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In the kidney, glucose in glomerular filtrate is reabsorbed primarily by sodium-glucose cotransporters 1 (SGLT1) and 2 (SGLT2) along the proximal tubules. SGLT2 has been characterized as a high capacity, low affinity pathway responsible for reabsorption of the majority of filtered glucose in the early part of proximal tubules, and SGLT1 reabsorbs the residual glucose in the distal part. Inhibition of SGLT2 is a viable mechanism for removing glucose from the body and improving glycemic control in patients with diabetes. Despite demonstrating high levels (in excess of 80%) of inhibition of glucose transport by SGLT2 in vitro, potent SGLT2 inhibitors, e.g., dapagliflozin and canagliflozin, inhibit renal glucose reabsorption by only 30-50% in clinical studies. Hypotheses for this apparent paradox are mostly focused on the compensatory effect of SGLT1. The paradox has been explained and the role of SGLT1 demonstrated in the mouse, but direct data in humans are lacking. To further explore the roles of SGLT1/2 in renal glucose reabsorption in humans, we developed a systems pharmacology model with emphasis on SGLT1/2 mediated glucose reabsorption and the effects of SGLT2 inhibition. The model was calibrated using robust clinical data in the absence or presence of dapagliflozin (DeFronzo et al., 2013), and evaluated against clinical data from the literature (Mogensen, 1971; Wolf et al., 2009; Polidori et al., 2013). The model adequately described all four data sets. Simulations using the model clarified the operating characteristics of SGLT1/2 in humans in the healthy and diabetic state with or without SGLT2 inhibition. The modeling and simulations support our proposition that the apparent moderate, 30-50% inhibition of renal glucose reabsorption observed with potent SGLT2 inhibitors is a combined result of two physiological determinants: SGLT1 compensation and residual SGLT2 activity. This model will enable in silico inferences and predictions related to SGLT1/2 modulation.

The empirical hypothesis generation and testing approach to pharmaceutical research and development (R&D), and biomedical research has proven very effective over the last half-century; resulting in tremendous increases productivity and the rates of approval for new drug applications at the Food and Drug Administration (FDA). However, as discovery of new therapeutic approaches for diseases with unmet medical need becomes more challenging, the productivity and efficiency of the traditional approach to drug discovery and development is diminishing. Innovative approaches are needed, such as those offered by Quantitative Systems Pharmacology (QSP) modeling and simulation. This "systems" approach to modeling and simulation can be used to guide the hypothesis generation and testing process in pharmaceutical R&D, in a manner similar to its adoption in other industries in the past. Embedding QSP into the existing processes within pharmaceutical discovery and development will be required in order to realize the full beneficial impact of this innovative approach.

Multiple software programs are available for designing and running large scale system-level pharmacology models used in the drug development process. Depending on the problem, scientists may be forced to use several modeling tools that could increase model development time, IT costs and so on. Therefore, it is desirable to have a single platform that allows setting up and running large-scale simulations for the models that have been developed with different modeling tools. We developed a workflow and a software platform in which a model file is compiled into a self-contained executable that is no longer dependent on the software that was used to create the model. At the same time the full model specifics is preserved by presenting all model parameters as input parameters for the executable. This platform was implemented as a model agnostic, therapeutic area agnostic and web-based application with a database back-end that can be used to configure, manage and execute large-scale simulations for multiple models by multiple users. The user interface is designed to be easily configurable to reflect the specifics of the model and the user's particular needs and the back-end database has been implemented to store and manage all aspects of the systems, such as Models, Virtual Patients, User Interface Settings, and Results. The platform can be adapted and deployed on an existing cluster or cloud computing environment. Its use was demonstrated with a metabolic disease systems pharmacology model that simulates the effects of two antidiabetic drugs, metformin and fasiglifam, in type 2 diabetes mellitus patients.

Use of tamoxifen is associated with a 50% reduction in breast cancer incidence and an increase in endometrial cancer incidence. Here, we documented tamoxifen-induced gene expression changes in cultured normal human mammary epithelial cells (strains 5, 16, and 40), established from tissue taken at reduction mammoplasty from three individuals. Cells exposed to 0, 10, or 50 micromol/L of tamoxifen for 48 hours were evaluated for (E)-alpha-(deoxyguanosine-N(2)-yl)-tamoxifen (dG-N(2)-TAM) adduct formation using TAM-DNA (DNA modified with dG-N(2)-TAM) chemiluminescence immunoassay, gene expression changes using National Cancer Institute DNA-oligonucleotide microarray, and real-time PCR. At 48 hours, cells exposed to 10 and 50 micromol/L of tamoxifen were 85.6% and 48.4% viable, respectively, and there were no measurable dG-N(2)-TAM adducts. For microarrays, cells were exposed to 10 micromol/L of tamoxifen and genes with expression changes of >3-fold were as follows: 13 genes up-regulated and 1 down-regulated for strain 16; 17 genes up-regulated for strain 5, and 11 genes up-regulated for strain 40. Interferon-inducible genes (IFITM1, IFIT1, MXI, and GIP3), and a potassium ion channel (KCNJ1) were up-regulated in all three strains. No significant expression changes were found for genes related to estrogen or xenobiotic metabolism. Real-time PCR revealed the up-regulation of IFNA1 and confirmed the tamoxifen-induced up-regulation of the five other genes identified by microarray, with the exception of GIP3 and MX1, which were not up-regulated in strain 40. Induction of IFN-related genes in the three normal human mammary epithelial cell strains suggests that, in addition to hormonal effects, tamoxifen exposure may enhance immune response in normal breast tissue.

Folylpolyglutamate synthase (FPGS) catalyzes the polyglutamation of folic acid, methotrexate, and pemetrexed to produce highly active metabolites. To characterize genetic variation in the FPGS gene, FPGS, have resequenced the gene in four different ethnic populations. Thirty-four single nucleotide polymorphisms were identified including five nonsynonymous coding single nucleotide polymorphisms that altered the FPGS protein sequence: F13L and V22I polymorphisms in the mitochondrial isoform of FPGS, and R466/424C, A489/447V, and S499/457F polymorphisms, which exist in both the mitochondrial and cytosolic isoforms. When expressed in AuxB1 cells, the A447V cytosolic variant was functionally similar to the wild-type cytosolic (WT Cyt) allozyme, whereas the R424C and S457F cytosolic variants were reduced by approximately 2-fold in protein expression compared with WT Cyt (P < 0.01). The intrinsic clearance of glutamate was reduced by 12.3-fold (R424C, P < 0.01) and 6.2-fold (S457F, P < 0.01), whereas the intrinsic clearance of methotrexate was reduced by 4.2-fold (R424C, P < 0.05) and 5.4-fold (S457F, P < 0.05) in these two cytosolic variants when compared with the WT Cyt isoform. Additionally, the in vitro enzyme velocity at saturating pemetrexed concentrations was reduced by 1.6-fold (R424C, P < 0.05) and 2.6-fold (S457F, P < 0.01) compared with WT Cyt. AuxB1 cells harboring these same cytosolic variant allozymes displayed significant increases in the EC(50) for folic acid and in the IC(50) values for both methotrexate and pemetrexed relative to the WT Cyt form of FPGS. These observations suggest that genetic variations in FPGS may alter the efficacy of antifolate therapy in cancer patients.

We investigated the ubiquitin-like modification of GABA(A) receptor-associated protein (GABARAP) and its function. A fusion protein of GABARAP with v5 in the N terminus and myc in the C terminus was expressed in rat cultured hippocampal neurons and PC12 cells. Western blotting with antibodies to v5 and myc revealed that the C terminus of GABARAP was cleaved off. Cleavage was blocked by mutating the C-terminal Gly116 to Ala, suggesting that G116 is required for the processing. Unlike ubiquitin, GABARAP was not incorporated covalently into higher-molecular-weight protein complexes. Nor was GABARAP degraded by ubiquitinylation through the proteasome, although GABARAP formed noncovalent dimers. Immunofluorescent confocal microscopy demonstrated that recombinantly expressed GABARAP was diffusely localized in PC12 cells. However, prevention of C-terminal processing in the mutant GABARAP(G116A) resulted in redistribution to the Golgi. In neurons, punctate cytoplasmic staining of GABARAP was seen in soma and processes, whereas GABARAP(G116A) was limited to soma. Compared with wild-type GABARAP, the colocalization and interaction of GABARAP(G116A) with GABA(A) receptors were significantly reduced, resulting in a reduction in expression of receptors in the plasma membrane. When alpha1beta2gamma2S-containing GABA(A) receptors were expressed in oocytes, the increased surface expression of GABA(A) receptors, as shown by increased GABA currents and surface-accessible GABA(A) receptor subunit polypeptides resulting from GABARAP coexpression, was prevented by mutation G116A. In addition, the distribution of NSF (N-ethylmaleimide-sensitive factor) was affected in GABARAP(G116A)-expressing neurons. These results suggest that glycine 116 is required for C-terminal processing of GABARAP and that processing is essential for the localization of GABARAP and its functions as a trafficking protein.

In response to DNA damage, cell cycle arrest, apoptosis, and DNA repair are mediated by a TP53 pathway that induces p21(WAF1/Cip1). The chemotherapeutic drug cis-diamminedichloroplatinum-II (cisplatin) damages cellular DNA by forming cis-diammineplatinum-N(7)-d[GpG] and cis-diammine-platinum-N(7)-d[ApG] adducts. To investigate the role of p21, skin keratinocytes from p21(WAF1/Cip1) wild-type (+/+), heterozygous (+/-), and null (-/-) mice, cultured in calcium levels designed to maintain a proliferating state, were exposed to 5 microM cisplatin continuously for 0, 8, 24, 48 and 72 h. At all time points the (+/-) cells had the fewest Pt-DNA adducts, and at 24 h mean Pt-DNA adduct levels were 541, 153 and 779 fmol adduct/mug DNA for p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively [P < 0.05 for (+/+) versus (+/-) and (-/-) versus (+/-)]. In order to understand underlying events, we examined p21(WAF1/Cip1) messenger RNA (mRNA), cell cycle arrest, and apoptosis in these cells. At 48 h of cisplatin exposure p21(WAF1/Cip1) mRNA expression was 2-fold higher in the (+/+) cells, compared to the (+/-) cells. At 24 h, the % of cells in S-phase in cisplatin-exposed cultures, compared to unexposed cultures, was decreased by 51, 40 and 11% in p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively (P = 0.04, ANOVA). At 24, 48 and 72 h the % of cisplatin-exposed (+/+) cells in apoptosis was 9.4-10.5%, while the cisplatin-exposed (+/-) and (-/-) cells had 1.2-3.7% of cells in apoptosis. The data support the interpretation that DNA replication arrest and apoptosis do not completely explain the low levels of Pt-DNA adducts in the (+/-) cells, and suggest that p21(WAF1/Cip1) controls activity resulting in either low Pt-DNA adduct formation or enhanced Pt-DNA adduct removal.

The trafficking of GABA(A) receptors is an important component of the pathway that regulates plasticity of inhibitory synapses. The 17 kDa GABA(A) receptor-associated protein (GABARAP) has been implicated in the trafficking of GABA(A) receptors because of its ability to interact not only with the gamma2 subunit of the receptor but also with microtubules and the N-ethylmaleimide-sensitive factor (NSF). To elucidate the role of GABARAP in the trafficking of GABA(A) receptors, we have constructed a yellow fluorescent protein (YFP) fusion protein of GABARAP and expressed it in neurons using adenovirus, so that its function may be examined. YFP-GABARAP colocalized with gamma2 subunit-containing GABA(A) receptors and NSF to the perinuclear cytoplasm in cultured hippocampal neurons and to the proximal regions of dendrites that are making synaptic contact. Expression of YFP-GABARAP in Cos7 cells and cultured hippocampal neurons was able to increase the level of GABA(A) receptors detected at the plasma membrane, even at low levels of YFP-GABARAP expression. This effect is specific to the function of GABARAP on GABA(A) receptor trafficking, because point mutations in the gamma2-binding domain of YFP-GABARAP interfered with the ability of YFP-GABARAP to increase GABA(A) receptor surface levels. These mutations also disrupted the colocalization of YFP-GABARAP with the gamma2 subunit and with NSF in hippocampal neurons. The results of this study show for the first time that GABARAP has a functional effect on the trafficking of GABA(A) receptors and provide decisive evidence for the role of GABARAP in transporting GABA(A) receptors to the plasma membrane in neurons.

Integrins link the extracellular matrix to the cellular cytoskeleton and serve important roles in cell growth, differentiation, migration, and survival. Ablation of beta1 integrin in all murine tissues results in peri-implantation embryonic lethality. To investigate the role of beta1 integrin in the myocardium, we used Cre-LoxP technology to inactivate the beta1 integrin gene exclusively in ventricular cardiac myocytes. Animals with homozygous ventricular myocyte beta1 integrin gene excision were born in appropriate numbers and grew into adulthood. These animals had 18% of control levels of beta1D integrin protein in the heart and displayed myocardial fibrosis. High-fidelity micromanometer-tipped catheterization of the intact 5-week-old beta1 integrin knockout mice showed depressed left ventricular basal and dobutamine-stimulated contractility and relaxation (LV dP/dt(max) and LV dP/dt(min)) as compared with control groups (n=8 to 10 of each, P<0.01). Hemodynamic loading imposed by 7 days of transverse aortic constriction showed that the beta1 integrin knockout mice were intolerant of this stress as they had 53% survival versus 88% in controls (n=15 each). By 6 months of age, mice with depressed ventricular expression of beta1 integrin developed a dilated cardiomyopathy that was not evident in any control animals and had patchy decrease in glucose metabolism as determined by positron emission tomography. Myocyte membrane integrity as determined via Evan's blue dye staining was disrupted in the beta1 integrin knockout mice. This model provides strong evidence for the importance of beta1 integrin in cardiac form and function and indicates that integrins can be linked to development of cardiomyopathies.