Pharmacokinetic Modeling of Ceftiofur Sodium Using Nonlinear Mixed-Effects in Healthy Beagle Dogs

Ceftiofur (CEF) sodium is a third-generation broad-spectrum cephalosporin commonly used in an extra-label manner in dogs for the treatment of respiratory and urinary system infections. To contribute to the literature supporting CEF use in companion animals, we have developed a compartmental, nonlinear mixed-effects (NLME) model of CEF pharmacokinetics in dogs (PK). We then used the mathematical model to predict (via Monte Carlo simulation) the duration of time for which plasma concentrations of CEF and its pharmacologically active metabolites remained above minimum inhibitory concentrations (respiratory tract Escherichia coli spp). Twelve healthy beagle dogs were administered either 2.2 mg/kg ceftiofur-sodium (CEF-Na) intravenously (I.V) or 2.2 mg/kg CEF-Na subcutaneously (S.C). Plasma samples were collected over a period of 72 hours post-administration. To produce a measurement of total CEF, both CEF and CEF metabolites were derivatized into desfuroylceftiofur acetamide (DCA) before analysis by UPLC-MS/MS. No adverse effects were reported after I.V or S.C dosing. The NLME PK models were parameterized using the stochastic approximation expectation maximization algorithm as implemented in Monolix 2018R2. A two-compartment mammillary model with first-order elimination and first-order S.C absorption best described the available kinetic data. Final parameter estimates indicate that CEF has a low systemic clearance (0.25 L/h/kg) associated with a low global extraction ratio E = 0.02) and a moderate volume of distribution (2.97 L/kg) in dogs. The absolute bioavailability after S.C administration was high (93.7%). Gender was determined to be a significant covariate in explaining the variability of S.C absorption. Our simulations predicted that a dose of 2.2 mg/kg CEF-Na S.C would produce median plasma concentrations of CEF of at least 0.5 µg/mL (MIC50) for approximately 30 hours

The First Case of Ischemia-Free Kidney Transplantation in Humans

Background: Ischemia-reperfusion injury (IRI) has been considered an inevitable event in organ transplantation since the first successful kidney transplant was performed in 1954. To avoid IRI, we have established a novel procedure called ischemia-free organ transplantation. Here, we describe the first case of ischemia-free kidney transplantation (IFKT). Materials and Methods: The kidney graft was donated by a 19-year-old brain-dead donor. The recipient was a 47-year-old man with end-stage diabetic nephropathy. The graft was procured, preserved, and implanted without cessation of blood supply using normothermic machine perfusion. Results: The graft appearance, perfusion flow, and urine production suggested that the kidney was functioning well-during the whole procedure. The creatinine dropped rapidly to normal range within 3 days post-transplantation. The levels of serum renal injury markers were low post-transplantation. No rejection or vascular or infectious complications occurred. The patient had an uneventful recovery. Conclusion: This paper marks the first case of IFKT in humans. This innovation may offer a unique solution to optimizing transplant outcomes in kidney transplantation

Improvement of polysaccharide and triterpenoid production of Ganoderma lucidum through mutagenesis of protoplasts

Ganoderma lucidum is a traditional medicinal macrofungus in China, which has two kinds of key bioactive compounds -- polysaccharides and triterpenoids. To improve the polysaccharide and triterpenoid production from G. lucidum, the preparation and regeneration conditions of protoplasts were optimized. This was done by systematic trials with various parameters, and protoplast mutation was subsequently performed. A mycelium that was cultivated for seven days and treated with 0.33 mL of 1% snailase and 0.66 mL of 0.5% cellulase solution for 2.5 h at 30 °C in the presence of osmotic pressure stabilizer mannitol (0.5 mol/L), had the best conditions, in which the resultant protoplasts were 6.40 × 105/mL and the regeneration rate was 6.25%. The resultant protoplasts were subjected to subsequent mutation by lithium chloride or by the combination of lithium chloride and Triton X-100. The highest yields of intracellular polysaccharide and triterpenoid in two mutant strains were 37.50 and 40.81 mg/g, which were increased with 568.45% and 373.43%, respectively, as compared to the original strain. Furthermore, the yields of intracellular polysaccharides and triterpenoids in the second generation and the third generation of the mutants were comparable to that of the first generation, which showed genetic stability of the mutants for the production of polysaccharides and triterpenoids

Pharmacokinetic Modeling of Ceftiofur Sodium Using Nonlinear Mixed-Effects in Healthy Beagle Dogs

Ceftiofur (CEF) sodium is a third-generation broad-spectrum cephalosporin commonly used in an extra-label manner in dogs for the treatment of respiratory and urinary system infections. To contribute to the literature supporting CEF use in companion animals, we have developed a compartmental, nonlinear mixed-effects (NLME) model of CEF pharmacokinetics in dogs (PK). We then used the mathematical model to predict (via Monte Carlo simulation) the duration of time for which plasma concentrations of CEF and its pharmacologically active metabolites remained above minimum inhibitory concentrations (respiratory tract Escherichia coli spp). Twelve healthy beagle dogs were administered either 2.2 mg/kg ceftiofur-sodium (CEF-Na) intravenously (I.V) or 2.2 mg/kg CEF-Na subcutaneously (S.C). Plasma samples were collected over a period of 72 hours post-administration. To produce a measurement of total CEF, both CEF and CEF metabolites were derivatized into desfuroylceftiofur acetamide (DCA) before analysis by UPLC-MS/MS. No adverse effects were reported after I.V or S.C dosing. The NLME PK models were parameterized using the stochastic approximation expectation maximization algorithm as implemented in Monolix 2018R2. A two-compartment mammillary model with first-order elimination and first-order S.C absorption best described the available kinetic data. Final parameter estimates indicate that CEF has a low systemic clearance (0.25 L/h/kg) associated with a low global extraction ratio E = 0.02) and a moderate volume of distribution (2.97 L/kg) in dogs. The absolute bioavailability after S.C administration was high (93.7%). Gender was determined to be a significant covariate in explaining the variability of S.C absorption. Our simulations predicted that a dose of 2.2 mg/kg CEF-Na S.C would produce median plasma concentrations of CEF of at least 0.5 µg/mL (MIC50) for approximately 30 hours.This is a pre-print of the article Wang, Jianzhong, Benjamin Schneider, Jiao Xue, Pan Sun, Jicheng Qiu, Jonathan Mochel, and Xingyuan Cao. "Pharmacokinetic Modeling of Ceftiofur Sodium Using Nonlinear Mixed-Effects in Healthy Beagle Dogs." (2019). Preprints (2019): 2019070322. DOI: 10.20944/preprints201907.0322.v1. Posted with permission.</p

Histone Deacetylase Inhibitors Inhibit the Proliferation of Gallbladder Carcinoma Cells by Suppressing AKT/mTOR Signaling.

Gallbladder carcinoma is an aggressive malignancy with high mortality mainly due to the limited potential for curative resection and its resistance to chemotherapeutic agents. Here, we show that the histone deacetylase inhibitors (HDACIs) trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA) reduce the proliferation and induce apoptosis of gallbladder carcinoma cells by suppressing the AKT/mammalian target of rapamycin (mTOR) signaling. Gallbladder carcinoma SGC-996 cells were treated with different concentrations of TSA and SAHA for different lengths of time. Cell proliferation and morphology were assessed with MTT assay and microscopy, respectively. Cell cycle distribution and cell apoptosis were analyzed with flow cytometry. Western blotting was used to detect the proteins related to apoptosis, cell cycle, and the AKT/mTOR signaling pathway. Our data showed that TSA and SAHA reduced SGC-996 cell viability and arrested cell cycle at the G1 phase in a dose- and time-dependent manner. TSA and SAHA promoted apoptosis of SGC-996 cells, down-regulated the expression of cyclin D1, c-Myc and Bmi1, and decreased the phosphorylation of AKT, mTOR p70S6K1, S6 and 4E-BP1. Additionally, the mTOR inhibitor rapamycin further reduced the cell viability of TSA- and SAHA-treated SGC-996 cells and the phosphorylation of mTOR, whereas the mTOR activator 1,2-dioctanoyl-sn-glycero-3-phosphate (C8-PA) exerted the opposite influence. Our results demonstrate that histone deacetylase inhibitors (HDACIs) suppress the proliferation of gallbladder carcinoma cell via inhibition of AKT/mTOR signaling. These findings offer a mechanistic rationale for the application of HDACIs in gallbladder carcinoma treatment

mTOR signaling involved in HDACIs-induced cell proliferation inhibition.

<p>(A) SGC-996 cells were treated with the indicated concentrations of rapamycin for 24, 48 or 72 h. Cell viability was measured using an MTT assay. *<i>P</i>< 0.05 vs. 24 h of the 0 nM group. (B) Western blot showing that p-mTOR expression and cell viability were affected by rapamycin and C8-PA in the HDACIs treated SGC-996 cells. Ctl, untreated control cells; TSA, cells treated with 0.4μM TSA; SAHA, cells treated with 10μM SAHA. RAP, rapamycin (50μM); PA, C8-PA (300μM). *<i>P</i>< 0.05 vs. Ctrl; a, <i>P</i>< 0.05 vs. TSA group; b, <i>P</i>< 0.05 vs. SAHA group.</p

HDACIs decreased cyclin D1, c-Myc and Bmi1 and increased histone H3 acetylation.

<p>(A) SGC-996 cells were treated with different concentrations of TSA or SAHA as indicated for 24 h. Total proteins were extracted for immunoblotting of cyclin D1, c-Myc and Bmi1with β-actin as the loading control. (B) Analysis of band intensity in A. *<i>P</i>< 0.05 vs. cyclin D1 in control. #<i>P</i>< 0.05 vs. c-Myc in control. Δ<i>p</i>< 0.05 vs Bmi1 in control. (C) Cells were treated as in A; histone H3 acetylation was determined by Western blots with β-actin as the loading control. (D) Analysis of band intensity in C. *<i>P</i>< 0.05 vs. control. Data shown represent three independent experiments.</p

HDACIs inhibited the activity of AKT/mTOR signaling.

<p>(A) SGC-996 cells were treated with 0.1, 0.4 and 0.8 μM TSA or 1, 5 and 10 μM SAHA for 24 h. Total proteins were extracted for immunoblotting with antibodies against the indicated proteins. β-actin served as the loading control.(B) Relative band intensity of p-AKT in A. (C) Relative band intensity of p-mTOR in A. Data shown represent three independent experiments. *<i>P</i>< 0.05 vs. control.</p

HDACIs reduced the cell viability of gallbladder carcinoma cells in a dose- and time-dependent manner.

<p>SGC-996 cells were treated with or without different concentrations of TSA or SAHA for 24 h. The cell morphology (A) and cell number (B) were recorded under a phase contrast microscope. Scale bars: 200 μm. (C) SGC-996 cells were treated with various concentrations of TSA for 48 h. Cell viability was measured by MTT assay. (D) SGC-996 cells were incubated with 0.4 μM TSA for the time indicated. Cell viability was measured by MTT assay. (E) SGC-996 cells were treated with various concentrations of SAHA for 48 h. Cell viability was measured by MTT assay. (F) SGC-996 cells were incubated with 10 μM SAHA for the time indicated. Cell viability was measured by MTT assay. The data are representatives of three independent experiments with SD for triplicates. Compared with the control group at 0 μM (h): *<i>P</i>< 0.05.</p