Radioactive labeling method for neuropeptide Y derivative compound and medicine for multi-type breast cancer therapy thereof

Abstract

The present invention provides a radioactive labeling method for neuropeptide Y (NPY) compound and a mammalian diagnostic radioactive targeting medicine with NPY peptide being modified at position 27.sup.th to 36.sup.th, and after binding with the chelating agent and labeling the radiation nucleus .sup.66Ga.sup.67Ga.sup.68Ga.sup.177Lu or .sup.111In to provide a radioactive targeting medicine for multi-type breast cancer diagnosis and treatment.

Claims

1. A neuropeptide Y (NPY) derivative compound of Formula 1, ##STR00005## wherein R1 is a metal chelating agent; EB*is Evans Blue or Evans Blue-lysine; R2 is a first linker selected from the group consisting of 1 to 10 amino acids and 4 to 10 polyethylene glycol (PEG); R2′ is a second linker selected from the group consisting of 1 to 10 amino acids and 4 to 10 polyethylene glycol (PEG); B1 is C.sub.4H.sub.11B.sub.10O.sub.3 or S.sub.10B.sub.12H.sub.11; and NPY derivative is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, and SEO ID NO. 3.

2. The neuropeptide Y (NPY) derivative compound of claim 1, wherein the metal chelating agent R1 is DOTA, NOTA, or DTPA.

3. The neuropeptide Y (NPY) derivative compound of claim 1, wherein the boron-containing molecule B1 at NH2 side is bonded with an amino acid K (Lys).

4. The neuropeptide Y (NPY) derivative compound of claim 1, wherein EB* is Evans Blue-lysine-.

5. The neuropeptide Y (NPY) derivative compound of claim 1, wherein NPY derivative is SEQ ID NO. 1 or SEQ ID NO. 2.

6. The neuropeptide Y (NPY) derivative compound of claim 1, wherein the NPY derivative is SEQ ID NO. 3.

7. The neuropeptide Y (NPY) derivative compound of claim 1, wherein the NPY derivative compound is DOTA-EB*-GSG-K(C.sub.4H.sub.11B.sub.10O.sub.3)-GK(C.sub.4H.sub.11B.sub.10O.sub.3) SEQ ID NO. 3 or DOTA-EB*-GSG-K(S.sub.10B.sub.12H.sub.11)-GK(S.sub.10B.sub.12H.sub.11) SEQ ID NO. 3.

8. A method of for radioactive labeling of the neuropeptide Y (NPY) derivative compound of claim 1, 2, 3, 4, 5, 6 or 7, comprising: dissolving an amount of the NPY derivative compound in solution; mixing said NPY derivative compound solution with a radioactive isotope selected from the group consisting of .sup.111In, .sup.68Ga and .sup.177Lu; wherein said NPY derivative compound is labeled with said radioactive isotope.

9. The method of claim 8, wherein the amount of NPY derivative compound is from 30μ to 100 μg.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a synthesis of the blood albumin affinity molecule.

(2) FIG. 2 is a flow chart of the synthesis of INER-NPY-9 of the present invention.

(3) FIG. 3 is a experimental result showing labeling of neuropeptide Y derivative compound of the present invention.

(4) FIG. 4A is a RP-HPLC analysis spectrum of free .sup.111In-INER-NPY-9.

(5) FIG. 4B is a RP-HPLC analysis spectrum of .sup.111In-INER-NPY-9.

(6) FIG. 5 is a 4T1 tumor animal model angiogram with alphabet “t” indicating tumor.

(7) FIG. 6 is a comparison of tumor absorption rate between medicines of commercial .sup.18F-FBPA and the .sup.111In-INER-NPY-9 of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(8) The present invention aims to develop a multi-type breast cancer diagnosis and treatment medicine NPY, and can be applied to radioactive targeting diagnosis and treatment and boron neutron capture treatment (BNCT) for breast cancer patients. A design of Neuropeptide Y (NPY) compound for the medicine of multi-type breast cancer diagnosis and treatment is shown in Table 1.

(9) The present invention aims to develop a multi-type breast cancer diagnosis and treatment medicine NPY, and can be applied to radioactive targeting diagnosis and treatment and boron neutron capture treatment (BNCT) for breast cancer patients. The NPY derivative compound of the present invention comprising INER-NPY-1 to INER-NPY-10 as shown in Table 1,

(10) The neuropeptide Y (NPY) derivative compound, namely INER-NPY-9, is provided by the present invention for breast cancer radioactive and boron neutron capture therapy, and the flow chart of the synthesis of INER-NPY-9 is shown in FIG. 2. A schematic structure of the NPY derivative compound of the present invention is shown in Formula 1 below for description:

(11) ##STR00001##

(12) in which, R1 is a metal chelating agent for radioactive labeling .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.177Lu, or .sup.111In; EB* is blood albumin affinity molecules for prolonging circulation time of the NPY derivative compound in vivo; each of R2 and R2′ is a linker consisting of 1 to 10 amino acids or 4 to 10 polyethylene glycol (PEG) to enhance hydrophilicity and circulation time of the NPY derivative compound in vivo; B1 is a boron-containing molecule m-carborane-1, 7-dicarboxylic acid (C.sub.4H.sub.11B.sub.10O.sub.3) or maleimide-(S.sub.10B.sub.12H.sub.11) and is bonded at NH.sub.2 side with amino acid including K (Lys), R(Arg), N(Asp), or Q (Glu) for boron neutron capture therapy; and the neuropeptide Y derivative includes amino acids at 27th to 36th positions in the sequence of neuropeptide Y, and the selected sequence is modified to YINLITRPRY (SEQ ID NO. 3), which is modified at 34th amino acid of NPY The NPY derivative can also be selected at the 28th to 36th amino acid in the NPY sequence and the selected sequence is modified to YNLITRPRY (SEQ ID NO. 1), which is modified at 28th and 34th amino acid of NPY, or INLITRPRY (SEQ ID NO. 2), which is modified at 34th amino acid of NPY.

(13) The structure of the NPY derivative compounds having each respective peptide sequence are shown in Table 1.

(14) TABLE-US-00001 TABLE 1 Code Nomenclature NPY Derivative Compound 1 INER- DOTA-GSG-YNLITRPPY (SEQ ID NO. 1) DOTA-Gly-Ser-Gly -Tyr-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr NPY-1 (SEQ ID NO. 1) 2 INER- DOTA-GSG-INLITRPRY (SEQ ID NO. 2) DOTA-Gly-Ser-Gly -Ile-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr NPY-2 (SEQ ID NO. 2) 3 INER- DOTA-PEG.sub.4-YNLIRPRY (SEQ ID NO. 1) DOTA-PEG.sub.4-Tyr-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr NPY-3 (SEQ ID NO. 1) 4 INER- DOTA-PEG.sub.4-INLITRPRY (SEQ ID NO. 2) DOTA-PEG.sub.4-Ile-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr NPY-4 (SEQ ID NO. 2) 5 INER- DOTA-GSG-K-YINLITRPRY (SEQ ID NO. 3) DOTA-Gly-Ser-Gly-Lys-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Pro- NPY-5 Arg-Tyr (SEQ ID NO. 3) 6 INER- DOTA-GSG-K(C.sub.4H.sub.11B.sub.10O.sub.3)-YINLITRPRY DOTA-Gly-Ser-Gly-Lys-(C.sub.4H.sub.11B.sub.10O.sub.3)-Tyr-Ile-Asn-Leu-Ile- NPY-6 (SEQ ID NO. 3) Thr-Arg-Pro-Arg-Tyr (SEQ ID NO. 3) 7 INER- DOTA-GSG-KGK-YINLITRPRY (SEQ ID NO. DOTA-Gly-Ser-Gly-Lys-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Pro- NPY-7 3) Arg-Tyr (SEQ ID NO. 3) 8 INER- DOTA-GSG-K(C.sub.4H.sub.11B.sub.10O.sub.3)-GK(C.sub.4H.sub.11B.sub.10O.sub.3-Y DOTA-Gly-Ser-Gly -Lys(C.sub.4H.sub.11B.sub.10O.sub.3)-Gly-Lys-(C.sub.4H.sub.11B.sub.10O.sub.3)- NPY-8 INLITRPRY (SEQ ID NO. 2) Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr (SEQ ID NO. 2) 9 INER- DOTA-EB*-GSG-K(C.sub.4H.sub.11B.sub.10O.sub.3)-GK(C.sub.4H.sub.11B.sub.10 DOTA-EB*-Gly-Ser-Gly-Lys-(C.sub.4H.sub.11B.sub.10O.sub.3)-Gly-Lys- NP7-9 O.sub.3)-YINLITRPRY (SEQ ID NO. 3) (C.sub.4H.sub.11B.sub.10O.sub.3)-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr (SEQ ID NO. 3) 10 INER- DOTA-EB*-GSG-K(malemide-S.sub.10B.sub.12H.sub.11)-GK DOTA-EB*-Gly-Ser-Glly-Lys-(maleimide-S.sub.10B.sub.12H.sub.11)-Gly- NPY-10 (malemide-S.sub.10B.sub.12H.sub.11)-YINLITRPRY Lys-(maleimide-S.sub.10B.sub.12H.sub.11)-Tyr-Ile-Asn-Leu-Ile-Thr-Arg- (SEQ ID NO. 3) Pro-Arg-Tyr (SEQ ID NO. 3)

(15) In the Table 1, the peptide sequences are shown in Formula 2a, 2b, and 2c below with respective schematic structure.

(16) 1) NPY derivative having peptide in sequence YNLITRPRY (SEQ ID NO. 1): Tyr-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr (SEQ ID NO. 1) is shown in Formula 2a:

(17) ##STR00002##

(18) 2) NPY derivative having peptide in sequence INLITRPRY (SEQ ID NO. 2): Ile-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr (SEQ ID NO. 2) is shown in Formula 2b:

(19) ##STR00003##

(20) 3) NPY derivative having peptide in sequence YINLITRPRY (SEQ ID NO. 3): Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr (SEQ ID NO. 3) is shown in Formula 2c:

(21) ##STR00004##

Embodiment 1: The Synthesis of the Blood Albumin Affinity Molecule

(22) The synthesis of the blood albumin affinity molecule is shown in FIG. 1, comprising steps: dissolving EB-NH.sub.2 and Fmoc-Lys(Boc)-OH in DMF solvent, and HATU and DIPEA are added and reacted at room temperature for 8 hours to form EB-Fmoc-Lys(Boc):

(23) adding EB-Fmoc-Lys (Boc) to Pipedine and DMF, and reacted at room temperature for 3 hours to form EB-Lys (Boc);

(24) dissolving EB-Lys (Boc) in DMF solvent, and DOTA chelating group, HATU, and DIPEA were added, and reacting at room temperature for 8 hours to form DOTA-EB-Fmoc-Lys (Boc); adding TFA and DMF to DOTA-EB-Fmoc-Lys(Boc) to form DOTA-EB-Fmoc-Lys; and adding DOTA-EB-Fmoc-Lys to the Maleimide derivative and reacting at room temperature for 3 hours to form an Evans Blue-lysine derivative.

Embodiment 2: The Synthesis of INER-NPY-9 of the Present Invention

(25) The synthesis of INER-NPY-9 of the present invention is shown in FIG. 2, comprising steps: synthesizing Fmoc-Tyr(tBu)-wang resin and neuropeptide amino acid by solid phase peptide synthesis (SPPS)

(26) removing the protecting group and the resin with 95% TFA/EDT/ddH.sub.2O lysate after the N-terminal NH.sub.2 is vulcanized to sulfide;

(27) adding HATU, DIPEA, and DMF to react at room temperature for 8 hours, and bonding the NH.sub.2 at lysine side with C.sub.4H.sub.11B.sub.10O.sub.3; and

(28) reacting of DOTA-EB-lysine in DMF solvent for 12 hours at room temperature to obtain the INER-NPY-9 peptide.

Embodiment 3: Experimental Result of Labeling NPY Derivative Compound and the Radiochemical Purity Analysis by Radio-HPLC

(29) The experimental result of labeling NPY derivative compound is shown in FIG. 3, comprising steps:

(30) providing 30 μg of one of from INER-NPY-1 to INER-NPY-9 peptide, and dissolving and mixing in 30 μL DMSO;

(31) adding 0.2M NaOAc buffer and a radioactive isotope from one of .sup.111In, .sup.68Ga, .sup.177Lu having activity from 3 to 12 mCi, a total reaction volume 300 μL, and pH of the solution from 5 to 6, to the solution;

(32) reacting the solution in a range from 85 to 100° C., preferably 95° C., for 10 to 30 minutes, preferably 15 minutes;

(33) taking 1 μL of the solution after cooling for efficiency analysis by Radio-TLC (using TLC (1*10 cm) ITLC-SG strip, and the developing solution is 0.1 M and Citric acid: Sodium Citrate=2:8 (V:V); taking the solution 20˜30 μCi and carrying out Radio-HPLC radiochemical purity analysis (waters HPLC system with radiation detector for radiochemical purity analysis, Column: Aglient, ZORBAX SB-C18, 5 μm, 80 Å, 4.6×250 mm, flow rate: 0.8 mL/min, analysis wavelength: 220 nm, mobile phase A: 20% ACN+0.1% TFA, mobile phase B: 80% water+0.1% TFA).

(34) The radiochemical purity analysis results of In-111-INER-NPY-9 by Radio-HPLC are shown in FIG. 3, in which the main signal has a residence time of about 11 minutes.

(35) The RP-HPLC analysis spectrum of free-.sup.111In INER-NPY-9 and .sup.111In-INER-NPY-9 are shown in FIG. 4A and FIG. 4B, respectively.

Embodiment 4: Radioactive Labeling Neuropeptide Y Derivative in 4T1 Tumor Animal Model with Radio-HPLC for Analyzing the Labeling Purity

(36) In animal studies, tumor xenografts were performed in 6-week-old female BALB/c mice by subcutaneous injection of 2×10.sup.6 4T1 cells, and .sup.111In-INER-NPY8 and .sup.111In-INER-NPY9 were injected into the tail vein, and Nano-SPECT/CT imaging was performed at 0.5, 2, 4, 24, and 48 hours, respectively, and compared with .sup.18F-FBPA for PET contrast images. The results are shown in FIG. 5 and FIG. 6, respectively.