Kelly Dong

Stability: recommendation for best practices and harmonization from the Global Bioanalysis Consortium Harmonization Team.

This paper provides a comprehensive overview of stability-related aspects of quantitative bioanalysis and recommends science-based best practices, covering small and large molecules as well as chromatographic and ligand-binding assays. It addresses general aspects, such as the use of reference values, transferability and treatment of failing stability results, and also focuses on specific types of stability assessment: bench-top, freeze/thaw and long-term frozen stability, stock stability, extract stability, stability in whole blood, tissue and urine, and stability of endogenous analytes, in special matrix types and in incurred samples.

In vitro, in vivo and in silico models of drug distribution into the brain

Achieving sufficient brain penetration to elicit efficacy in humans is one of the most challenging tasks for scientists in CNS Drug Discovery. Substantial progress has been made in the past decade in understanding the factors influencing the rate and extent of brain distribution via a variety of in vivo, in vitro and in silico methodologies, and hence, predict their likelihood of success in man. This purpose of this review is to summarize the current approaches with a special focus on parameters related to free drug concentrations in brain which are the most pharmacologically relevant for the majority of CNS disease targets. Due to the dynamic and complex nature of this targeted organ, it is inevitable that these approaches have not been able to provide a fully comprehensive assessment of brain distribution and are expected to evolve further in the years to come.

2-Aminopyrimidin-4(1H)-one as the novel bioisostere of urea: Discovery of novel and potent CXCR2 antagonists

2-Aminopyrimidin-4(1H)-one was proposed as the novel bioisostere of urea. Bioisosteric replacement of the reported urea series of the CXCR2 antagonists with 2-aminopyrimidin-4(1H)-ones led to the discovery of the novel and potent CXCR2 antagonist 3e. 2-Aminopyrimidin-4(1H)-one derivative 3e demonstrated a good developability profile (reasonable solubility and high permeability) and superior chemical stability especially in simulated gastric fluid (SGF) compared with ureas.

Correlation Between Membrane Protein Expression Levels and Transcellular Transport Activity for Breast Cancer Resistance Protein

Emerging evidence indicates an important role for the breast cancer resistance protein (BCRP) in limiting brain penetration of substrate drugs. While in vitro transwell assays can provide an indication of BCRP substrate potential, the predictability of these assays in relation to in vivo brain penetration is still under debate. The present study examined the correlation of BCRP membrane protein expression level and transcellular transport activity across Madin-Darby canine kidney (MDCK) II monolayers. We expressed human BCRP or murine BCRP1 in MDCKII wild-type cells using BacMam2 virus transduction. The selective P-glycoprotein (P-gp) inhibitor LY335979 (1 μM) was included in the transport medium to measure BCRP-mediated transcellular transport for P-gp and BCRP cosubstrates. The BCRP levels in membrane extracts from MDCKII-BCRP or MDCKII-Bcrp1 cells were quantified by liquid chromatography–tandem mass spectrometry. The results are summarized as follows: 1) the membrane protein expression levels correlate with the corrected efflux ratios of substrates for human BCRP and murine BCRP1 within the efflux ratios investigated; 2) we demonstrate good concordance in rank order between the BCRP and BCRP1-mediated efflux ratios for 12 drugs; and 3) we propose an approach to contextualize in vitro BCRP transport data of discovery compounds by comparing them to the in vitro and in vivo transport data of the reference drug dantrolene and taking into account interbatch variation in BCRP expression. This approach correctly predicted compromised brain penetration for 25 discovery compounds in rodents, which were BCRP substrates but not P-gp or weak P-gp substrates. These results suggest that BCRP-expressing MDCKII cells are useful in predicting the in vivo role of BCRP in brain penetration.

Discovery of 5-substituent-N-arylbenzamide derivatives as potent, selective and orally bioavailable LRRK2 inhibitors

Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic target for Parkinsons disease. Herein we report the discovery of 5-substituent-N-arylbenzamide derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds 8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome, good brain exposure, and high oral bioavailability.

Drug Distribution into Peripheral Nerve

Little is known about the impact of the blood–nerve barrier (BNB) on drug distribution into peripheral nerves. In this study, we examined the peripheral nerve penetration in rats of 11 small-molecule drugs possessing diverse physicochemical and transport properties and ProTx-II, a tarantula venom peptide with molecular mass of 3826 Daltons. Each drug was administered as constant rate intravenous infusion for 6 hours (small molecules) or 24 hours (ProTx-II). Blood and tissues including brain, spinal cord, sciatic nerve, and dorsal root ganglion (DRG) were collected for drug concentration measurements. Unbound fractions of a set of compounds were determined by equilibrium dialysis method in rat blood, brains, spinal cords, sciatic nerves, and DRG. We also investigated the influence of N-[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10H-acridine-4-carboxamide (GF120918), a P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) inhibitor, on the peripheral nerve and central nervous system (CNS) tissue penetration of imatinib. We found that: 1) the unbound fraction in brain tissue homogenate highly correlates with that in the spinal cord, sciatic nerve, and DRG for a set of compounds and thus provides a good surrogate for spinal cord and peripheral nerve tissues, 2) small-molecule drugs investigated can penetrate the DRG and sciatic nerve, 3) P-gp and BCRP have a limited impact on the distribution of small-molecule drugs into peripheral nerves, and 4) DRG is permeable to ProTx-II, but its distribution into sciatic nerve and CNS tissues is restricted. These results demonstrate that small-molecule drugs investigated can penetrate peripheral nerve tissues, and P-gp/BCRP may not be a limiting factor at the BNB. Biologics as large as ProTx-II can access the DRG but not sciatic nerve and CNS tissues.

Discovery of CNS Penetrant CXCR2 Antagonists for the Potential Treatment of CNS Demyelinating Disorders.

Structure-activity relationship exploration of the historical biarylurea series led to the identification of novel CNS penetrant CXCR2 antagonists with nanomolar potency, favorable PK profile, and good developability potentials. More importantly, the key compound 22 showed efficacy in a cuprizone-induced demyelination model with twice daily oral administration, thereby supporting CXCR2 to be a potential therapeutic target for the treatment of demyelinating diseases such as multiple sclerosis.

Quantitative Algorithm-Based Paired Imaging Measurement for Antibody-Triggered Endocytosis in Cultured Cells

Antibody-triggered endocytosis (ATE) is a biological mechanism on which many therapeutic strategies are grounded, such as delivery of antibody–drug conjugates (ADCs). Current methods monitoring ATE include confocal Z-stack analysis, acid wash, antibody quenching, and pH-sensitive dye labeling. However, those generate less quantifiable results with low throughput. Here we report a new method referred to as “paired imaging measurement” to analyze ATE using a quantitative algorithm in conjunction with high-content imaging. With two sequential measurements of cell surface antibody employing live cell staining and total antibody by immunostaining before and after cell permeabilization, intracellular antibody undergoing endocytosis can be quantified indirectly. Antibodies against CD98 and transferrin receptor were tested on hCMEC/D3 and hiPSC-derived endothelial cells. The maximal response and potency of endocytosed antibodies were generated with good assay robustness (Z′ > 0.6) and >5-fold signal/background ratio. Antibody endocytosis response ranking is consistent between batches (R2 > 0.9). The obtained results were confirmed by other traditional methods. In conclusion, we have developed a novel method using a quantitative imaging algorithm in conjunction with live cell staining for high-throughput investigation of ATE.

Discovery of 4-ethoxy-7 H -pyrrolo[2,3- d ]pyrimidin-2-amines as potent, selective and orally bioavailable LRRK2 inhibitors

Inhibition of LRRK2 kinase activity with small molecules has emerged as a potential novel therapeutic treatment for Parkinsons disease. Herein we disclose the discovery of a 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amine series as potent LRRK2 inhibitors identified through a kinase-focused set screening. Optimization of the physicochemical properties and kinase selectivity led to the discovery of compound 7, which exhibited potent in vitro inhibition of LRRK2 kinase activity, good physicochemical properties and kinase selectivity across the kinome. Moreover, compound 7 was able to penetrate into the CNS, and in vivo pharmacology studies revealed significant inhibition of Ser935 phosphorylation in the brain of both rats (30 and 100 mg/kg) and mice (45 mg/kg) following oral administration.