Michael Tanen

Induction of Adipocyte Complement-Related Protein of 30 Kilodaltons by PPARγ Agonists: A Potential Mechanism of Insulin Sensitization

Adipocyte complement-related protein of 30 kDa (Acrp30, adiponectin, or AdipoQ) is a fat-derived secreted protein that circulates in plasma. Adipose tissue expression of Acrp30 is lower in insulin-resistant states and it is implicated in the regulation of in vivo insulin sensitivity. Here we have characterized the ability of PPARγ agonists to modulate Acrp30 expression. After chronic treatment of obese-diabetic (db/db) mice with PPARγ agonists (11 d), mean plasma Acrp30 protein levels increased (>3×). Similar effects were noted in a nongenetic type 2 diabetes model (fat-fed and low-dose streptozotocin-treated mice). In contrast, treatment of mice (db/db or fat-fed) with metformin or a PPARα agonist did not affect plasma Acrp30 protein levels. In a cohort of normal human subjects, 14-d treatment with rosiglitazone also produced a 130% increase in circulating Acrp30 levels vs. placebo. In addition, circulating Acrp30 levels were suppressed 5-fold in patients with severe insulin resistance in association wit...

Pharmacokinetics and pharmacodynamics of sitagliptin, an inhibitor of dipeptidyl peptidase IV, in healthy subjects: results from two randomized, double-blind, placebo-controlled studies with single oral doses.

Sitagliptin (MK‐0431 [(2R)‐4‐oxo‐4‐(3‐[trifluoromethyl]‐5,6‐dihydro[1,2,4]triazolo[4,3‐a]pyrazin‐7[8H]‐yl)‐1‐(2,4,5‐trifluorophenyl)butan‐2‐amine]) is an orally active, potent, and selective inhibitor of dipeptidyl peptidase IV (DPP‐IV) currently in phase III development for the treatment of type 2 diabetes.

NOVEL PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) GAMMA AND PPARDELTA LIGANDS PRODUCE DISTINCT BIOLOGICAL EFFECTS

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARα, PPARδ, and PPARγ. PPARγ has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPARγ and PPARδ that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPARγ and PPARδ directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARγ agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabeticdb/db mice all PPARγ agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selectivein vivo activation of PPARδ did not significantly affect these parameters. In vivo PPARα activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPARγ and PPARδ; 2) ligand-dependent activation of PPARδ involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARγ activation (but not PPARδ or PPARα activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARγ agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARα activation is sufficient to affect triglyceride metabolism, PPARδ activation does not appear to modulate glucose or triglyceride levels.

Pharmacokinetics and Pharmacodynamic Effects of the Oral DPP-4 Inhibitor Sitagliptin in Middle-Aged Obese Subjects

Sitagliptin (MK‐0431) is an oral, potent, and selective dipeptidyl peptidase‐IV (DPP‐4) inhibitor developed for the treatment of type 2 diabetes. This multicenter, randomized, double‐blind, placebo‐controlled study examined the pharmacokinetic and pharmacodynamic effects of sitagliptin in obese subjects. Middle‐aged (45–63 years), nondiabetic, obese (body mass index: 30–40 kg/m2) men and women were randomized to sitagliptin 200 mg bid (n = 24) or placebo (n = 8) for 28 days. Steady‐state plasma concentrations of sitagliptin were achieved within 2 days of starting treatment, and >90% of the dose was excreted unchanged in urine. Sitagliptin treatment led to ∼90% inhibition of plasma DPP‐4 activity, increased active glucagon‐like peptide‐1 (GLP‐1) levels by 2.7‐fold (P < .001), and decreased post—oral glucose tolerance test glucose excursion by 35% (P < .050) compared to placebo. In nondiabetic obese subjects, treatment with sitagliptin 200 mg bid was generally well tolerated without associated hypoglycemia and led to maximal inhibition of plasma DPP‐4 activity, increased active GLP‐1, and reduced glycemic excursion.

Dipeptidyl Peptidase-4 Inhibitors Administered in Combination With Metformin Result in an Additive Increase in the Plasma Concentration of Active GLP-1

The aim of the study was to investigate the effects of a dipeptidyl peptidase‐4 (DPP‐4) inhibitor, of metformin, and of the combination of the two agents, on incretin hormone concentrations. Active and inactive (or total) incretin plasma concentrations, plasma DPP‐4 activity, and preproglucagon (GCG) gene expression were determined after administration of each agent alone or in combination to mice with diet‐induced obesity (DIO) and to healthy human subjects. In mice, metformin increased Gcg expression in the large intestine and elevated the plasma concentrations of inactive glucagon‐like peptide 1 (GLP‐1) (9–36) and glucagon. In healthy subjects, a DPP‐4 inhibitor elevated both active GLP‐1 and glucose dependent insulinotropic polypeptide (GIP), metformin increased total GLP‐1 (but not GIP), and the combination resulted in additive increases in active GLP‐1 plasma concentrations. Metformin did not inhibit plasma DPP‐4 activity either in vitro or in vivo. The study results show that metformin is not a DPP‐4 inhibitor but rather enhances precursor GCG expression in the large intestine, resulting in increased total GLP‐1 concentrations. DPP‐4 inhibitors and metformin have complementary mechanisms of action and additive effects with respect to increasing the concentrations of active GLP‐1 in plasma.

Localization of PPARδ in murine central nervous system: expression in oligodendrocytes and neurons

The peroxisome proliferator-activated receptors (PPARs), PPARdelta, PPARgamma and PPARalpha, comprise a subclass of the supergene family of nuclear receptors. As such they are ligand-regulated transcription factors whose major effects are mediated by altering expression of target genes. PPARdelta has been shown to be ubiquitously expressed in mammals. However, its primary biological role(s) has yet to be defined. Several recent studies have demonstrated that PPARdelta is the most highly expressed PPAR isoform in the central nervous system, but ambiguity still exists as to the specific brain sub-regions and cells in which it is expressed. Here, utilizing novel, isoform-selective PPARdelta riboprobes and an anti-peptide antibody, we performed a series of in situ hybridization and immunolocalization studies to determine the distribution of PPARdelta in the central nervous system (CNS) of mice. We found that PPARdelta mRNA and protein is expressed throughout the brain, with particularly high levels in the entorhinal cortex, hypothalamus and hippocampus, and lower levels in the corpus callosum and caudate putamen. At the cellular level, PPARdelta mRNA and protein were found to be expressed in oligodendrocytes and neurons but not astrocytes. Such results suggest a role for PPARdelta in both myelination and neuronal functioning within the CNS.

The BACE1 inhibitor verubecestat (MK-8931) reduces CNS β-amyloid in animal models and in Alzheimer’s disease patients

The BACE1 inhibitor verubecestat safely reduces β-amyloid deposition in rats, monkeys, healthy human subjects, and patients with Alzheimer’s disease. Getting to first BACE The discovery of BACE1 inhibitors that reduce β-amyloid peptides in Alzheimer’s disease (AD) patients has been an encouraging development in the quest for a disease-modifying therapy. Kennedy and colleagues now report the discovery of verubecestat, a structurally unique, orally bioavailable small molecule that potently inhibits brain BACE1 activity resulting in a reduction in Aβ peptides in the cerebrospinal fluid of animals, healthy volunteers, and AD patients. No dose-limiting toxicities were observed in chronic animal toxicology studies or in phase 1 human studies, thus reducing safety concerns raised by previous reports of BACE inhibitors and BACE1 knockout mice. β-Amyloid (Aβ) peptides are thought to be critically involved in the etiology of Alzheimer’s disease (AD). The aspartyl protease β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of Aβ, and BACE1 inhibition is thus an attractive target for the treatment of AD. We show that verubecestat (MK-8931) is a potent, selective, structurally unique BACE1 inhibitor that reduced plasma, cerebrospinal fluid (CSF), and brain concentrations of Aβ40, Aβ42, and sAPPβ (a direct product of BACE1 enzymatic activity) after acute and chronic administration to rats and monkeys. Chronic treatment of rats and monkeys with verubecestat achieved exposures >40-fold higher than those being tested in clinical trials in AD patients yet did not elicit many of the adverse effects previously attributed to BACE inhibition, such as reduced nerve myelination, neurodegeneration, altered glucose homeostasis, or hepatotoxicity. Fur hypopigmentation was observed in rabbits and mice but not in monkeys. Single and multiple doses were generally well tolerated and produced reductions in Aβ40, Aβ42, and sAPPβ in the CSF of both healthy human subjects and AD patients. The human data were fit to an amyloid pathway model that provided insight into the Aβ pools affected by BACE1 inhibition and guided the choice of doses for subsequent clinical trials.

Quantification of Tau in Cerebrospinal Fluid by Immunoaffinity Enrichment and Tandem Mass Spectrometry

BACKGROUND Cerebrospinal fluid (CSF) tau is a common biomarker for Alzheimer disease (AD). Measurements of tau have historically been performed using immunoassays. Given the molecular diversity of tau in CSF, the selectivity of these immunoassays has often been questioned. Therefore, we aimed to develop an analytically sensitive and selective immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) (IA-MS) assay. METHODS IA-MS sample analysis involved the addition of an internal standard, immunoaffinity purification of tau using a tau monoclonal antibody coupled to magnetic beads, trypsin digestion, and quantification of a surrogate tau peptide by LC-MS/MS using a Waters Trizaic nanoTile ultraperformance LC microfluidic device. Further characterization of tau peptides was performed by full-scan MS using a Thermo Orbitrap LC-MS. CSF samples from a cohort of age-matched controls and patients with AD were analyzed by the IA-MS method as well as a commercially available immunoassay. RESULTS The IA-MS assay had intra- and interassay imprecision values of 3.2% to 8.1% CV and 7.8% to 18.9% C, respectively, a mean recovery of 106%, and a limit of quantification of 0.25 pmol/L and was able to quantify tau concentrations in all human specimens tested. The IA-MS assay showed a correlation of R(2) = 0.950 against a total-tau immunoassay. In patients with AD, tau was increased approximately 2-fold. CONCLUSIONS Combining immunoaffinity enrichment with microflow LC-MS/MS analysis is an effective approach for the development of a highly selective assay to measure total tau and, potentially, other posttranslationally modified forms of tau in CSF.

Cerebrospinal Fluid Biomarkers Distinguish Postmortem-Confirmed Alzheimer's Disease from Other Dementias and Healthy Controls in the OPTIMA Cohort

Cerebrospinal fluid (CSF) amyloid-β (Aβ) and tau have been studied as markers of Alzheimers disease (AD). Combined Aβ42 and t-tau distinguishes AD from healthy controls with a sensitivity and specificity (sens/spec) near 89% across studies. This study examined these markers in the homogeneous OPTIMA cohort, using extensive longitudinal follow up and postmortem evaluation to confirm clinicopathological status. Baseline CSF was analyzed from 227 participants with AD (97% autopsy-confirmed), mild cognitive impairment (MCI; 73% confirmed), other dementia syndrome (ODS; 100% confirmed), and controls (CTL; 27% confirmed, follow up approximately 9-13 years). Biomarker concentrations were analyzed using validated ELISAs. AD patients had lower CSF Aβ42 and higher t-tau, p-tau, t-tau/Aβ42, and t-tau/Aβ40 compared to CTLs, with MCI intermediate. CTL and MCI participants who progressed to AD demonstrated more AD-like profiles. Aβ40, sAβPPα, and sAβPPβ were lower in AD compared to CTL. High-level discriminators of AD from CTL were t-tau/Aβ40 (AUROC 0.986, sens/spec of 92%/94%), p-tau/Aβ42 (AUROC 0.972, sens/spec of 94%/90%), and Aβ42 (AUROC 0.941, sens/spec of 88%). For discriminating AD from ODS, p-tau/Aβ42 demonstrated sens/spec of 88%/100% (95%/86% at the AD versus CTL cutoff) and Aβ42 demonstrated sens/spec of 84%/100% (88%/100% at the AD versus CTL cutoff). In a well-characterized, homogeneous population, a single cutoff for baseline CSF Aβ and tau markers can distinguish AD with a high level of sens/spec compared to other studies. It may be important to characterize sources of demographic and biological variability to support the effective use of CSF diagnostic assays in the broader AD population.

Kinetics of TH2 biomarkers in sputum of asthmatics following inhaled allergen

Background Allergen-induced late airway response offers important pharmacodynamic targets, including T helper 2 (TH2) biomarkers. However, detection of inflammatory markers has been limited in dithiothreitol-processed sputum. Objectives To test whether allergen-induced TH2 inflammatory markers can be reproducibly quantified by sensitive detection techniques in ultracentrifuged sputum and the effect of fluticasone (FP) on these endpoints. Methods Thirteen allergic asthmatics with dual allergen-induced airway responses, documented during a single-blind placebo run-in period, participated in a double-blind, two-period crossover study. Each period consisted of three consecutive days, separated by ≥3 weeks. Following randomization, subjects inhaled FP (500 µg bid, five doses total) or placebo. On Day 2 in each study period, allergen challenge was performed and airway response measured by forced expiratory volume in 1 sec (FEV1) until 7 h post-challenge. Sputum was induced 24 h pre-allergen and 7 and 24 h post-allergen. Sputum samples were split into two portions: TH2 biomarkers were quantified by Meso Scale multiplex platform following ultracentrifugation, and cell differentials were counted on Giemsa–May-Grünwald-stained cytospins. Allergen-induced changes in inflammatory endpoints were compared between FP and placebo using a mixed model ANCOVA. Results Inhaled allergen induced dual airway responses in all subjects during both placebo periods with reproducible late asthmatic response (LAR) and increased sputum inflammatory biomarkers (IL-2, IL-4, IL-13, and eotaxin-1) and eosinophil counts. FP effectively blunted both the LAR and the inflammatory biomarkers. Conclusions Combining novel, sensitive quantification methods with ultracentrifugation allows reproducible quantification of sputum biomarkers following allergen challenge, reversed by FP. This approach allows non-invasive identification of pharmacodynamic targets for anti-asthma therapies.