Radioactive labeled long-acting peptide targeted pharmaceutical and production method thereof

Abstract

A radioactive labeled long-acting peptide-targeting pharmaceutical and production method, in which the peptide targeted pharmaceutical is firstly dissolved in a solution, followed by labeling the radioactive at a high temperature, and the dosage of the pharmaceutical with radioactive labeling is expected to be reduced and labeling efficiency is improved, and no further purification by filtration is required, which shortens the preparation process and reduces personnel exposure in the working environment. The radioactive labeled long-acting peptide-targeting pharmaceutical can increase the specific binding capacity of tumors and reduce the non-specific accumulation in normal tissues. It can be applied to the field of tumor and nuclear medicine for diagnosis and treatment of tumors and/or tumor metastases with efficacy and precision treatment.

Claims

1. A radioactive labeled long-acting peptide targeted pharmaceutical consisting of radiolabeled DOTA-EB-cRGDfK peptide derivative, including a chelating agent, an Evans blue (EB), a cyclic peptide cRGDfK derivative; a chemical structure of DOTA-EB-cRGDfK peptide derivative is shown below: ##STR00001##

2. The radioactive labeled long-acting targeted peptide pharmaceutical according to claim 1, wherein the chelating agent comprises a metal chelate compound for labeling with radioisotope.

3. The radioactive labeled long-acting targeted peptide pharmaceutical according to claim 1, wherein the radioisotope is selected from one of radioactive isotopes .sup.68Ga, .sup.111In, and .sup.177Lu.

4. A kit of radioactive labeled long-acting targeted peptide pharmaceutical, comprising a cryostat bottle containing DOTA-EB-cRGDfK, a solution bottle, a filtering device, an adsorption device, is provided for containing a radioactive labeled long-acting targeted peptide pharmaceutical.

5. The kit of radioactive labeled long-acting targeted peptide pharmaceutical according to claim 4, wherein the solution bottle is provided with an acidic solution of pH 3.

6. The kit of radioactive labeled long-acting targeted peptide pharmaceutical according to claim 5, wherein the acidic solution in the solution bottle is one selected from the group consisting of hydrochloric acid, acetic acid and citric acid.

7. The kit of radioactive long-acting targeted peptide pharmaceutical according to claim 4, wherein the filter device is a 0.22 m PVDF filter.

8. The kit of radioactive long-acting targeted peptide pharmaceutical according to claim 4, wherein the DOTA-EB-cRGDfK contained cryostat bottle having main components of a single dose after mixing includes: DOTA-EB-cRGDfK peptide 60 to 120 g, dimethyl sulfoxide DMSO 2 to 10 L, and sodium acetate 80 to 100 mg.

9. The kit of radioactive long-acting targeted peptide pharmaceutical according to claim 4, wherein the adsorption device is a two-layer adsorption column, including the upper layer of the adsorption column of an enhanced anion exchange resin and the composition of Dowex 18-200, 100-200 mesh, 8% cross-linking and 0.5 g2 g dosage, and including the lower layer of the adsorption column includes titanium dioxide particles with size 75-300 m and zero dosage and the composition of rutile or anatase filled in a Biorad column (731-1550) column.

10. A method for using the kit of radiation-labeled long-acting targeted peptide pharmaceutical of claim 4, comprising following steps: injecting 1 to 2 mL of pH 3 acidic solution from a solution bottle into a cryostat bottle of DOTA-EB-cRGDfK; using 0.1 N HCl to wet an adsorption column of an adsorption device and placing the adsorption column on the cryostat bottle; injecting a radiation solution into the adsorption column, and the radiation solution flows into the cryostat bottle after purification; heating a chemical reaction bottle in a range from 85 to 95 C. for 10 to 30 minutes for further mixing the radiation solution, and then filtering through a filtering device to obtain an initial product; a final product of the radioactive labeled DOTA-EB-cRGDfK is produced after a Radio-ITLC labeling efficiency test.

11. The method for using a radioactive labeled long-acting targeted peptide pharmaceutical according to claim 10, wherein the radiation solution is .sup.111InCl.sub.3, .sup.68GaCl.sub.3, or .sup.177LuCl.sub.3 with solution activity 3 to 12 mCi and a volume 10 to 200 L.

12. The method for using a radioactive labeled long-acting targeted peptide pharmaceutical according to claim 10, wherein the final product of the radioactive labeled DOTA-EB-cRGDfK is .sup.111In-DOTA-EB-cRGDfK, .sup.68Ga-DOTA-EB-cRGDfK or .sup.177Lu-DOTA-EB-cRGDfK.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a chemical structural diagram of the DOTA-EB-cRGDfK of the present invention.

[0017] FIG. 2 is a graph (I) showing the effect of the DOTA-EB-cRGDfK of the present invention.

[0018] FIG. 3 is a graph (2) showing the effect of the DOTA-EB-cRGDfK of the present invention.

[0019] FIG. 4 is a graph showing the pharmacokinetic profile of 111In-DOTA-EB-cRGDfK of the present invention in U-87 MG tumor animal model.

[0020] FIG. 5 is a statistical diagram showing the distribution of tumor pharmaceutical in organs of U-87 MG of 111In-DOTA-EB-cRGDfK of the present invention.

[0021] FIG. 6 is a graph showing the biodistribution of the tumor pharmaceutical of U-87 MG of the present invention by 111In-DOTA-EB-cRGDfK.

[0022] FIG. 7 is a flow chart showing the method of using the kit of 111In-DOTA-EB-cRGDfK of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The chemical structure of DOTA-EB-cRGDfK of the present invention is shown In FIG. 1, The radioactive labeled DOTA-EB-cRGDfK is prepared by adding 30 g of DOTA-EB-cRGDfK peptide containing 20 g/L in DMSO solution, adding a radiation solution containing .sup.111In or .sup.68Ga or .sup.177Lu), and adding 1M NaOAc solution to make the volume of 300 L, the pH of the solution is 6-7 after mixing evenly. The radiation solution is placed in a 95 C. thermal reactor for 15 minutes and 1 L was taken for ITLC analysis after cooling. The labeling efficiency showed greater than 90%. The radiation solution was taken for Radio-HPLC analysis at 20-30 Ci and the main signal showed a retention time of 7 minute.

[0024] Please refer to FIG. 2, which is a diagram showing the effect of concentration and pH of the radioactive labeled DOTA-EB-cRGDfK of the present invention, and FIG. 3, which shows the effect of radioactive labeled DOTA-EB-cRGDfK of the present invention, and the activity and concentration statistics table indicate the result of the radioactive labeled DOTA-EB-cRGDfK under conditional test as follows:

[0025] 1) The content of DOTA-EB-cRGDfK is 40 to 60 g, the In.sup.111 activity is 6mCi, and the reaction is carried out at 95 C. for 10 to 20 minutes at pH 4.5-6, and the labeling efficiency is greater than 90%.

[0026] 2) The content of DOTA-EB-cRGDfK is 40 to 60 g, the In.sup.111 activity is 6 to 13mCi, and the reaction is carried out at 95 C. for 15 minutes at pH6, and the labeling efficiency is greater than 90%.

[0027] Referring to FIG. 4, it can be seen that the above 111In-DOTA-EB-cRGDfK of the present invention is administered in the U-87 MG tumor animal model for pharmacokinetic test, and the .sup.111In-DOTA-EB-cRGDfK is administered via the tail vein at different times and the blood was taken for pharmacokinetic analysis at different times. As a result, the pharmaceutical half-life (T1/2) was 77.9 hours, the clearance (CL) was 0.426 g/h, and the area under curve (AUC) was 242 h*% ID/g.

[0028] Referring to FIGS. 5 and 6, it can be seen that the above .sup.111In-DOTA-EB-cRGDfK of the present invention is administered in the U-87 MG tumor pharmaceutical for biodistribution test, and the .sup.111In-DOTA-EB-cRGDfK is administered via the tail vein at different times, and the tissue/organ of the sacrificial animal is taken for samples to perform a radiation dosimeter reading and converts it into organism distribution data (% ID/g). The results showed that the .sup.111In-DOTA-EB-cRGDfK had a significant accumulation in U-87 MG tumors up to approximately 25% ID/g and retained for at least 96 hours.

[0029] In the practical application, the above-mentioned radioactive labeled DOTA-EB-cRGDfK of the present invention can be prepared into a DOTA-EB-cRGDfK cryostat bottle, which is convenient for long-term storage and transportation, and can improve the convenience of use; DOTA- The preparation method of EB-cRGDfK cryostat bottle is as follows:

[0030] adding 4-5 L of DOTA-EB-cRGDfK/DMSO solution with concentration 20 g/L to 333 L in volume of 3M sodium acetate solution, adding injection water to obtain a volume of 1000 L with pH 7, and freeze-drying at 25 C. for 96 hours, refilling with nitrogen for about 1 minute, and sealing the cap with a plastic soft plug and aluminum cap.

[0031] In practical application, the DOTA-EB-cRGDfK cryostat bottle can be combined with a solution bottle, a filtering device and an adsorption device to form a kit suitable for production of the radioactive labeled DOTA-EB-cRGDfK of In.sup.111 and Ga.sup.68. The solution bottle has a content of 3 mL of hydrochloric acid or acetic acid of pH 3 for use as a conditioning agent and pH adjustment.

[0032] The components of the DOTA-EB-cRGDfK cryostat bottle include:

[0033] 1) a content of 60-120 g, preferably 100 g of DOTA-EB-cRGDfK peptide;

[0034] 2) dimethyl sulfoxide DMSO having a content of 4-10 L;

[0035] 3) sodium acetate in an amount of 80-100 mg.

[0036] The adsorption device has an adsorption column, the composition of which has two layers, and the upper layer is an enhanced anion exchange resin, and the composition thereof includes Dowex 18-200, 100-200 mesh, 8% cross-linking, the dosage is 0.5 g to 2 g, and the lower layer is titanium dioxide particles with a size 75-300 m and the amount is zero or 0.1 g to 1 g, and its composition includes rutile or anatase filled in a Biorad column (731-1550). The filtration device is a 0.22 m PVDF (polyvinylidene fluoride) filter.

[0037] Referring to FIG. 7, it can be seen that the above-mentioned .sup.111In-DOTA-EB-cRGDfK or .sup.68Ga-DOTA-EB-cRGDfK or .sup.177Lu-DOTA-EB-cRGDfK kit of the present invention has the following steps:

[0038] injecting 1 to 2 mL of pH 3 acidic solution from a solution bottle into a cryostat bottle of DOTA-EB-cRGDfK;

[0039] using 0.1 N HCl to wet an adsorption column of an adsorption device and placing the adsorption column on the cryostat bottle;

[0040] injecting radiation solution of .sup.68GaCl.sub.3 or .sup.111InCl.sub.3 or .sup.177LuCl.sub.3 having 3 to 12mCi radiation activity into the adsorption column, and the radiation solution flows into the cryostat bottle after purification;

[0041] heating a chemical reaction bottle from 85 to 95 C., preferably 95 C., for 10 to 30 minutes for further mixing the radiation solution, and then filtering through a filtering device to obtain an initial product;

[0042] a final product of the radioactive labeled .sup.111In-DOTA-EB-cRGDfK or .sup.68Ga-DOTA-EB-cRGDfK or .sup.177Lu-DOTA-EB-cRGDfK is produced after a Radio-ITLC labeling efficiency test.

[0043] The above DOTA-EB-cRGDfK cryostat was subjected to the In-111 labeling test, and the labeling efficiency and radiochemical purity test were carried out by ITLC and Radio-HPLC, and the 111In-DOTA-EB-cRGDfK labeling efficiency and radiochemical purity were greater than 90%, its cryostat bottle can still reach radiochemical purity and labeling efficiency of greater than 90% after maintaining at 25 C. for 1 month.

[0044] The above-mentioned radiation labeled long-acting targeted peptide pharmaceutical of the present invention has the following advantages:

[0045] 1) Increasing the amount of pharmaceutical specific to the tumor, and reducing the amount of pharmaceutical accumulation in the non-tumor tissue.

[0046] 2) Improving shortcomings of rapid elimination rate of peptide pharmaceuticals.

[0047] 3) The amount of salt composition is small to litigate the burden on the body.

[0048] 4) To be used for diagnosis and treatment.

[0049] 5) The preparation process is short and personnel exposure is reduced.

[0050] 6) Radiation species, such as, .sup.68Ga, .sup.111In, and .sup.177Lu that can be used for diagnosis and treatment.

[0051] In addition, the combination of its pharmaceutical and albumin in the blood can prolong the circulation time of the peptide pharmaceutical in the body, increase the specific binding amount of the tumor, and reduce the non-specific accumulation of normal tissues to meet the development trend of precision medicine.

[0052] In practical applications, the .sup.177Lu/.sup.90Y-DOTA-EB-cRGDfK for therapeutic use can be further developed in the future to achieve the goal of personalized medicine by diagnosis and screening first, and followed by precise treatment. The advantage is to accurately assess the distribution of breast cancer in the body and the size of the tumor, mainly for the surgical operation, ultrasound, tomography and those other methods, which can't be used accurately diagnose the systemic metastatic Integrin v3 tumor and thus increasing the diagnostic accuracy.