Development and validation of a major adverse limb events prediction model for peripheral arterial disease with frailty.

OBJECTIVE: To investigate the risk factors for major limb adverse events (MALE) in peripheral arterial disease (PAD) combined with frailty and to develop and validate a risk prediction model of MALE. METHODS: This prospective study was performed in the vascular surgery department of patients in six hospitals in southwest China. Prospective collection of patients with PAD combined with frailty from February 1 to December 20, 2021, with MALE as the primary outcome, and followed for 1 year. The cohort was divided into a development cohort and a validation cohort. In the development cohort, a multivariate risk prediction model was developed to predict MALE using random forests for variable selection and multivariable Cox regression analysis. The model is represented by a visualized nomogram and a web-based calculator. The model performance was tested with the validation cohort and assessed using the C-statistic and calibration plots. RESULTS: A total of 1179 patients were prospectively enrolled from February 1 to December 20, 2021. Among 816 patients with PAD who were included in the analysis, the median follow-up period for this study was 9 ± 4.07 months, the mean age was 74.64 ± 9.43 years, and 249 (30.5%) were women. Within 1 year, 222 patients (27.2%) developed MALE. Target lesion revascularizations were performed in 99 patients (12.1%), and amputations were performed in 131 patients (16.1%). The mortality rate within the whole cohort was 108 patients (13.2%). After controlling for competing risk events (death), the cumulative risk of developing MALE was not statistically different. Prealbumin (hazard ratio [HR], 0.6; 95% confidence interval [CI], 0.41-0.89; P = .010), percutaneous coronary intervention (HR, 2.31; 95% CI, 1.26-4.21; P = .006), Rutherford classification (HR, 1.77; 95% CI, 1.36-2.31; P < .001), white blood cell (HR, 1.85; 95% CI, 1.20-2.87; P = .005), high altitude area (HR, 3.1; 95% CI, 1.43-6.75; P = .004), endovascular treatment (HR, 10.2; 95% CI, 1.44-72.50; P = .020), and length of stay (HR, 1.01; 95% CI, 1.00-1.03; P = .012) were risk factors for MALE. The MALE prediction model had a C-statistic of 0.76 (95% CI, 0.70-0.79). The C-statistic was 0.68 for internal validation and 0.66 for external validation for the MALE prediction model. The MALE prediction model for PAD presented an interactive nomogram and a web-based network calculator. CONCLUSIONS: In this study, the MALE prediction model has a discriminative ability to predict MALE among patients with PAD in frailty. The MALE model can optimize clinical decision-making for patients with PAD in frailty.

Comparative Pharmacokinetic Study of Hesperetin after Oral Administration in Normal and Hyperuricemia Rats by UPLC-MS/MS.

BACKGROUND: Hesperetin has antihyperuricemia activity, and the pharmacokinetic profiles of hesperetin may be altered by hyperuricemia. This study aimed to develop a highly sensitive and specific method for the determination of hesperetin in normal and hyperuricemia rats, and to compare pharmacokinetic profiles of hesperetin after oral administration between normal and hyperuricemia rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into one normal group (group A) and four hyperuricemia groups (group B, C, D, and E). Groups A, B, C, and D received a single dose (9-81 mg/kg) of hesperetin on Day 28, respectively, while group E received multiple doses (27 mg/kg) of hesperetin once daily for 28 days. Blood samples were collected at 10 different time points post-dose, and hesperetin was determined by Ultra-high Performance Liquid Chromatography- tandem Mass Spectrometric (UPLC-MS/MS). RESULTS: Compared with normal condition of group A, hyperuricemia of group C induced 48.19% and 19.57% decreases in Cmax and CL/F, and resulted in 58.25% and 19.48% increases in Tmax and AUC0-t for hesperetin, respectively. After 28 days of hesperitin treatment, Cmax of group E was significantly elevated than that of group C (p < 0.05). Hesperetin exhibited nonlinear pharmacokinetic properties in the range of 9-81 mg/kg in hyperuricemia rats. CONCLUSION: The pharmacokinetic parameters of hesperetin in hyperuricemia rats were reported for the first time. Intestinal injury may be ameliorated by hesperetin in hyperuricemia rats after 28 days' treatment. These findings could provide more beneficial information to the mechanism and clinical applications of hesperetin.

Erratum: Uricase alkaline enzymosomes with enhanced stabilities and anti-hyperuricemia effects induced by favorable microenvironmental changes.

This corrects the article DOI: 10.1038/srep20136.

Nanocapsule assemblies as effective enzyme delivery systems against hyperuricemia.

The uricase nanocapsule assemblies (UNAs) were developed as effective delivery systems against hyperuricemia following parenteral enzyme therapy. UNAs were characterized in terms of micromorphology, size, catalytic activity, stability, and enzymatic kinetics. The pharmacokinetics/pharmacodynamics in rats after intravenous or subcutaneous injection was investigated. Immunogenicity, hemolysis and stimulation were determined. UNA was composed of many nanocapsules, and thus had higher loading efficiencies and stabilities than a single nanocapsule. The uricase molecules entrapped inside nanocapsules were separated from the circulating bloodstream to retain catalytic activities for a longer time than free uricase. UNAs increased the bioavailability and uric acid-lowering efficacy of uricase, while the immunogenicity and hemolysis rate of uricase were decreased. The superior properties of UNAs might be ascribed to the favorable conformational changes of uricase. Nanocapsule assemblies appeared to be able to deliver uricase effectively. This study also highlighted the importance of suitable systems for therapeutic enzyme delivery.

Uricase alkaline enzymosomes with enhanced stabilities and anti-hyperuricemia effects induced by favorable microenvironmental changes.

Enzyme therapy is an effective strategy to treat diseases. Three strategies were pursued to provide the favorable microenvironments for uricase (UCU) to eventually improve its features: using the right type of buffer to constitute the liquid media where catalyze reactions take place; entrapping UCU inside the selectively permeable lipid vesicle membranes; and entrapping catalase together with UCU inside the membranes. The nanosized alkaline enzymosomes containing UCU/(UCU and catalase) (ESU/ESUC) in bicine buffer had better thermal, hypothermal, acid-base and proteolytic stabilities, in vitro and in vivo kinetic characteristics, and uric acid lowering effects. The favorable microenvironments were conducive to the establishment of the enzymosomes with superior properties. It was the first time that two therapeutic enzymes were simultaneously entrapped into one enzymosome having the right type of buffer to achieve added treatment efficacy. The development of ESU/ESUC in bicine buffer provides valuable tactics in hypouricemic therapy and enzymosomal application.

[The Pharmacokinetics and Pharmacodynamics of Intravenous Uricase Multivesicular Liposomes].

OBJECTIVE: To determine and compare the pharmacokinetics and pharmacodynamics of uricase-multivesicular liposomes (UOMVLs) with free uricase (UOX) in rats. METHODS: UOMVLs were prepared by the double emulsion method and confirmed with its entrapment efficiency, size and Zeta potential. Twelve healthy rats were randomly divided into two groups: one with i. v. injection of UOMVLs, and the other with i. v. injection of UOX. Their serum activity of uricase was assayed. The pharmacokinetic parameters were calculated using software DAS 2. 1. 1. Another 24 male SD rats were enrolled, the rat model of hyperuricemia was established with hypoxanthine and potassium oxonate, while normal group (n=6) was set as control. Injection of UOMVLs (1 mL, 0. 47 U/mL), UOX (1 mL, 0. 47 U/mL) and nothiy were given 1 h later in UOMVLs group (n=6), UOX group (n=6) and model group (n=6), and their serum uric acid levels were determined 1, 2, 3, 5, 7, 9, 12, 24, 36, 48 h after the establishment of hyperuricemia model. RESULTS: The entrapment efficiency of UOMVLs was (63. 75 ± 3. 65) %, with an average particle size of (22. 56 ± 1. 70) µm and Zeta potential of (-41. 81±6. 59) mV. The pharmacokinetic parameters of UOMVLs and UOX were as follows, respectively: area under time-concentration curve from 0 to infinity time (AUC0-∞) (498. 83 ± 58. 85) U/L . h and (28. 49 ± 9. 95) U/L . h; time to peak concentration (Tmax) (1. 00±0. 00) h and (0. 00±0. 00) h; peak concentration (Cmax) (73. 04±6. 35) U/L and (31. 00±6. 03) U/L; elimination half-life (t1/2) (3. 49±0. 80) h and (1. 17±0. 33) h. The relative bioavailability of UOMVLs was (1 750. 90±206. 56) %. UOMVLs decreased serum uric to normal in 9 h; whereas it took 48 h for the UOX group and the model group to return to normal. CONCLUSION: UOMVLs can prolong tmax and t1/2 and improve the relative bioavailability. UOMVLs decrease serum uric acid levels in rats with hyperuricemia more effectively than UOX.

Nanosomal Microassemblies for Highly Efficient and Safe Delivery of Therapeutic Enzymes.

Enzyme therapy has unique advantages over traditional chemotherapies for the treatment of hyperuricemia, but overcoming the delivery obstacles of therapeutic enzymes is still a significant challenge. Here, we report a novel and superior system to effectively and safely deliver therapeutic enzymes. Nanosomal microassemblies loaded with uricase (NSU-MAs) are assembled with many individual nanosomes. Each nanosome contains uricase within the alkaline environment, which is beneficial for its catalytic reactions and keeps the uricase separate from the bloodstream to retain its high activity. Compared to free uricase, NSU-MAs exhibited much higher catalytic activity under physiological conditions and when subjected to different temperatures, pH values and trypsin. NSU-MAs displayed increased circulation time, improved bioavailability, and enhanced uric acid-lowering efficacy, while decreasing the immunogenicity. We also described the possible favorable conformational changes occurring in NSU-MAs that result in favorable outcomes. Thus, nanosomal microassemblies could serve as a valuable tool in constructing delivery systems for therapeutic enzymes that treat various diseases.

Azithromycin cationic non-lecithoid nano/microparticles improve bioavailability and targeting efficiency.

PURPOSE: The purpose of this study was to develop and evaluate the azithromycin cationic non-lecithoid nano/microparticles with high bioavailability and lung targeting efficiency. METHODS: The cationic niosomes with different sizes (AMCNS-S and AMCNS-L) along with varied built-in characteristics were produced to achieve high bioavailability and lung targeting efficiency of azithromycin (AM) via two administration routes widely used in clinical practice, i.e., oral and intravenous routes, instead of transdermal route (by which the only marketed niosome-based drug delivery dermatologic products were given). The possible explanations for improved bioavailability and lung targeting efficacy were put forward here. RESULTS: AMCNS-S (or AMCNS-L) had high bioavailability, for example, the oral (or intravenous) relative bioavailability of AMCNS-S (or AMCNS-L) to free AM increased to 273.19% (or 163.50%). After intravenous administration, AMCNS-S (or AMCNS-L) had obvious lung targeting efficiency, for example, the lung AM concentration of AMCNS-S (or AMCNS-L) increased 16 (or 28) times that of free AM at 12 h; the AM concentration of AMCNS-S (or AMCNS-L) in lung was higher than that in heart and kidney all the time. CONCLUSIONS: The development of niosome-based AM nanocarriers provides valuable tactics in antibacterial therapy and in non-lecithoid niosomal application.

[Correlation between in vitro release and in vivo absorption of sustained-releasing tablets of neostigmine bromide].

OBJECTIVE: To determine the correlation between in vitro release and in vivo absorption of sustained-releasing tablets of neostigmine bromide. METHODS: Water was used as dissolution medium to measured in vitro release of neostigmine bromide. After a single oral administration of 100 mg neostigmine bromide to rabbits, the plasma concentrations of neostigmine bromide in the rabbits were determined by HPLC. The compartment model and deconvolution method were employed to explain the in vitro-in vivo correlation. RESULTS: Using Y as cumulative in vitro release and Fa as percentage of absorption, the regression equation was established: Fa = 0.9298Y + 4.6074, r = 0.9961. The input function of R = 2.0163Y-11.242,r = 0.9270. CONCLUSION: The correlation between in vitro release and in vivo absorption of neostigmine bromide is good.

Improved biological properties and hypouricemic effects of uricase from Candida utilis loaded in novel alkaline enzymosomes.

OBJECTIVE: Previous studies on various enzymosomes (functional lipid vesicles encapsulating an enzyme) have been mostly carried out in vitro and have focused on preserving catalytic activity and improving the stability of the enzyme. Until now, few studies have focused on their in vivo fate. Similarly, although we have previously reported the increased in vitro uricolytic activity (about 2.2 times higher than that of free uricase, or three times higher than that of PEGylated uricase, Puricase(®), under physiological pH and temperature) and improved stability of the novel alkaline enzymosomes (functional lipid vesicles encapsulating uricase from Candida utilis: uricase-containing lipid vesicles, UOXLVs), it is still necessary to study the biological properties and hypouricemic effects of UOXLVs in vivo. METHODS: The enzyme kinetics, pharmacokinetics, pharmacodynamics, immunogenicity, and preliminary safety of UOXLVs were evaluated. RESULTS: The Michaelis constant (K(m)) value of the UOXLVs was slightly lower than that of the free enzyme. The enzyme release from the UOXLVs lasted over 12 hours and their circulation half-life was about sevenfold longer than that of the free uricase. Meanwhile, the UOXLVs had a 22-fold increase in the area under the curve compared with the free uricase. Furthermore, it took less than 3 hours for the UOXLVs to lower the plasma uric acid concentration from a high to a normal level, compared with 6 hours for the free uricase. In addition, the UOXLVs had much less immunogenicity than free uricase and were well tolerated by all animals throughout the observation period. CONCLUSION: The UOXLVs markedly improved the biological properties and enhanced the hypouricemic effects of uricase in vivo.

Uricase from Bacillus fastidious loaded in alkaline enzymosomes: enhanced biochemical and pharmacological characteristics in hypouricemic rats.

The aim of this study was to assess the potential of a novel alkaline enzymosome to deliver uricase from Bacillus fastidious (UBF) and enhance its biochemical and pharmacological characteristics. The in vitro catalytic activity of the UBF loaded in the novel alkaline enzymosomes (ESUBFs) was almost 3.8 times that of free UBF at the optimum pH or 1.5 times that of free UBF at the physiological pH. Following intravenous (i.v.) administration (2000 mU/kg) in rats, ESUBFs provided significantly higher (22.5-fold) area under the plasma concentration (AUC) and longer (8.2-fold) circulation half-life (t(1/2)) compared with free UBF, respectively. Further, it took only 4.5h (or 1.1h) for ESUBFs to lower the plasma uric acid concentration from a high level to the normal level of rat (or human beings), compared with 7.6h (or 5.4h) for free UBF. Our results showed that ESUBFs could efficiently deliver UBF and favorably modify its biochemical and pharmacological characteristics by increasing the AUC, t(1/2), and catalytic activity. Therefore, ESUBFs might be a preferred alternative to cure hyperuricemia and gout.

Design and evaluation of a novel evodiamine-phospholipid complex for improved oral bioavailability.

A novel evodiamine (EVO)-phospholipid complex (EPLC) was designed to improve the bioavailability of EVO. A central composite design approach was employed for process optimization. EPLC were characterized by differential scanning calorimetry, ultraviolet spectroscopy, Fourier transformed infrared spectroscopy, (1)H-NMR spectroscopy, matrix-assisted laser desorption/ionization time-of-flight spectroscopy, apparent solubility, and dissolution rate. After oral administration of EPLC, the concentrations of EVO at different time points were determined by high-performance liquid chromatography. The optimal formulation for EPLC was obtained where the values of X (1), X (2), and X (3) were 2, 0.5, and 2.5 mg/mL, respectively. The average particle size and zeta potential of EPLC with the optimized formulation were 246.1 nm and -26.94 mV, respectively. The EVO and phospholipids in the EPLC were associated with non-covalent interactions. The solubility of EPLC in water and the dissolution rate of EPLC in phosphate-buffered solution (pH 6.8) were substantially enhanced. The plasma EVO concentration-time curves of EPLC and free EVO were both in accordance with the two-compartment model. The peak concentration and AUC(0-∞) of EPLC were increased, and the relative bioavailability was significantly increased to 218.82 % compared with that of EVO.

[Pharmacokinetics and relative bioavailability of neostigmine bromide sustained-release tablets].

OBJECTIVE: To study the pharmacokinetics and relative bioavailability of neostigmine bromide conventional tablets and sustained-release tablets in rabbits. METHODS: Six healthy rabbits were randomly divided into two groups for a cross self-contrast trial. RP-HPLC was used to detect plasma concentrations of neostigmine bromide. The pharmacokinetic parameters were calculated with the aid of DAS 2.0 software. RESULTS: The main pharmacokinetics parameters of the sustained-release tablets and conventional tablets were as follows, respectively: T(max)(3.67 +/- 1.51) hand (1.58 +/- 0.38) h; C(max) (5.04 +/- 1.19) mg/L and (4.56 +/- 1.70) mg/L; AUC(0 --> infinity) (32.82 +/- 9.88) mg/L x h and (29.84 +/- 14.27) mg/L x h. The relative bioavailability of the neostigmine bromide sustained-release tablets was 115.4%. CONCLUSION: The pharmacokinetics of neostigmine bromide accords with two compartments model, showing constant plasma concentration and relatively high bioavailability.