Rapidly separating dissolving microneedles with sustained-release colchicine and stabilized uricase for simplified long-term gout management

Despite growing prevalence and incidence, the management of gout remains suboptimal. The intermittent nature of the gout makes the long-term urate-lowering therapy (ULT) particularly important for gout management. However, patients are reluctant to take medication day after day to manage incurable occasional gout flares, and suffer from possible long-term toxicity. Therefore, a safe and easy-to-operate drug delivery system with simple preparation for the long-term management of gout is very necessary. Here, a chitosan-containing sustained-release microneedle system co-loaded with colchicine and uricase liposomes were fabricated to achieve this goal. This microneedle system was confirmed to successfully deliver the drug to the skin and maintain a one-week drug retention. Furthermore, its powerful therapeutic potency to manage gout was investigated in both acute gouty and chronic gouty models. Besides, the drug co-delivery system could help avoid long-term daily oral colchicine, a drug with a narrow therapeutic index. This system also avoids mass injection of uricase by improving its stability, enhancing the clinical application value of uricase. In general, this two-drug system reduces the dosage of uricase and colchicine and improves the patient's compliance, which has a strong clinical translation.

Resonance light scattering spectroscopy can directly characterize protein solubility / 南方医科大学学报

OBJECTIVE@#To develop a fast, sensitive and cost-effective method based on resonance light scattering (RLS) for characterization of protein solubility to facilitate detection of changes in solubility of mutant proteins.@*METHODS@#We examined the response curve of RLS intensities to the protein concentrations in synchronous scanning mode. The curve intersection points were searched to predict the maximal concentrations of the protein in dispersion state, which defined the solubility of the protein in this given state. Bovine serum albumin (BSA, 0-50 g/L) was used as the model to investigate the influences of pH values (6.5, 7.0, and 7.4) and salt concentrations (0.05, 0.10, 0.15, and 0.20 mol/L) on the determined solubility. The solubility of glutathione S-transferase isoenzymes alpha (GSTA, 0-27.0 g/L) and Mμ (GSTM, 0-20.0 g/L) were estimated for comparison. The RLS-based method was used to determine the solubility of uricase (MGU, 0-0.4 g/L) to provide assistance in improving the solubility of its mutants.@*RESULTS@#We identified two intersection points in the RLS response curves of the tested proteins, among which the lower one represented an approximation of the maximal concentration (or the solubility of the protein) in single molecular dispersion, and the higher one the saturated concentration of the protein in multiple molecular aggregation. In HEPES buffer, the two intersection points of BSA (isoelectric point 4.6) both increased with the increase of pH (6.5-7.4), and their values were ~1.2 g/L and ~33 g/L at pH 7.4, respectively; the latter concentration approached the solubility of commercial BSA in the same buffer at the same pH. The addition of NaCl reduced the values of the two intersection points, and increasing salt ion concentration decreased the values of the lower intersection points. Further characterizations of GSTA and GSTM showed that the low concentration intersection points of the two proteins were ~0.7 g/L and ~0.8 g/L, and their high concentration intersection points were ~10 g/L and ~11 g/L, respectively, both lower than those of BSA, indicating the feasibility of the direct characterization of protein solubility by RLS. The two concentration intersection points of MGU were 0.24 g/L and 0.30 g/L, respectively, and the low concentration intersection point of its selected mutant was increased by 2 times.@*CONCLUSIONS@#RLS allows direct characterization of the solubility of macromolecular proteins. This method, which is simple and sensitive and needs only a small amount of proteins, has a unique advantage for rapid comparison of solubility of low-abundance protein mutants.

Progress on diagnosis and treatment of tophus / 中国骨伤

Tophi deposit in the peripheral bone joints or soft tissues are formed by uric acid and urate crystal. It does not only affect local appearance but also destroy the bone and joint structure, resulting in loss of function. Traditional medical treatment is an effective way to control the hyperuricemia, but it is ineffective to tophus. Surgery is a relatively effective method for the treatment of tophus. It can successfully reduces the content of uric acid in the body and improves the limbs function, but it causes surgical trauma and complications. As new medicine, Urcase is a hot spot in the present study. It can not only control the blood uric acid level but also dissolve part of tophus. But the cost is higher than traditional medicines. There is still dispute in treatment for advanced tophus.

Characteristics in vitro and preliminary pharmacodynamics of hyaluronic acid-uricase multivesicular liposomes / 第二军医大学学报

Objective To investigate the characteristics of hyaluronic acid-uricase multivesicular liposomes (UHMVLs) in vitro and the pharmacodynamics of UHMVLs in rats. Methods UHMVLs was prepared by multiple emulsion method. The entrapment efficiency and physicochemical properties were detected. Twelve healthy male SD rats were enrolled in this study. The rat model of hyperuricemia was established with hypoxanthine and oteracil potassium, while the normal rats (n=3) were set as controls. Intravenous UHMVLs, uricase (UC) and nothing were given to the rats of UHMVLs group (n=3), UC group (n=3) and hyperuricemia model group (n=3), respectively; the levels of serum uric acid (UA) were detected in rats of the 4 groups. Results The average entrapment efficiency of UHMVLs was (62.48±3.87)%. The optimum temperatures of UHMVLs and UC were 40°, while the optimum pH values of UHMVLs and free UC were 8.0 and 8.5, respectively. The activity of UC in UHMVLs was significantly higher than that in free UC at the same temperature (20-70°) and pH value (6.5-9.5) (P<0.05). UHMVLs was more effective than free UC in decreasing serum UA in rats with hyperuricemia at all time points (P<0.05), except for 1 h, 36 h and 48 h. Conclusion Under the same condition, UHMVLs can improve not only the activity, but also the stability of UC. UHMVLs is more effective in decreasing serum uric acid in rats compared with free UC, which may pave a way for clinical application of UC.

Property and pharmacodynamics of uricase-catalase liposomes / 中国药理学通报

Aim To observe the optimal temperature and optimal pH of uricase-catalase liposomes(UCALP) and free uricase(UAE), and study the abilities of UCALP to reduce uric acid and hydrogen peroxide in mice with hyperuricemia.Methods UCALP were prepared by reverse phase evaporation, optimal temperature and optimal pH of UCALP and UAE were determined, respectively.Mouse model of hyperuricemia was established by intraperitoneally injection of uric acid, and the model mice were intravenously injected UCALP and UAE, respectively, then the serum concentration of uric acid and hydrogen peroxide in mice at different time points were measured by the assay kits, respectively.Results Optimal temperature of UCALP and UAE was 40℃, and optimal pH was 8.0 and 8.5, respectively.UCALP could more significantly lower uric acid level of hyperuricemia mice than that of UAE, and the concentration of hydrogen peroxide in UCALP group was lower than in UAE group.Conclusion UCALP can effectively decrease the level of uric acid and control the level of hydrogen peroxide in mice with hyperuricemia.

Acido úrico: ¿un nuevo factor contribuyente al desarrollo de obesidad?

The excess of uric acid is recognized as a risk factor for diverse metabolic diseases which include gout, urolithiasis, type 2 diabetes, arterial hypertension as well as metabolic syndrome and cardiovascular disease. Current studies suggest that the exaggerated increment in fructose consumption, caused mainly by added suggars, is implicated in the high prevalence of hyperuricemia in the western population.The loss of uricase by mutation of its gene 15 million years ago in the large hominids, including man, has contributed to hyperuricemia, facilitated through the metabolism of fructose the formation of uric acid. It has been proposed that the elevation of uric acid in the remote past was an evolutionary benefit to intensify the lipogenic effects of fructose, allowing humans to survive periods of fruit shortages. However, today the high consumption of fructose and resultant hyperuricemia are a disadvantage, increasing the development of obesity and type 2 diabetes.

El exceso de ácido úrico es reconocido como un factor de riesgo para diversas enfermedades metabólicas, incluyendo: gota, urolitiasis, diabetes tipo 2, hipertensión arterial, síndrome metabólico y enfermedad cardiovascular. Estudios actuales sugieren que el incremento exagerado del consumo de fructosa, proveniente especialmente de los azúcares añadidos, está implicado en la alta prevalencia de hiperuricemia que muestra la población occidental. La pérdida de la uricasa por mutación de su gen hace 15 millones de años atrás en los grandes homínidos, incluyendo el hombre, ha contribuido a la hiperuricemia, facilitando a través del metabolismo de la fructosa la formación de ácido úrico. Se ha propuesto que la elevación del ácido úrico en épocas pasadas, fue una ventaja evolutiva al intensificar los efectos lipogénicos de la fructosa, permitiendo al ser humano sobrevivir en períodos de escasez de frutas. Sin embargo, hoy la alta ingesta de fructosa y su hiperuricemia resultante es una desventaja, promoviendo el desarrollo de obesidad y diabetes tipo 2.

New drugs for gont: Research advances / 国际药学研究杂志

Gout is a common disease in the elderly men. The prevalence rate of gout is rising worldwide in recent years, and gout has become a serious metabolic disease threatening human health. Moreover, gout is also closely related to incidence of many diseases and symptoms such as hypertension, hyperlipidemia, atherosclerosis, obesity and insulin resistance. Recently, investigation and development of new drugs have attracted increasing attention. This paper summarizes the research advances in new agents for treatment of gout, including the uric acid reduction drugs that target the key enzymes of purine metabolism, the drugs which target the renal tubular urate transporters to lower the uric acid level, the dual inhibitors of xanthine oxidoreductase(XOR) and renal tubular urate transporters, and the uricase.

Preparation and characterization of hyaluronic acid-uricase liposomes / 第二军医大学学报

Objective To prepare hyaluronic acid-nricase liposomes (UHLP) and to investigate the activity and stability of UHLP and uricase (UC) in vitro. Methods UHLP was prepared using reverse-phase evaporation and observed by transmission electron microscopy. The entrapment efficiency, particle size and zeta potential of UHLP were detected. The physical and chemical properties of UC in free UC and UHLP-including the optimum pi I and temperature-thermal stability-storage stability, pi I stability, stability to trypsinase, and stability to metal ions and organic compounds, were examined. In addition, the mechanism by which UHLP promotes UC activity was also explored. Results The mean entrapment efficiency of UIILP was (57. 2 ± 3. 93)% (n = 3), mean particle size was (322. 6 ± 8. 2) nm (n=3), and mean zeta potential was (–19.4 ± 1.7) mV (n = 3). UHLP was round or oval in shape and was evenly distributed under transmission electron microscopy. The optimum temperature of UC in UHLP and free UC was 40°C -and the optimum pH values of UC in UHLP and free UC was 8. 0 and 8. 5, respectively. Thermal stability, storage stability, pi I stability, stability to trypsin and stability to metal ions and organic compounds of UC in UHLP were better than those in free UC. The envelopment of UC with UHLP increased the activity of UC by reversing conformation and exposing active center of UC. Conclusion UHLP can not only enhance the activity but also improve the stability of UC in vitro.

New drugs for gont:research advances / 国际药学研究杂志

Gout is a common disease in the elderly men. The prevalence rate of gout is rising worldwide in recent years,and gout has become a serious metabolic disease threatening human health. Moreover,gout is also closely related to incidence of many dis?eases and symptoms such as hypertension,hyperlipidemia,atherosclerosis,obesity and insulin resistance. Recently,investigation and development of new drugs have attracted increasing attention. This paper summarizes the research advances in new agents for treat?ment of gout,including the uric acid reduction drugs that target the key enzymes of purine metabolism,the drugs which target the re?nal tubular urate transporters to lower the uric acid level,the dual inhibitors of xanthine oxidoreductase(XOR)and renal tubular urate transporters,and the uricase.

Research on PEGylated uricase by periodate oxidation and reductive- amination / 中国药科大学学报

@#PEGylated uricase was prepared with the N-terminal amino site-specific modification by periodate oxidation followed by reductive-amination. A monomethoxy poly(ethylene glycol)intermediate was synthesized by amidation from monomethoxy poly(ethylene glycol)amine hydrochloride 20000(mPEG20000-NH2 ·HCl)with the relative molecular mass of 20 kD and N-(tert-butoxycarbonyl)-L-serine(Boc-Ser-OH), and then the Boc group of the intermediate was removed by trifluoroacetic acid(TFA)to produce the desired product Ser-mPEG20000. This compound could be oxidated by periodate to obtain a new poly(ethylene glycol)aldehyde derivative with high activity, which could be used to modify proteins with the N-terminal amino site-specific PEGylation after ultrafiltration, and the modification conditions to uricase by Ser-mPEG20000 were optimized. The structures of poly(ethylene glycol)intermediate and the target product were characterized by IR and 1H NMR, and the overall yield of the target product was 72. 8%. The preliminary modification to uricase indicated that the desired product Ser-mPEG20000 could modify proteins easily and efficiently. The optimal modification conditions of uricase PEGylated by Ser-mPEG20000 were obtained as follows: the molar ratio of Ser-mPEG20000 to uricase was 2 ∶1; the pH value of solution was 5. 0; the reaction temperature was 25 °C and the reaction time was 6 h.

Isolation, partial purification and characterization of thermophilic uricase from Pseudomonas otitidis strain SN4

Aims: The present study aimed at isolating new source of uricase producers from Malaysian hot springs together with partial purification and characterization of thermophilic uricase from novel strain. Methodology and results: A bacteria strain, designated as SN4, was found to have the ability to degrade uric acid. 16S rRNA analysis identified SN4 as Pseudomonas otitidis. Uricase was then extracted from SN4 and purification was performed via ammonium sulphate precipitation. The effects of temperature, pH and metal ions on partially purified uricase were evaluated. Results showed that 70% ammonium sulphate concentration gave the highest uricase activity at 4.18 U/mL compared to other concentrations. The molecular weight of the partially purified uricase was 33 kilodalton (kDa). The optimum temperature for uricase was 45 °C and its activity was highest at pH 8.0. Calcium ions and copper ions enhanced uricase activity while cobalt ions reduced uricase activity. Conclusion, significance and impact of study: Isolation and investigation of uricase producers from new sources such as thermophiles would increase availability and thermal stability of the uricase that could be used for significant purposes such as in biochemical and clinical applications.

Isolation of a novel uric-acid-degrading microbe Comamonas sp. BT UA and rapid biosensing of uric acid from extracted uricase enzyme.

Uric-acid-utilizing soil bacteria were isolated, and 16s rRNA sequence was studied for strain identification. The most prominent uricase-producing bacterium was identified as Comamonas sp BT UA. Crude enzyme was extracted, freeze-dried and its Km and Vmax were determined as 40 μM and 0.047 μM min−1ml−1 using Line-weaver Burke plot. An activity of 80 U/mg of total protein was observed when cultured at 37°C for 84 h at pH 7. The purified enzyme was used to measure uric acid by spectrophotometric method and electrochemical biosensor. In the biosensing system the enzyme was immobilized on the platinum electrode with a biodegradable glutaraldehyde-crosslinked gelatin film having a swelling percentage of 109±3.08, and response was observed by amperometry applying fixed potential. The electrochemical process as obtained by the anodic peak current and scan rate relationship was further configured by electrochemical impedance spectroscopy (EIS). The polymer matrix on the working electrode gave capacitive response for the electrode–electrolyte interaction. The sensitivity of the biosensor was measured as 6.93 μAμM−1 with a sensor affinity [Km(app)] of 50 μM and 95% reproducibility after 50 measurements. The spectrophotometric method could be used in the range of 6–1000 μM, whereas the biosensor generated linear response in the 1.5– 1000 μM range with a response time of 24 s and limit of detection of 0.56 μM. Uric acid was estimated in human blood samples by the biosensor and satisfactory results were obtained.

Effect of active fractions from modified Simiao Wan on hyperuricemia and its mechanism / 中国药理学与毒理学杂志

OBJECTIVE Tostudytheeffectofeffectivefractions(EFC)frommodifiedSimiaoWan (MSW)onthelevelofuricacidinhyperuricemicratsandinvestigatethemechanism.METHODS Two types of hyperurice mic models were established.A persistant hyperurice mic model was prepared by giving rats oxonic acid 200 mg·kg -1 and feeding the m with hypoxanthine.The models were ig given with modified Simiaowan (MSW)50 g·kg -1 or EFC 1 2.5,25 and 50 g·kg -1 consecutively for 5 d.The models were treated with MSW or EFC 50 g·kg -1 for 3 d.After the final treatment,the uric acid concen-trations in seru m and urine were determined by an auto matic bioche mistry analyzer.The activity of xan-thine oxidase (XOD )in the serum and liver was determined by enzymic colorimetric method.The activity of purine nucleoside phosphorylase (PNP)and uricase was detected by spectrophotometry. RESULTS Comparedwithnormalcontrolgroup,theserumlevelofuricacidinbothmodelgroupswas remarkably increased(P<0.01 ).Compared to model control group,MSW 50 g·kg -1 and EFC 12.5, 25 and 50 g·kg -1 significantly reduced the serum level of uric acid(P<0.05,P<0.01 ),but increased the activity of erythrocyte PNP(P<0.01 )in the oxonic acid potassium-induced hyperuricemia rats. MSW 50 g·kg -1 and EFC 50 g·kg -1 elevated the activity of liver uricase in the nicotinic acid-induced hyperuricemia rats(P<0.05).EFC 50 g·kg -1 also significantly decreased the serum XOD activity of hyperuricemicrats.CONCLUSION EFCsignificantlyinhibitstheserumlevelofuricacidinhyperurice-mic rats,which might involve down-regulation of protein levels of serum XOD to inhibit the production of uric acid and activation of uricase to pro mote the deco mposition of uric acid.

Study on stability of N-terminal site-specific PEGylated uncase / 中国生化药物杂志

Purpose The research was conducted to evaluate the stability of N-terminal site-specific PEGylated uricase. Methods The enzyme activity is used as an index to evaluate the thermal stability (at 4-80 ℃) , the pH stability, the ability of avoiding the trypsin digestion and half-life in vivo of PEG-uricase, then it is compared with uricase. Results PEGylated uricase is thermally more stable than uricase between 4 ℃ and 60 ℃, but at 70 ℃, the enzyme activity of both PEG-uricase and uricase decreases sharply. In the test of pH stability, the curve of uricase shows an obvious decrease of enzyme activity at 5 .2-6.0 and 9.2-10.0. The behavior of PEG-uricase indicates that the destabilizing was prevented effectively by PEGylation. The test of anti-trypsin digestion suggests that PEG-uricase retains 70% of its enzyme activity,but uricase only 20% ,200 minutes after being reserved in trypsin solution. Stability in vivo indicates that the half-life of PEG-uricase is 1 530 min and uricase, only 45 min. Conclusion PEGylated uricase has improved thermal stability, the pH stability, ability of protecting from trypsin digestion and stability in vivo.

Comparison between Candida utilis uricase and Bacillus fastidious uricase / 基础医学与临床

Objective To identify the characteristics of Candida utilis uricase and that of Bacillus fastidious intra-cellular uricase.Methods The new strains were cultivated to prepare the uricase.Then the uricase was purified by ammonium sulfate fractionation precipitation,DEAE-cellulose 52 chromatography and preparative PAGE.The activity,characterization and subunit constitution were determined with the purified uricase.Results The two uricas-es were the iso-tetramer.The single peptide chain molecular weight and total molecular weight of Candida utilis uricase were 33.0 ku and 134.0 ku individually,the optimum pH was close to 8.8 and more than 50% activity was reserved at pH 7.4,the Michaelis-Menten constant was (32.8 ± 3.1) μmol/L (n = 10) and the inhibition constant of xanthine was (4.8 ± 0.2) μmol/L(n = 3) for this uricase.For Bacillus fastidious intracellular uricase,its single peptide chain molecular weight and total molecular weight were 35.7 ku and 151.0 ku,the optimum pH was over 9.0 and less than 30% activity left at pH 7.4,the Michaelis-Menten constant and the inhibition constant of xanthine were (204 ± 14) μmol/L(n = 8) and (41 ± 7) μmol/L(n = 5) individually for it.Conclusion Through the research,a significant theoretical basis was funded for constructing hybrid medicinal uricase to treat diseases as-sociated with hyperuricemia.

Gota en el anciano / No presenta

La gota es la artritis crónica más común en el anciano. Puede ser fácilmente diagnosticada por la presencia de cristales de urato monosódico al examen microscópico del líquido articular. Existen algunas diferencias clínicas entre la gota del adulto y la del anciano. El tratamiento es altamente efectivo pero puede ocasionar serias reacciones adversas si no se tiene en cuenta la comorbilidad de este grupo.

Gout is the most common chronic arthritis in older people that can lead to significant and severe disability. It can be easy diagnosed with the presence of crystals of urate monosodic in the articular fluid. The clinical stages of gout include asymptomatic hyperuricemia, intermittent gouty arthritis, intercritical period and chronic tophaceous gout. There are some differences necessary to recognize to avoid errors in the management. Treatment of acute gout involves the use of NSAIDs, colchicine, corticosteroids or corticotropin (adrenocorticotropic hormone). Profilactic treatment includes the use of allopurinol and uricosuric agents; but all of these drugs could cause serious reactions in the elderly.

Research on PEG modification of uricase / 中国药科大学学报

To modify uricase with PEG reagent in order to decrease uricase immunogenieity and increase its stability.Methods:The branched PEG of 40 kD was chosen to modify native uricase.The properties of the mod-ified uricase including the stabilities to protease,pH and temperature,in vivo half-life time,as well as the immu-nogenicity were evaluated.The pharmacokinetic profiles of the midofied uricase were studied in mice.Results:It is demonstrated that the conjugation of PEG to lysine residues of Candida utilis uricase resulted in higher tryp-sin resistance.reduced immune response.and prolonged in vivo half-life.PEG modified uricase retained 80% of the enzymatic activity of native uricase.In addition,it was found that half-life in serum of the intravenously injec-ted PEGylated uriease of up to 696 min was longer that that of native uficase of 45 min.Higher plasma drug con-centrations were also reached with dosing of the PEGylated uricase to mice.Furthermore,the binding affinity Was shown to be reduced for the PEG-uricase using ELISA assay.and it was one-eishth that of native uricase.Final-ly,it Was indicated that the PEG uficase induced a delayed immunoresponse in mice following repeated adminis-trations.Conclusion:These findings demonstrate that this chemically modified form of uricase may serve as a potentially effective drug to treat gout patients.

A preliminary study on the replication of continuity hyperuricemia rat model by administration and fed with hypoxanthine / 中国药理学通报

Aim To observe the fluctuation of uric acid in serum after replicating rat hyperuricemia model with hypoxanthine、uricase inhibitor combined,and to discuss the method of continuity hyperuricemia replication on rats. Method Hypoxanthine was administered via stomach or bait vessel,uricase inhibitor was injected subcutaneouslly,then the relative biochemical indicators of rats were inspected respectively at different periods of time after replicating the model. Results When hypoxanthine(50 g HX?kg-1bait vessel or 100 g HX?kg-1bait vessel) was administered via bait vessel,uricase inhibitor(200mg?kg-1) was injected subcutaneouslly,the uric acid in serum of different groups were much higher than control group(P

A preliminary study on the establishment of rat model with acute hyperuricemia / 中国药理学通报

Aim To observe the fluctuation of uric acid in serum after replicating rat hyperuricemia model with hypoxanthine, uricase inhibitor singly or combined. Discuss the method of acute hyperuricemia replication on rats. Method Hypoxanthine was administered via stomach, uricase inhibitor was injected intraperitoneally, then inspect the uric acid in serum at different periods of time. Results When we combined hypoxanthine with uricase inhibitor, the uric acid in serum of different dose groups was higher than control group after 3 hours(P