CN101787077A - Preparation of 131I-thyroid stimulating hormone (TSH) and application thereof - Google Patents

Abstract

The invention relates to the preparation of 131I-thyroid stimulating hormone (TSH) and an application thereof, belonging to the field of radionuclide therapeutic drugs. The compound 131I-thyroid stimulating hormone is a radioiodine marker for thyroid stimulating hormone with pathoclisis in vivo acting on thyrocyte. A preparation method of the 131I-thyroid stimulating hormone comprises the following steps of: carrying out iodine 131 marking on the thyroid stimulating hormone by utilizing a chloramine-T method, a peroxide oxidation method or an iodogen method, and introducing radionuclide iodine 131 for treatment into tyrosine residues in thyroid stimulating hormone molecules. The marking rate of the prepared 131I-TSH is 85.9 percent, the radiochemical purity after purification achieves more than 90 percent, and the prepared 131I-TSH can be stored stably for more than one week at room temperature; the 131I-TSH is mainly metabolized through the liver and excreted through the kidneys; the percentage of ID/g of the 131I-TSH in the thyroid gland is not obviously reduced in four hours; and the 131I-TSH can be concentrated in thyroid gland issues sealed by an iodine solution. Concerning low/undifferentiated thyroid tumours incapable of iodine uptake, the 131I-TSH can increase the radioiodine uptake of thyroid gland cancer cells, and the radioiodine 131 can be chosen for treatment.

Description

131The preparation of I-thyrotropic hormone and application thereof

Technical field

¹³¹The I-thyrotropic hormone ( ¹³¹I-TSH) preparation and the application in the low/poor differentiation/dedifferentiation thyroid cancer of treatment thereof the present invention relates to pharmaceutical field, are specifically related to ¹³¹The medicinal use of I-TSH.

Background technology

Thyroid carcinoma is present modal endocrine system malignant tumour, mainly contains treatment meanss such as excision, radioiodine, thyrotropic hormone inhibition clinically, and wherein the application of radioiodine more and more comes into one's own.But, there are partly low differentiation and poor differentiation/dedifferentiation thyroid cancer cell to lose its peculiar function and characteristic, descend, take the photograph iodine as sodium/iodine symport body (NIS) protein expression and reduce, take the photograph bad isolatism of iodine and diffusivity focus for these, the one, adopt ¹³¹The treatment of the blind method of I, the 2nd, take the tumour differentiating inducer, as all-trans-retinoic acid etc.But comprehensive research both at home and abroad, present method is for the thyroid carcinoma patient of these low/losing points, by ¹³¹The means of I do not reach the purpose that suppresses thyroid carcinoma cell growth, control and treatment thyroid carcinoma, and this has brought very big difficulty for clinical thyroid carcinoma treatment work, causes the waste of social resources and medical resource.

The thyroid function of human body is subjected to this axial line control of hypothalamus-pituitary gland-Tiroidina, hypothalamus is top, secretion throtropin releasing hormone (thyroid-stimulating hormone releasinghormone, TRH), and Tiroidina is the lowest layer, discharges thyroxine.In the middle of Anterior pituitary occupy, on be subjected to hypothalamus excretory TRH regulation and control, the feedback regulation that is subjected to Tiroidina excretory Triiodothyronine is arranged down.And thyrotropic hormone (thyroid-stimulating hormone TSH) is a kind of glycoprotein hormones of Anterior pituitary excretory, and it mainly acts on is exactly to control Tiroidina.It can promote the synthetic of Triiodothyronine, can also promote the Triiodothyronine that has generated to be released into blood.Growth and metabolism to Tiroidina itself also play an important role, if excised hypophysis, Tiroidina is with regard to atrophy, and blood flow reduces, and function reduces, and can not make and discharge Triiodothyronine.If inject thyrotropic hormone, Tiroidina is with regard to the hyperplasia hypertrophy, and blood flow increases, increased functionality, and the synthetic and release of Triiodothyronine increases.

Thyrotropic hormone control Tiroidina be by with the thyroid follicular cells basilar membrane on combining of thyrotropin receptor (TSH-R) realize.Human TSH (TSH-R) comprises functional zone, extracellular and 346 7 transmembrane segments that amino acid is formed that 398 amino acid are formed, and fragment in the born of the same parents of carboxyl terminal.Early stage thyroid tumor differentiation degree combines TSH with TSH-R ability is negative correlation.That is to say that low/undifferentiated thyroid carcinoma cell has and TSH bonded ability.

The radioiodide of thyrotropic hormone ¹³¹The thyrotropin receptor of I-TSH and thyroid cell surface expression (TSH-R) specific combination, thereby dense poly-Thyreoidine tissue, this for radioiodine therapy low/undifferentiated thyroid carcinoma provides new means.

Summary of the invention

The invention provides a kind of ¹³¹The preparation method of I-TSH, purpose make the application of radioiodination thyrotropic hormone in the low/undifferentiated thyroid carcinoma medicine of preparation treatment.The present invention introduces the treatment radioactive nuclide iodine in the thyrotropic hormone protein molecular, utilize the performance of thyrotropin receptor (TSH-R) specific combination on thyrotropic hormone and the thyroid follicular cells basilar membrane, feasible expression has low/undifferentiated thyroid carcinoma cell of TSH-R can dense poly-treatment radioactive nuclide iodine, thus reach nucleic 131 iodine therapies low/purpose of undifferentiated thyroid carcinoma.

Technical scheme of the present invention: the iodine label of thyrotropic hormone ¹³¹The I-thyrotropic hormone abbreviates as ¹³¹I-TSH is to introduce the treatment radioiodine in the tyrosine residues of thyrotropic hormone protein molecular ¹³¹I.

Described ¹³¹The preparation method of I-thyrotropic hormone carries out radioiodination with chloramine-t method, peroxide oxidation method or Iodogen method, comprises the steps:

(1) with thyrotropic hormone in 0.2mol/L pH 7.4PBS system with an amount of Na ¹³¹I and oxygenant be the oscillatory reaction certain hour at room temperature;

Described Na ¹³¹The I consumption is the Na of 5 μ g thyrotropic hormone and 0.5-2.0mCi ¹³¹The I reaction; Described oxygenant is selected a kind of among 20 μ L 1mg/mL chloramine-Ts, 20 μ L3% hydrogen peroxide or the 2 μ g Iodogen for use; The described reaction times is 30-60 second for chloramine-t method, is 10-25 minute for peroxide oxidation method or Iodogen rule;

(2) add excessive reductant to stop above-mentioned oxidizing reaction;

Described reductive agent is selected a kind of in Sodium Metabisulfite or the Sulfothiorine for use;

(3) above-mentioned reaction solution application of sample is extremely contained the PBS balance of 0.5%BSA and saturated PD-10 post through 50mmol/L pH 7.4, collect, get protein peak with 1mL/ pipe substep.

Described ¹³¹The application of I-thyrotropic hormone, the preparation radioiodine therapy low/application in the poor differentiation/dedifferentiation thyroid cancer medicine, ¹³¹I-TSH is in vivo mainly by hepatic metabolism, renal excretion; In the Tiroidina ¹³¹The %ID/g of I-TSH did not have obviously decline in 4 hours; Its " Tiroidina/flesh " ratio reaches more than 30 times, significantly concentrates in the Tiroidina that is closed by the iodine fluid-tight, ¹³¹I-TSH makes thyroid carcinoma cell increase the picked-up of radioiodine, makes the thyroid carcinoma of low/losing points can select radioactivity for use ¹³¹Iodine is treated;

Described %ID/g is that every gram is organized percentage injection dose rate; " Tiroidina/flesh " ratio descends the ratio of the %ID/g of Tiroidina and muscle mutually when being each; The iodine fluid-tight is closed into the 200 μ L 1%LugolShi iodine liquid that instiled in the experiment mice oral cavity in preceding 10 minutes in experiment, with the picked-up of sealing parathyroid tissue to free-iodine.

The present invention also is applicable to the radioiodine therapy that those express low/losing points tumour in the tissue of TSH-R gene equally at cell surface.

Beneficial effect of the present invention:

(1) the present invention prepares ¹³¹I-TSH, its preparation technology is simple, and marker is stable, is convenient to the further application of clinical, scientific research and drug development.Make ¹³¹The I-TSH mark rate is 85.9%, and putting is pure behind the purifying reaches more than 90%, and at room temperature can stablize and deposit more than 1 week.

(2) of the present invention ¹³¹I-TSH can densely gather low/undifferentiated thyroid carcinoma cell that TSH-R is arranged in expression, thereby reaches the purpose of the low/undifferentiated thyroid carcinoma of nucleic iodine 131 treatment.

Embodiment

Embodiment 1: ¹³¹The preparation of I-TSH (chloramine-t method)

5 μ g thyrotropic hormone are dissolved among the 20 μ L 0.2mol/L pH 7.4PBS, add 20 μ L Na ¹³¹I (0.5-2mCi), 20 μ L 1mg/mL chloramine-Ts, add the inclined to one side Sodium Pyrosulfite termination reaction of 100 μ L 2mg/mL at room temperature oscillatory reaction 30-60 second.Reaction solution is splined on abundant balance of 50mmol/L pH 7.4PBS (containing 0.5%BSA) and saturated PD-10 post, collects, get protein peak with 1mL/ pipe substep.

Embodiment 2: ¹³¹The preparation of I-TSH (peroxide oxidation method)

5 μ g thyrotropic hormone are dissolved among the 20 μ L 0.2mol/L pH 7.4PBS, add 20 μ L Na ¹³¹I (0.5-2mCi), 20 μ L, 3% hydrogen peroxide was placed 15 minutes room temperature oscillatory reaction 30-60 second, added the inclined to one side Sodium Pyrosulfite termination reaction of 100 μ L 2mg/mL.Reaction solution is splined on abundant balance of 50mmol/L pH 7.4PBS (containing 0.5%BSA) and saturated PD-10 post, collects, get protein peak with 1mL/ pipe substep.

Embodiment 3: ¹³¹The preparation of I-TSH (Iodogen method)

5 μ g thyrotropic hormone are dissolved among the 20 μ L 0.2mol/L pH 7.4PBS, add to be coated with in the Eppendorf pipe of 2 μ gIodogen, add 20 μ L Na ¹³¹I (0.5-2mCi), placed 20 minutes at room temperature oscillatory reaction 30-60 second, the sucking-off reaction solution is with termination reaction, reaction solution is splined on abundant balance of 50mmol/L pH7.4PBS (containing 0.5%BSA) and saturated PD-10 post, collects, get protein peak with 1mL/ pipe substep.

Embodiment 4: ¹³¹The evaluation of I-TSH and stability analysis

¹³¹After the I-TSH preparation is finished, get protein peak pipe 10 μ L, γ-Counter surveys its per minute radiocounting (cpm) and is grand total, adds the 50mmol/L pH 7.4PBS that 10 μ L contain 1%BSA, the Tricholroacetic Acid precipitation of 200 μ L 10%, mixing, centrifugal 20 minutes of 5000rpm abandons supernatant, surveys the cpm in the precipitation, so that counting is divided by grand total in the precipitation, the putting that calculates marked product is pure.Reach more than 95% as putting is single, and stable maintenance is more than 1 week.

Embodiment 5: ¹³¹I-TSH is in the intravital distribution of KM mouse

Get 40 of KM mouse, got Lu Ge Shi (Lugol) iodine drop in the mouse oral cavity (200 μ L) in preceding 10 minutes, sealing Tiroidina, tail vein injection in experiment ¹³¹I-TSH or Na ¹³¹I 200 μ L/ are (about 0.6MBq/ only) only, respectively at 10,60,240 and 1440min after put to death (n=5), get internal organs such as blood, liver,kidney,spleen, stomach, the heart, lung, brain, bone, flesh, intestines, Tiroidina, weigh, γ-Counter surveys cpm, calculates the %ID/g of each internal organs.The result shows: ¹³¹I-TSH is in vivo mainly by hepatic metabolism, renal excretion; In the Tiroidina ¹³¹The %ID/g of I-TSH did not have obviously decline in 4 hours; In experiment periods, ¹³¹" Tiroidina/flesh " ratio of I-TSH reaches more than 30, and Na ¹³¹" Tiroidina/flesh " ratio of I is then about 10; Equally, 24 hours ¹³¹I-TSH and Na ¹³¹" Tiroidina/blood " ratio of I is respectively 10.9 and 4.7.Disclose ¹³¹I-TSH can be long-time dense poly-in parathyroid tissue.Therefore for direct ingestion of iodine low/do not break up the radionuclide of thyroid tumor ¹³¹The treatment of I, ¹³¹I-TSH will have using value.

Claims (3)

1. the iodine label of thyrotropic hormone ¹³¹The I-thyrotropic hormone abbreviates as ¹³¹I-TSH is characterized in that introducing the treatment radioiodine in the tyrosine residues of thyrotropic hormone protein molecular ¹³¹I.

2. as claimed in claim 1 ¹³¹The preparation method of I-thyrotropic hormone is characterized in that carrying out radioiodination with chloramine-t method, peroxide oxidation method or Iodogen method, comprises the steps:

(1) with thyrotropic hormone in 0.2mol/L pH 7.4PBS system with an amount of Na ¹³¹I and oxygenant be the oscillatory reaction certain hour at room temperature;

Described Na ¹³¹The I consumption is the Na of 5 μ g thyrotropic hormone and 0.5-2.0mCi ¹³¹The I reaction; Described oxygenant is selected a kind of among 20 μ L 1mg/mL chloramine-Ts, 20 μ L3% hydrogen peroxide or the 2 μ g Iodogen for use; The described reaction times is 30-60 second for chloramine-t method, is 10-25 minute for peroxide oxidation method or Iodogen rule;

(2) add excessive reductant to stop above-mentioned oxidizing reaction;

Described reductive agent is selected a kind of in Sodium Metabisulfite or the Sulfothiorine for use;

(3) above-mentioned reaction solution application of sample is extremely contained the PBS balance of 0.5%BSA and saturated PD-10 post through 50mmol/L pH 7.4, collect, get protein peak with 1mL/ pipe substep.

3. as claimed in claim 1 ¹³¹The application of I-thyrotropic hormone, it is characterized in that the preparation radioiodine therapy low/application in the poor differentiation/dedifferentiation thyroid cancer medicine, ¹³¹I-TSH is in vivo mainly by hepatic metabolism, renal excretion; In the Tiroidina ¹³¹The %ID/g of I-TSH did not have obviously decline in 4 hours; Its " Tiroidina/flesh " ratio reaches more than 30 times, significantly concentrates in the Tiroidina that is closed by the iodine fluid-tight, ¹³¹I-TSH makes thyroid carcinoma cell increase the picked-up of radioiodine, makes the thyroid carcinoma of low/losing points can select radioactivity for use ¹³¹Iodine is treated;

Described %ID/g is that every gram is organized percentage injection dose rate; " Tiroidina/flesh " ratio descends the ratio of the %ID/g of Tiroidina and muscle mutually when being each; The iodine fluid-tight is closed into the 200 μ L 1%LugolShi iodine liquid that instiled in the experiment mice oral cavity in preceding 10 minutes in experiment, with the picked-up of sealing parathyroid tissue to free-iodine.