KR101658206B1 - GRP-R antagonistic 177-Lutetium-labeled bombesin analogue for diagnosis and treatment of prostate cancer - Google Patents

Abstract

It is an object of the present invention to provide a method for the diagnosis and treatment of prostate cancer by marking a radioisotope in a bombesin derivative capable of selectively targeting a target substance that is overexpressed in tumor cells to develop a method for effectively diagnosing and treating prostate- And to provide novel compounds which can be usefully used.

Description

[0001] The present invention relates to a novel bombesin derivative compound based on a gastrin-releasing peptide receptor antagonist for the diagnosis and treatment of prostate cancer.

The present invention relates to a novel bombesin derivative compound based on a gastrin-releasing peptide receptor (GRP-R) antagonist sequence for the diagnosis and treatment of prostate cancer.

Prostate cancer, one of the most common cancers in Koreans, is a very fatal disease for men. In the early stage of the disease, prostate removal or local radiation therapy is easy to treat. However, once the metastasis has occurred, the success rate of treatment is significantly lowered, so early detection and effective treatment are required.

Chemotherapy and surgery are the most common types of conventional chemotherapy. Chemotherapeutic chemotherapy has a serious side effect on normal cells, and it has a problem that the life and quality of the patient is poor because of long treatment period. In addition, conventional surgical procedures are not easy to apply to older patients and their prognosis is very low. Recently, a target treatment technique capable of specifically diagnosing tumor cells and selectively treating only tumor cells has been spotlighted, and many studies have been conducted. In particular, the target radioactive therapy technique using radioactive isotopes is very low in side effects compared to chemotherapeutic chemotherapy, and has a merit of being applicable to elderly or terminal cancer patients because of short treatment period.

In the case of prostate cancer, it is possible to target using GRP-R ([Gastrin-releasing peptide receptor], BB2, BRS-2), a receptor specifically over-expressed in cancer cell membranes. Therefore, it has been actively studied for a target treatment technique using a bombesin derivative capable of selectively targeting GRP-R (177Lu-AMBA: Synthesis and Characterization of a Selective 177Lu-Labeled GRP-R Agonist for Systemic Radiotherapy of Prostate Cancer, J Nucl Med 2006; 47: 11441152). However, it is very important that such a target technology has high therapeutic efficiency as well as more accurate diagnosis of diseases because of its excellent target efficiency. Therefore, the researchers felt the need for the development of better target radiotherapy technology compared to the conventional technology for target efficacy against tumors. Using the target material with excellent target efficiency for the above prostate cancer, The present invention has been completed on the development of a radiotherapeutic technique that is superior to, and further effective in, the therapeutic efficacy of prostate cancer.

In particular, it is possible to diagnose and treat prostate cancer by conjugating a radioisotope to a bombesin derivative that acts as an antagonist of GRP-R. Such targeted therapeutic techniques not only increase the accurate cancer detection rate as the target tumors accumulate, but also have a high therapeutic effect and can be applied as therapeutic agents with low side effects due to low accumulation in normal tissues.

Sandra B. (2014) 177 Luteum-labeled bombesin analogs for radiotherapy. U.S.A. Patent No. 988629

It is an object of the present invention to provide a method for the diagnosis and treatment of prostate cancer by marking a radioisotope in a bombesin derivative capable of selectively targeting a target substance that is overexpressed in tumor cells to develop a method for effectively diagnosing and treating prostate- And to provide novel compounds which can be usefully used.

The present invention provides a bombesin derivative compound represented by the following formula.

[Chemical Formula 1]

[In the above formula (1)

n is an integer of 1 to 3,

X is a metal chelator,

Y is a spacer,

A is a bombesin analog antagonist represented by the following sequence.

A: Gln-Trp-Ala- Val-N-methyl-Gly-His-Statine-Leu-NH 2

The present invention provides a metal chelator comprising at least one radionuclide metal, wherein the metal chelator is a metal chelator for a trivalent metal, and analogues thereof.

The present invention provides a metal chelator for the trivalent metal, a DOTA-based chelator and analogous bombesin derivative compounds thereof.

The invention the radionuclide metal is for radioactive ^{therapy, 176 Yb, 111 In, 169} Gd, 177 Lu, 105 Rh, 111 Ag, 64 Cu, 67 Ga, 68 Ga, 125 I, 123 I, 129 I and ¹⁶⁶ Ho. ≪ / RTI >

The present invention provides a bombesin derivative compound represented by the following formula (2).

(2)

[In the formula (2)

n is an integer of 1 to 5,

m is an integer of 1 to 10,

R ₁ and R ₂ are independently of each other hydrogen or (C 1 -C 5) alkyl,

R ₁₁ independently represents hydrogen, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, a hydrazine, a carboxyl group or a salt thereof, a sulfonic acid group or a salt thereof,

X is a metal chelator,

A is a bombesin analog antagonist represented by the following sequence.

Ala: Gln-Trp-Ala-Val-N-methyl-Gly-His-Statine-Leu-NH2]

The invention the spacer Y provides the bombesin derivatives _{Ala- (SO 3 H) -Aminooctanoic acid} .

And a step of synthesizing a compound of the following formula (2).

(2)

[In the formula (2)

n is an integer of 1 to 10,

R ₁ and R ₂ are independently of each other hydrogen or (C 1 -C 5) alkyl,

R ₁₁ independently represents hydrogen, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, a hydrazine, a carboxyl group or a salt thereof, a sulfonic acid group or a salt thereof,

X is a metal chelator,

A is a bombesin analog antagonist represented by the following sequence.

Ala: Gln-Trp-Ala-Val-N-methyl-Gly-His-Statine-Leu-NH2]

The present invention provides a composition for diagnosing and treating a prostate-related disease comprising the bombesin derivative compound.

The present invention relates to a bombesin derivative capable of efficiently and selectively targeting prostate cancer in which GRP-R is overexpressed. By using the bombesin derivative, the higher the tumor target efficiency, the more accurate diagnosis effect and the higher therapeutic effect You can expect.

In addition, the material provided in the present invention has an advantage of having excellent tumor targeting efficiency as compared with conventional bombesin derivatives having a similar mechanism of action, and by the characteristics of the radioactive isotope 177-Lu releasing beta and gamma energy Very accurate imaging and treatment is possible.

Therefore, the substance provided by the present invention can be more effectively used for diagnosis and treatment of prostate cancer that overexpresses GRP-R.

Figure 1 shows the purity of the preparation of 177Lu-DOTA-sBBNA.
Figure 2 shows the results of competition analysis evaluation of 125I- [Tyr ⁴ ] -bombesin in human prostate cancer PC-3 cells.
Figure 3 shows the results of the stability of 177Lu-DOTA-sBBNA in human prostate cancer PC-3 cells.
Figure 4 shows the results of intracellular inflow of 177Lu-DOTA-sBBNA in human prostate cancer PC-3 cells.
Figure 5 shows the in vivo distribution pattern of 177Lu-DOTA-sBBNA in a tumor mouse model transplanted with human prostate cancer PC-3.
Figure 6 compares the in vivo prostate cancer target efficacy with 177Lu-DOTA-sBBNA and a comparable agent with similar mechanism of action.

The present invention provides a bombesin derivative compound capable of targeting GRP-R overexpressing in prostate cancer.

The present invention relates to a novel bombesin derivative compound represented by the following general formula (1).

[Chemical Formula 1]

[In the above formula (1)

n is an integer of 1 to 3,

X is a metal chelator,

Y is a spacer,

A is a bombesin analog antagonist represented by the following sequence.

A: Gln-Trp-Ala-Val-N-methyl-Gly-His-Statine-Leu-NH2

In one embodiment of the present invention, there is provided a novel and improved compound for use in imaging and radiotherapy of prostate cancer.

It is most preferable that n = 1 in the above formula (1).

The compound may include a metal chelator. The compound includes an optically labeled moiety that can be synthesized by chelating a medically useful metal ion or radionuclide to a GRP-R targeting peptide having a bombesin analogue antagonist sequence by a linker or spacer group.

The metal chelator refers to a molecule that forms a complex with a metal atom, wherein the complex is stable under physiological conditions. More specifically, the metal chelator may be a molecule synthesized in the radionuclide metal and physiologically stable and having at least one reactive functional group for bonding with the spacer. In addition, the metal chelator may comprise a single amino acid between the metal chelator and the spacer to couple with the spacer.

The metal chelator is a metal chelator comprising one or more radionuclide metals, and the metal chelator may be a metal chelator for trivalent metals and analogues thereof.

Preferably, the metal chelator for the trivalent metal may be a DOTA-based killer and its analog. The most preferred chelator used in the compounds of the present invention is a metal chelator represented by the following formula (4).

[Chemical Formula 4]

The metal chelator may comprise one or more radionuclide metals and preferred radionuclide metals for imaging and radiotherapy include but are not limited to ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ¹¹¹ In, ¹⁰⁵ Rh, ¹⁷⁶ Yb, ¹⁶⁹ Gd, ¹⁷⁷ Lu, ¹¹¹ Ag, ¹²⁵ I, ¹²⁹ I and ¹⁶⁶ Ho. The choice of the radionuclide metal may be determined by diagnostic or therapeutic applications, with ¹⁷⁷ Lu being particularly preferred.

The bombesin derivative compound according to the present invention includes a newly improved spacer capable of linking an optical marker synthesized with a metal radionuclide to a GRP-R targeting peptide having a bombesin analog antagonist sequence.

The spacer of the present invention is represented by the following formula (2).

(2)

[In the formula (2)

n is an integer of 1 to 10,

R ₁ and R ₂ are independently of each other hydrogen or (C 1 -C 5) alkyl,

R ₁₁ independently represents hydrogen, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, a hydrazine, a carboxyl group or a salt thereof, a sulfonic acid group or a salt thereof,

X is a metal chelator,

A is a bombesin analog antagonist represented by the following sequence.

Ala: Gln-Trp-Ala-Val-N-methyl-Gly-His-Statine-Leu-NH2]

The spacer may be used to link a metal chelator comprising a metal radionuclide to a GRP-R targeting peptide having a bombesin analog analogue antagonist sequence.

According to an embodiment of the present invention, a metal chelator is connected to the N-terminus of the spacer, and a GRP-R targeting peptide having the Bombesin analog antagonist sequence of the present invention may be connected to the C-terminus of the spacer.

The spacer used in the present invention may preferably be a spacer comprising at least one amino acid. Specifically, in one embodiment of the present invention, the spacer may contain at least one amino acid, or may be a form in which a sulfonic acid group is added to an amino acid. For example, the amino acid is most preferably Ala.

The most preferred spacer used in the compounds of the invention may be a _{Ala- (SO 3 H) -Aminooctanoci acid} , to be represented by the formula (3).

(3)

에서 A를 의미한다)을 포함한다.The bombesin derivative compound according to the present invention is a Bombesin analog agonist sequence of the GRP-R targeting peptide

Quot; A ").

The GRP-R is a receptor for a gastrin-releasing peptide (GRP), which is a kind of peptide having physiological activity such as promoting gastrin secretion or secretion of pancreatic enzymes and cell proliferation, and is a peptide known to overexpress in the prostate cancer cell membrane , GRP-R can be used as a target peptide for prostate cancer.

As the GRP-R targeting peptide, the bombesin analog antagonist acts as a GRP-R antagonist. Bombesin analog antagonist binds with high affinity to GRP-R, which is overexpressed in cancer cell membrane, and acts opposite to the agonist. Thus, it does not induce the internalization of the agent into the cell while antagonizing the agonist effect, Lt; RTI ID = 0.0 > GRP-R < / RTI >

The bombesin analog antagonist may be a bombesin inducing substance for targeting GRP-R of prostate cancer and may be composed of several amino acid bonds. Most preferably Gln-Trp-Ala-Val-N-methyl-Gly-His-Statine-Leu-NH2 sequence.

Also, these analogues can be performed by modifying one or several amino acids with substitution, deletion or addition, which can be carried out as an antagonist of GRP-R, to the extent that the biological function is conserved.

Thus, in one embodiment of the present invention, the bombesin analog antagonist can be used to diagnose and treat prostate cancer by conjugating a radioisotope to a bombesin analog or derivative thereof as an antagonist of GRP-R .

Another aspect of the present invention provides a process for preparing a bombesin derivative represented by the following formula (1)

To a compound of the following formula (2).

(2)

[In the formula (2)

n is an integer of 1 to 5,

m is an integer of 1 to 10,

R ₁ and R ₂ are independently of each other hydrogen or (C 1 -C 5) alkyl,

R ₁₁ independently represents hydrogen, a halogen atom, a hydroxyl group, a cyano group, a nitro group, an amino group, a hydrazine, a carboxyl group or a salt thereof, a sulfonic acid group or a salt thereof,

X is a metal chelator,

A is a bombesin analog antagonist represented by the following sequence.

Ala: Gln-Trp-Ala-Val-N-methyl-Gly-His-Statine-Leu-NH2]

The method for preparing the bombesin derivative compound of the present invention can be most conveniently prepared by a solid phase synthesis method and specifically can be prepared by a solid phase peptide synthesis method which is generally established and widely used in the field of peptide bonding. Solid phase peptide synthesis involves the stepwise addition of amino acid residues to an insoluble support or peptide chain that grows linked to polystyrene. The C-terminal residue of the peptide is first protected with an N-protecting agent, a t-butyloxycarbonyl group (Boc) or a fluorophenylmethoxycarbonyl group (Fmoc). The amino protecting group is TFA in the case of Boc, in the case of Fmoc it is removed by piperidine and the amino acid residue (N-protected form) is then added with a coupling agent, for example HBTU.

It is most preferable that n = 1 in the above formula (1).

The spacer may then be coupled by reacting the amino group of the Gln residue of the Bombesin analog antagonist with the functional group of the spacer to form a junction.

The bombesin analog compound represented by the formula (2) can be finally prepared by reacting the bombesin analog antagonist with the spacer with a metal chelator containing a radioactive nucleus. This reaction can be carried out by a known chelating reaction. Through this chelation reaction, a chelator containing a radionuclide can be labeled with a bombesin analog analogue, and a bombesin derivative (hereinafter referred to as' 177Lu -DOTA-sBBNA ").

The present invention provides a composition for diagnosing and treating a prostate-related disease comprising the bombesin derivative compound.

 The present invention relates to a diagnostic method of prostate cancer which overexpresses GRP-R in a cancer cell membrane in the field of therapeutic techniques such as radiological imaging and radiotherapy, which are diagnostic or therapeutically useful by providing the improved bombesin derivative compound by the above- And can be used for treatment. The application of the target treatment technique using the improved bombesin derivative compound prepared according to the present invention can be expected to have a higher therapeutic effect because it is highly integrated in the targeted tumor site, while the accumulation in the normal tissue is low, Has an improved ability to be lowered. In addition, a large accumulation in the target tumor site can be accumulated in a large amount even in a small size tumor when diagnosing a lesion, so that the cancerous lesion can be diagnosed more accurately. In addition, since the bombesin derivative compound of the present invention acts as an antagonist of GRP-R, it can bind to GRP-R peptide with high affinity and be absorbed into cells as described above. Since the bombesin derivative compound binds to and absorbs tumor in vivo, it can exhibit a highly effective ability to target a prostate cancer tumor overexpressing GRP-R and to image it.

The targeted therapeutic application of the bombesin derivative compound may refer to a medicament that is used together with a chemotherapeutic agent that combines chemotherapy with the above compound in diagnosing and treating cancer.

Chemotherapeutic agents that can be used to diagnose and treat cancer are those that can serve both as a diagnostic agent and as a therapeutic agent. The chemotherapeutic agent may be an anticancer agent used in the field. More preferably, the antibody can be used for diagnosis and treatment by using a monoclonal antibody capable of binding to a cancer antigen.

The present invention can be administered to a patient as part of a composition containing excipients, diluents, stabilizers, and carriers well known in the art as a cancer treatment agent, in addition to the above therapeutic agents.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are for illustrative purposes only and are not intended to be limiting.

[Example 1] Synthesis of Bombesin Derivative Compound (DOTA-sBBNA).

The bombesin derivative compound (DOTA-sBBNA) of the present invention was prepared using solid phase synthesis as shown in the following reaction formula.

[Example 1] Synthesis of Bombesin Derivative Compound (DOTA-sBBNA).

The bombesin derivative compound (DOTA-sBBNA) of the present invention was prepared using solid phase synthesis as shown in the following reaction formula.

[Example 2] Preparation of 177Lu-DOTA-sBBNA

DOTA-sBBNA can be labeled with 177Lu and a high purity of 98% or more, and the results are shown in FIG.

[Example 3] In vitro target efficiency for prostate cancer.

3-1. Cell culture

PC-3 cells were used as human prostate cancer cells in order to confirm the in vitro target efficiency of DOTA-sBBNA and 177Lu-DOTA-sBBNA prepared according to Examples 1 and 2 for prostate cancer cells. Human prostate cancer cell line PC-3 cells were purchased from Korean Cell Line Bank. For cell culture, PC-3 cells were cultured in RPMI 1640 supplemented with 10% FBS (Fetal bovine serum) at 37 ° C and 5% CO ₂ .

The cells were cultured in a disposable flask for cell culture and cultured in a 96-well or 12-well cell culture plate according to the experimental method.

3-2. Evaluation of Stability of 177Lu-DOTA-sBBNA in PBS and Serum

Experiments were conducted to confirm the stability evaluation of 177Lu-DOTA-sBBNA prepared in Example 2 in vivo. When 177Lu-DOTA-sBBNA prepared in Example 2 was stored in phosphate buffered saline (PBS) at 37 ° C and human serum for 48 hours, the degree of decomposition of 177Lu-DOTA-sBBNA was confirmed. As a result, it was confirmed that even when stored for 48 hours in PBS and human serum, they had excellent stability without being decomposed (Fig. 2). Since 177Lu degraded in the body can be deposited in the bone marrow and cause various side effects, the high stability of 177Lu-DOTA-sBBNA in the body indicates that it can be useful for diagnosis and treatment of prostate cancer do.

3-3. DOTA-sBBNA prostate cancer cells in vitro ( in vitro ) Target efficiency

Human prostate cancer PC-3 cells in the 10 ^-6 - 10 ^-12 M concentration of the DOTA-sBBNA each of 20,000 CPM 125I- [Tyr4] -bombesin and co-cultured and reacted competition. The cell plate was incubated at 37 ° C for 1 hour, and the reaction solution was recovered. Cells were washed with PBS, and then recovered with 1N NaOH. The radioactivity of each solution was measured using a gamma counter. The competitive binding curve for 125I- [Tyr ⁴ ] -bombesin is shown in FIG. 3, and as a result, the IC ₅₀ value is 6.96 nM, which shows excellent target efficiency for the GRP-R. The results are shown in Fig.

In addition, 0.01-100 nM of 177Lu-DOTA-sBBNA prepared according to Example 2 was reacted with PC-3 cells, respectively. The cell plate was incubated at 37 ° C for 2 hours, and the reaction solution was recovered. Cells were washed with PBS, and then recovered with 1N NaOH. The radioactivity of each solution was measured using a gamma counter. The measured values were analyzed using the GraphPad Prism5 statistical program, and the dissociation constant (Kd) value was also 1.88 nM, indicating excellent target efficiency at the nanomolar level for GRP-R.

[Example 4] Intracellular influx of 177Lu-DOTA-sBBNA

Experiments were carried out to confirm the inflow characteristics of 177Lu-DOTA-sBBNA prepared in Example 2 into PC-3 cells. 25,000 CPM of 177Lu-DOTA-sBBNA was reacted at 37 ° C in PC-3 cells, human prostate cancer cells, and the reaction solution was recovered at 15 minutes, 30 minutes, 60 minutes, and 120 minutes, respectively. The cells were washed with PBS, and the radioactivity attached to the cell membrane was recovered with a pH 2.5 acetic acid solution. The radioactivity introduced into the cells was recovered with 1N NaOH solution, and the radioactivity of each solution was measured using a gamma counter.

The results are shown in Fig. 4, and about 17% of the 177Lu-DOTA-sBBNA is introduced into PC-3 cells within about 4 hours, allowing the radioisotope to stay in the cells. This is a high level of intracellular inflow compared to conventional materials of similar mechanism of targeting the antagonist of GRP-R and is therefore useful for the diagnosis and treatment of prostate cancer.

[Example 5] In vivo target efficiency of 177Lu-DOTA-sBBNA on prostate cancer

To evaluate the target efficiency of 177Lu-DOTA-sBBNA, the distribution of human tumor cells in a tumor model mouse model of human prostate cancer cell line PC-3 was evaluated in Balb / c nude mice (Nara Biotech). 1 × 10 ⁷ PC-3 cells were subcutaneously injected into the upper right shoulder region of a 7-week-old male Balb / c nude mouse and a tumor model was prepared by growing the tumor for 3 weeks. Intravenous injection of 370 KBq of 177Lu-DOTA-sBBNA was performed using the tail vein of the tumor mouse model. 1.5 hours after the injection, the animals were euthanized and the organs, blood, liver, kidney, spleen, heart, large intestine, small intestine, lung, stomach and pancreas were excised and the organ weight was measured. Respectively. The absorbed amount of 177Lu-DOTA-sBBNA by each organ was expressed as absorption / dose per unit weight (% ID / g). To confirm receptor-specific uptake, a group of 10 ^-8 moles of DOTA-sBBNA was added.

It was confirmed that prostate cancer cells were targeted at a high efficiency of 12.91 ± 3.43% ID / g at 1.5 hours after intravenous injection. The results are shown in Fig.

[Example 6] Comparison of target efficiency of prostate cancer in vivo with a comparative agent

A substance having a prostate cancer targeting mechanism similar to the bombesin derivative compound (177Lu-DOTA-sBBNA) utilizing a bombesin derivative which is an antagonist of GRP-R prepared according to Example 2 is included in the reference the Plus One, 7 (9); '177Lu-DOTA-4-amino 1-carboxymethyl-piperidine-D Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2' ( Comparative substance e44046 , Sandra B. (2014) 177), has been patented recently in Europe and the United States for its excellent target efficiency. The 177Lu-DOTA-sBBNA according to Example 2 exhibits a 2 to 3-fold higher prostate cancer target efficiency than the comparative substance, and has remarkable superiority in that it is removed more rapidly in blood . Therefore, it was proved that the 177Lu-DOTA-sBBNA exhibited an improved effect as a bombesin derivative compound as compared with the comparative substance, as shown in Fig.

Claims (8)

A bombesin derivative compound represented by the following formula:
[Chemical Formula 1]

[In the above formula (1)
n is an integer of 1 to 3,
X is a metal chelator,
Y is a spacer which Ala- (SO ₃ H) -Aminooctanonic acid and
A is a bombesin analog antagonist represented by the following sequence.
A: Gln-Trp-Ala-Val-N-methyl-Gly-His-Statine-Leu-NH2
The method according to claim 1,
Wherein X is a metal chelator comprising one or more radionuclide metals, wherein the metal chelator is a metal chelator for a trivalent metal and analogous thereto.
3. The method of claim 2,
Wherein the metal chelator for the trivalent metal is a DOTA-based chelator and analogous thereto.
3. The method of claim 2,
The radionuclide metal is for radiotherapy and may be used for radio therapy such as ¹⁷⁶ Yb, ¹¹¹ In, ¹⁶⁹ Gd, ¹⁷⁷ Lu, ¹⁰⁵ Rh, ¹¹¹ Ag, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ¹²⁵ I, ¹²³ I, ¹²⁹ I and ¹⁶⁶ Ho ≪ / RTI > or a pharmaceutically acceptable salt thereof.
delete delete A method for producing a bombesin derivative compound represented by the following formula:
[Chemical Formula 1]

(Wherein, n is an integer of 1 to 3, X is a metal chelator, Y is Ala- (SO ₃ H) -Aminooctanonic acid as a spacer, and A is a bombesin analog antagonist represented by the following sequence. A: Gln-Trp-Ala-Val-N-methyl-Gly-His-Statine-Leu-NH2)
A composition for the diagnosis of prostate-related diseases comprising the compound according to claim 1.

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