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 provided for breast cancer radioactive and boron neutron capture therapy, herein a structure of the NPY derivative compound is shown in Formula 1 below: ##STR00005## wherein 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; R2 is a linker consisting of an amino acid or a polymer to enhance hydrophilicity and circulation time of the NPY derivative compound in vivo; B1 is a boron-containing molecule (C.sub.4H.sub.11B.sub.10O.sub.3) or maleimide-(S.sup.10B.sub.12H.sub.11) for boron neutron capture therapy; wherein the neuropeptide Y derivative includes amino acids of position 27th to 36th in sequence of neuropeptide

2. The neuropeptide Y (NPY) derivative compound of claim 1, wherein the metal chelating agent is DOTA, NOTA, or DTPA for labeling .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.177Lu or .sup.111In

3. The neuropeptide Y (NPY) derivative compound of claim 1, wherein R2 is a linker consisting of 1 to 10 amino acids or 4 to 10 polyethylene glycol (PEG) enhancing hydrophobicity and circulation time of NPY derivative compound in vivo.

4. The neuropeptide Y (NPY) derivative compound of claim 1, wherein B1 is a boron-containing molecule (C.sub.4H.sub.11B.sub.10O.sub.3)n, wherein the number n is 0 or 1, or a molecule maleimide-(S.sub.10B.sub.12H.sub.11) for use as boron neutron capture therapy.

5. The neuropeptide Y (NPY) derivative compound of claim 1, wherein the boron-containing; molecule m-carborane-1,7-dicarboxylic acid (C.sub.4H.sub.11B.sub.10O.sub.3) or maleimide-(S.sup.10B.sub.12H.sub.11) is bonded with amino acid K (Lys).

6. The neuropeptide Y (NPY) derivative compound of claim 1, wherein the blood albumin affinity molecule EB* includes a metal chelating agent R1 and a linker R2.

7. The neuropeptide Y (NPY) derivative compound of claim 1, wherein the amino acid bonding to the boron-containing molecule B1 at NH.sub.2 side is K(Lys), R(Arg), N(Asp), or Q (Glu).

8. The neuropeptide Y (NPY) derivative compound of claim 1, wherein NPY derivative is selected at 28th to 36th position of amino acid in the NPY sequence, and the selected sequence is modified to YNLITRPRY (SEQ ID NO. 1) or INLITRPRY (SEQ ID NO. 2).

9. The neuropeptide Y (NPY) derivative compound of claim 1, wherein NPY derivative is selected at 27th to 36th position of amino acid in the NPY sequence, and the selected sequence is modified to YINLITRPRY (SEQ ID NO. 3).

10. The neuropeptide Y (NPY) derivative compound of claim 1, wherein the NPY derivative is selected from the group consisting of 1) DOTA-GSG-YNLITRPRY(SEQ ID NO. 1), 2) DOTA-GSG-INLITRPRY(SEQ ID NO. 2), 3) DOTA-PEG4-YNLITRPRY(SEQ ID NO. 1), 4) DOTA-PEG4-INLITRPRY(SEQ ID NO. 2), 5) DOTA-GSG-K-YINLITRPRY(SEQ ID NO. 3), 6) DOTA-(SSG-K(C.sub.4H.sub.11B.sub.10O.sub.3)-YINLITRPRY (SEQ ID NO. 3), 7) DOTA-GSG-KGK(C.sub.4H.sub.11B.sub.10O.sub.3)-YINLTIRPRY (SEQ ID NO. 3), 8) DOTA-GSG-K(C.sub.4H.sub.11B.sub.10O.sub.3)-GK(C.sub.4H.sub.11B.sub.10O.sub.3)-YINLITRPRY (SEQ ID NO. 3), 9) DOTA-EB*-GSG-K(C.sub.4H.sub.11B.sub.10O.sub.3)-GK(C.sub.4H.sub.11B.sub.10O.sub.3)-YINLITRPRY (SEQ ID NO. 3), and 10) DOTH-EB*-GSG-K(maleimide-S.sup.10B.sub.12H.sub.11)-GK(maleimide-S.sup.10B.sub.12H.sub.11)-YLNLITRPRY (SEQ ID NO. 3).

11. A method of radioactive labeling the neuropeptide Y (NPY) derivative compound, comprising steps: taking an amount from one of the NPY derivative group listed in claim 9 and dissolving in DMSO 30 L solution and mixing; adding 0.2 M 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 L, and pH of the solution from 5 to 6, to the solution; reacting the solution in a range from 85 to 100 C. for 10 to 30 minutes; and taking 1 L 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).

12. The method of radioactive labeling the neuropeptide Y (NPY) derivative compound of claim 11, wherein the amount taken from one of the NPY derivative group is in a range from 30 to 100.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a synthesis of the blood albumin affinity molecule.

[0016] FIG. 2 is a flow chart of the synthesis of INER-NPY-9 of the present invention.

[0017] FIG. 3 is a experimental result showing labeling of neuropeptide Y derivative compound of the present invention.

[0018] FIG. 4A is a RP-HPLC analysis spectrum of free .sup.111In-INER-NPY-9.

[0019] FIG. 4B is a RP-HPLC analysis spectrum of .sup.111In-INER-NPY-9.

[0020] FIG. 5 is a 4T1 tumor animal model angiogram with alphabet t indicating tumor.

[0021] 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

[0022] The present invention aims to develop a multi-type breast cancer diagnosis and treatment medicine NP&, 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.

[0023] 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,

[0024] 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:

##STR00001##

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; 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.sup.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.

[0025] The structure of the NPY derivative compounds having each respective peptide sequence are shown in Table 1.

TABLE-US-00001 TABLE1 Code Nomenclature NPYDerivativeCompound 1 INER-NP DOTA-GSG-YNLITRPPY(SEQIDNO.1) Y-1 DOTA-Gly-Ser-Gly-Tyr-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr 2 INER-NP DOTA-GSG-INLITRPRY(SEQIDNO.2) (SEQIDNO.1) Y-2 DOTA-Gly-Ser-Gly-Ile-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr 3 INER-NP DOTA-PEG.sub.4-YNLIRPRY(SEQIDNO.1) (SEQIDNO.2) Y-3 DOTA-PEG.sub.4-Tyr-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr 4 INER-NP DOTA-PEG.sub.4-INLITRPRY(SEQIDNO.2) (SEQIDNO.1) Y-4 DOTA-PEG.sub.4-Ile-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr 5 INER-NP DOTA-GSG-K-YINLITRPRY(SEQIDNO.3) (SEQIDNO.2) Y-5 DOTA-Gly-Ser-Gly-Lys-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Pro- Arg-Tyr(SEQIDNO.3) 6 INER-NP 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- Y-6 (SEQIDNO.3) Thr-Arg-Pro-Arg-Tyr(SEQIDNO.3) 7 INER-NP DOTA-GSG-KGK-YINLITRPRY(SEQIDNO. DOTA-Gly-Ser-Gly-Lys-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Pro- Y-7 3) Arg-Tyr(SEQIDNO.3) 8 INER-NP 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)- Y-8 INLITRPRY(SEQIDNO.2) Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr(SEQIDNO.2) 9 INER-NP 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- 7-9 O.sub.3)-YINLITRPRY(SEQIDNO.3) (C.sub.4H.sub.11B.sub.10O.sub.3)-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Tyr (SEQIDNO.3) 10 INER-NP DOTA-EB*-GSG-K(malemide-S.sub.10B.sub.12H.sub.11)-GK DOTA-EB*-Gly-Ser-Glly-Lys-(maleimide-S.sup.10B.sub.12H.sub.11)-Gly- Y-10 (malemide-S.sup.10B.sub.12H.sub.11)-YINLITRPRY(SEQ Lys-(maleimide-S.sup.10B.sub.12H.sub.11)-Tyr-Ile-Asn-Leu-Ile-Thr-Arg- IDNO.3) Pro-Arg-Tyr(SEQIDNO.3)

[0026] Table 1, the peptide sequences are shown in Formula 2a, 2b, and 2c below with respective schematic structure.

[0027] 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:

##STR00002##

[0028] 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:

##STR00003##

[0029] 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:

##STR00004##

Embodiment 1

The Synthesis of the Blood Albumin Affinity Molecule

[0030] The synthesis of the blood albumin affinity molecule is shown in FIG. 1, comprising steps:

[0031] dissolving EB-NH.sub.2 and Fmoc-Lys(Boc)-OH in DMF solvent, and HAW and DIPEA are added and reacted at room temperature for 8 hours to form EB-Fmoc-Lys(Boc);

[0032] adding EB-Fmoc-Lys (Boc) to Pipedine and DMF, and reacted at room temperature for 3 hours to form EB-Lys (Boc);

[0033] 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);

[0034] adding TFA and DMF to DOTA-EB-Fmoc-Lys(Boc) to form DOTA-EB-Fmoc-Lys; and

[0035] 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

[0036] The synthesis of INFER-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)

[0037] removing the protecting group and the resin with 95% TEA/EDT/ddH.sub.2O lysate after the N-terminal NH.sub.2 is vulcanized to sulfide;

[0038] adding HATU, DIPEA, and DI IF 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

[0039] 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

[0040] The experimental result of labeling NPY derivative compound is shown in FIG. 3, comprising steps:

[0041] providing 30 g of one of from INER-NPY-1 to INER-NPY-9 peptide, and dissolving and mixing in 30 L DMSO;

[0042] adding 0.2 M 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 L, and pH of the solution from 5 to 6, to the solution;

[0043] reacting the solution in a range from 85 to 100 C., preferably 95 C. for 10 to 30 minutes, preferably 15 minutes;

[0044] taking 1 L 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);

[0045] taking the solution 2030 Ci and carrying out Radio-HPLC radiochemical purity analysis (waters HPLC system with radiation detector for radiochemical purity analysis, Column: Aglient, ZORBAX SB-C1.8, 5 m, 80 , 4.6250 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).

[0046] 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.

[0047] 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

[0048] In animal studies, tumor xenografts were performed in 6-week-old female BALB/c mice by subcutaneous injection of 210.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.