CN109069584A - A kind of TRAIL albuminoid persistently inhibits the medication of growth of tumour cell - Google Patents

Abstract

Provide the medication that a kind of TRAIL albuminoid persistently inhibits growth of tumour cell, specific method is the administration at interval, repetition and full course for the treatment of drug exposure, by extending dosing interval, increase tumour cell in the drug exposure of the full course for the treatment of, it is unattenuated in the effect of the full course for the treatment of to make drug, to persistently inhibit tumour growth.

Description

A kind of TRAIL albuminoid persistently inhibits the medication of growth of tumour cell

Technical field

The present invention relates to field of medicaments, and in particular to a kind of TRAIL albuminoid persistently inhibits the administration of growth of tumour cell Method.

Background technique

TRAIL is the member of tumor necrosis factor (Tumor necrosis factor, TNF) superfamily, gene order It is obtained by Wiley et al. and 1996 by Pitti et al. independent cloning respectively at nineteen ninety-five, the latter is named as necrohormone 2 Ligand (Apo2ligand, Apo2L).Later research confirms that Apo2L and TRAIL are substantially same protein, therefore, Apo2L/TRAIL can be traditionally referred to as.The function of TRAIL is as organism is congenital or the tune of acquired immunity first Agent is saved, secondly plays antineoplastic action as immunosurveillance in the exogenous apoptosis pathway of cell.The great advantage of TRAIL is The property of can choose induced various types of tumors Apoptosis and to normal cell substantially without toxicity.Research data show no matter It is external or in vivo, human tumor cell line of the Apo2L/TRAIL for a variety of sources, including knot (straight) intestinal cancer, lung cancer, mammary gland Cancer, prostate cancer, cancer of pancreas, kidney, central nerve neuroma, thyroid cancer, lymthoma, leukaemia and multiple marrow Tumor etc. all has the function of apoptosis-induced.

In tumor patient body, the destruction of Apoptosis balance — that is, the increasing of the decrease of rush apoptotic signal and anti-apoptotic signal It is strong very common, therefore repair cell apoptosis balance out of control is a kind of important tumor therapeuticing method.For anti-tumor drug Understanding deeply recognize people, no matter cytotoxic drug, molecular targeted agents or monoclonal antibody, play a role During all refer to the activation of apoptosis of tumor cells approach, the signal path approach of inducing apoptosis of tumour cell is these medicines The hinge and key link that object plays a role, and it is exactly tumor development and drug resistant important mechanisms that apoptosis, which is escaped,.

In 20 years of discovery so far, TRAIL is developed as a kind of important potential anti-tumor drug always, The clinical trial of TRAIL entered for II phase in foreign countries, was completed for III phase at home.A large amount of internal and external tests confirm that TRAIL has There is tumor-specific cytotoxicity, especially shows significantly to cooperate with when it is with small dose chemotherapy drug combination and synergy is made With.On the contrary, fast-growth and transfer of the TRAIL tolerance caused by the missing of Apoptosis mechanism with tumour cell in research discovery body Clear correlation.

Nearest progress shows that relying only on Apo2L/TRAIL and treating a variety of different types of tumours is still far from No more.Although the agonistic monoclonal antibodies of recombined human Apo2L/TRAIL or TRAIL receptor DR4/DR5 are in I phase clinical treatment Obtain encouraging as a result, but not showing specific clinical benefit in the II phase clinical research then carried out.Face Bed research prompt, Apo2L/TRAIL class drug show as relatively limited therapeutic response (Limited tumor to kinds of tumors Responses), it is overall it is medium shows slightly disappointed (Disappointingly modest overall), some patientss without clinic by Beneficial (Lack of therapeutic benefit).

A large number of studies show that passage of tumor cell strain pair more than normal cell and approximately half of (even as high as 60%) TRAIL shows drug resistance.According to the summary of Roberta Di Pietro and Giorgio Zauli, Apo2L/TRAIL is to Research 92 plants primary or passage of tumor cell in 61 plants of sensitivities, Sensitivity rate 66.3%, and to remaining 31 plants of drug resistance is resistance to Medicine rate is 33.7%.A large amount of Research Literature concentrates in the drug resistance of TRAIL, and the research contents that adds up to is concentrated on lower section Face: (1) the high tumour cell of grade malignancy is more likely to drug resistance (Malignant tumors are resistant), and (2) are come Derived from the basic drug resistance of intracorporal primary tumor cell (Resistance in primary tumor cells), (3) it is congenital and Two kinds of situations of acquired resistance and deposit (Congenital and acquired resistance), (4) multiple target point and more way The resistance mechanism of diameter has illustrated (Resistance mechanisms to TRAIL), and (5) are effectively evaded drug resistant method and visited Rope is in Showed Very Brisk (Resistance and effective therapies).

Links and factor of the almost all of TRAIL Sensitive Tumor Cells in its apoptotic signal access all have phase As complete and function, and some links and factor of each TRAIL cells of resistant tumors in apoptotic signal access are deposited In defect and variation, these defects and variation are so that the raising extremely of these drug resistant apoptosis of tumor cells threshold values, is easier to escape and wither Removing is died, so that continued propagation is proliferated.

Restricting Apo2L/TRAIL and playing the factor of preferable clinical efficacy further includes rshTRAIL albumen itself, due to trimerization Body is the stabilizing active form of TRAIL, and the preparation of TRAIL tripolymer storage difficulty and heterogeneous (the Trimer has of structure height Low stability), the vivo biodistribution half-life period of TRAIL is shorter (Inherent short-half-life), does not have preferably Pharmacokinetic properties.

Numerous studies confirm, are applied alone Apo2L/TRAIL that many tumour cells are not generated with efficient inhibition and killing Effect.To find out its cause, apoptosis of tumor cells signal path is a sufficiently complex huge system, wherein both withering comprising many rush Factor is died, and includes a large amount of survivin, the interaction of these two aspects factor determines finally returning for tumour cell Place.The sound and function of apoptotic signal access is the necessary condition of apoptosis of tumor cells, but is not adequate condition.Tumor death The manifestations of signal path exist: (1) exogenous and endogenous apoptosis signal path, including promote antiapoptotic factors and anti-apoptotic because Son.Promoting antiapoptotic factors includes Caspases, DRs, FADD, Smac, Bax, Bak etc., anti-apoptosis factor include c-FLIP, XIAP, Bcl-2, Mcl-1 etc..(2) promote survival-signal access, including NF κ B, PI3K, Akt, MAPK etc..(3) apoptosis of tumor cells defect Heterogeneity, the different links in apoptotic signal access can occur for the apoptosis defect of tumour cell.The study found that TRAIL sheet Body biological function has diversity.TRAIL receptor stimulating agent, by classical signals access, is formed dead in conjunction with TRAIL receptor Die induction compound, active cell apoptosis.TRAIL receptor stimulating agent forms second level complex also in conjunction with TRAIL receptor, leads to Cross non-classical signal path, activation includes the different kinases such as I κ B/NF- κ B, MAPKs, PKC, PI3K/Akt, Src, directly or The non-apoptotic response of indirect induction.The activation of these signal paths is related to the proliferation of tumour and transfer.

TRAIL receptor stimulating agent needs that performance can be oriented by the regulation of some substances and enhances antineoplastic action. A variety of different types of drugs, molecule or Gene intervention can enhance TRAIL to the sensibility of tumour cell, these drugs include Different types of chemotherapeutics, natural products, small molecule kinase inhibitors etc..They pass through respectively strengthens extracellular apoptotic signal Access or mitochondrial apoptosis signal path inhibit the joint of other cells survival signal paths or several accesses and enhance TRAIL The apoptosis of tumor cells activity of induction.The effect link of exogenous apoptotic signal access includes up-regulation DRs, promotes DRs to Lipid Rafts The movement and aggregation of film micro area promote the recruitment of FADD, DISC to receptor-ligand complexes, enhance the activity of Caspases, press down The activity etc. of the apoptosis antagonism factor FLIP, XIAP, IAPs processed.The link of mitochondrial apoptosis signal path includes enhancing mitochondrial membrane Potential depolarising, promotes mitochondria to discharge Cytc, Smac or ARTS, promotes Bid to be cracked into tBid, promote Bax, Bad oligomerization, Inhibit factors such as apoptosis antagonism Bcl-2, Bcl-xL, Bcl-w, Mcl-1, Survivin etc..Inhibit other cells survival signals logical Road includes ERK/PI3K/Akt, MEK, Jak-STAT3, MAPK, NF- κ B etc..

Genentech Inc is most early in studying the pharmacokinetics of ApoL/TRAIL in several animal models. The plasma half-life of rodent single intravenous injection Apo2L/TRAIL is 3~5 minutes, and primate be then 23~ 31 minutes.In 1 hour intravenous injection Apo2L/TRAIL (0,10,30 or 100mg/kg) of machin, drug is quickly removed, repeatedly The plasma half-life that (maximum dose level 100mg/kg) is administered is 28~30 minutes, and multiple dosing does not change partly declining for drug Phase does not also cause drug accumulation.Apo2L/TRAIL linear pharmacokinetic properties in the dosage range of research, serum medicine The area under the curve (AUC) of object concentration and the increase (Cmax) of maximum plasma concentration and dosage increase in ratio.In chimpanzee (dosage 1,5,50mg/kg) also complies with above-mentioned rule in In vivo study.

The selection of Apo2L/TRAIL administration route.It is proved on one group of Nude Mouse Model, Apo2L/TRAIL vein The growth inhibition effect for tumour is injected better than intraperitoneal injection.On a variety of Nude Mouse Models, Apo2L/TRAIL is in short-term Between be injected intravenously (1 hour/day or 3 hours/day) or persistent intravenous injection (24 hour/day) and can obviously inhibit the life of tumour It is long, but the curative effect of short time intravenous injection is superior to long lasting for administration.The results show that the short time maintains relatively high blood Concentration is more stronger than the antitumor action being constantly exposed under lower drug concentration.1 hour/day is given with the intravenous injection of 3 hours/day The direct comparison of medicine shows, the administration mode activity at least having the same of the administration mode of 1 hour/day and 3 hours/day, because This clinically selects the medication of 1 hour/day.

The determination of Apo2L/TRAIL dose-effect relationship.It is proved on one group of Nude Mouse Model, Apo2L/TRAIL is in short-term Between to be injected intravenously the peak effective dose of (1 hour/day or 3 hours/day) be 30~90mg/kg, furthermore use 5 days short time The curative effect of continuous intravenous injection (once a day) is better than same dose lower 3 days short time continuous intravenous injections (once a day), And intravenous injection in 5 days is enough to inhibit the growth of tumour.In conjunction with Apo2L/TRAIL shorter drug metabolism half-life period (21~31 Minute), therefore finally determining dosage regimen is continuous intravenous injection 5 days once a day (1 hour/day), repeats one within every three weeks Secondary, this use course for the treatment of also with clinically Treated with Chemotherapeutic Drugs object is consistent.

Nevertheless, experimental data disclosed in Genentech Inc is shown, and once a day (1 hour/day), continuous vein Injection 5 days, the preclinical drug effect for being repeated once dosage regimen for every three weeks is unsatisfactory, this is also to cause Apo2L/TRAIL clinical The undesirable one of the major reasons of curative effect.Preclinical laboratory data show that cell highly sensitive for Apo2L/TRAIL is (as tied Colon-cancer cell COLO205, non-small cell lung cancer cell NCI-H2122 etc.) nude mouse xenograft tumor model, (1 is small once a day When/day), 5 days dosage regimens of continuous intravenous injection, tumour is obviously reduced in the 0th~5 (7) day, and tumour continues again after 10 days Increase.For medium sensitivity cell (such as non-small cell lung cancer NCI-H460) nude mouse xenograft tumor model, (1 is small once a day When/day), 5 days dosage regimens of continuous intravenous injection, tumour increasesd slowly at the 0th~5 day, and 5 days after tumour increase it is fast Speed, with negative control group indifference.It is above-mentioned the experimental results showed that, once a day (1 hour/day), give within continuous intravenous injection 5 days Prescription case can not continue to inhibit tumour growth (in 21 days treatment cycles), and this dosage regimen is not Apo2L/ The best dosage regimen of TRAIL.

The dosage regimen that Genentech Inc is determined at least haves the defects that 4 aspects: (1) different sensitivitys is swollen Oncocyte is different for the therapeutic response of Apo2L/TRAIL, and the data source that Apo2L/TRAIL dosage regimen determines is based primarily upon It is for highly sensitive and medium sensitivity tumour cell inhibiting effect, and it is thin to opposite drug-resistant tumor to represent it completely The practical function of born of the same parents, therefore the experimental basis that dosage regimen determines is not comprehensive enough, applicability is restricted.(2) even for height Degree is sensitive and the tumour cell of medium sensitivity, Apo2L/TRAIL intravenous injection, once a day, continuously 5 days dosage regimen still not It can continue the growth of inhibition tumour cell.Apo2L/TRAIL is obvious to the inhibiting effect of tumour growth during administration, and is discontinued It rapidly disappears afterwards to the inhibiting effect of tumour growth, the second half section of the course for the treatment of is practically under the effect of no drug effect.(3) Genentech Inc do not explore extend Apo2L/TRAIL administration time (intravenous injection, once a day, continuously 10 days or 15 It) with intravenous injection, once a day, the difference of continuous 5 days dosage regimen curative effects.According to our data, be injected intravenously, Once a day, continuously the tumor-inhibiting action of 10 days or 15 days dosage regimens better than intravenous injection, once a day, continuously gives prescription in 5 days Case.(4) Genentech Inc do not explore Apo2L/TRAIL intravenous injection, interval, repeat, multiple dosing and intravenous injection, Once a day, the continuous difference of 5 days dosage regimen curative effects.

Summary of the invention

And the dosage regimen for using interval, repetition, full course for the treatment of drug to expose is exactly that the present invention increases substantially TRAIL class egg The white key point for persistently inhibiting tumour growth effect.

Therefore, in view of the drawbacks of the prior art, the object of the present invention is to provide a kind of TRAIL albuminoids persistently to inhibit tumour The medication of cell growth, can increase substantially TRAIL albuminoid and persistently kinds of tumor cells be inhibited to grow.

The technical scheme of the present invention is realized as follows:

A kind of TRAIL albuminoid persistently inhibits the medication of growth of tumour cell, is interval, repetition and full course for the treatment of medicine The administration of object exposure, is extension dosing interval, increases tumour cell in the drug exposure of the full course for the treatment of, is treating drug entirely The effect of journey is unattenuated, to persistently inhibit tumour growth.

Further, the TRAIL albuminoid includes natural or recombination Apo2L/TRAIL albumen after birth 114-281aa Outer segment, TRAIL receptor-selective mutant, TRAIL cell-penetrating peptide sample mutant TRAIL-Mu3, TRAIL-MuR5 and TRAIL- The TRAIL-MuR5S4TR and TRAIL-MuR6S4TR of MuR6, TRAIL cell-penetrating peptide mutain and in other mutant one Kind is several, wherein the amino acid sequence of other mutant and the similarity of wild-type protein are 75% or more.

Further, the tumour cell is solid tumor or derived from bone marrow tumour, and the solid tumor includes lung cancer, Colon and rectum One or more of cancer, breast cancer, cancer of pancreas, liver cancer, gastric cancer, oophoroma, kidney, brain tumor, osteochondroma, prostate cancer； The derived from bone marrow tumour includes one or more of leukaemia, non-Hodgkin's lymphoma.

Further, persistently inhibiting growth of tumour cell includes the horizontal tumor-inhibiting action of cell in vitro and the suppression of animal experiment in vivo Tumor effect；In vitro experiment, TRAIL albuminoid observes 24-96 hours drugs pair in effective dosage ranges and cytosis The inhibiting rate of tumour cell；In the tumour cell of different sensibility, TRAIL albuminoid is small in 24-72 to growth of tumour cell When be in the peak of inhibition, for highly sensitive cell strain or higher activity, the rush hour of tumor suppression continues By 96 hours；In vivo experiment, the animal-transplanted tumor of different interval medication is in obvious growth inhibition shape in 21 days State, Relative tumor growth T/C is ≤40%.

Further, with daily medication compared with primary, continuous medication for five days therapy, in tumour cell transplanted tumor in nude mice mould Tumour inhibiting rate at least improves 20% or more in type, and acting duration extends 5 days or more in the course for the treatment of 21 days at one.

Further, the dosage regimen at interval, repetition and full course for the treatment of drug exposure includes any one following:

(1) TRAIL albuminoid be injected intravenously, the next day it is primary, from the course for the treatment of 0 day, administration time was respectively 0,2,4,6,8, 10,12,14,16,18 or respectively 0,2,4,6,8,10,12,14,16,18,20, every 21 days as a treatment course；

(2) TRAIL albuminoid is injected intravenously, and three-times-weekly, from the course for the treatment of 0 day, administration time was respectively 0,2,4,7,9, 11,14,16,18, every 21 days as a treatment course；

(3) TRAIL albuminoid is injected intravenously, and is administered once every three days, and from the course for the treatment of 0 day, administration time was respectively 0,3, 6,9,12,15,18, every 21 days as a treatment course；

(4) TRAIL albuminoid is injected intravenously, and is administered once within every four days, and from the course for the treatment of 0 day, administration time was respectively 0,4, 8,12,16,20, every 21 days as a treatment course.

Further, since the maximal tolerance dose that Apo2L/TRAIL drug is given in a single dose is more than 1500mg/kg, using height In the minimum activity that its inside and outside works, extend its dosing interval.

The rational use of medicines is a clinical pharmacy unfailing project for many years, and many pharmacy men have done largely thus thus Fruitful work, after making a definite diagnosis for disease, correctly select therapeutic agent, rationally determine dosing interval, be patient early stage The key point of rehabilitation.

Drug metabolism half-life period (t_1/2) it is also known as biological half-life and biological half effect phase, refer to plasma drug level by maximum value The required time, usually uses t when drop by half_1/2It indicates.Drug half-life is long to be indicated to eliminate slowly in vivo, and the residence time is long.Cause This pays attention to drug half-life, and for grasping drug, residence time, savings degree especially determine the treatment of medication repeatedly in vivo Delivery time, adjustment dosage regimen have very big value.Drug half-life determines dosing interval, and guarantee is used repeatedly The drug safety of medicine, avoids poisonous side effect of medicine caused by drug accumulation from playing a significant role.But drug half-life and drug are made Often inconsistent with the duration, drug half-life is not equal to the drug effect duration.Traditionally, medication interval time master Wanting reference drug half-life period (drug elimination rate) is a kind of way for more considering drug safety.In fact, partly being declined according to drug Phase determines that dosing interval is that have its adaptation range.Some delivery time appropriate values, with its drug t_1/2What numerical value coincide substantially Drug is suitable for according to t_1/2To determine dosing interval.Effect eliminates class drug (t in such as_1/2=4~8h), such as Ciprofloxacin, promise Oxygen sand star, atropine.Metoclopramide (metoclopramide), phenalgin petrin etc. can be administered 3 times or 4 times/d.Slow effect eliminates class drug (t_1/2=8~12h), such as sulfamethoxazole, gliclazide (Diamicron), ketoconazole (inner element is fragrant), amphotericin, often Day administration 2 times.It is super to eliminate class drug (t at a slow speed_1/2> is for 24 hours), such as piroxicam (feldene), Nitrazepam, digoxin, chlorine Third urea of sulphur etc. is administered daily 1 time.The especially short supper-fast removing class drug (t of drug half-life_1/2≤ 1h) and it is quickly clear Except class drug (t_1/2=1~4h), because it is eliminated fastly in vivo, it is intended to maintain required blood concentration, according to t_1/2It arranges between being administered Every the time, it is necessary to increase administration number of times, frequent drug administration, not only patient cannot receive in this way, and clinic is not accomplished yet.Such as penicillin It is metabolized and drains in vivo all very fast, half-life period only 0.5h.If pressing t_1/2Frequent drug administration is to maintain required blood concentration to show It is so worthless.Penicillin dosing interval is than its t on clinical practice_1/2It is much longer.This is because dosage is big, it is reached Blood concentration substantially exceed the minimal inhibitory concentration to most of microbe.In other words, the safety of penicillin is good, and treatment refers to Number is high, and administration number of times can be reduced suitably, and dosage is bigger, and dosing interval is longer.For t_1/2Especially long drug, it is such as super slow Speed eliminates class drug (t_1/2=200h) in foxalin, if press t_1/2It being administered once every 9 days, blood concentration fluctuation is big, Erious adverse reaction clinically often takes low dose by daily single, and the fluctuation of such blood concentration is small, and safe range expands. T in a word_1/2Reflect speed of the drug from internal release rate, for eliminating fast, t_1/2Short drug administration number ratio t_1/2Long Drug is frequent, and dosing interval can be appropriately extended in the drug high for therapeutic index.Biology of the Apo2L/TRAIL in rodent Half-life period is 3~5 minutes, is 23~31 minutes in the biological half-life of the animal of non-human primates, belongs to supper-fast removing class Drug.The quick removing of Apo2L/TRAIL is main to be completed by kidney, is removed highly relevant with glomerular filtration rate.Single The maximal tolerance dose for giving Apo2L/TRAIL drug is more than 1500mg/kg, and safety is very high, and therapeutic index is especially big. Apo2L/TRAIL is not appropriate for determining medication interval in strict accordance with its drug metabolism half-life period, therefore using higher than its inside and outside The minimum activity to work, so that it is feasible for extending its dosing interval.

More and more researchs disclose, the metabolic index of many drugs and its biological action duration not fully one It causes, there are drug aftereffects for some drugs.As i.e. there are drug withdrawal aftereffect (Post antibiotic for many antibiotics Effect, PAE).After PAE refers to bacterium and the of short duration contact of antibiotic, when drug concentration descends below minimum inhibitory concentration (MIC) Or after eliminating, effect of the bacterial growth by lasting inhibition.It is gradually extensive to the research of PAE both at home and abroad in the latest 20 years, And using PAE as the important evidence of the important parameter of evaluation antibiotic and design clinical dosing regimen, for instructing infectious disease The treatment of disease.For a long time it is believed that antibacterials must reach and maintain the good antibacterial of effective blood drug concentration competence exertion Effect.Therefore the previously use of antibiotic is mostly removed according to the medicaments insensitive degree of bacterium and drug effective blood drug concentration, half-life period The pharmacokinetic parameters such as rate determine dosage and dosing interval, and have ignored drug to the shadow of the growth and breeding rule of bacterium Loud and human immunity mechanism role during killing bacterium.It is observed that after bacterium and antibiotic effect, when After drug is removed, bacterium can be made to generate a variety of detectable variations: activity, the cellular morphology of such as enzyme and non-zymoprotein change Become.Bacterial metabolism and growth inhibition bacterial receptor change, again contacts to the change of the sensibility of phagocytosis and to antibiotic Sensibility change etc., in this way change be exactly antibiotic antibacterial aftereffect.Inhibition of the Apo2L/TRAIL for tumour cell Also there is specific tumor suppression aftereffect.We pass through the study found that Apo2L/TRAIL for different tumour cells have it is different Sensibility, for highly sensitive tumour cell, when Apo2L/TRAIL a certain concentration range and tumour cell effect 5 minutes, so Apo2L/TRAIL is eluted with culture medium afterwards, is cultivated 24~72 hours at 37 DEG C, the culture hole through eluting is compareed with normal effect Hole is compared, and does not have difference at 24~72 hours to the inhibiting effect of tumour.For relatively drug resistant tumour cell, work as Apo2L/ TRAIL is acted on 1 hour in higher concentration range with tumour cell, is then eluted Apo2L/TRAIL with culture medium, 37 DEG C culture 24~72 hours, culture hole through eluting compared with normally acting on control wells, to the inhibiting effect of tumour 24~ There is no difference within 72 hours.Above-mentioned experiment shows the death receptor that Apo2L/TRAIL can be quickly incorporated on Sensitive Tumor Cells film And causing the transduction of apoptotic signal access, once starting, subsequent process is no longer dependent on extracellular free the above process The presence of Apo2L/TRAIL, and for drug resistant tumour cell, the contact start time needed for the above process is longer.When Apo2L/TRAIL is acted within the scope of a certain concentration with tumour cell, observes inhibition of 24~96 hours drugs to tumour cell Rate, as a result, it has been found that, in the different kinds of tumor cells of susceptibility, Apo2L/TRAIL is higher using concentration, lasting to inhibit swollen The time of tumor cell proliferation is faster.For higher activity, Apo2L/TRAIL is small 24~96 to the inhibition of tumour cell When maintain drug effect peak, for lower activity, Apo2L/TRAIL reached high to the inhibition of tumour cell at 24 hours Peak drug effect is gradually reduced for 48~72 hours, and effect in 84 hours and 96 hours completely disappears.Above-mentioned experiment shows Apo2L/TRAIL With the effect of tumour cell, work rapidly, persistent.The combination of death receptor on Apo2L/TRAIL and tumor cell membrane Stringent concentration dependant and affine dependence, and with receptor number on initial tumour cell and non-correlation, this combination according to The medicaments insensitive degree of tumour cell and have differences, the combination of sensitive cells is very fast (5 minutes), and mdr cell combine Relatively slow (1 hour).Make receptor multimerization after the death receptor (DR4, DR5) on Apo2L/TRAIL and cell membrane combines, rapidly Cause aggregation and redistribution of the ligand/receptor compound in cell membrane Lipid Rafts film micro area, then ligand/receptor complex recruits by Body molecule Fas associated death domain (Adaptor molecule Fas-associated death domain, FADD) with And preceding Caspase-8；Form dead inducing signaling complex (Death-inducing signaling complex, DISC). Subsequent Caspase-8 activation causes the biological effect of apoptosis through mitochondria Dependent and non-mitochondria Dependent.Non-thread grain Body Dependent is mainly initial phase Caspases, if Caspase8,9,10 activate, causes Caspases protease cascade anti- It answers, the homology enzyme effective stage Caspases in further chain type hydrolysis activation downstream is finally activated such as Caspase6,7 Caspase-3.Mitochondria Dependent changes mitochondrial transmembrane potentials by various apoptosis precipitating factors, and it is penetrating to increase mitochondria Property, lead to the release and caspase activation of thick apoptotic cell pigment c (Cytochrome c, Cyt c) and Smac/DIABLO etc., Apoptotic proteins bcl-2 family is wherein important adjusting factor.Two apoptotic signal accesses finally meet at Caspase-3, then Being catalyzed many apoptosis correlation targeted molecular decomposition by Caspase-3 leads to Apoptosis.The transduction and transmitting of apoptotic signal access Process complexity is tediously long, the various time-consuming of the link of experience, this is point of Apo2L/TRAIL inducing apoptosis of tumour cell tumor suppression aftereffect Subsolution is released.

In vitro experiment, TRAIL albuminoid (TRAIL-Mu3 and TRAIL-MuR5S4TR) is in a certain concentration (dosage) model Enclose interior and cytosis, inhibiting rate of 24~96 hours drugs of observation to tumour cell.In the tumour cell of different sensibility, TRAIL albuminoid was in the peak of inhibition to growth of tumour cell at 24~72 hours, for highly sensitive cell strain (or Higher activity), the rush hour of tumor suppression continues to 96 hours.

Different dosing number animal experiment in vivo is shown, compares physiological saline group, taxol group, TRAIL is (once a day, Successive administration 5 days, once a day, successive administration 5 days, is spaced 2 days, then successive administration 5 days and once a day, and successive administration 5 days It being spaced 2 days successive administration 5 days again afterwards, 5 days three different dosing times of successive administration again after being spaced 2 days again) group is to people Lung cancer NCI-H460 nude mouse xenograft tumor has certain inhibiting effect, wherein be injected intravenously, once a day, successive administration 5 days, Interval 2 days, then 5 days groups of successive administration and be spaced 2 days successive administration 5 days again once a day, after successive administration 5 days, again between The curative effect of 5 days groups of successive administration is obviously superior to individually once a day again after 2 days, 5 days groups of successive administration, after administration, suppression There is significant difference in ratio of outflow.But the raising of the tumor-inhibiting action compared with scheme 2 of scheme 3 is not statistically significant (P > 0.05).It is real It tests and shows that increase administration number of times (extending administration time) can significantly improve drug to the tumour inhibiting rate of Nude Mouse Model, but give For medicine number at 15 times compared with 10 times, the raising of tumour inhibiting rate is unobvious.

Different dosing interval animal experiment in vivo shows, compares physiological saline group, taxol group, TRAIL-Mu3 group with MuR5S4TR each group has certain inhibiting effect to human lung cancer NCI-H460 nude mouse xenograft tumor, and after administration, tumour inhibiting rate goes out Existing significant difference, wherein TRAIL-Mu3 and MuR5S4TR two connective days administration group and three-times-weekly, the tumor suppression of three weeks administration groups Effect is superior to be administered daily, and continuous 5 days, totally two weeks groups.TRAIL-Mu3 and MuR5S4TR two connective days administration group and on every Wendesdays It is secondary, the tumor-inhibiting action therapeutic equivalence of three weeks administration groups.

Different dosing interval animal experiment in vivo shows that, compared to solvent group, CPT-11 group, TRAIL-Mu3 72mg/kg is every Zhou Sanci administration group, TRAIL-Mu3 93mg/kg are administered once group every three days, administration one in TRAIL-Mu3 108mg/kg every four days Secondary group, MuR5S4TR 105mg/kg administration group three-times-weekly, MuR5S4TR 135mg/kg is administered once group every three days, The group that is administered once for MuR5S4TR 158mg/kg every four days has human colon cancer cell HT-29 nude mouse xenograft tumor and has centainly Inhibiting effect, after administration, there is significant difference in tumour inhibiting rate；TRAIL-Mu3 and TRAIL-MuR5S4TR are administered every three days The tumor inhibitory effect of primary group and the group that is administered once for every four days is obviously superior to administration group three-times-weekly, and one is administered every three days There was no significant difference for the tumor inhibitory effect for the group that is administered once for secondary group and every four days.

Different dosing interval animal experiment in vivo is shown, compares solvent group, gemcitabine group, TRAIL-Mu3 72mg/kg Three-times-weekly, continuous three weeks groups, TRAIL-Mu3 93mg/kg once every three days, continuous three weeks groups, TRAIL-Mu3 108mg/kg Once every four days, continuous three weeks groups, MuR5S4TR 105mg/kg three-times-weekly, continuous three weeks groups, MuR5S4TR 135mg/kg Once every three days, continuous three weeks groups, once every four days, continuously three weeks groups are to human pancreatic cancer cell by MuR5S4TR 158mg/kg PANC-1 nude mouse xenograft tumor has and has apparent inhibiting effect, and after administration, significant difference occurs in tumour inhibiting rate；TRAIL- Mu3 72mg/kg three-times-weekly, continuous three weeks groups, TRAIL-Mu3 93mg/kg once every three days, continuous three weeks groups, TRAIL- Curative effect does not have difference to Mu3 108mg/kg between three weeks group each groups once every four days, continuously；MuR5S4TR 105mg/kg is on every Wendesdays Secondary, continuous three weeks groups, MuR5S4TR 135mg/kg once every three days, continuous three weeks groups, MuR5S4TR 158mg/kg every four days Once, continuously curative effect does not have difference between three weeks group each groups.

TRAIL albuminoid of the present invention persistently inhibits the medication of growth of tumour cell, it will be appreciated that be a kind of TRAIL albuminoid is in preparation for the purposes in the persistently drug of inhibition growth of tumour cell.And it can do when necessary With understanding or modification.

The beneficial effects of the present invention are:

The design of the scheme of the invention is not limited to drug metabolism half-life period, but sufficiently has studied the effect of drug inside and outside Duration and tumor suppression aftereffect, are used for the reasonable dosing interval of design, the medication finally obtained be interval, It repeats, the new dosage regimen of full course for the treatment of drug exposure.The program is extended compared with the method that prior document is reported between administration Every, tumour cell drug treating time in the full course for the treatment of is increased, thus with stronger inhibition tumour growth activity, and complete Acted in the course for the treatment of it is unattenuated, have the function of persistently inhibit tumour growth.Interval, repeat, the full course for the treatment of drug exposure give prescription Case is optimization TRAIL albuminoid medication, increases substantially TRAIL albuminoid and persistently inhibits kinds of tumor cells growth more For excellent dosage regimen.While improving curative effect and acting duration, the treatment pain of patient is reduced, improves patient's Compliance meets clinical application reality, has great operability, is convenient for clinical expansion.

Detailed description of the invention

Fig. 1 is that TRAIL-Mu3 changes over time relational graph to NCI-H460 cell-growth inhibitory effect.

TRAIL-Mu3 is co-cultured with various concentration (dosage) and lung carcinoma cell NCI-H460, observes 24,48,72,84 respectively Or 96 hours, measure the inhibition rate of tumor growth of each time point different pharmaceutical concentration (dosage).The results show that drug concentration (agent Amount) in 0.02~5ug/ml, peak plateau of the inhibition rate of tumor growth between 24~72 hours in Suppressive effect, is (most Low concentration tumour inhibiting rate is that 80.33%), in decaying in 96 hours, still unobvious (minimum concentration tumour inhibiting rate was inhibition rate of tumor growth 46.30%).

Fig. 2 is that TRAIL-MuR5S4TR changes over time relational graph to NCI-H460 cell-growth inhibitory effect.

TRAIL-MuR5S4TR is co-cultured with various concentration (dosage) and lung carcinoma cell NCI-H460, observe 24 respectively, 48, 72,84 or 96 hours, the inhibition rate of tumor growth of each time point different pharmaceutical concentration (dosage) is measured.The results show that drug is dense (dosage) is spent in 0.04~10ug/ml, and peak of the inhibition rate of tumor growth between 24~72 hours in Suppressive effect is flat The platform phase (minimum concentration tumour inhibiting rate is 88.93%), inhibition rate of tumor growth was in 96 hours still unobvious (minimum concentration tumor suppressions of decaying 68.76%) rate is.

Fig. 3 is that TRAIL-Mu3 changes over time relational graph to Calu-1 cell-growth inhibitory effect.

TRAIL-Mu3 is co-cultured with various concentration (dosage) and lung carcinoma cell Calu-1, observe respectively 24,48,72,84 or 96 hours, measure the inhibition rate of tumor growth of each time point different pharmaceutical concentration (dosage).The results show that drug concentration (dosage) In 33.3~100ug/ml, peak plateau of the inhibition rate of tumor growth between 24~72 hours in Suppressive effect, is (most Low concentration tumour inhibiting rate is that 69.76%), in decaying in 96 hours, still unobvious (minimum concentration tumour inhibiting rate was inhibition rate of tumor growth 78.28%).

Fig. 4 is that TRAIL-MuR5S4TR changes over time relational graph to Calu-1 cell-growth inhibitory effect.

TRAIL-MuR5S4TR is co-cultured with various concentration (dosage) and lung carcinoma cell Calu-1, observe 24 respectively, 48, 72,84 or 96 hours, the inhibition rate of tumor growth of each time point different pharmaceutical concentration (dosage) is measured.The results show that drug is dense (dosage) is spent in 33.3~100ug/ml, and peak of the inhibition rate of tumor growth between 24~72 hours in Suppressive effect is flat The platform phase (minimum concentration tumour inhibiting rate is 80.75%), inhibition rate of tumor growth was in 96 hours still unobvious (minimum concentration tumor suppressions of decaying 90.89%) rate is.

Fig. 5 is that TRAIL-Mu3 changes over time relational graph to NCI-H1299 cell-growth inhibitory effect.

TRAIL-Mu3 is co-cultured with various concentration (dosage) and lung carcinoma cell NCI-H1299, observe 24 respectively, 48,72, 84 or 96 hours, measure the inhibition rate of tumor growth of each time point different pharmaceutical concentration (dosage).The results show that drug concentration (dosage) in 5.55~50ug/ml, inhibition rate of tumor growth is in the peak platform of Suppressive effect between 24~72 hours Phase (minimum concentration tumour inhibiting rate is 87.56%), inhibition rate of tumor growth was in 96 hours still unobvious (minimum concentration tumour inhibiting rates of decaying For 71.20%).

Fig. 6 is that TRAIL-MuR5S4TR changes over time relational graph to NCI-H1299 cell-growth inhibitory effect.

TRAIL-MuR5S4TR is co-cultured with various concentration (dosage) and lung carcinoma cell NCI-H1299, observe 24 respectively, 48,72,84 or 96 hours, the inhibition rate of tumor growth of each time point different pharmaceutical concentration (dosage) is measured.The results show that drug For concentration (dosage) in 5.55~50ug/ml, inhibition rate of tumor growth is in the peak of Suppressive effect between 24~72 hours Plateau (minimum concentration tumour inhibiting rate is 88.88%), inhibition rate of tumor growth is in still unobvious (the minimum concentration suppression of decaying in 96 hours 75.15%) ratio of outflow is.

Fig. 7 is that TRAIL different dosing number is long-pending to knurl in animal in human lung cancer NCI-H460 Nude Mouse Model Influence diagram.

With solvent group ratio, taxol (25mg/kg) has human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect aobvious Sex differernce is write, in Day10, Relative tumor proliferation rate T/C is 34.88%；When to Day21, T/C 46.12%.

Compared to solvent group, in this experiment, three different dosing group of frequencies TRAIL-Mu3 are different to human lung cancer NCI-H460 nude mice The kind equal conspicuousness inhibiting effect of transplantable tumor.

TRAIL (once a day, continuous to give 5 days, scheme 1: totally 5 times) group, to human lung cancer NCI-H460 nude mouse xenograft There is significant difference in Day3 in tumor inhibiting effect, and Relative tumor proliferation rate T/C is 39.30% (P < 0.001), in Day10 When, Relative tumor proliferation rate T/C is 54.99% (P < 0.001), arrives Day21, and Relative tumor proliferation rate T/C is 56.31% (P < 0.01).

TRAIL (once a day, successive administration 5 days are spaced 2 days, then successive administration 5 days, scheme 2: totally 10 times) group, to people There is significant difference in Day3 in lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect, and Relative tumor proliferation rate T/C is 32.68% (P < 0.001), in Day10, Relative tumor appreciation rate T/C is 34.12% (P < 0.001), arrives Day21, relatively Tumour appreciation rate T/C is 37.48% (P < 0.001).

TRAIL (once a day, is spaced 2 days successive administration 5 days again, after being spaced 2 days again again after successive administration 5 days Successive administration 5 days, scheme 3: totally 15 times) group, occur to human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect in Day3 Significant difference, Relative tumor proliferation rate T/C are 36.79% (P < 0.001), and in Day10, Relative tumor appreciation rate T/C is 34.46% (P < 0.001), arrives Day21, and Relative tumor appreciation rate T/C is 32.87% (P < 0.001).

Fig. 8 is for TRAIL different dosing number to tumor weight in animal in human lung cancer NCI-H460 Nude Mouse Model Influence diagram.

Day21 experiment terminates, and all animals are put to death with after gross tumor volume weighing in, and tumour is separated from animal, And it weighs.Physiological saline group, taxol group, TRAIL (once a day, successive administration 5 days, once a day, successive administration 5 days, Every 2 days, then successive administration 5 days or it is spaced 2 days successive administration 5 days again once a day, after successive administration 5 days, is spaced 2 days again 5 days three different dosing times of successive administration again afterwards) exemplary embodiment lock of group each group is 1.532g, 0.728g respectively, 0.845g, 0.646g and 0.602g.

Fig. 9 is that TRAIL-Mu3 and TRAIL-MuR5S4TR different dosing is spaced in human lung cancer NCI-H460 transplanted tumor in nude mice To the influence diagram of knurl product in animal in model.

With solvent group ratio, taxol (20mg/kg) has human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect aobvious Sex differernce is write, but in experimentation, Relative tumor appreciation rate T/C > 40%.

Compared to solvent group, in this experiment, three different dosing group of frequencies TRAIL-Mu3 are different to human lung cancer NCI-H460 nude mice The kind equal conspicuousness inhibiting effect of transplantable tumor.

TRAIL-Mu3 (once a day, continuous to give 5 days, altogether to two weeks) group, to human lung cancer NCI-H460 nude mouse xenograft There is significant difference in Day4 in tumor inhibiting effect, and Relative tumor appreciation rate T/C is 43.97% (P < 0.001), in Day11 When, Relative tumor appreciation rate T/C is minimum, is 24.75% (P < 0.001), arrives Day21, Relative tumor appreciation rate T/C is 33.5% (P < 0.01).

TRAIL-Mu3 (2 days primary, totally 10 times) group, exists to human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect There is significant difference in Day4, and Relative tumor appreciation rate T/C is 51.77% (P < 0.001), and in Day14, Relative tumor increases Value rate T/C is minimum, is 16.16% (P < 0.001), arrives Day21, and Relative tumor appreciation rate T/C is 17.63% (P < 0.001).

TRAIL-Mu3 (one week 3 times, altogether to 3 weeks) group, exists to human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect There is significant difference in Day4, and Relative tumor appreciation rate T/C is 48.94% (P < 0.001), and in Day18, Relative tumor increases Value rate T/C is minimum, is 16.61% (P < 0.001), arrives Day21, and Relative tumor appreciation rate T/C is 16.75% (P < 0.001).

Similar to TRAIL-Mu3, three different dosing group of frequencies MuR5S4TR move human lung cancer NCI-H460 bare mouse different species Plant the equal conspicuousness inhibiting effect of tumor.

MuR5S4TR (once a day, continuous to give 5 days, altogether to two weeks) group, to human lung cancer NCI-H460 nude mouse xenograft There is significant difference in Day4 in tumor inhibiting effect, and Relative tumor appreciation rate T/C is 59.57% (P < 0.001), arrives Day21, Relative tumor appreciation rate T/C is 38.92% (P < 0.01).

MuR5S4TR (2 days primary, totally 10 times) group, exists to human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect There is significant difference in Day4, and Relative tumor appreciation rate T/C is 54.61% (P < 0.001), arrives Day21, Relative tumor increment Rate T/C is minimum, is 24.43% (P < 0.001).

MuR5S4TR (one week 3 times, altogether to 3 weeks) group, exists to human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect There is significant difference in Day4, and Relative tumor appreciation rate T/C is 56.03% (P < 0.001), and in Day21, Relative tumor increases Value rate T/C is minimum, is 24.43% (P < 0.001).

Figure 10 is that TRAIL-Mu3 and TRAIL-MuR5S4TR different dosing is spaced in human lung cancer NCI-H460 transplanted tumor in nude mice To the influence diagram of tumor weight in animal in model.

Day21 experiment terminates, and all animals are put to death with after gross tumor volume weighing in, and tumour is separated from animal, And it weighs.Physiological saline group, taxol group, TRAIL-Mu3 (three different dosing frequencies) group and MuR5S4TR (three differences Administration frequency) group, the exemplary embodiment lock of each group is 0.911g, 0.658g, 0.366g, 0.170g, 0.170g respectively, 0.416g, 0.249 and 0.237g.

Figure 11 is that TRAIL-Mu3 and TRAIL-MuR5S4TR different dosing is spaced in human colon carcinoma HT-29 transplanted tumor in nude mice To the influence diagram of knurl product in animal in model.

With solvent group ratio, CPT-11, there is certain inhibiting effect to human colon cancer cell HT-29 nude mouse xenograft tumor, There is significant difference in tumour inhibiting rate, and in Day3, significant difference occurs in T/C, is 88.74% (P < 0.05)；In Day21, T/C is minimum, is 29.14% (P < 0.001)

TRAIL-Mu3 72mg/kg administration group three-times-weekly, has one to human colon cancer cell HT-29 nude mouse xenograft tumor Fixed inhibiting effect, in Day7, there is significant difference in T/C, is 54.69% (P < 0.05)；In Day21, T/C is minimum, For 36.43% (P < 0.01).

The TRAIL-Mu3 93mg/kg secondary group that is administered once every three days, to human colon cancer cell HT-29 nude mouse xenograft tumor There is certain inhibiting effect, in Day3, significant difference occurs in T/C, is 81.84% (P < 0.05)；In Day21, T/C Minimum is 28.00% (P < 0.01).

The every four days groups that are administered once of TRAIL-Mu3 108mg/kg, to human colon cancer cell HT-29 nude mouse xenograft tumor There is certain inhibiting effect, in Day3, significant difference occurs in T/C, is 85.49% (P < 0.05)；In Day21, T/C Minimum is 28.69% (P < 0.01).

MuR5S4TR 105mg/kg weekly administration group three times, has one to human colon cancer cell HT-29 nude mouse xenograft tumor Fixed inhibiting effect, in Day3, there is significant difference in T/C, is 88.26% (P < 0.05)；In Day21, T/C is minimum, For 42.86% (P < 0.01).

MuR5S4TR 135mg/kg is administered once group every three days, has to human colon cancer cell HT-29 nude mouse xenograft tumor Certain inhibiting effect, in Day7, there is significant difference in T/C, is 53.26% (P < 0.05)；In Day21, T/C is most It is small, it is 29.03% (P < 0.01).

The every four days groups that are administered once of MuR5S4TR 158mg/kg, there is human colon cancer cell HT-29 nude mouse xenograft tumor Certain inhibiting effect, in Day7, there is significant difference in T/C, is 51.53% (P < 0.05)；In Day21, T/C is most It is small, it is 28.87% (P < 0.01).

Figure 12 is that TRAIL-Mu3 and TRAIL-MuR5S4TR different dosing is spaced in human colon carcinoma HT-29 transplanted tumor in nude mice To the influence diagram of tumor weight in animal in model.

Day21 experiment terminates, and all animals are put to death with after gross tumor volume weighing in, and tumour is separated from animal, And it weighs.Solvent group, CPT-11 group, TRAIL-Mu3 72mg/kg administration group three-times-weekly, TRAIL-Mu3 93mg/kg every three Its group that is administered once, the every four days groups that are administered once of TRAIL-Mu3 108mg/kg, MuR5S4TR 105mg/kg are administered three-times-weekly Group, MuR5S4TR 135mg/kg are administered once group every three days, the tumour for the group that is administered once for MuR5S4TR 158mg/kg every four days Average weight is 1.339 grams, 0.811 gram, 0.898 gram, 0.796 gram, 0.805 gram, 0.936 gram, 0.823 gram and 0.812 respectively Gram.

Figure 13 is that TRAIL-Mu3 and TRAIL-MuR5S4TR different dosing is spaced in human pancreas cancer PANC-1 transplanted tumor in nude mice To the influence diagram of knurl product in animal in model.

With solvent group ratio, gemcitabine has certain inhibition to make human pancreatic cancer cell PANC-1 nude mouse xenograft tumor With significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 80.28% (P < 0.05)；In Day21 When, T/C is minimum, is 36.14% (P < 0.001).

Three-times-weekly, continuous three weeks groups are to human pancreatic cancer cell PANC-1 nude mouse xenograft by TRAIL-Mu3 72mg/kg Tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 73.94% (P < 0.01)；In Day21, T/C is minimum, is 22.34% (P < 0.001).

Once every three days, continuous three weeks groups move human pancreatic cancer cell PANC-1 bare mouse different species to TRAIL-Mu3 93mg/kg Planting tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 63.38% (P < 0.01)；In Day21, T/C is minimum, is 23.25% (P < 0.001).

Once every four days, continuous three weeks groups move human pancreatic cancer cell PANC-1 bare mouse different species to TRAIL-Mu3 108mg/kg Planting tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 61.27% (P < 0.01)；In Day21, T/C is minimum, is 23.13% (P < 0.001).

MuR5S4TR 105mg/kg three-times-weekly, continuous three weeks groups, to human pancreatic cancer cell PANC-1 nude mouse xenograft Tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 65.49% (P < 0.001)；In Day21, T/C is minimum, is 24.82% (P < 0.001).

Once every three days, continuous three weeks groups move human pancreatic cancer cell PANC-1 bare mouse different species to MuR5S4TR 135mg/kg Planting tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 70.42% (P < 0.001)；In Day21, T/C is minimum, is 27.00% (P < 0.001).

Once every four days, continuous three weeks groups move human pancreatic cancer cell PANC-1 bare mouse different species to MuR5S4TR 158mg/kg Planting tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 73.24% (P < 0.001)；In Day21, T/C is minimum, is 27.02% (P < 0.001).

Figure 14 is that TRAIL-Mu3 and TRAIL-MuR5S4TR different dosing is spaced in human pancreas cancer PANC-1 transplanted tumor in nude mice To the influence diagram of tumor weight in animal in model.

Day21 experiment terminates, and all animals are put to death with after gross tumor volume weighing in, and tumour is separated from animal, And it weighs.Solvent group, gemcitabine group (n=7), TRAIL-Mu3 72mg/kg three-times-weekly, continuous three weeks groups, TRAIL-Mu3 93mg/kg once every three days, continuous three weeks groups, TRAIL-Mu3 108mg/kg once every four days, continuous three weeks groups, MuR5S4TR 105mg/kg three-times-weekly, continuous three weeks groups, MuR5S4TR 135mg/kg once every three days, continuous three weeks groups, MuR5S4TR Once every four days, continuously the exemplary embodiment lock of three weeks groups is 0.131 gram respectively to 158mg/kg, 0.075 gram, 0.042 gram, 0.044 gram, 0.043 gram, 0.047 gram, 0.050 gram and 0.052 gram.

Specific embodiment

Below by embodiment, the present invention is further explained, but scope of protection of the present invention is not limited thereto.

Embodiment 1

The research of TRAIL-Mu3 and TRAIL-MuR5S4TR extracorporeal suppression tumor cell growth time-effect relationship

1. research purpose

The TRAIL-Mu3 and TRAIL-MuR5S4TR of various concentration (dosage) are studied in vitro for human lung carcinoma cell NCI- The tumor-inhibiting action of H460, Calu-1, NCI-H1299 and its relationship changed over time.

2. experimental material, reagent and instrument and equipment

2.1 preliminary experiment

2.1.1 cell selects: by experimental data before, the cell that (being shown in Table 1) selects three plants of sensitivitys different is used In formal experiment.

External tumor-inhibiting action of the table 1.TRAIL-Mu3 and TRAIL-MuR5S4TR to 7 kinds of lung cancer cell lines

From the point of view of the data of upper table, the effect of comprehensive TRAIL-Mu3 and TRAIL-MuR5S4TR selects one plant of sensitive strain: NCI-H460；One plant of insensitive strain: NCI-H1299；One plant of medium sensitive strain: calu-1.

2.1.2 show that this three plants of cells are shown in drug in 20170314-LJ1 experiment (preliminary experiment in advance) data In the identical situation of activity, extracorporeal anti-tumor cell proliferation inhibition rate does not occur extending and declining at any time before 72h Trend.Therefore by drug treating time interval be adjusted to for 24 hours, 48h, 72h, 84h, 96h, so as to further observing time With the relationship of drug dose.

2.1.3 in 20170314-LJ1 experiment, when microscopically observation cell growth status, is found, negative in 72h Hole cell density is excessive, leads to have cell dead situation occur.Therefore 96 orifice plates of three plants of cells are connect in this experiment Kind density suitably reduces, and is separately adjusted to angularly NCI-H460 (6 × 10³)、NCI-H1299(4×10³)、Calu-1(6×10⁴)。

2.2 material

TRAIL-Mu3, TRAIL-MuR5S4TR are provided by Chengdu Hua Chuan biotech company, lot number: 20160822.

2.3 reagent

2.4 instrument and equipment

3. experimental method and step

3.1 methods: see each reagent specification

3.2 steps:

One, experimental procedure

1. cell culture

Cell NCI-H460, Calu-1, NCI-H1299 to be cultivated, culture medium and condition of culture see the table below, and 2~3 days It changes the liquid once, 0.25% pancreatin mixes (1:1) had digestive transfer culture with 0.02%EDTA.Logarithmic growth phase cell connects 96 holes when experiment Plate.

2.IC₅₀Experiment

(1) logarithmic growth phase cell is collected, is counted, with complete medium again suspension cell, adjusts cell density to conjunction Suitable density NCI-H460 (6 × 10³)、Calu-1(6×10⁴)、NCI-H1299(4×10³) 96 orifice plates of inoculation, every hole adds 100 μ l Cell suspension.Cell is at 37 DEG C, 100% relative humidity, 5%CO₂It is incubated for 24 hours in incubator.

(2) after prerun protein sample being diluted to following table concentration with cell corresponding culture medium, gradient dilution 10 times, 3 Times gradient dilution, totally 10 concentration points, are added cell by 25 holes μ l/.

(3) cell is placed in 37 DEG C, 100% relative humidity, 5%CO₂In incubator, every kind of cell be incubated for respectively for 24 hours, It is inhaled after 48h, 72h, 84h, 96h and abandons culture medium, the complete medium containing 10%CCK-8 is added, then be placed in 37 DEG C of incubators and incubate It educates.

(4) detected at microplate reader 450nm wavelength when negative hole OD value be 1 or so when, gently shake after in microplate reader The absorbance at 450nm wavelength is measured on (Infinite F50), calculates inhibiting rate.

Two, data processing

Drug is calculated as follows to the inhibiting rate of growth of tumour cell: growth of tumour cell inhibiting rate %=[(Ac-As)/ (Ac-Ab)] × 100%

As: the OA/RLU (cell+CCK-8+ untested compound) of sample

Ac: the OA/RLU (cell+CCK-8) of negative control

Ab: the OA/RLU (culture medium+CCK-8) of positive control

4. experimental result

Suppressive effect of the 4.1TRAIL-Mu3 and TRAIL-MuR5S4TR to the NCI-H460 cell different role time

Tumour inhibiting rate of the table 2.TRAIL-Mu3 and TRAIL-MuR5S4TR to the NCI-H460 cell different role time

Tumour inhibiting rate of the table 3.TRAIL-Mu3 and TRAIL-MuR5S4TR to the Calu-1 cell different role time

Tumour inhibiting rate of the table 4.TRAIL-Mu3 and TRAIL-MuR5S4TR to the NCI-H1299 cell different role time

5. experiment conclusion

In vitro experiment, TRAIL albuminoid (TRAIL-Mu3 and TRAIL-MuR5S4TR) is in a certain concentration (dosage) model Enclose interior and cytosis, inhibiting rate of 24~96 hours drugs of observation to tumour cell.In the tumour cell of different sensibility, TRAIL albuminoid was in the peak of inhibition to growth of tumour cell at 24~72 hours, for highly sensitive cell strain (or Higher activity), the rush hour of tumor suppression continues to 96 hours.TRAIL-Mu3 and TRAIL-MuR5S4TR is to three plants The relationship of the Suppressive effect time to time change of the different lung carcinoma cell of sensitivity is detailed in attached drawing 1~6.

Embodiment 2

TRAIL different dosing number is to human lung cancer NCI-H460 cell nude mouse xenograft anti-tumor Effect study

1. experiment purpose

It is injected intravenously using the dosage of 60mg/kg, once a day, continuous injection 5 days (scheme 1: totally 5 times), 60mg/kg Dosage intravenous injection, once a day, continuous injection be spaced after 5 days continuously inject again within 2 days 5 days (schemes 2: totally 10 times) or The dosage of 60mg/kg is injected intravenously, and once a day, continuous injection is spaced 2 days continuous injection 5 days again after 5 days, is spaced 2 again 5 days (scheme 3: totally 15 times) three kinds of different dosing regimes are continuously injected after it again, compare three kinds of different dosing times for people The difference of anti-tumor activity in lung cancer NCI-H460 Nude Mouse Model body.

2. experimental animal

2.1 animal species

Mouse.

2.2 kind

Balb/c nude mice.

2.3 gender

Female.

2.4 quantity

Inoculation 60 chooses 40.

2.5. the age

4~6 weeks.

2.6. weight

± 20% weight mean value of 16~18g.

2.7. animal origin (supplier)

Shanghai western Poole-Bi Kai experimental animal Co., Ltd (BK), credit number SCXK (Shanghai) 2013-0016, animal are closed Lattice card number: 2008001661519.

2.8. management of laboratory animal

2.8.1 animal identification identification method

Each equal wearing of mouse cage has the information such as experiment numbers, experiment group, experimenter's name, mouse strain and gender Identity card, mouse are marked with ear tag method.

2.8.2 random grouping

It is grouped when gross tumor volume reaches 100~200mm3 with randomized blocks, guarantees gross tumor volume and mouse between each group Weight is uniform, and the mean value difference of the mean value of each group gross tumor volume and all experimental animal tumor volumes is no more than ± 10%.

2.8.3 operational administrative specification

The operation and management of all experimental animals strictly observe Shanghai Mei Dixi experimental animal and use and management guidance original Then.

2.8.4 rearing conditions

Living conditions: 3, every cage

Temperature: 20 DEG C~26 DEG C

Humidity: 40%~70%

Illumination: day alternates with night within 12 hours

2.8.5 feed

Size mouse feed is irradiated, is pulled together feed corporation,Ltd purchased from Beijing Australia of section.Ad lib.

2.8.6 drinking-water

City tap-water is drunk after filtering high pressure sterilization.

2.8.7 padding

Corncob, Shanghai Mao Sheng derivative Science and Technology Ltd. use after high pressure sterilization, change padding twice weekly.

2.8.8 the laundering period

Give mouse most short one week environment laundering period before experiment.

3. experimental material

3.1 test drugs

Tester TRAIL, taxol information are as follows:

3.2 other chemical materials

3.2.1 asepsis injector

1ml asepsis injector is purchased from purchase Shanghai Kindly Enterprise Development Group (Shanghai, China).

3.2.2 cell strain

Human lung carcinoma cell line NCI-H460 is purchased from cell biological research institute of the Shanghai Chinese Academy of Sciences.

NCI-H460 is incubated at F12-K culture medium (GIBCO, the U.S.), (GIBCO, the U.S.) containing 10% fetal calf serum FBS. It is incubated at containing 5%CO₂37 DEG C of incubators.

3.2.3 matrigel (BD Matrigel)

Matrigel Matrigel is purchased from U.S. company BD

4. experimental design

Establish human lung cancer NCI-H460 Xenografts in nude mice model, every animal inoculation pvaccination 3 × 10⁶A cell, inoculum Product is 0.1ml/ animal, contains 50%Matrigel in cell suspension.

This independent experiment designs dosage and dosage regimen is as follows.

The TRAIL different dosing time is to human lung cancer NCI-H460 nude mouse xenograft tumor dosage regimen

5. experimental method

NCI-H460 cell culture contains 10% fetal calf serum FBS in RPMI-1640.Cell is placed in 5%CO₂Incubator 37 DEG C of cultures.

Cell inoculation method establishes tumour nude mice by subcutaneous transplantation model: collecting the tumour cell of logarithmic growth phase, weight after counting It is suspended from 1 × PBS, Matrigel, adjustment concentration of cell suspension to 3 × 10 is added in 1:1⁷/ml.Existed with 1ml syringe (No. 4 syringe needles) Dorsal sc inoculated tumour cell on the right side of nude mice, 3 × 10⁶/ 0.1ml/ mouse is inoculated with 60 altogether.

Reach 100~200mm in gross tumor volume³When, animal is grouped at random by randomized blocks, makes each group tumour Difference is less than the 10% of mean value, and every group of 8 mouse, the grouping same day is denoted as Day 0, and starts to be administered according to average weight.

The weight of animals twice and tumor size are measured during experiment weekly.Clinical symptoms are observed and recorded daily.All animals Experimental implementation strictly observes the use of Shanghai Medixi Biomedicine Co., Ltd. animal and management regulation.The meter of tumour relevant parameter It calculates and refers to China SFDA " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle "^[1]。

The evaluation index of anti-tumor activity is Relative tumor appreciation rate T/C (%), calculation formula are as follows: T/C (%)=TRTV/ CRTV*100%.(TRTV: treatment group RTV；CRTV: negative control group RTV)；Relative tumour volume (relative tumor Volume, RTV), calculation formula are as follows: RTV=Vt/V0.(Day0) measurement gained gross tumor volume when wherein V0 is sub-cage administration, Vt is gross tumor volume when measuring each time.

The changes of weight (%) of tumor animal calculates as follows: weight when (weight when weight-grouping when measurement)/be grouped × 100。

According to Chinese SFDA " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle " (in November, 2006), T/ C (%)≤40% and be effective through statistical analysis P < 0.05.If the weight loss of mouse is more than that 20% or drug are relevant dead Number is died more than 20%, then it is assumed that the drug dose has serious toxicity.

6. data are analyzed

Using time point as X-axis, gross tumor volume is that Y-axis draws tumor growth curve；Using time point as X-axis, the weight of animals becomes Change value (%) is that Y-axis draws body weight increase change curve.Comparison among groups are examined using t-, and P < 0.05 is significant difference, P < 0.01 is extremely significant sex differernce.

7. result

Influence of the 7.1 each tested materials to human lung cancer NCI-H460 Nude Mice the weight of animals

In experiment, vehicle control group is administered 21 days, and animal average weight is not influenced by solvent.Within the entire test period, Animal average weight constantly increases.Compared with Day 0, animal average weight has gone up 8.34% (i.e. 2.12 grams) when Day21.

Animal shows obviously the toxicity that taxol (25mg/kg, IV) acts on, and distinguishes after second and third time are administered There is an animal dead.Obvious in the 7th~14 day weight loss of entire test period, weight limit decreases by Weight gradually increases after 18.52%, Day14, until weight is restored substantially when off-test.

Animal is resistant to the TRAIL (scheme 1: totally 5 times, scheme 2: totally 10 times, scheme 3: totally 15 times) of different dosing time By within the entire test period, animal average weight constantly increases.Compared with Day 0, three different dosing frequencies when Day21 Group animal average weight has gone up 7.68% (i.e. 1.97 grams), 5.25% (i.e. 1.20 grams) and 5.34% (1.18 grams) respectively.

Influence of the 7.2 each tested materials to human lung cancer NCI-H460 Nude Mice animal tumor volume

With solvent group ratio, taxol (25mg/kg) has human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect aobvious Sex differernce is write, in Day10, Relative tumor proliferation rate T/C is 34.88%；When to Day21, T/C 46.12%.

Compared to solvent group, in this experiment, three different dosing group of frequencies TRAIL-Mu3 are different to human lung cancer NCI-H460 nude mice The kind equal conspicuousness inhibiting effect of transplantable tumor.

TRAIL (once a day, continuous to give 5 days, scheme 1: totally 5 times) group, to human lung cancer NCI-H460 nude mouse xenograft There is significant difference in Day3 in tumor inhibiting effect, and Relative tumor proliferation rate T/C is 39.30% (P < 0.001), in Day10 When, Relative tumor proliferation rate T/C is 54.99% (P < 0.001), arrives Day21, and Relative tumor proliferation rate T/C is 56.31% (P < 0.01).

TRAIL (once a day, successive administration 5 days are spaced 2 days, then successive administration 5 days, scheme 2: totally 10 times) group, to people There is significant difference in Day3 in lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect, and Relative tumor proliferation rate T/C is 32.68% (P < 0.001), in Day10, Relative tumor appreciation rate T/C is 34.12% (P < 0.001), arrives Day21, relatively Tumour appreciation rate T/C is 37.48% (P < 0.001).

TRAIL (once a day, is spaced 2 days successive administration 5 days again, after being spaced 2 days again again after successive administration 5 days Successive administration 5 days, scheme 3: totally 15 times) group, occur to human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect in Day3 Significant difference, Relative tumor proliferation rate T/C are 36.79% (P < 0.001), and in Day10, Relative tumor appreciation rate T/C is 34.46% (P < 0.001), arrives Day21, and Relative tumor appreciation rate T/C is 32.87% (P < 0.001).

Detailed results are shown in Table experimental result and are shown in Table 5, attached drawing 7.

Influence of the 7.3 each tested materials to human lung cancer NCI-H460 Nude Mice animal tumor weight

Day21 experiment terminates, and all animals are put to death with after gross tumor volume weighing in, and tumour separates simultaneously from animal Tumor tissues are collected in weighing, and each tumor tissues are divided into two parts: it saves after a liquid nitrogen Rapid-Freezing Method and is saved with -80 degree refrigerators, For subsequent analysis；Portion carries out paraffin embedding after being fixed with formaldehyde, is used for subsequent analysis.Physiological saline group, taxol group, (once a day, successive administration 5 days, once a day, successive administration 5 days, is spaced 2 days TRAIL, then successive administration 5 days or daily one It is secondary, 2 days successive administration 5 days again are spaced after successive administration 5 days, 5 days three differences of successive administration are given again after being spaced 2 days again The medicine time) group each group exemplary embodiment lock be 1.532g, 0.728g, 0.845g, 0.646g and 0.602g respectively.

Detailed results are shown in Table 5, attached drawing 8.

The table 5.TRAIL different dosing time is big to animal tumor in human lung cancer NCI-H460 nude mouse xenograft tumor model Small influence

*: P < 0.05；*: P < 0.01；*: P < 0.001 of * is compared with solvent group

8. brief summary

In this experiment, taxol similar to physiological saline group, TRAIL (once a day, successive administration 5 days, daily one It is secondary, it successive administration 5 days, is spaced 2 days, then successive administration 5 days or once a day, is spaced 2 days after successive administration 5 days and continuously gives again Medicine 5 days, 5 days three different dosing times of successive administration again after being spaced 2 days again) group has little effect the weight of animals, Small toxicity, it is safer；Taxol group the weight of animals is declined slightly, gradually stable in experimentation, safer.Compared to physiology Salt water group, taxol group, (once a day, successive administration 5 days, once a day, successive administration 5 days, is spaced 2 days, then connect TRAIL It is continuous to be administered 5 days and be spaced 2 days successive administration 5 days again once a day, after successive administration 5 days, it is continuous again after being spaced 2 days again 5 days three different dosing times of administration) group has certain inhibiting effect to human lung cancer NCI-H460 nude mouse xenograft tumor, wherein Intravenous injection successive administration 5 days, is spaced 2 days, then 5 days groups of successive administration and once a day, after successive administration 5 days once a day 2 days successive administration 5 days again are spaced, the curative effect of 5 days groups of successive administration is obviously superior to individually daily again after being spaced 2 days again Once, 5 days groups of successive administration, after administration, there is significant difference in tumour inhibiting rate.But the tumor-inhibiting action compared with scheme 2 of scheme 3 Improve not statistically significant (P > 0.05).Experiment shows that drug pair can be significantly improved by increasing administration number of times (extending administration time) The tumour inhibiting rate of Nude Mouse Model, but administration number of times at 15 times compared with 10 times, the raising of tumour inhibiting rate is unobvious.

9. bibliography

[1] " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle " in November, 2006

Embodiment 3

TRAIL-Mu3 and TRAIL-MuR5S4TR different dosing interval is to human lung cancer NCI-H460 nude mouse xenograft tumor Therapeutic effect research

1. experiment purpose

This research uses human lung cancer NCI-H460 nude mouse xenograft tumor model, evaluates different dosing interval TRAIL-Mu3 With the internal anti-tumor activity difference of TRAIL-MuR5S4TR.

2. experimental animal

2.1 animal species

Mouse.

2.2 kind

Balb/c nude mice.

2.3 gender

Female.

2.4 quantity

Inoculation 90 chooses 64.

2.5. the age

4-6 weeks.

2.6. weight

± 20% weight mean value of 16~18g.

2.7. animal origin (supplier)

Shanghai western Poole-Bi Kai experimental animal Co., Ltd (BK), credit number SCXK (Shanghai) 2013-0016, animal are closed Lattice card number: 2008001661519.

2.8. management of laboratory animal

2.8.1 animal identification identification method

Each equal wearing of mouse cage has the information such as experiment numbers, experiment group, experimenter's name, mouse strain and gender Identity card, mouse are marked with ear tag method.

2.8.2 random grouping

When gross tumor volume reaches 100~200mm³When be grouped with randomized blocks, guarantee each group between gross tumor volume and mouse Weight is uniform, and the mean value difference of the mean value of each group gross tumor volume and all experimental animal tumor volumes is no more than ± 10%.

2.8.3 operational administrative specification

The operation and management of all experimental animals strictly observe Shanghai Mei Dixi experimental animal and use and management guidance original Then.

2.8.4 rearing conditions

Living conditions: 3, every cage

Temperature: 20 DEG C~26 DEG C

Humidity: 40%~70%

Illumination: day alternates with night within 12 hours

2.8.5 feed

Size mouse feed is irradiated, is pulled together feed corporation,Ltd purchased from Beijing Australia of section.Ad lib.

2.8.6 drinking-water

City tap-water is drunk after filtering high pressure sterilization.

2.8.7 padding

Corncob, Shanghai Mao Sheng derivative Science and Technology Ltd. use after high pressure sterilization.Padding twice is changed weekly.

2.8.8 the laundering period

Give mouse most short one week environment laundering period before experiment.

3. experimental material

3.1 test drugs

Tester TRAIL-Mu3 and TRAIL-MuR5S4TR, taxol information are as follows:

3.2 other chemical materials

3.2.1 asepsis injector

1ml asepsis injector is purchased from purchase Shanghai Kindly Enterprise Development Group (Shanghai, China).

3.2.2 cell strain

Human lung carcinoma cell line NCI-H460 is purchased from cell biological research institute of the Shanghai Chinese Academy of Sciences.

NCI-H460 is incubated at F12-K culture medium (GIBCO, the U.S.), (GIBCO, the U.S.) containing 10% fetal calf serum FBS. It is incubated at containing 5%CO₂37 DEG C of incubators.

3.2.3 matrigel (BD Matrigel)

Matrigel Matrigel is purchased from U.S. company BD

4. experimental design

Establish human lung cancer NCI-H460 Xenografts in nude mice model, every animal inoculation pvaccination 3 × 10⁶A cell, inoculum Product is 0.1ml/ animal, contains 50%Matrigel in cell suspension.

This independent experiment designs dosage and dosage regimen is as follows:

Antitumor action of the TRAIL-Mu3 and MuR5S4TR in human lung cancer NCI-H460 Nude Mouse Model

5. experimental method

NCI-H460 cell culture contains 10% fetal calf serum FBS in RPMI-1640.Cell is placed in 5%CO₂Incubator 37 DEG C of cultures.

Cell inoculation method establishes tumour nude mice by subcutaneous transplantation model: collecting the tumour cell of logarithmic growth phase, weight after counting It is suspended from 1 × PBS, Matrigel, adjustment concentration of cell suspension to 3 × 10 is added in 1:1⁷/ml.Existed with 1ml syringe (No. 4 syringe needles) Dorsal sc inoculated tumour cell on the right side of nude mice, 3 × 10⁶/ 0.1ml/ mouse is inoculated with 90 altogether.

Reach 100~200mm in gross tumor volume³When, animal is grouped at random by randomized blocks, makes each group tumour Difference is less than the 10% of mean value, and every group of 8 mouse, the grouping same day is denoted as Day0, and starts to be administered according to average weight.

The weight of animals twice and tumor size are measured during experiment weekly.Clinical symptoms are observed and recorded daily.All animals Experimental implementation strictly observes the use of Shanghai Medixi Biomedicine Co., Ltd. animal and management regulation.The meter of tumour relevant parameter It calculates and refers to China SFDA " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle "^[1]。

The evaluation index of anti-tumor activity is Relative tumor appreciation rate T/C (%), calculation formula are as follows: T/C (%)=TRTV/ CRTV*100%.(TRTV: treatment group RTV；CRTV: negative control group RTV)；Relative tumour volume (relative tumor Volume, RTV), calculation formula are as follows: RTV=Vt/V0.(Day0) measurement gained gross tumor volume when wherein V0 is sub-cage administration, Vt is gross tumor volume when measuring each time.

The changes of weight (%) of tumor animal calculates as follows: weight when (weight when weight-grouping when measurement)/be grouped × 100。

According to Chinese SFDA " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle " (in November, 2006), T/ C (%)≤40% and be effective through statistical analysis P < 0.05.If the weight loss of mouse is more than that 20% or drug are relevant dead Number is died more than 20%, then it is assumed that the drug dose has serious toxicity.

6. data are analyzed

Using time point as X-axis, gross tumor volume is that Y-axis draws tumor growth curve；Using time point as X-axis, the weight of animals becomes Change value (%) is that Y-axis draws body weight increase change curve.Comparison among groups are examined using t-, and P < 0.05 is significant difference, P < 0.01 is extremely significant sex differernce.

7. result

Influence of the 7.1 each tested materials to human lung cancer NCI-H460 Nude Mice the weight of animals

In experiment, vehicle control group is administered 21 days, and animal average weight is not influenced by solvent.Within the entire test period, Animal average weight constantly increases.Compared with Day 0, animal average weight has gone up 9.27% (i.e. 2.22 grams) when Day21.

Animal is resistant to taxol (20mg/kg, IV), and weight is declined slightly within the entire test period, when Day21, This group of animal average weight has dropped 2.32% (i.e. 0.52g).

Animal (once a day, continuously gives 5 days, altogether to two weeks to the TRAIL-Mu3 of different dosing frequency；2 days primary to altogether 10 times；One week 3 times, altogether to 3 weeks；60mg/kg, IV) it is resistant to, within the entire test period, animal average weight constantly increases. Compared with Day 0, three different dosing group of frequencies animal average weights have gone up 8.93% (i.e. 2.06 grams) respectively when Day21, 5.38% (i.e. 1.23 grams) and 7.72% (i.e. 1.78 grams).

Animal (once a day, continuously gives 5 days, altogether to two weeks to the MuR5S4TR of different dosing frequency；2 days primary to altogether 10 times；One week 3 times to 3 weeks；60mg/kg, IV) it is resistant to, within the entire test period, animal average weight constantly increases.With Day 0 is compared, and three different dosing group of frequencies animal average weights have gone up 8.02% (i.e. 1.79 grams) respectively when Day21, 6.80% (i.e. 1.56 grams) and 6.68% (i.e. 1.52 grams).

Influence of the 7.2 each tested materials to human lung cancer NCI-H460 Nude Mice animal tumor volume

With solvent group ratio, taxol (20mg/kg) has human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect aobvious Sex differernce is write, but in experimentation, Relative tumor appreciation rate T/C > 40%.

Compared to solvent group, in this experiment, three different dosing group of frequencies TRAIL-Mu3 are different to human lung cancer NCI-H460 nude mice The kind equal conspicuousness inhibiting effect of transplantable tumor.

TRAIL-Mu3 (once a day, continuous to give 5 days, altogether to two weeks) group, to human lung cancer NCI-H460 nude mouse xenograft There is significant difference in Day4 in tumor inhibiting effect, and Relative tumor appreciation rate T/C is 43.97% (P < 0.001), in Day11 When, Relative tumor appreciation rate T/C is minimum, is 24.75% (P < 0.001), arrives Day21, Relative tumor appreciation rate T/C is 33.5% (P < 0.01).

TRAIL-Mu3 (2 days primary, totally 10 times) group, exists to human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect There is significant difference in Day4, and Relative tumor appreciation rate T/C is 51.77% (P < 0.001), and in Day14, Relative tumor increases Value rate T/C is minimum, is 16.16% (P < 0.001), arrives Day21, and Relative tumor appreciation rate T/C is 17.63% (P < 0.001).

TRAIL-Mu3 (one week 3 times, altogether to 3 weeks) group, exists to human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect There is significant difference in Day4, and Relative tumor appreciation rate T/C is 48.94% (P < 0.001), and in Day18, Relative tumor increases Value rate T/C is minimum, is 16.61% (P < 0.001), arrives Day21, and Relative tumor appreciation rate T/C is 16.75% (P < 0.001).

Similar to TRAIL-Mu3, three different dosing group of frequencies MuR5S4TR move human lung cancer NCI-H460 bare mouse different species Plant the equal conspicuousness inhibiting effect of tumor.

MuR5S4TR (once a day, continuous to give 5 days, altogether to two weeks) group, to human lung cancer NCI-H460 nude mouse xenograft There is significant difference in Day4 in tumor inhibiting effect, and Relative tumor appreciation rate T/C is 59.57% (P < 0.001), arrives Day21, Relative tumor appreciation rate T/C is 38.92% (P < 0.01).

MuR5S4TR (2 days primary, totally 10 times) group, exists to human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect There is significant difference in Day4, and Relative tumor appreciation rate T/C is 54.61% (P < 0.001), arrives Day21, Relative tumor increment Rate T/C is minimum, is 24.43% (P < 0.001).

MuR5S4TR (one week 3 times, altogether to 3 weeks) group, exists to human lung cancer NCI-H460 nude mouse xenograft tumor inhibiting effect There is significant difference in Day4, and Relative tumor appreciation rate T/C is 56.03% (P < 0.001), and in Day21, Relative tumor increases Value rate T/C is minimum, is 24.43% (P < 0.001).

Detailed results are shown in Table experimental result and are shown in Table 6 and attached drawing 9.

Influence of the 7.3 each tested materials to human lung cancer NCI-H460 Nude Mice animal tumor weight

Day21 experiment terminates, and all animals are put to death with after gross tumor volume weighing in, and tumour separates simultaneously from animal Tumor tissues are collected in weighing, and each tumor tissues are divided into two parts: it saves after a liquid nitrogen Rapid-Freezing Method and is saved with -80 degree refrigerators, For subsequent analysis；Portion carries out paraffin embedding after being fixed with formaldehyde, is used for subsequent analysis.Physiological saline group, taxol group, TRAIL-Mu3 (three different dosing frequencies) group and MuR5S4TR (three different dosing frequencies) group, the tumour of each group are averagely heavy Amount is 0.911g, 0.658g, 0.366g, 0.170g, 0.170g, 0.416g, 0.249 and 0.237g respectively.

Detailed results are shown in Table 6 and attached drawing 10.

Table 6.TRAIL-Mu3 and MuR5S4TR is in human lung cancer NCI-H460 nude mouse xenograft tumor model to animal tumor Size influences

*: P < 0.05；*: P < 0.01；*: P < 0.001 of * is compared with solvent group

8. brief summary

In this experiment, taxol similar to physiological saline group, TRAIL-Mu3 group and MuR5S4TR group are to the weight of animals It has little effect, small toxicity is safer；Taxol group the weight of animals is declined slightly, gradually stable in experimentation, more Safety.

It is naked to human lung cancer NCI-H460 compared to physiological saline group, taxol group, TRAIL-Mu3 group and MuR5S4TR each group Mouse xenograft tumor has certain inhibiting effect, and after administration, significant difference occurs in tumour inhibiting rate, wherein TRAIL-Mu3 and MuR5S4TR two connective days administration group and three-times-weekly, the tumor-inhibiting action of three weeks administration groups is superior to be administered daily, and continuous 5 days, Totally two weeks groups.TRAIL-Mu3 and MuR5S4TR two connective days administration group and three-times-weekly, the tumor-inhibiting action curative effect of three weeks administration groups Quite.

9. bibliography

[1] " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle " in November, 2006

Embodiment 4

TRAIL-Mu3 and TRAIL-MuR5S4TR different dosing interval is to human colon cancer cell HT-29 nude mouse xenograft The therapeutic effect of tumor is studied

1. experiment purpose

This research uses human colon cancer cell HT-29 nude mouse xenograft tumor model, evaluates TRAIL-Mu3 and TRAIL- The internal anti-tumor activity at MuR5S4TR different dosing interval.

2. experimental animal

2.1 animal species

Mouse.

2.2 kind

Balb/c nude mice.

2.3 gender

Female.

2.4 quantity

Experiment uses 64.

2.5 the age

4~6 weeks.

2.6 weight

± 20% weight mean value of 16~18g.

2.7 animal origins (supplier)

Shanghai western Poole-Bi Kai experimental animal Co., Ltd (BK), credit number SCXK (Shanghai) 2013-0016, animal are closed Lattice card number: 2008001665079.

2.8 management of laboratory animal

2.8.1 animal identification identification method

Each equal wearing of mouse cage has the information such as experiment numbers, experiment group, experimenter's name, mouse strain and gender Identity card, mouse are marked with ear tag method.

2.8.2 random grouping

It is grouped when gross tumor volume averagely reaches 200mm3 or so with randomized blocks, guarantees gross tumor volume and mouse between each group Weight is uniform, and the mean value difference of the mean value of each group gross tumor volume and all experimental animal tumor volumes is no more than ± 10%.

2.8.3 operational administrative specification

The operation and management of all experimental animals strictly observe Shanghai Mei Dixi experimental animal and use and management guidance original Then.

2.8.4 rearing conditions

Living conditions: 3, every cage.

Temperature: 20 DEG C~26 DEG C

Humidity: 40%~70%

Illumination: day alternates with night within 12 hours

2.8.5 feed

Size mouse feed is irradiated, is pulled together feed corporation,Ltd purchased from Beijing Australia of section.Ad lib.

2.8.6 drinking-water

City tap-water is drunk after filtering high pressure sterilization.

2.8.7 padding

Corncob, Shanghai Mao Sheng derivative Science and Technology Ltd. use after high pressure sterilization.Padding twice is changed weekly.

2.8.8 the laundering period

Give mouse most short one week environment laundering period before experiment.

3. experimental material

3.1 test drugs

Tester TRAIL-Mu3 and MuR5S4TR information

3.2 cell strain

Human colon cancer cell cell strain HT-29 is purchased from cell biological research institute of the Shanghai Chinese Academy of Sciences.

3.3 reagent

McCoy's 5a culture medium (GIBCO, the U.S.)

Fetal calf serum FBS (GIBCO, the U.S.)

Pancreatin Trypsin-EDTA (is purchased from GIBCO, the U.S.)

Trypan blue Trypan Blue (is purchased from GIBCO, the U.S.)

Physiological saline is purchased from Huayu (Wuxi) Pharmaceutical Co., Ltd. (Jiangsu, China).

3.4 instrument

Biohazard Safety Equipment (model: AC2-6E1) is purchased from ESCO；

CO₂Water proof cell incubator (model: 3111), is purchased from Thermo Scientific Forma；

Inverted microscope (model: CKX41SF) is purchased from Olympus；

Electric suction apparatus (model YX930D) is purchased from Shanghai Medical appliance industry (group) Co., Ltd；

Low speed centrifuge (model LD5-2A) is purchased from Beijing Lei Boer centrifuge Co., Ltd.

3.5 other

1ml asepsis injector is purchased from purchase Shanghai Kindly Enterprise Development Group (Shanghai, China).

4. experimental design

Establish human colon cancer cell HT-29 Xenografts in nude mice model, every animal inoculation pvaccination 3 × 10⁶A cell, inoculation Volume is 0.1ml/ animal.

This independent experiment designs dosage and dosage regimen is as follows:

TRAIL-Mu3 and MuR5S4TR different dosing is spaced in human colon cancer cell HT-29 Nude Mouse Model Antitumor action

5. experimental method

5.1 test medicine preparations are prepared

5.1.1CPT-11 2.5mg/ml 2ml

CPT-11 is dispensed with stoste (20mg/ml), every pipe 0.25ml.Set room temperature preservation.

Take the CPT-11 stoste (20mg/ml) one of above-mentioned packing.

1.75ml physiological saline is added, mixes.

It is ready-to-use, before use, being saved in 18~25 DEG C.

5.1.2TRAIL-Mu3 10.8mg/ml 2.4ml

Solvent: 0.9% physiological saline: water for injection=1:1

TRAIL-Mu3 stoste (24mg/ml) 1.08ml is taken,

The above-mentioned solvent of 1.32ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours.

5.1.3TRAIL-Mu3 9.3mg/ml 2.4ml

Solvent: 0.9% physiological saline: water for injection=1:1

TRAIL-Mu3 stoste (24mg/ml) 0.93ml is taken,

The above-mentioned solvent of 1.47ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours.

5.1.4TRAIL-Mu3 7.2mg/ml 2.4ml

Take TRAIL-Mu3 stoste (24mg/ml) 0.72ml.

The above-mentioned solvent of 1.68ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours.

5.1.5MuR5S4TR 15.8mg/ml 2.4ml

Solvent: 0.9% physiological saline: water for injection=1:1

Take MuR5S4TR stoste (24mg/ml) 1.58ml.

The above-mentioned solvent of 0.82ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours.

5.1.6MuR5S4TR 13.5mg/ml 2.4ml

Solvent: 0.9% physiological saline: water for injection=1:1

Take MuR5S4TR stoste (24mg/ml) 1.35ml.

The above-mentioned solvent of 1.05ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours.

5.1.7MuR5S4TR 10.5mg/ml 2.4ml

Take MuR5S4TR stoste (24mg/ml) 1.05ml.

The above-mentioned solvent of 1.35ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours

5.2 experimental method

5.2.1 cell culture

HT-29 cell culture contains 10% fetal calf serum FBS in McCoy's 5a culture medium.It is incubated at containing 5%CO₂37 DEG C incubator.It after cell recovery, expands and passes on by cell, collect enough cells for animal inoculation pvaccination.

5.2.2 cell inoculation method establishes tumour Xenografts in nude mice model

The tumour cell for collecting logarithmic growth phase is resuspended in serum-free McCoy's 5a culture medium after counting, adjust cell Suspension concentration is to 3 × 10⁷/mL.With 1mL syringe (No. 4 syringe needles) the dorsal sc inoculated tumour cell on the right side of nude mice, 3 × 10⁶/ 0.1mL/ mouse is inoculated with 92 animals altogether.

5.2.3 grouping administration

Reach 200mm3 or so in gross tumor volume, animal is grouped at random by randomized blocks, keeps each group tumour poor Different 10% less than mean value, every group 8, totally 8 groups.The grouping same day is denoted as Day 0.It is administered by " 4. experimental design ".

5.2.4 Indexs measure

The weight of animals twice and tumor size are measured during experiment weekly, observes and records clinical symptoms, Day21 measurement daily After put to death all animals, strip tumour, weigh knurl weight and simultaneously take pictures.

All zoopery operations strictly observe Shanghai Mei Dixi Biomedics Inc. animal and use and manage Specification.The calculating of tumour relevant parameter refers to China CFDA " cell toxicant series antineoplastic medicament non-clinical study technological guidance's principle 》^[1]。

5.3 calculating

The calculating of tumour relevant parameter:

The calculation formula of gross tumor volume (Tumor volume, TV) are as follows: TV=a × b2/2.Wherein a, b respectively represent tumour Measurement is long and wide.

The evaluation index of anti-tumor activity is Relative tumor appreciation rate T/C (%) and tumour inhibiting rate (%), calculation formula difference Are as follows: T/C (%)=TRTV/CRTV*100%.(TRTV: treatment group RTV；CRTV: negative control group RTV)；Relative tumour volume (relative tumor volume, RTV), calculation formula are as follows: RTV=Vt/V0.When wherein V0 is sub-cage administration (Day 0) Measurement gained gross tumor volume, Vt are gross tumor volume when measuring each time.

The changes of weight (%) of tumor animal calculates as follows: weight when (weight when weight-grouping when measurement)/be grouped × 100。

According to Chinese CFDA " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle " (in November, 2006), T/ C (%)≤40% and be effective through statistical analysis P < 0.05.If the weight loss of mouse is more than that 20% or drug are relevant dead Number is died more than 20%, then it is assumed that the drug dose has serious toxicity.

6. data are analyzed

Using time point as X-axis, gross tumor volume is that Y-axis draws tumor growth curve；Using time point as X-axis, animal is averaged body Weight (g) is that Y-axis draws changes of weight curve.Comparison among groups are examined using t-, and P < 0.05 is significant difference, and P < 0.01 is pole Significant difference.

7. result

Influence of the 7.1 each tested materials to human colon cancer cell HT-29 transplanted tumor in nude mice the weight of animals

Within this entire experimental period, solvent group animal average weight is not influenced by solvent solvent.Animal average weight Constantly increase.Compared with the first day, that is, Day 0 is administered, animal average weight has gone up 7.01% (i.e. 1.64 grams) when Day21.

Animal is resistant to CPT-11 25mg/kg, within entire experimental period, compared with the first day, that is, Day0 is administered, When Day21, this group of animal average weight has gone up 9.23% (i.e. 2.17 grams).

Three times to TRAIL-Mu3 72mg/kg weekly administration, continuous three weeks dosage regimens are resistant to animal, are entirely being tested In period, compared with Day0, when Day21, this group of animal average weight has gone up 10.62% (i.e. 2.46g).

Animal is administered once every three days to TRAIL-Mu3 93mg/kg, and continuous three weeks dosage regimens are resistant to, entire real It tests in the period, compared with Day0, when Day21, this group of animal average weight has gone up 5.83% (i.e. 1.44 grams).

It is administered once within animal four days every to TRAIL-Mu3 108mg/kg, continuous three weeks dosage regimens are resistant to, entire In experimental period, compared with Day0, when Day21, this group of animal average weight has gone up 7.85% (i.e. 1.82 grams).

Three times to MuR5S4TR 105mg/kg weekly administration, continuous three weeks dosage regimens are resistant to animal, are entirely being tested In period, compared with Day0, when Day21, this group of animal average weight has gone up 6.76% (i.e. 1.60 grams).

Animal is administered once every three days to MuR5S4TR 135mg/kg, and continuous three weeks dosage regimens are resistant to, entire real It tests in the period, compared with Day0, when Day21, this group of animal average weight has gone up 5.62% (i.e. 1.30 grams).

It is administered once within animal four days every to MuR5S4TR 158mg/kg, continuous three weeks dosage regimens are resistant to, entire real It tests in the period, compared with Day0, when Day21, this group of animal average weight has gone up 7.60% (i.e. 1.78 grams).

Influence of the 7.2 each tested materials to human colon cancer cell HT-29 transplanted tumor in nude mice animal tumor volume

With solvent group ratio, CPT-11, there is certain inhibiting effect to human colon cancer cell HT-29 nude mouse xenograft tumor, There is significant difference in tumour inhibiting rate, and in Day3, significant difference occurs in T/C, is 88.74% (P < 0.05)；In Day21, T/C is minimum, is 29.14% (P < 0.001)

TRAIL-Mu3 72mg/kg administration group three-times-weekly, has one to human colon cancer cell HT-29 nude mouse xenograft tumor Fixed inhibiting effect, in Day7, there is significant difference in T/C, is 54.69% (P < 0.05)；In Day21, T/C is minimum, For 36.43% (P < 0.01).

The TRAIL-Mu3 93mg/kg secondary group that is administered once every three days, to human colon cancer cell HT-29 nude mouse xenograft tumor There is certain inhibiting effect, in Day3, significant difference occurs in T/C, is 81.84% (P < 0.05)；In Day21, T/C Minimum is 28.00% (P < 0.01).

The every four days groups that are administered once of TRAIL-Mu3 108mg/kg, to human colon cancer cell HT-29 nude mouse xenograft tumor There is certain inhibiting effect, in Day3, significant difference occurs in T/C, is 85.49% (P < 0.05)；In Day21, T/C Minimum is 28.69% (P < 0.01).

MuR5S4TR 105mg/kg weekly administration group three times, has one to human colon cancer cell HT-29 nude mouse xenograft tumor Fixed inhibiting effect, in Day3, there is significant difference in T/C, is 88.26% (P < 0.05)；In Day21, T/C is minimum, For 42.86% (P < 0.01).

MuR5S4TR 135mg/kg is administered once group every three days, has to human colon cancer cell HT-29 nude mouse xenograft tumor Certain inhibiting effect, in Day7, there is significant difference in T/C, is 53.26% (P < 0.05)；In Day21, T/C is most It is small, it is 29.03% (P < 0.01).

The every four days groups that are administered once of MuR5S4TR 158mg/kg, there is human colon cancer cell HT-29 nude mouse xenograft tumor Certain inhibiting effect, in Day7, there is significant difference in T/C, is 51.53% (P < 0.05)；In Day21, T/C is most It is small, it is 28.87% (P < 0.01).

Experimental result is shown in Table 7 and attached drawing 11.

Influence of the 7.3 each tested materials to human colon cancer cell HT-29 transplanted tumor in nude mice animal tumor weight

Day21 experiment terminates, and all animals are put to death with after gross tumor volume weighing in, and tumour is separated from animal, And it weighs.Solvent group, CPT-11 group, TRAIL-Mu3 72mg/kg administration group three-times-weekly, TRAIL-Mu3 93mg/kg every three Its group that is administered once, the every four days groups that are administered once of TRAIL-Mu3 108mg/kg, MuR5S4TR 105mg/kg are administered three-times-weekly Group, MuR5S4TR 135mg/kg are administered once group every three days, the tumour for the group that is administered once for MuR5S4TR 158mg/kg every four days Average weight is 1.339 grams, 0.811 gram, 0.898 gram, 0.796 gram, 0.805 gram, 0.936 gram, 0.823 gram and 0.812 respectively Gram.Experimental result is shown in Table 7 and attached drawing 12.

Table 7.TRAIL-Mu3 and MuR5S4TR is in human colon cancer cell HT-29 nude mouse xenograft tumor model to animal Tumor size influences

8. brief summary

In this experiment, CPT-11 group similar to solvent group, TRAIL-Mu3 72mg/kg administration group three-times-weekly, TRAIL-Mu3 93mg/kg is administered once group every three days, the every four days groups that are administered once of TRAIL-Mu3 108mg/kg, MuR5S4TR 105mg/kg administration group three-times-weekly, MuR5S4TR 135mg/kg are administered once group every three days, and MuR5S4TR 158mg/kg is every Four days groups that are administered once, have little effect the weight of animals.

Compared to solvent group, CPT-11 group, TRAIL-Mu3 72mg/kg administration group three-times-weekly, TRAIL-Mu3 93mg/kg Be administered once group every three days, and the every four days groups that are administered once of TRAIL-Mu3 108mg/kg, MuR5S4TR 105mg/kg is three-times-weekly Administration group, MuR5S4TR 135mg/kg are administered once group every three days, and be administered once group pair for MuR5S4TR 158mg/kg every four days Human colon cancer cell HT-29 nude mouse xenograft tumor has and has certain inhibiting effect, and after administration, conspicuousness occurs in tumour inhibiting rate Difference.TRAIL-Mu3 and TRAIL-MuR5S4TR be administered once every three days group and be administered once within every four days group tumor suppression effect It should be obviously superior to administration group three-times-weekly, and the tumor inhibitory effect of be administered once every three days group and the group that is administered once for every four days There was no significant difference.

9. bibliography

[1] " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle " in November, 2006

Embodiment 5

TRAIL-Mu3 and TRAIL-MuR5S4TR different dosing interval is to human pancreatic cancer cell PANC-1 nude mouse xenograft The therapeutic effect of tumor is studied

1. experiment purpose

This research uses human pancreatic cancer cell PANC-1 nude mouse xenograft tumor model, evaluates TRAIL-Mu3 and TRAIL- The internal anti-tumor activity at MuR5S4TR different dosing interval.

2. experimental animal

2.1 animal species

Mouse.

2.2 kind

Balb/c nude mice.

2.3 gender

Female.

2.4 quantity

Experiment uses 64.

2.5 the age

4~6 weeks.

2.6 weight

± 20% weight mean value of 16~18g.

2.7 animal origins (supplier)

Shanghai western Poole-Bi Kai experimental animal Co., Ltd (BK), credit number SCXK (Shanghai) 2013-0016, animal are closed Lattice card number: 2008001665079.

2.8 management of laboratory animal

2.8.1 animal identification identification method

Each equal wearing of mouse cage has the information such as experiment numbers, experiment group, experimenter's name, mouse strain and gender Identity card, mouse are marked with ear tag method.

2.8.2 random grouping

It is grouped when gross tumor volume averagely reaches 160mm3 or so with randomized blocks, guarantees gross tumor volume and mouse between each group Weight is uniform, and the mean value difference of the mean value of each group gross tumor volume and all experimental animal tumor volumes is no more than ± 10%.

2.8.3 operational administrative specification

The operation and management of all experimental animals strictly observe Shanghai Mei Dixi experimental animal and use and management guidance original Then.

2.8.4 rearing conditions

Living conditions: 3, every cage.

Temperature: 20 DEG C~26 DEG C

Humidity: 40%~70%

Illumination: day alternates with night within 12 hours

2.8.5 feed

Size mouse feed is irradiated, is pulled together feed corporation,Ltd purchased from Beijing Australia of section.Ad lib.

2.8.6 drinking-water

City tap-water is drunk after filtering high pressure sterilization.

2.8.7 padding

Corncob, Shanghai Mao Sheng derivative Science and Technology Ltd. use after high pressure sterilization.Padding twice is changed weekly.

2.8.8 the laundering period

Give mouse most short one week environment laundering period before experiment.

3. experimental material

3.1 test drugs

Tester TRAIL-Mu3 and MuR5S4TR information

3.2 cell strain

Human pancreatic cancer cell cell strain PANC-1 is purchased from cell biological research institute of the Shanghai Chinese Academy of Sciences.

3.3 reagent

DMEM culture medium (GIBCO, the U.S.)

Fetal calf serum FBS (GIBCO, the U.S.)

Pancreatin Trypsin-EDTA (is purchased from the U.S. GIBCO)

Trypan blue Trypan Blue (is purchased from the U.S. GIBCO)

3.4 instrument

Biohazard Safety Equipment (model: AC2-6E1) is purchased from ESCO；

CO₂Water proof cell incubator (model: 3111), is purchased from Thermo Scientific Forma；

Inverted microscope (model: CKX41SF) is purchased from Olympus；

Electric suction apparatus (model YX930D) is purchased from Shanghai Medical appliance industry (group) Co., Ltd；

Low speed centrifuge (model LD5-2A) is purchased from Beijing Lei Boer centrifuge Co., Ltd.

3.5 other

1ml asepsis injector is purchased from purchase Shanghai Kindly Enterprise Development Group (Shanghai, China).

4. experimental design

Human pancreatic cancer cell PANC-1 Xenografts in nude mice model is established, 5 × 106 cells of every animal inoculation pvaccination connect Kind volume is 0.1ml/ animal.

This independent experiment designs dosage and dosage regimen is as follows:

Antitumor action of the TRAIL-Mu3 and MuR5S4TR in human pancreatic cancer cell PANC-1 Nude Mouse Model

5. experimental method

5.1 test medicine preparations are prepared

5.1.1 gemcitabine 4mg/ml 2ml

Gemcitabine stoste (20mg/ml)

Gemcitabine (200mg) is dispensed, and four pipes is divided into (every pipe contains gemcitabine 50mg).4 degree of refrigerators are set to protect It deposits.

The gemcitabine (50mg) one of above-mentioned packing is taken to manage.

2.5ml physiological saline is added, mixes, is dissolved to clarification, is configured to 20mg/ml gemcitabine stoste.

Take gemcitabine stoste (20mg/ml) 0.4ml.

1.6ml physiological saline is added, mixes.

It is ready-to-use.

5.1.2TRAIL-Mu3 10.8mg/ml 2.4ml

Solvent: 0.9% physiological saline: water for injection=1:1

TRAIL-Mu3 stoste (24mg/ml) 1.08ml is taken,

The above-mentioned solvent of 1.32ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours.

5.1.3TRAIL-Mu3 9.3mg/ml 2.4ml

Solvent: 0.9% physiological saline: water for injection=1:1

TRAIL-Mu3 stoste (24mg/ml) 0.93ml is taken,

The above-mentioned solvent of 1.47ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours.5.1.4TRAIL-Mu3 7.2mg/ml 2.4ml

Take TRAIL-Mu3 stoste (24mg/ml) 0.72ml.

The above-mentioned solvent of 1.68ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours.

5.1.5MuR5S4TR 15.8mg/ml 2.4ml

Solvent: 0.9% physiological saline: water for injection=1:1

Take MuR5S4TR stoste (24mg/ml) 1.58ml.

The above-mentioned solvent of 0.82ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours.

5.1.6MuR5S4TR 13.5mg/ml 2.4ml

Solvent: 0.9% physiological saline: water for injection=1:1

Take MuR5S4TR stoste (24mg/ml) 1.35ml.

The above-mentioned solvent of 1.05ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours.

5.1.7MuR5S4TR 10.5mg/ml 2.4ml

Take MuR5S4TR stoste (24mg/ml) 1.05ml.

The above-mentioned solvent of 1.35ml is added, mixes.

It is ready-to-use, it is maintained at 4 DEG C before use, is used in 4 hours

5.2 experimental method

5.2.1 cell culture

PANC-1 cell culture contains 10% fetal calf serum FBS in DMEM culture medium.It is incubated at 37 DEG C of cultures containing 5%CO2 Case.It after cell recovery, expands and passes on by cell, collect enough cells for animal inoculation pvaccination.

5.2.2 cell inoculation method establishes tumour Xenografts in nude mice model

The tumour cell for collecting logarithmic growth phase, is resuspended in plasma-free DMEM medium after counting, adjustment cell suspension is dense It spends to 5 × 10⁷/mL.With 1mL syringe (No. 4 syringe needles) the dorsal sc inoculated tumour cell on the right side of nude mice, 5 × 10⁶/0.1mL/ Mouse is inoculated with 90 animals altogether.

5.2.3 grouping administration

Reach 160mm3 or so in gross tumor volume, animal is grouped at random by randomized blocks, keeps each group tumour poor Different 10% less than mean value, every group 8, totally 8 groups.The grouping same day is denoted as Day0.It is administered by " 4. experimental design ".

5.2.4 Indexs measure

The weight of animals twice and tumor size are measured during experiment weekly, observes and records clinical symptoms, Day21 measurement daily After put to death all animals, strip tumour, weigh knurl weight and simultaneously take pictures.

All zoopery operations strictly observe Shanghai Mei Dixi Biomedics Inc. animal and use and manage Specification.The calculating of tumour relevant parameter refers to China CFDA " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle " [1]。

5.3 calculating

The calculating of tumour relevant parameter:

The calculation formula of gross tumor volume (Tumor volume, TV) are as follows: TV=a × b2/2.Wherein a, b respectively represent tumour Measurement is long and wide.

The evaluation index of anti-tumor activity is Relative tumor appreciation rate T/C (%) and tumour inhibiting rate (%), calculation formula difference Are as follows: T/C (%)=TRTV/CRTV*100%.(TRTV: treatment group RTV；CRTV: negative control group RTV)；Relative tumour volume (relative tumor volume, RTV), calculation formula are as follows: RTV=Vt/V0.When wherein V0 is sub-cage administration (Day0) Measurement gained gross tumor volume, Vt are gross tumor volume when measuring each time.

The changes of weight (%) of tumor animal calculates as follows: weight when (weight when weight-grouping when measurement)/be grouped × 100。

According to Chinese CFDA " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle " (in November, 2006), T/ C (%)≤40% and be effective through statistical analysis P < 0.05.If the weight loss of mouse is more than that 20% or drug are relevant dead Number is died more than 20%, then it is assumed that the drug dose has serious toxicity.

6. data are analyzed

Using time point as X-axis, gross tumor volume is that Y-axis draws tumor growth curve；Using time point as X-axis, animal is averaged body Weight (g) is that Y-axis draws changes of weight curve.Comparison among groups are examined using t-, and P < 0.05 is significant difference, and P < 0.01 is pole Significant difference.

7. result

Influence of the 7.1 each tested materials to human pancreatic cancer cell PANC-1 transplanted tumor in nude mice the weight of animals

Within this entire experimental period, solvent group animal average weight is not influenced by solvent solvent.Animal average weight Constantly increase.Compared with the first day, that is, Day 0 is administered, animal average weight has gone up 8.42% (i.e. 1.93 grams) when Day21.

Gemcitabine (40mg/kg, IV, Day0,2,4) group, at administration initial stage, the weight of animals declines to a great extent, and gives The medicine first day, that is, Day0 is compared, and when Day7, this group of animal average weight has dropped 16.92% (i.e. 3.18 grams), while when Day7, out An existing animal dead；As dosage period terminates, this group of the weight of animals starts to restore and go up, in Day21, this group of animal Average weight has gone up 8.57% (i.e. 1.93 grams).

Three times to TRAIL-Mu3 72mg/kg weekly administration, continuous three weeks dosage regimens are resistant to animal, are entirely being tested In period, compared with Day0, when Day21, this group of animal average weight has gone up 6.09% (i.e. 1.36g).

Animal is administered once every three days to TRAIL-Mu3 93mg/kg, and continuous three weeks dosage regimens are resistant to, entire real It tests in the period, compared with Day0, when Day21, this group of animal average weight has gone up 5.76% (i.e. 1.35g).

It is administered once within animal four days every to TRAIL-Mu3 108mg/kg, continuous three weeks dosage regimens are resistant to, entire In experimental period, compared with Day0, when Day21, this group of animal average weight has gone up 5.63% (i.e. 1.26g).

Three times to MuR5S4TR 105mg/kg weekly administration, continuous three weeks dosage regimens are resistant to animal, are entirely being tested In period, compared with Day0, when Day21, this group of animal average weight has gone up 5.53% (i.e. 1.24g).

Animal is administered once every three days to MuR5S4TR 135mg/kg, and continuous three weeks dosage regimens are resistant to, entire real It tests in the period, compared with Day0, when Day21, this group of animal average weight has gone up 5.09% (i.e. 1.12g).

It is administered once within animal four days every to MuR5S4TR 158mg/kg, continuous three weeks dosage regimens are resistant to, entire real It tests in the period, compared with Day0, when Day21, this group of animal average weight has gone up 5.89% (i.e. 1.33g).

Influence of the 7.2 each tested materials to human pancreatic cancer cell PANC-1 transplanted tumor in nude mice animal tumor volume

With solvent group ratio, gemcitabine has certain inhibition to make human pancreatic cancer cell PANC-1 nude mouse xenograft tumor With significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 80.28% (P < 0.05)；In Day21 When, T/C is minimum, is 36.14% (P < 0.001).

Three-times-weekly, continuous three weeks groups are to human pancreatic cancer cell PANC-1 nude mouse xenograft by TRAIL-Mu3 72mg/kg Tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 73.94% (P < 0.01)；In Day21, T/C is minimum, is 22.34% (P < 0.001).

Once every three days, continuous three weeks groups move human pancreatic cancer cell PANC-1 bare mouse different species to TRAIL-Mu3 93mg/kg Planting tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 63.38% (P < 0.01)；In Day21, T/C is minimum, is 23.25% (P < 0.001).

Once every four days, continuous three weeks groups move human pancreatic cancer cell PANC-1 bare mouse different species to TRAIL-Mu3 108mg/kg Planting tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 61.27% (P < 0.01)；In Day21, T/C is minimum, is 23.13% (P < 0.001).

MuR5S4TR 105mg/kg three-times-weekly, continuous three weeks groups, to human pancreatic cancer cell PANC-1 nude mouse xenograft Tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 65.49% (P < 0.001)；In Day21, T/C is minimum, is 24.82% (P < 0.001).

Once every three days, continuous three weeks groups move human pancreatic cancer cell PANC-1 bare mouse different species to MuR5S4TR 135mg/kg Planting tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 70.42% (P < 0.001)；In Day21, T/C is minimum, is 27.00% (P < 0.001).

Once every four days, continuous three weeks groups move human pancreatic cancer cell PANC-1 bare mouse different species to MuR5S4TR 158mg/kg Planting tumor has certain inhibiting effect, and significant difference occurs in tumour inhibiting rate, and in Day4, significant difference occurs in T/C, is 73.24% (P < 0.001)；In Day21, T/C is minimum, is 27.02% (P < 0.001).

Experimental result is shown in Table 8 and attached drawing 13.

Influence of the 7.3 each tested materials to human pancreatic cancer cell PANC-1 transplanted tumor in nude mice animal tumor weight

Day21 experiment terminates, and all animals are put to death with after gross tumor volume weighing in, and tumour is separated from animal, And it weighs.Solvent group, gemcitabine group (n=7), TRAIL-Mu3 72mg/kg three-times-weekly, continuous three weeks groups, TRAIL-Mu3 93mg/kg once every three days, continuous three weeks groups, TRAIL-Mu3108mg/kg once every four days, continuous three weeks groups, MuR5S4TR 105mg/kg three-times-weekly, continuous three weeks groups, MuR5S4TR 135mg/kg once every three days, continuous three weeks groups, MuR5S4TR Once every four days, continuously the exemplary embodiment lock of three weeks groups is 0.131 gram respectively to 158mg/kg, 0.075 gram, 0.042 gram, 0.044 gram, 0.043 gram, 0.047 gram, 0.050 gram and 0.052 gram.Experimental result is shown in Table 8 and attached drawing 14.

Table 8.TRAIL-Mu3 and MuR5S4TR is in human pancreatic cancer cell PANC-1 nude mouse xenograft tumor model to animal Tumor size influences

8. brief summary

It is similar to solvent group in this experiment, TRAIL-Mu3 72mg/kg three-times-weekly, continuous three weeks groups, TRAIL- Mu3 93mg/kg once every three days, continuous three days groups, TRAIL-Mu3 108mg/kg once every four days, continuous three weeks groups, MuR5S4TR 105mg/kg three-times-weekly, continuous three weeks groups, MuR5S4TR135mg/kg once every three days, continuous three weeks groups, MuR5S4TR 158mg/kg once every four days, continuous three weeks groups, the weight of animals is had little effect.

Gemcitabine group occurs an animal dead in Day7.

Compared to solvent group, gemcitabine group, TRAIL-Mu3 72mg/kg three-times-weekly, continuous three weeks groups, TRAIL-Mu3 93mg/kg once every three days, continuous three weeks groups, TRAIL-Mu3 108mg/kg once every four days, continuous three weeks groups, MuR5S4TR 105mg/kg three-times-weekly, continuous three weeks groups, MuR5S4TR135mg/kg once every three days, continuous three weeks groups, MuR5S4TR Once every four days, continuously three weeks groups have human pancreatic cancer cell PANC-1 nude mouse xenograft tumor to 158mg/kg apparent suppression Production is used, and after administration, significant difference occurs in tumour inhibiting rate.TRAIL-Mu3 72mg/kg three-times-weekly, continuous three weeks groups, TRAIL-Mu3 93mg/kg once every three days, continuous three weeks groups, TRAIL-Mu3 108mg/kg once every four days, continuously three weeks Curative effect does not have difference between group each group.MuR5S4TR 105mg/kg three-times-weekly, continuous three weeks groups, MuR5S4TR 135mg/kg Once every three days, continuous three weeks groups, curative effect is not poor between three weeks group each groups once every four days, continuously by MuR5S4TR158mg/kg It is different.

9. bibliography

[1] " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle " in November, 2006

The series of detailed descriptions listed above are illustrated only for possible embodiments of the invention, The protection scope that they are not intended to limit the invention, it is all without departing from equivalent embodiment made by technical spirit of the present invention or change It should all be included in the protection scope of the present invention.

Claims (10)

1. the medication that a kind of TRAIL albuminoid persistently inhibits growth of tumour cell, which is characterized in that it is interval, repeats And the administration of full course for the treatment of drug exposure, it is extension dosing interval, increases tumour cell in the drug exposure of the full course for the treatment of, make Drug is unattenuated in the effect of the full course for the treatment of, to persistently inhibit tumour growth.

2. TRAIL albuminoid according to claim 1 persistently inhibits the medication of growth of tumour cell, feature exists In, the TRAIL albuminoid include natural or recombination Apo2L/TRAIL albumen after birth outer segment 114-281aa, TRAIL by Body selective mutation body, TRAIL cell-penetrating peptide sample mutant TRAIL-Mu3, TRAIL-MuR5 and TRAIL-MuR6, TRAIL wear film One or more of polypeptide mutant albumen TRAIL-MuR5S4TR and TRAIL-MuR6S4TR and other mutant, wherein described The amino acid sequence of other mutant and the similarity of wild-type protein are 75% or more.

3. TRAIL albuminoid according to claim 1 persistently inhibits the medication of growth of tumour cell, feature exists In the tumour cell is solid tumor or derived from bone marrow tumour, and the solid tumor includes lung cancer, colorectal cancer, breast cancer, pancreas One or more of cancer, liver cancer, gastric cancer, oophoroma, kidney, brain tumor, osteochondroma, prostate cancer；The derived from bone marrow is swollen Tumor includes one or more of leukaemia, non-Hodgkin's lymphoma.

4. TRAIL albuminoid according to claim 1 persistently inhibits the medication of growth of tumour cell, feature exists In persistently inhibiting growth of tumour cell includes the horizontal tumor-inhibiting action of cell in vitro and animal experiment in vivo tumor-inhibiting action；In vitro In experiment, TRAIL albuminoid observes suppression of the 24-96 hours drugs to tumour cell in effective dosage ranges and cytosis Rate processed；In the tumour cell of different sensibility, TRAIL albuminoid was in inhibition at 24-72 hours to growth of tumour cell Peak, for highly sensitive cell strain or higher activity, the rush hour of tumor suppression continues to 96 hours；In body In interior experiment, the animal-transplanted tumor of different interval medication is in obvious growth inhibition state, Relative tumor in 21 days Growth rate T/C is ≤40%.

5. TRAIL albuminoid according to claim 1 persistently inhibits the medication of growth of tumour cell, feature exists In with daily medication compared with primary, continuous medication for five days therapy, tumour inhibiting rate is at least on tumour cell Nude Mouse Model 20% or more is improved, acting duration extends 5 days or more in the course for the treatment of 21 days at one.

6. TRAIL albuminoid according to claim 1 persistently inhibits the medication of growth of tumour cell, feature exists In the dosage regimen at interval, repetition and full course for the treatment of drug exposure includes any one following:

(1) TRAIL albuminoid be injected intravenously, the next day it is primary, from the course for the treatment of 0 day, administration time was respectively 0,2,4,6,8,10, 12,14,16,18 or respectively 0,2,4,6,8,10,12,14,16,18,20, every 21 days as a treatment course；

(2) TRAIL albuminoid is injected intravenously, and three-times-weekly, from the course for the treatment of 0 day, administration time was respectively 0,2,4,7,9,11, 14,16,18, every 21 days as a treatment course；

(3) TRAIL albuminoid is injected intravenously, and is administered once every three days, and from the course for the treatment of 0 day, administration time was respectively 0,3,6,9, 12,15,18, every 21 days as a treatment course；

(4) TRAIL albuminoid is injected intravenously, and is administered once within every four days, and from the course for the treatment of 0 day, administration time was respectively 0,4,8,12, 16,20, every 21 days as a treatment course.

7. TRAIL albuminoid according to claim 1 persistently inhibits the medication of growth of tumour cell, feature exists In, using be higher than its inside and outside work minimum activity, extend its dosing interval.

8. TRAIL albuminoid according to claim 2 persistently inhibits the medication of growth of tumour cell, feature exists In, TRAIL-Mu3 group and MuR5S4TR each group have certain inhibiting effect to human lung cancer NCI-H460 nude mouse xenograft tumor, After administration, there is significant difference in tumour inhibiting rate, wherein TRAIL-Mu3 and MuR5S4TR two connective days administration group and on every Wendesdays Secondary, the tumor-inhibiting action of three weeks administration groups is superior to be administered daily, and continuous 5 days, totally two weeks groups；TRAIL-Mu3 and MuR5S4TR two Connective day administration group and three-times-weekly, the tumor-inhibiting action therapeutic equivalence of three weeks administration groups.

9. TRAIL albuminoid according to claim 2 persistently inhibits the medication of growth of tumour cell, feature exists In, TRAIL-Mu3 72mg/kg administration group three-times-weekly, TRAIL-Mu3 93mg/kg is administered once group every three days, TRAIL- The every four days groups that are administered once of Mu3 108mg/kg, MuR5S4TR 105mg/kg administration group three-times-weekly, MuR5S4TR 135mg/ Kg is administered once group every three days, and the group that is administered once is different to human colon cancer cell HT-29 nude mice within MuR5S4TR 158mg/kg every four days Kind transplantable tumor has and has certain inhibiting effect, and after administration, significant difference occurs in tumour inhibiting rate；TRAIL-Mu3 and TRAIL- MuR5S4TR be administered once every three days group and be administered once within every four days group tumor inhibitory effect be obviously superior to three-times-weekly to Medicine group, and be administered once every three days group and be administered once within every four days group tumor inhibitory effect there was no significant difference.

10. TRAIL albuminoid according to claim 2 persistently inhibits the medication of growth of tumour cell, feature exists In, TRAIL-Mu3 72mg/kg three-times-weekly, continuous three weeks groups, TRAIL-Mu393mg/kg once every three days, continuously three weeks Group, TRAIL-Mu3 108mg/kg once every four days, continuous three weeks groups, MuR5S4TR 105mg/kg three-times-weekly, continuously three weeks Group, MuR5S4TR 135mg/kg once every three days, continuous three weeks groups, MuR5S4TR 158mg/kg once every four days, continuously three All group has human pancreatic cancer cell PANC-1 nude mouse xenograft tumor and has apparent inhibiting effect, and after administration, tumour inhibiting rate goes out Existing significant difference；TRAIL-Mu3 72mg/kg three-times-weekly, continuous three weeks groups, TRAIL-Mu393mg/kg once every three days, Continuous three weeks groups, curative effect does not have difference to TRAIL-Mu3 108mg/kg between three weeks group each groups once every four days, continuously； MuR5S4TR 105mg/kg three-times-weekly, continuous three weeks groups, MuR5S4TR 135mg/kg once every three days, continuous three weeks groups, Curative effect does not have difference to MuR5S4TR 158mg/kg between three weeks group each groups once every four days, continuously.