Extension of the Mechanistic Tissue Distribution Model of Rodgers and Rowland by Systematic Incorporation of Lysosomal Trapping: Impact on Unbound Partition Coefficient and Volume of Distribution Predictions in the Rat.

Physiologically based pharmacokinetic modeling has become a standard tool to predict drug distribution in early stages of drug discovery; however, this does not currently encompass lysosomal trapping. For basic lipophilic compounds, lysosomal sequestration is known to potentially influence intracellular as well as tissue distribution. The aim of our research was to reliably predict the lysosomal drug content and ultimately integrate this mechanism into pharmacokinetic prediction models. First, we further validated our previously presented method to predict the lysosomal drug content (Schmitt et al., 2019) for a larger set of compounds (n = 41) showing a very good predictivity. Using the lysosomal marker lipid bis(monoacylglycero)phosphate, we estimated the lysosomal volume fraction for all major tissues in the rat, ranging from 0.03% for adipose up to 5.3% for spleen. The pH-driven lysosomal trapping was then estimated and fully integrated into the mechanistic distribution model published by Rodgers et al. (2005) Predictions of Kpu improved for all lysosome-rich tissues. For instance, Kpu increased for nicotine 4-fold (spleen) and 2-fold (lung and kidney) and for quinidine 1.8-fold (brain), although for most other drugs the effects were much less (≤7%). Overall, the effect was strongest for basic compounds with a lower lipophilicity, such as nicotine, for which the unbound volume of distribution at steady-state prediction changed from 1.34 to 1.58 l/kg. For more lipophilic (basic) compounds or those that already show strong interactions with acidic phospholipids, the additional contribution of lysosomal trapping was less pronounced. Nevertheless, lysosomal trapping will also affect intracellular distribution of such compounds. SIGNIFICANCE STATEMENT: The estimation of the lysosomal content in all body tissues facilitated the incorporation of lysosomal sequestration into a general physiologically based pharmacokinetic model, leading to improved predictions as well as elucidating its influence on tissue and subcellular distribution in the rat.

Quantitation of Lysosomal Trapping of Basic Lipophilic Compounds Using In Vitro Assays and In Silico Predictions Based on the Determination of the Full pH Profile of the Endo-/Lysosomal System in Rat Hepatocytes.

Lysosomal sequestration may affect the pharmacokinetics, efficacy, and safety of new basic lipophilic drug candidates potentially impacting their intracellular concentrations and tissue distribution. It may also be involved in drug-drug interactions, drug resistance, and phospholipidosis. However, currently there are no assays to evaluate the lysosomotropic behavior of compounds in a setting fully meeting the needs of drug discovery. We have, therefore, integrated a set of methods to reliably rank order, quantify, and calculate the extent of lysosomal sequestration in rat hepatocytes. An indirect fluorescence-based assay monitors the displacement of the fluorescence probe LysoTracker Red by test compounds. Using a lysosomal-specific evaluation algorithm allows one to generate IC50 values at lower than previously reported concentrations. The concentration range directly agrees with the concentration dependency of the lysosomal drug content itself directly quantified by liquid chromatography-tandem mass spectrometry and thus permits a quantitative link between the indirect and the direct trapping assay. Furthermore, we have determined the full pH profile and corresponding volume fractions of the endo-/lysosomal system in plated rat hepatocytes, enabling a more accurate in silico prediction of the extent of lysosomal trapping based only on pK a values as input, allowing early predictions even prior to chemical synthesis. The concentration dependency-i.e., the saturability of the trapping-can then be determined by the IC50 values generated in vitro. Thereby, a more quantitative assessment of the susceptibility of basic lipophilic compounds for lysosomal trapping is possible.

Role of renal function in risk assessment of target non-attainment after standard dosing of meropenem in critically ill patients: a prospective observational study.

BACKGROUND: Severe bacterial infections remain a major challenge in intensive care units because of their high prevalence and mortality. Adequate antibiotic exposure has been associated with clinical success in critically ill patients. The objective of this study was to investigate the target attainment of standard meropenem dosing in a heterogeneous critically ill population, to quantify the impact of the full renal function spectrum on meropenem exposure and target attainment, and ultimately to translate the findings into a tool for practical application. METHODS: A prospective observational single-centre study was performed with critically ill patients with severe infections receiving standard dosing of meropenem. Serial blood samples were drawn over 4 study days to determine meropenem serum concentrations. Renal function was assessed by creatinine clearance according to the Cockcroft and Gault equation (CLCRCG). Variability in meropenem serum concentrations was quantified at the middle and end of each monitored dosing interval. The attainment of two pharmacokinetic/pharmacodynamic targets (100%T>MIC, 50%T>4×MIC) was evaluated for minimum inhibitory concentration (MIC) values of 2 mg/L and 8 mg/L and standard meropenem dosing (1000 mg, 30-minute infusion, every 8 h). Furthermore, we assessed the impact of CLCRCG on meropenem concentrations and target attainment and developed a tool for risk assessment of target non-attainment. RESULTS: Large inter- and intra-patient variability in meropenem concentrations was observed in the critically ill population (n = 48). Attainment of the target 100%T>MIC was merely 48.4% and 20.6%, given MIC values of 2 mg/L and 8 mg/L, respectively, and similar for the target 50%T>4×MIC. A hyperbolic relationship between CLCRCG (25-255 ml/minute) and meropenem serum concentrations at the end of the dosing interval (C8h) was derived. For infections with pathogens of MIC 2 mg/L, mild renal impairment up to augmented renal function was identified as a risk factor for target non-attainment (for MIC 8 mg/L, additionally, moderate renal impairment). CONCLUSIONS: The investigated standard meropenem dosing regimen appeared to result in insufficient meropenem exposure in a considerable fraction of critically ill patients. An easy- and free-to-use tool (the MeroRisk Calculator) for assessing the risk of target non-attainment for a given renal function and MIC value was developed. TRIAL REGISTRATION: Clinicaltrials.gov, NCT01793012 . Registered on 24 January 2013.

Quantitative molecular tissue atlas of Bis(monoacylglycero)phosphate and phosphatidylglycerol membrane lipids in rodent organs generated by methylation assisted high resolution mass spectrometry.

Bis(monoacylglycero)phosphate (BMP) and phosphatidylglycerol (PG) are structural isomeric phospholipids with very different properties and biological functions. Due to their isomeric nature, it has thus far been challenging to simultaneously quantify BMP and PG lipids in tissue samples by mass spectrometry. Therefore, we have developed a sensitive LC-MS/MS based approach with prior methylation derivatization that is able to handle large batches of samples. Using this high throughput platform, a simulated MS/MS database was established for confident lipid assignment. In this work, we have simultaneously identified and quantified BMP and PG lipid molecules in different body tissues of rats and mice. We report for the first time a quantitative molecular atlas of BMP and PG lipids for 14 different tissues and organs in Wistar rats, NMRI and CD1 mice. Organ- and species-specificity was analyzed and compared for both lipid molecule classes. A total of 34 BMP and 10 PG molecules were quantified, with PG concentrations being generally much higher across tissues than BMP, but BMP lipids showing a much higher molecular diversity between animal organs. The large diversity of the BMP lipids with regard to their abundance and molecular composition suggests distinct biological function(s) of the individual BMP molecules in different tissues and organs of body. Particularly high tissue levels of BMP were seen in spleen, lung, liver, kidney and small intestines, i.e. tissues that are known for their high abundance and/or activity level of lysosomes late and endosomes. Elevated BMP levels in brain tissue of APP/PSEN transgenic compared to age matched wild-type mice were also observed using this platform. This analytical methodology presented a high throughput LC-based approach incorporating simulated MS/MS database to identify and quantify BMP lipids as well as PG molecules.