Multiple doses of tenofovir disoproxil fumarate (TDF) together with emtricitabine is effective for HIV preexposure prophylaxis (PrEP). TDF is converted to tenofovir (TFV) in circulation, which is ...subsequently cleared via tubular secretion by organic ion transporters (OATs; OAT1 and OAT3). Using in vitro kinetic parameters for TFV and the OAT1 and OAT3 inhibitor probenecid, a bottom‐up physiologically‐based pharmacokinetic model was successfully developed for the first time that accurately describes the probenecid–TFV interaction. This model predicted an increase in TFV plasma exposure by 60%, which was within 15% of the observed clinical pharmacokinetic data, and a threefold decrease in renal cells exposure following coadministration of a 600 mg TDF dose with 2 g probenecid. When compared with multiple‐dose regimens, a single‐dose probenecid‐boosted TDF regimen may be effective for HIV PrEP and improve adherence and safety by minimizing TFV‐induced nephrotoxicity by reducing TFV accumulation in renal cells.
Recent progress in ultrafast spectroscopy and semiconductor technology is enabling unique applications in screening, detection, and diagnostics in the Terahertz (T‐ray) regime. The promise of ...efficaciously operation in this spectral region is tempered by the lack of devices that can spectrally analyze samples at sufficient temporal and spatial resolution. Real‐time, multispectral T‐ray (Mul‐T) imaging is reported by designing and demonstrating hyperspectral metamaterial focal plane array (MM‐FPA) interfaces allowing multiband (and individually tunable) responses without compromising on the pixel size. These MM‐FPAs are fully compatible with existing microfabrication technologies and have low noise when operating in the ambient environment. When tested with a set of frequency switchable quantum cascade lasers (QCLs) for multicolor illumination, both MM‐FPAs and QCLs can be tuned to operate at multiple discrete THz frequencies to match analyte “fingerprints.” Versatile imaging capabilities are presented, including unambiguous identification of concealed substances with intrinsic and/or human‐engineered THz characteristics as well as effective diagnosis of cancerous tissues without notable spectral signatures in the THz range, underscoring the utility of applying multispectral approaches in this compelling wavelength range for sensing/identification and medical imaging.
A real‐time, multispectral T‐ray (Mul‐T) imager based on multispectral metamaterial focal plane array and frequency switchable quantum cascade lasers is reported. Non‐destructive material identification and defect inspection for concealed tablets based on the Mul‐T imager are implemented successfully.
Integrase strand transfer inhibitor (INSTI)-based regimens dominate initial human immunodeficiency virus treatment. Most INSTIs are metabolized predominantly via UDP-glucuronosyltransferases (UGTs). ...For drugs predominantly metabolized by UGTs, including INSTIs, in vitro data recovered from human liver microsomes (HLMs) alone often underpredict human oral clearance. While several factors may contribute, extrahepatic glucuronidation may contribute to this underprediction. Thus, we comprehensively characterized the kinetics for the glucuronidation of INSTIs (cabotegravir, dolutegravir, and raltegravir) using pooled human microsomal preparations from liver (HLMs), intestine (HIMs), and kidney (HKMs) tissues; human embryonic kidney 293 cells expressing individual UGTs; and recombinant UGTs. In vitro glucuronidation of cabotegravir (HLMs≈HKMs>>>HIMs), dolutegravir (HLMs>HIMs>>HKMs), and raltegravir (HLMs>HKMs>> HIMs) occurred in hepatic and extrahepatic tissues. The kinetic data from expression systems suggested the major enzymes in each tissue: hepatic UGT1A9 > UGT1A1 (dolutegravir and raltegravir) and UGT1A1 (cabotegravir), intestinal UGT1A3 > UGT1A8 > UGT1A1 (dolutegravir) and UGT1A8 > UGT1A1 (raltegravir), and renal UGT1A9 (dolutegravir and raltegravir). Enzymes catalyzing cabotegravir glucuronidation in the kidney and intestine could not be identified unequivocally. Using data from dolutegravir glucuronidation as a prototype, a "bottom-up" physiologically based pharmacokinetic model was developed in a stepwise approach and predicted dolutegravir oral clearance within 4.5-fold (hepatic data only), 2-fold (hepatic and intestinal data), and 32% (hepatic, intestinal, and renal data). These results suggest clinically meaningful glucuronidation of dolutegravir in tissues other than the liver. Incorporation of additional novel mechanistic and physiologic underpinnings of dolutegravir metabolism along with in silico approaches appears to be a powerful tool to accurately predict the clearance of dolutegravir from in vitro data.
Abstract
OBJECTIVE
To determine the dose of alfaxalone for IM administration combined with dexmedetomidine and hydromorphone that would allow endoscopic-guided orotracheal intubation in rabbits ...without causing a decrease in respiratory rate or apnea.
ANIMALS
15 sexually intact (9 females and 6 males) healthy Miniature Lop rabbits weighing a mean ± SD of 2.3 ± 0.3 kg and ranging in age from 4 to 9 months.
PROCEDURES
In a randomized, controlled clinical trial, rabbits received 0.1 mg of hydro-morphone/kg and 0.005 mg of dexmedetomidine/kg, plus alfaxalone at either 2 mg/kg (5 rabbits), 5 mg/kg (5 rabbits), or 7 mg/kg (5 rabbits). Drugs were mixed in a single syringe and administered IM. Semiquantitative rating scales were used to evaluate quality of anesthesia and intubation. Orotracheal intubation was attempted with endoscopy and confirmed by capnography.
RESULTS
The number of successful intubations was 0, 3, and 4 in rabbits receiving 2, 5, and 7 mg of alfaxalone/kg, respectively. Median (range) anesthesia quality scores (scale, 0 to 12; 12 = deepest anesthesia) were 3 (2 to 5), 6 (5 to 6), and 6 (4 to 9) for rabbits receiving 2, 5, and 7 mg of alfaxalone/kg, respectively. The median (range) intubation quality scores (scale, 0 to 3 ie, intubation not possible to easiest intubation) were 0 (0 to 0), 2 (0 to 3), and 2 (0 to 3) for rabbits receiving 2, 5, and 7 mg of alfaxalone/kg, respectively. None of the rabbits experienced a decrease in respiratory rate or apnea.
CONCLUSIONS AND CLINICAL RELEVANCE
Increasing doses of alfaxalone combined with hydromorphone and dexmedetomidine increased the success rate of endoscopic-guided orotracheal intubation. Increasing the dose of alfaxalone had no effect on respiratory rate.
PERJETA (pertuzumab), administered with Herceptin (trastuzumab), is used in the treatment of human epidermal growth factor receptor 2‐positive breast cancer. Pertuzumab is currently approved with an ...initial loading dose of 840 mg, followed by a 420‐mg maintenance dose intravenously every 3 weeks. A reloading dose is required if there is a ≥6‐week delay in treatment. In response to the potential treatment disruption due to COVID‐19, the impact of dose delays and alternative dosing regimens on intravenous pertuzumab for human epidermal growth factor receptor 2‐positive breast cancer treatment is presented. Simulations were conducted by using the validated population pharmacokinetic model for pertuzumab, and included (1) 4‐, 6‐, and 9‐week dose delays of the 840 mg/420 mg every 3 weeks dosing regimen and (2) 840 mg/420 mg every 4 weeks and 840 mg every 6 weeks alternative dosing regimens. Simulations were compared with the currently approved pertuzumab dosing regimen. The simulations in 1000 virtual patients showed that a dose reload (840 mg) is required following a dose delay of ≥6 weeks to maintain comparable Ctrough (lowest concentration before the next dose is given) levels to clinical trials. The 840 mg/420 mg every 4 weeks and 840 mg every 6 weeks alternative dosing regimens decrease median steady‐state Ctrough by ≈40% compared with the approved regimen, and <90% of patients will be above the target Ctrough. Thus, the alternative 840 mg/420 mg every 4 weeks and 840 mg every 6 weeks pertuzumab dosing regimens are not recommended. Flexibility for intravenous PERJETA‐based regimens is available with an alternative route of pertuzumab administration (subcutaneous vs intravenous).
EBV latent membrane protein 1 (LMP1) is released from latently infected tumor cells in small membrane-enclosed extracellular vesicles (EVs). Accumulating evidence suggests that LMP1 is a major driver ...of EV content and functions. LMP1-modified EVs have been shown to influence recipient cell growth, migration, differentiation, and regulation of immune cell function. Despite the significance of LMP1-modified exosomes, very little is known about how this viral protein enters or manipulates the host EV pathway. In this study, LMP1 deletion mutants were generated to assess protein regions required for EV trafficking. Following transfection of LMP1 or mutant plasmids, EVs were collected by differential centrifugation, and the levels of specific cargo were evaluated by immunoblot analysis. The results demonstrate that, together, the N terminus and transmembrane region 1 of LMP1 are sufficient for efficient sorting into EVs. Consistent with these findings, a mutant lacking the N terminus and transmembrane domains 1 through 4 (TM5-6) failed to be packaged into EVs, and exhibited higher colocalization with endoplasmic reticulum and early endosome markers than the wild-type protein. Surprisingly, TM5-6 maintained the ability to colocalize and form a complex with CD63, an abundant exosome protein that is important for the incorporation of LMP1 into EVs. Other mutations within LMP1 resulted in enhanced levels of secretion, pointing to potential positive and negative regulatory mechanisms for extracellular vesicle sorting of LMP1. These data suggest new functions of the N terminus and transmembrane domains in LMP1 intra- and extracellular trafficking that are likely downstream of an interaction with CD63.
EBV infection contributes to the development of cancers, such as nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin's disease, and posttransplant lymphomas, in immunocompromised or genetically susceptible individuals. LMP1 is an important viral protein expressed by EBV in these cancers. LMP1 is secreted in extracellular vesicles (EVs), and the transfer of LMP1-modified EVs to uninfected cells can alter their physiology. Understanding the cellular machinery responsible for sorting LMP1 into EVs is limited, despite the importance of LMP1-modified EVs. Here, we illustrate the roles of different regions of LMP1 in EV packaging. Our results show that the N terminus and TM1 are sufficient to drive LMP1 EV trafficking. We further show the existence of potential positive and negative regulatory mechanisms for LMP1 vesicle sorting. These findings provide a better basis for future investigations to identify the mechanisms of LMP1 targeting to EVs, which could have broad implications in understanding EV cargo sorting.
Epstein-Barr virus LMP1 is an oncoprotein required for immortalizing B lymphocytes and also plays important roles in transforming non-lymphoid tissue. The discovery of LMP1 protein interactions will ...likely generate targets to treat EBV-associated cancers. Here, we define the broader LMP1 interactome using the recently developed BioID method. Combined with mass spectrometry, we identified over 1000 proteins across seven independent experiments with direct or indirect relationships to LMP1. Pathway analysis suggests that a significant number of the proteins identified are involved in signal transduction and protein or vesicle trafficking. Interestingly, a large number of proteins thought to be important in the formation of exosomes and protein targeting were recognized as probable LMP1 interacting partners, including CD63, syntenin-1, ALIX, TSG101, HRS, CHMPs, and sorting nexins. Therefore, it is likely that LMP1 modifies protein trafficking and exosome biogenesis pathways. In support of this, knock-down of syntenin-1 and ALIX resulted in reduced exosomal LMP1.
•Identification of over 1000 direct and proximal LMP1 protein-protein interactions.•Many interactors are involved in signal transduction and protein/vesicle trafficking.•Novel validated interactions include HSC70, ITGB1, syntenin-1, TSG101, and STAT3.
In a randomized, crossover pharmacokinetic study in healthy volunteers (N = 14), a single dose of 2 g probenecid (PRO)‐boosted 600 mg tenofovir disoproxil fumarate (TDF)/400 mg emtricitabine (FTC) ...(test (T) +PRO) was compared with the current on‐demand HIV preexposure prophylaxis from the IPERGAY study (a 600 mg TDF/400 mg FTC on day 1 and 300 mg TDF/200 mg FTC on days 2 and 3) (control, C IPERGAY). PRO increased mean single‐dose area under the plasma concentration‐time curve extrapolated to infinity (AUC0–∞,SD) of tenofovir (TFV) and FTC by 61% and 68%, respectively. The TFV‐diphosphate (TFV‐DP) concentrations in peripheral blood mononuclear cells were higher (~30%) at 24 hours in T +PRO but then fell significantly lower (~40%) at 72 hours compared with C IPERGAY. The interaction between FTC and PRO was unexpected and novel. Further study is needed to determine if this PRO‐boosted TDF/FTC regimen would be clinically effective.
PDF
