A composition based on tree shrew ugt enzyme and its application in promoting alcohol metabolism

Abstract

The application discloses a high-activity UGT1A1 enzyme based on tree shrew and a feed composition for enhancing alcohol metabolism. The catalytic efficiency of the recombinant tree shrew UGT1A1 enzyme on ethanol is significantly higher than that of human isozyme. The core of the application is to provide a complex feed system containing the enzyme, which solves the key bottleneck of poor stability and low delivery efficiency of exogenous enzymes in application through the synergistic effect of enzyme stabilizers, cell penetration enhancers, enteric coatings and coenzyme supply agents. Animal experiments show that the composition can safely and effectively accelerate in vivo alcohol elimination and reduce blood ethanol concentration, and can be used for developing alcoholism products, acute alcoholism treatment drugs and alcoholic liver disease prevention preparations.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medicine, and in particular to the recombinant expression of a highly active uridine diphosphate glucuronide transferase (UGT) family member derived from tree shrews (Tupaia belangeri), especially the UGT1A1 enzyme, its stabilizing excipient composition, and the application of the composition in enhancing the ability of mammals (including humans) to metabolize alcohol (ethanol), alleviating acute alcohol poisoning, and preventing alcoholic liver injury. Background Technology

[0002] The metabolism of alcohol (ethanol) in the body primarily relies on the liver's alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1) to oxidize it into toxic acetaldehyde, which is then converted to acetic acid by acetaldehyde dehydrogenase (ALDH). Acetaldehyde produced through this pathway is the key substance causing intoxication and liver toxicity. However, there is also a safer non-oxidative metabolic pathway for ethanol: catalyzed by UGT enzymes, ethanol directly binds to uridine diphosphate glucuronide (UDPGA) to generate non-toxic ethyl glucuronide (EtG), which is excreted in the urine. This pathway does not produce acetaldehyde and is an ideal detoxification route.

[0003] While the human UGT1A1 enzyme can catalyze this reaction, its catalytic efficiency (Vmax / Km) for ethanol is low, and its application potential is limited by significant individual variability (e.g., influenced by UGT1A1 * 28 polymorphism). Through comparative genomics and enzymological analysis, the inventors discovered that tree shrews, whose alcohol metabolism physiology is similar to that of humans, possess a UGT1A1 homolog that exhibits exceptionally high affinity and catalytic efficiency for ethanol, with its in vitro catalytic activity approximately 3-5 times that of the wild-type human UGT1A1.

[0004] However, directly applying recombinant enzymes in vivo faces challenges such as poor stability, short half-life, and susceptibility to protease degradation. Therefore, developing a stable excipient composition that can effectively deliver and maintain the function of this highly active enzyme is key to realizing its application value. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a highly active tree shrew-derived UGT1A1 enzyme, its coding sequence, and an excipient composition that can significantly enhance the stability and efficacy of the enzyme in vivo and in vitro.

[0006] To address the aforementioned technical problems, the present invention provides an isolated polynucleotide encoded with an amino acid sequence as shown in SEQ IDNO: 1.

[0007] The present invention also provides a recombinant expression vector comprising the above-described polynucleotides.

[0008] The present invention also provides a recombinant tree shrew UGT1A1 enzyme, which is produced by expression in host cells using the above-mentioned vector.

[0009] The present invention also provides a composition for promoting alcohol metabolism, comprising the above-described recombinant tree shrew UGT1A1 enzyme, and pharmaceutically acceptable excipients, said excipients comprising at least an enzyme stabilizer and a cell penetration enhancer.

[0010] Furthermore, the enzyme stabilizer is selected from one or more of trehalose, sucrose, or glycerol; the cell penetration enhancer is selected from one or more of chitosan and its derivatives, cell-penetrating peptides, or liposome nanocarriers.

[0011] Furthermore, it also includes a coenzyme supplier, which is uridine diphosphate glucuronic acid or its precursor.

[0012] Furthermore, it is an oral enteric-coated formulation and contains enteric coating material and / or protease inhibitors.

[0013] The present invention also provides the use of the above-described recombinant tree shrew UGT1A1 enzyme or composition in the preparation of products for promoting alcohol metabolism in mammals.

[0014] Furthermore, the product is used to prevent or treat acute alcohol poisoning.

[0015] Furthermore, the product is used to prevent alcoholic liver damage.

[0016] Compared with the prior art, the present invention has the following advantages: 1. High enzyme activity: The recombinant tree shrew UGT1A1 enzyme has a significantly higher catalytic efficiency for ethanol than the human isoenzyme, providing a more efficient basis for detoxification.

[0017] 2. Good stability: Through a unique excipient system, especially the synergistic effect of enteric coating and enzyme stabilizer, the key problem of easy inactivation of exogenous enzymes during storage, transportation and in vivo delivery has been solved.

[0018] 3. Targeted delivery: The composition is designed to effectively protect enzymes from damage by gastric acid and proteases and promote their absorption in the intestines, achieving efficient in vivo delivery.

[0019] 4. High safety: It metabolizes alcohol through a non-oxidative pathway, avoiding the accumulation of toxic acetaldehyde and minimizing side effects. Attached Figure Description

[0020] Figure 1 Comparison of the enzymatic kinetic curves (Km and Vmax values) of recombinant tree shrew UGT1A1 enzyme and human UGT1A1 enzyme with ethanol.

[0021] Figure 2: Comparison of residual enzyme activity between the oral microcapsule composition of the present invention and the free enzyme after incubation in rat gastric juice (pH 2.0).

[0022] Figure 3 : Blood ethanol concentration-time curve of mice after ethanol gavage following pretreatment with the composition of the present invention (compared with the control group). Detailed Implementation

[0023] The present invention will now be further described with reference to the accompanying drawings and embodiments.

[0024] In a first aspect, the present invention provides an isolated polynucleotide encoding a tree shrew UGT1A1 protein having the amino acid sequence shown in SEQ ID NO: 1, or a variant thereof having at least 90% sequence identity and retaining UGT glycoluronidation activity.

[0025] Secondly, the present invention provides a recombinant protein produced by expression of the polynucleotide in an expression system. The expression system is preferably *Escherichia coli* BL21(DE3), *Pichia pastoris*, or an insect cell-baculovirus system.

[0026] Thirdly, an enzyme-excipient composition for promoting alcohol metabolism is provided, comprising an active ingredient and an excipient system; the active ingredient is an effective dose of the recombinant tree shrew UGT1A1 enzyme as described in the second aspect. The excipient system is composed as follows: Enzyme stabilizers: selected from one or more of trehalose (10-50 mM), sucrose (20-100 mM) or glycerol (5-20% v / v), used to prevent enzyme denaturation during storage and transportation.

[0027] Cell penetration enhancers: selected from chitosan and its derivatives (0.01-0.1% w / v), cell-penetrating peptides (CPP, such as TAT ​​peptide, 1-10 μM), or liposome nanocarriers, to promote enzymes across the intestinal mucosa or cell membrane.

[0028] Coenzyme supplier: uridine diphosphate glucuronic acid (UDPGA, 1-10 mM) or its precursor (such as glucuronic acid, 5-20 mM) provides the necessary substrate for the enzymatic reaction.

[0029] Intestinal protectants: for oral dosage forms, including pH-sensitive enteric coating materials (such as Eucerin® L100) to ensure enzyme release in a neutral pH environment in the intestine; and protease inhibitors (such as aprotinin, at low concentrations of 0.001-0.01% w / v) to reduce enzyme degradation by digestive tract proteases.

[0030] Pharmaceutically acceptable carriers include fillers, disintegrants, lubricants (for tablets or capsules), or saline solution for injection and buffer solution (for injections).

[0031] Preferred excipient composition formulation (oral microcapsule granules): Core ingredient: Recombinant tree shrew UGT1A1 enzyme lyophilized powder (each capsule contains 5000-20000 U of enzyme activity); Trehalose: 30 mM (as a freeze-drying protectant and stabilizer); TAT cell-penetrating peptide: 5 μM (covalently linked to the enzyme); Outer layer: Enteric coating using pH-sensitive hydroxypropyl methylcellulose phthalate (HPMCP).

[0032] Fourthly, the present invention provides the use of the said composition in the preparation of pharmaceuticals or health products for the following purposes: 1. Accelerates the metabolism of alcohol in the body and quickly reduces the concentration of blood ethanol; 2. Prevention or treatment of acute alcohol poisoning; 3. Prevent alcoholic liver damage caused by long-term alcohol consumption; 4. As a dietary supplement for people with low alcohol metabolism.

[0033] Example 1: Cloning and Recombinant Expression of the Tree Shredder UGT1A1 Gene Total RNA was extracted from tree shrew liver tissue, and cDNA was obtained by reverse transcription. Primers were designed based on the predicted coding sequence (SEQ ID NO: 2), and after PCR amplification, the DNA was cloned into the pET-28a(+) vector and transformed into E. coli BL21(DE3). IPTG-induced expression was performed, and high-purity recombinant protein was obtained by purification using a Ni-NTA affinity chromatography column.

[0034] Example 2: Determination of Enzymatic Properties Using an in vitro enzyme activity assay system with ethanol as the substrate and UDPGA as the coenzyme, the generation rate of the product EtG was detected. The Km value of recombinant tree shrew UGT1A1 enzyme with ethanol was measured to be 12 ± 2 mM, and the Vmax was 85 ± 5 nmol / min / mg. Its catalytic efficiency (Vmax / Km) was significantly higher than that of human recombinant UGT1A1 (Km: 45 ± 5 mM, Vmax: 40 ± 3 nmol / min / mg).

[0035] Example 3: Preparation and in vitro stability testing of the oral composition The purified enzyme was mixed with trehalose and TAT peptide and then freeze-dried to prepare microparticle cores. Enteric coating was performed using fluidized bed coating technology with HPMCP ethanol solution. After treatment with simulated gastric juice (pH 2.0, containing pepsin) for 2 hours, the coated enzyme retained >85% of its activity, while the uncoated free enzyme completely lost its activity.

[0036] Example 4: In vivo efficacy verification in animals Mice were randomly divided into three groups: a control group (physiological saline), a free enzyme group, and a group containing the composition of this invention (coated microcapsules). One hour after gavage administration of the corresponding sample, mice were uniformly administered ethanol (3 g / kg body weight) by gavage. Blood ethanol concentration was measured periodically by tail vein sampling.

[0037] Results: Compared with the control group, the peak blood ethanol concentrations in the composition group of the present invention were reduced by 35% and 50% at 60 minutes and 120 minutes, respectively, and the ethanol clearance rate was significantly accelerated. The free enzyme group showed no significant effect, indicating that the excipient system is crucial for protecting enzyme activity and achieving in vivo delivery.

[0038] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be defined by the claims.

Claims

1. An isolated polynucleotide, characterized in that, Its encoding is the amino acid sequence shown in SEQ ID NO:

1.

2. A recombinant expression vector, characterized in that, It comprises the polynucleotide of claim 1.

3. A recombinant tree shrew UGT1A1 enzyme, characterized in that, It is produced by expression in host cells using the vector described in claim 2.

4. A composition for promoting alcohol metabolism, characterized in that, It comprises the recombinant tree shrew UGT1A1 enzyme of claim 3, and pharmaceutically acceptable excipients, said excipients comprising at least an enzyme stabilizer and a cell penetration enhancer.

5. The composition according to claim 4, characterized in that, The enzyme stabilizer is selected from one or more of trehalose, sucrose, or glycerol; the cell penetration enhancer is selected from one or more of chitosan and its derivatives, cell-penetrating peptides, or liposome nanocarriers.

6. The composition according to claim 4, characterized in that, It also contains a coenzyme supplier, wherein the coenzyme supplier is uridine diphosphate glucuronic acid or its precursor.

7. The composition according to claim 4, characterized in that, It is an oral enteric-coated formulation and contains enteric coating material and / or protease inhibitors.

8. The use of the recombinant tree shrew UGT1A1 enzyme of claim 3 or the composition of any one of claims 4-7 in the preparation of a product for promoting alcohol metabolism in mammals.

9. The application according to claim 8, characterized in that, The product is used to prevent or treat acute alcohol poisoning.

10. The application according to claim 8, characterized in that, The product is used to prevent alcoholic liver damage.

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