KR102646267B1 - Gadolinium-based compound, mri contrast agent comprising the same - Google Patents

Abstract

A gadolinium-based compound and an MRI contrast agent containing the same are disclosed. The gadolinium-based compound may be a DO3A-based compound to which coumaric acid is bound. The MRI contrast agent may include the compound.

Description

Gadolinium-based compound, MRI contrast agent containing the same {GADOLINIUM-BASED COMPOUND, MRI CONTRAST AGENT COMPRISING THE SAME}

The present invention relates to a gadolinium-based compound and an MRI contrast agent containing the same.

Today, due to the aging of the population, the number of patients with degenerative brain diseases is increasing, and accordingly, the need for early detection of the diseases is emerging. Degenerative brain diseases include Parkinson's disease, vascular dementia, and Alzheimer's disease, and neurotoxicity due to excessive accumulation of amyloid beta polymer (oligomeric Aβ) is considered one of the causes of the disease.

Amyloid beta (Aβ) is the main component of amyloid plaques found in the brains of Alzheimer's patients, and refers to a 36 to 43 amino acid peptide that is critically involved in Alzheimer's disease. The peptide is derived from amyloid precursor protein (APP).

The amyloid beta molecule can aggregate to form a soluble polymer that can exist in various forms. The formed amyloid beta polymer (oligomeric Aβ) is toxic to nerve cells and accumulates excessively in the brain, directly contributing to the development of Alzheimer's disease. It is known to do so. Therefore, it was expected that detecting changes in the concentration of amyloid beta polymer would enable early diagnosis of degenerative brain diseases.

Meanwhile, Magnetic Resonance Image (MRI) is a method of obtaining images of anatomical, physiological, and biochemical information of the body by using the phenomenon of hydrogen atoms being relaxed in a magnetic field due to differences in the distribution of hydrogen atoms between tissues in the body. . Unlike CT or PET, MRI does not use radiation harmful to the human body, but uses magnetic field gradients and radio waves under a strong magnetic field to create images inside the body, so it is non-invasive, has high resolution, and is excellent for soft tissue examination.

In order to utilize such MRI equipment more precisely, a contrast agent is injected into the subject to obtain an MRI image. The contrast between tissues on an MRI image is a phenomenon that occurs because the relaxation of the nuclear spins of water molecules within the tissue to return to an equilibrium state is different for each tissue. The contrast agent uses a paramagnetic or superparamagnetic material to affect the relaxation effect, widening the difference in relaxation between tissues and causing changes in MRI signals to make the contrast between tissues clearer.

Currently, the most commonly used contrast agent clinically is a contrast agent based on gadolinium (Gd) chelate. Currently, Gd-DTPA (Magnevist®), Gd-DOTA (Dotaram®), Gd(DTPA-BMA) (Omniscan®), Gd(DO3A-HP) (ProHance®), Gd(BOPTA) (MultiHance®), etc. It is being used. However, most of the commercially available contrast agents are non-specific contrast agents that are distributed into the extracellular space (extracellular fluid, ECF). As a specific contrast agent, a special contrast agent is used. Recent research is promoting the development of contrast agents that have specific targeting or can show signal enhancement due to physiological activity (pH change, enzyme activity), but to date, MRI contrast agents with specific targeting, especially for degenerative brain Sufficient results have not been obtained for disease-specific MRI contrast agents.

One object of the present invention is to provide a compound that specifically binds to amyloid beta polymer.

Another object of the present invention is to provide an MRI contrast agent containing the above compound.

In one aspect, the present invention provides a compound having the formula (1):

(One)

here,

L is *-(CH ₂ ) _x -A ¹ -(CH ₂ ) _y -A ² -(CH ₂ ) _z -*,

x, y and z are each independently selected as random integers from 0 to 5,

A ¹ and A ² are single bonds, in the group containing *-COO-*, *-CO-*, *-NH-*, *-CH ₂ -*, *-CONH-* and *-O-* Each is one or more independently selected structures,

X is a structure having the following formula (2):

(2)

* is the binding site.

In one embodiment, A ² may be *-NH-*.

In one embodiment, A ¹ may be *-CONH-*.

In one embodiment, x may be 1.

In one embodiment, y may be 2.

In one embodiment, z may be 0.

In one embodiment, the compound may have the following formula (3).

(3)

In one embodiment, the gadolinium (Gd) may coordinate with one or more water molecules.

In one embodiment, the compound can specifically bind to mammalian amyloid beta polymer (oligomeric Aβ).

In one embodiment, the compound may have a relaxation rate of 4.8 to 5.2 s ^-1 .

In one embodiment, the compound can pass through the blood-brain-barrier (BBB) when injected intravenously.

In another aspect, the present invention provides an MRI contrast agent containing the above compound.

In one embodiment, the MRI contrast agent can be used for diagnosis of degenerative brain disease.

In one embodiment, the MRI contrast agent can be used to diagnose Alzheimer's disease.

Compounds according to embodiments of the present invention can specifically bind to amyloid beta polymer.

The MRI contrast agent according to an embodiment of the present invention can be used in the diagnosis of degenerative brain diseases including Alzheimer's.

1 to 8 are diagrams showing experimental results according to experimental examples of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. Since the present invention can be subject to various changes and can have various forms, specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, and should be understood to include all changes, equivalents, and substitutes included in the spirit and technical scope of the present invention. While describing each drawing, similar reference numerals are used for similar components. In the attached drawings, the dimensions of the structures are enlarged from the actual size for clarity of the present invention.

The terms used in this application are only used to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, or a combination thereof described in the specification, but are not intended to indicate the presence of one or more other features or numbers. It should be understood that this does not exclude in advance the possibility of the existence or addition of steps, operations, components, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the technical field to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted in an ideal or excessively formal sense unless explicitly defined in the present application. No.

Compounds according to embodiments of the present invention may have the following formula (1).

(One)

In the formula (1), the gadolinium ion (Gd ³⁺ ) can form a complex by coordinating with the carboxylate (COO ^- ) group in the formula (1).

In the formula (1), L may be *-(CH ₂ ) _x -A ¹ -(CH ₂ ) _y -A ² -(CH ₂ ) _z -*, and x, y and z are any of 0 to 5. can be each independently selected as an integer, and A ¹ and A ² are a single bond, *-COO-*, *-CO-*, *-NH-*, *-CH ₂ -*, *-CONH-* and *-O-*. * is the binding site.

The L may be a linker connecting the nitrogen and the X in the cyclic structure of the compound. The A ¹ and A ² may determine the functional group determined by the method or the method by which the linker connects the nitrogen and the X in the ring-shaped structure of the compound. The x, y and z may determine the length of the chain connecting A ¹ and A ² in the linker. In one embodiment, A ² may be *-NH-*. In one embodiment, A ¹ may be *-CONH-*. In one embodiment, x may be 1. In one embodiment, y may be 2. In one embodiment, z may be 0.

As long as the compound according to the embodiment of the present invention substantially has the structure of Formula (1), bonding or removal of bonding acceptable to those skilled in the art is not excluded from the scope of the present invention. For example, in one embodiment, the gadolinium (Gd) in the compound may coordinate with one or more water molecules.

In the above formula (1), X may have a structure having the following formula (2).

(2)

* is the binding site.

In one embodiment, the compound may have the following formula (3).

(3)

In one embodiment, the compound can specifically bind to mammalian amyloid beta polymer (oligomeric Aβ). In one embodiment, the compound may have a relaxation rate of 4.8 to 5.2 s ^-1 . In one embodiment, the compound can pass through the blood-brain-barrier (BBB) when injected intravenously.

As discussed above, the compound according to the embodiment of the present invention can specifically bind to amyloid beta polymer.

An MRI contrast agent according to an embodiment of the present invention may include the above compound. In one embodiment, the MRI contrast agent can be used for diagnosis of degenerative brain disease. In one embodiment, the MRI contrast agent can be used to diagnose Alzheimer's disease.

As discussed above, the MRI contrast agent according to an embodiment of the present invention can be used in the diagnosis of degenerative brain diseases including Alzheimer's.

Hereinafter, embodiments of the present invention will be described in detail. However, the examples described below are only some embodiments of the present invention, and the scope of the present invention is not limited to the following examples.

<Synthesis of Gd-DO3A-Cou>

Gd-DO3A-Cou compound was prepared by stepwise synthesis.

1) tri-tert-butyl 2,2',2''-(10-(2-((2-(3-(4-hydroxyphenyl)acrylamido)ethyl)amino)-2-oxoethyl)-1,4, Synthesis of 7,10-tetraazacyclododecane-1,4,7-triyl)(E)-triacetate, 1

Coumaric aicd (0.2 g, 1.22 mmol), N, N-diisopropylethylamine (0.32 mL, 2.44 mmol), and HOBt (0.28 g, 1.83 mmol) were dissolved in DMF and stirred for 30 minutes. HBTU (0.93 g, 2.44 mmol) and DO3AtBU-N-(2-aminoethyl) ethanamide (0.75 g, 1.22 mmol) were added to the mixture and stirred at room temperature for 20 hours. The reaction was stopped by adding 10% NaHCO ₃ and extracted using EA (Ethyl acetate, 3 x 50 mL). The obtained organic layer was washed with brine solution, Na ₂ SO ₄ was added to remove remaining moisture, and dried under vacuum to obtain an oily product. Yield: 78%

2) (E)-2,2',2''-(10-(2-((2-(3-(4-hydroxyphenyl)acrylamido)ethyl)amino)-2-oxoethyl)-1,4,7 ,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

1 (0.8 g, 1.05 mmol) was dissolved in a mixed solution of TFA and CH ₂ Cl ₂ under an ice-bath and stirred for 22 hours until a clear solution was formed at room temperature. The reaction of the reactants was confirmed by TLC. After the reaction had sufficiently progressed, the solvent was removed and ethyl ether was added to obtain a precipitate. The precipitate was filtered, washed several times with ethyl ether, and dried under vacuum to obtain a pale yellow-white solid. Yield: 90%

3) Synthesis of GdL

L (0.5 g, 0.84 mmol) was dissolved in DI water and 1M NaOH solution was added to adjust pH to 7. GdCl ₃ ·6H ₂ O (0.31 g, 0.84 mmol) dissolved in DI water was slowly added to the above solution and stirred at room temperature for 22 hours. The pH was maintained at 7.0-7.4 while stirring. The reaction mixture was obtained by filtering and further purified using HPLC. Yield: 35%

<ESI-MS analysis results of Gd-DO3A-Cou>

Gd-DO3A-Cou was analyzed through ESI-MS. Figure 1 is a diagram showing the results. Referring to Figure 1, it can be seen that a peak (783.4634 m/z) corresponding to the peak (783.4632 m/z) predicted for Gd-DO3A-Cou appears.

<HR-ESI-MS of Gd-DO3A-Cou and ^One H NMR analysis results>

Gd-DO3A-Cou was analyzed by HR-ESI-MS and ¹ H NMR. Figures 2 and 3 are diagrams showing the results. Referring to Figure 2, it can be seen that a peak (593.2947 m/z) corresponding to the peak (593.2935 m/z) predicted for Gd-DO3A-Cou appears. Referring to Figure 3, it can be seen that the following ¹ H NMR results are obtained for Gd-DO3A-Cou.

<HR-MS and purity analysis results of Gd-DO3A-Cou>

Gd-DO3A-Cou was analyzed through HR-MS. Figure 4 is a diagram showing the results. Referring to Figure 4, it can be seen that a peak (748.1941 m/z) corresponding to the peak (748.1943 m/z) predicted for Gd-DO3A-Cou appears.

Additionally, the purity of Gd-DO3A-Cou was analyzed using HPLC. Figure 5 is a diagram showing the results. Referring to Figure 5, it can be seen that the synthesized compound has a purity of 96.7%.

<Gd-DO3A-Cou performance evaluation method and results>

1) Measurement of relaxation rate

The synthesized compounds were diluted in distilled water to concentrations of 0.0625, 0.125, 0.25, 0.5, and 1 mM, respectively, and then a phantom was produced. Measured values were compared with Gd-DO3A-BT, a commercial contrast agent. T ₁ (relaxation time) was measured in 3T MRI. The self-relaxation ratio ( r _{1 ,} r ₂ ) results of GdL synthesized through linear regression analysis are summarized in Table 1 below. In all solutions tested, GdL was measured to be similar to Gd-DO3A-BT. The self-relaxation rate is sufficient for clinical use, and the synthesized GdL can be used as an MRI contrast agent.

Compound Water PBS r _1mM ^-1 s ^-1 r _2mM ^-1 s ^-1 r _1mM ^-1 s ^-1 r _2mM ^-1 s ^-1 GdL 4.98±0.10 5.18±0.10 4.77±0.03 6.05±0.06 Gd-DO3A-BT 4.52±0.07 5.29±0.08 4.27±0.05 5.50±0.07

2) Stability measurement

2-1) Kinetic stability

- When a gadolinium-coordinated complex enters the human body, gadolinium ions undergo metal exchange with ions such as zinc and calcium in the human body, and may dissociate into free Gd. Since dissociated Gd ions cause toxicity in the body, the mechanical stability of GdL is very important.

- The synthesized GdL was prepared with a 2.5mM concentration, a PBS solution, and ZnCl ₂ with a 250mM concentration, a PBS solution. A commercial contrast agent, Gd-DO3A-BT, was used as a comparison group. Relaxation rates ( R ₂ ) were measured in 3T MRI for 4 days. If the change in relaxation rate (R ₂ ^p (t)/R ₂ ^p (0)) remains constant at 1, it can be considered stable. Figure 6 is a diagram showing the results. Referring to Figure 6, it was confirmed that GdL was stable by maintaining a constant level with Gd-DO3A-BT.

2-2) pH stability

- This is an experiment to check whether the synthesized GdL maintains stability at various pH. A phantom was prepared by dissolving 1mM GdL in pH buffer (1, 4, 7, 10). Relaxation rate ( R ₂ ) was measured on 3T MRI for 3 days. Figure 7 is a diagram showing the results. Referring to Figure 7, it was confirmed that the relaxation rate was maintained constant and stable.

3-1) Phantom experiment method to confirm amyloid beta polymer targeting

① 221.5 uL of HFIP was added to 1 mg of amyloid beta at a concentration of 1 mM, and preaggregation was removed using a shaker for 1 hour at room temperature.

② After drying in the air, add 221.5 uL of DMSO to make a 1mM concentration. Use a mixer and sonicator to make a suspension, then add 878.5 uL of PBS (1X, pH 7.4) to make a 0.2mM concentration.

③ After culturing at 37 ℃ for 4 days using a shaker, dispense 200 uL of the resulting amyloid beta polymer, add 20 uL of GdL dissolved in PBS at a concentration of 2 mM and Gadovist ^® , a commercial contrast agent, and then shaker. and cultured at 37°C for 24 hours.

④ 24 hours after the culture was completed, centrifugation was performed to remove the supernatant, washed off the amyloid beta polymer using PBS, and 200 uL of a solution of PBS:DMSO = 9:1 was added to the pellet to prepare a phantom sample.

3-2) Evaluation of amyloid beta polymer targeting ability

An experiment to evaluate amyloid beta polymer targeting ability was conducted on a 9.5T MR equipment. Figure 8(b) is a diagram showing the results.

The contrast enhancement effect was similar in the group cultured with only amyloid beta polymer and the group cultured with commercial contrast agent. This means that commercial contrast agents do not have a targeting effect on amyloid beta polymer. The group treated with the synthesized compound showed a high contrast enhancement effect compared to commercial contrast agents. As a result, it is determined that the synthesized compound has a targeting effect on amyloid beta polymer. You can check the difference through the MR signal comparison graph of the MR T1 phantom. The synthesized GdL can be evaluated to have a function as an amyloid beta polymer targeting contrast agent.

Although the present invention has been described above with reference to preferred embodiments, those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the present invention as set forth in the following patent claims. You will understand that it is possible.

Claims (12)

Having the following formula (1),
compound:
(One)
here,
L is *-(CH ₂ ) _x -A ¹ -(CH ₂ ) _y -A ² -(CH ₂ ) _z -*,
x, y and z are each independently selected as random integers from 0 to 5,
A ¹ and A ² are single bonds, in the group containing *-COO-*, *-CO-*, *-NH-*, *-CH ₂ -*, *-CONH-* and *-O-* Each is one or more independently selected structures,
X is a structure having the following formula (2):
(2)
* is the binding site. According to paragraph 1,
The A ² is *-NH-*,
compound. According to paragraph 2,
The A ¹ is *-CONH-*,
compound. According to paragraph 3,
where x is 1,
where y is 2,
where z is 0,
compound. According to paragraph 1,
Having the following formula (3),
(3)
compound. According to paragraph 1,
The gadolinium (Gd) coordinates with one or more water molecules,
compound. According to paragraph 1,
The compound specifically binds to mammalian amyloid beta polymer (oligomeric Aβ),
compound. According to paragraph 1,
The compound has a relaxation rate of 4.8 to 5.2 s ^-1 ,
compound. According to paragraph 1,
The compound crosses the blood-brain-barrier (BBB) when injected intravenously.
compound. Comprising a compound according to any one of claims 1 to 9,
MRI contrast agent. According to clause 10,
The MRI contrast agent is used for diagnosis of degenerative brain diseases,
MRI contrast agent. According to clause 11,
The MRI contrast agent is used in the diagnosis of Alzheimer's disease,
MRI contrast agent.