M a quinin hydrochloride aqueous solution when, although we have also made some technical improvement, and result of the eligible result still with prior art is consistent, and tumour inhibiting rate is less than 30%.And reach 75% as the tumour inhibiting rate of the 5-FU of positive control.

When we are injected directly into a kind of preparation on animal knurl body to dilute the standby quinin hydrochlorides of 0.25M mono- (containing 2% anodyne), unexpected result is but occurred in that.(tumour inhibiting rate is for example set to increase although target area administration may improve the efficiency of systemic administration as you know, generally improve 10-30%), and drug effect has been brought up to mysterious height (can improve more than 90%) by our achievement in research.Conversely, when said composition passes through tail vein injection, tumour inhibiting rate is again below 30%.

The administration of knurl area depends not only on drug effect, also depends on its security.Therefore, we compare the animal tail vein injection of the quinin hydrochloride injections of 0.25M mono- and the maximum tolerated dose (Maximum tolerated dose, MTO) of Lump body injection, method is as follows：

Experimental animal is lotus HCC nude mice, it is grouped into tail vein injection various dose group (four groups) and Lump body injection various dose group (four groups) at random, 5 nude mices of each dosage group, injection dosage is respectively 250mg/kg, 300mg/kg, 350mg/kg and 400mg/kg.Every other day administration, continuous five times.

Naked mouse changes of weight and Survival are observed in experiment, while toxicity of the record with obvious physiological significance, Ru Kang Blighted scurfs, dehydration, drowsiness, incoordination and is short of breath.If there is Mouse Weight decline>30% continues more than 3 days or then stops the increase of trial drug dosage because of toxic death, determines previous group dosage for maximum tolerated dose, is within 21 days the experimental observation cycle, puts to death within the 22nd day animal, and autopsy is visually observed and whether there is lesion.MTO in tail vein injection is less than 250mg/kg, and the MTO in Lump body injection may be up to 300mg/kg.Using same dosage, the injection of knurl area looks also more smaller than the toxicity being injected intravenously.

Further investigation revealed that, also there is similar performance in the animal experiment of some other tumours.Some other quinin hydrochloride injections prepared by embodiment 1 also have similar performance.Therefore, it is main application mode that application of the invention have selected target area administration.

3) administration concentrations scope

Experimental animal is lotus HCC nude mice, is divided into negative control group, positive controls and drug research group.The conventional antitumor medicine 5-FU of positive controls injection, drug research group injects a quinin hydrochloride aqueous solution.Positive controls application method is still tail vein injection, and negative control group and drug research group are Lump body injection.Positive controls application method is still tail vein injection, and negative control group and drug research group are Lump body injection.The preparation method of the injection of each drug research group (B, C, D, E, F, G, H, I group) is identical with the preparation method of A2 in table 1, includes 2% phenmethylol but contains the required quinin hydrochloride of various concentrations one.One quinin hydrochloride Drug level designs anomaly gradient design, and uses logarithm gradient design, i.e., with reference to its IC₅₀The negative control group (A groups) of inactive concentration (5 μM) is set, with its 10²、10³、5×10³、10⁴、3×10⁴、6×10⁴、8×10⁴、10⁵Experimental concentration is set again, and the injection concentration of a quinin hydrochloride is respectively 0.0005M, 0.005M, 0.025M, 0.05M, 0.15M, 0.3M, 0.4M, 0.5M.Drug research Zu Liu areas injection dosage is less than mono- quinin hydrochlorides of 300mg/kg, and volume injected is determined by injection dosage and concentration, and administration number of times is 5 times, and administration frequency is per once two days.Concrete outcome is as shown in Figure 1 and is described as follows.

It is in very big concentration range (difference of X is more than 3), i.e., foreseeable more than IC in general knowledge₅₀Tens times, in the range of even thousands of times of Drug level, do not observe pharmacodynamic change corresponding with this change in concentration, tumour inhibiting rate is less than 25% always.Compared with negative control group, tumour inhibiting rate no significant difference (P<0.05).And at 80mM (X=4) place, tumour inhibiting rate mutation rises to 55%.Yi liu rates are raised to 78% at 0.15M, and then (difference of X is less than 1) reaches 87% in the range of a Drug level for relative narrower, occurs in that breakthrough.At 0.4M, declining occurs again in tumour inhibiting rate.

Within whole experimental period, negative control group gross tumor volume shows a rising trend.Drug research group in the range of the injection concentration of the quinin hydrochlorides of 0.15M-0.4M mono- and 3-9% ethanol compared with negative control group, the statistically significant (P of tumour inhibiting rate difference<0.05).The tumour inhibiting rate of positive control is 75%, the statistically significant (P of difference compared with negative control group<0.05).

Sum it up, when having its Lump body injection concentration only more than a certain critical value, a quinin hydrochloride injection just shows that drug effect, to the extremely sensitive of concentration, then becomes less sensitive again, and even tumour inhibiting rate is likely to occur decline.

General status, changes of weight, the death rate that we have been observed after the injection of animal subject knurl area.During testing, each seminar of a quinin hydrochloride injection has no that nude mice is dead, and average weight and negative control group changes of weight represent medicine under experimental conditions without obvious toxic reaction close to (being dropped by less than 10%).Compared with positive controls, general status are similar to for seminar.But positive controls average weight declines bigger (being more than 20%), points out a stronger toxic reaction.

In view of, not only containing a quinin hydrochloride but also containing organic solvent, we go back the partial denaturization that emphasis has observed medication area in thing in combination of the invention.During the medication of knurl area, compared with negative control group, in seminar, especially high concentration (>0.4M) most of injection site has obvious color and luster to change in group, or even with myodegeneration, scope is more within 10mm.This points out the partial injury that the application conditions cause myocyte really, injection site to have inflammatory cell infiltration, but does not influence normal function.After stopping is administered, the musculature outward appearance of seminar nude mice injection site is done step-by-step normalization.The injection site for cuing open the nude mice for killing after stopping to administration for 30 days has carried out check pathological section, as a result shows that seminar is obviously reduced with the difference of negative control group, tends to normal.

Additionally, we also utilize the effect of one quinin hydrochloride injection of the identical technique study knurl body otherwise varied for the development of inoculation time difference thus cancer cell.Drug research group is B groups and C groups.10 days more late than the C groups animals of inoculation human liver cancer cell of B groups.10 days after last group inoculation, each group selects knurl body average external volume to respectively may be about 28mm respectively³(B groups) and 126mm³8 tumor bearing nude mices injection of (C groups).The quinin hydrochlorides of Lump body injection 0.25M mono-, injection dosage is 300mg/kg, and volume injected is determined by injection dosage and concentration.Every other day administration, continuous 6 times.

From experiment the 5th day, each drug research group tumor-bearing mice tumour relative tumour volume was substantially reduced；Tumour relative volume reaches minimum at 17 days；From 32 days, rising occurs in C groups relative tumour volume, and has the trend of recurrence；B groups then effect always clearly, temporarily without the trend of recurrence.At 38 days, C groups are more significantly greater than the knurl weight of B groups, and the average knurl weight of B groups is close to 0.

This experimental result illustrates that under the optimum condition in the anti-knurl application that the present invention is provided, a quinin hydrochloride injection can be applied to treating malignant tumor, even can be used for the tumour that removal completely has certain carcinogenic risk, to prevent it from developing into malignant tumour.

In some documents, a quinin hydrochloride is also considered curing agent.We have selected it has been generally acknowledged that a kind of more effectively curing agent (polidocanol) has carried out comparative study.At identical conditions, the concentration of polidocanol is that, until 6w/v% (concentration causes the obvious bubble in knurl area and poisoning symptom occurs), its tumour inhibiting rate 30% or so, finds no similar concentration-tumour inhibiting rate relation trend always from 1.5w/v%.The result explanation of this control experiment, composition of the invention is clearly distinguished from the application based on curing agent mechanism.

Similar results are also obtained with some other the quinin hydrochloride injections (such as the injection in table 1) prepared in embodiment 1.Additionally, in the examples below, similar results are also obtained when a quinin hydrochloride injection is applied to the zoopery of other gland gland diseases.

According to these pharmacodynamic results and local side reaction result, optimum condition of the quinin hydrochloride injection that the present invention is provided in gland gland disease treatment use is as follows：Target area is administered, and the concentration of a quinin hydrochloride is 0.15-0.4M, more preferably preferably 0.15-0.35M, 0.2-0.3M in injection；The concentration of the miscible organic solvent of water is 3-20%, more preferably preferably 5-12%, 5-9%.

Additionally, another battery of tests shows that under same concentration, a larger injection volume shows more preferable pharmacological effect to pharmaceutical composition of the invention.It is control dosage if Gross Target Volume is too big, branch's treatment can be carried out to target area.Each administered volume of injection is necessary for more than the 70% of part volume to be processed, preferably more than 110%.When the target block of target area is lump (such as tumour, non-struma block) and its is individual sufficiently small, part volume to be processed is single lump volume.

Embodiment 3：Application in medicinal breast disease treatment

Clinically, most common mammary gland disease is mammary gland enlargement, including tumour and non-struma it is big.In the present embodiment, the preparation method of the injection of each drug research group (B, C, D, E, F group) is identical with the preparation method of A2 in table 1,2% phenmethylol is included, a quinin hydrochloride and water-miscible organic solvent injection concentration are respectively 0.1M and 5%, 0.15M and 6%, 0.20M and 7%, 0.30M and 9%, 0.4M and 12%.Injection dosage is less than mono- quinin hydrochlorides of 300mg/kg animals, and volume injected is determined by injection dosage and concentration.

1. tumor of breast treatment

Experimental animal is lotus breast cancer cell nude mice, is divided into negative control group, positive controls and drug research group.Positive controls tail vein injection routine antitumor medicine adriamycin (7mg/kg), negative control group Lump body injection physiological saline.Drug research Zu Liu areas inject, and administration number of times is 5 times, and administration frequency is per once two days.

Within whole experimental period, negative control group gross tumor volume shows a rising trend；From experiment the 5th day, positive controls and each drug research group tumor-bearing mice gross tumor volume were reduced compared with negative control group, the statistically significant (P of difference<0.05).

Until the 17th day from testing the 7th day, the Relative tumor proliferation rate (T/C) of each drug research group tumor-bearing mice is respectively less than 40%；From testing the 17th day, B group Relative tumors proliferation rate rises, and has the trend of recurrence；The Relative tumor proliferation rate (T/C) of C, D, E, F group is still reducing, and illustrates that this four groups still have obvious tumor growth inhibitory effect after drug withdrawal.From testing the 22nd day, B, F group Relative tumor proliferation rate rise, and have recurrence trend；And D, E group Relative tumor proliferation rate remain close to 10%, effect always clearly, temporarily without recurrence trend.During testing, suppression ratio D, the E group of positive controls Relative tumor proliferation rate is late in time, significantly larger in data (40% or so).

The 28th day after administration, the knurl weight of each drug research group tumor-bearing mice is significantly reduced compared with negative control group, and with significant difference (P<0.05), wherein with D, E group tumour inhibiting rate highest (more than 85%)；Other group tumour inhibiting rates are less than 70%.Positive controls tumour inhibiting rate is 53%.

In addition, under optimum condition in the anti-knurl application that the present invention is provided, the effect of the one quinin hydrochloride injection knurl body otherwise varied for the development of inoculation time difference thus cancer cell, it is also the better shorter tumour tumor killing effect of inoculation time, it is except can be applied to breast cancer treatment, even can be used for the removal completely of the nonmalignant tumor for having larger carcinogenic risk, to prevent it to develop into malignant tumor.

During testing, each seminar has no that nude mice is dead, and average weight and negative control group changes of weight represent medicine under experimental conditions without obvious toxic reaction close to (being dropped by less than 10%).Compared with positive controls, general status are similar to for seminar.But positive controls average weight declines bigger (being more than 20%), points out a stronger toxic reaction.

During the medication of target area, compared with negative control group, in seminar, especially in E, F group most of injection site have obvious color and luster change, even with myodegeneration, scope is more within 10mm.Further research explanation, these denaturation are that quinin hydrochloride effect is caused.After stopping is administered, the musculature outward appearance of experimental group nude mice injection site is done step-by-step normalization.The injection site for cuing open the nude mice for killing after stopping to administration for 30 days has carried out check pathological section, as a result shows that test group is obviously reduced with the difference of negative control group, tends to normal.

2. the non-knurl hyperplasia of mammary gland (non-struma is big) treatment

The proliferation of mammary gland is a kind of non-tumour benign proliferative enlargement, and clinical manifestation is characterized with different degrees of breast pain and lump in breast.Zoopery is summarized as follows：

Animal used：SPF grades of non-pregnant rat, all females of sex, the weight of animals is 150-180g during packet.

Animal models and is grouped：By above-mentioned rat muscle injection oestradiol benzoate 0.5mg/kg, 1 times/day, continuous 20 days.Then intramuscular injection progesterone 5mg/kg, 1 times/day, continuous 5 days.After modeling terminates, 6 rats are taken at random and are put to death, take second pair of breast biopsy, see lobule of mammary gland and acinus showed increased, ductal epithelium increase, cell growth it is active, mammary gland is in secretor state, represents modeling success.Random district's groups are carried out to animal using the softwares of PEMS 3.2, is divided into blank control group (end modeling animal) and modeling successfully with the following group：Positive controls, negative control group (A groups), drug research group.

Administration：The packet same day starts administration.The injected material of negative control group is 9% ethanol.Positive control is RUZENGNING PIAN, is gavaged, and administration number of times is 27 times, and administration frequency is for once a day, dosage is each 1g/kg.Each drug research group is injected in lump, and administration number of times is 5 times, and administration frequency is per once two days.

Observation, measurement and analysis：

1) general states observation：Observation 1 time daily, self administration of medication starts to experiment the 28th day, and observation index or content include but is not limited to situations such as animal administration part, appearance sign, general behavior activity, the state of mind, death and other Novel presentations.

2) body weight determinations：Determined 1 time before administration and administration phase is determined weekly 2 times.

3) food rations are determined：Administration phase is determined weekly 1 time.

4) breast local form：Respectively at the 1st, 3,5,7,11,16,21,27 days after administration with second pair or so two papilla diameters (being designated as D) of slide measure accurate measurement rat.

It is with respect to papilla diameter computing formula：RTD=D_t/D₀, D in formula₀It is the packet gained papilla diameter of administration measurement for the first time, D_tPapilla diameter during to measure each time.

It is with respect to nipple Magnification computing formula：T/C (%)=T_RTD/C_RTD× 100, T in formula_RTDIt is positive controls or drug research group RTD, C_RTDIt is negative control group RTD.

The evaluating drug effect standard of drug research group is：T/C (%)>50 for inactive, T/C (%)≤50 (but>And Analysis of variance P compared with negative control group 25%)<0.05 for active, T/C (%)≤25 and Analysis of variance P compared with negative control group<0.05 is have preferred activity.

5) breast tissues pathology changes：Put to death animal within second day after last dose, win rat second to mammary gland, paraffin section, in the change of light Microscopic observation breast tissue pathology form after HE dyeing.By observing the form of leaflet and acinus, each group rat breast tissue pathology is integrated, 4 grades are divided into by pathological proliferation degree：Lobule of mammary gland not hyperplasia, body of gland quantity is few, and acinus is not expanded, and remembers 0 point；Without obvious hyperplasia, indivedual acinuses have slight hyperplasia to lobule of mammary gland, but without expansion, remember 1 point；Lobule of mammary gland major part hyperplasia, part acinus is substantially expanded, and remembers 2 points；The obvious hyperplasia of lobule of mammary gland, acinus is in extreme expansion state, and glandular epithelium has substantial amounts of secretion in flat in acinus and in conduit, remember 3 points；Mammary gland alveolus, conduit, leaflet pathological proliferation substantially, remember 4 points.

The evaluating drug effect standard of drug research group is：Pathology integration >=3 for inactive, 1<Pathology is integrated<3 and Analysis of variance P compared with negative control group<0.05 is active, and pathology integrates≤1 and Analysis of variance P compared with negative control group<0.05 is have preferred activity.

The different degrees of appearance fur after modeling of animal is owed gloss, depilation, apocleisis, easily enrages etc. symptom.The all tumor-bearing mice body weight of drug research group and food ration show no obvious abnormalities change, the not statistically significant (P of difference compared with negative control group>0.05).

The packet same day, animal teat enlargement is upright, congested obvious, and indivedual visible nipple surfaces have yellow-white secretion to remain.Within whole experimental period, negative control group papilla diameter shows a rising trend；From experiment the 7th day, each drug research group papilla diameter was reduced compared with negative control group in addition to B groups, the statistically significant (P of difference<0.05).Until the 19th day from testing the 10th day, each drug research group is respectively less than 50% with respect to nipple Magnification (T/C) beyond B groups；From testing the 19th day, it is slow with respect to the decline of nipple Magnification that C, D group are respectively less than 20%, E with respect to nipple Magnification.During testing, positive controls are late in time with respect to suppression ratio C, D, the E groups of nipple Magnification, significantly larger in data (being more than 30%).The 20th day after administration, C, D, E pathology integration≤1, and with significant difference (P<0.05) and positive controls pathology integration be more than 2.

More than during each experiment, each seminar has no animal dead, average weight and negative control group changes of weight close to (being dropped by less than 10%), represents medicine under experimental conditions without obvious toxic reaction.Compared with positive controls, general status are similar to for seminar.But transplantable tumor tests positives control group average weight and declines bigger (being more than 20%), points out a stronger toxic reaction.

During the medication of target area, compared with negative control group, in seminar, especially in maximum concentration group most of injection site have obvious color and luster change, even with myodegeneration, scope is more within 10mm.Further research explanation, these denaturation are that quinin hydrochloride effect is caused.After stopping is administered, the musculature outward appearance of experimental group nude mice injection site is done step-by-step normalization.The injection site for cuing open the nude mice for killing after stopping to administration for 30 days has carried out check pathological section, as a result shows that test group is obviously reduced with the difference of negative control group, tends to normal.

Similar results are also obtained with some other the quinin hydrochloride injections (such as the injection in table 1) prepared in embodiment 1.According to the above pharmacodynamic result and local side reaction result, optimum condition of the pharmaceutical composition that the present invention is provided in mammary gland disease application is treated is as follows：Target area is administered, and the concentration of a quinin hydrochloride is 0.15-0.4M, more preferably preferably 0.15-0.35M, 0.2-0.3M in injection；The concentration of the miscible organic solvent of water is 3-20%, more preferably preferably 5-12%, 5-9%.

Embodiment 4：Application in liver disease treatment

Experimental animal is lotus HCC nude mice, positive controls tail vein injection routine antitumor medicine 5-FU.Each drug research group (B, C, D, E, F group) and other experiment conditions are identical with the test method that the tumor of breast described in embodiment 3 is treated.Its experimental result is as follows.

From experiment the 5th day, each drug research group tumor-bearing mice gross tumor volume was reduced compared with negative control group, the statistically significant (P of difference<0.05).Until the 17th day from testing the 7th day, the Relative tumor proliferation rate (T/C) of each drug research group tumor-bearing mice is respectively less than 40%.From testing the 17th day, the Relative tumor proliferation rate (T/C) of D, E, F group is respectively less than 15%, illustrates that they still have obvious tumor growth inhibitory effect after drug withdrawal.

The 28th day after administration, the knurl weight of each drug research group tumor-bearing mice is significantly reduced compared with negative control group, and with significant difference (P<0.05).The tumour inhibiting rate of each drug research group tumor-bearing mice is all higher than>40%, the tumour inhibiting rate of wherein D, E group may be up to more than 90%, and other group tumour inhibiting rates are less than 75%.During testing, in time late, significantly larger in data (35% or so), its tumour inhibiting rate is then significantly smaller (72%) for suppression ratio D, E, E, F group of positive controls Relative tumor proliferation rate.

In addition, the model similar to embodiment 2 is also illustrated, under optimum condition in the anti-knurl application that the present invention is provided, the effect of the one quinin hydrochloride injection knurl body otherwise varied for the development of inoculation time difference thus tumour cell, it is also the better shorter tumour tumor killing effect of inoculation time, it even can be used for the removal completely of the nonmalignant tumor for having larger carcinogenic risk except can be applied to treatment of cancer, to prevent it to develop into malignant tumor.

During being tested more than, each seminar has no animal dead, average weight and negative control group changes of weight close to (being dropped by less than 10%), represents medicine under experimental conditions without obvious toxic reaction.Compared with positive controls, general status are similar to for seminar.But transplantable tumor tests positives control group average weight and declines bigger (being more than 20%), points out a stronger toxic reaction.

During the medication of target area, compared with negative control group, in seminar, especially in F groups most of injection site have obvious color and luster change, even with myodegeneration, scope is more within 10mm.Further research explanation, these denaturation are that quinin hydrochloride effect is caused.After stopping is administered, the musculature outward appearance of experimental group nude mice injection site is done step-by-step normalization.The injection site for cuing open the nude mice for killing after stopping to administration for 30 days has carried out check pathological section, as a result shows that test group is obviously reduced with the difference of negative control group, tends to normal.

Similar results are also obtained with other quinin hydrochloride injections.According to these pharmacodynamic results and local side reaction result, optimum condition of the quinin hydrochloride that the present invention is provided in liver disease treatment use is as follows：Target area is administered, and the concentration of a quinin hydrochloride is 0.15-0.4M, more preferably preferably 0.15-0.35M, 0.2-0.3M in injection；The concentration of the miscible organic solvent of water is 3-20%, more preferably preferably 5-12%, 5-9%.

Embodiment 5：Application in pancreatic disease treatment

Experimental animal is pancreatic tumor borne cell nude mice.Positive controls tail vein injection routine antitumor medicine gemcitabine (100mg/kg).Each drug research group (B, C, D, E, F group) and other experiment conditions are identical with the method that the tumor of breast described in embodiment 3 is treated.Its experimental result is as follows.

From experiment the 5th day, each drug research group tumor-bearing mice gross tumor volume was reduced compared with negative control group, the statistically significant (P of difference<0.05).Until the 17th day from testing the 7th day, the Relative tumor proliferation rate (T/C) of each drug research group tumor-bearing mice is respectively less than 40%.From testing the 17th day, the Relative tumor proliferation rate (T/C) of C, D, E group is respectively less than 15%, illustrates that they still have obvious tumor growth inhibitory effect after drug withdrawal.

The 28th day after administration, the knurl weight of each drug research group tumor-bearing mice is significantly reduced compared with negative control group, and with significant difference (P<0.05).The tumour inhibiting rate of each drug research group tumor-bearing mice is all higher than>40%, the tumour inhibiting rate of wherein D, E group may be up to more than 90%.Other group tumour inhibiting rates are less than 70%.During testing, in time late, significantly larger in data (35% or so), its tumour inhibiting rate is then significantly smaller (62%) for suppression ratio D, E, E, F group of positive controls Relative tumor proliferation rate.

In addition, the model similar to embodiment 2 is also illustrated, under optimum condition in the anti-knurl application that the present invention is provided, the effect of the one quinin hydrochloride injection knurl body otherwise varied for the development of inoculation time difference thus cancer cell, it is also the better shorter tumour tumor killing effect of inoculation time, it even can be used for the removal completely of the nonmalignant tumor for having larger carcinogenic risk except can be applied to treatment of cancer, to prevent it to develop into malignant tumor.

Some other quinin hydrochloride injections prepared using embodiment 1, it is also possible to obtain similar results.According to these pharmacodynamic results and local side reaction result, optimum condition of the quinin hydrochloride that the present invention is provided in pancreatic disease treatment use is as follows：Target area is administered, and the concentration of a quinin hydrochloride is 0.15-0.4M, more preferably preferably 0.15-0.35M, 0.2-0.3M in injection；The concentration of the miscible organic solvent of water is 3-20%, more preferably preferably 5-12%, 5-9%.

Embodiment 6：Application in thyroid disease treatment

Clinically, most common thyroid disease is Thyroid Gland Swell and hyperthyroidism.In the present embodiment, the injection and dosage of each drug research group (B, C, D, E, F group) are same as Example 3.

1. thyroid tumors treatment

Experimental animal is lotus thyroid carcinoma cell nude mice, positive controls tail vein injection routine antitumor medicine adriamycin (4mg/kg), negative control group Lump body injection physiological saline.Other experiment conditions are identical with the method for tumor of breast Experiment on therapy in embodiment 3.Its experimental result is as follows.

The 28th day after administration, the knurl weight of each drug research group tumor-bearing mice is significantly reduced compared with negative control group, and with significant difference (P<0.05).The tumour inhibiting rate of each drug research group tumor-bearing mice is all higher than>40%, the tumour inhibiting rate of wherein D, E group may be up to more than 92%, and other group tumour inhibiting rates are less than 75%.During testing, in time late, significantly larger in data (35% or so), its tumour inhibiting rate is then significantly smaller (61%) for suppression ratio D, E, the F groups of positive controls Relative tumor proliferation rate.

In addition, the effect of the injection knurl body otherwise varied for the development of inoculation time difference thus cancer cell, it is also the better shorter tumour tumor killing effect of inoculation time, it is except can be applied to treatment of cancer, even can be used for the removal completely of the non-malignant tumors for having larger carcinogenic risk, to prevent it to develop into malignant tumor.

2. goitre treatment

Simple goiter (simple goiter), also known as non-inflammatory goitre, is to cause thyroid gland compensatory enlargement caused by thyroxine dyssynthesis by non-inflammatory or tumprigenicity reason.By setting up goiter due to iodine deficiency animal model, this research has been carried out.

Experimental animal：From adult rat, male and female are not limited, body weight 100-200g.

Modeling and packet：During beginning, animal is raised with iodine-deficient forage (amount of iodine 50ng/kg weight in wet base feeds mainly contain corn 55%, soybean 15%, yeast 10%, milk powder 10%, low iodine mutton 9%, inorganic salts vitamin 1%).Progressively remove low iodine mutton and milk powder, rat is adapted to Low iodine diet without causing underfed generation.Rat is raised more than 3 months in the environment of iodine-deficient forage nursing.It is decreased obviously with urinating iodine, the obvious enlargement of thyroid gland is to model successfully.Random district's groups are carried out to animal using the softwares of PEMS 3.2, is divided into blank control group (end modeling animal) and modeling successfully with the following group：Positive controls, negative control group (A groups), drug research group, every group 6.

Administration：The packet same day starts administration.Positive control is Potassiumiodate KIO₃, gavaging, administration number of times is 27 times, and administration frequency is for once a day, dosage is every time 0.4 μ g/kg.The injected material of negative control group is corresponding solvent.Drug research group and negative control group are Thyroid Tumors and periphery injection, and administration number of times is 10 times, and administration frequency is per once two days.

Observation, measurement and analysis：

1) general states observation：Observation 1 time daily, self administration of medication starts to experiment the 22nd day, and observation index or content include but is not limited to situations such as animal administration part, appearance sign, general behavior activity, the state of mind, death and other Novel presentations.

4) local forms：Respectively at observation rat thyroid enlargement in the 1st, 6,11,16,21 days after administration.

5) .24h iodine discharge rate：24h urines and excrement are collected, 24h urine iodine and excrement iodine discharge rate is determined and calculate.Urine iodide determination is using gentle acid nitrification arsenic-cerium reaction AAS.Excrement iodide determination uses sode ash method.

6) pathological examinations：Put to death within the 10th day after last dose animal, observation thyroid morphology, fibr tissue form between folliculus form, epithelial cell form, leaflet.

In the whole test period, animal without death, without abnormal body temperature.

24h iodine discharge rate detection display before packet, modeling animal is substantially reduced with not modeling animal and compare its iodine discharge rate, the obvious enlargement of thyroid gland.From experiment the 11st day, each drug research group Thyroid Gland Swell was obviously improved compared with negative control group, and the reduction of iodine discharge rate is relative to ease up.Wherein, D, E group iodine discharge rate and blank control group reach unanimity (difference<25%), effect is substantially than positive controls (with blank control group difference>35%) it is good.

In pathological examination, negative control group can be observed the obvious enlargement of thyroid gland, and folliculus is intensive, and it is in high column that epithelial hyperplasia is loose, it is seen that the cell mass rope of hyperplasia, interfollicular blood vessel showed increased, lumen distention is congested, and fibr tissue increases between leaflet.Can be observed to be obviously improved in each drug research group.Wherein, D, E group are in thyroid size, folliculus form size, the aspect such as fibr tissue tends to normal between epithelial cell, leaflet, hence it is evident that than positive controls closer to the state for not modeling rat.

3. Treatment of Hyperthyroidism

Hyperthyroidism (hyperthyroidism, HT) (abbreviation hyperthyroidism in the present invention), refer to cause internal thyroxine secretion excessively by many factors, it is the main one group of general name of disease for showing to cause to increase with hypermetabolism with system stimulants such as nerve, circulation, digestion.Therefore, hyperthyroidism is a kind of clinical syndrome.

Experimental animal：Adult male SD rats, body weight 300-400g, 36-40 age in days.

Modeling and packet：Levothyrocine is dissolved in physiological saline, daily intraperitoneal injection, injection dosage is 50 μ g/100g body weight, continuous injection 10 days.Then Serological testing and pathological examination are carried out, to judge whether to model successfully.Random district's groups are carried out to animal using the softwares of PEMS 3.2, is divided into blank control group (end modeling animal) and modeling successfully with the following group：Positive controls, negative control group (A groups), drug research group, every group 6.

Administration：The packet same day starts administration.Positive control is methimazol, is gavaged, and administration number of times is 27 times, and administration frequency is for once a day, dosage is each 2mg/kg.The injected material of negative control group is physiological saline.Drug research group and negative control group are thyroid body injection, and administration number of times is 10 times, and administration frequency is per once two days.

Observation, measurement and analysis：

1) general states observation：Observe 1 time within every 2 days, self administration of medication starts to administration the 30th day, observation index or content include but is not limited to situations such as animal administration part, appearance sign, general behavior activity, the state of mind, death and other Novel presentations.

4) Serological testing：Respectively at blood 5ml is extracted from animal to be checked within the 30th day before administration and after being administered, its serum T is determined using the method for exempting from (RIA) is put₃、T₄And thyrotropic hormone (TSH) concentration value.

5) pathological examinations：Respectively at administration before and administration after the 30th day by blood drawing after animal to be checked put to death, observe thyroid morphology, fibr tissue form between folliculus form, epithelial cell form, leaflet.Thyroid gland is visually observed and increased in diffusivity in various degree；Microscopic observation is in high column to follicular epithelium hyperplasia, and has small folliculus to be formed；There are many epithelial cells not of uniform size and absorbs vacuole, interstitial rich blood vessel, hyperemia, lymphadenia in folliculus periphery.

Serological testing before packet shows that modeling animal compares its serum T with animal is not modeled₃、T₄Significantly raised (raising more than 100%), TSH is then decreased obviously, and illustrates to model successfully.Upon administration in the Serological testing of the 30th day, each drug research group serum T₃、T₄The T compared with negative control group₃、T₄Rising eases up, and TSH declines and tends to improving.Wherein, the Serological testing result of D, E group is less than 25% with the blood serum values difference for not modeling animal, and the difference of positive controls is more than 35%.

In pathological examination, negative control group can be observed thyroid gland and increase in diffusivity, and follicular epithelium hyperplasia is in high column and has small folliculus to be formed, and folliculus periphery many epithelial cells not of uniform size occurs and absorbs vacuole, interstitial rich blood vessel, hyperemia, lymphadenia.Compared with negative control group, the thyroid gland diffusivity of each drug research group has gone down, and the situation that follicular epithelium hyperplasia and small folliculus are formed is significantly reduced；The epithelial cell that folliculus periphery occurs absorbs vacuole, the number of interstitial blood vessel and congested and lymphadenia have been weakened.Each group thyroid gland diffusivity is in disappearance trend, and other pathological phenomenas relevant with hyperthyroidism have regression trend, close to the state for not modeling animal, hence it is evident that better than positive controls.

During the medication of target area, compared with negative control group, in seminar, especially in F groups most of injection site have obvious color and luster change, even with myodegeneration, scope is more within 10mm.Further research explanation, these denaturation are that the collective effect of a quinin hydrochloride and ethanol is caused.After stopping is administered, the musculature outward appearance of experimental group nude mice injection site is done step-by-step normalization.The injection site for cuing open the nude mice for killing after stopping to administration for 30 days has carried out check pathological section, as a result shows that test group is obviously reduced with the difference of negative control group, tends to normal.

Some other quinin hydrochloride injections prepared using embodiment 1, it is also possible to obtain similar results.According to these pharmacodynamic results and local side reaction result, optimum condition of the quinin hydrochloride that the present invention is provided in thyroid disease treatment use is as follows：Target area is administered, and the concentration of a quinin hydrochloride is 0.15-0.4M, more preferably preferably 0.15-0.35M, 0.2-0.3M in injection；The concentration of the miscible organic solvent of water is 3-20%, more preferably preferably 5-12%, 5-9%.

Embodiment 7：Application in prostata disease treating

Clinically, most common prostatic disorders are prostatitis adenoncus, including tumour and non-struma it is big.In the present embodiment, the injection and dosage of each drug research group (B, C, D, E, F group) are same as Example 3.

1. tumor of prostate treatment

Experimental animal is lotus prostate gland cancer cell nude mice, positive controls tail vein injection routine antitumor medicine adriamycin (4mg/kg), negative control group Lump body injection physiological saline.Other experiment conditions are identical with the method for tumor of breast Experiment on therapy in embodiment 3.Its experimental result is as follows.

Within whole experimental period, negative control group gross tumor volume shows a rising trend；From experiment the 5th day, each drug research group tumor-bearing mice gross tumor volume beyond positive controls and B groups was reduced compared with negative control group, the statistically significant (P of difference<0.05).

In administration phase, the Relative tumor proliferation rate of each drug research group tumor-bearing mice declines；From testing the 17th day, each drug research group Relative tumor proliferation rate beyond B groups very low level (<40%).From testing the 22nd day, C, D group Relative tumor proliferation rate are less than 15%, effect always clearly, temporarily without recurrence trend.During testing, each drug research group beyond the suppression ratio B groups of positive controls Relative tumor proliferation rate is late in time, significantly larger in data (being more than 35%).

The 28th day after administration, the knurl weight of each drug research group tumor-bearing mice beyond B groups is significantly reduced compared with negative control group, and with significant difference (P<0.05), wherein with C, D group tumour inhibiting rate highest (more than 90%), other group tumour inhibiting rates are less than 80%.Positive controls tumour inhibiting rate is 63%.

In addition, the effect of the injection knurl body otherwise varied for the development of inoculation time difference thus tumour cell, it is also the better shorter tumour tumor killing effect of inoculation time, except can be applied to relatively late prostate cancer and early prostate cancer, even can be used for the removal completely of the nonmalignant tumor for having larger carcinogenic risk, to prevent it to develop into malignant tumor.

2. hyperplasia of prostate (hypertrophy) treatment

Hyperplasia of prostate (benign prostatic hypertrophia, BPH), is called hypertrophy of the prostate, and the research of its etiology and pathogenesis is more, but can not extremely determine so far, and it can cause bladder-neck obstruction, influences the normal function of urinary system.

Animal model：Kunming mouse, male, body weight 20-25g.

Modeling and packet：First bilateral testes are extractd through scrotum, Post operation the 3rd day is again to plucking the daily hypodermic injection testosterone propionate 5mg/kg/d (being dissolved in soybean oil) of the successful animal of testis, continuous 3 weeks, Serological testing and pathological examination are then carried out, to judge whether to model successfully.Random district's groups are carried out to animal using PEMS3.2 softwares, is divided into blank control group (end modeling animal) and modeling successfully with the following group：Positive controls, negative control group (A groups), drug research group, every group 6.

Administration：The packet same day starts administration.Positive control is the easypro suspension of the retention of urine, and gavage, administration number of times is 27 times, and administration frequency is for once a day, dosage is each 0.3g/kg.The target area of each drug research group is hyperplasia of prostate lump, and administration number of times is 8 times, and administration frequency is per once two days.The injected material of negative control group is physiological saline.

Observation, measurement and analysis：

4) Serological testing：Respectively at blood is taken from animal eye socket to be checked within the 30th day before administration and after administration, its serum testosterone (T), estradiol (E is determined₂) level.The measure of T in serum, with mouse T antibody ELISA detection methods.E in serum₂Measure mouse E₂Antibody ELISA detection method.

5) pathological examinations：Animal to be checked is put to death after the 28th day eye socket after the administration same day and administration takes blood, prostata tissue, thymus gland, kidney and spleen are taken rapidly, claim prostate wet weight, and calculate prostate index (prostate index=prostate wet weight mg/ Mouse Weights g)；Prostata tissue, thymus gland, kidney and spleen are fixed in 10% formalin solution, FFPE, cut into slices, HE dyeing, the change of light Microscopic observation each group prostatic histomorphology.

In Serological testing before packet, animal is compared with animal is not modeled for modeling, and T levels significantly raise (P ＜ 0.01), E in its serum₂Level significantly reduces (P ＜ 0.01), illustrates that hyperplasia of prostate is modeled successfully.Upon administration in the Serological testing of the 28th day, compared with negative control group, the serum T level of each drug research group significantly returns drop, E₂Level is significantly gone up.Wherein, the Serological testing result of D, E group is less than 22% with the blood serum values difference for not modeling animal, and the difference of positive controls is more than 41%.

In pathological examination before packet, animal is compared with animal is not modeled for modeling, the wet quality of its prostate and prostate index substantially increase (P ＜ 0.01) and prostatic histomorphology (galandular epithelium and interstitial have a large amount of proliferations of fibrous tissue, interstitial have cell infiltration) extremely, illustrate that hyperplasia of prostate is modeled successfully.Upon administration in the pathological examination of the 28th day, compared with negative control group, the wet quality of prostate and prostate index of each drug research group are substantially returned drop (P ＜ 0.01), prostatic histomorphology and are occurred be clearly better (P ＜ 0.01) extremely.The result of drug research group has concentration-effect relation, and the exception of the wet quality of D, E group prostate, prostate index and prostatic histomorphology is in disappearance trend, close to the state for not modeling mouse, hence it is evident that better than positive controls.

Some other quinin hydrochloride injections prepared using embodiment 1, it is also possible to obtain similar results.According to these pharmacodynamic results and local side reaction result, optimum condition of the quinin hydrochloride that the present invention is provided in prostata disease treating application is as follows：Target area is administered, and the concentration of a quinin hydrochloride is 0.15-0.4M, more preferably preferably 0.15-0.35M, 0.2-0.3M in injection；The concentration of the miscible organic solvent of water is 3-20%, more preferably preferably 5-12%, 5-9%.

Claims

1. it is a kind of for treat secretion gland disease pharmaceutical composition, it includes a quinin hydrochloride and the pharmaceutically miscible organic solvent of acceptable water, and be the formulation for being suitable for being administered through target area, wherein the concentration of a quinin hydrochloride is 0.15-0.4M and the concentration of the miscible organic solvent of water is 3-20% in described pharmaceutical composition when being administered through target area.

2. the pharmaceutical composition described in claim 1, wherein the concentration of a quinin hydrochloride is 0.15-0.35M, is preferably 0.2-0.3M and the concentration of the miscible organic solvent of the water is 5-12%, preferably 5-9%.

3. the pharmaceutical composition described in claim 1, wherein when target area is administered, the volume of the injection is not less than the 70% of the target block product for being intended to process, is preferably not less than 110%.

4. the pharmaceutical composition described in claim 1, it includes a quinin hydrochloride, the pharmaceutically acceptable miscible organic solvent of water and water.

5. the pharmaceutical composition described in claim 1, wherein the pharmaceutically acceptable miscible organic solvent of water includes one below kind or various：Ethanol, propane diols, PEG, isopropanol.

6. the pharmaceutical composition described in claim 1, its medicine and/or synergist that gland disease is also secreted comprising anodyne, other treatment, the anodyne preferably includes one below kind or various：Phenmethylol, procaine hydrochloride, anesin, hydrochloric acid benefit card, and the synergist preferably includes curing agent and/or immunologic adjuvant, the curing agent preferably includes one below kind or various：Lauromacrogol, sodium morrhuate, EO, polidocanol, bleomycin A5.

7. the pharmaceutical composition described in claim 1, wherein the gland disease includes secretion adenoncus.

8. the pharmaceutical composition described in claim 7, wherein the gland enlargement includes big malignant tumour, non-malignant tumors, non-struma or non-knurl hyperplasia, tumour.

9. the pharmaceutical composition described in claim 1, wherein the gland disease includes hyperthyroidism.

10. the pharmaceutical composition described in claim 8, wherein the malignant tumour includes breast cancer, liver cancer, cancer of pancreas, thyroid cancer, prostate cancer, lung cancer, head and neck cancer, colon cancer, nasopharyngeal carcinoma.