Microphysiological systems in early stage drug development: Perspectives on current applications and future impact.

Microphysiological systems (MPS) are making advances to provide more standardized and predictive physiologically relevant responses to test articles in living tissues and organ systems. The excitement surrounding the potential of MPS to better predict human responses to medicines and improving clinical translation is overshadowed by their relatively slow adoption by the pharmaceutical industry and regulators. Collaboration between multiorganizational consortia and regulators is necessary to build an understanding of the strengths and limitations of MPS models and closing the current gaps. Here, we review some of the advances in MPS research, focusing on liver, intestine, vascular system, kidney and lung and present examples highlighting the context of use for these systems. For MPS to gain a foothold in drug development, they must have added value over existing approaches. Ideally, the application of MPS will augment in vivo studies and reduce the use of animals via tiered screening with less reliance on exploratory toxicology studies to screen compounds. Because MPS support multiple cell types (e.g. primary or stem-cell derived cells) and organ systems, identifying when MPS are more appropriate than simple 2D in vitro models for understanding physiological responses to test articles is necessary. Once identified, MPS models require qualification for that specific context of use and must be reproducible to allow future validation. Ultimately, the challenges of balancing complexity with reproducibility will inform the promise of advancing the MPS field and are critical for realization of the goal to reduce, refine and replace (3Rs) the use of animals in nonclinical research.

An in vitro alveolar epithelial cell model recapitulates LRRK2 inhibitor-induced increases in lamellar body size observed in preclinical models.

Alveolar type II (ATII) epithelial cells contain lamellar bodies (LBs) which synthesize and store lung surfactants. In animals, the inhibition or knockout of leucine-rich repeat kinase 2 (LRRK2) causes abnormal enlargement of LBs in ATII cells. This effect of LRRK2 inhibition in lung is largely accepted as being mediated directly through blocking of the kinase function; however, downstream consequences in the lung remain unknown. In this work we established an in vitro alveolar epithelial cell (AEC) model that recapitulates the in vivo phenotype of ATII cells and developed an assay to quantify changes in LB size in response to LRRK2 inhibitors. Culture of primary human AECs at the air-liquid interface on matrigel and collagen-coated transwell inserts in the presence of growth factors promoted the LB formation and apical microvilli and induced expression of LRRK2 and ATII cell markers. Treatment with a selective LRRK2 inhibitor resulted in pharmacological reduction of phospho-LRRK2 and a significant increase in LB size; effects previously reported in lungs of non-human primates treated with LRRK2 inhibitor. In summary, our human in vitro AEC model recapitulates the abnormal lung findings observed in LRRK2-perturbed animals and holds the potential for expanding current understanding of LRRK2 function in the lung.

Single nucleotide polymorphisms in the FcγR3A and TAP1 genes impact ADCC in cynomolgus monkey PBMCs.

Phenotypic variability is often observed in cynomolgus monkeys on preclinical studies and may, in part, be driven by genetic variability. However, the role of monkey genetic variation remains largely unexplored in the context of drug response. This study evaluated genetic variation in cynomolgus monkey FcγR3A and TAP1 genes and the potential impact of identified polymorphisms on antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Studies in humans have demonstrated that a single nucleotide polymorphism (SNP), F158V, in FcγR3A can influence response to rituximab through altered ADCC and that SNPs in TAP1/2 decrease natural killer (NK) cell activity against major histocompatibility complex (MHC) class I deficient cells, potentially through altered ADCC. Monkeys were genotyped for FcγR3A and TAP1 SNPs, and ADCC was assessed in vitro using peripheral blood mononuclear cells (PBMCs) treated with trastuzumab in the presence of NCI-N87 cells. FcγR3A g.1134A>C (exonic S42R), FcγR3A g.5027A>G (intronic), and TAP1 g.1A>G (start codon loss) SNPs were all significantly associated with decreased ADCC for at least one trastuzumab concentration ≥0.0001 µM when compared with wild type (WT). Regression analysis demonstrated significant association of the SNP-SNP pairs FcγR3A g.1134A>C/TAP1 g.1A>G and FcγR3A g.5027A>G/TAP1 g.1A>G with a combinatorial decrease on ADCC. Mechanisms underlying the decreased ADCC were investigated by measuring FcγR3A/IgG binding affinity and expression of FcγR3A and TAP1 in PBMCs; however, no functional associations were observed. These data demonstrate that genetic variation in cynomolgus monkeys is reflective of known human genetic variation and may potentially contribute to variable drug response in preclinical studies.

Functional interaction between the Fanconi Anemia D2 protein and proliferating cell nuclear antigen (PCNA) via a conserved putative PCNA interaction motif.

Fanconi Anemia (FA) is a rare recessive disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The FA proteins and the familial breast cancer susceptibility gene products, BRCA1 and FANCD1/BRCA2, function cooperatively in the FA-BRCA pathway to repair damaged DNA and to prevent cellular transformation. Activation of this pathway occurs via the mono-ubiquitination of the FANCD2 protein, targeting it to nuclear foci where it co-localizes with FANCD1/BRCA2, RAD51, and PCNA. The regulation of the mono-ubiquitination of FANCD2, as well as its function in DNA repair remain poorly understood. In this study, we have further characterized the interaction between the FANCD2 and PCNA proteins. We have identified a highly conserved, putative FANCD2 PCNA interaction motif (PIP-box), and demonstrate that mutation of this motif disrupts FANCD2-PCNA binding and precludes the mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant protein fails to correct the mitomycin C hypersensitivity of FA-D2 patient cells. Our results suggest that PCNA may function as a molecular platform to facilitate the mono-ubiquitination of FANCD2 and activation of the FA-BRCA pathway.

Insulin-like stimulation of cardiac fuel metabolism by physiological levels of glucagon: involvement of PI3K but not cAMP.

At concentrations around 10(-9) M or higher, glucagon increases cardiac contractility by activating adenylate cyclase/cyclic adenosine monophosphate (AC/cAMP). However, blood levels in vivo, in rats or humans, rarely exceed 10(-10) M. We investigated whether physiological concentrations of glucagon, not sufficient to increase contractility or ventricular cAMP levels, can influence fuel metabolism in perfused working rat hearts. Two distinct glucagon dose-response curves emerged. One was an expected increase in left ventricular pressure (LVP) occurring between 10(-9.5) and 10(-8) M. The elevations in both LVP and ventricular cAMP levels produced by the maximal concentration (10(-8) M) were blocked by the AC inhibitor NKY80 (20 microM). The other curve, generated at much lower glucagon concentrations and overlapping normal blood levels (10(-11) to 10(-10) M), consisted of a dose-dependent and marked stimulation of glycolysis with no change in LVP. In addition to stimulating glycolysis, glucagon (10(-10) M) also increased glucose oxidation and suppressed palmitate oxidation, mimicking known effects of insulin, without altering ventricular cAMP levels. Elevations in glycolytic flux produced by either glucagon (10(-10) M) or insulin (4 x 10(-10) M) were abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (10 microM) but not significantly affected by NKY80. Glucagon also, like insulin, enhanced the phosphorylation of Akt/PKB, a downstream target of PI3K, and these effects were also abolished by LY-294002. The results are consistent with the hypothesis that physiological levels of glucagon produce insulin-like increases in cardiac glucose utilization in vivo through activation of PI3K and not AC/cAMP.