Freeze-dried and redissolved iron oxide MRI contrast agent preparation and stabilizer composition thereof

Abstract

The invention discloses a freeze-dried and redissolved iron oxide MRI (Magnetic Resonance Imaging) contrast agent preparation and a stabilizer composition thereof. The preparation comprises superparamagnetic iron oxide nanoparticles and the stabilizer composition consisting of mannitol, trehalose, a polyethylene glycol derivative and glycine in a specific concentration ratio. According to the composition, through the synergistic effect of multiple components, the ice crystal size can be effectively controlled in the freeze-drying process, coordination aggregation of iron ions among particles is blocked, and the collapse temperature of a system is increased, so that a freeze-dried cake body with a complete structure is obtained; and the particle size and the magnetic resonance relaxation performance (r2) of the redissolved superparamagnetic iron oxide nanoparticles are highly maintained. The preparation solves the technical bottleneck that the existing superparamagnetic iron oxide contrast agent is poor in stability, depends on a cold chain and is short in shelf life, realizes long-term stable storage at room temperature and clinical rapid redissolution use, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of medical nanomaterials and pharmaceutical formulation technology, specifically relating to a freeze-dried and reconstituteable iron oxide MRI contrast agent formulation and its stabilizer composition. Background Technology

[0002] In magnetic resonance imaging (MRI), superparamagnetic iron oxide (SPIO) nanoparticles were once an important contrast agent, but they have been withdrawn from the market for many years due to defects such as insufficient formulation stability, the need for stringent cold chain storage and transportation, and short shelf life. Currently, SPIO is still regarded as a potential alternative to gadolinium-based contrast agents due to its good biocompatibility, strong magnetism, and biodegradability, but existing technical solutions all have significant bottlenecks.

[0003] Currently, SPIO formulations are mostly in the form of aqueous dispersions. Due to their high surface energy and static magnetic interaction, the nanoparticles are prone to severe aggregation during storage (aggregation rate >25% within 12 months), leading to a 30-40% decrease in relaxation performance. Furthermore, they require cold chain storage and transportation at 2-8°C, as irreversible aggregation occurs once frozen, resulting in a shelf life typically less than 12 months, high clinical cost, and poor accessibility. To address this issue, freeze-drying technology has been attempted. However, traditional freeze-drying processes fail in SPIO systems due to differences in material properties: large ice crystals (>50 μm) generated during the pre-freezing stage mechanically compress the particles, causing Fe-O-Fe bridging and a surge in particle size; during drying, the hydration layer on the particle surface is removed, exposing empty Fe³⁺ orbitals that lead to irreversible coordination aggregation between particles, resulting in a magnetic property loss >50%; simultaneously, the collapse temperature of SPIO dispersions is extremely low (<-35°C), which conventional freeze-drying protectants cannot effectively raise, resulting in a freeze-dried cake collapse rate >60%. Existing improved solutions, such as patent CN103316361A which uses single hydroxyethyl starch for protection, still suffer from problems such as particle size increase >120% after reconstitution, r2 loss rate 35%, and reconstitution time >10 minutes, failing to meet the clinical requirements for rapid and stable use. Therefore, developing a novel formulation technology that can achieve stable lyophilization, rapid reconstitution, and excellent performance maintenance of SPIO nanoparticles has become an urgent need in the industry. Summary of the Invention

[0004] The present invention aims to provide a freeze-dried and reconstituteable iron oxide MRI contrast agent formulation and its stabilizer composition, which solves the problems of existing superparamagnetic iron oxide (SPIO) contrast agents, such as poor physical stability, reliance on cold chain storage and transportation, short shelf life, and the inability of traditional freeze-drying technology to be applied to the SPIO system, resulting in nanoparticle agglomeration, decreased magnetic properties, and collapse of freeze-dried cakes after reconstitution.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A lyophilized and reconstituteable iron oxide MRI contrast agent formulation comprises superparamagnetic iron oxide nanoparticles and a stabilizer composition; the stabilizer composition comprises the following components based on the concentration of the main component in the formulation solution: Mannitol: 1.5%-3.0% (w / v); Trehalose: 1.0%-2.0% (w / v); Polyethylene glycol derivatives: 0.3%-0.8% (w / v); Glycine: 0.2%-0.5% (w / v).

[0006] Furthermore, the iron content concentration of the superparamagnetic iron oxide nanoparticles is 1.5 mg / mL to 2.5 mg / mL.

[0007] Furthermore, the concentrations of each component in the stabilizer composition are mannitol 2.0% (w / v), trehalose 1.5% (w / v), the polyethylene glycol derivative 0.5% (w / v), glycine 0.3% (w / v), and the iron concentration of the superparamagnetic iron oxide nanoparticles is 2.0 mg / mL.

[0008] Furthermore, the molecular weight of the polyethylene glycol derivative is 1000-5000 Da.

[0009] Furthermore, the polyethylene glycol derivative is methoxy polyethylene glycol 2000.

[0010] Furthermore, the particle size of the superparamagnetic iron oxide nanoparticles is 1 nm to 20 nm.

[0011] Furthermore, the superparamagnetic iron oxide nanoparticles have a particle size of 3 nm to 8 nm.

[0012] A stabilizer composition for a freeze-dried and reconstituteable iron oxide MRI contrast agent formulation, comprising mannitol, trehalose, a polyethylene glycol derivative, and glycine, wherein the concentrations of each component in the final formulation are: mannitol 1.5%-3.0% (w / v), trehalose 1.0%-2.0% (w / v), polyethylene glycol derivative 0.3%-0.8% (w / v), and glycine 0.2%-0.5% (w / v).

[0013] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention effectively inhibits the mechanical damage of ice crystals and the aggregation of dehydration stress during the freeze-drying process by using specific concentrations of mannitol, trehalose, polyethylene glycol derivatives and glycine, and controls the particle size increase of nanoparticles after reconstitution to ≤5.5% and the relaxation rate (r2) retention rate to ≥92%, thus solving the industry problem of freeze-drying agglomeration and performance degradation of SPIO nanoparticles. (2) The resulting lyophilized formulation can be stored stably at room temperature for a long time (predicted shelf life of up to 24 months), eliminating the absolute dependence on the cold chain and achieving rapid and complete reconstitution within 30 seconds, which greatly improves the convenience and accessibility of clinical use. (3) The stabilizer composition exhibits good protective effect on SPIO particles of different particle sizes (1-20 nm), and has a wide formulation process window, good compatibility with existing freeze-drying and injection production lines, and has the potential for large-scale industrial production. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] The present invention will be further described in detail below with reference to the embodiments.

[0016] Example 1: Preparation and performance testing of an optimally formulated lyophilized and reconstituteable iron oxide MRI contrast agent. A lyophilized and reconstituteable iron oxide MRI contrast agent formulation comprises superparamagnetic iron oxide nanoparticles and a stabilizer composition. The superparamagnetic iron oxide nanoparticles are polyethylene glycol-modified superparamagnetic iron oxide nanoparticles with a hydrated particle size of 4-6 nm, a core crystal size of approximately 5 nm, an iron concentration of 2.0 mg / mL in the stock solution, and a transverse relaxation rate r² = 51.3 mM. -1 ·s -1 The longitudinal relaxation rate r1 = 8.6 mM -1 ·s -1 The ratio of r2 to r1 is approximately 6.0.

[0017] Based on the final volume of the solution, the stabilizer composition comprises the following components: Mannitol (injection grade): 2.0% (w / v), Trehalose (HPLC grade, purity ≥99.5%): 1.5% (w / v), Methoxy polyethylene glycol 2000 (mPEG2000): 0.5% (w / v), Glycine (USP standard): 0.3% (w / v).

[0018] A process for preparing a lyophilized and reconstituteable iron oxide MRI contrast agent formulation includes the following steps: (1) Solution preparation: The superparamagnetic iron oxide stock solution and the aqueous solution containing the above four excipients were sterile filtered through a 0.22μm polyethersulfone (PES) filter membrane; (2) Mixing: At 4°C, the filtered superparamagnetic iron oxide stock solution and the auxiliary material solution are mixed in proportion and placed on a magnetic stirrer and stirred at low speed (about 200 rpm) for 30 minutes to ensure uniform mixing; (3) pH adjustment: Use dilute sodium hydroxide solution or dilute hydrochloric acid solution to precisely adjust the pH value of the mixture to 7.2 ± 0.1; (4) High-pressure homogenization: The mixture was transferred to a high-pressure homogenizer and homogenized at 8000 rpm for 3 minutes, with a shear rate of approximately 5000 s⁻¹. -1 To obtain a highly dispersed and uniform suspension; (5) Filling: The homogenized liquid is aseptically filled into vials, with each vial containing 2 mL. (6) Freeze-drying: The following process parameters were used for freeze-drying: First, the pre-freezing stage was carried out by placing the filled vials on the freeze dryer partition and cooling them to -45°C at a cooling rate of 1.8°C / min and holding them for 2 hours. The supercooling in this process was greater than 15°C, and the average size of the ice crystals formed was less than 10 μm. Second, the primary drying (sublimation) stage was carried out by starting the vacuum pump and controlling the chamber pressure at 20 Pa. The partition temperature was raised from -25°C to -15°C at a slow heating rate of 0.5°C / h. This stage lasted for about 12 hours. Third, the secondary drying (desorption) stage was carried out by further increasing the partition temperature to 25°C and holding it for 4 hours, while reducing the chamber pressure to about 10 Pa. The drying endpoint was determined by the pressure rise method to ensure that the residual moisture content was less than 2%. After freeze-drying, the vials were sealed with caps to obtain a white, loose, cake-like solid with an intact structure and no collapse.

[0019] The reconstitution of the obtained lyophilized formulation (adding 2 mL of water for injection and shaking manually) and its performance characterization are shown below: Resolution performance: The resolution time is approximately 25-30 seconds, and the solution is clear with no visible particles or precipitate. Physical stability: The hydrated particle size of the reconstituted nanoparticles was determined using dynamic light scattering (DLS). The results showed that the average increase in particle size was only +3.2%, indicating that particle aggregation was effectively suppressed.

[0020] Magnetic properties: Relaxation rate was measured on a 1.5 T clinical MRI scanner. After rehydration, r1 retention was 96%, and r2 retention was 95%. Signal-to-noise ratio (SNR) retention was 97.2%.

[0021] Appearance of the formulation: The freeze-dried cake is 100% intact, with no shrinkage, collapse or melting.

[0022] Accelerated stability testing: The lyophilized formulation was subjected to accelerated stability testing for 30 days in a constant temperature and humidity chamber at 40°C and 75% relative humidity (40°C / 75% RH). The test results showed that the r2 retention rate remained as high as 96%. Based on the Arrhenius equation, the theoretical shelf life of this formulation under normal storage conditions at 25°C is estimated to be up to 24 months, with a predicted r2 retention rate of over 91%.

[0023] Example 2: Verification of Formulation Parameter Range To demonstrate that the formulation of this invention has a broad and robust process window, the concentrations of each component of the stabilizer composition and the concentration of superparamagnetic iron oxide were adjusted to the limits and intermediate values ​​within the scope of the claims, based on Example 1, for verification. The preparation process was the same as in Example 1, and the key parameters and results are summarized in the table below: Parameter categories Minimum solution intermediate value scheme Maximum value solution Mannitol (w / v) 1.5％ 2.0％ 3.0％ Trehalose (w / v) 1.0％ 1.5％ 2.0％ PEG derivatives (w / v) 0.3％ 0.5％ 0.8％ Glycine (w / v) 0.2％ 0.3％ 0.5％ SPIO concentration Fe 1.5 mg / mL Fe 2.0 mg / mL Fe 2.5 mg / mL <![CDATA[r2 before lyophilization]]> <![CDATA[48.5 mM -1 ·s -1 ]]> <![CDATA[51.3 mM -1 ·s -1 ]]> <![CDATA[53.8 mM -1 ·s -1 ]]> <![CDATA[Retention rate of r2 after reconstitution]]> 92％ 95％ 94％ Reconstitution time 35 s 30 s 32 s Particle size increase +4.8％ +3.2％ +5.5％ biscuit integrity 95％ 100％ 98％ Even when the concentrations of each component and the SPIO concentration are at the limits of the claims, the prepared lyophilized formulation still meets the key performance requirements of short reconstitution time (≤35 seconds), small particle size increase (≤+5.5%), high relaxation rate retention (≥92%), and intact cake structure (integrity ≥95%). This indicates that the formulation window of the present invention is broad, the process is robust, and it is easy to achieve industrial production and quality control.

[0024] Example 3: Verification of the universality of SPIO particles with different sizes To verify the universal protective effect of the stabilizer composition of this invention on SPIO nanoparticles of different sizes, four different particle size ranges of SPIO particles were selected. The freeze-dried formulations were prepared and tested using the stabilizer formulation (mannitol 2.0%, trehalose 1.5%, mPEG2000 0.5%, glycine 0.3%) and process described in Example 1. The results are shown in the table below: Particle size range <![CDATA[r1 before lyophilization]]> <![CDATA[r2 before lyophilization]]> <![CDATA[r1 retention rate]]> <![CDATA[r2 retention rate]]> Reconstitution time shelf life 1-3 nm 5.2±0.8 24.5±3.2 94% 92% 25 s 18 months 3-8 nm 8.6±1.2 51.3±5.8 96% 95% 30 s 24 months 8-15 nm 4.1±0.6 98.7±12.4 93% 94% 35 s 18 months 15-20 nm 2.8±0.5 156.2±18.6 91% 92% 40 s 12 months The stabilizer composition of this invention exhibits excellent lyophilization protection for superparamagnetic iron oxide nanoparticles with particle sizes ranging from 1 nm to 20 nm. Particularly within the preferred particle size range of 3-8 nm, it not only demonstrates the optimal r1 / r2 contrast ratio (approximately 6.0), but also exhibits the best retention of relaxation rate, reconstitution rate, and predicted stability after lyophilization and reconstitution. This proves that the technical solution of this invention has broad applicability and can cover the core size requirements of mainstream SPIO contrast agent products.

[0025] It should be noted that, in this document, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0026] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A lyophilized and reconstituteable iron oxide MRI contrast agent formulation, characterized in that, The formulation comprises a composition of superparamagnetic iron oxide nanoparticles and a stabilizer; based on the concentration of the main component in the solution, the stabilizer composition comprises the following components: Mannitol: 1.5%-3.0% (w / v); Trehalose: 1.0%-2.0% (w / v); Polyethylene glycol derivatives: 0.3%-0.8% (w / v); Glycine: 0.2%-0.5% (w / v).

2. The lyophilized and reconstituteable iron oxide MRI contrast agent formulation according to claim 1, characterized in that, The iron content concentration of the superparamagnetic iron oxide nanoparticles is 1.5 mg / mL to 2.5 mg / mL.

3. The lyophilized and reconstituteable iron oxide MRI contrast agent formulation according to claim 2, characterized in that, The stabilizer composition comprises mannitol 2.0% (w / v), trehalose 1.5% (w / v), the polyethylene glycol derivative 0.5% (w / v), glycine 0.3% (w / v), and the superparamagnetic iron oxide nanoparticles have an iron concentration of 2.0 mg / mL.

4. The lyophilized and reconstituteable iron oxide MRI contrast agent formulation according to claim 3, characterized in that, The molecular weight of the polyethylene glycol derivative is 1000-5000 Da.

5. The lyophilized and reconstituteable iron oxide MRI contrast agent formulation according to claim 4, characterized in that, The polyethylene glycol derivative is methoxy polyethylene glycol 2000.

6. The lyophilized and reconstituteable iron oxide MRI contrast agent formulation according to claim 3, characterized in that: The superparamagnetic iron oxide nanoparticles have a particle size of 1 nm to 20 nm.

7. The lyophilizable and reconstituteable iron oxide MRI contrast agent formulation according to claim 6, characterized in that: The superparamagnetic iron oxide nanoparticles have a particle size of 3 nm to 8 nm.

8. A stabilizer composition for a lyophilized and reconstituteable iron oxide MRI contrast agent formulation, characterized in that, It is composed of mannitol, trehalose, polyethylene glycol derivative and glycine, wherein the concentrations of each component in the final formulation are as follows: mannitol 1.5%-3.0% (w / v), trehalose 1.0%-2.0% (w / v), polyethylene glycol derivative 0.3%-0.8% (w / v) and glycine 0.2%-0.5% (w / v).