KR20230014658A - 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 caffeic acid is bound. The MRI contrast agent may include the compound. One object of the present invention is to provide the compound that specifically binds to an amyloid beta polymer.

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.

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

Amyloid beta (Aβ) is a major 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 is directly involved in the development of Alzheimer's disease as it accumulates excessively in the brain. It is known to do Therefore, it was expected that detecting changes in the concentration of amyloid beta polymer would enable early diagnosis of degenerative brain diseases.

On the other hand, Magnetic Resonance Image (MRI) is a method of obtaining anatomical, physiological, and biochemical information images of the body by using a phenomenon in which the distribution of hydrogen atoms is different between tissues in the body and the hydrogen atoms are relaxed in a magnetic field. . Unlike CT or PET, MRI does not use radiation that is harmful to the human body and uses radio waves and the gradient of a magnetic field under a strong magnetic field to create images of the inside of the body.

In order to use such MRI equipment more precisely, a contrast agent is injected into an object to obtain an MRI image. The contrast between the tissues on the MRI image is a phenomenon that occurs because the relaxation action in which the nuclear spin of water molecules in the tissue returns to an equilibrium state is different for each tissue. The contrast agent uses a paramagnetic or superparamagnetic material to affect the relaxation effect, thereby widening the difference in relaxation between tissues and causing a change in the MRI signal, thereby making the contrast between tissues clearer.

Currently, the most commonly used contrast agent clinically is a contrast agent based on a 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 commercially available contrast agents are non-specific contrast agents that are distributed in the extracellular space (ECF). As a specific contrast agent, only a liver-specific contrast agent is used. Recent studies have been promoting the development of contrast agents that have specific targets or can exhibit signal enhancement by physiological activity (pH change, enzyme activity), but so far, MRI contrast agents with specific targets, 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 compound.

In one aspect, the present invention provides a compound having Formula (1) below.

(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 any integer from 0 to 5;

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

X is a structure having formula (2):

(2)

(2)

* is a bonding site.

In one embodiment, the A ² may be *-NH-*.

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

In one embodiment, the x may be 1.

In one embodiment, the y may be 2.

In one embodiment, the z may be 0.

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

(3)

(3)

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

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

In one embodiment, the compound may have a magnetic relaxation rate (relaxivity) of 8.5 to 8.9 s ^-1 in water.

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

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

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

In one embodiment, the MRI contrast agent may be used for diagnosis of Alzheimer's disease.

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

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

1 to 7 are views 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 accompanying drawings. Since the present invention can have various changes and 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 form disclosed, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. Like reference numerals have been used for like elements throughout the description of each figure. In the accompanying drawings, the dimensions of the structures are shown enlarged than actual for clarity of the present invention.

Terms used in this application are only used to describe specific embodiments, and are not intended to limit the present 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 that there is a feature, number, step, operation, component, or combination thereof described in the specification, but one or more other features or numbers However, it should be understood that it does not preclude the presence or addition of steps, operations, components, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

A compound according to an embodiment of the present invention may have the following formula (1).

(1)

(One)

In the above formula (1), the gadolinium ion (Gd ³⁺ ) may coordinate with the carboxylate (COO ⁻ ) group of the above formula (1) to form a complex compound.

In 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 Each can be independently selected as an integer of, A ¹ and A ² are single bonds, *-COO-*, *-CO-*, *-NH-*, *-CH ₂ -*, *-CONH-* And *-O-* may be one or more structures each independently selected from the group containing. * is a bonding site.

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

As long as the compounds according to the embodiments of the present invention substantially have the structure of Formula (1) above, binding or removal of binding 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 Formula (1), X may have a structure having the following Formula (2).

(2)

(2)

* is a bonding site.

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

(3)

(3)

In one embodiment, the compound may specifically bind to mammalian amyloid beta polymer (oligomeric Aβ). In one embodiment, the compound may have a magnetic relaxation rate (relaxivity) of 8.5 to 8.9 s ^-1 in water. In one embodiment, the compound can cross the blood-brain barrier (BBB) when injected intravenously.

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

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

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

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

<Synthesis of Gd-DO3A-Caf>

A Gd-DO3A-Caf compound was prepared by stepwise synthesis.

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

Caffeic acid (0.2 g, 1.11 mmol), N,N-diisopropylethylamine (0.28 mL, 2.22 mmol), and HOBt (Hydroxybenzotriazole, 0.25 g, 1.50 mmol) were dissolved in DMF (Dimethylformamide, 10 mL) and stirred for 30 minutes. HBTU ((2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, (0.84 g, 2.22 mmol) and DO3A-NH ₂ (0.68 g, 1.11 mmol) were added to the mixture. 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 a brine solution and then Na ₂ SO ₄ was added The remaining moisture was removed and dried under vacuum to obtain an oily product. Yield: 74%

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

1 (0.9 g, 1.16 mmol) was dissolved in a mixed solution of TFA and CH ₂ Cl ₂ under an ice-bath, stirred at room temperature until it became a clear solution, and the reaction of the reactants was confirmed by TLC. After the reaction proceeded sufficiently, the solvent was removed and ethyl ether was added to obtain a precipitate. The precipitate was filtered, washed with ethyl ether several times, and vacuum dried to obtain a pale yellowish white solid. Yield: 88%

3) Synthesis of GdL

L (0.5 g, 0.82 mmol) was dissolved in DI water (10 mL), and 1M NaOH solution was added to the pH to 7. GdCl ₃ 6H ₂ O (0.30 g, 0.82 mmol) dissolved in DI water (5 mL) was slowly added to the above solution, followed by stirring at room temperature for one day. During stirring, the pH was maintained at 7-7.3. A reaction mixture was obtained by filtering and further purification was performed using HPLC. Yield: 38%

<Results of ESI-MS analysis of Gd-DO3A-Caf>

Gd-DO3A-Caf was analyzed through ESI-MS. 1 is a diagram showing the result. Referring to FIG. 1, it can be confirmed that a peak (776.07 m/z) corresponding to the predicted peak (776.47 m/z) for the Gd-DO3A-Caf appears.

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

Gd-DO3A-Caf was analyzed by HR-ESI-MS. 2 is a diagram showing the result. Referring to FIG. 2, it can be confirmed that peaks (609.2885 m/z, 631.2702 m/z) corresponding to the peaks (609.2884 m/z, 631.2704 m/z) predicted for the Gd-DO3A-Caf appear. In addition, Gd-DO3A-Caf was analyzed through ¹ H NMR, and the following results were obtained.

<Results of HR-MS and purity analysis of Gd-DO3A-Caf>

Gd-DO3A-Caf was analyzed by HR-MS. 3 is a diagram showing the result. Referring to FIG. 3, it can be seen that a peak (765.1894 m/z) corresponding to the predicted peak (764.1890 m/z) appears for the Gd-DO3A-Caf.

The purity of Gd-DO3A-Caf was analyzed using HPLC. 4 is a diagram showing the result. Referring to FIG. 4, it can be confirmed that Gd-DO3A-Caf has a purity of 95.1%.

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

1) Measurement of relaxation rate

After diluting the synthesized Gd-DO3A-Caf in distilled water to concentrations of 0.0625, 0.125, 0.25, 0.5, and 1 mM, respectively, phantoms were fabricated. The measured values were compared with Gd-DO3A-BT, a commercial contrast agent. Relaxation time ( T ₁ , T ₂ ) was measured in 3T MRI. Table 1 summarizes the results of the self-relaxation rates ( r _{1 ,} r ₂ ) of GdL synthesized through linear regression analysis. For water and PBS, r _{1 and} r ₂ of GdL were measured higher than those of Gd-DO3A-BT. The self-relaxation rate is sufficient for clinical use, and synthesized GdL can be used as an MRI contrast agent.

Compound Water PBS r ₁ mM ^-1 s ^-1 r ₂ mM ^-1 s ^-1 r ₁ mM ^-1 s ^-1 r ₂ mM ^-1 s ^-1 GdL 8.41±0.07 12.2±0.05 5.09±0.04 8.61±0.05 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, the gadolinium ion undergoes metal exchange with ions such as zinc and calcium in the human body and can be dissociated into free Gd. Since dissociated Gd ions cause toxicity in the body, the mechanical stability of GdL is very important.

The synthesized 2.5 mM GdL was prepared as a PBS solution, 250 mM ZnCl ₂ concentration, and a PBS solution, and Gd-DO3A-BT, a commercial contrast agent, was used as a comparison group. Relaxation rates ( R ₂ ) in 3T MRI were measured for 4 days. 5 is a diagram showing the result. If the change in relaxation rate (R ₂ ^p (t)/R ₂ ^p (0)) remains constant at 1, it can be considered stable. Referring to FIG. 5, it was confirmed that GdL was stable with Gd-DO3A-BT.

2-2) pH stability

It was confirmed whether the synthesized GdL maintains its stability at various pHs. A phantom was prepared by dissolving 1 mM GdL in various pH buffers (1, 4, 7, 10). Relaxation rate ( R ₂ ) in 3T MRI was measured for 3 days. 6 is a diagram showing the result. Referring to FIG. 6, it was confirmed that the pH was stable even when the pH was changed through the constant relaxation rate.

3-1) Phantom test method for confirming amyloid beta polymer targeting

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

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

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

④ After 24 hours of incubation, the supernatant was removed by centrifugation, and the amyloid beta polymer was washed away using PBS, and 200 uL of PBS: DMSO = 9: 1 solution was added to the pellet to prepare a phantom sample.

3-2) Experimental results of structure of GdL and targeting effect of amyloid beta polymer

Amyloid beta polymer targeting ability evaluation experiment was conducted in a 9.4T MR equipment. Figure 7 (b) is a diagram showing the result.

It was confirmed by dividing into three groups. The group in which only amyloid beta polymer was cultured and the group treated with a commercial contrast agent showed similar contrasting effects, but the group treated with the synthesized compound showed a higher contrasting effect. Through this, it can be evaluated that the synthesized compound has an amyloid beta targeting effect, and the difference was confirmed through the R1 relaxation rate experiment.

Although the above has been described with reference to preferred embodiments of the present invention, those skilled in the art can variously modify and change the present invention without departing from the spirit and scope of the present invention described in the claims below. You will understand that you can.

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 any integer from 0 to 5;
A ¹ and A ² are a single bond, in the group containing *-COO-*, *-CO-*, *-NH-*, *-CH ₂ -*, *-CONH-* and *-O-* one or more structures, each independently selected,
X is a structure having formula (2):

(2)
* is a bonding site. According to claim 1,
Wherein A ² is *-NH-*;
compound. According to claim 2,
Wherein A ¹ is *-CONH-*,
compound. According to claim 3,
wherein x is 1,
wherein y is 2,
wherein z is 0;
compound. According to claim 1,
Having the following formula (3),

(3)
compound. According to claim 1,
The gadolinium (Gd) coordinates with one or more water molecules,
compound. According to claim 1,
The compound specifically binds to mammalian amyloid beta polymer (oligomeric Aβ),
compound. According to claim 1,
The compound has a relaxation rate of 8.5 to 8.9 s ^-1 in water,
compound. According to claim 1,
The compound, when injected via intravenous injection, crosses the blood-brain barrier (BBB),
compound. Comprising the compound according to any one of claims 1 to 9,
MRI contrast agent. According to claim 10,
The MRI contrast agent is used for diagnosis of degenerative brain disease,
MRI contrast agent. According to claim 11,
The MRI contrast agent is used for diagnosis of Alzheimer's disease,
MRI contrast agents.

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