Efavirenz Is Predicted To Accumulate in Brain Tissue: an In Silico, In Vitro, and In Vivo Investigation

Adequate concentrations of efavirenz in the central nervous system (CNS) are necessary to suppress viral replication, but high concentrations may increase the likelihood of CNS adverse drug reactions. The aim of this investigation was to evaluate the efavirenz distribution in the cerebrospinal fluid (CSF) and the brain by using a physiologically based pharmacokinetic (PBPK) simulation for comparison with rodent and human data. The efavirenz CNS distribution was calculated using a permeability-limited model on a virtual cohort of 100 patients receiving efavirenz (600 mg once daily). Simulation data were then compared with human data from the literature and with rodent data. Wistar rats were administered efavirenz (10 mg kg of body weight(−1)) once daily over 5 weeks. Plasma and brain tissue were collected for analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The median maximum concentrations of drug (C(max)) were predicted to be 3,184 ng ml(−1) (interquartile range [IQR], 2,219 to 4,851 ng ml(−1)), 49.9 ng ml(−1) (IQR, 36.6 to 69.7 ng ml(−1)), and 50,343 ng ml(−1) (IQR, 38,351 to 65,799 ng ml(−1)) in plasma, CSF, and brain tissue, respectively, giving a tissue-to-plasma ratio of 15.8. Following 5 weeks of oral dosing of efavirenz (10 mg kg(−1)), the median plasma and brain tissue concentrations in rats were 69.7 ng ml(−1) (IQR, 44.9 to 130.6 ng ml(−1)) and 702.9 ng ml(−1) (IQR, 475.5 to 1,018.0 ng ml(−1)), respectively, and the median tissue-to-plasma ratio was 9.5 (IQR, 7.0 to 10.9). Although it is useful, measurement of CSF concentrations may give an underestimation of the penetration of antiretrovirals into the brain. The limitations associated with obtaining tissue biopsy specimens and paired plasma and CSF samples from patients make PBPK modeling an attractive tool for probing drug distribution

Long-Acting Injectable Statins-Is It Time for a Paradigm Shift?

In recent years, advances in pharmaceutical processing technologies have resulted in development of medicines that provide therapeutic pharmacokinetic exposure for a period ranging from weeks to months following a single parenteral administration. Benefits for adherence, dose and patient satisfaction have been witnessed across a range of indications from contraception to schizophrenia, with a range of long-acting medicines also in development for infectious diseases such as HIV. Existing drugs that have successfully been formulated as long-acting injectable formulations have long pharmacokinetic half-lives, low target plasma exposures, and low aqueous solubility. Of the statins that are clinically used currently, atorvastatin, rosuvastatin, and pitavastatin may have compatibility with this approach. The case for development of long-acting injectable statins is set out within this manuscript for this important class of life-saving drugs. An overview of some of the potential development and implementation challenges is also presented

<i>In vitro</i> evaluation of physicochemical-dependent effects of polymeric nanoparticles on their cellular uptake and co-localization using pulmonary calu-3 cell lines.

ObjectiveThe study evaluated physicochemical properties of eight different polymeric nanoparticles (NPs) and their interaction with lung barrier and their suitability for pulmonary drug delivery.MethodsEight physiochemically different NPs were fabricated from Poly lactic-co-glycolic acid (PLGA, PL) and Poly glycerol adipate-co-ω-pentadecalactone (PGA-co-PDL, PG) via emulsification-solvent evaporation. Pulmonary barrier integrity was investigated in vitro using Calu-3 under air-liquid interface. NPs internalization was investigated using a group of pharmacological inhibitors with subsequent microscopic visual confirmation.ResultsEight NPs were successfully formulated from two polymers using emulsion-solvent evaporation; 200, 500 and 800 nm, negatively-charged and positively-charged. All different NPs did not alter tight junctions and PG NPs showed similar behavior to PL NPs, indicating its suitability for pulmonary drug delivery. Active endocytosis uptake mechanisms with physicochemical dependent manner were observed. In addition, NPs internalization and co-localization with lysosomes were visually confirmed indicating their vesicular transport.ConclusionPG and PL NPs had shown no or low harmful effects on the barrier integrity, and with effective internalization and vesicular transport, thus, prospectively can be designed for pulmonary delivery applications

Firefly luciferase offers superior performance to AkaLuc for tracking the fate of administered cell therapies

IntroductionA novel, red-shifted bioluminescence imaging (BLI) system called AkaBLI has been recently developed for cell tracking in preclinical models and to date, limited data is available on how it performs in relation to existing systems.PurposeTo systematically compare the performance of AkaBLI and the standard Firefly luciferase (FLuc) systems to monitor the biodistribution and fate of cell therapies in rodents.MethodsUmbilical cord mesenchymal stromal cells (MSCs) were transduced to produce two genetically engineered populations, expressing either AkaLuc or the engineered FLuc luc2. The bioluminescence of AkaLuc+ and FLuc+ cells was assessed both in vitro (emission spectra, saturation kinetics and light emission per cell) and in vivo (substrate kinetics following intraperitoneal and subcutaneous administration and biodistribution of the cells up to day 7).ResultsIntroduction of the reporter genes has no effect on MSC phenotype. For BLI, the FLuc system is superior to AkaBLI in terms of (i) light output, producing a stronger signal after subcutaneous substrate delivery and more consistent signal kinetics when delivered intraperitoneally; (ii) absence of hepatic background; and (iii) safety, where the AkaLuc substrate was associated with a reaction in the skin of the mice in vivo.ConclusionWe conclude that there is no advantage in using the AkaBLI system to track the biodistribution of systemically administered cell-based regenerative medicine therapies in vivo

Exposure of the basophilic cell line KU812 to liposomes reveals activation profiles associated with potential anaphylactic responses linked to physico-chemical characteristics

Lipidic nanoparticles (LNP), particularly liposomes, have been proven to be a successful and versatile platform for intracellular drug delivery for decades. Whilst primarily developed for small molecule delivery, liposomes have recently undergone a renaissance due to their success in vaccination strategies, delivering nucleic acids, in the COVID-19 pandemic. As such, liposomes are increasingly being investigated for the delivery of nucleic acids, beyond mRNA, as non-viral gene delivery vectors. Although not generally considered toxic, liposomes are increasingly shown to not be immunologically inert, which may have advantages in vaccine applications but may limit their use in other conditions where immunological responses may lead to adverse events, particularly those associated with complement activation. We sought to assess a small panel of liposomes varying in a number of physico-chemical characteristics associated with complement activation and inflammatory responses, and examine how basophil-like cells may respond to them. Basophils, as well as other cell types, are involved in the anaphylactic responses to liposomes but are difficult to isolate in sufficient numbers to conduct large scale analysis. Here, we report the use of the human KU812 cell line as a surrogate for primary basophils. Multiple phenotypic markers of activation were assessed, as well as the release of histamine and inflammasome activity within the cells. We found that larger liposomes were more likely to result in KU812 activation, and that non-PEGylated liposomes were potent stimulators of inflammasome activity (four-fold greater IL-1β secretion than untreated controls), and a lower ratio of cholesterol to lipid was also associated with greater IL-1β secretion ([Cholesterol:DSPC ratio] 1:10; 0.35 pg/mL IL-1β vs. 5:10; 0.1 pg/mL). Additionally, PEGylation appeared to be associated with direct KU812 activation. These results suggest possible mechanisms related to the consequences of complement activation that may be underpinned by basophilic cells, in addition to other immune cell types. Investigation of the mechanisms behind these responses, and their impact on use in vivo, are now warranted

Preclinical Evaluation of Long-Acting Emtricitabine Semi-Solid Prodrug Nanoparticle Formulations.

Long-acting injectable (LAI) formulations promise to deliver patient benefits by overcoming issues associated with non-adherence. A preclinical assessment of semi-solid prodrug nanoparticle (SSPN) LAI formulations of emtricitabine (FTC) is reported here. Pharmacokinetics over 28 days were assessed in Wistar rats, New Zealand white rabbits, and Balb/C mice following intramuscular injection. Two lead formulations were assessed for the prevention of an HIV infection in NSG-cmah-/- humanised mice to ensure antiviral activities were as anticipated according to the pharmacokinetics. Cmax was reached by 12, 48, and 24 h in rats, rabbits, and mice, respectively. Plasma concentrations were below the limit of detection (2 ng/mL) by 21 days in rats and rabbits, and 28 days in mice. Mice treated with SSPN formulations demonstrated undetectable viral loads (700 copies/mL detection limit), and HIV RNA remained undetectable 28 days post-infection in plasma, spleen, lung, and liver. The in vivo data presented here demonstrate that the combined prodrug/SSPN approach can provide a dramatically extended pharmacokinetic half-life across multiple preclinical species. Species differences in renal clearance of FTC mean that longer exposures are likely to be achievable in humans than in preclinical models

Dose prediction for repurposing nitazoxanide in SARS-CoV-2 treatment or chemoprophylaxis

Background Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has been declared a global pandemic and urgent treatment and prevention strategies are needed. Nitazoxanide, an anthelmintic drug has been shown to exhibit in vitro activity against SARS‐CoV‐2. The present study used physiologically‐based pharmacokinetic (PBPK) modelling to inform optimal doses of nitazoxanide capable of maintaining plasma and lung tizoxanide exposures above the reported SARS‐CoV‐2 EC90. Methods A whole‐body PBPK model was validated against available pharmacokinetic data for healthy individuals receiving single and multiple doses between 500–4000 mg with and without food. The validated model was used to predict doses expected to maintain tizoxanide plasma and lung concentrations above the EC90 in >90% of the simulated population. PopDes was used to estimate an optimal sparse sampling strategy for future clinical trials. Results The PBPK model was successfully validated against the reported human pharmacokinetics. The model predicted optimal doses of 1200 mg QID, 1600 mg TID, 2900 mg BID in the fasted state and 700 mg QID, 900 mg TID and 1400 mg BID when given with food. For BID regimens an optimal sparse sampling strategy of 0.25, 1, 3 and 12h post dose was estimated. Conclusion The PBPK model predicted tizoxanide concentrations within doses of nitazoxanide already given to humans previously. The reported dosing strategies provide a rational basis for design of clinical trials with nitazoxanide for the treatment or prevention of SARS‐CoV‐2 infection. A concordant higher dose of nitazoxanide is now planned for investigation in the seamless phase I/IIa AGILE trial (www.agiletrial.net)

Functionalized superparamagnetic iron oxide nanoparticles provide highly efficient iron-labelling in macrophages for magnetic resonance-based detection in vivo

Tracking cells during regenerative cytotherapy is crucial for monitoring their safety and efficacy. Macrophages are an emerging cell-based regenerative therapy for liver disease and can be readily labeled for medical imaging. A reliable, clinically applicable cell-tracking agent would be a powerful tool to study cell biodistribution.Using a recently described chemical design, we set out to functionalize, optimize and characterize a new set of superparamagnetic iron oxide nanoparticles (SPIONs) to efficiently label macrophages for magnetic resonance imaging-based cell tracking in vivo.A series of cell health and iron uptake assays determined that positively charged SPIONs (+16.8 mV) could safely label macrophages more efficiently than the formerly approved ferumoxide (-6.7 mV; Endorem) and at least 10 times more efficiently than the clinically approved SPION ferumoxytol (-24.2 mV; Rienso). An optimal labeling time of 4 h at 25 µg/mL was demonstrated to label macrophages of mouse and human origin without any adverse effects on cell viability whilst providing substantial iron uptake (>5 pg Fe/cell) that was retained for 7 days in vitro. SPION labeling caused no significant reduction in phagocytic activity and a shift toward a reversible M1-like phenotype in bone marrow-derived macrophages (BMDMs). Finally, we show that SPION-labeled BMDMs delivered via the hepatic portal vein to mice are localized in the hepatic parenchyma resulting in a 50% drop in T2* in the liver. Engraftment of exogenous cells was confirmed via immunohistochemistry up to 3 weeks posttransplantation.A positively charged dextran-coated SPION is a promising tool to noninvasively track hepatic macrophage localization for therapeutic monitoring