Integrated PK/PD Modeling Relates Smoothened Inhibitor Biomarkers to The Heterogeneous Intratumor Disposition of Cetuximab in Pancreatic Cancer Tumor Models.

Therapeutic antibodies have shown little efficacy in the treatment of pancreatic ductal adenocarcinomas (PDAC). Tumor desmoplasia, hypovascularity, and poor perfusion result in insufficient tumor cell exposure, contributing to treatment failure. Smoothened inhibitors of hedgehog signaling (sHHi) increase PDAC tumor permeability, perfusion, and drug delivery, and provide a tool to develop a quantitative, mechanistic understanding as to how the temporal dynamics of tumor priming can impact intratumor distribution of monoclonal antibodies (mAb). A linked pharmacokinetic (PK)/pharmacodynamic (PD) model was developed to integrate the plasma and tumor PK of a sHHi priming agent with its effects upon downstream stromal biomarkers Gli1, hyaluronic acid, and interstitial fluid pressure in PDAC patient-derived xenograft (PDX) tumors. In parallel, in situ tumor concentrations of cetuximab (CTX: anti-epidermal growth factor receptor; EGFR) were quantified as a marker for tumor delivery of mAb or antibody-drug conjugates. A minimal, physiologically-based pharmacokinetic (mPBPK) model was constructed to link sHHi effects upon mechanistic effectors of tumor barrier compromise with the intratumor distribution of CTX, and CTX occupancy of EGFR in tumors. Integration of the mPBPK model of mAb deposition and intratumor distribution with the PK/PD model of tumor responses to priming not only identified physiological parameters that are critical for tumor antibody distribution, but also provides insight into dosing regimens that could achieve maximal tumor disposition of therapeutic antibodies under conditions of transient PDAC tumor permeability barrier compromise that mechanistically-diverse tumor priming strategies may achieve.

SAR of L-ABBA analogs for GGT1 inhibitory activity and L-ABBA's effect on plasma cysteine and GSH species.

Gamma-glutamyl transferase 1 (GGT1) is a critical enzyme involved in the hydrolysis and/or transfer of gamma-glutamyl groups of glutathione, which helps maintain cysteine levels in plasma. In this study, we synthesized L-ABBA analogs to investigate their inhibitory effect on GGT1 hydrolysis and transpeptidase activity, with the goal of defining the pharmacophore of L-ABBA. Our structure-activity relationship (SAR) study revealed that an α-COO- and α-NH3+ group, as well as a two-CH2 unit distance between α-C and boronic acid, are essential for the activity. The addition of an R (alkyl) group at the α-C reduced the activity of GGT1 inhibition, with L-ABBA being the most potent inhibitor among the analogs. Next, we investigated the impact of L-ABBA on plasma levels of cysteine and GSH species, with the expectation of observing reduced cysteine levels and enhanced GSH levels due to its GGT1 inhibition. We administered L-ABBA intraperitoneally and determined the plasma levels of cysteine, cystine, GSH, and GSSG using LCMS. Our results showed time- and dose-dependent L-ABBA changes in total plasma cysteine and GSH levels. This study is the first to demonstrate the regulation of plasma thiol species upon GGT1 inhibition, with plasma cystine levels reduced by up to âˆ¼ 75 % with L-ABBA (0.3 mg/dose). Cancer cells are highly dependent on the uptake of cysteine from plasma for maintaining high levels of intracellular glutathione. Thus, our findings suggest that GGT1 inhibitors, such as L-ABBA, have the potential to be used in GSH reduction thereby inducing oxidative stress in cancer cells and reducing their resistance to many chemotherapeutic agents.

Defining the Intravital Renal Disposition of Fluorescence-Quenched Exenatide.

Despite the understanding that renal clearance is pivotal for driving the pharmacokinetics of numerous therapeutic proteins and peptides, the specific processes that occur following glomerular filtration remain poorly defined. For instance, sites of catabolism within the proximal tubule can occur at the brush border, within lysosomes following endocytosis, or even within the tubule lumen itself. The objective of the current study was to address these limitations and develop methodology to study the kidney disposition of a model therapeutic protein. Exenatide is a peptide used to treat type 2 diabetes mellitus. Glomerular filtration and ensuing renal catabolism have been shown to be its principal clearance pathway. Here, we designed and validated a Förster resonance energy transfer-quenched exenatide derivative to provide critical information on the renal handling of exenatide. A combination of in vitro techniques was used to confirm substantial fluorescence quenching of intact peptide that was released upon proteolytic cleavage. This evaluation was then followed by an assessment of the in vivo disposition of quenched exenatide directly within kidneys of living rats via intravital two-photon microscopy. Live imaging demonstrated rapid glomerular filtration and identified exenatide metabolism occurred within the subapical regions of the proximal tubule epithelia, with subsequent intracellular trafficking of cleaved fragments. These results provide a novel examination into the real-time, intravital disposition of a protein therapeutic within the kidney and offer a platform to build upon for future work.

Unconjugated p-cresol activates macrophage macropinocytosis leading to increased LDL uptake.

Patients with chronic kidney disease (CKD) and end-stage renal disease suffer from increased cardiovascular events and cardiac mortality. Prior studies have demonstrated that a portion of this enhanced risk can be attributed to the accumulation of microbiota-derived toxic metabolites, with most studies focusing on the sulfonated form of p-cresol (PCS). However, unconjugated p-cresol (uPC) itself was never assessed due to rapid and extensive first-pass metabolism that results in negligible serum concentrations of uPC. These reports thus failed to consider the host exposure to uPC prior to hepatic metabolism. In the current study, not only did we measure the effect of altering the intestinal microbiota on lipid accumulation in coronary arteries, but we also examined macrophage lipid uptake and handling pathways in response to uPC. We found that atherosclerosis-prone mice fed a high-fat diet exhibited significantly higher coronary artery lipid deposits upon receiving fecal material from CKD mice. Furthermore, treatment with uPC increased total cholesterol, triglycerides, and hepatic and aortic fatty deposits in non-CKD mice. Studies employing an in vitro macrophage model demonstrated that uPC exposure increased apoptosis whereas PCS did not. Additionally, uPC exhibited higher potency than PCS to stimulate LDL uptake and only uPC induced endocytosis- and pinocytosis-related genes. Pharmacological inhibition of varying cholesterol influx and efflux systems indicated that uPC increased macrophage LDL uptake by activating macropinocytosis. Overall, these findings indicate that uPC itself had a distinct effect on macrophage biology that might have contributed to increased cardiovascular risk in patients with CKD.

Comparison of the composition and in vitro activity of polymyxin B products.

A number of companies manufacture polymyxin B using United States Pharmacopeia (USP) metrics, rather than chemical composition, to report biological activity. Given that polymyxin B contains several different components, it is unknown whether pharmacokinetic and pharmacodynamic variability exists between the different brands and whether USP metrics capture this variability. Here we investigated the composition of polymyxin B obtained from four manufacturers (Sigma-Aldrich, AK Scientific, USP and MP Biomedicals) and evaluated their rate and extent of killing against multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae using in vitro static time-kill experiments. Ultraviolet (UV) fingerprinting and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed similarities and differences between component distributions. The significant differences between products, based on UV fingerprinting and LC-MS/MS, did not translate into pharmacodynamic differences at the three concentrations evaluated. The aggregate polymyxin B concentration, rather than that of the individual components, influences overall bacterial killing.

Simultaneous determination of the flavonoids robinin and kaempferol in human breast cancer cells by liquid chromatography-tandem mass spectrometry.

An accurate, precise and sensitive method was developed and validated for the simultaneous quantification of the flavonoid glycoside robinin, and its algycone kaempferol in human breast cancer MCF-7 cells. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C(18) column using a mobile phase consisting of (A) water with 0.025% formic acid and 1mM ammonium formate and (B) acetonitrile with 0.025% formic acid. Rutin was used as the internal standard for robinin and fisetin as the internal standard for kaempferol. The assay had a limit of detection of 0.1ng/ml for both compounds when present in cell lysate. The calibration curves were linear from 1 to 250ng/ml (r>0.999) for each compound. The intra- and inter-day coefficients of variation were less than 10% and intra- and inter-day accuracies were within 11%. This assay was successfully applied in a robinin cellular uptake study to determine the intracellular concentrations of robinin in MCF-7 cells.