Alcohol detoxification

Abstract

The present invention relates to the field of alcoholism and its treatment and discloses novel hybrid materials and pharmaceutical compositions, methods of their preparation and their use in cases of excessive alcohol consumption. The disclosed hybrid materials comprise (a) an amyloid, such as a fibrillar amyloid, and (b) a plurality of single-site iron coordinated to the amyloid. These hybrid materials can be converted into hydrogels, pharmaceutical compositions comprising such hydrogels and oral dosage forms comprising such hydrogels. Further disclosed are methods of manufacturing such hybrid materials and hydrogels and methods of using such hybrid materials and hydrogels for the treatment of alcoholism.

Description

Technical Field

[0001] This invention relates to the field of alcohol poisoning and its treatment, including detoxification and prevention. Therefore, this invention discloses novel materials and compositions, methods for their preparation, and their use in cases of excessive alcohol consumption. Background Technology

[0002] Providing strategies to reduce the global health impacts of widespread alcohol consumption is a challenging and provocative task. While positive effects have been observed with intravenous administration of natural enzyme complexes, their insufficient activity and complex administration often lead to the accumulation of toxic acetaldehyde, raising significant clinical concerns and highlighting the urgent need for stable oral strategies.

[0003] While various therapies exist to mitigate the effects of alcohol, they primarily constitute supportive measures, relying on endogenous enzymes to metabolize alcohol. These substances, acting as alcohol detoxifiers, only temporarily relieve symptoms such as nausea and headaches, but fail to address other significant problems, such as drowsiness, fatigue, and chronic alcoholism. In recent years, nanocomposites composed of multiple complementary liver enzymes have emerged as a promising approach to accelerate alcohol metabolism in the body. Despite this promising outlook, a major obstacle lies in the insufficient activity of commercially available aldehyde dehydrogenase (ALDH) or aldehyde oxidase (ADOx) enzymes, leading to the accumulation of the more dangerous intermediate acetaldehyde and further damage to human organs.

[0004] For example, Xie et al. (Journal of Ethnopharmacology 282 (2022): 114593) discussed the protective effect and mechanism of a modified mung bean and licorice (MLG) decoction against acute alcohol poisoning in mice.

[0005] In addition, Xu et al. (Advanced Materials 30.22 (2018): 1707443) evaluated a liver cell-mimicking detoxifier by co-delivering nanocapsules of alcohol oxidase (AOx), catalase (CAT), and aldehyde dehydrogenase (ALDH) to the liver, where AOx and CAT catalyze the oxidation of alcohol to acetaldehyde, while ALDH catalyzes the oxidation of acetaldehyde to acetate.

[0006] In addition, Chen et al. (Alcoholism: Clinical and Experimental Research 38.7(2014): 1839-1846) evaluated the effects of kudzu root extract (FPE) on alcohol metabolism, liver damage and memory impairment in mice after acute ethanol (EtOH) poisoning.

[0007] Furthermore, Liu et al. (Nature nanotechnology 8.3 (2013): 187-192) demonstrated that two or more enzymes with complementary functions can be assembled and encapsulated within a thin polymer shell to form enzyme nanocomposites. Compared to free enzymes, these nanocomposites exhibit improved catalytic efficiency and enhanced stability. These nanocomposites are considered potential applications as an antidote and preventative measure against alcohol poisoning.

[0008] While the aforementioned prior art is considered a promising method for treating alcohol poisoning, it requires non-oral administration of the disclosed composition. It would be beneficial to provide a safe and effective oral administration route for treating alcohol poisoning.

[0009] Nanozymes have been proposed as a possible strategy for alcohol detoxification. For example, Geng et al. ( ACS Nano (2023, 17, 8, 7443-7455) Alcohol detoxification was achieved in a mouse model using mesoporous metal-organic framework nanozymes conjugated with enzymes. Graphene oxide quantum dots have also been proposed as nanozymes with peroxidase-like activity for alcohol poisoning, as described by Sun et al. (2023, 17, 8, 7443-7455). ACS Appl. Mater. Interfaces (2017, 9, 14, 12241-12252). However, safety concerns associated with these materials keep their practical application to humans far off.

[0010] In the context of nutritional materials, Bolisetty et al. (WO2018 / 166947) disclosed a composite material comprising amyloid protoplasmic fibers and nanoparticles of iron minerals (such as iron oxides, hydroxides, and salts). This literature demonstrated improved bioavailability of iron as a food supplement and confirmed that the disclosed composite material could be rapidly digested by enzymes; however, its therapeutic effect on alcohol poisoning remained undisclosed.

[0011] Similarly, Shen et al. ( NATURE NANOTECHNOLOGY Volume 12, No. 7, April 24, 2017, pp. 642-647 discusses iron deficiency anemia as a global public health problem and suggests iron-amyloid fibrillary hybrids as a novel and efficient iron delivery system in both solid and liquid matrices. The hybrids disclosed in that document differ from those of this invention in that the iron is not present as a single-site iron but rather as nanoparticles, see, for example... Figure 1 e and 1g. This literature remains silent on the treatment of alcohol poisoning.

[0012] Remondetto et al. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY(Volume 52, No. 26, December 1, 2004, pp. 8137-8143) A study in vitro discussed the availability of iron in whey protein hydrogels and concluded that filamentous gels show promise as a matrix for transporting iron and promoting its absorption, and therefore should be of primary interest in the development of innovative functional foods. This literature remains silent on the properties of iron within the gel. Instead, it focuses on the influence of gel structure and gel microstructure on iron release. This literature also remains silent on the treatment of alcohol poisoning.

[0013] Choi et al. ANGEWANDTE CHEMIE Int. Ed. Volume 50, No. 27, May 20, 2011, pp. 6070-6074) discusses a hybrid material comprising photoelectric protein nanofibrils of α-synuclein with embedded iron and phthalocyanine tetrasulfonate. The authors demonstrate that the porphyrin compound phthalocyanine tetrasulfonate (PcTS) selectively localizes in the protein matrix of amyloid protofibrils made of α-synuclein in the presence of iron, leading to the transformation of the dielectric protein nanofibrils into photoinduced conductive hybrid nanomaterials. Unlike the hybrid materials disclosed in this invention, the nanofibrils in Choi et al. are spherical, and the protofibrils are formed by the linkage of phthalocyanine tetrasulfonate ligands (see Scheme 1 of Choi et al.). Therefore, the hybrid material of Choi et al. differs from the hybrid material of this invention. Furthermore, the hybrid material was prepared in the nonpolar organic solvent hexane, while the synthetic scheme of this invention requires the presence of a polar organic solvent. Additionally, as with other literature, Choi et al. remain silent on the treatment of alcohol poisoning. Summary of the Invention

[0014] The invention will now be described in more detail with reference to its first, second, and third aspects. The first aspect relates to novel materials (hybrid materials and hydrogels), the second aspect relates to pharmaceutical compositions and their uses, and the third aspect relates to the manufacture of the aforementioned novel materials and pharmaceutical compositions. It should be understood that the various embodiments, preferred embodiments, and scopes provided / disclosed in this specification can be combined in any way. Furthermore, depending on the specific implementation, certain definitions, embodiments, or scopes may be selectively excluded.

[0015] Unless otherwise stated, the following definitions shall apply to this specification:

[0016] As used herein, the terms “a”, “the”, and similar terms used in the context of this invention (particularly in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by the context. Furthermore, as used herein, the terms “comprising,” “containing,” and “including” are used in their open, non-limiting sense. The term “containing” should include “comprising,” “substantially consisting of,” and “consisting of.”

[0017] Pharmaceutical composition: This term, as is known in the art, describes any composition suitable for treating, preventing, or delaying the progression of disease in a subject of need. This term particularly includes formulations suitable for oral administration, such as liquid or solid dosage forms. Suitable liquid dosage forms include oral solutions / suspensions, such as hydrogels dissolved in pharmaceutically acceptable diluents. Suitable solid dosage forms include capsules, such as encapsulated hydrogels.

[0018] The dosage of the minerals discussed may vary; those skilled in the art can prepare pharmaceutical compositions that meet these standards. For example, the hydrogel of the present invention can be applied in the range of 1 mL kg⁻¹ to 30 mL kg⁻¹ body weight (BW), such as 10 mL kg⁻¹ BW.

[0019] Alcohol poisoning: This term is known in the art and includes alcohol abuse, alcohol dependence, and alcohol use disorder. The term should include both acute and chronic alcohol poisoning in subjects, particularly humans. The materials and methods described herein are specifically designed to mitigate the negative effects of ethanol consumption on the body. Attached Figure Description

[0020] The invention will be better understood in conjunction with the accompanying drawings.

[0021] Figure 1 The synthesis process of Fe1@FibBLG is explained.

[0022] Figure 2 The analytical details of the hybrid material of the present invention are shown.

[0023] a. Normalized XANES spectrum of Fe1@FibBLG at Fe K-edge and reference sample. [x-axis photon energy (eV), y-axis XANES (au)]

[0024] b. EXAFS fitting curves of Fe1@FibBLG in r-space and K-space (inset). [x-axis R (Å), y-axis FT (Å)]

[0025] Figure 3 The present invention's hybrid materials, Fe1@FibBLG [circle] and Fe1@BLG [square], which have multiple single-site iron, are compared with the hybrid material FeNP@FibBLG [triangle] which has multiple nanoparticle iron.

[0026] a., b. Typical Michaelis curves of Fe1@FibBLG [circle], Fe1@BLG [square], and FeNP@FibBLG [triangle] obtained by changing the concentrations of ethanol (a) and acetaldehyde (b) in the presence of H2O2.

[0027] c., d. Comparison of the specific activities of Fe1@FibBLG, Fe1@BLG and FeNP@FibBLG for the oxidation of ethanol (c) and acetaldehyde (d) in the presence of H2O2.

[0028] One nanozyme activity unit (U) is defined as the amount of nanozyme that catalyzes 1 μmol of product per minute. The specific activity (U mg⁻¹) is determined by plotting the nanozyme activity against its weight and measuring the gradient of the fitted curve. ¹H NMR spectra of the reaction products of Fe1@FibBLG catalyzing the oxidation of ethanol (inset c) and acetaldehyde (inset d).

[0029] Figure 4 The effect of Fe1@AH as a preventive agent for acute alcohol poisoning was demonstrated.

[0030] a, Visualization of Na+-induced Fe1@FibBLG hydrogel (Fe1@AH) (I), microstructure of Fe1@AH under polarized light (II), and injectability test (III).

[0031] b, Schematic diagram of the construction of an acute alcohol poisoning model.

[0032] c. Effects of different treatments (PBS, AH, and Fe1@AH) on alcohol tolerance time and sobering time in C57BL / 6 mice.

[0033] d, e. Average concentrations of alcohol (d) and acetaldehyde (e) in the blood of alcohol-poisoned mice treated with PBS, AH, and Fe1@AH.

[0034] Data are presented as mean ± standard deviation, n=8. The p-value was determined using a one-way ANOVA followed by a Tukey-Kramer test. p<0.05, p<0.01, p<0.001, p<0.0001.

[0035] Figure 5 Chronic alcoholism

[0036] a. Schematic diagram of the construction of a chronic alcohol poisoning model.

[0037] b. Changes in body weight of the four groups of mice during the feeding period.

[0038] c. Histopathological evaluation of liver tissue from different groups of mice based on H&E staining.

[0039] d. Histopathological evaluation of colon tissue from different groups of mice based on H&E staining. Data are presented as mean ± standard deviation, n=8.

[0040] For histopathological and physiological parameters (b, c, and d), the p-values ​​were tested using one-way ANOVA followed by the Tukey-Kramer test. p<0.05, p<0.01, p<0.001. Invention Details

[0042] More broadly, in a first aspect, the present invention provides novel materials containing (i.e., including or comprising) (a) amyloid protein (preferably amyloid fibrils as described below); and (b) iron (preferably in the form of single-site iron as described below). These novel materials may exist in the form of hybrid materials as defined herein, or in the form of hydrogels as defined herein.

[0043] Hybrid Material: According to the present invention, components (a) and (b) are in close contact. The individual components remain separate and distinct in the finished structure, but are thoroughly and randomly mixed. This is ensured by the manufacturing process defined below. The material exhibits properties of both amyloid fibrils and single-site iron, and is therefore referred to as a hybrid material. Figure 1 A schematic diagram is provided, showing amyloid protein (left, iron-free), amyloid fibrils (middle, primordial, i.e., iron-free), and a hybrid material containing amyloid fibrils (a) and a single site of iron (b) (right).

[0044] Advantageously, the ratio (b) / (a) is in the range of 0.2 / 100 to 7 / 100, preferably in the range of 0.5 / 100 to 5 / 100 (w / w, dry weight basis, measured by ICP-MS).

[0045] Amyloid fibrils: The term “amyloid fibrils” (or “fibrillated amyloid”) is generally known in the art and specifically describes fibrils made of proteins or peptides that are predominantly found to have a β-sheet secondary structure. Therefore, the term amyloid fibrils excludes native proteins and other types of non-amyloid aggregates.

[0046] Amyloid fibrils can be obtained from several different protein sources, such as plant-based and animal-based proteins. The appropriate protein source is called amyloid-producing protein.

[0047] In the implementation scheme, amyloid fibrils are obtained from plant-based proteomes, preferably:

[0048] Zea prolysin (e.g., obtained from corn).

[0049] Rice protein (e.g., obtained from rice bran).

[0050] cruciferin and napin (e.g., obtained from rapeseed).

[0051] Sunflower protein (e.g., obtained from sunflower seed meal), and

[0052] Glycine globulin and β-conglycine globulin (e.g., obtained from soybean residue / soy milk), and

[0053] In the implementation scheme, amyloid fibrils are obtained from animal-based proteomes, preferably:

[0054] β-lactoglobulin (FibBLG, for example, obtained from whey),

[0055] Keratin (e.g., obtained from feathers).

[0056] Lysozyme (e.g., obtained from egg white), and

[0057] BSA (e.g., obtained from whey).

[0058] Unbound by theory, the fibrils derived from these proteins are believed to possess a wide range of functional properties, and due to their remarkable characteristics, particularly the large number of nitrogen atoms available for coordination, they have been found to be particularly suitable for preparing the inventive materials described herein. Amyloid fibrils stabilize atomically dispersed iron, thereby acting as nanocarriers.

[0059] As an example, amyloid fibrils are derived from animal-based proteomes, such as whey. Whey is a rich source of protein, the most abundant of which is β-lactoglobulin, a globular protein that can readily self-assemble into amyloid fibrils. In a preferred embodiment, the amyloid fibrils are β-lactoglobulin (FibBLG).

[0060] Advantageously, the amyloid fibrils have a high aspect ratio, typically with a radius ≤50 nm and a length ≥0.3 μm, preferably with a radius ≤10 nm and a length ≥0.5 μm, and particularly preferably with a radius ≤5 nm and a length ≥1 μm.

[0061] Advantageously, amyloid fibrils possess a highly charged surface. The term "highly charged surface" is generally known in the art and specifically describes a surface exhibiting an electrophoretic mobility of approximately 2 μm·cm / V·s at pH 4. Electrophoretic mobility can be determined from zeta potential measurements; i.e., colloidal drift mobility under an electric field.

[0062] Single-site iron: The hybrid material of the present invention contains multiple single-site irons that coordinate with the amyloid protein.

[0063] Based on the detailed analysis below, the conclusion is that iron at a single site is Fe +2 and / or Fe +3 It exists in the form of and is coordinated with nitrogen and / or oxygen and / or sulfur atoms present in the amyloid protein.

[0064] The coordination environment and oxidation state of Fe were investigated using β-lactoglobulin amyloid fibrils (FibBLG), and the corresponding hybrid material was designated Fe1@FibBLG. X-ray absorption fine structure (XAFS) spectroscopy was used to study Fe1@FibBLG. Fe-edge X-ray absorption near-edge structure (XANES) spectroscopy revealed that the leading edge of Fe1@FibBLG is located between Fe foil (metallic iron) and Fe2O3, while the white line region at higher binding energies indicates a lower oxidation state than Fe2O3 and a different coordination environment. XANES features are valuable for identifying site symmetry around iron (Fe) in high molecular weight complexes. The prominent leading edge feature before 7120 eV indicates the presence of ferrous iron (Fe2+) in FePc. 2+ Fe1@FibBLG exhibits slightly weakened characteristics due to its deviation from perfect tetragonality, while Fe1@FibBLG demonstrates planar tetragonality. Notably, the XANES spectrum of Fe1@FibBLG (…) Figure 2 (a, illustration) The spectra are closely aligned with those of iron(II) phthalocyanine (FePc). These findings suggest that Fe within Fe1@FibBLG exists in a positively charged ionic state (Feδ+, where the average δ is close to 2). Fourier transform (FT) extended X-ray absorption fine structure (EXAFS) spectroscopy provides further insights in R space. Figure 2 b) revealed a single peak at approximately 1.4 Å. According to the reference material, this peak is attributed to backscattering between Fe and lighter atoms (primarily nitrogen (N)). Figure 2(b) This observation strongly supports the atomic-level dispersion of Fe sites within Fe1@FibBLG, confirming the results of the HAADF-STEM analysis. Furthermore, wavelet transform (WT) analysis, a tool that simultaneously provides radial distance and k-space resolution, was performed to discern differences between Fe1@FibBLG and the reference material. Unlike the Fe foil and Fe2O3 reference, the WT analysis of Fe1@FibBLG showed a maximum intensity at approximately 4 Å⁻¹ and 1.4 Å, likely due to the contribution of Fe-N. The fitting results indicate a coordination number (CN) of 4.5 for Fe-N.

[0065] Further analysis showed that BLG fibrils have effective binding sites that can capture Fe atoms through Fe-N coordination, thereby forming the active Fe center of Fe1@FibBLG.

[0066] X-ray photoelectron spectroscopy (XPS) was used to determine the binding states of carbon (C), nitrogen (N), and iron (Fe) in Fe1@FibBLG. Analysis of these XPS data indicated that approximately 62% of the single-site Fe in Fe1@FibBLG is in Fe... 2+ The state exists. Fe 2+ The presence of Fe is attributed to the reduction effect of BLG fibrils. In summary, these XPS results not only confirm the presence of Fe species within BLG fibrils but also quantify the charge state of Fe within the BLG fibril framework.

[0067] These findings collectively confirm that the Fe in the hybrid material of this invention exists as a single-site Fe, primarily coordinated with the N atom of amyloid protein, but also to some extent with the O and / or S atoms of amyloid protein. These findings further confirm that iron exists as Fe. +2 and / or Fe +3 It exists in the form of . It is further believed that Fe does not have any crystalline or iron oxide metallic structure.

[0068] Hydrogel: The above-mentioned hybrid material can be converted into an amyloid protein hydrogel (“Fe1@AH”). Studies have found that this hydrogel retains its bioactivity while providing a convenient oral dosage form. The typical concentration of the hybrid material in the hydrogel is 0.1-8 wt.%, preferably 0.5-5 wt.%.

[0069] Hydrogels are known in themselves. Typically, hydrogels are networks of hydrophilic, cross-linked polymer chains that can thus bind aqueous fluids. They are primarily identified by the monomeric units that form the polymer, which can form a hydrogel upon contact with a fluid. Typically, the fluid is an aqueous fluid, such as water, and the polymer is an amyloidogen-producing protein.

[0070] The Fe1@AH of this invention exhibits typical self-supporting ability and displays a distinct nanofibril structure under polarized light. Furthermore, good injectability was observed. Fe1@AH is used... 18 F-fluoro-2-deoxyglucose ( 18 Fe1@AH hydrogels were labeled with F-FDG and their transport was visualized in C57BL / 6 mice using microPET-CT scanning. The Fe1@AH formed by the hydrogel persisted in the upper gastrointestinal tract for more than 6 hours after gavage, indicating a prolonged retention time in vivo, which is attributed to the properties of the hydrogel. Figure 4 b).

[0071] In a second aspect, the present invention relates to pharmaceutical compositions comprising the novel materials described herein (first aspect), and methods of using such compositions as medicines, particularly in the context of alcohol detoxification. Those skilled in the art will understand that the compositions described herein and the methods of using such compositions offer significant advantages over the prior art.

[0072] This invention provides a novel method for alcohol detoxification by employing a hybrid material (preferably in hydrogel form), considered a biomimetic nanozyme, as a catalytic platform for oral administration. Unbound by theory, amyloid proteins are believed to be rich in coordinating nitrogen atoms, thus acting as nanocarriers to stabilize atomically dispersed Fe. The resulting single-site iron nanozyme effectively catalyzes the alcohol oxidation reaction, with the significant advantage of producing less toxic acetic acid. Application of the hydrogel described herein to mice affected by alcohol poisoning significantly reduced their blood alcohol levels without causing additional acetaldehyde accumulation, superior to existing therapies based on natural enzymes. Furthermore, the hydrogel of this invention exhibits hepatoprotective effects while mitigating intestinal damage and dysbiosis associated with chronic alcohol consumption, opening a promising new avenue for effective alcohol detoxification strategies, including preventative treatment. Accordingly, this invention provides pharmaceutical compositions comprising the hybrid material and hydrogel described herein (first aspect). In summary, the pharmaceutical compositions described herein are simple to manufacture, safe to use, and effective in treating alcohol poisoning, particularly effective in preventing / avoiding alcohol poisoning. Therefore, this invention also relates to:

[0073] The hybrid materials and hydrogels described in this article (Aspect 1) are used as pharmaceuticals;

[0074] The hybrid materials and hydrogels described in this article (Aspect 1) are used to treat alcohol poisoning;

[0075] The hybrid materials and hydrogels described herein (First Aspect) are used to manufacture a drug for treating alcohol poisoning;

[0076] A pharmaceutical composition comprising an effective amount of a hybrid material or hydrogel as defined herein (First Aspect) for the prevention, treatment or delay of the progression of alcohol poisoning (“alcohol poisoning”) in subjects (particularly humans) in need.

[0077] The pharmaceutical composition can be administered in any suitable dosage form. A significant advantage is that the dosage form can be an oral formulation, such as capsules, sugar-coated pills, syrups, or the like, each containing the hybrid material of the present invention or the hydrogel of the present invention.

[0078] The pharmaceutical composition can be used to prevent, treat, or delay the progression of alcohol poisoning (“alcohol poisoning”). In one embodiment, the pharmaceutical composition can be used to treat acute alcohol poisoning. In another embodiment, the pharmaceutical composition can be used to treat chronic alcohol poisoning.

[0079] Prevention: To assess whether oral administration of Fe1@AH conferred any preventive benefit in an animal model of poisoning, mice were orally administered PBS, AH, or Fe1@AH of the present invention (0.01 mL / g body weight) 30 minutes prior to a single gavage of ethanol (12 mg g⁻¹ body weight; n=8 per group). Figure 4 c). A group of mice without ethanol but administered PBS by gavage served as a negative control (blank; n=8). All mice administered ethanol by gavage were in a state of toxic sleep. Although they struggled for a longer time to wake up (approximately 40 minutes), the Fe1@AH mice woke up significantly earlier (approximately 2 hours) than the other toxic groups. Figure 4 d). Then, we performed the Morris water maze (MWM) test 6 hours after drinking to quantitatively assess the mice's spatial reference memory ( Figure 4 e). The average swimming speed of the alcohol-exposed mice was comparable to that of the control group, indicating that their basic activity had recovered. However, compared with mice on a non-alcoholic diet, mice treated with PBS and AH required more time and swam longer distances to find the hidden platform; while the administration of Fe1@AH of the present invention showed a significant reduction in both distance and time. Figure 4 (f and 4g). In addition, "marginal" and "random" were the main search strategies in the PBS and AH groups (37.5%), while mice belonging to the Fe1@AH and blank groups preferred "linear" and "trend".

[0080] Chronic alcoholism: To confirm the long-term beneficial effects of the hydrogel (“Fe1@AH”) of the present invention, we based our findings on those of Bertola et al. ( Nat ProtocThe protocol proposed in 8, 627-637 (2013) was followed, and the NIAAA model (chronic plus binge-feeding ethanol mouse model) was performed as further outlined in the examples. This model involved oral administration of Fe1@AH to mice. The model was successfully induced, characterized by a significant decrease in body weight, increased liver damage (ballooning degeneration and multifocal inflammatory cell infiltration), and increased hepatic lipid accumulation between the control and PBS groups. Figure 5 (b and 5c). Notably, Fe1@AH-rescued mice showed significantly reduced weight loss, less liver damage, and re-regulation of hepatic lipid metabolism after poisoning. Figure 5 (b and 5c). Furthermore, mice treated with Fe1@AH had lower BA levels than mice treated with PBS and AH.

[0081] In a third aspect, the present invention relates to a method for manufacturing the hybrid materials described herein (first aspect) and the compositions described herein (second aspect).

[0082] Manufacturing of Hybrid Materials: The hybrid materials of this invention are simple to manufacture; large-scale production using readily available starting materials is promising. Generally, manufacturing involves a wet impregnation process (…). Figure 1 a) includes dispersing fibrous amyloid protein in a mixture of a polar organic solvent (e.g., ethanol) and a dispersant (e.g., PEG200), and exposing it to Fe containing the dispersant (e.g., PEG200). 3+ Solution (e.g., Fe(NO3)3). After multiple rounds of centrifugation and washing, the resulting precipitate was freeze-dried to promote the secure fixation of Fe atoms within the protofibrillary structure. Following synthesis, the hybrid material underwent comprehensive characterization and testing. The morphology of the material maintained a nanometer diameter consistent with pure amyloid protofibrils, indicating that Fe integration did not affect the structural characteristics of the amyloid protofibrils. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and energy-dispersive X-ray spectroscopy (EDS) mapping analysis revealed a significant overlap between the Fe-K profile and the elemental composition of the amyloid protofibrils, demonstrating uniform dispersion of Fe throughout the entire amyloid protofibrillary framework. The height of the protofibrils was determined using atomic force microscopy (AFM). The results showed consistent height before and after Fe integration, confirming the absence of crystalline Fe or oxide species within the detection limits of the analytical methods.

[0083] In the implementation scheme, the manufacturing of the hybrid material includes the following steps (i), (ii) and (iii).

[0084] Step (i): The synthesis of amyloid proteins and fibrillary amyloid proteins is known. Suitable starting materials are food-grade proteins that are structurally stable, readily available, and inexpensive. Such starting materials allow for the preparation of amyloid fibrils, such as β-lactoglobulin. The self-assembly process is simple and controllable. For fibrils, proteolysis followed by β-sheet-driven fibrillation is particularly suitable, as exemplified by Jung et al. (…). Biomacromolecules As described in (2008, 9, 2477-2486). The process is carried out under acidic conditions (e.g., pH 2) for an extended period (e.g., 5 hours) at a high temperature (e.g., 90°C). The resulting fibrous material can be further processed.

[0085] Typically, freeze-dried materials are used as starting materials for step (ii).

[0086] Step (ii) Dissolve / finely disperse the material from step (i) in a diluent containing a polar organic solvent (such as an alcohol, e.g., ethanol) and a dispersant (such as a polyol, e.g., PEG or ethylene glycol). Degas the resulting first solution and then subject it to UV irradiation to form free radicals.

[0087] A second solution containing a soluble Fe(III) compound, a polar organic solvent, and a dispersant is added, and the solution is allowed to react with the first solution. Suitable Fe+3 compounds are known and commercially available, such as ferric nitrate.

[0088] The reaction is typically carried out over a longer period of time, usually more than one hour, for example 8-24 hours, and at a moderate temperature of 20-50°C, usually at room temperature, to obtain the hybrid material of the invention in a dissolved or finely dispersed form.

[0089] Step (iii) As is known in the art, the hybrid material can be separated from the solvent and other components, for example, by a combination of centrifugation and washing steps, followed by freeze drying.

[0090] Preparation of hydrogels: It is known to convert amyloid protofilaments and fibrous amyloid proteins into hydrogels themselves. Advantageously, hydrogel formation is achieved by adding ions (e.g., sodium ions in the form of NaCl). In another advantageous embodiment, Au nanoparticles are incorporated as a catalyst for the glucose oxidation reaction to produce H2O2.

[0091] In the embodiments, the hydrogel manufacturing of the present invention includes the following steps (iv), (v) and (vi).

[0092] Step (iv) provides the hybrid material of the present invention dissolved in an aqueous medium. For example, the material obtained from step (iii) can be used. Alternatively, the freeze-dried hybrid material can be dissolved in an aqueous medium. Typically, pure water is used as the aqueous medium.

[0093] Step (v) provides a solution containing finely dispersed gold nanoparticles. The finely dispersed gold nanoparticles, preferably stabilized by amyloid protein as is known in the art, are used as an auxiliary agent. For example, amyloid protofibrils are bound to a HAUCl4 solution, typically in a ratio of amyloid protofibrils to Au precursor >10:1. The mixture is subjected to a reduction reaction, for example, by adding NaBH4, followed by purification, for example, by dialysis, to obtain finely dispersed Au nanoparticles on the surface of the amyloid protofibrils.

[0094] Step (vi), gelation. The materials from steps (iv) and (v) are combined (“Fifth Solution”) and a gelation reaction is carried out. This gelation reaction is known per se but has not been applied to the starting materials described herein. Suitably, the gelation reaction is carried out by slowly combining the Fifth Solution with an acidic saline solution over a prolonged period to obtain a homogeneous hydrogel. A suitable reaction time is at least 1 hour, preferably at least 12 hours. Suitable conditions include pH = 7.4. Suitable low temperatures include 0-10°C, for example, 4°C. The Fifth Solution and the acidic saline solution can be contacted via a membrane.

[0095] To further illustrate the present invention, the following embodiments are provided. These embodiments are not intended to limit the scope of the invention.

[0096] I. Hybrid Materials

[0097] Starting materials

[0098] (β-lactoglobulin (BLG; >98%) was purchased from Davisco Foods International, Inc. and purified.)

[0099] Synthesis of the hybrid material Fe1@FibBLG of this invention:

[0100] For the synthesis of Fe1@FibBLG, 100 mg of lyophilized BLG fibrillation powder was dispersed in a mixture of 8.0 mL ethanol and 1.9 mL polyethylene glycol 200 (PEG 200). The dispersion was then bubbled with argon for 30 minutes to remove dissolved oxygen, followed by illumination under a xenon lamp with a UV filter (250-380 nm, 27.9 mW cm⁻¹). -2 Irradiation with a PLS-SXE300CUV for 10 minutes was performed to generate free radicals. Subsequently, under magnetic stirring, 0.1 mL of 108.21 mg / mL solution was added. -1 Fe(NO3)3·9H2O PEG 200 solution was added dropwise to the dispersion of BLG fibrils, and the reaction was carried out for 12 hours at 25°C.

[0101] Synthesis of the hybrid material Fe1@BLG of this invention:

[0102] Except for replacing BLG fibril powder with an equal amount of BLG powder, Fe1@BLG was prepared using the same synthesis procedure as Fe1@FibBLG.

[0103] Synthesis of the comparative material FeNP@FibBLG:

[0104] For the synthesis of FeNP@FibBLG, the Fe1@FibBLG dispersion obtained above was further irradiated with UV for 18 minutes under anaerobic conditions to reduce Fe ions.

[0105] Post-processing

[0106] Samples were collected by centrifugation at 11,100 × g for 10 min at 4°C, washed with ethanol (10.0 mL × 6 times), and resuspended in 5.0 mL of deionized water (pH 2). Fe1@FibBLG, Fe1@BLG, and FeNP@FibBLG were obtained as powders by freeze-drying and stored at 4°C.

[0107] analyze

[0108] Analysis results as follows Figure 2 As shown, and discussed above.

[0109] II. Hydrogel

[0110] Starting material (BLG fibrils with attached AuNPs)

[0111] For the synthesis of AuNPs, all glassware was cleaned with freshly prepared aqua regia (HCl:HNO3 = 3:1, v / v) and then rinsed thoroughly with water. 2 mL of BLG fibril solution (2.0 wt%, pH 2) was mixed with 40 mM HAuCl4 solution to achieve a final protein:Au mass ratio of 14.7:1. The mixture was chemically reduced by dropwise addition of NaBH4 solution (0.8 mL) under a nitrogen atmosphere. The resulting solution was then dialyzed to remove any residual NaBH4 and concentrated to 2 mL using a dialysis membrane (Spectra / Por®, MWCO 6-8 kDa, Spectrum Laboratories, Inc.) at pH 2 against a 6 wt% polyethylene glycol solution (Mr~35,000, Sigma-Aldrich). TEM images of AuNPs stabilized by BLG fibrils show three-dimensional particles with an average size of 1.32 nm.

[0112] Fe1@AH was prepared using fibrous amyloid protein.

[0113] For the preparation of Fe1@AH, 2 g of Fe1@FibBLG powder obtained according to the above protocol was dissolved in 2 mL of the above starting material (a solution of BLG fibrils with attached AuNPs). The mixture was then transferred to a plastic syringe, the tip of which had been previously cut off. The plastic syringe was covered with a section of dialysis tubing (Spectra / Por®, MWCO 6-8 kDa), and the syringe tip was in direct contact with an excess of 300 mM NaCl solution (pH 7.4). The syringe was placed in a cold room at 4°C for at least 16 hours to promote gelation. The resulting hydrogel sample was stored at 4°C.

[0114] Working hydrogels, i.e. samples intended for direct oral administration, are freshly prepared by mixing the above-mentioned hydrogel with 0.1 mL of glucose solution (8.0 M) prior to further characterization or detoxification.

[0115] Fe1@AH was prepared using amyloid protein.

[0116] The same procedure was used to obtain BLG fibrillated hydrogels, except that an equal amount of BLG fibrillated dispersion was used instead of Fe1@FibBLG.

[0117] III. In vitro toxicity assessment

[0118] Cell viability assay. Caco-2 cells (2 mL and 3 × 10⁶ cells) were seeded in six-well plates. 5 Cells / well were treated with AH or Fe1@AH at 24 hours after resuscitation under the following conditions: DMEM containing 10% (v / v) fetal bovine serum (FBS), non-essential amino acids, and 1% (v / v) penicillin-streptomycin (10,000 U / mL penicillin and 10 mg / mL streptomycin), and cultured at 37°C, 5% CO2. The same volume (4.0 g / mL, 40 μL) of AH or Fe1@AH was then incubated with the cells for another 24 hours at 37°C, 5% CO2. Cells treated with PBS served as a negative control. After exposure, cells were washed three times in cold PBS, centrifuged at 1500 rpm for 5 minutes, and resuspended in 500 μL binding buffer. Cells were then subjected to Annexin V-FITC (Invitrogen) assays. TM Cells were stained and their fluorescence was detected using a flow cytometer (FACSCalibur 2, USA) equipped with an argon ion laser with an excitation wavelength of 488 nm. Green and red fluorescence were measured at 530 / 30 nm (FL1) and 670 nm (FL3), respectively.

[0119] IV mouse model

[0120] General situation

[0121] Male wild-type (WT) C57BL / 6 mice, 20-25 g, 8-10 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mouse experiments in this study were approved under the "Beijing Municipal Regulations on the Management of Laboratory Animals" (Approval No.: AW40803202-5-1) and conducted according to the guidelines developed by the Institutional Animal Care and Use Committee of China Agricultural University.

[0122] In vivo safety assessment.

[0123] According to Section 2, male C57BL / 6 mice (n=6) were subcutaneously injected with hydrogels to assess their sensitization and toxicity. Mice were anesthetized by intramuscular injection of ketamine (85 mg kg⁻¹ BW) and toluenethiazide (10 mg kg⁻¹ BW) in the hind leg. After shaving the hair on the back, mice were subcutaneously injected with 50 μl of AH or Fe1@AH (1 mg / mL) at time points 0 and 24 hours, respectively. Mice injected with H₂O₂ solution or PBS buffer served as positive and negative controls, respectively. After skin testing, mice were sacrificed, and skin tissue was collected and fixed for further analysis. Skin samples were stained with hematoxylin and eosin (H&E), and apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL, Roche Molecular Biochemicals). Images were acquired using either a Zeiss Axio Vert AI or a Zeiss LSM980 (ZEN 2012) confocal microscope and processed using CaseViewer 3.3 software.

[0124] Acute model.

[0125] Thirty-two male C57BL / 6 mice were randomly divided into four groups after a 12-hour fast. Mice were orally administered AH and Fe1@AH (10 mL kg⁻¹ BW), while the control group received the same volume of PBS. After a 20-minute acclimatization period, mice in the AH, Fe1@AH, and PBS groups were orally administered an alcoholic liquid diet (10 g kg⁻¹ BW), while the control group received the same volume of PBS. All mice were euthanized after 6 hours.

[0126] Chronic model.

[0127] A mouse model of chronic plus binge-feeding ethanol was established according to the protocol proposed by Bertola et al. (NIAAA model). In short, after acclimatization to a Lieber-DeCarli diet for 5 days, 32 mice were randomly divided into four groups: (1) a control group (Con), with mice paired and fed the control diet; (2) an ethanol diet group (EtOH); (3) an ethanol diet group supplemented with 10 mL kg⁻¹ AH; and (4) an ethanol diet group supplemented with 10 mL kg⁻¹ Fe1@AH. The ethanol-fed groups received an unrestricted Lieber-DeCarli diet containing 5% (vol / vol) ethanol for 10 days, and were additionally gavaged with PBS, AH, or Fe1@AH each morning (9:00 AM). The control group was paired and fed an isocaloric control diet and daily control fluid gavage. On day 16, mice in both the ethanol-fed and paired-fed groups were given a single oral dose of ethanol (5 g kg⁻¹ BW) or isocaloric maltodextrin at 9:30 a.m., and were euthanized 6 hours later. Mouse weight was recorded every 2 days.

[0128] MicroPET-CT

[0129] After fasting overnight, mice were administered 0.1 mL of F-fluorodeoxyglucose (18F-FDG)-labeled Fe1@AH via gavage. The mice were then anesthetized with oxygen containing 2% isoflurane and placed prone in the imaging chamber. Time-series images were acquired using an Inveon MicroPET / CT scanner (Siemens) with 15 minutes of CT scanning (80 kVp, 500 μA, 1100 ms exposure time), followed by 10 minutes of PET acquisition. Quantitative image analysis was performed using AMIDE software 30.

[0130] Alcohol tolerance test

[0131] Approximately 10 μl of blood was collected from the mandibular vein at 30, 60, 90, 120, 180, and 300 minutes after alcohol exposure. In the chronic model, sampling was performed after binge-feeding with ethanol. Blood alcohol concentration (BAC) was determined using a kit from Abcam Biotechnology (ab65343). As previously described, BAC was normalized to mouse body weight. The normalized BAC (BACnor) was calculated using the following formula: BACnor = BACi × (BWTi / BWTave), where BACi and BWTi represent the mouse's blood alcohol level and body weight, respectively, and BWTave represents the average body weight of all mice in each experimental group. Blood acetaldehyde concentration (BAceC) was quantified using a kit from Abcam Biotechnology (ab308327), with normalization performed using the same method as for BAC.

[0132] Alcohol tolerance time is the duration from alcohol administration to the disappearance of the righting reflex, while the duration of the disappearance of the righting reflex is recorded as sobering time. In mice, ataxia was considered as loss of the righting reflex, and the mice were then placed in a supine position. The time it took for the mouse to return to a normal upright posture marked the recovery of its righting reflex.

[0133] Morris Water Maze (MWM) Test

[0134] MWM testing was conducted. Specifically, the MWM apparatus consisted of a large circular pool (120 cm in diameter and 40 cm in height) filled with TiO2-stained water at a constant temperature of 25°C, with a 10 cm diameter platform positioned and fixed 2 cm below the water level. Prior to acute ethanol exposure, mice underwent four rounds of training daily for six days. Each test was limited to 60 seconds, and the time required for the mouse to successfully locate the platform was recorded. On day 7, five hours after ethanol feeding (when all mice had regained consciousness and mobility), the mice were retested (without the platform). Test items included trajectory, path length, escape latency, and swimming speed (MWM animal behavior video tracking system, Morris V2.0).

Claims

1. A hybrid material comprising (a) amyloid protein and (b) a plurality of single-site iron molecules coordinated to said amyloid protein, The single-site iron mentioned above is atomically dispersed iron, lacking any crystalline or iron oxide metallic structure; and The amyloid protein mentioned therein is fibrous amyloid protein.

2. The hybrid material according to claim 1, wherein the amyloid protein is fibrous amyloid protein, and its fibril radius is ≤50 nm and its length is ≥0.3 μm.

3. The hybrid material according to claim 1 or 2, characterized in that, The amyloid protein (a) is - Plant-based proteins, preferably zein, rice protein, brassinolide and brassinolide, sunflower protein, and soy globulin; or - Animal-based proteins, preferably BLG, lysozyme, ovalbumin, and serum protein.

4. The hybrid material according to any one of the preceding claims, characterized in that, The single-site iron(b) - with Fe +2 and / or Fe +3 It exists in the form of, and - Coordinated with nitrogen and / or oxygen and / or sulfur atoms present in the amyloid protein (a).

5. The hybrid material according to any one of the preceding claims, characterized in that... - The ratio (b) / (a) is in the range of 0.2 / 100 to 7 / 100 (w / w, dry weight basis, measured by ICP-MS); and / or - The amyloid protein (a) showed an electrophoretic mobility of about 2 μm·cm / V·s at pH 4 (determined by zeta potential measurement).

6. A hydrogel comprising the hybrid material according to any one of the preceding claims.

7. A pharmaceutical composition comprising a hybrid material according to any one of claims 1-5 or a hydrogel according to claim 6.

8. An oral dosage form comprising the pharmaceutical composition according to claim 7.

9. The hybrid material according to any one of claims 1-5, or the hydrogel according to claim 6, is used as a pharmaceutical.

10. Use of the pharmaceutical composition according to claim 7 or the oral dosage form according to claim 8, wherein... - Used to prevent, treat, or slow the progression of alcohol poisoning ("alcoholism"); - Used to treat acute alcohol poisoning; or - Used to treat or improve the effects of chronic alcoholism.

11. A method for manufacturing a hybrid material according to any one of claims 1-5, comprising exposing a dispersion ("first solution") of (fibrous) amyloid protein in a mixture of a polar organic solvent and a dispersant to Fe containing the dispersant. 3+ The steps of preparing the solution ("second solution").

12. The method for manufacturing a hybrid material according to claim 11, comprising the following steps: Step (i) provides a powder of amyloid or fibrous amyloid; Step (ii) A first solution is prepared by dissolving the powder from step (i) in a diluent containing a polar organic solvent and a dispersant, followed by degassing and UV irradiation; as well as The first solution is combined with the second solution, the second solution containing soluble Fe. +3 Compounds, polar organic solvents and dispersants, The mixture thus obtained is allowed to react, preferably at room temperature for at least one hour, so as to obtain the hybrid material in a dissolved form; Step (iii) may optionally separate the hybrid material from the solvent and other components, preferably by freeze drying; This leads to the acquisition of hybrid materials.

13. A method of manufacturing the hydrogel according to claim 6, comprising the step of exposing a solution ("fifth solution") comprising a hybrid material and finely dispersed Au nanoparticles according to any one of claims 1-5 to an acidic aqueous salt solution to allow gelation.

14. The method for manufacturing a hydrogel according to claim 13, comprising the following steps: Step (iv) provides a hybrid material ("third solution") dissolved in an aqueous medium according to any one of claims 1-5; as well as Step (v) provides a solution containing gold nanoparticles, preferably gold nanoparticles finely dispersed on amyloid protofibrils ("fourth solution"); as well as Step (vi) Combine the third and fourth solutions to obtain a fifth solution, and allow gelation of the fifth solution to occur by slowly combining the fifth solution with an acidic salt solution at low temperature for an extended period of time. This results in a uniform hydrogel.

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