CN101940797B

CN101940797B - Cladded nuclide labeled protein microsphere, preparation method and applications thereof

Info

Publication number: CN101940797B
Application number: CN2010102738445A
Authority: CN
Inventors: 陈晓理; 李林; 夏传琴; 马宇
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2010-09-06
Filing date: 2010-09-06
Publication date: 2012-07-04
Anticipated expiration: 2030-09-06
Also published as: CN101940797A

Abstract

The invention provides a cladded nuclide labeled protein microsphere, comprising the following bulk pharmaceutical chemicals and auxiliary materials in parts by weight: 1-5 parts of Na131I, 1-5 parts of Na125I, 300-1000 parts of chitosan, 100-500 parts of sodium alginate and 300-800 parts of gelatin, wherein, the specific radioactivity concentrations of the Na131I and the Na125I are both 37GBq/ml. The invention also provides a preparation method and applications of the cladded nuclide labeled protein microsphere; the cladded nuclide labeled protein microsphere adopts the inclusion of the chitosan and the sodium alginate; the actual half-life period of the cladded nuclide microsphere in the bodies of animals is longer, and is close to the half-life period of the nuclide; and the blood gamma counts and urine gamma counts of two microspheres are compared to discover that the nuclide released by the cladded nuclide microsphere is more stable than the nuclide released by the uncladded nuclide microsphere, thereby reliving the effect of the explosive release, thus the cladding can protect the nuclide microsphere, reduce the label peeling, and prolong the degrading time of the microsphere in the bodies.

Description

A kind of isotope labeling protein microsphere of involucrum

Technical field

The present invention relates to a kind of isotope labeling protein microsphere of involucrum.Belong to drug world.

Background technology

According to The World Health Organization's data, the whole world has 1,000 ten thousand New Development tumor patients, 6,000,000 people to die from malignant tumor every year approximately, and the sickness rate of malignant tumor also increases progressively in the amplitude with annual 1.8-4%.Malignant tumor has become the cardiovascular and cerebrovascular disease that continues, occupies the tertiary cause of death after the infectious disease.Even through effort for many years, the prognosis of many malignant tumor does not have significant improvement yet, and malignant tumor remains global difficult medical problem in present and a very long time from now on.In China, because hepatitis B popular in the past, make hepatitis B have the sickness rate of primary hepatocarcinoma of direct etiology and pathology contact high; Along with Chinese people's living standard raising, life style is westernization gradually, and the sickness rate of the malignant tumor of cancer of pancreas and cancer of bile ducts and other systems is also in rising trend; Under existing treatment means; The malignant tumor patient of most liver and gall pancreas system is dead in morbidity 1 year, like 30.6 ten thousand people that fall ill in the hepatocarcinoma year of China, and year dead 30.0 ten thousand people; 3.3 ten thousand people that fall ill in the cancer of pancreas year of the U.S., year dead 3.2 ten thousand people.How to treat emphasis and difficult point that malignant tumor is medical research always.

The Therapeutic Method of malignant tumor mainly comprises excision at present, X-ray therapy, chemotherapy and various comprehensive treatment, and wherein excision is first-selected Therapeutic Method; But most of malignant tumor patients have been in the middle and advanced stage of tumor when diagnosing, and the chance of ocal resection is little, and for example primary hepatocarcinoma excision rate is less than 10%; Cancer of pancreas is less than 15%; And excision not only expense is high, painful big, the prognosis of postoperative is also undesirable.5 years survival rates of postoperative of hepatocarcinoma are at 20-45% at present; And 5 years survival rates of the postoperative of cancer of pancreas are less than 15%; Most of patients can only be treated through non-operative treatment, and the malignant tumor of liver and gall pancreas system is dark because of the position, and conventional outer radiation is relatively poor because of the shielding effect of rib; Again because of liver and digestive tract to the very responsive radiation damage that suffers easily of lonizing radiation; Make liver and gall pancreas system become outer radiocurable relative forbidden zone, liver and gall pancreas malignant tumor is low to the sensitivity of most of chemotherapeutics, and for example the approval of American National Food and Drug Admistraton is used to treat the standard drug gemcitabine of cancer of pancreas; The new drug Ge Lifeini of treatment primary hepatocarcinoma, its clinical effective rate is all less than 20%.Therefore press for and seek non-operative treatment new method evident in efficacy.

Most of nucleic have strong toxicity, ⁹⁰Y, ⁸⁹Sr has intensive close bone property, in case disengage being gathered in the osseous tissue, destroys myeloid tissue, and the probability that causes aplastic anemia is arranged; ³²P can be by the most of tissue absorption of body, and cause that body damages widely, thus at present in the world the carrier of plesioradiotherapy all adopt metal, glass, pottery etc.; The nucleic sintering is buried in it; To reduce the release of nucleic, the nucleic distributivity is poor, and carrier in vivo can not be degraded; This technical operation is complicated, and costs an arm and a leg.The nucleic medicine that is used at present to treat tumor abroad has: ⁹⁰The Y glass microsphere

In 1999 through U.S. food Drug Administration (FDA) authentication, be used to treat can't excision liver cancer patient. ⁹⁰The Y resin microsphere

Passed through U.S. food drug surveilance pipe (FDA) authentication in 2002, with the metastatic liver cancer of fluorouracil therapeutic alliance colorectal carcinoma. ¹²⁵I nucleic particle is diverted to treatment of prostate cancer through U.S. food Drug Administration (FDA) authentication. ⁹⁰The Y glass microsphere half-life is lacked (64.1 hours), and specific radioactivity is not high. ⁹⁰The Y glass microsphere discharges pure beta ray can't externally monitor, thus must use simultaneously technetium-99 ( ^99mTc) protein microsphere of labelling confirms that the blood of tumor supplies and radioactivity tumor liver ratio, ⁹⁰The Y glass microsphere must could obtain radioactive activity after the activation in the linear accelerator of special use, its short half-life has limited it with expensive activation equipment and can only use in bigger medical center. ⁹⁰The Y glass microsphere is the foreign patent product, costs an arm and a leg, and domestic being difficult to promotes.Above-mentioned nucleic particle or microsphere are carrier with metal or glass all, because the nucleic sintering buries in carrier, the mark situation can not occur taking off, and still, specific gravity of glass is big, and perfusion is difficulty relatively, and it is not good to distribute, the radioactivity tumor: liver is than (T: N) relatively low.Metal or glass can't be degraded, and are stranded in for a long time in vivo, have both influenced perfusion therapy once more, and its tissue injury's effect at a specified future date also remains to be observed; Such as ³²The P glass microsphere can cause liver portal area connective tissue to thicken, and insufferable abdominal distention symptom appears in the patient clinically.

Domestic in recent years part large hospital begins to introduce that u s company produces ¹²⁵I metal casing nucleic particle; Mode through tumor is implanted into is treated multiple solid malignant; Particularly cancer of pancreas, hepatocarcinoma, bone tumor etc. have obtained effect preferably, but this particle is very expensive, one of more than 400 yuan of RMB of domestic price; A patient accomplishes the common total cost of treatment and reaches 2-3 ten thousand, and the said firm has reached more than 1,000,000,000 yuan in the annual sales amount of China at present.

For solve the safety of metal casing nucleic particle low, can't degrade in vivo, problem that cost is high, bibliographical information is arranged at present with active nucleus labelling gelatine microsphere treatment tumor, like Chen Xiaoli, etc., ¹³¹The gelatine microsphere intratumor injection of I and ametycin is to the therapeutic effect of mouse experiment property hepatocarcinoma, and " cancer " rolled up for 5 phases in 1992 11, adopted gelatine microsphere as carrier, and biodegradable in vivo, clinical practice is safer.But, ¹³¹The I label has bigger defective as drug disposition, as: have the energy height, strong to the lethal effect of tumor cell, environmental pollution is big, must use in special radioactivity protection ward, and ¹³¹Half-life of I short (8 days), is prone to take off iodine at poor stability, and its application is restricted.

Behind gelatin microsphere process isotope labeling, if directly be used for pathological changes or tumor locus, the part radionuclide can be from the microsphere surface stripping; Break away from the labelling microsphere and also get into blood, this situation is called " taking off mark ", take off mark after; Radionuclide is brought to other positions with blood circulation, not only has a strong impact on the effect of isotope labeling protein microsphere, but also can cause harmful effect to the patient; Therefore, when the isotope labeling protein microsphere is used for the close-range treatment of malignant entity tumor, should guarantee that the active nucleus clinical practice is safer; Prevent to take off in the body mark again, be the problem that is perplexing clinicist and researcher always.

Chitosan/sodium alginate bio-microcapsule is as a good Atrigel commonly used; Have excellent biological compatibility and mechanical strength; Raw material is easy to get, low price; But preparation technology is simple and suitability for industrialized production, through chitosan/sodium alginate enclose medicine, can reach slow releasing function preferably.

The material medicine that can be used as slow releasing preparation of ordinary meaning and radionuclide are two types of materials of different nature, chitosan/sodium alginate are not used to wrap up active nucleus at present, take off target pertinent literature report to prevent active nucleus.

Summary of the invention

Technical scheme of the present invention has provided the isotope labeling protein microsphere of involucrum, and another technical scheme of the present invention has provided the method for preparing and the purposes of this protein microsphere.

The invention provides a kind of isotope labeling protein microsphere of involucrum, it is the microsphere that is prepared from following raw materials by weight proportions and adjuvant:

Na ¹³¹I 1-5 part, Na ¹²⁵I 1-5 part, chitosan 300-1000 part, sodium alginate 100-500 part, gelatin 300-800 part, wherein Na ¹³¹I, Na ¹²⁵The specific radioactivity concentration of I is: 37GBq/ml.

Further, it is the microsphere that is prepared from the following weight proportion raw material:

Na ¹³¹2 parts of I, Na ¹²⁵4 parts of I, 800 parts of chitosans, 300 parts of sodium alginates, 300 parts in gelatin.

Wherein, contain in every 50mg gelatine microsphere ¹³¹The radioactivity of I is 0.422-0.429mCi, ¹²⁵The radioactivity of I is 0.83855-0.872mCi.

Further preferably, contain in every 50mg gelatine microsphere ¹³¹The radioactivity of I is 0.4253 ± 0.003mCi, ¹²⁵The radioactivity of I is 0.8514 ± 0.01285mCi.

Wherein, the particle diameter of described microsphere is 100-200 μ m.

The present invention also provides a kind of method for preparing the isotope labeling protein microsphere of this involucrum, and it comprises the steps:

A. the preparation of gelatine microsphere:

Get liquid paraffin, add Arlacel-80, put in 55 ℃ of water-baths; 10% the gelatin solution speed with 30 droplets/minute under 550 rev/mins mechanical agitation of getting slowly splashes in the paraffin liquid; Stir and after 15 minutes aforesaid liquid is cooled to 4 ℃ rapidly, stir the adding glutaraldehyde cross-linking, add acetone dehydration back sucking filtration again, thus obtained microsphere is used acetone, sucking filtration, and the screening preservation of the air-dry back of room temperature is subsequent use;

B, microsphere labelling

0.2mmol/L sodium dihydrogen phosphate and 0.2mmol/L sodium hydrogen phosphate are mixed with the buffer solution PBS of pH=7.0, the amp-T of preparation 20mg/ml chlorine, 15mg/ml sodium metabisulfite; Get microsphere to reaction tube, add the PBS swelling; With Na ¹³¹I and Na ¹²⁵I presses 1-4 part: 5 parts weight proportion adds, and adds the amp-T of chlorine again, and the reaction of room temperature magnetic agitation adds the sodium metabisulfite stopped reaction; 4400 rev/mins centrifugal 4 minutes, abandon supernatant, gained labelling microsphere with normal saline clean, air-dry, it is subsequent use to sterilize;

C, parcel:

Preparation 0.5% sodium alginate soln is mixed with 0.5% chitosan solution with 3% acetum, and the labelling microsphere of b step preparation is dropped in the sodium alginate soln, wraps up the labelling microsphere, drops into chitosan solution after separating microsphere again; Or the labelling microsphere of b step preparation put in the chitosan solution, parcel labelling microsphere drops into sodium alginate soln after separating microsphere again; 4-7 time repeatedly, promptly get the isotope labeling protein microsphere of involucrum of the present invention.

The envelop rate of microsphere of the present invention can reach 61.37%.

The present invention also provides the purposes of isotope labeling protein microsphere in the medicine of preparation treatment hepatocarcinoma, renal carcinoma, breast carcinoma, thyroid carcinoma, cancer of pancreas, gastrointestinal solid tumor or orthopaedics tumor of this involucrum.

The present invention also provides the purposes of isotope labeling protein microsphere in the agent of preparation neoplasm tracing of described involucrum.

The present invention adopts ¹³¹I with ¹²⁵I is clinical nucleic of treating tumor for many years; Compare with other nucleic, ¹³¹I with ¹²⁵The great advantage of I is that toxicity is low: the main in vivo passive armor shape glandular tissue of iodine absorbs, and seldom stops at its hetero-organization, is not mainly excreted through urinary system by the iodine of thyroid picked-up in the serum.Oral Na when clinical treatment first cancer, hyperthyroidism ¹³¹I (iodine 131 sodium) millicuries up to a hundred do not have serious tissue injury yet.On the other hand, give the oral sealing parathyroid tissue of IodineSodium Solution (Compound Iodine Solutlon) before the art and can stop its reuptake radioiodine effectively, thereby prevent the thyroid damage.In addition, thyroid has stronger self repair ability, and the radiolesion of certain limit can be by himself rapid reparation, and therefore, the radioiodine nucleic is safer than other nucleic.The present invention will adopt degradable protein gelatin microsphere as nucleic and pharmaceutical carrier; Gelatin is the product of collagen protein partial hydrolysis, but good biocompatibility, the multiple radionuclide of biodegradable stable bond and medicine (like chemotherapeutics, bioactive macromolecule etc.) are arranged; , combine with the radioiodine nucleic as carrier with gelatine microsphere, the employing group is established route of administration, can carry out plesioradiotherapy to the malignant tumor of multiple character; Microsphere can distribute between tumor tissues through the method for tumor body injection more equably, can comprise hepatocarcinoma to various solid tumors; Renal carcinoma, breast carcinoma, thyroid carcinoma; Cancer of pancreas, the gastrointestinal solid tumor, the orthopaedics tumor is carried out tumor body implantable brachytherapy radiotherapy etc.After nucleic had decayed, gelatin is degraded gradually in vivo, and the nucleic that has decayed excretes through urinary system.But gelatine microsphere high concentration binding radioactivity iodine, ¹³¹The effect of I by force but ofer short duration, ¹²⁵The adding of I can reduce ¹³¹The consumption of I, thus the difficulty of environmental pollution and protection reduced, and ¹²⁵I has the characteristics of long half time (60 days), thus the recurrence and the transfer of partial radiation inhibition tumor for a long time. ¹³¹I with ¹²⁵The mixing of I, under specific consumption proportion, the ray characteristics of two kinds of isotope releases are replenished each other, make it better meet the oncotherapy principle. ¹³¹The high-energy gamma ray of I can clearly video picture on SPECT, thus help microsphere that the doctor accurately understands implantation in tumor with intravital distribution; Will ¹³¹I with ¹²⁵I mixed mark nucleic protein microsphere is implanted hepatocarcinoma through getting involved promoting the circulation of blood pipe perfusion thromboembolism or injection, in the pancreatic tumour tissue and microsphere more stable bonded ¹³¹I with ¹²⁵I discharges gamma-rays in the part, can reach directed and lasting tumor internal radiotherapy and as the purpose of tracer.

Simultaneously, the isotope labeling protein microsphere of involucrum of the present invention adopts chitosan and sodium alginate enclose, and employing has the nucleic microsphere actual half-life in animal body of involucrum longer, more near half-life of nucleic itself.Contrast two kinds of microsphere blood, urine γ counting, finding to have the nucleic microsphere of involucrum to discharge nucleic, not have the nucleic microsphere of involucrum steady, alleviated " outburst discharges " effect.Therefore, involucrum can be protected the nucleic microsphere, reduces it and " takes off mark "; Can prolong microsphere degradation time in vivo, and after the nucleic decay, the equal metabolism of chitosan and sodium alginate is to external; Clinical use is safer, for clinical nucleic uses a kind of new selection is provided.

Description of drawings

Fig. 1: the involucrum nucleic microsphere sketch map of preparation, wherein 1 is A type gelatin, and 2 is the Type B gelatin, and 3 is the sodium alginate involucrum, and 4 is the chitosan involucrum.

Fig. 2: outturn sample

Fig. 3: ¹³¹The I channel measurement ¹²⁵I radioactivity linear graph (γ=1.7142x, R ²=1)

Fig. 4: ¹²⁵The I channel measurement ¹³¹I radioactivity linear graph (γ=0.5828x, R ²=0.9999)

Fig. 5: postoperative rabbit 100ul serum r counting

Fig. 6: postoperative rabbit 100ul urine γ counting

Fig. 7: postoperative ¹³¹The peak SPECT video picture of I ability, 306KeV, window width 15%, developing time 2 minutes.A: postoperative 1 day, B: postoperative 8 days, C: postoperative 16 days, D: postoperative 24 days, E: postoperative 32 days, F: postoperative 48 days was the radionuclide imaging that does not have outstanding background

Fig. 8: postoperative double labelling microsphere liver implantation T-A curve ( ¹³¹The ability peak of I)

Fig. 9: postoperative adopted in 48 days ¹²⁵I's can scan 35KeV, window width 15%, developing time 2 minutes by the capable SPECT in peak.Find Hepar Leporis seu Oryctolagi position video picture.Be marked with two kinds of radioiodine nucleic simultaneously on the proof microsphere.

Figure 10: microsphere results of interventional treatment of the present invention: wherein, A: hepatic arteriography, conduit accurately get into Hepatic artery.B: radiography behind the thromboembolism, see that tiny tremulous pulse does not develop (arrow indication among the figure) in lines, explains that it is by the microsphere thromboembolism.

Figure 11: rabbit CT scan three-dimensional reconstruction, shown in " ten " word, the plantation tumor is low-density among the figure, and the border is clear.

Figure 12: SPECT/CT merges 3-D view behind the hepatic artery interventional therapy, shows that radionuclide concentrates in tumor locus (among the figure shown in the cross), and A is 16 days, and B is 24 days.

Figure 13: A: near the necrosis of liver tissue band involucrum nucleic microsphere injection site, near B: the necrosis of liver tissue no involucrum nucleic microsphere injection site.

Figure 14: the SPECT contrast (ability peak 306KeV, window width 15%, 2 minute) of involucrum (A, C, E, G, I, K) and no involucrum (B, D, F, H, J, L) nucleic protein microsphere is arranged; A, B are postoperative 1 day; C, D are postoperative 8 days, and E, F are postoperative 16 days, and G, H are postoperative 24 days; I, J are postoperative 32 days, and K, L are postoperative 48 days.Explain the involucrum microsphere more not involucrum microsphere nucleic come off fewly, metabolism gets slower, can stop in vivo the longer time, and is significant to reducing disengaging of nucleic.

Figure 15: the T-A curve after two kinds of microsphere Hepar Leporis seu Oryctolagi implantations.Can find out have involucrum group liver T-A curve to descend not have the involucrum group slow.

Figure 16: serum r counting after two kinds of microsphere implantations.Observable difference is little.

Figure 17: urine γ counting after two kinds of microsphere Hepar Leporis seu Oryctolagi implantations.It is thus clear that the radioactivity, particularly postoperative later stage of involucrum group in most of the time point urine arranged, and it is strong not have the involucrum group.

Figure 18: pathological section, be HE dyeing, no involucrum group is A: postoperative 1 day (* 200), C: postoperative 4 days (* 200), E: postoperative 16 days (* 200), seeing microsphere has degraded, G: postoperative 32 days (* 400), I: postoperative 48 days (* 400); It is B that the involucrum group is arranged: postoperative 1 day (* 200), D: postoperative 4 days (* 200), F: postoperative 16 days (* 200); H: postoperative 32 days (* 400), the injection site is by fibers encapsulation; J: postoperative 48 days (* 400), see the cavity that the microsphere degraded stays, but form is still complete.

The specific embodiment

The method for preparing of embodiment 1 microsphere of the present invention

One, crude drug:

Na ¹³¹I, Na ¹²⁵I (sodium iodide)

Medical Type B gelatin: Sigma A type gelatin 50g microsphere adopts the preparation of improvement emulsifying condensation method.Bottle behind the chloramine-t method labelling

Labelling adopts Na ¹³¹I is 2mCi, i.e. 2ul; Use Na ¹²⁵I is 4mCi, i.e. 4ul; Use gelatine microsphere 0.8g; Using sodium alginate is 0.3g; Using chitosan is 0.3g.Therefore the ratio of this each component of test involucrum nucleic microsphere is: 0.002: 0.004: 0.8: 0.3: 0.3.

Na wherein ¹³¹I, Na ¹²⁵The specific radioactivity concentration of I is: 37GBq/ml.

Concrete method for preparing is:

1. microsphere is made

Adopt the preparation of improvement emulsifying condensation method.Get the 80ml liquid paraffin, add the 0.8ml Arlacel-80, put into round-bottomed flask, put in 55 ℃ of water-baths.10% the gelatin solution 10ml speed with 30 droplets/minute under 550 rev/mins mechanical agitation of getting slowly splashes in the paraffin liquid.Stir and after 15 minutes aforesaid liquid is cooled to 4 ℃ rapidly, continue to stir 8 minutes adding 3ml glutaraldehyde cross-linkings, continue to stir 22 minutes; Add 30ml acetone dehydration sucking filtration after 15 minutes again; Thus obtained microsphere is with acetone twice, sucking filtration, and room temperature air-dry back screening is preserved subsequent use.

2. microsphere labelling

Adopt the chloramine-t method labelling ¹³¹I, ¹²⁵I.0.2mmol/L sodium dihydrogen phosphate and 0.2mmol/L sodium hydrogen phosphate are mixed with the buffer solution (PBS) of pH=7.0, the amp-T of preparation 20mg/ml chlorine, 15mg/ml sodium metabisulfite.Take by weighing the 50mg microsphere to reaction tube, added 190ul PBS swelling 10 minutes.With Na ¹³¹I and Na ¹²⁵I adds in 1: 2 ratio, adds the 200ul chloramine-T again, and room temperature magnetic agitation reaction 30 minutes adds 200ul sodium metabisulfite stopped reaction.4400 rev/mins centrifugal 4 minutes, abandon supernatant, gained labelling microsphere is air-dry after cleaning 4 times with normal saline, the Co60 illumination-based disinfection is subsequent use.

3, packaging method:

Principle: A type gelatin is electronegative when pH=7, and the Type B gelatin is positively charged at pH=7; Sodium alginate is positively charged, and chitosan is dissolved in acid, and is electronegative; Therefore, because electric charge is different, two kinds of materials can combine.

The sodium alginate soln of preparation 0.5% is with the chitosan solution of 3% acetum preparation 0.5%.The A type gelatin nucleic microsphere for preparing is dropped in the sodium alginate soln; Electric charge attracts each other and makes sodium alginate be deposited in microsphere surface; With dropping into chitosan solution again after the microsphere separation, in like manner, chitosan is deposited on the microsphere that is surrounded by sodium alginate; For several times, then several layers of involucrum have been added so repeatedly for microsphere.If the Type B gelatine microsphere of preparation then drops into chitosan solution with it earlier, drop into sodium alginate soln again.The microsphere of accomplishing double labelling is moved to a 50ml beaker, after microspheres drying, in beaker, add sodium alginate soln or chitosan solution, through parcel, drying, parcel is dry more again ... So wrap up four layers of after scouring.

The involucrum nucleic microsphere of preparation is as shown in Figure 1.If want the slower of microsphere degraded, can increase the parcel number of plies and realize; Otherwise, as the same.

Two, main component and toxic and side effects:

Gelatine microsphere: being made up of animal collagen, is that prolonged application is in clinical biomaterial.Mainly engulf processing by the intravital protease hydrolysis absorption of people or by phagocyte.No obvious toxic-side effects.

¹³¹ I: have two kinds of lonizing radiation of β and γ.With the β ray is master's (accounting for more than 99%), and gamma-rays is lower than 1%.

¹²⁵ I: have low-yield γ and X ray, long half time, basic non-environmental-pollution.

Three, the dosage form of medicine and specification:

Dosage form: pacify bottled dried powder preparation (Fig. 2), soak the back with normal saline before using and supply injection

The dry microspheres nt wt net weight; 1000mg (milligram)

Radioactivity: 25.64-26.02mCi (millicurie) ( ¹³¹I with ¹²⁵The radioactivity ratio of I is at 20-30%: between the 80-70%)

Four, principal indication, usage, consumption:

A) be mainly used in the hepatocarcinoma of each phase clinically, cancer of pancreas, breast carcinoma, thyroid carcinoma, renal carcinoma, gynecological and urinary system malignant tumor etc.

B) adopt the intervention method through arterial thrombosis (like hepatocarcinoma and renal carcinoma), or through CT or B ultrasonic guiding injected method tumor is implanted into or abdominal in direct-view injected method tumor be implanted into (like cancer of pancreas, breast carcinoma etc.).To pacify bottle before the use earlier and press routine disinfection, and, soak and made the abundant imbibition of microsphere in 10 minutes with normal saline 10ml injection ampere.Adopt perfusion of ordinary syringe tremulous pulse fixed point or intratumor injection then.

C) dosage of implanting is reference with the radioactivity.With 1 millicurie/1cm ³(tumor tissue) is effective dose.According to tumor size decision radioactivity accumulated dose.Big tumor can adopt the mode of interval procedure to carry out.

Embodiment 2 double labelling microspheres of the present invention can be measured at the peak

The microsphere labelling of the no involucrum of embodiment 1 step 2 preparation is carried out ability peak measure, concrete outcome is following, and wherein the labelling microsphere cleans 4 times with normal saline, and is right after each the cleaning ¹³¹I with ¹²⁵I activity is measured, and concrete assay method and result are following:

Radiological dose is measured:

When using the U.S. CAPINTEC.INC company medical activity meter of CRC-15R to measure the mixed mark thing, select ¹³¹The I channel measurement ¹³¹During the radioactivity of I ¹²⁵I can produce interference, and is same, selects ¹²⁵The I channel measurement ¹²⁵During the radioactivity of I ¹³¹I also can produce interference, therefore, has studied the medical activity meter measured value of the CRC-15R of U.S. CAPINTEC.INC company with actual ¹³¹I with ¹²⁵Relation between the I activity, and measured value is reduced to their actual activity.

Method:

1, employing is put for two groups and is exempted from pipe, and the A group adds ¹³¹I, the B group adds ¹²⁵I.Record A group, B organize radioactive activity.

2, two groups are put exempt from pipe and put into the medical activity meter of CRC-15R simultaneously, select respectively ¹³¹I with ¹²⁵I channel measurement and record.

3, find out relation between measured value and the actual radiation property activity.

4, this group measured value is reduced to actual radiation property activity.

Table 1 shows that the medical activity meter of use uses respectively in labelling and the washing process ¹³¹I with ¹²⁵The result of I channel measurement." just add " iodine 131 that refers to add in the reaction system, the original doses of iodine 125; After " after mixing " refers to that two kinds of radioiodines mix, before the reaction beginning, use the activity of activity meter measurement iodine 131 and iodine 125." mark back " uses activity meter to measure the activity of iodine 131 and iodine 125 after referring to react completion." one washes ", " two wash ", " three wash ", " four wash " refer to wash respectively uses activity meter to measure the activity of iodine 131 and iodine 125 after 1,2,3,4 time.

Table 1: the double labelling gelatine microsphere is measured

But the measured value that is shown in the table 1 is not that microsphere is entrained ¹³¹I with ¹²⁵The actual radiation intensity of I.Because mixing to produce mutually, disturb two kinds of nucleic.Therefore need find out the relation between measured value and the actual value.Further carry out following test.

1. actual sample value and the measured value result of adding:

1.1 use ¹³¹The I channel measurement ¹²⁵I radioactivity: the actual activity of iodine 125 samples is measured record, put it into activity meter again and select iodine 131 nucleic passage to measure (table 2).

Table 2: use ¹³¹The I channel measurement ¹²⁵The I radioactivity

It is linear to do the scatterplot discovery, fitting result such as Fig. 3:

1.2 use ¹²⁵The I channel measurement ¹³¹I radioactivity: in like manner the actual activity of iodine 131 sample is measured record, put it into activity meter again and select iodine 125 nucleic passages to measure (table 3).

Table 3: use ¹²⁵The I channel measurement ¹³¹The I radioactivity

It is linear to do the scatterplot discovery, fitting result such as Fig. 4:

1.3 because ¹³¹I with ¹²⁵The I chemical property is identical, so according to adding at 1: 2 behind the primary quantity labelling ¹³¹I with ¹²⁵The ratio of I still is 1: 2.Will ¹³¹I with ¹²⁵I according to 1: 2 ratio be respectively charged into A, B puts and exempts from pipe, uses medical activity meter after the mixing respectively ¹³¹The I passage with ¹²⁵I channel measurement (table 4).

Table 4: use activity meter to measure the activity of iodine 131 and iodine 125 by 1: 2 mixing back iodine 131 and iodine 125

Can find to establish actual adding ¹³¹I is m, the actual adding ¹²⁵I is n, mixes the back and uses ¹³¹I channel measurement value is M, uses ¹²⁵I channel measurement value is N, then has following relation:

M＝m+1.7142n

N＝n+0.5828m

m∶n＝1∶2

With above-mentioned relation substitution table 1, draw double labelling ¹³¹I with ¹²⁵The actual value of I is (table 5) as follows:

Table 5: double labelling ¹³¹I with ¹²⁵The actual value of I

Mark rate is 33.94%

With the measured value that uses activity meter to measure iodine 131 and iodine 125 behind the double labelling nucleic microsphere involucrum is (table 6):

Table 6: the measured value of iodine 131 and iodine 125 behind the involucrum

Bag back I131 passage	1.88	1.9	1.88	1.87	1.87	1.9
							Bag back I125 passage	1.1	1.11	1.085	1.085	1.09	1.127

Convert actual value into, be about to above-mentioned formula and bring table 6 into, be by (table 7):

Table 7: the actual activity of iodine 131 and iodine 125 behind the involucrum

Bag back I131 passage	0.424533	0.429049	0.424533	0.422274	0.422274	0.429049
							Bag back I125 passage	0.851789	0.859532	0.840173	0.840173	0.844045	0.872696

Calculating the package rate thus is: 61.37%

Can know by table, contain in every 50mg gelatine microsphere ¹³¹The radioactivity of I is 0.422-0.429mCi, ¹²⁵The radioactivity of I is 0.83855-0.872mCi.

Show through pharmacodynamics test, in the described 50mg gelatine microsphere ¹³¹The radioactivity of I is 0.4253 ± 0.003mCi, ¹²⁵The radioactivity of I is the scope of 0.8514 ± 0.013mCi, has best drug effect and spike effect simultaneously.

Below prove beneficial effect of the present invention through concrete effect experiment.

Test Example 1 medicine effect experiment of the present invention

1. distribute in the animal body of iodine isotope double labelling protein microsphere

Every laboratory animal rabbit is all accepted abdominal.With the double labelling gelatine microsphere of the no involucrum of embodiment 1 preparation ( ¹³¹I is 1.744 ± 0.171mCi, ¹²⁵I is 2.477 ± 0.243mCi) implantation rabbit livers.

1.1 rabbit anteserum radioactivity monitoring

4 rabbit ear edge of postoperative picked at random every day venous blood collection 1ml accurately gets supernatant 100 μ l after centrifugal for 4400 rev/mins, measures the γ counting.Result such as Fig. 5:

According to the numerical value of serum time-γ counting each point, find that it meets pharmacokinetic two-compartment model, adopt 1stOpt software to carry out curve fitting, try to achieve its pharmacokinetics lonizing radiation blood level formula and be:

C＝6281.0321e ^-0.5382t+548.1884e ^-0.0411t

t _{(1/2) α}=1.2876 (my god)

t _{(1/2) β}=16.86 (my god)

K ₂₁＝0.0810

K ₁₀＝0.2731

K ₁₂＝0.2252

1.2 rabbit urine radioactivity monitoring result such as Fig. 6, radiocounting promptly obviously descended during postoperative was urinated in 7 days:

1.3SPECT total body opacification

Postoperative 4 hours and the 1st, 4,8,16,24,32,48 day each group adopt Philip Skylight SPECT total body opacification by West China Nuclear Medicine Department of hospital, ability peak 364KeV, window width 15%; 1 times of amplification; Matrix 64 * 64, preceding bit scan, acquisition time 2 minutes (Fig. 7: ¹³¹I SPECT video picture).The ROI program is used in each time imaging, delineates liver, measures its radiocounting, and is abscissa with time, and ordinate value is pressed following calculating: (each time point liver counting-background count)/(liver counting-background count in first day), draw ¹³¹Time-radioactivity of I (time-activity curve, T-A) curve.Measure each time point counting of thyroid and draw T-A curve (Fig. 8) with identical ROI.Postoperative 85,96,105 days, promptly ¹³¹I through 10 half-life (80 days) after, at this moment ¹³¹I can ignore, and adopts Philip Skylight SPECT video picture, ability peak 35KeV, all the other parameter constants.Can find this moment ¹²⁵I still concentrate in the liver injection site (Fig. 9: ¹²⁵I SPECT video picture).

SPECT video picture prompting, postoperative all can be known in 24 days and see that microsphere injection Hepar Leporis seu Oryctolagi position has that radioactivity is dense gathers.Postoperative was seen the thyroid faint development in zone in the 8th day and the 16th day.

Time-activity curve shows, As time goes on the radioactivity at liver position descends gradually, during to 24 days near background.The liver area radiocounting descended very fast at 1-8 days, descended slow relatively in 16-32 days.And the radioactivity in thyroid zone is very low from the beginning.Carry out curve fitting according to each time point data of ROI, the regression equation of asking is following:

Y=31875.51*0.832^x obtains the nucleic microsphere in liver organization ¹³¹The actual half-life of I is 4.449 days.

2. iodine isotope double labelling protein microsphere is at VX ₂Application in the Hepar Leporis seu Oryctolagi carcinoma transplanted model

With VX ₂The animal via femoral arteriography of Hepar Leporis seu Oryctolagi carcinoma transplanted model injects iodine isotope double labelling protein microsphere to Hepatic artery, and concrete grammar is:

The rabbit preserved skin is fixed in after the anesthesia on the intervene operation platform, dissects left side or right side inguinal region, isolates femoral artery.Insert the F-18 conduit, until the Hepar Leporis seu Oryctolagi tremulous pulse, inject the suspension of an amount of nucleic microsphere and 25% glucose, i.e. TAE (transcatheter arterial embolization is through the conduit arterial thrombosis) art (Figure 10 A).The capable immediately radiography of postoperative is found Hepatic artery tiny branch thromboembolism (Figure 10 B).Row SPECT-CT inspection after accomplishing, Figure 11 shows the VX in the Hepar Leporis seu Oryctolagi ₂Transplanted tumor.

Experiment can be colonizated in implant site after showing that iodine isotope double labelling protein microsphere is implanted in the animal body; At trans-hepatic artery interventional therapy VX ₂In the model of Hepar Leporis seu Oryctolagi carcinoma transplanted, can be good at being gathered in tumor locus (Figure 12).Isotope iodide does not all stop in its hetero-organization in two tests.Think that thus the treatment that iodine isotope double labelling protein microsphere is used for entity tumor is safely and effectively.

The effect experiment of Test Example 2 direct liver injection microspheres of the present invention

With the nucleic microsphere of the no involucrum of embodiment 1 preparation with have 1 day Hepar Leporis seu Oryctolagi injection site of the direct liver injection microsphere of involucrum nucleic microsphere postoperative to see necrosis, shown in figure 13.Therefore, with microsphere treatment tumor of the present invention, can cause tumor tissue necrosis soon.

Report at present ¹³¹The gelatine microsphere intratumor injection of I and ametycin is to the therapeutic effect of mouse experiment property hepatocarcinoma (Chen Xiaoli, etc., " cancer ", 1992 11 5 phases of volume), wherein ¹³¹The consumption of I is according to known reduction formula, and its consumption is 81.25 μ Ci/cm ³, in the tumor after the injection 7 days, ¹³¹I-MMC-GM is 58.7% to the suppression ratio of tumor growth; Because this dosage is to mice, if this dosage is used for rabbit, if press the conversion coefficient of medicine dose,equivalent between each animal and the people; The coefficient of mice and rabbit is 2.7, and then the consumption of rabbit should be 81.25*2.7=219.375 μ Ci; In the pharmaceutical composition of the present invention ¹³¹The amount that I is used for rabbit is about 82 μ Ci/cm ³, with ¹²⁵The I compatibility uses, ¹³¹The consumption of I obviously reduces, and has brought into play synergistic function, with prior art report ¹³¹The I-MMC-GM gelatine microsphere is compared, and onset is obviously accelerated.

That above-mentioned evidence, medicine microspheres of the present invention can reach simultaneously is directed, continue the tumor internal radiotherapy and as the purpose of tracer.

The test of pesticide effectiveness of Test Example 3 involucrum isotope labeling protein microspheres of the present invention

1 laboratory animal

This group planning of experiment has passed through the approval of animal Ethics Committee of Sichuan University.Adopt 30 of healthy new zealand white rabbits, body weight 1.8-2.5Kg is divided into matched group (A group: no involucrum ¹³¹I double labelling gelatine microsphere group) and experimental group (B group: involucrum is arranged ¹³¹I double labelling gelatine microsphere group), A, B group respectively are divided into 6 groups (A1-A6 and B1-B6).

Implant between the hepatic tissue of 2 microspheres

Every animal is all accepted abdominal.After speed is slept new II 0.1-0.2ml/Kg intramuscular injection anesthesia, be fixed on the rabbit operating-table, epigastrium is shaved hair, sterilization, drape.Upper abdomen median line otch is about after 5cm opens abdomen, and left index finger stretches into the abdominal cavity, lifts Hepar Leporis seu Oryctolagi left middle lobe or right middle lobe, and with the fixing lobe of the liver of left hand thumb.With the 1ml empty needle extract ready double labelling gelatine microsphere ( ¹³¹I be 1.744 ± 0.171mCi) with the mixed liquor of 1ml25% Glucose Liquid; Puncture into the other hepatic tissue of gallbladder with No. 4 syringe needles, slow injectable drug was not injected 2-3 some when pumpback had blood back to liver; Each some injection 0.3～0.5ml pulled out behind the pin compressing injection point about 1 minute.Observing the Hepar Leporis seu Oryctolagi injection site does not have pass, hemorrhage back abdomen, finishes operation.Postoperative gives gentamycin 40,000 units intramuscular injection 1 time immediately.

3 rabbit anteserum radioactivity monitorings

Postoperative picked at random every day A group and each 4 rabbit ear edge venous blood collection 1ml of B group are accurately got supernatant 100 μ l after centrifugal, are measured γ and count for 4400 rev/mins.

4 rabbit urine radioactivity monitorings

Postoperative A every day, B respectively choose 4 rabbits at random for two groups and raise in rabbit metabolism cage; Collect urine preceding 32 day every day; Whenever collected 1 urine at a distance from 3 days, collected urine later on weekly 1 time, and collected 5ml at every turn in 64 days in 32 days to 64 days; Accurately get supernatant 100 μ l after centrifugal for 4400 rev/mins, measure the γ counting.

The 5ECT total body opacification

Postoperative 4 hours and the 1st, 4,8,16,24,32,48 day each group adopt Philip Skylight SPECT total body opacification by West China Nuclear Medicine Department of hospital, ability peak 360KeV, window width 15%; 1 times of amplification; Matrix 64 * 64, preceding bit scan, acquisition time 2 minutes.64 days acquisition times of postoperative 4 minutes.The ROI program is used in each time imaging; Delineate liver; Measure its radiocounting, and be abscissa with time, ordinate value is pressed following calculating: (each time point liver counting-background count)/(liver counting-background count in first day); Drafting time-radioactivity (time-activity curve, T-A) curve.Measure each time point counting of thyroid and draw the T-A curve with identical ROI.

6 liver functions, the inspection of first merit

The dead A in postoperative the 1st, 4,16,24,32,64 natural gift other places, two group the 1st of B, 2,3,4,5,6 treated animals.The animal hearts blood sampling 8ml that puts to death adopted the heart blood collection method to extract 4 animal 8ml blood on the 8th, 48 day, and institute's blood-sample withdrawal makes liver function by experimental medicine section of West China hospital clinical biochemical laboratory and hormone test experience chamber respectively and thyroid function detects.

7 pathological examinations

The liver specimens of putting to death animal was placed 10% formalin solution fixing 24～48 hours, FFPE, section, the pathology inspection is done in HE dyeing.

8 statistical procedures

Adopt SPSS 13.0 softwares, descriptive statistical analysis, measurement data meets two groups of data of parametric test condition and relatively adopts the t check, and enumeration data adopts X ²Check.

9 results

9.1 surgical outcome

All animal surgeries are success all.No animal unexpected death in the operation process.

9.2 the nucleic microsphere is to the destruction observed result of normal liver tissue

Postoperative was put to death each 1 of implantation belt involucrum and no involucrum nucleic microsphere experimental rabbit in 1 day, got its liver, all saw the hepatic tissue generation coagulation necrosis of the about 1cm in microsphere injection site near, and all the other do not inject nucleic microsphere place does not have necrosis region, Figure 13 B.

9.3ECT video picture result

Two treated animals show all that after surgery nucleic concentrates in the liver injection position.Two treated animals find all in the time of 4 days after surgery that the thyroid place has a small amount of nucleic to assemble.The involucrum group did not have video picture (Figure 14 J, L) in the injection site after 32 days; Involucrum group postoperative 32 days, even 48 days, visible nucleic is dense to gather in the injection site (Figure 14 I, K).

9.4T-A curve

Open counting average such as table 8 that the ROI program records two kinds of microspheres, table 9, Figure 15.

Table 8: each time point Hepar Leporis seu Oryctolagi ROI counting average after the no involucrum nucleic microsphere implantation

Natural law	1	2	3	4	5
						CPM	28127	23831	18526	16826	14764
Natural law	8	16	24	32
						CPM	6437	1373	226.5	149.5

Table 9: a time point Hepar Leporis seu Oryctolagi ROI counted average after involucrum nucleic microsphere implantation was arranged

Natural law	1	2	3	4	5	8
							Mean	42674	37522.25	34702.75	27923.5	26255.25	22220.5
Natural law	16	24	32	48	64
							Mean	9952.25	4541.25	1092.25	341.3333	145.5

Carry out curve fitting according to each time point data of ROI, it is following to obtain after the no involucrum microsphere Hepar Leporis seu Oryctolagi implantation regression equation of radioactive decay:

Y=31875.51*0.832^x r ²Value (coefficient of determination)=0.98, F value=349.0747, df=7 obtains no involucrum nucleic microsphere in liver organization ¹³¹The actual half-life of I is 4.449 days.

It is following to obtain after the involucrum microsphere Hepar Leporis seu Oryctolagi implantation regression equation of radioactive decay:

Y=42692.77*0.909^x r ²Value (coefficient of determination)=0.984, F value=558.95, df=9 obtains involucrum nucleic microsphere in liver organization ¹³¹The actual half-life of I is 7.27 days.

It is long that its actual half-life of nucleic microsphere that this shows involucrum does not have the nucleic microsphere of involucrum.

Above-mentioned " the actual half-life " is also referred to as effective half-life (effective half-life); It is meant that the radionuclide that in tissue, exists makes radioactivity reduce aspect spontaneous decay and bio-metabolic process two, this two process causes jointly, the 1/2 o'clock required time when making radioactive intensity reduce to beginning.

9.5 two kinds of nucleic microspheres are implanted back serum r counting like table 10, Figure 16

Table 10: serum r counting after two kinds of microsphere implantations

9.6 two kinds of microspheres are implanted back urine γ count results and are seen table 11, Figure 17.

Urine γ counting after two kinds of microsphere Hepar Leporis seu Oryctolagi of table 11 implantation.

9.7 pathological examination results

Two treated animal pathologic examination after operation find that microsphere rests near the injection site.Postoperative treated animal hepatic pathology section in the 1st day two has a small amount of inflammatory cell to hold (Figure 18 A, B) around seeing microsphere.Postoperative treated animal hepatic pathology section in the 4th day two is seen microsphere by more inflammatory cell parcels, and normal liver cell begins necrosis (Figure 18 C, D).The section of the 16th day two treated animal hepatic pathology of postoperative sees that the cavity that stays after the degraded appears in indivedual microspheres, but whole microsphere form globulate still, the injection site periphery has formed fine and close fibrous tissue and has held (Figure 18 E, F).The 32nd day pathological section of postoperative is still seen a large amount of microspheres, and microsphere is further degraded, and the injection site has formed the fibrosis involucrum.With interior big the apoptosis of hepatocyte of involucrum, only leave over hepatic cords (Figure 18 G, H) around the involucrum.No involucrum group the 48th day after surgery, it is residual still to observe microsphere, but volume less (Figure 18 I); It is more clear and legible that the involucrum group is arranged the 48th day after surgery microsphere does not have the involucrum group, though degraded is in various degree arranged, do not have the sign that disappears, and it is big that its volume does not have the involucrum group, and form does not have involucrum group rule (Figure 18 J).It is slow to find out that from pathologic finding the microsphere that involucrum is arranged does not have the microsphere degraded of involucrum.

To sum up, contrast has involucrum nucleic microsphere and no involucrum nucleic microsphere, and two kinds of microspheres all can play the effect of good destruction implant site tissue.There is the nucleic microsphere actual half-life in animal body of involucrum longer, more near half-life of nucleic itself.Contrast two kinds of microsphere blood, urine γ counting, finding to have the nucleic microsphere of involucrum to discharge nucleic, not have the nucleic microsphere of involucrum steady, alleviated " outburst discharges " effect.Therefore, involucrum can be protected the nucleic microsphere, reduces it and " takes off mark ", and prolong microsphere degradation time in vivo.

Claims

1. the isotope labeling protein microsphere of an involucrum, it is characterized in that: it is the microsphere that is prepared from the following weight proportion raw material:

Na ¹³¹I 1-5 part, Na ¹²⁵I 1-5 part, chitosan 300-1000 part, sodium alginate 100-500 part, gelatin 300-800 part, wherein Na ¹³¹I, Na ¹²⁵The specific radioactivity concentration of I is: 37 GBq/mL;

It is to be prepared from following steps:

A. the preparation of gelatine microsphere:

B, microsphere labelling

0.2mmol/L sodium dihydrogen phosphate and 0.2mmol/ L sodium hydrogen phosphate are mixed with the buffer solution PBS of pH=7.0, preparation 20mg/mL chloramine-T, 15mg/mL sodium metabisulfite; Get microsphere to reaction tube, add the PBS swelling; With Na ¹³¹I and Na ¹²⁵The weight proportion that I presses 5 parts of 1-5 Fen ︰ adds, and adds chloramine-T again, and the reaction of room temperature magnetic agitation adds the sodium metabisulfite stopped reaction; 4400 rev/mins centrifugal 4 minutes, abandon supernatant, gained labelling microsphere with normal saline clean, air-dry, it is subsequent use to sterilize;

C, parcel:

Preparation 0.5% sodium alginate soln is mixed with 0.5% chitosan solution with 3% acetum, and the labelling microsphere of b step preparation is dropped in the sodium alginate soln, wraps up the labelling microsphere, drops into chitosan solution after separating microsphere again; Or the labelling microsphere of b step preparation put in the chitosan solution, parcel labelling microsphere drops into sodium alginate soln after separating microsphere again; 4-7 time repeatedly, promptly get the isotope labeling protein microsphere of involucrum.

2. the isotope labeling protein microsphere of involucrum according to claim 1, it is characterized in that: it is the microsphere that is prepared from the following weight proportion raw material:

3, the isotope labeling protein microsphere of involucrum according to claim 1 and 2 is characterized in that: contain in every 50mg gelatine microsphere ¹³¹The radioactivity of I is 0.422-0.429mCi, ¹²⁵The radioactivity of I is 0.83855-0.872mCi.

4, the isotope labeling protein microsphere of involucrum according to claim 3 is characterized in that: contain in every 50mg gelatine microsphere ¹³¹The radioactivity of I is 0.4253 ± 0.003mCi, ¹²⁵The radioactivity of I is 0.8514 ± 0.01285mCi.

5, the isotope labeling protein microsphere of involucrum according to claim 1 and 2 is characterized in that: the particle diameter of described microsphere is 100-200 μ m.

6, a kind of method for preparing the isotope labeling protein microsphere of any described involucrum of claim 1-5, it comprises the steps:

A. the preparation of gelatine microsphere:

B, microsphere labelling

C, parcel:

7, the purposes of the isotope labeling protein microsphere of any described involucrum of claim 1-5 in the medicine of preparation treatment hepatocarcinoma, renal carcinoma, breast carcinoma, thyroid carcinoma, cancer of pancreas, gastrointestinal solid tumor or orthopaedics tumor.

8, the purposes of the isotope labeling protein microsphere of any described involucrum of claim 1-5 in the agent of preparation neoplasm tracing.