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.

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.

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aβ in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aβ EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a β-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aβ in guinea pigs. The therapeutic index between Aβ reductions and changes in B-cell populations were studied for compound 10 h.

Many forms of synaptic plasticity are critically dependent upon production of cGMP to trigger activity-dependent increases in synaptic size and strength. Phosphodiesterase 9A (PDE9A) is a high affinity, cGMP-specific phosphodiesterase with widespread distribution in the central nervous system. Inhibition of PDE9A results in significant accumulation of cGMP in brain tissue and CSF of rodents and also increases CSF cGMP in human volunteers (Schmidt et al., 2009 ICAD; Nicholas et al 2009 ICAD). We hypothesize that chronic exposure to a PDE9 inhibitor, and the associated elevations in brain cGMP could provide a therapeutic benefit to vulnerable synapses chronically exposed to Abeta in transgenic amyloid overexpressing mice. A total of N = 20 animals per group of 4 month old Tg2576+ and non-transgenic animals were implanted with Alzet osmotic minipumps to deliver vehicle or the PDE9A inhibitor PF-4447943, currently in clinical trials for the treatment of mild to moderate Alzheimer's Disease. Neurobehavioral outcomes were measured as conditioned fear response after 30 days of treatment and subsequently the brains were harvested for measurement of Abeta, gene expression profiling or synaptic density as assessed by Golgi staining of dendrites. Dendritic spine density on apical dendrites of CA1 neurons exhibited a small but significant deficit in the density of dendritic spines in vehicle treated transgenic animals as compared to non-transgenic animals. This deficit was ameliorated by 30 days of exposure to PF-04447943. No significant drug effect was observed in the non-transgenic animals. No significant effects of drug treatment were observed on Abeta levels in Tg2576 mice. Behavioral analysis of transgenic animals showed deficits in fear conditioning as early as 2 months of age, and therefore were considered unlikely to be due to the accumulation of oligomeric Abeta. These deficits were not affected by drug treatment. Transcriptional profiles of animals treated with drug compared to vehicle showed evidence of regulation of pathways related to synaptic plasticity and remodeling of the dendritic cytoskeleton, consistent with stabilization of vulnerable spine structure. This data supports the hypothesis that PDE9 inhibition can stabilize vulnerable synapses early in the Alzheimer's disease process.

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel gamma-secretase inhibitor that reduces amyloid-beta (Abeta) production with an in vitro IC(50) of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC(50) of 2.1 microM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Abeta in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Abeta. To further characterize Abeta dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Abeta, and the magnitude and duration of Abeta lowering exceeded those of the reductions in B-cell endpoints. Other gamma-secretase inhibitors have shown high potency at elevating Abeta in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Abeta11-40 and Abeta1-43 at doses that potently inhibited Abeta1-40 and Abeta1-42. PF-3084014, like previously described gamma-secretase inhibitors, preferentially reduced Abeta1-40 relative to Abeta1-42. Potency at Abeta relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.

Reducing levels of Aβ, the principal component of amyloid plaques, has been proposed as a disease-modifying approach for the treatment of Alzheimer's disease. Here we report the efficacy and exposure over time of PF-3084014, a novel potent γ-secretase inhibitor. PF-3084014 was dosed at 0.03–10 mg/kg subcutaneously in guinea pigs at multiple time points ranging from 0.75 to 30 hours. Aβ1-X was measured in brain, CSF, and plasma at each dose and time point, and PF-3084014 levels were measured in brain and plasma at selected time points. Dose-response relationships were observed in all compartments. At 10 mg/kg, Aβ levels were reduced by 70% in brain and plasma, and 50% in CSF, which was maintained at 30 hours post-dose. At all time points assessed, a linear relationship was observed between efficacy and exposure. PF-3084014 exhibited a brain-to-plasma ratio of approximately 1, and brain and plasma Aβ levels were reduced to a similar degree. Other γ-secretase inhibitors have been shown to strongly elevate plasma Aβ at low doses or in a rebound following a period of inhibition in guinea pigs; however, this pattern was not observed with PF-3084014. To further determine changes in specific Aβ isoforms, PF-3084014 was administered to young (plaque-free) Tg2576 mice. Brain, CSF, and plasma were harvested following PF-3084014 dosing at 1–18 mg/kg. In mice, a lower brain-to-plasma ratio correlated with greater Aβ reductions in plasma versus brain. Aβ1-X, Aβ1–40, and Aβ1–42 were reduced in a dose-responsive manner; at 18 mg/kg Aβ levels were reduced by 78% in brain, 72% in CSF, and 92% in plasma. As in guinea pigs, a linear efficacy-exposure relationship across doses was observed in mice. Aβ1–40 was most potently inhibited in all compartments, followed closely by Aβ1-X. Aβ1–42 showed approximately 20% less reduction than Aβ1–40 in all compartments, a feature shared by other published “nonselective” γ-secretase inhibitors. PF-3084014 effectively reduces Aβ levels in brain, plasma, and CSF, and exhibits a linear efficacy-exposure relationship across doses.

Postoperative cognitive dysfunction, confusion, and delirium are common after general anesthesia in the elderly, with symptoms persisting for months or years in some patients. Even middle-aged patients are likely to have postoperative cognitive dysfunction for months after surgery, and Alzheimer's disease (AD) patients appear to be particularly at risk of deterioration after anesthesia. Several investigators have thus examined whether general anesthesia is associated with AD, with some studies suggesting that exposure to anesthetics may increase the risk of AD. However, little is known on the biochemical consequences of anesthesia on pathogenic pathways in vivo. Here, we investigated the effect of anesthesia on tau phosphorylation and amyloid precursor protein (APP) metabolism in mouse brain. We found that, regardless of the anesthetic used, anesthesia induced rapid and massive hyperphosphorylation of tau, rapid and prolonged hypothermia, inhibition of Ser/Thr PP2A (protein phosphatase 2A), but no changes in APP metabolism or Abeta (beta-amyloid peptide) accumulation. Reestablishing normothermia during anesthesia completely rescued tau phosphorylation to normal levels. Our results indicate that changes in tau phosphorylation were not a result of anesthesia per se, but a consequence of anesthesia-induced hypothermia, which led to inhibition of phosphatase activity and subsequent hyperphosphorylation of tau. These findings call for careful monitoring of core temperature during anesthesia in laboratory animals to avoid artifactual elevation of protein phosphorylation. Furthermore, a thorough examination of the effect of anesthesia-induced hypothermia on the risk and progression of AD is warranted.

LY-450139 is a gamma-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-beta (Abeta) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate Abeta responses to LY-450139 in the guinea pig, a nontransgenic model that has an Abeta sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma Abeta levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma Abeta levels at early time points, with return to baseline within hours. Higher doses inhibited Abeta levels in all compartments at early time points, but elevated plasma Abeta levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma Abeta was significantly inhibited at 10-30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of Abeta elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted Abeta were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the gamma-secretase complex, eliciting Abeta lowering at high concentrations but Abeta elevation at low concentrations.

ABSTRACT Analysis of additions to reserves compared to cumulative exploratory well completions since the beginning of the U. S. petroleum industry clearly exhibits a close correlation between maximum exploratory effectiveness and applied technology. Surges of increased exploration results can be readily identified as those periods of optimum application of surface geology, reconnaissance subsurface and geophysical techniques, and prospect definition by intensive refraction and reflection seismic methods. The domestic petroleum industry can be optimistic about the opportunities for future effective exploration. Current technology is well advanced. Continuous refinements have improved its definitive capabilities and broadened its opportunity for application. Additional technological development can be expected from the heavy expenditures on research and development currently being made by the petroleum industry, as well as associated industries and the Federal Government. There should be no concern about the opportunities to apply this technology when the volume of unexplored sedimentary section of the United States is considered. Tremendous opportunities for new reserves lie in the 350,000 square miles of potential and almost totally unexplored continental shelf surrounding the North American continent. The exploration professions should place more emphasis on how to apply existing and future technology to generate a maximum of exploratory opportunity. This will require conceptual adjustments in exploration approach. Exploration staffs must have available full technical capability of all the skills and talents which can contribute to more effective exploration. Well-conceived programs must utilize each of these professional skills to the extent it can contribute, as opposed to the frequent practice of attempting to employ one technique beyond its capability and failing to utilize others which could make significant contributions. Organization must be compatible with optimum developments of technical capability and provide sufficient flexibility to bring the maximum of talents and judgment to bear in the development of concepts and formulation of programs. Management must confidently support the application of technology in the discovery of new reserves. They must be knowledgable of the inherent risk in exploration and willing to include in their planning sufficient financial support to provide for adequate exposure to exploration opportunities to offset this inherent risk and take maximum advantage of applied technology. End_of_Record - Last_Page 1---------