A novel antibiotic class targeting the lipopolysaccharide transporter.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Novel Indane-Containing NBTIs with Potent Anti-Gram-Negative Activity and Minimal hERG Inhibition.

Novel bacterial topoisomerase inhibitors (NBTIs) make up a promising new class of antibiotics with the potential to combat the growing threat of antimicrobial resistance. Two key challenges in the development of NBTIs have been to obtain broad spectrum activity against multidrug-resistant Gram-negative bacteria and to diminish inhibition of the hERG cardiac ion channel. Here we report the optimization of a series of NBTIs bearing a novel indane DNA intercalating moiety. The addition of a basic, polar side chain connected to the indane by an ether or an N-linked secondary amide linkage together with a lipophilicity-lowering modification of the enzyme binding moiety led to compounds such as 2a and 2g which showed excellent broad spectrum potency and minimal hERG inhibition. Compound 2a demonstrated robust bactericidal in vivo activity in a mouse lung infection model with the strain P. aeruginosa ATCC 27853 which is resistant to several clinically relevant antibiotics. Rodent pharmacokinetic studies with 2a revealed an unusual profile characterized by rapid tissue distribution and a prolonged, flat terminal phase. This profile precluded further development of these compounds as potential new antibiotics.

Evaluation of the drug disposition of RO7049389 with in vitro data and human mass balance supported by physiologically based pharmacokinetic modelling.

AIMS: RO7049389 (linvencorvir) is a developmental oral treatment for chronic hepatitis B virus infection. The aim of this work was to conduct mass balance (MB) and absolute bioavailability (BA) analyses in healthy volunteers, alongside in vitro evaluations of the metabolism of RO7049389 and a major circulating active metabolite M5 in human hepatocytes, and physiologically based pharmacokinetic (PBPK) modelling to refine the underlying drug disposition paradigm. METHODS: Participants in the clinical study (MB: Caucasian, male, n = 6; BA: Caucasian and Asian, male and female, n = 16, 8 in each ethnic groups) received oral [14 C] or unlabelled RO7049389 (600/1000 mg) followed by 100 µg intravenous [13 C]RO7049389. Metabolic pathways with fractions metabolized-obtained from the in vitro incubation results of 10 µM [14 C]RO7049389 and 1 µM M5 with (long-term cocultured) human hepatocytes in the absence and presence of the cytochrome P450 3A4 (CYP3A4) inhibitor itraconazole-were used to complement the PBPK models, alongside the clinical MB and BA data. RESULTS: The model performance in predicting the pharmacokinetic profiles of RO7049389 and M5 aligned with clinical observations in Caucasians and was also successfully applied to Asians. Accordingly, the drug disposition pathways for RO7049389 were postulated with newly characterized estimates of the fractions: biliary excretion by P-glycoprotein (~41%), direct glucuronidation via uridine 5'-diphosphoglucuronosyltransferase 1A3 (~11%), hexose conjugation (~6%), oxidation by CYP3A4 (~28%) and other oxidation reactions (~9%). CONCLUSION: These results support the ongoing clinical development program for RO7049389 and highlight the broader value of PBPK and MB analyses in drug development.

Estimating Unbound Drug Concentrations in Simulated Human Lung Fluid: Relevance to Lung Antibiotic PKPD.

The use of unbound drug concentrations is crucial for the prediction of efficacious doses. Hence, dose predictions for antibiotics targeting respiratory pathogens should be based on free, rather than the currently used, total drug concentrations in epithelial lining fluid (ELF). In this work, we describe an assay to estimate the percent unbound of drugs in ELF using simulated epithelial lining fluid (sELF) containing the most abundant components of ELF in healthy humans. A diverse set of 85 compounds showed a broad range of unbound values ranging from <0.01 to 100%. Binding in sELF was influenced by ionization, with basic compounds typically resulting in a stronger binding than neutral and acidic compounds (median percent unbound values 17, 50, and 62%, respectively). A permanent positive charge further increased binding (median percent unbound 11%), while zwitterions showed a lower binding (median percent unbound 69%). In lipid-free sELF, the binding of basic compounds was less pronounced, while compounds of other ionization classes were less impacted, indicating that lipids are involved in the binding of bases. A reasonable correlation was found between binding in sELF and human plasma (R2 = 0.75); however, plasma binding poorly predicted sELF binding for basic compounds (R2 = 0.50). Bases are an important compound class for antibacterial drug development since positive charges affect permeability into Gram-negative bacteria, which are important in terms of bacterial pneumonia. To evaluate in vivo activity, we selected two bases, for which strong sELF binding was observed (percent unbound <1 and 7%) and conducted an analysis of antibacterial efficacy in the neutropenic murine lung efficacy model and total vs free ELF drug concentrations. In both cases, the total ELF resulted in an overprediction of expected efficacy, while the corrected free ELF explained the observed in vivo efficacy. This supports that free, and not total, ELF concentrations should be used for the efficacious dose prediction for pneumonia and highlights the importance of determining binding in this matrix.

Calculation of an Apical Efflux Ratio from P-Glycoprotein (P-gp) In Vitro Transport Experiments Shows an Improved Correlation with In Vivo Cerebrospinal Fluid Measurements in Rats: Impact on P-gp Screening and Compound Optimization.

P-glycoprotein (P-gp) is a major blood-brain barrier (BBB) efflux transporter. In vitro approaches, including bidirectional efflux ratio (ER), are used to measure P-gp-mediated transport, but findings can be inconsistent across models. We propose a novel, more physiologically relevant, in vitro model: unidirectional apical efflux ratio (AP-ER)-a ratio of permeability rates at the apical side of the BBB with and without P-gp inhibitor. To test our approach, ER and AP-ER were calculated for 3227 structurally diverse compounds in porcine kidney epithelial cells (LLC-PK1) overexpressing human or mouse P-gp and classified based on their passive transcellular P-gp permeability or charged properties. In vivo rat infusion studies were performed for selected compounds with high ER but low AP-ER. One-third of the 3227 compounds had bidirectional ER that was much higher than AP-ER; very few had AP-ER higher than ER. Compounds with a large difference between AP-ER and ER were typically basic compounds with low-to-medium passive permeability and high lipophilicity and/or amphiphilicity, leading to strong membrane binding. Outcomes in the human model were similar to those in mice, suggesting AP-ER/ER ratios may be conserved for at least two species. AP-ER predicted measured cerebrospinal fluid (CSF) concentration better than ER for the five compounds tested in our in vivo rat infusion studies. We report superior estimations of the CSF concentrations of the compounds when based on less resource-intensive AP-ER versus classic ER. Better understanding of the properties leading to high P-gp-mediated efflux in vivo could support more efficient brain-penetrant compound screening and optimization. SIGNIFICANCE STATEMENT: To address inconsistencies associated with the historical, bidirectional efflux ratio (ER) calculation of P-glycoprotein-mediated transport, we propose to use the novel, more physiologically relevant, unidirectional apical efflux ratio (AP-ER) model. In vitro experiments suggested that compounds with strong membrane binding showed the largest difference between AP-ER and ER, and in vivo infusion studies showed that AP-ER predicted cerebrospinal fluid concentrations of compounds better than ER; outcomes in the human model were similar to those in mice.

Kinetics of metabolism of deltamethrin and cis- and trans-permethrin in vitro. Studies using rat and human liver microsomes, isolated rat hepatocytes and rat liver cytosol.

The kinetics of metabolism of deltamethrin (DLM) and cis- and trans-permethrin (CPM and TPM) was studied in male Sprague-Dawley rat and human liver microsomes. DLM metabolism kinetics was also studied in isolated rat hepatocytes, liver microsomes and cytosol. Apparent intrinsic clearance (CLint) values for the metabolism of DLM, CPM and TPM by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes in rat and human liver microsomes decreased with increasing microsomal protein concentration. However, when apparent CLint values were corrected for nonspecific binding to allow calculation of unbound (i.e., corrected) CLint values, the unbound values did not vary greatly with microsomal protein concentration. Unbound CLint values for metabolism of 0.05-1 µM DLM in rat liver microsomes (CYP and CES enzymes) and cytosol (CES enzymes) were not significantly different from rates of DLM metabolism in isolated rat hepatocytes. This study demonstrates that the nonspecific binding of these highly lipophilic compounds needs to be taken into account in order to obtain accurate estimates of rates of in vitro metabolism of these pyrethroids. While DLM is rapidly metabolised in vitro, the hepatocyte membrane does not appear to represent a barrier to the absorption and hence subsequent hepatic metabolism of this pyrethroid.

Application of the Extended Clearance Classification System (ECCS) in Drug Discovery and Development: Selection of Appropriate In Vitro Tools and Clearance Prediction.

In vitro to in vivo extrapolation (IVIVE) to predict human hepatic clearance, including metabolism and transport, requires extensive experimental resources. In addition, there may be technical challenges to measure low clearance values. Therefore, prospective identification of rate-determining step(s) in hepatic clearance through application of the Extended Clearance Classification System (ECCS) could be beneficial for optimal compound characterization. IVIVE for hepatic intrinsic clearance (CLint,h) prediction is conducted for a set of 36 marketed drugs with low-to-high in vivo clearance, which are substrates of metabolic enzymes and active uptake transporters in the liver. The compounds were assigned to the ECCS classes, and CLint,h, estimated with HepatoPac (a micropatterned hepatocyte coculture system), was compared with values calculated based on suspended hepatocyte incubates. An apparent permeability threshold (apical to basal) of 50 nm/s in LLC-PK1 cells proved optimal for ECCS classification. A reasonable performance of the IVIVE for compounds across multiple classes using HepatoPac was achieved (with 2-3-fold error), except for substrates of uptake transporters (class 3b), for which scaling of uptake clearance using plated hepatocytes is more appropriate. Irrespective of the ECCS assignment, metabolic clearance can be estimated well using HepatoPac. The validation and approach elaborated in the present study can result in proposed decision trees for the selection of the optimal in vitro assays guided by ECCS class assignment, to support compound optimization and candidate selection. SIGNIFICANCE STATEMENT: Characterization of the rate-determining step(s) in hepatic elimination could be on the critical path of compound optimization during drug discovery. This study demonstrated that HepatoPac and plated hepatocytes are suitable tools for the estimation of metabolic and active uptake clearance, respectively, for a larger set of marketed drugs, supporting a comprehensive strategy to select optimal in vitro tools and to achieve Extended Clearance Classification System-dependent in vitro to in vivo extrapolation for human clearance prediction.

Fundamental aspects of DMPK optimization of targeted protein degraders.

Targeted protein degraders are an emerging modality. Their properties fall outside the traditional small-molecule property space and are in the 'beyond rule of 5' space. Consequently, drug discovery programs focused on developing orally bioavailable degraders are expected to face complex drug metabolism and pharmacokinetics (DMPK) challenges compared with traditional small molecules. Nevertheless, little information is available on the DMPK optimization of oral degraders. Therefore, in this review, we discuss our experience of these DMPK optimization challenges and present methodologies and strategies to overcome the hurdles dealing with this new small-molecule modality, specifically in developing oral degraders to treat cancer.

Establishment of an In Vitro-In Vivo Correlation for Melanin Binding and the Extension of the Ocular Half-Life of Small-Molecule Drugs.

A large variety of drugs bind effectively to melanin, and this binding influences their ocular pharmacokinetic and distribution profiles. We aimed to establish a correlation between in vitro melanin binding and in vivo ocular pharmacokinetics (PK). The extent of melanin binding in vitro was determined for a set of model drugs; binding kinetics and binding isotherms were generated and fitted to a mechanistic model to derive the drug-melanin binding parameters (Bmax, KD, kon, and koff). In addition, in vitro ADME properties such as cellular permeability, P-glycoprotein-mediated efflux, plasma protein binding, and octanol partition coefficients were determined. Moreover, cellular uptake was measured in the nonpigmented ARPE-19 cells and in lightly pigmented human epidermal melanocytes. Finally, in vivo ocular PK studies were performed in albino and pigmented rats using intravenous injections. Substantial drug enrichment accompanied by a very long residence time was observed in pigmented ocular tissues, which could be linked to the melanin binding determined in vitro and to the intracellular drug uptake into the pigmented cells. The resulting ocular PK profile is shown to be a consequence of the interplay of melanin binding with concurrent processes such as systemic clearance, plasma protein binding, cellular permeation, P-glycoprotein efflux, pH partitioning, and tissue binding. Understanding this interplay at a mechanistic level could help in the rational design and development of new small-molecule drug candidates with the desired PK/pharmacodynamic profile to target the back of the eye.

Predicting Human Clearance of Organic Anion Transporting Polypeptide Substrates Using Cynomolgus Monkey: In Vitro-In Vivo Scaling of Hepatic Uptake Clearance.

This work explores the utility of the cynomolgus monkey as a preclinical model to predict hepatic uptake clearance mediated by organic anion transporting polypeptide (OATP) transporters. Nine OATP substrates (rosuvastatin, pravastatin, repaglinide, fexofenadine, cerivastatin, telmisartan, pitavastatin, bosentan, and valsartan) were investigated in plated cynomolgus monkey and human hepatocytes. Total uptake clearance and passive diffusion were measured in vitro from initial rates in the absence and presence of the OATP inhibitor rifamycin SV , respectively. Total uptake clearance values in plated hepatocytes ranged over three orders of magnitude in both species, with a similar rank order and good agreement in the relative contribution of active transport to total uptake between cynomolgus monkey and human. In vivo hepatic clearance for these nine drugs was determined in cynomolgus monkey after intravenous dosing. Hepatic clearances showed a range similar to human parameters and good predictions from respective hepatocyte parameters (with 2.7- and 3.8-fold bias on average, respectively). The use of cross-species empirical scaling factors (determined from cynomolgus monkey data either as the data set average or individual drug values) improved prediction (less bias, better concordance) of human hepatic clearance from human hepatocyte data alone. In vitro intracellular binding in hepatocytes also correlated well between species. It is concluded that the minimal species differences observed for the current data set between cynomolgus monkey and human hepatocyte uptake, both in vitro and in vivo, support future use of this preclinical model to delineate drug hepatic uptake and enable prediction of human in vivo intrinsic hepatic clearance.

Understanding the Interplay Between Uptake and Efflux Transporters Within In Vitro Systems in Defining Hepatocellular Drug Concentrations.

One of the most holistic in vitro systems for prediction of intracellular drug concentrations is sandwich-cultured hepatocytes (SCH); however, a comprehensive evaluation of the utility of SCH to estimate uptake and biliary clearances and the need for additional kinetic parameters has yet to be carried out. Toward this end, we have selected 9 compounds (rosuvastatin, valsartan, fexofenadine, pravastatin, repaglinide, telmisartan, atorvastatin, saquinavir, and quinidine) to provide a range of physicochemical and hepatic disposition properties. Uptake clearances were determined in SCH and compared with conventional monolayer and suspension hepatocyte systems, previously reported by our laboratory. CLuptake ranged from 1 to 41 µL/min/106 cells in SCH which were significantly lower (1%-10%) compared with the other hepatocyte models. The hepatocyte-to-media unbound concentration ratio (Kpu) has been assessed and ranged 0.7-59, lower compared with other hepatocyte systems (8-280). Estimates of in vitro biliary clearance (CLbile) for 4 drugs were determined and were scaled to predict in vivo values using both intracellular concentration and media drug concentrations. These studies demonstrate that reduced uptake in rat SCH may limit drug access to canalicular efflux transport proteins and highlight the importance of elucidating the interplay between these proteins for accurate prediction of hepatic clearance.

An immortalised astrocyte cell line maintains the in vivo phenotype of a primary porcine in vitro blood-brain barrier model.

Whilst it is well documented that all components of the neurovascular unit contribute to the restrictive nature of the blood-brain barrier (BBB), astrocytes have been identified as the cellular component most likely to play an essential role in maintaining the barrier properties. The aim of this study was to examine the impact of the rat astrocyte cell line, CTX-TNA2, on the structural and functional characteristics of an in vitro BBB and determine the capacity of this astrocyte cell line to maintain the BBB phenotype. Co-culture of the CTX-TNA2 cells with primary porcine brain endothelial cells produced an in vitro BBB model which retains key features of the in vivo BBB. High transendothelial electrical resistances, comparable to those reported in vivo, were obtained. Ultrastructural analysis revealed distinct intercellular tight junction protein complexes and immunocytochemistry confirmed expression of the tight junction proteins ZO-1 and occludin. Western blotting and fluorescent tracer assays confirmed expression and functional activity of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) efflux transporters. Studies employing Alexa-fluor 555-conjugated human transferrin revealed temperature-sensitive internalisation indicating the BBB model retains functional receptor-mediated transferrin uptake. The findings of this study indicate that a robust BBB model has been produced and this is the first report of the inductive capacity of the CTX-TNA2 cell line. Since this in vitro BBB model possesses many key characteristics of the BBB in vivo it has the potential to be a valuable tool for the study of biochemical and physiological processes associated with the BBB.