CN101816630B - Uricase lipid nanoparticle and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of medicinal preparation. The invention discloses a formula of an uricase lipid nanoparticle and a preparation method thereof. The content of the uricase is 0.1U/ml-1U/ml, and other materials are the medical auxiliary material or the additive of the lipid nanoparticle. The uricase lipid nanoparticle can be used for curing different diseases which are characterized in the increase of the level of the circulated uric acid, wherein the diseases comprise but are not limited to the hyperuricemia and the tumor disintegration syndrome.

Description

Uricase lipid nanoparticles and preparation method thereof

technical field

The invention belongs to the field of medicine, and relates to uricase lipid nanoparticles and a preparation method thereof.

Background technique

Purines generated during the decomposition of nucleic acids and nucleotides in cells are converted into uric acid under the action of xanthine oxidase. Uricase (Uricase, UC) can catalyze the oxidation of uric acid to produce hydrogen peroxide (H2O2) and allantoin (allantion), which are excreted from the body. However, due to the lack of UC in the human body, purine catabolism can only generate uric acid and excrete it through the kidneys. When uric acid production exceeds renal excretion, it will cause hyperuricemia (the concentration of uric acid in the blood is higher than 0.42mmol/L). Hyperuricemia can cause or exacerbate a variety of diseases: hyperuricemia caused by massive lysis of tumor cells during chemotherapy for malignant tumors can cause metabolic disorders, renal failure and even death, that is, tumor lysis syndrome; uric acid deposition in joints can cause Gout, deposition in renal tubules causes renal dysfunction, renal tubular damage, and inflammation of IgA nephropathy; uric acid stimulates the proliferation of vascular smooth muscle cells and leads to endothelial cell dysfunction; hyperuricemia is an important risk factor for cardiovascular diseases such as atherosclerosis.

The current common prevention and treatment plan for hyperuricemia has the following defects: (1) allopurinol can block the production of uric acid, but it will increase the renal excretion of uric acid precursors (hypoxanthine and The load of xanthine, which is more insoluble than uric acid in urine), is worse for patients with high concentrations of blood xanthine associated with hyperuricemia; Ineffective; (3) Allopurinol and probenecid drugs have obvious liver and kidney toxicity, and can easily cause allergic reactions.

The excellent water solubility of allantoin and the efficient excretion of allantoin by the kidneys make UC an ideal drug for the treatment of hyperuricemia. Clinical studies have shown that UC can quickly and efficiently reduce blood uric acid with high specificity. In the treatment of tumor lysis syndrome, UC is safer and more effective than allopurinol. More than 20 years ago, France and Italy approved Aspergillus flavus UC (Uricozyme ), and the United States and the European Union approved gene recombinant UC (Rasburicase) at the beginning of this century for the prevention and treatment of hyperuricemia caused by tumor chemotherapy. UC can also be used to treat gout. There is a great need for the use of UC as a drug to lower plasma uric acid.

However, all natural UCs have inherent defects: (1) The pH value of the external environment has a great influence on the catalytic ability of UC. The optimum pH value of UC is alkaline (8.5～9.2), the catalytic ability is low when pH 7.4 (such as in blood plasma) (the UC in Bacillus fastidiosa is only 20% of the activity when the optimum pH value), pH 5 (such as In the renal tubules) the catalytic ability is lower. This results in lower activity of UC in vivo, higher dosage required for treatment, higher treatment cost and lower safety. (2) The half-life in vivo circulation is short. UC is unstable in vivo and is easily deactivated by protease hydrolysis. (3) It is easy to induce the body's immune response, resulting in immunogenicity and antigenic response. The incidence of severe allergic reactions of Uricozyme is 5%, and adverse reactions such as vomiting (~50%), fever (~46%), and nausea (~27%) often occur when using UC.

Aiming at the defects of natural enzymes, there are the following studies: UC is adsorbed and covalently bound to chitosan or dextran, UC is embedded in carboxymethyl amylose or encapsulated in red blood cells, and UC is cross-linked with albumin. Linking, encapsulating PEGylated UC in liposomes, encapsulating UC in alginate microcapsules, immobilizing UC on the surface of metal chelating beads (150-200 μm), covalently binding UC to immunoliposomes Or ordinary liposome surface, etc. However, none of the above methods can enable UC to exhibit catalytic activity in an optimal or near-optimal pH environment, and most of them have disadvantages such as easy enzyme detachment, low biocompatibility, easy to cause blood coagulation, unsatisfactory stability and specificity, etc. .

In recent years, people have made some progress in modifying UC with PEG. Puricase, which is currently undergoing Phase III clinical trials in North America, is the most successful example of PEGylated UC research. Although PEGylated UC has certain advantages over natural enzymes in prolonging the half-life of the enzyme, reducing immunogenicity and antigen reactivity, there are still some disadvantages: (1) it is still at pH 7.4 instead of the optimum pH value of UC (8.5～ 9.2) function, the activity of UC is lower, and the activity after PEGylation is lower (retained specific activity is 10%-40%). (2) Even with the optimized Puricase, the bioavailability after intravenous injection is lower than that of the natural enzyme (partially due to the high molecular weight of the enzyme-PEG polymer). (3) PEGylated UC will be immunogenic as long as a small amount of macromolecular aggregates is present. (4) PEGylated UC has high technical requirements, complicated process and high cost.

So far, studies on enzyme-mediated semipermeable microreaction systems have mainly focused on PEGylated polylactic/glycolic acid (PLGA) nanoparticles, PLGA microspheres, polyisobutylcyanoacrylate (PIBCA) nanoparticles, and lipid Nanoparticles, the microenvironment of the enzyme constructed is mostly neutral or slightly acidic, which is not suitable for UC (see Figure 1).

After querying patents and literature, no one uses alkaline (pH8-9) buffer pairs to construct a semi-permeable alkaline micro-reaction system mediating uricase. Moreover, the design ideas, formulations, preparation methods, preparations obtained and their characteristics of this patent are all different from previous studies.

Contents of the invention

The technical problem to be solved by the present invention is to provide a uricase lipid nanoparticle and a preparation method thereof. The preparation is aimed at the exact clinical needs and can overcome the shortcomings of other uricase preparations developed so far. The uricase lipid nanoparticles prepared in this study can significantly improve the activity of uricase at pH 7.4 in body fluid, prolong the half-life, have no immunogenicity, reduce dosage, and improve stability. The prepared preparation can be used clinically as a dosage form for intravenous administration.

The uricase lipid nanoparticle of the present invention is a colloid drug delivery system, including a therapeutically effective dose of uricase, phospholipids, cholesterol, a pH-adjusting buffer, a stabilizer bovine serum albumin, and the preparation of the lipid nanoparticle. other additives. The particle size of lipid nanoparticles is 100-1000nm, and the preferred prescription is: 1 part of lecithin, 1 part of cholesterol, 0.05 parts of polyethylene glycol-type long-acting lipids such as DSPE-PEG20000, and 0.3 U/kg of uricase. ml, bovine serum albumin 0.05mg/ml, 50mM boric acid-borax buffer solution pH 8.5. The particle size of the optimized lipid nanoparticles is about 200nm. The preparation method of the uricase lipid nanoparticles of the present invention is as follows: dissolving the formula amount lecithin and cholesterol in an appropriate amount of organic solvent, evaporating the organic solvent under reduced pressure with a rotary evaporator, adding an appropriate amount of organic solvent, adding 50 mmol/l of UC and BSA L boric acid-borax buffer buffer solution (pH 8-9), ultrasonically in a water bath until a uniform dispersion system with opalescence is formed, continue to evaporate under reduced pressure until the gel collapses into a milky white uniform liquid with opalescence , add an appropriate amount of boric acid buffer solution, continue to rotate under reduced pressure, and place the resulting suspension at 4°C in the refrigerator for 24 hours, and pass through a 0.22 μm microporous membrane to obtain the obtained suspension. The total number of lipid nanoparticles obtained in the present invention with particle diameters less than 1 μm shall not be lower than 95%, the total number of lipid nanoparticles greater than 1 μm shall not exceed 3%, and nanoparticles larger than 5 μm shall not be detected. The lipid nanoparticles obtained in the present invention can be separated and removed from the free enzymes not encapsulated in the lipid nanoparticles by Sephadex column chromatography. The average particle size of the uricase nanoparticles obtained by the optimized formula and process is about 200nm, the Zeta potential is <-30mV, and the encapsulation efficiency is >88%. The lipid nanoparticles obtained in the present invention can be injected intravenously for clinical use, or prepared into powder and freeze-dried formulations for administration by methods recognized in the art.

Under the condition of medium pH7.4, the same dose of UC mediated by the lipid nanoparticles (prepared with pH 8.5 boric acid buffer) mediated by the present invention will reduce uric acid from high concentration (0.6mM) to normal level ( 0.24mM), the time required is significantly shorter than that of natural UC, down to the low level of other uric acid, and the time required for UC mediated by lipid nanoparticles is also significantly lower than that of natural UC. The uricase lipid nanoparticles prepared in this study can significantly improve the activity of uricase at pH 7.4 in body fluid (the activity of preparation-mediated UC retention can reach 80-150% of the activity of natural enzymes at the optimum pH, which requires The intracellular UC of Bacillus is only 20% of the optimum pH value, and the activity of PEGylated UC is 10%-40% when the body fluid pH is 7.4), and has no immunogenicity (the complete Freund's containing UC is injected subcutaneously). Prepare rabbit antiserum with adjuvant emulsion, incubate uricase lipid nanoparticles with different amounts of antiserum at 37°C for 30 minutes, then investigate antigenicity, uricase lipid nanoparticles do not react with antiserum, the same dose of natural enzyme highly immunogenic). The uricase lipid nanoparticles prepared by adding polyethylene glycol-type long-lasting lipids can significantly prolong the half-life of uricase in vivo (from 4 hours to 18.5 hours of natural enzyme). Uricase lipid nanoparticles are non-irritating to blood vessels (select 20 healthy Kunming mice, inject 0.2ml uricase lipid nanoparticles into the tail vein, 1 hour after administration, the mice do not have any abnormal behavior, no stimulation Sexual response, mouse activity and diet were not affected). Uricase lipid nanoparticles have no coagulation and no erythrocyte agglutination (one big-eared white rabbit, take blood from the heart, prepare 2% erythrocyte suspension in each test tube, and then add different amounts of test drug solution and normal saline Add only physiological saline to the positive control tube, add only deionized water to the positive control tube, place in a 37°C incubator, and observe once every 20 minutes for a total of 6 hours. Add lipid nanoparticles to each tube, Neither the supernatant of the negative control tube nor the transparent red and reddish-brown flocculent precipitates appeared, and the positive control tube was completely hemolyzed).

In this patent, for the first time, a semi-permeable lipid nanoparticle microreaction system mediating uricase was constructed with an alkaline (pH 8-9) buffer, and the microenvironment of the nanoparticle mediating uricase was determined to be alkaline with a fluorescent probe. Nature, which is different from the acidic or neutral microenvironment of conventional lipid nanoparticles. The prepared uricase lipid nanoparticle can well retain the activity of uricase, has no immunogenicity, reduces dosage, improves stability, and the lipid nanoparticle prepared by adding long-acting lipid can obviously prolong the half-life of uricase in vivo.

Description of drawings:

Fig. 1 is under the condition of medium pH7.4, the situation that uricase lipid nanoparticle of the present invention and natural enzyme reduce uric acid (the uric acid concentration at the beginning of the system is 0.60mmol/L). Preparation 1 is the situation that the uricase lipid nanoparticle preparation prepared by the present invention (prepared with boric acid-borax buffer solution with pH of 9) reduces uric acid (the uric acid concentration at the beginning of the system is 0.60mmol/L), and preparation 2 is natural The PBS buffer preparation of uricase (pH is 7.4) reduces the situation of uric acid (the uric acid concentration at the beginning of the system is 0.60mmol/L), preparation 3 is the borate buffer preparation of natural uricase (pH is 9) (reduces uric acid (system The initial uric acid concentration is 0.60mmol/L) Determination method: add 6mL of 0.60mmol/L uric acid PBS (pH is 7.4) to 60μL sample (preparation), mix well, shake at 37°C constant temperature, and take samples at different time points. Time Points: 0, 0.083h, 0.167h, 15min-0.25h, 0.5h, 0.75h, 1h, 2h, 4h, 8h, 12h, 16h, 24h. Measure the concentration of uric acid, take time as the abscissa, and the remaining uric acid in the system The percentage of is plotted on the ordinate.)

Fig. 2 is the electron micrograph of uricase lipid nanoparticle of the present invention

Fig. 3 is the particle size distribution of uricase lipid nanoparticle of the present invention

Fig. 4 is the Zeta potential of uricase lipid nanoparticle of the present invention

Fig. 5 is the standard curve of FITC fluorescent probe (emission wavelength is 522nm). It is recorded that the ratio (λ495/409) of the fluorescence intensity _FEM of the lipid nanoparticles constructed by the boric acid-borax buffer solution at pH 8.5 is 12.76. From the FITC fluorescent probe standard curve, it can be known that the uricase lipid nanoparticles constructed by the present invention The particle microenvironment is about 8.0, which is slightly alkaline.

In order to further illustrate the present invention and its advantages, the following specific examples are given, and it should be understood that these examples are only for illustration and not as limitation of the scope of the present invention.

Example 1:

Formula: 0.8 parts of lecithin, 0.8 parts of cholesterol, 0.07 parts of DSPE-PEG2000, appropriate amount of uricase and bovine serum albumin (making the content in the prepared preparation 0.3U/ml, bovine serum albumin 0.05mg/ml), 50mM Tris buffer The liquid pH is 8. The preparation method is: dissolve the formula amount of lecithin and cholesterol in 10ml of ether, evaporate the chloroform under reduced pressure with a rotary evaporator, add 15ml of ether, add 50mmol/L boric acid-borax buffer solution containing UC and BSA, and ultrasonic in a water bath To form a uniform dispersion system with opalescence, continue to evaporate under reduced pressure until the gel collapses into a milky white opalescent uniform liquid, add an appropriate amount of boric acid buffer, continue to rotate under reduced pressure, and the resulting suspension is stored in the refrigerator for 4 After standing at ℃ for 24 hours, pass through a 0.22 μm microporous membrane to obtain the product.

Example 2:

Formula: 1.5 parts of lecithin, 1 part of cholesterol, 0.05 part of DSPE-PEG5000, appropriate amount of uricase and bovine serum albumin (making the content in the prepared preparation 0.2U/ml, bovine serum albumin 0.08mg/ml), 10mM boric acid - Borax buffer (pH 8.5). The preparation method is: dissolve the formula amount of lecithin and cholesterol in 20ml of chloroform, evaporate the chloroform to dryness under reduced pressure with a rotary evaporator, add 30ml of ether, add 50mmol/L boric acid-borax buffer solution containing UC and BSA, and the rest of the operations are the same as in the examples 1.

Example 3:

Formula: 2 parts of lecithin, 2 parts of cholesterol, 0.05 part of DSPE-PEG2000, appropriate amount of uricase and bovine serum albumin (making the content in the prepared preparation 0.1U/ml, bovine serum albumin 0.1mg/ml), 100mMBicine buffer solution (pH 9). The preparation method is as follows: dissolve the formulated amount of lecithin and cholesterol in 30ml of ether, evaporate to dryness under reduced pressure with a rotary evaporator, add an appropriate amount of ether, add 100mmol/LBicine buffer solution containing UC and BSA, and the rest of the operations are the same as in Example 1.

Example 4:

Formula: 1 part of lecithin, 1 part of cholesterol, 0.06 part of DSPE-PEG5000, appropriate amount of uricase and bovine serum albumin (making the content in the prepared preparation 0.5U/ml, bovine serum albumin 0.15mg/ml), 50mM Tris buffer solution (pH 8.5). The preparation method is as follows: dissolve the formula amount of lecithin and cholesterol in 50ml of chloroform, evaporate the chloroform to dryness under reduced pressure with a rotary evaporator, add an appropriate amount of ether, add 50mmol/LTris buffer solution containing UC and BSA, and the rest of the operations are the same as in Example 1.

Example 5:

Formula: 1.2 parts of lecithin, 2.8 parts of cholesterol, 0.05 parts of DSPE-PEG2000, appropriate amount of uricase and bovine serum albumin (making the content in the prepared preparation 0.8U/ml, bovine serum albumin 0.15mg/ml), 50mM boric acid - Borax buffer (pH 8.5). The preparation method is as follows: dissolve the formula amount of lecithin and cholesterol in 50ml of chloroform, evaporate the chloroform to dryness under reduced pressure with a rotary evaporator, add an appropriate amount of ether, add 50mmol/L boric acid-borax buffer solution containing UC and BSA, and the rest of the operations are the same as in the examples 1.

Embodiment 6:

Recipe: 3 parts of lecithin, 1.5 parts of cholesterol, appropriate amount of uricase and bovine serum albumin (making the content in the prepared preparation be 1U/ml, bovine serum albumin 0.1mg/ml), 30mM Bicine buffer solution (pH is 8.5). The preparation method is as follows: dissolve the formula amount of lecithin and cholesterol in 15ml of chloroform, evaporate the chloroform to dryness under reduced pressure with a rotary evaporator, add 8ml of ether, add 30mmol/LBicine buffer solution containing UC and BSA, and the rest of the operations are the same as in Example 1.

Claims (4)

1. A uricase lipid nanoparticle, characterized in that in the preparation, the uricase content is 0.1～1U/mL, bovine serum albumin is 0.02～0.15mg/ml, and the used buffer pH for preparing the preparation is 8～9, The ionic strength is 10-100mM, and the weight ratio of the other components is: 1-3 parts of phospholipid and 1-3 parts of cholesterol. 2. The uricase lipid nanoparticle according to claim 1 is characterized in that the used buffer for preparing preparations is boric acid-borax buffer, Tris buffer, Bicine buffer, ionic strength is 10～100mM, and the pH value range Between 8 and 9. 3. The uricase lipid nanoparticle according to claim 1, characterized in that the phospholipid used in the preparation of the preparation is one or more of lecithin for injection, soybean lecithin, polyethylene glycol type long-acting lipid. 4. The uricase lipid nanoparticle according to claim 1, characterized in that: the average particle diameter is less than 1 μm, the Zeta potential is less than -20, the microenvironment of the lipid nanoparticle-mediated enzyme is slightly alkaline, and when the pH of the body fluid is 7.4 , compared with natural uricase, the catalytic activity of uricase lipid nanoparticles is increased, the half-life is prolonged, and the immunogenicity is decreased.

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