Future Pharmacokinetic Studies Research Articles

RGB Trichromatic Whiteness Assessment of Bio Analytical Chromatographic Tool Using Fluorescence for Quantitation of Semaglutide: Application to Pharmaceutical Preparations and Spiked Plasma.

Semaglutide (SEMG) is one of the most widely used and trending medications to treat type II diabetes and obesity.This work aimed to develop a liquid chromatography with spectroflourimetric detection (HPLC-flourimetry) analysis of SEMG in both its tablet dosage form and plasma. The power of fluorescence detection coupled with HPLC proved its capability as a bioanalytical tool to assay SEMG in plasma samples owing to its simplicity and sensitivity which reached below the Cmax of SEMG. Separation was done using a C18 column with mobile phase of acetonitrile and water acidified with orthophosphoric acid (pH 3.5) (1.41 × 10-5M) in isocratic mode in ratio 57:43 and 1mL/min flow rate after extraction using protein precipitation. Detection was carried out at λ excitation of 238nm and λ emission of 416 and 307nm for SEMG and the internal standard, respectively. Evaluation of greenness of the proposed method was done using AGREE (Analytical GREEnness Metric Approach), ComplexGAPI (Complementary Green Analytical Procedure Index) & the new algorithm RGB 12 model (Red-Green-Blue). They showed that these methods can be a greener alternative with acceptable sensitivity for analysis of SEMG. The developed seven min-assay was validated per ICH as well as FDA bio analytical methods' guidelines to prove its applicability for routine sample analysis and future pharmacokinetic studies.

Development, validation and application of a high-performance liquid chromatography method for the detection of ibuprofen in elephant plasma

A simple and accurate method for the determination of ibuprofen concentrations in elephant plasma was developed and validated using reverse phase high-performance liquid chromatography (HPLC). Utilizing a liquid-liquid extraction with ethyl acetate, methanol, and phosphoric acid, samples were separated on an XBridge C18 column. The mobile phase consisted of 0.005 M ammonium phosphate and acetonitrile (40:60, v/v). Both ibuprofen and the internal standard flurbiprofen were quantitated using an ultraviolet (UV) detector set at 214 nm. The lower limit of quantification (LLOQ) was 0.05 μg/mL in 100 μL of plasma. The average recovery was over 95 % while the intra-assay variability ranged from 0.8 to 9.9 % and the inter-assay variability ranged from 2.7 to 9.8 %. The method was used successfully in a therapeutic drug monitoring program for elephants and could be used in future pharmacokinetic studies.

Novel drug sampling technique: portal vein catheterization in steers.

To determine the feasibility of catheterizing the portal vein to obtain serial portal vein blood samples in steers. We hypothesized that the portal vein catheterization would be a successful continuous sampling technique with minimal adverse effects in steers. 2 groups of steers were used: a pilot group (n = 2) and experimental group (n = 6). In both groups, steers were sedated with xylazine. The right rib spaces were clipped and aseptically prepped. The portal vein was visualized via ultrasound, and a 14-gauge catheter was placed percutaneously and advanced into the portal vein. A guide wire was passed through the catheter, followed by a tissue dilator and then a vascular balloon catheter. In the pilot group, blood chemistries were performed prior to portal vein catheterization and then again once the catheter was placed (with samples from both the jugular vein and portal catheter). The liver was also examined at necropsy for any gross lesions in both groups. All steers tolerated the portal vein catheters well, with the catheters lasting for the full length of the study period (7 days). The only observed adverse reaction was a superficial abscess at the catheter site (n = 3). On necropsy, 1 liver had gross discoloration, but no other abnormalities were noted. There were no significant changes in biochemistry profiles before or after portal vein catheterization. Portal vein catheterization is a novel and feasible serial sampling technique of the portal vein. This technique can be used in future pharmacokinetic, nutrition, metabolism, or toxicity studies.

A New and Rapid LC-MS/MS Method for the Determination of Cysteamine Plasma Levels in Cystinosis Patients.

Cystinosis is a rare lysosomal storage disorder caused by autosomal recessive mutations in the CTNS gene that encodes for the cystine transporter cystinosin, which is expressed on the lysosomal membrane mediating the efflux of cystine. Cysteamine bitartrate is a cystine-depleting aminothiol agent approved for the treatment of cystinosis in children and adults. In this study, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of cysteamine levels in plasma samples. This LC-MS/MS method was validated according to the European Medicines Agency (EMA)'s guidelines for bioanalytical method validation. An ultra-performance liquid chromatograph (UPLC) coupled with a 6470 mass spectrometry system was used for cysteamine determination. Our validated method was applied to plasma samples from n = 8 cystinosis patients (median, interquartile range (IQR) = 20.5, 8.5-26.0 years). The samples were collected before cysteamine oral administration (pre-dose) and 1 h after (post-dose). Our bioanalytical method fulfilled the regulatory guidelines for method validation. The cysteamine plasma levels in pre-dose samples were 2.57 and 1.50-3.31 μM (median and IQR, respectively), whereas the post-dose samples reported a cysteamine median concentration of 28.00 μM (IQR: 17.60-36.61). Our method allows the rapid determination of cysteamine plasma levels. This method was successfully used in cystinosis patients and, therefore, could be a useful tool for the evaluation of therapy adherence and for future pharmacokinetic (PK) studies involving a higher number of subjects.

Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of the antimalarial drug pyronaridine in human whole blood

Malaria remains a major health concern, aggravated by emerging resistance of the parasite to existing treatments. The World Health Organization recently endorsed the use of artesunate-pyronaridine to treat uncomplicated malaria. However, there is a lack of clinical pharmacokinetic (PK) data of pyronaridine, particularly in special populations such as children and pregnant women. Existing methods for the quantification of pyronaridine in biological matrices to support PK studies exhibit several drawbacks. These include limited sensitivity, a large sample volume required, and extensive analysis time. To overcome these limitations, an ultra-performance reversed-phase liquid chromatography tandem-mass spectrometry method to determine pyronaridine was developed and validated according to international guidelines. The method enabled fast and accurate quantification of pyronaridine in whole blood across a clinically relevant concentration range of 0.500–500 ng/mL (r2 ≥ 0.9963), with a required sample volume of 50 µL. Pyronaridine was extracted from whole blood using liquid-liquid extraction, effectively eliminating the matrix effect and preventing ion enhancement or suppression. The method achieved a satisfactory reproducible sample preparation recovery of 77%, accuracy (as bias) and precision were within ±8.2% and ≤5.3%, respectively. Stability experiments demonstrated that pyronaridine was stable for up to 315 days when stored at −70°C. Adjustments to the chromatographic system substantially reduced carry-over and improved sensitivity compared to prior methods. The method was successfully applied to quantify pyronaridine in whole blood samples from a selection of pregnant malaria patients participating in the PYRAPREG clinical trial (PACTR202011812241529) in the Democratic Republic of the Congo, demonstrating its suitability to support future PK studies. Furthermore, the enhanced sensitivity allows for the determination of pyronaridine up to 42 days post-treatment initiation, enabling assessment of the terminal elimination half-life.

LC-MS/MS determination of N-acetyl-l-cysteine in chicken plasma.

N-acetyl-l-cysteine (NAC) shows beneficial effects in cases of aflatoxicosis and heat stress in poultry but little is known about its pharmacokinetics in chickens. Therefore, the study aimed to develop and validate a sensitive LC-MS/MS analytical method for quantitative analysis of NAC in chicken plasma. A split calibration curve approach was used for determination of NAC in chicken plasma. Standard curves for low (0.05-2.5μg/ml) and high (2.5-100 μg/ml) ranges of concentrations were prepared. The standard curves for low (r2 = 0.9987) and high (r2 = 0.9899) concentrations were linear within the tested range. The limits of detection (LOD) and of quantification (LOQ) for the standard at low concentrations were 0.093 and 0.28 μg/ml, respectively. The accuracy was from 97.35 to 101.33%. The values of LOD and LOQ for the standard at high concentrations were 0.76 and 2.30 μg/ml, respectively. The accuracy was between 99.77 and 112.14%. The intra- and inter-day precisions for all concentrations from both standards did not exceed 8.57% and 10.69%, respectively. The recovery for all concentrations was between 92.45 and 105.52%. The validated method for determination of NAC in chicken plasma can be applied in future pharmacokinetic studies in chickens without dilution of samples and their repeated analysis.

Statins Ticagrelor and Rhabdomyolysis: A Coincidence or a Drug Interaction?

Statins play a key role in the management of atherosclerotic cardiovascular disease for both primary and secondary prevention. However, their increasing usage has correspondingly led to a higher incidence of adverse effects, with muscle symptoms being the most common. An intriguing drug interaction exists between ticagrelor and high-intensity statins, which may exacerbate the adverse effects of statin-induced rhabdomyolysis, leading to significant consequences. This study was conducted to examine the profile of patients who have experienced statin-induced rhabdomyolysis while undergoing percutaneous transluminal coronary angioplasty (PTCA). This was an observational study that included 1,862 patients who underwent PTCA at our institute over the course of 1 year. Over a 1-year period, we encountered four patients who were being treated with high-intensity statin therapy following acute coronary syndrome. These patients presented with muscle weakness and kidney injury. A notable commonality among all patients was the co-prescription of ticagrelor. Two patients died, while the other 2 were successfully managed through hydration, electrolyte balance, dialysis, and alternative lipid management drugs. The concomitant use of ticagrelor and high-intensity statins should be carefully considered due to the additional risk of rhabdomyolysis and kidney injury. Future pharmacokinetic studies are needed to establish a causal relationship and predict potential drug interactions, which, if not avoided, could be fatal.

Microdialysis as a sampling tool for the chemotherapeutic agent Doxorubicin

Doxorubicin is a chemotherapeutic agent used for more than fifty years to treat a great variety of cancers in both children and adults. Despite hereof, pharmacokinetic knowledge is almost solely based on systemic plasma concentrations. Microdialysis is a catheter-based pharmacokinetic sampling tool enabling simultaneous target site sampling of unbound molecules of interest. The aim of this study was to thoroughly evaluate the feasibility of applying microdialysis for sampling of Doxorubicin in both in vitro experiments and an in vivo setting. Doxorubicin relative recovery by gain and by loss was tested for different catheter types, perfusion fluids, concentrations and collection vials. Adsorption tests revealed polystyrene/santoprene vials to be the biggest contributor of unwanted adsorption between Doxorubicin and the microdialysis equipment, and confirmed LoBind Eppendorf tubes to be a suitable alternative. The methodological combination of polyamide membranes, saline as perfusion fluid and LoBind Eppendorf sampling tubes demonstrated no statistically significant differences for relative recovery by gain and by loss, and the relative recovery was also found to be concentration independent. We conclude, that a proper microdialysis set-up can be used to collect samples containing concentrations of the chemotherapeutic drug Doxorubicin in vitro and in vivo, which encourage future pharmacokinetic studies to evaluate current treatment regimens to find the most effective and least toxic anti-neoplastic treatment for the patients.

Development, validation and clinical implementation of a UPLC-MS/MS bioanalytical method for simultaneous quantification of cabotegravir and rilpivirine E-isomer in human plasma

A reversed phase ultra-high performance liquid chromatography method was developed for the simultaneous quantification of cabotegravir (CAB) and the E-isomer of rilpivirine (RPV) in human EDTA plasma, also considering RPV E-isomer instability. Because of the instability of RPV (and CAB) in all light conditions, the (RPV Z-isomer/total RPV)-isomer ratio of RPV was determined for each stock, calibration curve standard, quality control sample and patient sample. [2H3]-CAB and [13C6]-RPV were used as internal standard. Sample preparation involved protein precipitation of plasma using methanol. An HSS T3 column with a guard column (set at 40 °C) was used for analyte separation. The mobile phase components were 65 % 0.1 % formic acid in water (A) and 35 % 0.1 % formic acid in acetonitrile (B) and the flow rate was 0.5 mL/min. Detection was performed with tandem mass spectrometry (MS/MS) in a total runtime of 3.0 min. The assay was validated over the concentration range of 0.0500 – 10.0 mg/L for CAB and 0.00300 – 3.00 mg/L for RPV. The average within-day and between-day accuracies for the assay in plasma were 101 % and 101 % for CAB and 97.6 % and 98.5 % voor RPV, respectively. Within-day and between-day precision in coefficients of variations (CV) were 5.0 %. Extraction recovery was 99 % and 102 % for CAB and its internal standard and 95 % and 97 % for RPV and its internal standard. As our aim was that the (Z-isomer RPV/total RPV) response ratio in patient samples had to be less than 10 % to give reliable results, the (Z-isomer RPV/total RPV) response ratio in stocks, calibration curve standards and internal quality control samples were also taken into account being maximal 0.9 % and 2.3 % respectively. This assay has been successfully used in our Therapeutic Drug Monitoring (TDM) service for people living with HIV on long-acting injectable therapy with CAB/RPV and will also be used in future pharmacokinetic studies.

Changes in the degree of substitution of HES in vivo and their influence on methods for determining HES concentrations in plasma.

The degree of substitution (DS) of HES describes the average proportion of substituted glucose molecules in a starch molecule. Although no quantitative studies of the in vivo behavior of the DS have been conducted so far, most pharmacokinetic studies to date have measured HES concentrations using the enzymatic method. This method assumes that at any point in time after an infusion, the DS in a serum remains constant and is identical to the DS in the infused solution. In the present study, we examined the changes in the DS of HES 130/0.42 in vivo in an animal model and compared two methods of measuring HES concentrations in plasma (the enzymatic and the o-Toluidine method). We randomized 22 pigs into 2 groups. After induction of anesthesia, the pigs received 500 ml or 1000 ml of HES 130/0.42 (Tetraspan®). The DS was measured directly after the infusion, then after 30, 60, 120 and 240 minutes. In determining the DS, the hydroxyethyl starch was extracted from the plasma and hydrolyzed with hydrochloric acid to form non-substituted glucose and hydroxyethyl glucose. Subsequently, the concentration of free unsubstituted glucose was determined enzymatically and the total concentration of all (i.e., substituted and unsubstituted) glucose molecules was determined using the o-Toluidine method. From this, the concentration of the substituted glucose (hydroxyethyl glucose) and the DS could be calculated. In addition, the HES concentration was measured first in vitro and then in vivo at any point after the infusion by both the enzymatic method and the o-Toluidine method. The DS increased significantly directly after the infusion from 0.42 to 0.53 (for 500ml) or to 0.50 (for 1000ml); 4 hours later this had further increased to 0.55 and 0.54, respectively (p <0.0001). The HES concentration in vitro showed no significant difference (p = 0.17) when determined with the enzymatic and the o-Toluidine method. In contrast, the serum concentrations in vivo displayed significant differences (p<0.0001) between the two measurement methods. Immediately after the infusion of 500ml HES, the concentration measured with the o-Toluidine method was 31% higher than the one measured with the enzymatic method; 4 hours later, this discrepancy was still at 25%. For 1000 ml HES, the differences amounted to 16% and 25%, respectively. The DS of HES in vivo increases significantly over time. As a result, an HES concentration measured with the enzymatic method in vivo will be significantly lower than the same concentration determined with the o-Toluidine method. In future pharmacokinetic studies, HES concentrations should be measured using a method that takes into account changes in the DS in vivo.

Representative Rodent Models for Renal Transporter Alterations in Human Nonalcoholic Steatohepatitis.

Alterations in renal elimination processes of glomerular filtration and active tubular secretion by renal transporters can result in adverse drug reactions. Nonalcoholic steatohepatitis (NASH) alters hepatic transporter expression and xenobiotic elimination, but until recently, renal transporter alterations in NASH were unknown. This study investigates renal transporter changes in rodent models of NASH to identify a model that recapitulates human alterations. Quantitative protein expression by surrogate peptide liquid chromatography-coupled mass spectrometry (LC-MS/MS) on renal biopsies from NASH patients was used for concordance analysis with rodent models, including methionine/choline deficient (MCD), atherogenic (Athero), or control rats and Leprdb/db MCD (db/db), C57BL/6J fast-food thioacetamide (FFDTH), American lifestyle-induced obesity syndrome (ALIOS), or control mice. Demonstrating clinical similarity to NASH patients, db/db, FFDTH, and ALIOS showed decreases in glomerular filtration rate (GFR) by 76%, 28%, and 24%. Organic anion transporter 3 (OAT3) showed an upward trend in all models except the FFDTH (from 3.20 to 2.39 pmol/mg protein), making the latter the only model to represent human OAT3 changes. OAT5, a functional ortholog of human OAT4, significantly decreased in db/db, FFDTH, and ALIOS (from 4.59 to 0.45, 1.59, and 2.83 pmol/mg protein, respectively) but significantly increased for MCD (1.67 to 4.17 pmol/mg protein), suggesting that the mouse models are comparable to human for these specific transport processes. These data suggest that variations in rodent renal transporter expression are elicited by NASH, and the concordance analysis enables appropriate model selection for future pharmacokinetic studies based on transporter specificity. These models provide a valuable resource to extrapolate the consequences of human variability in renal drug elimination. SIGNIFICANCE STATEMENT: Rodent models of nonalcoholic steatohepatitis that recapitulate human renal transporter alterations are identified for future transporter-specific pharmacokinetic studies to facilitate the prevention of adverse drug reactions due to human variability.

PHLPP Inhibitor NSC74429 Is Neuroprotective in Rodent Models of Cardiac Arrest and Traumatic Brain Injury.

Pleckstrin homology domain and leucine rich repeat protein phosphatase (PHLPP) knockout mice have improved outcomes after a stroke, traumatic brain injury (TBI), and decreased maladaptive vascular remodeling following vascular injury. Thus, small-molecule PHLPP inhibitors have the potential to improve neurological outcomes in a variety of conditions. There is a paucity of data on the efficacy of the known experimental PHLPP inhibitors, and not all may be suited for targeting acute brain injury. Here, we assessed several PHLPP inhibitors not previously explored for neuroprotection (NSC13378, NSC25247, and NSC74429) that had favorable predicted chemistries for targeting the central nervous system (CNS). Neuronal culture studies in staurosporine (apoptosis), glutamate (excitotoxicity), and hydrogen peroxide (necrosis/oxidative stress) revealed that NSC74429 at micromolar concentrations was the most neuroprotective. Subsequent testing in a rat model of asphyxial cardiac arrest, and in a mouse model of severe TBI, showed that serial dosing of 1 mg/kg of NSC74429 over 3 days improved hippocampal survival in both models. Taken together, NSC74429 is neuroprotective across multiple insult mechanisms. Future pharmacokinetic and pharmacodynamic (PK/PD) studies are warranted to optimize dosing, and mechanistic studies are needed to determine the percentage of neuroprotection mediated by PHLPP1/2 inhibition, or potentially from the modulation of PHLPP-independent targets.

Quality by design approach for development and validation of a RP-HPLC method for simultaneous estimation of xipamide and valsartan in human plasma

A new rapid, simple, and sensitive RP-HPLC method was carried out through applying Quality by Design approach for determination of xipamide and valsartan in Human plasma. Fractional factorial design was used for screening of four independent factors: pH, flow rate, detection wavelength, and % of MeOH. Analysis of variance (ANOVA) confirmed that flow rate and % of MeOH were only significant. Chromatographic conditions optimization was carried out through using central composite design. Method analysis was performed using BDS Hypersil C8 column (250 × 4.6 mm, 5 μm) and an isocratic mobile phase of MeOH and 0.05 M KH2PO4 buffer pH 3 (64.5:35.5, v/v) at 1.2 mL/min flow rate with UV detection at 240 nm and 10 μL injection volume. According to FDA guidelines, the method was then validated for the determination of the two drugs clinically in human plasma in respect of future pharmacokinetic and bioequivalence simulation studies. The standard curve was linear in the concentration range of 5–100 µg/mL for both drugs, with a determination coefficient (R2) of 0.999. Also, the average recoveries lied within the range from 99.89 to 100.03%. The proposed method showed good predictability and robustness.

Quantitative Determination of 5-Aminoisoquinoline, a PARP-1 Inhibitor by UPLC-MS/MS: In Silico ADME Profile and In Vitro Metabolic Stability Study

5-Aminoisoquinoline (5-AIQ) is a water-soluble, potent and selective Poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor, widely used as a biochemical and pharmacological tool to study the inhibitory effect of PARPs enzyme. In this study, a simple, selective and reliable ultra-performance liquid chromatography-tandem mass spectrometry assay has been developed for the quantitative analysis of 5-AIQ in plasma using pantoprazole as an internal standard (IS). Both 5-AIQ and IS were separated on an Acquity CSH18 (2.1 × 100 mm; 1.7 µm) column after chromatographic elution of mobile phase comprising of 10 mM ammonium acetate and acetonitrile (35:65; v/v) at a flow rate of 0.3 mL/min. Electrospray ionization in positive mode was used for sample ionization and precursor to product ion transitions of 145.0 &gt; 91.0; 145.0 &gt; 117.4 for 5-AIQ and 384.0 &gt; 138.1 for IS were used for detection and quantification in multiple reaction monitoring mode. The assay was linear in the concentration range of 1.0 to 666 ng/mL with correlation coefficient of ≥0.995. The precision and bias were within the acceptable limits of ≤12.68% and −8.6 to 5.9%, respectively, with mean recovery of 79.1% from plasma and negligible matrix effects (92.4%). In silico ADME prediction, 5-AIQ showed to be very soluble in water and high gastrointestinal absorption along with blood–brain barrier (BBB) permeability. The validated assay was successfully applied in a metabolic stability study, and 5-AIQ was moderately metabolized by human liver microsomes with an in vitro half-life of 14.5 min and intrinsic clearance of 47.6 µL/min/mg. The validated method can be utilized for future pharmacokinetic and bio-distribution studies.

Mechanism-Based Pharmacokinetic Model for the Deglycosylation Kinetics of 20(S)-Ginsenosides Rh2.

Aim: The 20(S)-ginsenoside Rh2 (Rh2) is being developed as a new antitumor drug. However, to date, little is known about the kinetics of its deglycosylation metabolite (protopanoxadiol) (PPD) following Rh2 administration. The aim of this work was to 1) simultaneously characterise the pharmacokinetics of Rh2 and PPD following intravenous and oral Rh2 administration, 2) develop and validate a mechanism-based pharmacokinetic model to describe the deglycosylation kinetics and 3) predict the percentage of Rh2 entering the systemic circulation in PPD form. Methods: Plasma samples were collected from rats after the I.V. or P.O. administration of Rh2. The plasma Rh2 and PPD concentrations were determined using HPLC-MS. The transformation from Rh2 to PPD, its absorption, and elimination were integrated into the mechanism based pharmacokinetic model to describe the pharmacokinetics of Rh2 and PPD simultaneously at 10 mg/kg. The concentration data collected following a 20 mg/kg dose of Rh2 was used for model validation. Results: Following Rh2 administration, PPD exhibited high exposure and atypical double peaks. The model described the abnormal kinetics well and was further validated using external data. A total of 11% of the administered Rh2 was predicted to be transformed into PPD and enter the systemic circulation after I.V. administration, and a total of 20% of Rh2 was predicted to be absorbed into the systemic circulation in PPD form after P.O. administration of Rh2. Conclusion: The developed model provides a useful tool to quantitatively study the deglycosylation kinetics of Rh2 and thus, provides a valuable resource for future pharmacokinetic studies of glycosides with similar deglycosylation metabolism.

Determination of the Monoclonal Antibody Tocilizumab by a Validated Micellar Electrokinetic Chromatography Method

Tocilizumab is a monoclonal antibody used in the treatment of several inflammatory and autoimmune diseases as well as cancers. Tocilizumab improves clinical outcomes and reduce mortality rates in patients with COVID-19 disease. A novel, simple and reliable method was developed to determine tocilizumab using micellar electrokinetic chromatography (MEKC). Separation of tocilizumab and the internal standard, methotrexate, was achieved with a background electrolyte consisting of phosphoric acid buffer and sodium dodecyl sulfate (SDS) with UV detection at 195 nm. The method was linear in the concentration range from 10 to 250 µg/mL with correlation coefficient greater than 0.995. The method was successfully applied to the analysis of human and rat plasma samples with good recoveries. Sample preparation involved protein precipitation followed by dilution of the supernatant. The intra‐ and inter-day precisions were less than 5%, the accuracy varied from − 2.71 to 3.84%. The proposed method has acceptable analytical performance and could be applied in future clinical and pharmacokinetic studies including anticancer therapy.

Repurposing the Veterinary Antibiotic Apramycin for Antibacterial and Antibiofilm Activity Against Pseudomonas aeruginosa From Cystic Fibrosis Patients.

Objectives:To evaluate the in vitro antibacterial, antibiofilm, and antivirulence activities of apramycin, comparatively to tobramycin, against a set of P. aeruginosa from chronically infected cystic fibrosis (CF) patients.MethodsThe activity of antibiotics against planktonic cells was assessed by performing MIC, MBC, and time-kill assays. The activity against mature biofilms was evaluated, in a microtiter plate, both in terms of dispersion (crystal violet assay) and residual viability (viable cell count). The effect of drug exposure on selected P. aeruginosa virulence genes expression was assessed by real-time Reverse Transcription quantitative PCR (RT-qPCR).ResultsApramycin MIC90 and MBC90 values were found at least fourfold lower than those for tobramycin. A comparable trend was observed for mucoid strains. Only 4 out of 24 strains (16.6%) showed an apramycin MIC higher than the epidemiological cut-off value of 64 mg/L, whereas a higher resistance rate was observed for tobramycin (62.5%; p < 0.01 vs. apramycin). In time-kill analyses, both aminoglycosides were found bactericidal, although apramycin showed a more rapid effect and did not allow for regrowth. Apramycin generally stimulated biofilm biomass formation, whereas tobramycin showed opposite trends depending on the strain tested. Both drugs caused a highly significant, dose-dependent reduction of biofilm viability, regardless of strain and concentration tested. The exposure to apramycin and tobramycin caused increased expression of mexA and mexC (multidrug efflux pumps), whereas tobramycin specifically increased the expression of aprA (alkaline protease) and toxA (exotoxin A). Neither apramycin nor tobramycin showed cytotoxic potential toward IB3-1 bronchial epithelial CF cells.ConclusionOur results warrant future pharmacokinetic and pharmacodynamic studies for supporting the rationale to repurpose apramycin, a veterinary aminoglycoside, for CF lung infections.

Comparative Study of Teramnus labialis and Xanthium strumarium for Anti-Diabetic Property

Introduction: Plants have always been a very good source of drugs. Diabetes mellitus (DM) is one of the common metabolic disorders acquiring around 2.8% of the world’s population. Hence in the present study, we intend to evaluate the antidiabetic property of the Teramnus labialis, Xanthium strumarium. Materials and Methods: T. labialis, Methyl extract Teramnus labialis (METL) and X. strumarium Methyl extract Xanthium strumarium (MEXS) were collected from hills of Tirupathi, dried, and powdered. Healthy male Wistar albino rats induced DM with the Streptozotocin-Nicotinamide and Alloxan induced model were studied on days 1, 7, 14, 21, 28 for the serum glucose (SG), total cholesterol, triglycerides, high-density lipoprotein and low-density lipoprotein levels. The observations were compared with the standard drugs Glibenclamide. Results: When extracts administered at low dose in combination exhibited a valuable synergistic activity on mean blood glucose levels, which were almost similar when these results compared with that of standard drug-treated animals. Conclusion: Both the plant extracts were safe in the combination and do not shown any toxicities or drug-related interactions but future pharmacokinetic studies required to validate the given result.

Flumequine-loaded titanate nanotubes as antibacterial agents for aquaculture farms.

Flumequine (FLUM), a quinolone-derived antibiotic is one of the most prescribed drugs in aquaculture farms. However, its intensive use becomes worrisome because of its environmental risks and the emergence of FLUM-resistant bacteria. To overcome these problems we propose in this study the encapsulation and the delivery of FLUM by titanate nanotubes (TiNTs). Optimal FLUM loading was reached by suspending the dehydrated powder nanomaterials (FLUM : TiNTs ratio = 1 : 5) in ethanol. The drug entrapment efficiency was calculated to be 80% approximately with a sustained release in PBS at 37 °C up to 5 days. Then FLUM@TiNTs was evaluated for both its in vitro drug release and antimicrobial activity against Escherichia coli (E. coli). Spectacularly high antibacterial activity compared to those of free FLUM antibiotic was obtained confirming the efficiency of TiNTs to protect FLUM from rapid degradation and transformation within bacteria improving thereby its antibacterial effect. Indeed FLUM@TiNTs was efficient to decrease gradually the bacterial viability to reach ≈5% after 5 days versus ≈75% with free FLUM. Finally, the ex vivo permeation experiments on sea bass (Dicentrachus labrax) intestine shows that TiNTs act to increase the intestinal permeation of FLUM during the experiment. Indeed the encapsulated FLUM flux increased 12 fold (1.46 μg cm2 h−1) compared to the free antibiotic (0.18 μg cm2 h−1). Thanks to its physical properties (diameter 10 nm, tubular shape…) and its high stability in the simulated intestinal medium, TiNTs are easy internalized by enterocytes, thus involving an endocytosis mechanism, and then improve intestinal permeation of FLUM. Taken together, FLUM@TiNTs hold potential as an effective approach for enhancing the antimicrobial activity of FLUM and pave the way not only for future pharmacokinetic studies in the treatment and targeting of fish infections but also for instating of novel strategies that overcome the challenges associated with the abusive use of antibiotics in fish farming.

Pharmacokinetics of Single Domain Antibodies and Conjugated Nanoparticles Using a Hybrid near Infrared Method.

Iron oxide nanoparticles and single domain antibodies from camelids (VHHs) have been increasingly recognized for their potential uses for medical diagnosis and treatment. However, there have been relatively few detailed characterizations of their pharmacokinetics (PK). The aim of this study was to develop imaging methods and pharmacokinetic models to aid the future development of a novel family of brain MRI molecular contrast agents. An efficient near-infrared (NIR) imaging method was established to monitor VHH and VHH conjugated nanoparticle kinetics in mice using a hybrid approach: kinetics in blood were assessed by direct sampling, and kinetics in kidney, liver, and brain were assessed by serial in vivo NIR imaging. These studies were performed under “basal” circumstances in which the VHH constructs and VHH-conjugated nanoparticles do not substantially interact with targets nor cross the blood brain barrier. Using this approach, we constructed a five-compartment PK model that fits the data well for single VHHs, engineered VHH trimers, and iron oxide nanoparticles conjugated to VHH trimers. The establishment of the feasibility of these methods lays a foundation for future PK studies of candidate brain MRI molecular contrast agents.