Comprehensive stability analysis of 13 ß-lactams and ß-lactamase inhibitors in in vitro media, and novel supplement dosing strategy to mitigate thermal drug degradation.

Non-clinical antibiotic development relies on in vitro susceptibility and infection model studies. Validating the achievement of the targeted drug concentrations is essential to avoid under-estimation of drug effects and over-estimation of resistance emergence. While certain ß-lactams (e.g., imipenem) and ß-lactamase inhibitors (BLIs; clavulanic acid) are believed to be relatively unstable, limited tangible data on their stability in commonly used in vitro media are known. We aimed to determine the thermal stability of 10 ß-lactams and 3 BLIs via LC-MS/MS in cation-adjusted Mueller Hinton broth at 25 and 36°C as well as agar at 4 and 37°C, and in water at -20, 4, and 25°C. Supplement dosing algorithms were developed to achieve broth concentrations close to their target over 24 h. During incubation in broth (pH 7.25)/agar, degradation half-lives were 16.9/21.8 h for imipenem, 20.7/31.6 h for biapenem, 29.0 h for clavulanic acid (studied in broth only), 23.1/71.6 h for cefsulodin, 40.6/57.9 h for doripenem, 46.5/64.6 h for meropenem, 50.8/97.7 h for cefepime, 61.5/99.5 h for piperacillin, and >120 h for all other compounds. Broth stability decreased at higher pH. All drugs were ≥90% stable for 72 h in agar at 4°C. Degradation half-lives in water at 25°C were >200 h for all drugs except imipenem (14.7 h, at 1,000 mg/L) and doripenem (59.5 h). One imipenem supplement dose allowed concentrations to stay within ±31% of their target concentration. This study provides comprehensive stability data on ß-lactams and BLIs in relevant in vitro media using LC-MS/MS. Future studies are warranted applying these data to antimicrobial susceptibility testing and assessing the impact of ß-lactamase-related degradation.

Improved characterization of aminoglycoside penetration into human lung epithelial lining fluid via population pharmacokinetics.

Aminoglycosides are important treatment options for serious lung infections, but modeling analyses to quantify their human lung epithelial lining fluid (ELF) penetration are lacking. We estimated the extent and rate of penetration for five aminoglycosides via population pharmacokinetics from eight published studies. The area under the curve in ELF vs plasma ranged from 50% to 100% and equilibration half-lives from 0.61 to 5.80 h, indicating extensive system hysteresis. Aminoglycoside ELF peak concentrations were blunted, but overall exposures were moderately high.

KIFC1: A Reliable Prognostic Biomarker in Rb-positive Triple-negative Breast Cancer Patients Treated With Doxorubicin in Combination With Abemaciclib.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) prevalence and risk of relapse are greatest in African American (AA) patients. Doxorubicin (DOX) and abemaciclib (ABE) synergism in Rb-positive TNBC cells (MDA-MB-231), and antagonism in Rb-negative TNBC cells (MDA-MB-468) have been previously shown. Here, we assessed Kinesin-like protein 1 (KIFC1) as an ethnic-specific prognostic biomarker of the DOX+ABE combination for the Rb-status in TNBC. MATERIALS AND METHODS: Literature search for TNBC prognostic biomarkers in the AA population was conducted. MDA-MB-231 and MDA-MB-468 cells were exposed over 72 h to four treatment arms: 1) control (medium without drug), 2) DOX at 50% inhibitory concentration in MDA-MB-231 (0.565 µM) and MDA-MB-468 (0.121 µM), 3) ABE alone (2 µM), and 4) DOX+ABE combination at their corresponding concentrations in each cell-line. KIFC1 protein expression and temporal changes were quantified in MDA-MB-231 cells using western blot. RESULTS: KIFC1, Kaiso, and Annexin A2 are literature-identified AA-specific TNBC prognostic biomarkers. KIFC1 was found to be uncorrelated to other proposed biomarkers, suggesting it may predict risk independently of other TNBC biomarkers. In both cell lines, DOX alone did not significantly change KIFC1 expression relative to control. Conversely, ABE reduced KIFC1 expression in MDA-MB-231 but not in MDA-MB-468 cells. The combination DOX+ABE resulted in a greatest reduction in KIFC1 in MDA-MB-231 cells with a more rapid time-to-full inhibition of KIFC1 compared to ABE alone. CONCLUSION: Change in KIFC1 expression is primarily driven by ABE in Rb-positive TNBC cells. DOX increases ABE speed to achieve a full inhibition of KIFC1 in Rb-positive, yet, without influencing its expression in Rb-negative TNBC cells.

In vitro to Clinical Translation of Combinatorial Effects of Doxorubicin and Abemaciclib in Rb-Positive Triple Negative Breast Cancer: A Systems-Based Pharmacokinetic/Pharmacodynamic Modeling Approach.

BACKGROUND: Doxorubicin (DOX) and its pegylated liposomal formulation (L_DOX) are the standard of care for triple-negative breast cancer (TNBC). However, resistance to DOX often occurs, motivating the search for alternative treatment approaches. The retinoblastoma protein (Rb) is a potential pharmacological target for TNBC treatment since its expression has been associated with resistance to DOX-based therapy. METHODS: DOX (0.01-20 µM) combination with abemaciclib (ABE, 1-6 µM) was evaluated over 72 hours on Rb-positive (MDA-MB-231) and Rb-negative (MDA-MB-468) TNBC cells. Combination indices (CI) for DOX+ABE were calculated using Compusyn software. The TNBC cell viability time-course and fold-change from the control of phosphorylated-Rb (pRb) protein expression were measured with CCK8-kit and enzyme-linked immunosorbent assay. A cell-based pharmacodynamic (PD) model was developed, where pRb protein dynamics drove cell viability response. Clinical pharmacokinetic (PK) models for DOX, L_DOX, and ABE were developed using data extracted from the literature. After scaling cancer cell growth to clinical TNBC tumor growth, the time-to-tumor progression (TTP) was predicted for human dosing regimens of DOX, ABE, and DOX+ABE. RESULTS: DOX and ABE combinations were synergistic (CI<1) in MDA-MB-231 and antagonistic (CI>1) in MDA-MB-468. The maximum inhibitory effects (Imax) for both drugs were set to one. The drug concentrations producing 50% of Imax for DOX and ABE were 0.565 and 2.31 µM (MDA-MB-231) and 0.121 and 1.61 µM (MDA-MB-468). The first-orders rate constants of abemaciclib absorption (ka) and doxorubicin release from L_DOX (kRel) were estimated at 0.31 and 0.013 h-1. Their linear clearances were 21.7 (ABE) and 32.1 L/h (DOX). The estimated TTP for intravenous DOX (75 mg/m2 every 21 days), intravenous L_DOX (50 mg/m2 every 28 days), and oral ABE (200 mg twice a day) were 125, 31.2, and 8.6 days shorter than drug-free control. The TTP for DOX+ABE and L_DOX+ABE were 312 days and 47.5 days shorter than control, both larger than single-agent DOX, suggesting improved activity with the DOX+ABE combination. CONCLUSION: The developed translational systems-based PK/PD model provides an in vitro-to-clinic modeling platform for DOX+ABE in TNBC. Although model-based simulations suggest improved outcomes with combination over monotherapy, tumor relapse was not prevented with the combination. Hence, DOX+ABE may not be an effective treatment combination for TNBC.

Combating Multidrug-Resistant Bacteria by Integrating a Novel Target Site Penetration and Receptor Binding Assay Platform Into Translational Modeling.

Multidrug-resistant bacteria are causing a serious global health crisis. A dramatic decline in antibiotic discovery and development investment by pharmaceutical industry over the last decades has slowed the adoption of new technologies. It is imperative that we create new mechanistic insights based on latest technologies, and use translational strategies to optimize patient therapy. Although drug development has relied on minimal inhibitory concentration testing and established in vitro and mouse infection models, the limited understanding of outer membrane permeability in Gram-negative bacteria presents major challenges. Our team has developed a platform using the latest technologies to characterize target site penetration and receptor binding in intact bacteria that inform translational modeling and guide new discovery. Enhanced assays can quantify the outer membrane permeability of ß-lactam antibiotics and ß-lactamase inhibitors using multiplex liquid chromatography tandem mass spectrometry. While ß-lactam antibiotics are known to bind to multiple different penicillin-binding proteins (PBPs), their binding profiles are almost always studied in lysed bacteria. Novel assays for PBP binding in the periplasm of intact bacteria were developed and proteins identified via proteomics. To characterize bacterial morphology changes in response to PBP binding, high-throughput flow cytometry and time-lapse confocal microscopy with fluorescent probes provide unprecedented mechanistic insights. Moreover, novel assays to quantify cytosolic receptor binding and intracellular drug concentrations inform target site occupancy. These mechanistic data are integrated by quantitative and systems pharmacology modeling to maximize bacterial killing and minimize resistance in in vitro and mouse infection models. This translational approach holds promise to identify antibiotic combination dosing strategies for patients with serious infections.

First Penicillin-Binding Protein Occupancy Patterns for 15 ß-Lactams and ß-Lactamase Inhibitors in Mycobacterium abscessus.

Mycobacterium abscessus causes serious infections that often require over 18 months of antibiotic combination therapy. There is no standard regimen for the treatment of M. abscessus infections, and the multitude of combinations that have been used clinically have had low success rates and high rates of toxicities. With ß-lactam antibiotics being safe, double ß-lactam and ß-lactam/ß-lactamase inhibitor combinations are of interest for improving the treatment of M. abscessus infections and minimizing toxicity. However, a mechanistic approach for building these combinations is lacking since little is known about which penicillin-binding protein (PBP) target receptors are inactivated by different ß-lactams in M. abscessus We determined the preferred PBP targets of 13 ß-lactams and 2 ß-lactamase inhibitors in two M. abscessus strains and identified PBP sequences by proteomics. The Bocillin FL binding assay was used to determine the ß-lactam concentrations that half-maximally inhibited Bocillin binding (50% inhibitory concentrations [IC50s]). Principal component analysis identified four clusters of PBP occupancy patterns. Carbapenems inactivated all PBPs at low concentrations (0.016 to 0.5 mg/liter) (cluster 1). Cephalosporins (cluster 2) inactivated PonA2, PonA1, and PbpA at low (0.031 to 1 mg/liter) (ceftriaxone and cefotaxime) or intermediate (0.35 to 16 mg/liter) (ceftazidime and cefoxitin) concentrations. Sulbactam, aztreonam, carumonam, mecillinam, and avibactam (cluster 3) inactivated the same PBPs as cephalosporins but required higher concentrations. Other penicillins (cluster 4) specifically targeted PbpA at 2 to 16 mg/liter. Carbapenems, ceftriaxone, and cefotaxime were the most promising ß-lactams since they inactivated most or all PBPs at clinically relevant concentrations. These first PBP occupancy patterns in M. abscessus provide a mechanistic foundation for selecting and optimizing safe and effective combination therapies with ß-lactams.

Chloroquine sensitizes MDA-MB-231 cells to osimertinib through autophagy-apoptosis crosstalk pathway.

Background: Triple-negative breast cancer (TNBC) is a breast cancer that tests negative for estrogen receptor (ER), progesterone receptors, and human epidermal growth factor receptors 2 (HER2). It is aggressive and invasive in nature and lacks targeted therapy. Purpose: The EGFR is frequently overexpressed in TNBC, and the EGFR-overexpressing TNBC presumably escapes EGFR inhibitor therapy by upregulating autophagy and inhibiting apoptosis. Methods: To parse the autophagy-apoptosis crosstalk pathway as a potential targeted therapy in TNBC, the activity of an EGFR inhibitor, osimertinib, alone and in combination with an autophagy inhibitor, chloroquine, was examined in EGFR-overexpressing TNBC cell line, MDA-MB-231. The nature of interaction between both drugs at various concentrations was determined by calculating combination indexes (CI) using CompuSyn software. Temporal changes in the expression of the autophagy marker, LC3B-II, and several apoptosis signaling molecules were measured using Western blot and luminex assay with MAGPIX® after exposure to drugs. A synergistic interaction (CI <1) was identified with combinations of 4-6.5 µM osimertinib with 30-75 µM chloroquine. Results: A combination of osimertinib (6 µM) with chloroquine (30 µM) resulted in a 6-fold increase of LC3B-II relative to control compared to 2.5-fold increase for either drug alone. The caspase-3 expression increased 2-fold compared to a 0.5-fold decrease with chloroquine and 1.5-fold increase with osimertinib. Conclusion: Our results indicate that inhibition of the autophagic flux via chloroquine improves the effectiveness of osimertinib in TNBC cancer cells, warranting further investigations of this combination in vivo.