阅读说明：本技术 生长抑素类似物及其应用 (Somatostatin analogs and uses thereof ) 是由 成伟华 高菲 董瑞林 宋志浩 张蕴瀚 陈孟毅 邱珊珊 于 2021-09-09 设计创作，主要内容包括：本发明提供一种生长抑素类似物及其应用。所述生长抑素类似物的结构如式I)所示：其结构中保留了对生长抑素受体具有高亲和力的Phe-D-Trp-Lys-Thr基序,通过不同螯合剂和不同核素对探针结构进行修饰,设计出广谱型靶向生长抑素受体的放射性标记生长抑素类分子探针。靶向所有亚型的生长抑素受体,将其作为广谱型的神经内分泌肿瘤的早期诊断探针。以实现对神经内分泌肿瘤的早诊断、早发现、早治疗的精准化医疗。(The invention provides a somatostatin analogue and application thereof. The somatostatin analogue has a structure shown in a formula I):)

1. A somatostatin analogue characterized in that the structure of the somatostatin analogue is represented by formula I):

wherein R is a bifunctional chelating group, and R1 is a radionuclide;

the bifunctional chelating group is-DTPA, -NOTA, -p-SCN-Bn-NOTA, -DOTA or-MPAA-NODA;

r1 is selected from¹⁸F、⁶⁴Cu、⁶⁸At least one of Ga.

2. A process for the preparation of a somatostatin analogue according to claim 1, which comprises the steps of:

A. modifying the polypeptide KE108 by a bifunctional chelating agent to obtain a labeled precursor;

B. labeling the labeled precursor with a radionuclide;

wherein the bifunctional chelating agent is DTPA, NOTA, p-SCN-Bn-NOTA, DOTA or MPAA-NODA;

the radionuclide is selected from¹⁸F、⁶⁴Cu、⁶⁸At least one of Ga.

3. The method according to claim 2, wherein in step B, the-COOH group in the bifunctional chelating agent reacts with-NH in Tyr in the polypeptide KE108 by condensation reaction₂The groups are linked.

4. The method of claim 2, whichCharacterized in that when the bifunctional chelating agent is NODA and the radionuclide is¹⁸When F, the preparation method comprises the following steps: adding AlCl containing pH3-5 to 10-50 μ L of labeled precursor NODA-KE108 with concentration of 0.5mg/mL₃The acetic acid buffer solution of (1), (500) μ L and 3.7-370MBq¹⁸F]F^-Reacting the normal saline solution at 80-120 ℃ for 10-30min, and then separating and purifying by using a Sep-pakC18 column; wherein contains AlCl₃In acetic acid buffer solution of (2), AlCl₃The concentration of (A) is 1-30mM, and the concentration of the acetic acid buffer solution is 0.1M.

5. The method of claim 2, wherein when the bifunctional chelating agent is NODA and the radionuclide is⁶⁸Ga, the preparation method comprises the following steps: to 10-50. mu.L of the labeled precursor NODA-KE108 at a concentration of 0.5mg/mL was added 92.5MBq in sequence⁶⁸GaCl₃Reacting the eluate with 0.1-10M sodium formate solution 10-100 μ L at 80-120 deg.C for 10-30min, and separating and purifying with Sep-pakC18 column when the labeling rate is less than 90%.

6. Use of any one of the following somatostatin analogues of claim 1:

1) for preparing cancer detection reagents or kits;

2) for the preparation of an anti-cancer medicament or composition;

3) preparing a molecular probe for targeting cancer;

wherein the cancer is a cancer expressing a somatostatin receptor.

7. A tumor imaging agent characterized in that the effective ingredient is the somatostatin analog according to claim 1.

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a somatostatin analogue and application thereof.

Background

Neuroendocrine tumors (NETs) are a heterogeneous group of tumors originating in different neuroendocrine organs, ranging from classical carcinoids with good prognosis to the wide spectrum of highly malignant undifferentiated neuroendocrine cancers, 85% of which are derived from the digestive tract and 10% from the lungs, as yet visible in the larynx, thymus, adrenal gland, ovary, skin, prostate gland, etc. Studies have shown that up to 94% of NETs cell surface have high expression of somatostatin receptors (SSTRs), even on the surface of some poorly differentiated tumor cells. Due to tumor heterogeneity, different tumor cell surfaces express different SSTR subtypes (i.e., SSTR1-5), as well as tumors of the same species. The expression of SSTR1 on the cell surface of prostate cancer and sarcoma, the expression of SSTR2 on glioma, lymphoma, pheochromocytoma and small cell lung cancer cells, the expression of SSTR3 in most inactive pituitary tumors, and the expression of one or two of SSTR1 and SSTR2 in gastroenteropancreatic neuroendocrine tumors, gastric cancer and ependymoma.

Somatostatin is an endogenous regulatory peptide with a wide range of physiological functions. Natural somatostatin has good affinity for five subtypes of SSTR. Currently, a series of somatostatin analogues obtained by modifying a natural somatostatin structure, such as classical somatostatin analogues Octreotide (Octreotide) and Lanreotide (Lanreotide), have been widely used for diagnosing and treating the SSTR positive tumors such as neuroendocrine tumors, gastrointestinal tumors, thyroid cancer and the like, but have higher affinity only for the SSTR2 positive tumors, have low specificity for other subtype positive tumors, and are easy to cause missed diagnosis. Therefore, the development of a somatostatin analogue probe with broad spectrum has important significance for the diagnosis and treatment of SSTR positive tumors.

Disclosure of Invention

The invention aims to provide a novel somatostatin analogue and application thereof.

To achieve the object of the present invention, in a first aspect, the present invention provides a somatostatin analogue having a structure represented by formula I):

wherein R is a bifunctional chelating group, and R1 is a radionuclide.

The bifunctional chelating group is-DTPA, -NOTA, -p-SCN-Bn-NOTA, -DOTA or-MPAA-NODA and the like.

R1 can be selected from¹⁸F、⁶⁴Cu、⁶⁸At least one radionuclide such as Ga.

The amino acid sequence of the somatostatin analogue is Tyr⁰-cyclo-[D-Dab-Arg-Phe-Phe-D-Trp-Lys-Thr-Phe]。

From the polypeptide structure, the Phe-D-Trp-Lys-Thr motif with high affinity for somatostatin receptors is retained in the KE108 structure. Wherein D represents the configuration of the amino acid.

In a second aspect, the present invention provides a process for the preparation of said somatostatin analogue, comprising the steps of:

A. modifying the polypeptide KE108 by a bifunctional chelating agent to obtain a labeled precursor;

B. radionuclide labeling is performed on the labeled precursor.

Wherein the bifunctional chelating agent is DTPA, NOTA, p-SCN-Bn-NOTA, DOTA or MPAA-NODA and the like.

The radionuclide may be selected from¹⁸F、⁶⁴Cu、⁶⁸Ga and the like.

The aforementioned method, step B, wherein the-COOH group of said bifunctional chelating agent is condensedReaction with-NH in Tyr in polypeptide KE108₂The groups are linked.

Preferably, when the bifunctional chelating agent is NODA, the radionuclide is¹⁸When F, the preparation method comprises the following steps: adding AlCl containing pH3-5 to 10-50 μ L of labeled precursor NODA-KE108 with concentration of 0.5mg/mL₃The acetic acid buffer solution of (1), (500) μ L and 3.7-370MBq¹⁸F]F^-Reacting the physiological saline solution at 80-120 ℃ for 10-30min, and then separating and purifying by using a Sep-pakC18 column. Wherein contains AlCl₃In acetic acid buffer solution of (2), AlCl₃The concentration of (A) is 1-30mM, and the concentration of the acetic acid buffer solution is 0.1M.

Preferably, when the bifunctional chelating agent is NODA, the radionuclide is⁶⁸Ga, the preparation method comprises the following steps: to 10-50. mu.L of the labeled precursor NODA-KE108 at a concentration of 0.5mg/mL was added 92.5MBq in sequence⁶⁸GaCl₃Reacting the eluate with 0.1-10M sodium formate solution 10-100 μ L at 80-120 deg.C for 10-30min, and separating and purifying with Sep-pakC18 column when the labeling rate is less than 90%.

In a third aspect, the present invention provides any one of the following uses of the somatostatin analogue:

1) for preparing cancer detection reagents or kits;

2) for the preparation of an anti-cancer medicament or composition;

3) is used for preparing molecular probes for targeting cancers.

In a fourth aspect, the present invention provides a tumor imaging agent comprising as an active ingredient a somatostatin analogue of formula I). Can be used for PET imaging.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the Phe-D-Trp-Lys-Thr motif with high affinity to somatostatin receptors is reserved in the structure of the polypeptide KE108 derivative, and the probe structure is modified by different chelating agents and different nuclides to design a broad-spectrum radiolabeled somatostatin molecular probe targeting the somatostatin receptors. All subtypes of somatostatin receptors are targeted, which serves as a more sensitive probe for early diagnosis of neuroendocrine tumors. So as to realize the accurate medical treatment of early diagnosis, early discovery and early treatment of neuroendocrine tumors.

The preparation method of the somatostatin analogue provided by the invention has the characteristics of short marking time, mild conditions and the like, and is beneficial to the commercial application and clinical popularization of the marker.

The invention has imaging in somatostatin receptor positive tumor-bearing mice, and obvious radioactive uptake at the tumor visible part 30min after administration, which indicates that the probe has good targeting property.

Drawings

FIG. 1 is a scheme showing the synthesis of the NODA-MPAA-KE108 polypeptide in the preferred embodiment of the present invention.

FIG. 2 is a mass spectrum of NODA-MPAA-KE108 polypeptide in a preferred embodiment of the present invention.

FIG. 3 is an HPLC chromatogram of the NODA-MPAA-KE108 polypeptide in a preferred embodiment of the present invention.

FIG. 4 shows [ Al ] in the preferred embodiment of the present invention¹⁸F]NODA-MPAA-KE108 mark rate plot.

FIG. 5 is a diagram of the preferred embodiment of the present invention⁶⁸Ga]NODA-MPAA-KE108 mark rate plot.

FIG. 6 shows [ Al ] in the preferred embodiment of the present invention¹⁸F]In vitro stability chromatograms of the NODA-MPAA-KE108 probe in physiological saline (a) and 10% fetal bovine serum (b).

FIG. 7 shows [ Al ] in the preferred embodiment of the present invention¹⁸F]The NODA-MPAA-KE108 micro-PET image in AR42J tumor-bearing mice.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products. EXAMPLE 1 preparation of somatostatin analogue NODA-MPAA-KE108 and its labeled compound

The somatostatin analogue provided in this example has the structure shown in formula I):

wherein R is a bifunctional chelating group, and R1 is a radionuclide.

The bifunctional chelating group is-DTPA, -NOTA, -p-SCN-Bn-NOTA, -DOTA or-MPAA-NODA and the like.

R1 can be selected from¹⁸F、⁶⁴Cu、⁶⁸At least one radionuclide such as Ga.

1. The preparation method of the somatostatin analogue comprises the following steps:

(1) synthesis of NODA-MPAA-KE108 polypeptide (see FIG. 1 for the synthetic procedure)

Deprotection and washing of resin: weighing about 5g of solid-phase synthetic Resin Fmoc-Phe-CTC Resin (0.3-0.5mmol/g), placing the solid-phase synthetic Resin Fmoc-Phe-CTC Resin in a synthesis tube, adding dimethyl amide (DMF), soaking for 1-2h, draining the solution, repeatedly washing with DMF for 3 times, adding 20% piperidine (PIP)/DMF, reacting for 10min, draining the solution, repeating twice, carrying out deprotection process for about 30min, repeatedly washing with DMF for 5-6 times, and draining for later use.

And (2) deprotection complete detection: a small amount of resin was removed from the synthesis tube and checked by ninhydrin reaction to show that the Fmoc protecting group had been removed if the resin was dark blue in color.

③ coupling of amino acids Fmoc-Tyr (tBu) -OH: weighing 1g of Fmoc-Tyr (tBu) -OH, dissolving in Dichloromethane (DCM), adding 1.2 equivalents of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), 1-Hydroxybenzotriazole (HOBT) and 2 equivalents of N-methylmorpholine (NMM), stirring for reacting overnight, and performing rotary evaporation and concentration to obtain a cyclized crude product.

And fourthly, repeating the first step and the second step.

Fifthly, continuously connecting NODA by the method of the third step to obtain a crude product NODA-Tyr (tBu) -Cyclo (D-Dab-Arg (NO)₂)-Phe-Phe-D-Trp(Boc)-Lys(Z)-Thr(tBu)-Phe)。

Sixthly, Pd/C hydrogenation reaction: dissolving the crude product in the fifth step in methanol, adding Pd/C to hydrogenate to obtain crude product NODA-Tyr (tBu) -Cyclo (D-Dab-Arg-Phe-Phe-D-Trp (Boc) -Lys-Thr (tBu) -Phe).

Cutting and washing: dissolving the crude product in step (4)Acetic acid (TFA)/1, 2-Ethanedithiol (EDT)/water (H)₂O) is 95/2.5/2.5, and the temperature of a shaking table is controlled for 3 hours; adding into 6 times volume of glacial ethyl ether, precipitating with a centrifuge, collecting solid, and washing the precipitated crude product with ethyl ether for 3 times to obtain the final crude product;

drying and purifying: placing the crude product in the step (c) in a drying pot, drying in vacuum overnight, and purifying by reversed phase preparative HPLC, wherein the mobile phase is as follows: a: 0.1% aqueous TFA; b: 0.1% TFA in 80% Acetonitrile (ACN), gradient elution, detection wavelength 220nm, collection of the main peak.

Ninthly, freeze drying: collecting the main peak solution, adding a small amount of purified water, freezing at-20 ℃ overnight, then freeze-drying for 48-72h by using a freeze dryer, taking out a sample when the content is white powder, subpackaging the white powder into a freezing tube for storage according to requirements, and taking a small amount of sample for Mass Spectrum (MS) and HPLC detection.

Quality analysis of r

And (3) detecting the molecular weight: the mass spectra were obtained using a Waters SYNAPT G2 HDMS system. The high resolution mass spectrum of NODA-MPAA-KE108 is shown in fig. 2, M/z is 551.2[ (M +3H)/3], and the theoretical molecular weight of NODA-MPAA-KE108 is: 1650.8 Da. The molecular weight of the prepared polypeptide is consistent with the theoretical molecular weight of NODA-MPAA-KE 108.

And (3) purity detection:

the HPLC analysis conditions were as follows:

a chromatographic column: agilent ZORBAX XDB-C18, 5 μm, 4.6 mm. times.250 mm

Detection wavelength: 220nm, run time: and 15 min.

Mobile phase: a: 0.1% aqueous TFA; b: 0.1% TFA in 80% ACN.

Gradient elution:

purity > 95% was confirmed by HPLC and the results are shown in FIG. 3.

(2) Labeling and purification

The radionuclide is¹⁸At F timeThe preparation method comprises the following steps: adding 100 mu L of 0.1M acetic acid buffer solution (pH4) into 10-50 mu L of labeled precursor NODA-KE108 with the concentration of 0.5mg/mL, wherein 100 mu L of labeled precursor NODA-KE contains AlCl₃Acetic acid buffer solution (1mM), of 3.7MBq [, [ 2 ]¹⁸F]F^-Reacting with normal saline solution at 100 deg.C for 10min, and separating and purifying with Sep-pakC18 column to obtain [ Al ]¹⁸F]NODA-MPAA-KE108 Probe, [ Al ]¹⁸F]The NODA-MPAA-KE108 mark rate plot is shown in FIG. 4.

The radionuclide is⁶⁸Ga, the preparation method comprises the following steps: to 10. mu.L of the labeled precursor NODA-KE108 at a concentration of 0.5mg/mL was added 92.5MBq in sequence⁶⁸GaCl₃Eluting with 10 μ L of 0.1M sodium formate solution, reacting at 80 deg.C for 10min, and separating and purifying with Sep-pakC18 column when the labeling rate is less than 90%⁶⁸Ga]NODA-MPAA-KE108。[⁶⁸Ga]The NODA-MPAA-KE108 mark rate plot is shown in FIG. 5.

Example 2 affinity testing with SSTR

The affinity studies of somatostatin analogues with various subtypes of SSTR were tested using Surface Plasmon Resonance (SPR). The SSTR1-5 subtype receptors are subjected to detris treatment by adopting ultrafiltration centrifugation, a dextran chip is utilized, each chip is provided with a reference channel, and other channels are used for coupling SSTR proteins. SSTR proteins were formulated with a 200. mu.g/mL solution in pH4.5 sodium acetate buffer (10mM) for coupling to the chip. Somatostatin analogues (1mM) were serially diluted in multiples of phosphonate Tween solution (PBST) to 10 gradients (0.1-100. mu.M), with a blank PBST added every 5 gradients, and the samples were loaded onto the machine for testing starting at low concentrations. The test results are shown in table 1.

TABLE 1 affinity constants K for somatostatin and its analogs and five protein subtypes_dValue (M)

Note: SST14 and SST28 are described in Gao J, Tong H, Huang Z, et al, affinity analysis of and receptor by surface plasma response [ J ]. Analytical Methods,2013,5(13):3201.

Test results show that the affinity constant of each subtype of KE108 and NODA-MPAA-KE108 and SSTR obtained by modification through detection by SPR method is 10^-4～10^-5About M. The research literature adopts an SPR method to test that the affinity constants of natural somatostatin SST14 and 28 to SSTR2 and SSTR 4 are 10^-4～10^-6Within the range. Thus indicating that KE108 and the modified NODA-MPAA-KE108 have good affinity to SSTR subtypes.

Example 3 in vitro stability study

Taking 100 mu L of [ Al¹⁸F]Adding NODA-MPAA-KE108 into physiological saline solution (pH7.4), incubating at room temperature for 0.5h, 1h, 2h, detecting by HPLC, and collecting 100 μ L of [ Al¹⁸F]Adding NODA-MPAA-KE108 into 1.9mL 10% fetal calf serum, incubating at 37 deg.C for 0.5h, 1h, and 2h, adding 100 μ L acetonitrile into 400 μ L above serum, mixing, vortexing, centrifuging, and performing HPLC detection. The results are shown in FIG. 6(a and b), and the results show that [ Al ]¹⁸F]The NODA-MPAA-KE108 maintains better stability in normal saline (a) and 10% fetal calf serum (b), and the radiochemical purity is more than 95%.

Example 4 PET imaging of tumor-bearing mice

Injecting AR42J tumor-bearing mice (BALB/c nude mice, male, about 20 g) with tumor diameter of about 0.5-1cm into tail vein¹⁸F]NODA-MPAA-KE108(3.7 MBq/100. mu.L. only), PET imaged 0.5h and 1h after administration, respectively, with mice in prone position and scanned under general anesthesia using isoflurane and oxygen. The result is shown in figure 7, the high radioactivity uptake at the tumor part is obvious, the uptake value is 2.57 +/-0.60% ID/g, the muscle uptake value is 1.22 +/-0.50% ID/g, and the target-to-non-target ratio is better. Block control group was treated with [ Al ] through tail vein¹⁸F]NODA-MPAA-KE108 and NODA-MPAA-KE108 polypeptides were co-injected into AR42J tumor-bearing mice, PET imaged 1h after administration, mice were in prone position, and scanned under general anesthesia using isoflurane and oxygen. As shown in FIG. 7, there was no radioactive uptake at the tumor site, and the uptake was 0.53. + -. 0.24% ID/g, indicating that the probe had good specificity.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.