Diet-resistant obesity is characterized by a distinct plasma proteomic signature and impaired muscle fiber metabolism.

BACKGROUND/OBJECTIVES: Inter-individual variability in weight loss during obesity treatment is complex and poorly understood. Here we use whole body and tissue approaches to investigate fuel oxidation characteristics in skeletal muscle fibers, cells and distinct circulating protein biomarkers before and after a high fat meal (HFM) challenge in those who lost the most (obese diet-sensitive; ODS) vs the least (obese diet-resistant; ODR) amount of weight in a highly controlled weight management program. SUBJECTS/METHODS: In 20 weight stable-matched ODS and ODR women who previously completed a standardized clinical weight loss program, we analyzed whole-body energetics and metabolic parameters in vastus lateralis biopsies and plasma samples that were obtained in the fasting state and 6 h after a defined HFM, equivalent to 35% of total daily energy requirements. RESULTS: At baseline (fasting) and post-HFM, muscle fatty acid oxidation and maximal oxidative phosphorylation were significantly greater in ODS vs ODR, as was reactive oxygen species emission. Plasma proteomics of 1130 proteins pre and 1, 2, 5 and 6 h after the HFM demonstrated distinct group and interaction differences. Group differences identified S-formyl glutathione hydratase, heat shock 70 kDA protein 1A/B (HSP72), and eukaryotic translation initiation factor 5 (eIF5) to be higher in ODS vs ODR. Group-time differences included aryl hydrocarbon interacting protein (AIP), peptidylpropyl isomerase D (PPID) and tyrosine protein-kinase Fgr, which increased in ODR vs ODS over time. HSP72 levels correlated with muscle oxidation and citrate synthase activity. These proteins circulate in exosomes; exosomes isolated from ODS plasma increased resting, leak and maximal respiration rates in C2C12 myotubes by 58%, 21% and 51%, respectively, vs those isolated from ODR plasma. CONCLUSIONS: Findings demonstrate distinct muscle metabolism and plasma proteomics in fasting and post-HFM states corresponding in diet-sensitive vs diet-resistant obese women.

A prospective randomized evaluation of a pharmacogenomic approach to antiplatelet therapy among patients with ST-elevation myocardial infarction: the RAPID STEMI study.

Treatment of carriers of the CYP2C19*2 allele and ABCB1 TT genotype with clopidogrel is associated with increased ischemic complications after percutaneous coronary intervention (PCI). We sought to evaluate a pharmacogenomic strategy among patients undergoing PCI for ST-elevation myocardial infarction (STEMI), by performing a randomized trial, enrolling 102 patients. Point-of-care genetic testing for CYP2C19*2, ABCB1 TT and CYP2C19*17 was performed with carriers of either the CYP2C19*2 allele or ABCB1 TT genotype randomly assigned to a strategy of prasugrel 10 mg daily or an augmented dosing strategy of clopidogrel (150 mg daily for 6 days then 75 mg daily). The primary end point was the proportion of at-risk carriers exhibiting high on-treatment platelet reactivity (HPR), a marker associated with increased adverse cardiovascular events, after 1 month. Fifty-nine subjects (57.8%) were identified as carriers of at least one at-risk variant. Treatment with prasugrel significantly reduced HPR compared with clopidogrel by P2Y12 reaction unit (PRU) thresholds of >234 (0 vs 24.1%, P=0.0046) and PRU>208 (3.3 vs 34.5%, P=0.0025). The sensitivity of point-of-care testing was 100% (95% CI 88.0-100), 100% (86.3-100) and 96.9% (82.0-99.8) and specificity was 97.0% (88.5-99.5), 97.1% (89.0-99.5) and 98.5% (90.9-99.9) for identifying CYP2C19*2, ABCB1 TT and CYP2C19*17, respectively. Logistic regression confirmed carriers as a strong predictor of HPR (OR=6.58, 95% CI 1.24-34.92; P=0.03). We confirmed that concurrent identification of three separate genetic variants in patients with STEMI receiving PCI is feasible at the bedside. Among carriers of at-risk genotypes, treatment with prasugrel was superior to an augmented dosing strategy of clopidogrel in reducing HPR.

Outcomes of extremely low birth weight infants given early high-dose erythropoietin.

OBJECTIVE: To evaluate long-term outcomes of 60 extremely low birth weight (ELBW) infants treated with or without three injections of high-dose erythropoietin (Epo). STUDY DESIGN: A retrospective analysis of anthropometric and neurodevelopmental outcome data comparing 30 ELBW infants enrolled in a phase I/II study examining the pharmacokinetics of high-dose Epo (500, 1000 and 2500 U/kg × 3 doses) administered to 30 concurrent controls. RESULT: Birth characteristics and growth from 4 to 36 months were similar for untreated and Epo-treated patients. Multiple linear regression analysis of neurodevelopmental follow-up scores from 17/25 Epo-treated and 18/26 control infants identified that Epo correlated with improvement of cognitive (R=0.22, P=0.044) and motor (R=0.15, P=0.026) scores. No negative long-term effects of Epo treatment were evident. CONCLUSION: Retrospective analysis of the only available long-term follow-up data from ELBW infants given high-dose Epo treatment suggests that Epo treatment is safe and correlates with modest improvement of neurodevelopmental outcomes.

1-((3S,4S)-4-amino-1-(4-substituted-1,3,5-triazin-2-yl) pyrrolidin-3-yl)-5,5-difluoropiperidin-2-one inhibitors of DPP-4 for the treatment of type 2 diabetes.

A 3-amino-4-substituted pyrrolidine series of dipeptidyl peptidase IV (DPP-4) inhibitors was rapidly developed into a candidate series by identification of a polar valerolactam replacement for the lipophilic 2,4,5-trifluorophenyl pharmacophore. The addition of a gem-difluoro substituent to the lactam improved overall DPP-4 inhibition and an efficient asymmetric route to 3,4-diaminopyrrolidines was developed. Advanced profiling of a subset of analogs identified 5o with an acceptable human DPP-4 inhibition profile based on a rat PK/PD model and a projected human dose that was suitable for clinical development.

Discovery of PF-04620110, a Potent, Selective, and Orally Bioavailable Inhibitor of DGAT-1.

Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final committed step in the biosynthesis of triglycerides. DGAT-1 knockout mice have been shown to be resistant to diet-induced obesity and have increased insulin sensitivity. Thus, inhibition of DGAT-1 may represent an attractive target for the treatment of obesity or type II diabetes. Herein, we report the discovery and characterization of a potent and selective DGAT-1 inhibitor PF-04620110 (3). Compound 3 inhibits DGAT-1 with an IC50 of 19 nM and shows high selectivity versus a broad panel of off-target pharmacologic end points. In vivo DGAT-1 inhibition has been demonstrated through reduction of plasma triglyceride levels in rodents at doses of ≥0.1 mg/kg following a lipid challenge. On the basis of this pharmacologic and pharmacokinetic profile, compound 3 has been advanced to human clinical studies.

Naturally occurring R225W mutation of the gene encoding AMP-activated protein kinase (AMPK)gamma(3) results in increased oxidative capacity and glucose uptake in human primary myotubes.

AIMS/HYPOTHESIS: AMP-activated protein kinase (AMPK) has a broad role in the regulation of glucose and lipid metabolism making it a promising target in the treatment of type 2 diabetes mellitus. We therefore sought to characterise for the first time the effects of chronic AMPK activation on skeletal muscle carbohydrate metabolism in carriers of the rare gain-of-function mutation of the gene encoding AMPKgamma(3) subunit, PRKAG3 R225W. METHODS: Aspects of fuel metabolism were studied in vitro in myocytes isolated from vastus lateralis of PRKAG3 R225W carriers and matched control participants. In vivo, muscular strength and fatigue were evaluated by isokinetic dynamometer and surface electromyography, respectively. Glucose uptake in exercising quadriceps was determined using [(18)F]fluorodeoxyglucose and positron emission tomography. RESULTS: Myotubes from PRKAG3 R225W carriers had threefold higher mitochondrial content (p < 0.01) and oxidative capacity, higher leak-dependent respiration (1.6-fold, p < 0.05), higher basal glucose uptake (twofold, p < 0.01) and higher glycogen synthesis rates (twofold, p < 0.05) than control myotubes. They also had higher levels of intracellular glycogen (p < 0.01) and a trend for lower intramuscular triacylglycerol stores. R225W carriers showed remarkable resistance to muscular fatigue and a trend for increased glucose uptake in exercising muscle in vivo. CONCLUSIONS/INTERPRETATION: Through the enhancement of skeletal muscle glucose uptake and increased mitochondrial content, the R225W mutation may significantly enhance exercise performance. These findings are also consistent with the hypothesis that the gamma(3) subunit of AMPK is a promising tissue-specific target for the treatment of type 2 diabetes mellitus, a condition in which glucose uptake and mitochondrial function are impaired.

Discovery of small molecule isozyme non-specific inhibitors of mammalian acetyl-CoA carboxylase 1 and 2.

Screening Pfizer's compound library resulted in the identification of weak acetyl-CoA carboxylase inhibitors, from which were obtained rACC1 CT-domain co-crystal structures. Utilizing HTS hits and structure-based drug discovery, a more rigid inhibitor was designed and led to the discovery of sub-micromolar, spirochromanone non-specific ACC inhibitors. Low nanomolar, non-specific ACC-isozyme inhibitors that exhibited good rat pharmacokinetics were obtained from this chemotype.

Testing a scalable community-based approach to improve maternal and neonatal health in rural Nepal.

OBJECTIVE: The aim of the study was to determine the feasibility of improved maternal-neonatal care-seeking and household practices using an approach scalable under Nepal's primary health-care services. STUDY DESIGN: Impact was assessed by pre- and post-intervention surveys of women delivering within the previous 12 months. Each district sample comprised 30 clusters, each with 30 respondents. The intervention consisted primarily of community-based antenatal counseling and dispensing and an early postnatal home visit; most activities were carried out by community-based health volunteers. RESULT: There were notable improvements in most household practice and service utilization indicators, although results regarding care-seeking for danger signs were mixed. CONCLUSION: It is feasible in a Nepal setting to significantly improve utilization of maternal-neonatal services and household practices, using the resources available under the government primary health-care system. This has the potential to significantly reduce neonatal mortality.

Increased susceptibility to oxidative damage in post-diabetic human myotubes.

AIMS/HYPOTHESIS: Obesity is an important risk factor for the development of type 2 diabetes, but not all obese individuals develop this complication. The clinical signs of type 2 diabetes can often be reversed with weight loss; however, it is unknown whether the skeletal muscle oxidative stress associated with type 2 diabetes remains after weight loss. We hypothesised that chronic exposure to high glucose and insulin would re-elicit impaired metabolism in primary myotubes from patients with a history of type 2 diabetes. METHODS: Obese participants with or without type 2 diabetes completed a standardised weight loss protocol, following which all participants were euglycaemic and had similar indices of insulin sensitivity. Satellite cells were isolated from muscle biopsies and differentiated under low or high glucose and insulin conditions (HGI). RESULTS: Cells from participants with no history of type 2 diabetes showed robust increases in mitochondrial content, citrate synthase and cytochrome c oxidase activities when exposed to HGI. This increase in oxidative capacity was absent in cells from patients with a history of type 2 diabetes. High glucose and insulin caused increased oxidative damage in cells from the latter, despite higher superoxide dismutase expression. Cells from patients with a history of type 2 diabetes were unable to decrease mitochondrial membrane potential in response to HGI, potentially due to lower levels of uncoupling protein-3. CONCLUSIONS/INTERPRETATION: This is the first report to note that primary myotubes from patients with a history of type 2 diabetes are unable to adapt to a hyperglycaemic-hyperinsulinaemic challenge. We have demonstrated that impaired mitochondrial biogenesis and an inability to manage oxidative stress define a muscle phenotype at risk of obesity-associated type 2 diabetes.

(3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone: a potent, selective, orally active dipeptidyl peptidase IV inhibitor.

A series of 4-substituted proline amides was synthesized and evaluated as inhibitors of dipeptidyl pepdidase IV for the treatment of type 2 diabetes. (3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone (5) emerged as a potent (IC(50) = 13 nM) and selective compound, with high oral bioavailability in preclinical species and low plasma protein binding. Compound 5, PF-00734200, was selected for development as a potential new treatment for type 2 diabetes.

Enhancement of the activity of phenoxodiol by cisplatin in prostate cancer cells.

Phenoxodiol is a novel isoflav-3-ene, currently undergoing clinical trials, that has a broad in vitro activity against a number of human cancer cell lines. Phenoxodiol alone inhibited DU145 and PC3 in a dose- and time-dependent manner with IC(50) values of 8+/-1 and 38+/-9 microM, respectively. The combination of phenoxodiol and cisplatin was synergistic in DU145, and additive in PC3, as assessed by the Chou-Talalay method. Carboplatin was also synergistic in combination with phenoxodiol in DU145 cells. The activity of the phenoxodiol and cisplatin combination was confirmed in vivo using a DU145 xenograft model in nude mice. Pharmacokinetic data from these mice suggest that the mechanism of synergy may occur through a pharmacodynamic mechanism. An intracellular cisplatin accumulation assay showed a 35% (P<0.05) increase in the uptake of cisplatin when it was combined in a ratio of 1 microM:5 microM phenoxodiol, resulting in a 300% (P<0.05) increase in DNA adducts. Taken together, our results suggest that phenoxodiol has interesting properties that make combination therapy with cisplatin or carboplatin appealing.

Vascular calcifications in homozygote familial hypercholesterolemia.

BACKGROUND: Patients with homozygous familial hypercholesterolemia (hmzFH) attributable to LDL receptor gene mutations have shown a remarkable increase in survival over the last 20 years. Early onset coronary heart disease (CHD) and calcific aortic valve stenosis are the major complications of this disorder. We now report extensive premature calcification of the aorta in patients with hmzFH. METHODS AND RESULTS: We examined 25 hmzFH patients from Canada; mean age was 32 years (range 5 to 54), and mean baseline cholesterol before treatment was 19+/-5 mmol/L (737+/-206 mg/dL). Aortic calcification was quantified using computed tomography (CT). An elevated mean calcium score was found in patients by age 20 and correlated with age (r(2)=0.53, P=0.001). One quarter (24%) of patients underwent aortic valve surgery. CONCLUSIONS: We document premature severe aortic calcifications in all adult hmzFH patients studied. These presented considerable surgical management challenges. Strategies to identify and monitor aortic calcification in hmzFH by noninvasive techniques are required, as are clinical trials to determine whether additional or more intensive therapies will prevent the progression of such calcifications. Whether vascular calcifications in hmzFH subjects are related to sustained increases in LDL-C levels or to other mechanisms, such as abnormal osteoblast activity, remains to be determined.

Comparative effects of 10-mg versus 80-mg Atorvastatin on high-sensitivity C-reactive protein in patients with stable coronary artery disease: results of the CAP (Comparative Atorvastatin Pleiotropic effects) study.

BACKGROUND: The major beneficial effect of statins- reducing the risk for coronary events-has primarily been ascribed to reductions in low-density lipoprotein cholesterol (LDL-C) but may in part be related to a direct antiinflammatory action (ie, decreased high-sensitivity C-reactive protein [hs-CRP] concentration). OBJECTIVES: The objectives of this CAP (Comparative Atorvastatin Pleiotropic Effects) study were to compare the effects of low- versus high-dose atorvastatin on hs-CRP concentrations and to determine the relationship between changes in LDL-C and hs-CRP concentrations in patients with coronary artery disease (CAD), low-grade inflammation, and normal lipoprotein concentrations. METHODS: This multicenter, prospective, randomized, double-blind, double-dummy study was conducted at 65 centers across Canada and Europe. Patients with documented CAD, low-grade inflammation (hs-CRP concentration, 1.5-15.0 mg/L), and a normal-range lipid profile (LDL-C concentration, 1.29-3.87 mmol/L [50-150 mg/dL]; triglyceride [TG] concentration, <4.56 mmol/L [<400 mg/dL]) were randomly assigned to receive 26-week double-blind treatment with atorvastatin 10 or 80 mg QD. Investigators were to aim for the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) LDL-C target of <2.59 mmol/L (<100 mg/dL). The primary end point was the percentage change from baseline in hs-CRP, as measured at baseline and weeks 5, 13, and 26 using high-sensitivity, latex microparticle-enhanced immunoturbidimetric assay. Changes from baseline in LDL-C, as measured directly in serum at the same time points, were also calculated. The secondary efficacy variables included the percentage changes from baseline in lipid parameters (LDL-C, high-density lipoprotein cholesterol [HDL-C], total cholesterol [TC], TG, apolipoprotein B, non-HDL-C, and TC:HDL-C ratio) at 5, 13, and 26 weeks of treatment. Tolerability was assessed using physical examination, including vital sign measurement, and laboratory analyses. RESULTS: A total of 339 patients were enrolled (283 men, 56 women; mean age, 62.5 years; weight, 81.3 kg; 10-mg/d group, 170 patients; 80-mg/d group, 169). No significant differences in baseline demographic or clinical data were found between the 2 treatment arms. In the 10-mg group, hs-CRP was decreased by 25.0% at 5 weeks and remained stable thereafter (%Delta at week 26, -24.3%; P < 0.01). In the 80-mg group, hs-CRP was decreased by 36.4% at 5 weeks and continued to be decreased over the study period (%Delta, -57.1% at week 26; P < 0.001 vs baseline). At 5 weeks, LDL-C was decreased by 35.9% in the 10-mg group and by 52.7% in the 80-mg group (P < 0.001 between groups) and remained stable thereafter (%Delta at week 26, -34.8% and -51.3%, respectively; P < 0.001 between groups). The NCEP ATP III LDL-C target of <2.59 mmol/L (<100 mg/dL) was reached in 77.1% of patients treated with atorvastatin 10 mg and 92.3% of those treated with 80 mg (P < 0.001). Dual targets of hs-CRP <2 mg/L and LDL-C <1.81 mmol/L (<70 mg/dL) were reached in a significantly greater proportion of patients in the 80-mg group compared with the 10-mg group (55.6% vs 13.5%; P < 0.001). The decrease in hs-CRP was largely independent of baseline LDL-C and change in LDL-C. Two serious adverse events were reported by the investigator as treatment related: acute hepatitis in the 10-mg group and intrahepatic cholestasis in the 80-mg group, in 2 patients with multiple comorbidities. Two deaths occurred during the study, both in the atorvastatin 80-mg group (1, myocardial infarction; 1, sudden death), neither of which was deemed treatment related by the investigator. CONCLUSIONS: In these patients with documented CAD, evidence of low-grade inflammation, and normal range lipid profiles, the effects of atorvastatin on changes in hs-CRP were dose dependent, with the high dose (80 mg) being associated with significantly greater reductions in hs-CRP concentrations. Both doses were associated with a significant and progressive decline in hs-CRP largely independent of changes in LDL-C, HDL-C, and TG. Clinical Trials Identification Number: NCT00163202.

Design and synthesis of potent amido- and benzyl-substituted cis-3-amino-4-(2-cyanopyrrolidide)pyrrolidinyl DPP-IV inhibitors.

The cis-3-amino-4-(2-cyanopyrrolidide)-pyrrolidine template has been shown to afford low nanomolar inhibitors of human DPP-IV that exhibit a robust PK/PD profile. An X-ray co-crystal structure of 5 confirmed the proposed mode of binding. The potent single digit DPP-IV inhibitor 53 exhibited a preferred PK/PD profile in preclinical animal models and was selected for additional profiling.

(3R,4S)-4-(2,4,5-Trifluorophenyl)-pyrrolidin-3-ylamine inhibitors of dipeptidyl peptidase IV: synthesis, in vitro, in vivo, and X-ray crystallographic characterization.

A series of pyrrolidine based inhibitors of dipeptidyl peptidase IV were developed from a high throughput screening hit for the treatment of type 2 diabetes. Potency, selectivity, and pharmacokinetic properties were optimized resulting in the identification of a pre-clinical candidate for further profiling.

Automated sample exchange and tracking system for neutron research at cryogenic temperatures.

An automated system for sample exchange and tracking in a cryogenic environment and under remote computer control was developed. Up to 24 sample "cans" per cycle can be inserted and retrieved in a programed sequence. A video camera acquires a unique identification marked on the sample can to provide a record of the sequence. All operations are coordinated via a LABVIEW program that can be operated locally or over a network. The samples are contained in vanadium cans of 6-10 mm in diameter and equipped with a hermetically sealed lid that interfaces with the sample handler. The system uses a closed-cycle refrigerator (CCR) for cooling. The sample was delivered to a precooling location that was at a temperature of approximately 25 K, after several minutes, it was moved onto a "landing pad" at approximately 10 K that locates the sample in the probe beam. After the sample was released onto the landing pad, the sample handler was retracted. Reading the sample identification and the exchange operation takes approximately 2 min. The time to cool the sample from ambient temperature to approximately 10 K was approximately 7 min including precooling time. The cooling time increases to approximately 12 min if precooling is not used. Small differences in cooling rate were observed between sample materials and for different sample can sizes. Filling the sample well and the sample can with low pressure helium is essential to provide heat transfer and to achieve useful cooling rates. A resistive heating coil can be used to offset the refrigeration so that temperatures up to approximately 350 K can be accessed and controlled using a proportional-integral-derivative control loop. The time for the landing pad to cool to approximately 10 K after it has been heated to approximately 240 K was approximately 20 min.

Safety of Triamcinolone acetonide (TA)-assisted pars plana vitrectomy in macular hole surgery.

AIM: To evaluate whether Triamcinolone acetonide (TA)-assisted pars plana vitrectomy for visualisation of posterior hyaloid during macular hole surgery has any adverse effects on macular hole closure rate and intraocular pressure (IOP). METHODS: Case series comparing outcomes and adverse effects in patients who had surgery for macular holes with ILM peel, with and without the use of TA-assisted vitrectomy. RESULTS: During the study period, 29 patients had vitrectomy for macular holes. In 18 patients (group 1), TA was used intraoperatively to facilitate visualisation of the posterior hyaloid and in 11 patients (group 2) no TA was used. There was no statistically significant difference in the macular hole closure rates and the improvement in visual acuity between the two groups. No long-term increase in IOP was recorded in any of the 29 patients. The total anatomical success rate in both groups was 85.6% and the average improvement in visual acuity in both groups was two Snellen lines. CONCLUSIONS: TA is safe and there is no contraindication for its use as an intraoperative aid to facilitate vitreous visualisation in macular hole surgery.

cis-2,5-dicyanopyrrolidine inhibitors of dipeptidyl peptidase IV: synthesis and in vitro, in vivo, and X-ray crystallographic characterization.

Inhibitors of the glucagon-like peptide-1 (GLP-1) degrading enzyme dipeptidyl peptidase IV (DPP-IV) have been shown to be effective treatments for type 2 diabetes in animal models and in human subjects. A novel series of cis-2,5-dicyanopyrrolidine alpha-amino amides were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV) for the treatment of type 2 diabetes. 1-({[1-(Hydroxymethyl)cyclopentyl]amino}acetyl)pyrrolidine-2,5-cis-dicarbonitrile (1c) is an achiral, slow-binding (time-dependent) inhibitor of DPP-IV that is selective for DPP-IV over other DPP isozymes and proline specific serine proteases, and which has oral bioavailability in preclinical species and in vivo efficacy in animal models. The mode of binding of the cis-2,5-dicyanopyrrolidine moiety was determined by X-ray crystallography. The hydrochloride salt of 1c was further profiled for development as a potential new treatment for type 2 diabetes.