Oliver P. Flint

Discovery of Dapagliflozin: A Potent, Selective Renal Sodium-Dependent Glucose Cotransporter 2 (SGLT2) Inhibitor for the Treatment of Type 2 Diabetes

The C-aryl glucoside 6 (dapagliflozin) was identified as a potent and selective hSGLT2 inhibitor which reduced blood glucose levels in a dose-dependent manner by as much as 55% in hyperglycemic streptozotocin (STZ) rats. These findings, combined with a favorable ADME profile, have prompted clinical evaluation of dapagliflozin for the treatment of type 2 diabetes.

Effects of atazanavir/ritonavir and lopinavir/ritonavir on glucose uptake and insulin sensitivity: demonstrable differences in vitro and clinically.

Background:The HIV protease inhibitor (PI) atazanavir does not impair insulin sensitivity acutely but ritonavir and lopinavir induce insulin resistance at therapeutic concentrations. Objective:To test the hypothesis that atazanavir combined with a lower dose of ritonavir would have significantly less effect on glucose metabolism than lopinavir/ritonavir in vitro and clinically. Methods:Glucose uptake was measured following insulin stimulation in differentiated human adipocytes in the presence of ritonavir (2 μmol/l) combined with either atazanavir or lopinavir (3–30 μmol/l). These data were examined clinically using the hyperinsulinemic euglycemic clamp and oral glucose tolerance testing (OGTT) in 26 healthy HIV-negative men treated with atazanavir/ritonavir (300/100 mg once daily) and lopinavir/ritonavir (400/100 mg twice daily) for 10 days in a randomized cross-over study. Results:Atazanavir inhibited glucose uptake in vitro significantly less than lopinavir and ritonavir at all concentrations. Ritonavir (2 μmol/l) combined with either atazanavir or lopinavir (3–30 μmol/l) did not further inhibit glucose uptake. During euglycemic clamp, there was no significant change from baseline insulin sensitivity with atazanavir/ritonavir (P = 0.132), while insulin sensitivity significantly decreased with lopinavir/ritonavir from the baseline (−25%; P < 0.001) and from that seen with atazanavir/ritonavir (−18%; P = 0.023). During OGTT, the HOMA insulin resistance index significantly increased from baseline at 120 min with atazanavir/ritonavir and at 150 min with lopinavir/ritonavir. The area under the curve of glucose increased significantly with lopinavir/ritonavir but not with atazanavir/ritonavir. Conclusions:Both glucose uptake in vitro and clinical insulin sensitivity in healthy volunteers demonstrate differential effects on glucose metabolism by the combination PI atazanavir/ritonavir and lopinavir/ritonavir.

Developing High-Fidelity Hepatotoxicity Models From Pluripotent Stem Cells

Faithfully recapitulating human physiology “in a dish” from a renewable source remains a holy grail for medicine and pharma. Many procedures have been described that, to a limited extent, exhibit human tissue‐specific function in vitro. In particular, incomplete cellular differentiation and/or the loss of cell phenotype postdifferentiation play a major part in this void. We have developed an interdisciplinary approach to address this problem, using skill sets in cell biology, materials chemistry, and pharmacology. Pluripotent stem cells were differentiated to hepatocytes before being replated onto a synthetic surface. Our approach yielded metabolically active hepatocyte populations that displayed stable function for more than 2 weeks in vitro. Although metabolic activity was an important indication of cell utility, the accurate prediction of cellular toxicity in response to specific pharmacological compounds represented our goal. Therefore, detailed analysis of hepatocellular toxicity was performed in response to a custom‐built and well‐defined compound set and compared with primary human hepatocytes. Importantly, stem cell‐derived hepatocytes displayed equivalence to primary human material. Moreover, we demonstrated that our approach was capable of modeling metabolic differences observed in the population. In conclusion, we report that pluripotent stem cell‐derived hepatocytes will model toxicity predictably and in a manner comparable to current gold standard assays, representing a major advance in the field.

Accurate Prediction of Drug-Induced Liver Injury Using Stem Cell-Derived Populations

Despite major progress in the knowledge and management of human liver injury, there are millions of people suffering from chronic liver disease. Currently, the only cure for end‐stage liver disease is orthotopic liver transplantation; however, this approach is severely limited by organ donation. Alternative approaches to restoring liver function have therefore been pursued, including the use of somatic and stem cell populations. Although such approaches are essential in developing scalable treatments, there is also an imperative to develop predictive human systems that more effectively study and/or prevent the onset of liver disease and decompensated organ function. We used a renewable human stem cell resource, from defined genetic backgrounds, and drove them through developmental intermediates to yield highly active, drug‐inducible, and predictive human hepatocyte populations. Most importantly, stem cell‐derived hepatocytes displayed equivalence to primary adult hepatocytes, following incubation with known hepatotoxins. In summary, we have developed a serum‐free, scalable, and shippable cell‐based model that faithfully predicts the potential for human liver injury. Such a resource has direct application in human modeling and, in the future, could play an important role in developing renewable cell‐based therapies.

The Role of Protease Inhibitors in the Pathogenesis of HIV-Associated Lipodystrophy: Cellular Mechanisms and Clinical Implications

Metabolic complications associated with HIV infection and treatment frequently present as a relative lack of peripheral adipose tissue associated with dyslipidemia and insulin resistance. In this review we explain the connection between abnormalities of intermediary metabolism, observed either in vitro or in vivo, and this group of metabolic effects. We review molecular mechanisms by which the HIV protease inhibitor (PI) class of drugs may affect the normal stimulatory effect of insulin on glucose and fat storage. We then propose that both chronic inflammation from HIV infection and treatment with some drugs in this class trigger cellular homeostatic stress responses with adverse effects on intermediary metabolism. The physiologic outcome is such that total adipocyte storage capacity is decreased, and the remaining adipocytes resist further fat storage. The excess circulating and dietary lipid metabolites, normally “absorbed” by adipose tissue, are deposited ectopically in lean (muscle and liver) tissue, where they impair insulin action. This process leads to a pathologic cycle of lipotoxicity and lipoatrophy and a clinical phenotype of body fat distribution with elevated waist-to-hip ratio similar to the metabolic syndrome.

In vitro tests for teratogens: Desirable endpoints, test batteries and current status of the micromass teratogen test

Information from in vitro tests can be usefully used as a component of the risk/hazard assessment process. In vivo studies will be required to confirm the in vitro data. If the in vitro test system is designed around endpoints that reflect changes following in vivo toxic insult then it may be possible to modify the in vitro system to account for some of the discrepancies observed between in vivo and in vitro outcomes. When the discrepancy can be accounted for by low bioavailability in vivo, pharmacokinetic studies may be required to determine the relevance of the in vitro toxic concentrations. Reproductive hazard, especially teratogenicity, has been the subject of intensive in vitro test development. The observation of teratogenicity may affect the development of new products more significantly than any other type or category of reproductive toxicity. The micromass test, involving culture of differentiating rat embryo limb and midbrain cells exposed to test agents, may be useful as part of a battery of in vitro tests for teratogens. The most recent protocol for the micromass test is described, followed by a summary of validation and mechanistic studies confirming its usefulness. The test is robust in its transfer to new laboratories. Interlaboratory variability is small.

Genetic analysis implicates resistin in HIV lipodystrophy.

Objectives:To investigate the role of genetic variation in influencing the risk of metabolic complications associated with highly active antiretroviral therapy (HAART). Methods:Cluster analysis of metabolic traits of 189 patients enrolled in ACTG5005s, the metabolic substudy of ACTG384, a clinical trial of HAART, was performed to identify a subgroup of individuals with increased risk of developing a cluster of metabolic abnormalities after exposure to HAART. Almost 300 single nucleotide polymorphisms in 135 candidate genes were evaluated for their association with this subgroup. Results:A subgroup of patients was identified that had a normal metabolic profile at baseline but developed significantly elevated lipids and insulin resistance on HAART. This high-risk subgroup of patients also experienced significant body composition changes, particularly limb fat loss. Candidate gene analysis revealed that a single nucleotide polymorphism in resistin, a gene previously implicated in obesity and insulin resistance, was associated with this high-risk group (P = 0.0003). Conclusion:Genetic variation in resistin is associated with metabolic complications caused by HAART.

Establishment of a Molecular Embryonic Stem Cell Developmental Toxicity Assay

The mouse embryonic stem cell test (EST) is a 10-day screen for teratogenic potential developed to reduce animal use for embryotoxicity testing of chemicals (Spielmann, 2005; Spielmann et al., 1997). In this study, we used the cytotoxicity IC(50) values and transcriptional expression changes as primary endpoints in a shorter 4-day version of the EST, the molecular embryonic stem cell assay. Mouse D3 embryonic stem cells were used for cytotoxicity assessment (monolayers) or grown as embryoid bodies in low attachment plates for transcriptional profiling. Sixty-five compounds with known in vivo teratogenicity (33 teratogens and 32 nonteratogens) were evaluated to develop a model for classifying compounds with teratogenic potential. The expression of 12 developmentally regulated gene targets (nanog, fgf5, gsc, cd34, axin2, apln, chst7, lhx1, fgf8, sox17, foxa2, and cxcr4) was measured following exposure of embryoid bodies to a single compound concentration (0.1 × the cytotoxicity IC(20)) for 4 days. In the decision-tree model, compounds with IC(50) values < 22 µM were categorized as teratogens, whereas compounds in the two groups with IC(50) values between 22-200 µM and > 200 µM were categorized as teratogens if ≥ 8 and 12 genes, respectively, were deregulated by at least 10%. Forty-seven of 65 compounds of the training set were correctly identified (72% total concordance). In a test set of 12 additional compounds (5 teratogens, 7 nonteratogens), 10 were correctly classified by this approach (83% concordance). The false positive rate in the training and test sets was 24 and 0%, respectively, indicating that this assay has potential to identify teratogens.

Fluid shear stress modulation of hepatocyte-like cell function

Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype.

Polymer Supported Directed Differentiation Reveals a Unique Gene Signature Predicting Stable Hepatocyte Performance

In theory, pluripotent stem cells can give rise to all somatic cell types found in the human body. The ability to generate renewable sources of human cells has enormous potential to improve human health and wealth. One major obstacle to the routine deployment of stem cell-derived cells is their instability in culture. To tackle this issue a synthetic polymer surface is used.