CN101182349A - Recombinant octreotide and uses thereof - Google Patents
Recombinant octreotide and uses thereof Download PDFInfo
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- CN101182349A CN101182349A CNA2007101328475A CN200710132847A CN101182349A CN 101182349 A CN101182349 A CN 101182349A CN A2007101328475 A CNA2007101328475 A CN A2007101328475A CN 200710132847 A CN200710132847 A CN 200710132847A CN 101182349 A CN101182349 A CN 101182349A
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Abstract
The invention belongs to the biological engineering field and discloses a recombinant octreotide and an application thereof. Aiming at the problems that the prior octreotide can not realize targeting short-distance irradiation towards tumour cells and can not be applied to the preparation of targeting curative drug, the invention provides a recombinant octreotide, the amino acid sequence of which is shown as SEQ ID NO.2. The recombinant octreotide is in favor of radioactive isotope <131>I marking and can be applied to the preparations of antitumor drug or targeting diagnosis reagent.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of recombinant octreotide and application thereof.
Background technology
Micromolecule polypeptide is strong because of its acceptor specificity, be easy to synthetic and modification, few, the easy penetrate tissue of immune response and blood are removed characteristics such as fast, become the focus of nucleic targeting diagnosis and treatment research in recent years
[1-3](somatostatin is a kind of neuropeptide of round-robin in vivo SST) to Somatostatin, and it can suppress the release of multiple hormone as a kind of brain neurotransmitter
[4-5], as tethelin, Regular Insulin, hyperglycemic-glycogenolytic factor, gastrin etc.Somatostatin and analogue thereof are to use more sophisticated a kind of micromolecule polypeptide up to now, suppress cel l proliferation by resisting signal conduction performance, Sostatin (Octreotide) is the analogue of Somatostatin, be 8 peptides of spontaneous growth chalone molecule being modified synthetic, have the inhibition neuroendocrine function
[4-5]Its physiologically active is than spontaneous growth chalone height, its molecular structure is: D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-L-cysteinyl-L-ammonia alcohol (2 → 7) epidithio thing (SEQ ID NO.1) of reviving, 1,4 is D type amino acid, and 8 is amino alcohol, is difficult for by the rapid hydrolysis of proteolytic enzyme, transformation period has prolonged 30 times than the spontaneous growth chalone, is about 1.7 hours
[6], it mainly suppresses the pathologic supersecretion of tethelin, thyrotropin, gi tract and pancreatic secretion hormone, and noumenal tumour is also had certain restraining effect.Tumor tissues high expression level somatostatin receptor (SSTR) and high affinity is arranged with Octreotide makes micromolecule polypeptide mediation nucleic targeting diagnosis and treatment tumour bring into play potential.
Micromolecule polypeptide mediation radionuclide targeted therapy can utilize its polypeptide to combine with the tumor surface specific receptors on the one hand and play the effect that suppresses tumor growth, can bring into play the irridiation injury effect of nucleic on the other hand.Reported this micromolecule polypeptide (Octreotide) at present and in clinical study, obtained tumor killing effect preferably nonsmall-cell lung cancer, neurospongioma, carcinoma of the pancreas, mammary cancer etc.
[7-10]This seminar adopts
99mTc-Octretide to carcinoma of the pancreas, mammary cancer carried out video picture research and and the somatostatin receptor expression level made correlation research, the result shows
99mTc-Octreotide has the effect of target video picture preferably to lung cancer, carcinoma of the pancreas, mammary cancer, and tumor group is woven with higher expression of receptor level
[11-14]But
99mTc-Octreotide only is used for video picture, can not realize the purpose of target irradiation at short distance tumour cell, therefore can not be used to prepare the medicine of targeted therapy.
Summary of the invention
The purpose of this invention is to provide a kind of recombinant octreotide.
Another object of the present invention provides the application of above-mentioned recombinant octreotide.
The objective of the invention is to realize by following technical measures:
A kind of recombinant octreotide, its aminoacid sequence is shown in SEQ ID NO.2.
The encode nucleotide sequence of described recombinant octreotide.
Radio isotope
131The above-mentioned recombinant octreotide of I mark.
The application of described recombinant octreotide in the preparation antitumor drug.
The nucleotides sequence of described recombinant octreotide of encoding is listed in the application in the preparation antitumor drug.
The application of described recombinant octreotide in preparation targeting diagnosis reagent.
The nucleotides sequence of described recombinant octreotide of encoding is listed in the application in the preparation targeting diagnosis reagent.
Beneficial effect of the present invention:
The present invention is replaced into tyrosine (be abbreviated as Tyr-Octreotide, down with) with the L-phenylalanine in the Sostatin molecule the 3rd, and the recombinant octreotide pharmacological action of gained is similar to natural hormone, and its cancer target is still stronger in conjunction with restraining effect
[6], but be convenient to the iodate of polypeptide.Observe it in the intravital bio distribution of normal mouse, understand drug distribution and dynamic metabolism and measure, carry out nonsmall-cell lung cancer mice with tumor model SPECT video picture.The SPECT radionuclide imaging shows that tumor locus has significantly higher radioactivity to assemble, and than offside normal muscle tissue radiation height, confirms
131I-Tyr-Octreotide has higher affinity to tumor tissues.
Provided by the invention
131Mainly through liver, the metabolism of kidney system, remove fast, and tumor tissues is had avidity preferably by blood in the mouse body for I-Tyr-Octreotide, can be used for preparing the molecular medicine that the good biological targeting with the Somatostatin mediation is diagnosed and treated.
The present invention adopts the Ch-T oxidation style that iodide (NaI) are oxidized to iodine molecule, with the hydroxyl ortho position generation iodination reaction of tyrosine residues, obtains the radioactivity micromolecule polypeptide of efficient stable
131I-Tyr-Octreotide need not to be further purified.Ch-T method mark rate height, good reproducibility, method is easy, rapid, and reagent cheaply is easy to get, and is the more iodine labeling method of using at present.
Description of drawings
Fig. 1 is the colloidal content of the different amount of mark Tyr-Octreotide.
Fig. 2 is the mark rate of different content Tyr-Octreotide.
Fig. 3 is the influence of pH value to mark rate.
Fig. 4 is
131The vitro stability of I-Tyr-Octreotide.
Fig. 5 is Plasma Concentration-time curve.Wherein the data in the square frame are radiopharmaceuticals removing speed in the blood, and display speed is fast, and background interference is little.
Fig. 6 is
131The gathering of tumor locus radioactivity is shown in the I-Tyr-Octreotide video picture.A-F is followed successively by injection 0.2 mCi
1311d, 2d, 4d, 6d, 8d, 2W nonsmall-cell lung cancer mice with tumor model visualization behind the I-octreotide are along with the dense poly-degree of radioactivity of the prolongation tumor locus of time lowers gradually.Arrow is depicted as tumor locus.
Embodiment
The invention will be further elaborated by the following examples.
General explanation:
Chloramine-T, Na
2S
2O
5Provided by Tianjin Ke Miou chemical reagents corporation, bovine serum albumin is provided by Shanghai biochemical research institute,
131I is provided by Beijing Atom High Tech Co., Ltd., the video picture instrument is the two detector SPECT instrument of GE Millennium VGHawkeye, the γ well-type counter is Perkin Elmer Wizard 1470, γ radioimmunity counter GC1200 is available from good branch office in the Keda Innovation Co., Ltd, and the nonsmall-cell lung cancer model is provided by the institute of oncology, Shanghai.
Embodiment 1: preparation reorganization Tyr-Octreotide
Adopting technique known synthesized micromolecule polypeptide according to the sequence SEQ ID NO.2 that provides is the technology that those of ordinary skills know, the Tyr-Octreotide that among the present invention phenylalanine is replaced as tyrosine is prepared by the Huada Gene Research Center, Beijing, the small molecular protein Tyr-Octreotide of preparation has carried out high performance liquid chromatography (HPLC) analysis, through determining of molecular weight, be desirable proteins, purity＞95%.Sequencing analysis is SEQ ID NO.2.
Embodiment 2: preparation
131I-Tyr-Octreotide
131The mark of I-Tyr-Octreotide: get 0.2mol/L NaH
2PO
4With 0.2mol/L Na
2HPO
4Be configured to 0.2mol/L PB; Take by weighing Ch-T, the Na of 0.3g respectively
2S
2O
5Be configured to the reaction solution of 30 μ g/ μ L; Configuration concentration is the Tyr-Octreotide of 1mg/ml.Under 25 ℃, get the PB of 150 μ L, add Octreotide10 μ L, Na successively
131I (0.15mCi) 0.1ml, the Ch-T 4 μ L reaction 3min and the mixing that vibrates rapidly add Na afterwards
2S
2O
55 μ L, flag condition: the 1. pH value from 6.0 to 9.2 of phosphoric acid buffer; 2. the amount of Tyr-Octreotide is respectively 1 μ g, 5 μ g, 10 μ g, 15 μ g, 20 μ g; 3. temperature of reaction is 25 ℃, and 4. the reaction times is 2-3min.Reference numerals minute promptly
131I-Tyr-Octreotide.
The mensuration preparation
131I-Tyr-Octreotide radiochemicsl purity and vitro stability: adopt two individual system: system 1 adopts No. 1 chromatographic paper of Xinhua as stationary phase, with physiological saline as developping agent,
131I-Tyr-Octreotide and colloid Rf value (Rf) are 0,
131IRf=0.7-0.8; System 2 was soaked No. 1 chromatographic paper of nature air dried Xinhua as stationary phase with 2.5% bovine serum albumin, with ethanol: ammoniacal liquor: water (2: 1: 5v/v) launch to measure colloidal content as developping agent, colloid Rf=0, and
131I-Tyr-Octreotide reaches
131The Rf=0.7-0.8 of I.Room temperature is placed the radiochemicsl purity of 30min, 1,2,4,6,8,15,20,24h marked product after measuring mark respectively and finishing.Mark rate=
131I-Tyr-Octreotide radiocounting rate value/(
131I-Tyr-Octreotide radiocounting rate value+
131The radiocounting rate value of I) * 100%.
The result shows, adopts the ply of paper analysis system to measure marked product radiochemicsl purity under the variant reaction conditions, and protrude mark rate can reach 97%, and room temperature is placed 8h and still remained on 81%, and minimum colloidal content is 1.0%.Room temperature is placed the mark rate of colloidal content, different pH value and the protein content of 30min, 1,2,4,6,8,15,20,24h product radiochemicsl purity, the different protein content products of mark and is seen (Fig. 1-4).
Embodiment 3:
131The distribution of I-Tyr-Octreotide in the normal mouse body tissue
30 of Kunming mouses (20-25g) are divided into 6 groups at random, 5 every group.Tail vein injection
131I-Tyr-Octreotide0.15mL (3.7MBq), 30min after administration, 4,8,12,24,48h carries out video picture, pluck eyeball afterwards and get blood and put to death mouse.Get the vital tissue organ, comprise 13 histoorgans such as heart, liver, spleen, lung, kidney, stomach, small intestine (apart from stomach 1cm), large intestine, pancreas, femur, muscle, brain, Tiroidina, weigh and survey radiocounting, calculate %ID/g (every gram tissue radiation counting/be injected into gross activity counting * 100% in the mouse body).With time is X-coordinate, and radiocounting is that ordinate zou is drawn blood clearance curve, with the match of Origin5.0 software, calculates, analyzes it at the intravital pharmacokinetic parameter of normal mouse.
Behind the tail vein injection, different time points is measured its each internal organs in the normal mouse body and is distributed.Kidney is right
131The picked-up of I-Tyr-Octreotide is the highest, and removes comparatively fast, secondly be liver, lung, stomach and intestine, spleen, and brain does not absorb (seeing Table 1) substantially,
131I-Tyr-Octreotide removes rapidly at each internal organs.According to time dependent radiocounting in the blood, with Origin5.0 software matchization, draw Plasma Concentration-time curve, the result shows that Plasma Concentration-time meets the three-compartment model feature, removing phase transformation period (T
1/2) be 141.12min, Plasma Concentration-time curve is seen Fig. 5.
Table 1:
131I-Tyr-Octreotide is the radioactive uptake mark (%ID/g) of each internal organs mutually simultaneously not
(％ID/g＝X±S，N＝5)
Embodiment 4:
131The distribution of I-Tyr-Octreotide in tumor tissues.
With labeled drug
131I-Tyr-Octreotide through tail vein injection in the mice with tumor body, injected dose is 0.2mL (3.7MBq), it is got the ventricumbent position is fixed on the plate, four limbs are fully stretched, avoid limbs overlapping, adopt the low pinhole collimator of differentiating of high energy, 4h carries out IMAQ in the injection back, and acquisition time is 10min.
The radioactive uptake of each organ such as the visible heart of injection back 4h, liver, kidney, spleen distributes identical with normal mouse body radioactivity bio distribution, as seen tumor locus has significantly higher radioactivity to assemble, than offside normal muscle tissue radiation height,
131I-Tyr-Octreotide has higher affinity to tumor tissues, sees Fig. 6.
＜110〉king is from positive Wang Feng Du few magnificent Qu Wei of letter Lee together
＜120〉a kind of recombinant octreotide and application thereof
<160>2
<210>1
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉recombinant human endothelial statin
<400>1
Phe?Cys?Phe?Trp?Lys?Thr?Cys?Thr
1 5
<210>2
<211>8
<212>PRT
＜213〉artificial sequence
<220>
＜223〉recombinant human endothelial statin
<400>2
Phe?Cys?Tyr?Trp?Lys?Thr?Cys?Thr
1 5
Claims (7)
1. recombinant octreotide, its aminoacid sequence is shown in SEQ ID NO.2.
2. the nucleotide sequence of coding claim 1 described recombinant octreotide.
3. radio isotope
131The described recombinant octreotide of the claim 1 of I mark.
4. claim 1 or the 3 described recombinant octreotides application in the preparation antitumor drug.
5. the nucleotides sequence of the described recombinant octreotide of claim 2 is listed in the application in the preparation antitumor drug.
6. claim 1 or the 3 described recombinant octreotides application in preparation targeting diagnosis reagent.
7. the nucleotides sequence of the described recombinant octreotide of claim 2 is listed in the application in the preparation targeting diagnosis reagent.
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| CNA2007101328475A CN101182349A (en) | 2007-10-08 | 2007-10-08 | Recombinant octreotide and uses thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102260353A (en) * | 2010-05-28 | 2011-11-30 | 山东先声麦得津生物制药有限公司 | Targeted interleukin fusion protein and preparation method as well as application thereof |
| CN102260352A (en) * | 2010-05-28 | 2011-11-30 | 山东先声麦得津生物制药有限公司 | Targeted interleukin fusion protein as well as preparation method thereof and application thereof |
| CN108440664A (en) * | 2018-03-27 | 2018-08-24 | 上海欣科医药有限公司 | A kind of SMS 201-995 and its preparation method and application for cancer detection |
-
2007
- 2007-10-08 CN CNA2007101328475A patent/CN101182349A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102260353A (en) * | 2010-05-28 | 2011-11-30 | 山东先声麦得津生物制药有限公司 | Targeted interleukin fusion protein and preparation method as well as application thereof |
| CN102260352A (en) * | 2010-05-28 | 2011-11-30 | 山东先声麦得津生物制药有限公司 | Targeted interleukin fusion protein as well as preparation method thereof and application thereof |
| WO2011147138A1 (en) * | 2010-05-28 | 2011-12-01 | 山东先声麦得津生物制药有限公司 | Targeting fusion protein of interleukin and preparation method and use thereof |
| CN102260352B (en) * | 2010-05-28 | 2013-11-20 | 山东先声麦得津生物制药有限公司 | Targeted interleukin fusion protein as well as preparation method thereof and application thereof |
| CN108440664A (en) * | 2018-03-27 | 2018-08-24 | 上海欣科医药有限公司 | A kind of SMS 201-995 and its preparation method and application for cancer detection |
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Open date: 20080521 |