In Vitro Simulation of Tissue Back-Pressure for Pen Injectors and Auto-Injectors

The aim of this project was to show that tissue back-pressure can be measured in vitro using a simple pneumatic model. A thorough literature study revealed 4 relevant papers all describing in vivo studies. One of these studies where the subcutaneous tissue back-pressure was determined in 11 patients was used as a reference for the present work. A pneumatic model capable of simulating the back-pressure and the diffusion of drug during subcutaneous injection was developed. The in vitro model was tested using the same type of pen injector as used in the reference study. Comparison of the results revealed that the measured pressure in the in vitro experiments was similar to the subcutaneous tissue back-pressure measured in vivo. G30 0.3 × 8.0 mm and G32 0.23/0.25 × 4.0 mm needles were used for the in vitro experiments, whereas a G31 0.25 × 6.0 mm needle was used for the in vivo experiments. This is one possible explanation of approximately 30 μL/s higher flow rates for the in vitro experiments compared to the in vivo experiments. The low-complexity model allows repeated measurements and provides a stable data output paving the way for measuring subcutaneous back-pressure in vitro.     

In Vivo Reoxidation Kinetics of Free Thiols in Multiple Domains of IgG1 Antibodies in Rats

While free thiols in monoclonal antibodies (mAbs) have been extensively characterized by in vitro studies to probe its effect on antibody function and stability, their in vivo biotransformation has not been comprehensively studied. In this study, a panel of five recombinant IgG1 mAbs with elevated free thiols in the VH, VL, and CH2 domains were intravenously administered into Wistar rats. In vivo biotransformation of thirty-five free thiol sites in total (7 disulfide pairs in VL, CL, VH, CH1, HH, CH2, CH3 domains across the 5 mAbs) were monitored using a denaturing differential isotopic tagging procedure on immunopurified timepoints followed by LC-MS of tryptic digests. The free thiol levels in two VH domain and one CH2 domain disulfide sites decreased in vivo following first order kinetics. Free thiol levels of the remaining 32 sites were remarkably stable in vivo. Further analytical characterization highlighted a positive association between a free thiol's solvent accessibility and a free thiol's reoxidation propensity. The data and discussion presented here shed valuable insights into the in vivo fate of free thiols in several recombinant IgG1s and its implications for free thiols as a product quality attribute in therapeutic mAb products.     

Prevalence,Enterotoxigenic Potential and Antimicrobial Resistance of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from Algerian Ready to Eat Foods

Staphylococcus aureus causes a foodborne intoxication due to the production of enterotoxins and shows antimicrobial resistance, as in the case of methicillin-resistant strains (MRSA). Herein, we analyzed 207 ready-to-eat foods collected in Algeria, reporting a S. aureus prevalence of 23.2% (48/207) and respective loads of coagulase positive staphylococci (CPS) ranging from 1.00 ± 0.5 to 5.11 ± 0.24 Log CFU/g. The 48 S. aureus isolates were widely characterized by staphylococcal enterotoxin gene (SEg)-typing and 16S-23S rDNA intergenic spacer region (ISR)-PCR, as well as by detecting tst and mecA genes, genetic determinants of toxic shock syndrome toxin-1 and methicillin resistance, respectively. We found that the S. aureus isolates belonged to seven different SEg-types harboring the following combinations of genes: (1) selW, selX; (2) egc (seG, seI, seM, seN, seO), selW, selX; (3) seA, seH, seK, seQ, selW, selX; (4) seB, selW, selX; (5) seD, selJ, seR, selW, selX; (6) seH, selW, selX, selY; and (7) seA, egc, selW, selX, while among these, 2.1% and 4.2% were tst- and mecA- (staphylococcal chromosomal cassette mec-type IV) positive, respectively. Selected strains belonging to the 12 detected ISR-types were resistant towards antimicrobials including benzylpenicillin, ofloxacin, erythromycin, lincomycin, tetracyclin, kanamycin, oxacillin, and cefoxitin; 8.3% (1/12) were confirmed as MRSA and 16.7% (2/12) were multidrug resistant. The present study shows the heterogeneity of the S. aureus population in Algerian ready-to-eat foods as for their toxigenic potential and antimicrobial resistance, shedding the light on the quality and safety related to the consume of ready-to-eat foods in Algeria.     

Fabrication and Characterization of Surface Engineered Rifampicin Loaded Lipid Nanoparticulate Systems for the Potential Treatment of Tuberculosis: An In Vitro and In Vivo Evaluation

The main aim of the present investigation highlights the development of mannose appended rifampicin containing solid lipid nanoparticles (Mn-RIF-SLNs) for the management of pulmonary TB. The developed Mn-RIF-SLNs showed particle size of Mn-RIF-SLNs (479 ± 13 nm) which was found to be greater than that of unconjugated SLNs (456 ± 11 nm), with marginal reduction in percentage entrapment efficiency (79.41 ± 2.42%). The in vitro dissolution studies depicted an initial burst release followed by sustained release profile indicating biphasic release pattern, close-fitting Weibull model having least F-value. The cytotoxicity studies using J774A.1 cell line represented that the developed SLNs were non-toxic and safe as compared to free drug. Fluorescence imaging and flow cytometric (FACS) analysis depicted significant (1.79-folds) intracellular uptake of coumarin-6 (fluorescent marker) loaded Mn–C6-SLNs. The in vivo pharmacokinetic studies in sprague-dawley rats were performed and Mn-RIF-SLNs showed remarkable enhancement in terms of relative bioavailability (~17-folds) as compared to its drug solution via oral administration. The biodistribution studies revealed higher lung accumulation (1.8-folds) of Mn-RIF-SLNs as compared to the Un-RIF-SLNs. In conclusion, the developed Mn-RIF-SLNs could serve as a promising tool for delivering the drug cargo to the site of infection (lungs) in the treatment of TB.     

耐甲氧西林金黄色葡萄球菌肺炎小鼠感染模型的建立与评估

目的 本研究采用临床分离鉴定的ST239型耐甲氧西林金黄葡萄球菌(MRSA)感染BALB/c小鼠,建立小鼠肺炎模型,对小鼠的临床症状、肺载菌量与组织病理学变化进行时相性监测,并用该模型验证万古霉素对小鼠的治疗效果.方法 取35只小鼠随机分成MRSA感染组、药物组和对照组,通过滴鼻方式分别滴入50μL细菌(前两组)与PBS(对照组);感染一天后对药物组用同样的方式滴入万古霉素,CT检测肺部的变化,并对肺载菌量和组织病理学变化进行观察.结果 与对照组比较,CT和细菌计数等结果表明MRSA感染组和药物组的肺组织有明显的炎症反应,载菌量较高;与感染组相比,药物组小鼠肺部的载菌量明显降低,气管和肺部的组织病理学症状明显减轻.结论 本实验结果表明小鼠MRSA肺炎模型成功建立,并可用于药物疗效的比较评估.该模型的建立,将为进一步研究临床分离的MRSA的病原特性、发病机理、治疗方法等,提供可靠的小动物模型.     

Efficacy and safety of linezolid in methicillin-resistant Staphylococcus aureus (MRSA) complicated skin and soft tissue infection (cSSTI): a meta-analysis

Abstract

Objective:

To evaluate the clinical and microbiological outcomes of linezolid versus vancomycin in methicillin-resistant Staphylococcus aureus (MRSA) complicated skin and soft-tissue infection (cSSTI) using a meta-analysis.     

Comparison of intravitreal vancomycin and daptomycin application in experimental methicillin-resistant Staphylococcus aureus (MRSA) endophthalmitis in rabbits

Abstract

Purpose: To compare bactericidal activities of daptomycin and vancomycin in an experimental rabbit model of methicillin-resistant Staphylococcus aureus (MRSA) endophthalmitis.

Methods: The right eyes of 19 New Zealand rabbits weighing 2 to 2.5?kg were used. Each eye was inoculated with 1000 colony-forming units (cfu) of MRSA into the vitreous cavity. 24?h after the inoculation, the rabbits were randomly distributed into three groups: control group (n?=?5) was given 0.1?ml of balanced saline solution, daptomycin group 2 (n?=?7) was given 0.2?mg/0.1?ml daptomycin and vancomycin group 3 (n?=?7) was given 1?mg/0.1?ml vancomycin intravitreally. Clinical examination scores were recorded and vitreous aspirates were obtained for microbiological analysis on days 2 and 3 after MRSA inoculation. Rabbits were sacrificed, and the eyes were enucleated for histopathological examination.

Results: There was no difference between the daptomycin group, vancomycin group and control in terms of the clinical grading of endophthalmitis 24?h after the inoculation. In all treatment groups, mean number of cfu and histopathological scores were significantly lower compared to the control group. There was no difference between the daptomycin and vancomycin group in terms of the histopathological and clinical examination scores. Culture negativity achieved on day 3 was 71.4% and 57.1% in the daptomycin treatment group and the vancomycin treatment group, respectively.

Conclusions: Although both daptomycin and vancomycin are effective in treatment of experimental MRSA endophthalmitis, daptomycin has superior bactericidal activity 72?h after inoculation.     

Plasma and Tissue Concentrations of Methylergometrine (Methylergonovine) in the Rabbit

Abstract The concentrations of methylergometrine (M) (methylergonovine) in the plasma, uterus, liver, and kidneys of the rabbit were determined by a radioimmunoassay after a single 0.2 mg/kg intravenous injection and the drug responce was studied in the uterus in situ. M disappeared quickly from the plasma with a mean distribution phase half-life of 0.91 min. According to the fast uterine tissue uptake of M the drug response in this effector organ began quickly. The simulated concentrations in the peripheral compartment of the two-compartment open model can be useful in the understanding of the rapid drug effect, but they do not describe the real situation in any particular tissue.     

Comparison of microbiological and clinical characteristics based on SCCmec typing in patients with community-onset meticillin-resistant Staphylococcus aureus (MRSA) bacteraemia

Molecular identification methods based on the staphylococcal cassette chromosome mec (SCCmec) genotype are more reliable than clinical risk factors and demographic data for differentiating community-acquired and healthcare-associated (HCA) meticillin-resistant Staphylococcus aureus (MRSA). However, patients with community-onset (CO) MRSA infections, defined as a culture-positive sample obtained <48 h after admission and from patients with HCA risk factors, have been infrequently studied. This study compared the clinical profiles of different SCCmec genotypes in this group of patients. From 2004 to 2008, the clinical profiles of 122 non-repetitive patients with CO-MRSA infections at a tertiary medical centre in Taiwan were retrospectively recorded and the molecular characteristics of the isolates were examined. The proportion of SCCmec IV/V genotypes increased from 9.5% to 35.3% from 2004 to 2008. There were no differences in demographic data, underlying diseases, invasive procedures or outcomes between the SCCmec II/III and IV/V groups, except that patients with SCCmec II/III genotypes tended to have more HCA risk factors (3.1 vs. 2.4; P = 0.008). Multivariate logistic regression analysis revealed that having at least four HCA risk factors was independently associated with SCCmec II/III. The sensitivity of recovering SCCmec IV/V genotypes from patients with less than four HCA risk factors was 89.3%. This study revealed the emergence of SCCmec IV/V genotypes in CO-MRSA infections. Although the clinical characteristic boundaries between SCCmec II/III and IV/V diminished, having at least four HCA risk factors made the presence of SCCmec IV/V genotypes less likely in patients with CO-MRSA infections.     

Comparison of the effect of ciprofloxacin and Tazocin on the incidence of meticillin-resistant Staphylococcus aureus (MRSA) in an Intensive Care Unit

Meticillin-resistant Staphylococcus aureus (MRSA) is a very significant agent of recalcitrant healthcare-associated infections. A major risk of acquiring such infections is thought to be modulated by the use of particular antimicrobial therapies. The aim of this research was to evaluate prospectively the impact of using either ciprofloxacin or Tazocin (piperacillin+tazobactam) on the incidence of MRSA in an Intensive Care Unit (ICU). The 1-year (2 x 6 months) cross-over study was carried out in a medium-sized (426 beds) teaching hospital. During the first 6-month period, ciprofloxacin was used as the first-line broad-spectrum antibiotic therapy of choice. During the second 6-month period, Tazocin was used as first-line therapy. The incidence of hospital-acquired MRSA (i.e. colonised and/or infected) and rates of compliance of the ICU healthcare workers to optimal hand hygiene practices were recorded throughout the study. The study observed no statistically significant differences (P = 0.1) between MRSA incidence rates in the ICU during the ciprofloxacin (4.4/1000 bed-days) or Tazocin (11.4/1000 bed-days) arms of the study. Interestingly, observing healthcare workers' hand hygiene practices throughout the entire study showed that healthcare workers adhered to these practices 59.2% of the time during the ciprofloxacin arm and 66.0% during the Tazocin arm. The low incidence rates within the unit demonstrated the importance of infection control in limiting the spread of MRSA despite the extensive use of antibiotics in a high-risk setting.     

Comparison of disposition behavior and de-coppering effect of triethylenetetramine in animal model for Wilson's disease (Long-Evans Cinnamon rat) with normal Wistar rat.

The disposition behaviors and de-coppering effect of triethylenetetramine dihydrochloride (trientine), a selective chelating agent for copper and an 'orphan drug' for Wilson's disease, have been evaluated in an animal model, Long-Evans Cinnamon (LEC) rats, and normal rats (Wistar). In LEC rats, urinary excretion of trientine was remarkably lower than that of Wistar rats. The absorption rates from the jejunal loop and in vitro metabolism in the liver S9 fraction (supernatant of 9000 x g) were approximately the same for both strains. The decline of urinary excretion of trientine in LEC rats is thought to be due mainly to the lowering of the functional activity of the kidney, because urinary excretion of creatinine and phenolsulfonphthalein were significantly lower in LEC rats than those in Wistar rats. Both acceleration of urinary excretion of copper and reduction of hepatic copper levels were observed with treatment of trientine in LEC rats aged 6 weeks. In LEC rats aged 13 weeks, however, no de-coppering effect from the liver was observed, though urinary excretion of copper was increased. These results suggest that trientine has a pharmacological effect in disease state, especially in the early stages of hepatitis.     

ELISA法对神经生长因子在大鼠体内药代动力学的研究

目的研究神经生长因子（ＮＧＦ）在大鼠体内的药代动力学。方法制备兔抗ＮＧＦ抗体,用双抗体夹心酶联免疫吸附实验（ＥＬＩＳＡ）法测定大鼠血浆ＮＧＦ浓度。结果大鼠ｉｖ３２μｇ·ｋｇ－１后血药浓度时间曲线符合二室开放模型,Ｔ１／２α为０３３０ｈ,Ｔ１／２β为１５ｈ。大鼠ＮＧＦｉｍ３２μｇ·ｋｇ－１后血药浓度时间曲线符合一室一级吸收,血浆ＮＧＦ浓度达峰时间为３７１ｈ,Ｔ１／２（Ｋｅ）为４３０ｈ,２４ｈ吸收百分率为６９１％。结论ＮＧＦ肌注吸收良好,血浆ＮＧＦ浓度可在较长时间内维持在有效浓度以上。     

ELISA法对神经生长因子在大鼠体内药代动力学的研究

目的研究神经生长因子（ＮＧＦ）在大鼠体内的药代动力学。方法制备兔抗ＮＧＦ抗体,用双抗体夹心酶联免疫吸附实验（ＥＬＩＳＡ）法测定大鼠血浆ＮＧＦ浓度。结果大鼠ｉｖ３２μｇ·ｋｇ－１后血药浓度 时间曲线符合二室开放模型,Ｔ１／２α为０３３０ｈ,Ｔ１／２β为１５ｈ。大鼠ＮＧＦｉｍ３２μｇ·ｋｇ－１后血药浓度 时间曲线符合一室一级吸收,血浆ＮＧＦ浓度达峰时间为３７１ｈ,Ｔ１／２（Ｋｅ）为４３０ｈ,２４ｈ吸收百分率为６９１％。结论ＮＧＦ肌注吸收良好,血浆ＮＧＦ浓度可在较长时间内维持在有效浓度以上。     

Physicochemical,Pharmacokinetic and Pharmacodynamic Evaluation of Liposomal Tacrolimus (FK 506) in Rats

Purpose. Tacrolimus (FK 506) is a new potent immunosuppressant. Because of poor water solubility, the conventional intravenous dosage forms of FK 506 (C-FK 506) contain surfactants such as HCO-60 which may cause adverse effects. We sought a liposomal formulation of FK 506 (L-FK 506) containing endogenous phospholipids to target drug to the spleen, a major organ controlling the immune system. Methods. L-FK 506, consisting of 0.1 µm diameter vesicles of phosphatidylcholine and phosphatidylglycerol (molar ratio 9:1) and 7.5 mole% drug, was evaluated for in vitro stability. The intravenous disposition profile, spleen distribution, and immunosuppression of L-FK 506 was compared with that of C-FK 506 in the rat after single doses of 0.3 mg/kg. Results. The L-FK 506 showed good in vitro stability. L-FK 506 exhibited an increased volume of distribution at steady-state (V_ss) (from 3.41 to 14.71 L/kg) and increased mean residence time (MRT) (from 2.83 to 16.07 hr). FK 506 concentrations in spleen were increased by 40% at 10 hr after administration of the liposomal formulation. The pharmacodynamics of L-FK 506, evaluated by the extent of inhibition of splenocyte proliferation, was comparable to that of C-FK 506. Conclusions. Liposomal FK 506 may be an improved dosage form for parenteral use.     

夫西地酸钠与万古霉素治疗ICU病房耐甲氧西林金黄色葡萄球菌的疗效与安全性研究

目的评价夫西地酸钠与万古霉素治疗ICU病房耐甲氧西林金黄色葡萄球菌(MRSA)的疗效与安全性。方法将2011年1月至2013年1月入住我院ICU病房的MRSA感染病例61例,随机分为2组,对照组34例,给予万古霉素1 g,静脉滴注,2次/d;实验组27例,给予夫西地酸钠0.5 g,静脉滴注,3次/d;两组药溶媒均为0.9%氯化钠注射液100 mL,每次给药时间均>60 min,疗程均为5²0 d,具体根据病情严重程度和患者临床反应而定。比较两组抗菌药物治疗前后的临床疗效、细菌清除率及安全性。结果实验组与对照组的临床有效性、细菌清除率和不良反应发生率分别为81.48%、70.37%、11.11%和85.29%、79.41%、17.65%,两组比较差异均无统计学意义(P均>0.05)。结论夫西地酸钠治疗MRSA感染临床疗效与细菌清除率与万古霉素相当,且不良反应发生率低,因此可用于替代万古霉素用于ICU病房MRSA感染的治疗。     

A Self-Nanoemulsifying Drug Delivery System for Enhancing the Oral Bioavailability of Candesartan Cilexetil: Ex Vivo and In Vivo Evaluation

The drug delivery of candesartan cilexetil encounters an obstacle of low absolute oral bioavailability which is attributed mainly to its low aqueous solubility and efflux by intestinal P-glycoprotein (P-gp) transporters. However, the extent of P-gp contribution in the reduced oral bioavailability of candesartan cilexetil is not clear. In this study, a previously developed candesartan cilexetil–loaded self-nanoemulsifying drug delivery system (SNEDDS) was evaluated for its ability to increase the drug oral bioavailability via the inhibition of intestinal P-gp transporters. Despite the developed SNEDDS showing P-gp inhibition activity, P-gp–mediated efflux was found to have a minor role in the reduced oral bioavailability of candesartan cilexetil. On the other hand, the high surfactant concentration used in SNEDDS formulation represents a major challenge toward their widespread application especially for chronically administered drugs. The designed acute and subacute toxicity studies revealed that the degree of intestinal mucosal damage decreases as the treatment period increases. The latter observation was attributed to the reversibility of surfactant-induced mucosal damage. Thus, the developed SNEDDS could be considered as a promising delivery system for enhancing the oral bioavailability of chronically administered drugs.     

Virulence Factors Found in Nasal Colonization and Infection of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates and Their Ability to Form a Biofilm

Hospitalizations related to Methicillin-resistant Staphylococcus aureus (MRSA) are frequent, increasing mortality and health costs. In this way, this study aimed to compare the genotypic and phenotypic characteristics of MRSA isolates that colonize and infect patients seen at two hospitals in the city of Niterói—Rio de Janeiro, Brazil. A total of 147 samples collected between March 2013 and December 2015 were phenotyped and genotyped to identify the protein A (SPA) gene, the mec staphylococcal chromosomal cassette (SCCmec), mecA, Panton-Valentine Leucocidin (PVL), icaC, icaR, ACME, and hla virulence genes. The strength of biofilm formation has also been exploited. The prevalence of SCCmec type IV (77.1%) was observed in the colonization group; however, in the invasive infection group, SCCmec type II was prevalent (62.9%). The Multilocus Sequence Typing (MLST), ST5/ST30, and ST5/ST239 analyses were the most frequent clones in colonization, and invasive infection isolates, respectively. Among the isolates selected to assess the ability to form a biofilm, 51.06% were classified as strong biofilm builders. Surprisingly, we observed that isolates other than the Brazilian Epidemic Clone (BEC) have appeared in Brazilian hospitals. The virulence profile has changed among these isolates since the ACME type I and II genes were also identified in this collection.     

Evaluation of Prediction Accuracy for Volume of Distribution in Rat and Human Using In Vitro,In Vivo,PBPK and QSAR Methods

Volume of distribution at steady state (V_ss) is an important pharmacokinetic parameter of a drug candidate. In this study, V_ss prediction accuracy was evaluated by using: (1) seven methods for rat with 56 compounds, (2) four methods for human with 1276 compounds, and (3) four in vivo methods and three Kp (partition coefficient) scalar methods from scaling of three preclinical species with 125 compounds. The results showed that the global QSAR models outperformed the PBPK methods. Tissue fraction unbound (f_u,t) method with adipose and muscle also provided high V_ss prediction accuracy. Overall, the high performing methods for human V_ss prediction are the global QSAR models, Øie-Tozer and equivalency methods from scaling of preclinical species, as well as PBPK methods with Kp scalar from preclinical species. Certain input parameter ranges rendered PBPK models inaccurate due to mass balance issues. These were addressed using appropriate theoretical limit checks. Prediction accuracy of tissue Kp were also examined. The f_u,t method predicted Kp values more accurately than the PBPK methods for adipose, heart and muscle. All the methods overpredicted brain Kp and underpredicted liver Kp due to transporter effects. Successful V_ss prediction involves strategic integration of in silico, in vitro and in vivo approaches.     

Disposition of styrene-acrylonitrile (SAN) trimer in female rats: single dose intravenous and gavage studies

Styrene-acrylonitrile trimer (SAN Trimer), a mixture of six isomers (four isomers of 4-cyano-1,2,3,4-tetrahydro-alpha-methyl-1-naphthaleneacetonitrile [THAN] and two isomers of 4-cyano-1,2,3,4-tetrahydro-1-naphthaleneproprionitrile [THNP]), is a by-product of a specific production process of styrene-acrylonitrile polymer. Disposition studies in female rats were conducted to evaluate the pharmacokinetic behavior of [3H]SAN Trimer following a single intravenous administration (26 mg/kg) to nonpregnant rats; a single gavage administration (nominal doses of 25 mg/kg, 75 mg/kg, or 200 mg/kg in corn oil) to nonpregnant rats; and a single gavage administration (nominal dose of 200 mg/kg in corn oil) to pregnant and lactating rats. SAN Trimer was rapidly eliminated from blood (T1/2 approximately 1h) following a single intravenous dose and following single oral doses (T1/2 approximately 3-4h). SAN Trimer was also rapidly excreted in the urine and feces following single oral doses, while total radioactivity was cleared more slowly. In pregnant rats, the concentrations of both radioactivity and SAN Trimer 2h after dosing were highest in the blood, followed by the placenta, with the lowest levels in the fetus. In lactating rats, the concentrations of both radioactivity and SAN Trimer were higher in milk than in maternal blood. Total radioactivity and SAN Trimer blood concentrations in nonpregnant, pregnant, and lactating rats were both higher in lactating rats compared to nonpregnant and pregnant rats.