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Objective: Examine real-world effectiveness and hypoglycemia cost burden in patients with type 2 diabetes newly initiated on exenatide or insulin glargine.Design and Methods: Retrospective cohort study describing patient characteristics, drug adherence patterns, and 1-year hypoglycemia rates with associated costs using an administrative claims database. Adult subjects with type 2 diabetes had an initial claim for exenatide or insulin glargine between May 1, 2005 and June 30, 2007, and had continuous eligibility for >or= 6 months pre- and >or= 12 months post-initiation.Results: The exenatide cohort (n = 3262) was 53 +/- 10 years (+/-SD); 54% female. The insulin glargine cohort (n = 3038) was 56 +/- 12 years; 41% female. The mean Deyo-Charlson comorbidity index score was 1.45 for exenatide versus 1.82 for insulin glargine (p < 0.001). Baseline OAD use rates for exenatide and insulin glargine, respectively, were 77% versus 69% metformin; 47% versus 65% sulfonylurea; 50% versus 49% thiazolidinedione; 56% versus 60% multiple OAD. For patients with two or more pharmacy claims for exenatide or insulin glargine, the 12-month medication possession ratio (MPR) was 68 +/- 29% for exenatide and 58 +/- 28% for insulin glargine (p < 0.001). MPR >or= 80% was higher for exenatide (p < 0.001) and fewer patients discontinued therapy (p < 0.001). The probability of a hypoglycemic event was significantly lower for exenatide (p < 0.005), resulting in lower associated annual costs.Conclusions: This study provides the first real-world observational comparison of type 2 diabetes patients newly initiated on exenatide or insulin glargine. Exenatide patients had a lower comorbidity burden, better drug adherence, and a lower rate of hypoglycemic events with associated costs. Retrospective database analyses examine medical care utilization in large populations using a relatively inexpensive and expedient approach. However, data are only representative of a commercial health-care plan with limited information on multiple variables usually collected during clinical trials. [ABSTRACT FROM AUTHOR]

Background: Exenatide, an incretin mimetic for adjunctive treatment of type 2 diabetes (T2DM), reduced hemoglobin A(1c) (A1C) and weight in clinical trials. The objective of this study was to evaluate the effects of > or = 3 years exenatide therapy on glycemic control, body weight, cardiometabolic markers, and safety.Methods: Patients from three placebo-controlled trials and their open-label extensions were enrolled into one open-ended, open-label clinical trial. Patients were randomized to twice daily (BID) placebo, 5 mug exenatide, or 10 mug exenatide for 30 weeks, followed by 5 mug exenatide BID for 4 weeks, then 10 mug exenatide BID for > or = 3 years of exenatide exposure. Patients continued metformin and/or sulfonylureas.Results: 217 patients (64% male, age 58 +/- 10 years, weight 99 +/- 18 kg, BMI 34 +/- 5 kg/m(2), A1C 8.2 +/- 1.0% [mean +/- SD]) completed 3 years of exenatide exposure. Reductions in A1C from baseline to week 12 (-1.1 +/- 0.1% [mean +/- SEM]) were sustained to 3 years (-1.0 +/- 0.1%; p < 0.0001), with 46% achieving A1C < or = 7%. Exenatide progressively reduced body weight from baseline (-5.3 +/- 0.4 kg at 3 years; p < 0.0001). Patients with elevated serum alanine aminotransferase (ALT) at baseline (n = 116) had reduced ALT (-10.4 +/- 1.5 IU/L; p < 0.0001) and 41% achieved normal ALT. Patients with elevated ALT at baseline tended to lose more weight than patients with normal ALT at baseline (-6.1 +/- 0.6 kg vs. -4.4 +/- 0.5 kg; p = 0.03), however weight change was minimally correlated with baseline ALT (r = -0.01) or ALT change (r = 0.31). Homeostasis Model Assessment B (HOMA-B), blood pressure, and aspartate aminotransferase (AST) all improved. A subset achieved 3.5 years of exenatide exposure and had serum lipids available for analysis (n = 151). Triglycerides decreased 12% (p = 0.0003), total cholesterol decreased 5% (p = 0.0007), LDL-C decreased 6% (p < 0.0001), and HDL-C increased 24% (p < 0.0001). Exenatide was generally well tolerated. The most frequent adverse event was mild-to-moderate nausea. The main limitation of this study is the open-label, uncontrolled nature of the study design which does not provide a placebo group for comparison.Conclusion: Adjunctive exenatide treatment for > or = 3 years in T2DM patients resulted in sustained improvements in glycemic control, cardiovascular risk factors, and hepatic biomarkers, coupled with progressive weight reduction. [ABSTRACT FROM AUTHOR]

Background and Purpose: Proton therapy for breast cancer is usually given in free breathing (FB). With the use of deep inspiration breath-hold (DIBH) technique, the location of the heart is displaced inferiorly, away from the internal mammary nodes and, thus, the dose to the heart can potentially be reduced. The aim of this study was to explore the potential benefit of proton therapy in DIBH compared to FB for highly selected patients to reduce exposure of the heart and other organs at risk. We aimed at creating proton plans with delivery times feasible with treatment in DIBH., Material and Methods: Sixteen patients with left-sided breast cancer receiving loco-regional proton therapy were included. The FB and DIBH plans were created for each patient using spot-scanning proton therapy with 2-3 fields, robust and single field optimization. For the DIBH plans, minimum monitor unit per spot and spot spacing were increased to reduce treatment delivery time., Results: All plans complied with target coverage constraints. The median mean heart dose was statistically significant reduced from 1.1 to 0.6 Gy relative biological effectiveness (RBE) by applying DIBH. No statistical significant difference was seen for mean dose and V17Gy RBE to the ipsilateral lung. The median treatment delivery time for the DIBH plans was reduced by 27% compared to the FB plans without compromising the plan quality., Interpretation: The median absolute reduction in dose to the heart was limited. Proton treatment in DIBH may only be relevant for a subset of these patients with the largest reduction in heart exposure.

Aim: To compare the efficacy and safety of once-weekly (OW) semaglutide versus thrice-daily (TID) insulin aspart (IAsp) in participants with inadequately controlled type 2 diabetes (T2D) treated with insulin glargine (IGlar) and metformin., Materials and Methods: SUSTAIN 11 (NCT03689374) was a randomized (1:1), parallel, open-label, multinational, phase 3b trial. After a 12-week run-in to optimize once-daily IGlar U100, 1748 adults with T2D (HbA1c >7.5% to ≤10.0%) were randomized to OW semaglutide or TID IAsp as add-on to optimized IGlar and metformin for 52 weeks. The primary outcome was change in HbA1c from randomization to week 52. Confirmatory secondary endpoints included the occurrence of severe hypoglycaemic episodes and change in body weight (BW). Safety was assessed., Results: HbA1c (randomization: 8.6% [70.0 mmol/mol]) decreased by 1.5% points (16.6 mmol/mol) and 1.2% points (13.4 mmol/mol) with semaglutide (n = 874) and IAsp (n = 874), respectively (estimated treatment difference [ETD] -0.29% points [95% confidence interval {CI} -0.38; -0.20]; P < .0001 for non-inferiority). Few severe hypoglycaemic episodes were recorded in either group, with no statistically significant difference between the groups. Change in BW from randomization (87.9 kg) to week 52 was in favour of semaglutide (-4.1 kg) versus IAsp (+2.8 kg) (ETD -6.99 kg [95% CI -7.41; -6.57]). A higher proportion of participants experienced adverse events with semaglutide (58.5%) versus IAsp (52.1%); most were mild to moderate., Conclusions: In this basal insulin-treated population, OW semaglutide improved glycaemic control to a greater extent than TID IAsp and provided numerically greater weight loss., (© 2022 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Behaviors that rely on the hippocampus are particularly susceptible to chronological aging, with many aged animals (including humans) maintaining cognition at a young adult-like level, but many others the same age showing marked impairments. It is unclear whether the ability to maintain cognition over time is attributable to brain maintenance, sufficient cognitive reserve, compensatory changes in network function, or some combination thereof. While network dysfunction within the hippocampal circuit of aged, learning-impaired animals is well-documented, its neurobiological substrates remain elusive. Here we show that the synaptic architecture of hippocampal regions CA1 and CA3 is maintained in a young adult-like state in aged rats that performed comparably to their young adult counterparts in both trace eyeblink conditioning and Morris water maze learning. In contrast, among learning-impaired, but equally aged rats, we found that a redistribution of synaptic weights amplifies the influence of autoassociational connections among CA3 pyramidal neurons, yet reduces the synaptic input onto these same neurons from the dentate gyrus. Notably, synapses within hippocampal region CA1 showed no group differences regardless of cognitive ability. Taking the data together, we find the imbalanced synaptic weights within hippocampal CA3 provide a substrate that can explain the abnormal firing characteristics of both CA3 and CA1 pyramidal neurons in aged, learning-impaired rats. Furthermore, our work provides some clarity with regard to how some animals cognitively age successfully, while others' lifespans outlast their "mindspans.", Competing Interests: The authors declare no competing interest.

Introduction: Once-weekly (OW) subcutaneous (s.c.) semaglutide is an injectable glucagon-like peptide-1 (GLP-1) analogue approved for the treatment of type 2 diabetes. This trial was designed to assess the pharmacokinetics, safety and tolerability of OW s.c. semaglutide in healthy Chinese subjects., Methods: In this single-centre, randomised, double-blind, placebo-controlled trial, 36 healthy subjects were randomised to OW s.c. semaglutide 0.5 mg (n = 12), 1.0 mg (n = 12), or placebo (n = 12). Treatment (semaglutide or placebo) was blinded for the subjects, investigators and sponsor. The primary endpoint was steady-state semaglutide exposure, defined as the area under the curve over a dosing interval at steady state (AUC 0-168 h,SS )., Results: In total, 34 subjects completed the trial. The steady-state exposure of semaglutide was higher for subjects treated with 1.0 mg semaglutide (AUC 0-168 h,ss : 7961 nmol h/l and C max,ss : 55.9 nmol/l) compared to 0.5 mg semaglutide (AUC 0-168 h,ss : 4000 nmol h/l and C max,ss : 28.8 nmol/l). The total exposure of semaglutide increased in a dose-proportional manner in healthy Chinese subjects; the treatment ratio (1.0 mg/0.5 mg) [95% confidence interval] for AUC 0-168 h,SS was 1.99 [1.78; 2.23]. Treatment with OW s.c. semaglutide was well tolerated in healthy Chinese subjects. As expected for the GLP-1 receptor agonist class, the most common adverse events were gastrointestinal, and no new safety signals were identified., Conclusion: The pharmacokinetics, safety and tolerability of OW s.c. semaglutide in healthy Chinese subjects were consistent with previous clinical pharmacology trials of OW s.c. semaglutide in other populations. The results suggest that no dose adjustment is necessary for semaglutide in Chinese patients with T2D., Trial Registration: ClinicalTrials.gov, identifier NCT03288740.

Objective: To conduct a systematic review on the effect of posterior tilt on reoperations, patient reported outcome measures (PROM) and functional outcome following osteosynthesis of undisplaced FNFs (uFNF)., Material and Methods: A search string was developed with the aid of a scientific librarian and the search was performed in PubMed, CINAHL and Embase. The studies were screened independently by two authors using Covidence. Data were extracted by two authors and quality assessment was performed using Robins-I tool. The meta-analyses were performed in STATA IC 16 using Risk Ratio as the primary effect estimate., Results: In total, 617 studies were screened and ten studies were included with a total of 3,131 patients. The mean age ranged from 68.3 to 85.0 years and the prevalence of posterior tilt ≥20° in the studies ranged from 4.5% to 27.6%. There were 10.3% reoperations when posterior tilt was <20° whereas there were 24.5% when posterior tilt was ≥20° The meta-analysis therefore demonstrated an overall risk ratio of 0.11 (95% confidence interval; 0.04-0.18). Only one study investigated functional outcome, using ADL as measurement, but found no significant difference. No studies investigated PROM. In general, the studies were assessed to be of poor quality mainly due possible bias and confounding., Conclusion: A posterior tilt ≥20° leads to a higher risk of reoperations in uFNF. Even though the studies were of poor quality, the results suggest that we should include the measurement of posterior tilt in national guidelines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Endurance athletes participating in sporting events may be required to complete multiple training sessions a day or on successive days with a limited recovery time. Nutritional interventions that enhance the restoration of endogenous fuel stores (e.g., liver and muscle glycogen) and improve muscle damage repair have received a lot of attention. The purpose of this review is to investigate the effect of ingesting carbohydrate (CHO) and protein (PRO) on athletic performance. Studies were identified by searching the electronic databases PubMed and EMBASE. Random-effects meta-analyses were conducted to examine the intervention efficacy. A total of 30 randomized controlled trials (RCT), comprising 43 trials and 326 participants in total, were included in this review. The meta-analysis showed an overall significant effect in Time-To-Exhaustion (TTE) and Time-Trial (TT) performance, when ingesting carbohydrates and proteins (CHO-PRO) compared to CHO-only ( p = 0.03 and p = 0.0007, respectively). A subgroup analysis demonstrated a significant effect in TTE by ingesting CHO-PRO compared to CHO, when supplements were provided during and/or following an exercise bout. CHO-PRO significantly improved TTE compared to CHO-only, when a long-term recovery (i.e., ≥8 h) was implemented ( p = 0.001). However, no effect was found when the recovery time was short-term (i.e., ≤8 h). No significant effect was observed when CHO-PRO and CHO-only supplements were isocaloric. However, a significant improved TTE was evident with CHO-PRO compared to CHO-only, when the supplements were matched for carbohydrate content ( p < 0.00001). In conclusion, co-ingesting carbohydrates and proteins appears to enhance TTE and TT performance compared to CHO-only and presents a compelling alternate feeding strategy for athletes.

We introduce a remote interface to control and optimize the experimental production of Bose-Einstein condensates (BECs) and find improved solutions using two distinct implementations. First, a team of theoreticians used a remote version of their dressed chopped random basis optimization algorithm (RedCRAB), and second, a gamified interface allowed 600 citizen scientists from around the world to participate in real-time optimization. Quantitative studies of player search behavior demonstrated that they collectively engage in a combination of local and global searches. This form of multiagent adaptive search prevents premature convergence by the explorative behavior of low-performing players while high-performing players locally refine their solutions. In addition, many successful citizen science games have relied on a problem representation that directly engaged the visual or experiential intuition of the players. Here we demonstrate that citizen scientists can also be successful in an entirely abstract problem visualization. This is encouraging because a much wider range of challenges could potentially be opened to gamification in the future., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation.

Myelin - the multilayer membrane that envelops axons - is a facilitator of rapid nerve conduction. Oligodendrocytes form CNS myelin; the prevailing hypothesis being that they do it by extending a process that circumnavigates the axon. It is pertinent to ask how myelin is built because oligodendrocyte plasma membrane and myelin are compositionally different. To this end, we examined oligodendrocyte cultures and embryonic avian optic nerves by electron microscopy, immuno-electron microscopy and three-dimensional electron tomography. The results support three novel concepts. Myelin membranes are synthesized as tubules and packaged into "myelinophore organelles" in the oligodendrocyte perikaryon. Myelin membranes are matured in and transported by myelinophore organelles within an oligodendrocyte process. The myelin sheath is generated by myelin membrane fusion inside an oligodendrocyte process. These findings abrogate the dogma of myelin resulting from a wrapping motion of an oligodendrocyte process and open up new avenues in the quest for understanding myelination in health and disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide library approach with a peptide-HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C-specific peptide-binding data and update our pan-specific NetMHCpan predictor, whose predictive performance was considerably improved with respect to peptide binding to HLA-C. The updated predictor was used to assess the specificities of HLA-C molecules, which were found to cover a more limited sequence space than HLA-A and -B molecules. Assessing the functional significance of these new tools, HLA-C*07:01 transgenic mice were immunized with stable HLA-C*07:01 binders; six of six tested stable peptide binders were immunogenic. Finally, we generated HLA-C tetramers and labeled human CD8(+) T cells and NK cells. These new resources should support future research on the biology of HLA-C molecules. The data are deposited at the Immune Epitope Database, and the updated NetMHCpan predictor is available at the Center for Biological Sequence Analysis and the Immune Epitope Database., (Copyright © 2014 by The American Association of Immunologists, Inc.)

2-Monoacylglycerols are gaining increasing interest as signaling lipids, beyond endocannabinoids, for example, as ligands for the receptor GPR119 and as mediators of insulin secretion. In the vascular system, they are formed by the action of lipoprotein lipase (LPL); however, their further disposition is unclear. Assuming similar affinity for uptake and incorporation into tissues of 2-oleoylglycerol and 2-oleylglyceryl ether, we have synthesized a (3)H-labeled 2-ether analog of triolein (labeled in alkyl group) and compared its disposition with (14)C-labeled triolein (labeled in glycerol) 20 min after intravenous coadministration in a ratio of 1:1 to mice. We found that peripheral tissues and the liver in particular are able to take up 2-monoacylglycerols as seen from (3)H uptake. In muscle and adipose tissue, 2-monoacylglycerols are probably further hydrolyzed as seen by an increased (3)H/(14)C ratio, whereas in the liver and the heart, data suggest that they are also subjected to re-esterification to triacylglycerol, as seen by an unchanged (3)H/(14)C ratio in the lipid fraction of the tissues. Our findings suggest that LPL-generated 2-monoacylglycerol is likely to be stable in the vascular system and thus have a potential to circulate or at least exert effects in tissues where it may be locally produced., (© 2014 International Union of Biochemistry and Molecular Biology.)

Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides. As a general approach to class II tetramer generation, this method should support rational CD4+ T cell epitope discovery as well as enable specific monitoring and manipulation of CD4+ T cell responses.

Background: Patients with cancer experience side effects related to their antineoplastic treatment. Demands for efficiency limit the time patients spend with health professionals. This requires that professionals are able to offer adequate support to patients in coping with side effects of treatment in everyday life. However, it appears that the level of information about and support in living with side effects may be insufficient., Objective: To explore patients' experiences of how side effects from chemotherapy and radiotherapy impact everyday life as well as the information needs in coping with these side effects., Methods: Nine patients' experiences were analysed and interpreted using a phenomenological-hermeneutical approach. Data were collected through semi-structured interviews., Findings: The patients are deeply impacted by side effects in their everyday life. They struggle to take power but experience loss of control. They feel inexperienced without knowledge in an 'ongoing system', and because of lack of continuity, they do not come up with their real needs. To avoid pitfalls in the information, the patients need individualized support from the professionals., Conclusion: If the patients have to be supported during everyday life with side effects from cancer treatment in an individualized way, the professional must be aware that the patients risk adapting to the busy healthcare system. To empower the patient, the professional must be able to sense the patients' appeal for help and enter into caring relationships., Implications for Practice: To ensure targeted information, the relationship between nurse and patient must build on nursing values including the patients' illness - perspective. However, the professionals must take a critical look at how they provide information and how they act to empower the patients., (© 2012 The Authors Scandinavian Journal of Caring Sciences © 2012 Nordic College of Caring Science.)

The objective was to describe clinical findings and pain patterns following the provocation by activities of daily living (ADL) in women suffering from pregnancy-related post-natal pelvic pain 6-12 months after delivery. Forty-one informants answered questionnaires regarding pain characteristics in respect to 14 different ADL. All informants had bilateral pain, and typically it was described as an aching pain in the areas of the posterior superior iliac spines and in the symphyseal region. Running was the most provocative activity, followed by domestic work and by activities involving pushing and pulling. Duration of the activity had a great impact on the tolerance for all activities, and for the majority of the women, menstruation and ovulation caused an exacerbation of the symptoms.

Aim: This study evaluated changes in clinical effectiveness measures of patients with type 2 diabetes initiating exenatide therapy in a real-world setting., Methods: Eligible patients identified in the General Electric (GE) electronic medical record (EMR) research database from 1 January 2000 through 31 December 2007 were > or =18 years old with type 2 diabetes. Patients had prescription orders in the previous 395 days for metformin, a sulfonylurea, or a thiazolidinedione as monotherapy or in combination, and had at least 6 months of follow-up activity. Baseline clinical measures were documented from 45 days prior up to 15 days after exenatide initiation and follow-up measures documented at 6 months +/- 45 days., Results: A total of 1709 patients were identified for study inclusion. The overall mean A1C reduction (s.e.m.) at 6 months was -0.8% (0.05) (p<0.001), weight loss was -3.2 kg (0.14) (p<0.001), blood pressure (BP) lowering was -1.9 mmHg (0.46) systolic blood pressure (SBP) (p<0.001) and -0.5 mmHg (0.27) diastolic blood pressure (DBP) (p = 0.078). Changes in low-density lipoprotein (LDL), triglycerides and HDL were -7.4 mg/dl (1.7) (p<0.001), -23.2 mg/dl (6.7) (p = 0.001) and -0.8 mg/dl (0.33) (p = 0.012) respectively. In a quartile analysis by weight loss, mean A1C reduction ranged from -1.1 to -0.65% in the highest to lowest weight loss quartiles respectively., Conclusions: In a real-world setting, exenatide initiation is associated with significant improvements in the measures of clinical effectiveness for type 2 diabetes. These reductions were comparable to those reported in randomized, controlled registration trials after 6 months of therapy.

Background: Molecules of the class II major histocompability complex (MHC-II) specifically bind and present exogenously derived peptide epitopes to CD4+ T helper cells. The extreme polymorphism of the MHC-II hampers the complete analysis of peptide binding. It is also a significant hurdle in the generation of MHC-II molecules as reagents to study and manipulate specific T helper cell responses. Methods to generate functional MHC-II molecules recombinantly, and measure their interaction with peptides, would be highly desirable; however, no consensus methodology has yet emerged., Results: We generated alpha and beta MHC-II chain constructs, where the membrane-spanning regions were replaced by dimerization motifs, and the C-terminal of the beta chains was fused to a biotinylation signal peptide (BSP) allowing for in vivo biotinylation. These chains were produced separately as inclusion bodies in E. coli , extracted into urea, and purified under denaturing and non-reducing conditions using conventional column chromatography. Subsequently, diluting the two chains into a folding reaction with appropriate peptide resulted in efficient peptide-MHC-II complex formation. Several different formats of peptide-binding assay were developed including a homogeneous, non-radioactive, high-throughput (HTS) binding assay. Binding isotherms were generated allowing the affinities of interaction to be determined. The affinities of the best binders were found to be in the low nanomolar range. Recombinant MHC-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately., Conclusion: We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools.

Fusion tags add desirable properties to recombinant proteins, but they are not necessarily acceptable in the final products. Ideally, fusion tags should be removed releasing the intact native protein with no trace of the tag. Unique endoproteinases with the ability to cleave outside their own recognition sequence can potentially cleave at the boundary of any native protein. Chymosin was recently shown to cleave a pro-chymosin derived fusion tag releasing native target proteins. In our hands, however, not all proteins are chymosin-resistant under the acidic cleavage conditions (pH 4.5) used in this system. Here, we have modified the pro-chymosin fusion tag and demonstrated that chymosin can remove this tag at more neutral pH (pH 6.2); conditions, that are less prone to compromise the integrity of target proteins. Chymosin was successfully used to produce intact native target protein both at the level of small and large-scale preparations. Using short peptide substrates, we further examined the influence of P1' amino acid (the N-terminus of the native target protein) and found that chymosin accepts many different, although not all, amino acids. We conclude that chymosin has several appealing characteristics for the exact removal of fusion tags. It is readily available in highly purified recombinant versions approved by the FDA for preparation of food for human consumption. We suggest that one should consider extending the use of chymosin to the preparation of pharmaceutical proteins.

A synthesis of the hasubanan alkaloids hasubanonine, runanine, and aknadinine via a unified route was attempted. Construction of key phenanthrene intermediates by a Suzuki coupling-Wittig olefination-ring-closing metathesis sequence allowed a convergent and flexible approach. Conversion of the phenanthrenes into the target structures was projected to involve six steps including phenolic oxidation, ketone allylation, anionic oxy-Cope rearrangement, and acid-promoted cyclization. The final step was thwarted by a pinacol-like rearrangement that delivered the unnatural isohasubanan alkaloid skeleton. The structures of the products were established by exhaustive NMR experiments and confirmed by GIAO (13)C NMR calculations of runanine, isorunanine, and three other isomers. These computations revealed some inconsistencies with the benzene solvent correction which suggest that caution should be used in employing this algorithm. The racemic synthesis of isohasubanonine was transformed into an enantioselective synthesis by the discovery that Nakamura's chiral bisoxazoline-ligated allylzinc reagent mediates the enantioselective allylation of ketone 19 in 93% ee. This method could be extended to three other structurally related ketones (92-96% ee), and the enantioselective syntheses of two other isohasubanan alkaloids, isorunanine and isoaknadinine, were accomplished. Racemic isohasubanonine was found to be an ineffective analgesic agent.

Exenatide is a 39-amino acid peptide incretin mimetic approved for adjunctive treatment of type 2 diabetes. It shares several glucoregulatory activities with the mammalian hormone, glucagon-like peptide-1 (GLP-1). In clinical use, subcutaneous exenatide injections demonstrate glucoregulatory and weight loss effects with sustained plasma concentrations in the 50-100 pM range. We investigated the pharmacokinetics of exenatide in normoglycemic rats and biological activity in diabetic db/db mice after delivery to various epithelial surfaces of the intestinal and respiratory tracts. In rats, elimination kinetics were similar for all routes of administration (median k(e) 0.017 min(-1)). Bioavailability (versus intravenous administration) and C(max) per unit dose differed markedly. For gastrointestinal administration, sublingual administration invoked the highest bioavailability (0.37%); in db/db mice, potentially therapeutic concentrations were obtainable. In contrast, intraduodenal bioavailability was low (0.0053%). In regard to respiratory surfaces, bioavailability of intratracheal exenatide was up to 13.6%, and for nasal administration, 1.68%. Both routes of administration produced therapeutic plasma concentrations and glucose-lowering in db/db mice. At high doses, aerosolized exenatide also achieved effective concentrations and glucose-lowering. In summary, the intestinal tract seems to have limited potential as a route of exenatide administration, with sublingual being most promising. In contrast, the respiratory tract appears to be more viable, comparing favorably with the clinically approved subcutaneous route. Despite little optimization of the delivery formulation, exenatide bioavailability compared favorable to that of several commercially available bioactive peptides.

Obesity increases the risk of diabetes up to 90-fold and worsens hyperglycemia, hyperinsulinemia, insulin resistance, dyslipidemia, and nonalcoholic fatty liver disease. For patients with type 2 diabetes, weight loss can trigger improvements in all these conditions and decrease the need for glucose-lowering agents. The incretin mimetic exenatide shares many glucoregulatory properties with native glucagon-like peptide-1, including enhancement of glucose-dependent insulin secretion, glucose-dependent suppression of inappropriately high glucagon secretion, slowing of gastric emptying, and reduction of food intake in patients with type 2 diabetes. Exenatide treatment was associated with progressive weight loss in the majority of patients in clinical trials. In addition, patients with elevated markers of liver injury at baseline showed improvements. Therefore, exenatide represents a unique option for adjunctive therapy for patients with type 2 diabetes not achieving adequate glycemic control on oral antidiabetic agents, especially in patients for whom weight gain would be an additional contraindication.

Introduction: Hospital admissions due to exacerbations of COPD are frequent. UK studies have shown that early supported discharge (ESD) for patients with exacerbations of COPD can reduce the length of stay without adversely affecting mortality or readmission rates. However, experience of ESD in Denmark has not been reported., Materials and Methods: Hospital admissions due to exacerbations of COPD at Hvidovre Hospital in a 1-year period were reviewed., Results: In the study period, the number of admissions due to exacerbations was 875 and the mean length of stay was 6.3 days in contrast to 5.8 days the previous year; 159 (18.2%) admissions in 108 patients were suitable for ESD. Prior to ESD, the mean duration of in-hospital stay was 4.0 days. Patients selected for ESD had severe COPD with FEV1 31.8% (7-89%) of predicted value. They had on average 3.8 (1-11) home visits in a mean period of 10.5 (1-29) days. While being cared for at home, one patient died and readmission was necessary in 19 (17.6%) cases. Within three months 51.4% of the patients were readmitted and 14.8% died. The income and costs related to ESD were approximately 120,000 EUR and 75,000 EUR, respectively., Conclusion: Almost 20% of all admissions were eligible for ESD. Compared to British studies on ESD, our patients had more severe COPD. We cannot determine whether this model of care has reduced days in hospital, but costs and income seem to balance.

Background: Exenatide, an incretin mimetic for adjunctive treatment of type 2 diabetes mellitus (T2DM), reduced glycosylated hemoglobin (HbA(1c)) and weight in 30-week placebo-controlled trials. Some patients were followed up in open-label extensions to provide 'real-world' exenatide clinical experience., Objective: The purpose of this study was to examine the metabolic effects of 2 years of exenatide treatment in patients with T2DM., Methods: For this interim analysis, data were pooled from patients who completed 1 of three 30-week, multicenter, double-blind, placebo-controlled trials and their open-label extensions. In the initial trials, subjects were randomized to BID 5-microg exenatide, 10-microg exenatide, or placebo for 30 weeks. All subjects who enrolled in the extension phase then received 5-pg exenatide BID for 4 weeks, followed by open-label treatment with 10-pg exenatide BID. Subjects continued their existing metformin and/or sulfonylurea regimens. Analyses were conducted on data from all subjects who had the opportunity to achieve 2 years of exenatide exposure, irrespective of their treatment arm in the 30-week placebo-controlled trials., Results: A total of 974 patients entered the open-label, extension phase of the trial. Two hundred eighty-three subjects (mean [SD] age, 57 [10] years; mean [SD] weight, 100[19] kg; sex, 63% male; mean [SD] body mass index, 34 [6] kg/m(2); mean [SD] HbA(1c), 8.3% [1.0%]) completed 2 years of exenatide treatment. Reductions in mean (SE) HbA(1c) from baseline to week 30 (-0.9% [0.1%]) were sustained through 2 years (-1.1% [0.1%]; P < 0.05 vs baseline), with 50% of the population achieving HbA(1c) < or = 7%. At week 30, exenatide was associated with a significant reduction in mean (SD) body weight from baseline (-2.1 [0.2] kg), with progressive reductions after 2 years (-4.7 [0.3] kg; P < 0.001 vs baseline). Patients with normal baseline alanine aminotransferase (ALT) (132/283 [47%]; normal: female < or =19 IU/L; male < or =30 IU/L) had no significant ALT change. However, patients with elevated ALT at baseline (151/283 [53%]) had a mean (SEM) reduction of ALT (-11 [1] IU/L from baseline 38 [1] IU/1; P < 0.05) and 39% achieved normal ALT by week 104. Patients with elevated ALT at baseline lost significantly more weight than patients with normal ALT at baseline (P = 0.04). However, weight change was minimally correlated with baseline ALT (r = -0.09) or ALT change (r = 0.31). Also, homeostasis model assessment of the beta-cell function (HOMA-B), blood pressure, and aspartate aminotransferase (AST) all improved. The most frequently reported adverse event was mild-to-moderate nausea., Conclusions: In these patients with T2DM, adjunctive exenatide treatment for 2 years was generally well tolerated and resulted in a sustained reduction of HbA(1c), progressive reduction in weight, and improvements in HOMA-B, blood pressure, and the hepatic injury biomarkers, AST and ALT.

The incretin mimetic exenatide improved glycemic control and reduced body weight in patients with type 2 diabetes inadequately controlled with metformin+/-a sulfonylurea. We assessed postprandial beta-cell function by mathematical modeling, independent of confounding effects from differing ambient glucose levels among treatments. Subjects were 63% males, 55+/-10 years, BMI 33+/-6 kg/m2, HbA1C 8.1+/-1.1% (+/- SD) randomized to 5 microg exenatide or placebo twice daily for 4 weeks. Subsequently, one arm remained at 5 microg twice daily, one arm escalated to 10 microg twice daily, and one treatment arm remained on placebo for 26 weeks. Subjects continued metformin+/-a sulfonylurea. A subset with meal tests at baseline and week 30 were analyzed (n=73). Outcome measures were the model-based beta-cell function parameters dose-response relating insulin secretion to glucose concentration, rate sensitivity, and potentiation. Exenatide reduced postprandial glucose excursions. Modeling predicted an upward shift of the beta-cell dose-response. Model-predicted insulin secretion rate at a reference glucose concentration increased 72% (10 microg), increased 40% (5 microg), or decreased 21% (placebo) at week 30 [ p=0.015 (10 microg); p=0.045 (5 microg); vs. placebo]. At week 30, the 2-hour post-meal to basal potentiation factor ratio was increased to 1.53+/-0.10 (10 microg; p=0.0142 vs. placebo) or 1.40+/-0.08 (5 microg; p=0.0402 vs. placebo) compared with 1.15+/-0.06 (placebo). Exenatide caused an upward shift of the beta-cell dose-response and enhanced potentiation of insulin secretion. This model suggests exenatide improved beta-cell function in patients with type 2 diabetes treated with metformin+/-a sulfonylurea.

Incretin mimetics are a new class of pharmacological agents with multiple antihyperglycemic actions that mimic several of the actions of incretin hormones originating in the gut, such as glucagon-like peptide (GLP)-1. Dipeptidyl peptidase-IV (DPP-IV) inhibitors suppress the degradation of many peptides, including GLP-1, thereby extending their bioactivity. These agents seem to have multiple mechanisms of action for the treatment of type 2 diabetes mellitus (T2DM), including some or all the following: enhancement of glucose-dependent insulin secretion, suppression of inappropriately elevated glucagon secretion, slowing of gastric emptying, and decreased food intake. Exenatide (BYETTA) is the first incretin mimetic approved for clinical use by the US Food and Drug Administration. In phase 3 clinical trials, exenatide reduced HbA(1c) by approximately 1% and body weight by approximately 2 kg in T2DM patients failing to achieve glycemic control with metformin and/or a sulfonylurea, with mild-to-moderate nausea the most common side effect. Several GLP-1 analogues and DPP-IV inhibitors are in late-stage clinical testing and may soon become available for treating T2DM patients. The use of these agents may provide an opportunity to bring about new improvements in diabetes care.

Aim: The ability of the incretin mimetic exenatide to improve glycaemic control and reduce body weight was assessed over 82 weeks in patients with type 2 diabetes failing to achieve glycaemic control with maximally effective doses of metformin., Methods: In this interim 82-week analysis, 150 (total cohort) of an eligible population of 183 patients opted to continue exenatide treatment in an uncontrolled open-label extension of a 30-week double-blind, placebo-controlled trial. Of these, 92 patients (completer cohort) achieved 82 weeks of exenatide therapy. Patients continued metformin throughout the study., Results: At the end of the placebo-controlled trial, exenatide resulted in an haemoglobin A1c (HbA1c) reduction from baseline of -1.0 +/- 0.1% (mean +/- SE) (exenatide treatment arms), with durable HbA1c reductions after 82 weeks of -1.3 +/- 0.1%. The percent of patients who achieved HbA1c < or = 7% at weeks 30 and 82 was 46 and 59% respectively. After 30 weeks, exenatide caused a reduction in weight from baseline of -3.0 +/- 0.6 kg, with a progressive reduction in weight of -5.3 +/- 0.8 kg after 82 weeks. In addition, exenatide treatment produced clinically significant improvements in cardiovascular risk factors after 82 weeks. The most frequent adverse event after 30 and 82 weeks of exenatide was nausea, which was generally of mild-or-moderate intensity. It decreased in incidence after initiation in the controlled trial and the uncontrolled open-label extension. Hypoglycaemia was rare, with no severe events., Conclusion: Exenatide was generally well tolerated, producing a durable reduction in HbA1c and a progressive reduction in weight over 82 weeks in patients with type 2 diabetes failing to achieve glycaemic control with metformin.

Objectives: To evaluate the long term effects of perioperative beta blockade on mortality and cardiac morbidity in patients with diabetes undergoing major non-cardiac surgery., Design: Randomised placebo controlled and blinded multicentre trial. Analyses were by intention to treat., Setting: University anaesthesia and surgical centres and one coordinating centre., Participants: 921 patients aged > 39 scheduled for major non-cardiac surgery., Interventions: 100 mg metoprolol controlled and extended release or placebo administered from the day before surgery to a maximum of eight perioperative days., Main Outcome Measures: The composite primary outcome measure was time to all cause mortality, acute myocardial infarction, unstable angina, or congestive heart failure. Secondary outcome measures were time to all cause mortality, cardiac mortality, and non-fatal cardiac morbidity., Results: Mean duration of intervention was 4.6 days in the metoprolol group and 4.9 days in the placebo group. Metoprolol significantly reduced the mean heart rate by 11% (95% confidence interval 9% to 13%) and mean blood pressure by 3% (1% to 5%). The primary outcome occurred in 99 of 462 patients in the metoprolol group (21%) and 93 of 459 patients in the placebo group (20%) (hazard ratio 1.06, 0.80 to 1.41) during a median follow-up of 18 months (range 6-30). All cause mortality was 16% (74/462) in the metoprolol group and 16% (72/459) in the placebo group (1.03, 0.74 to 1.42). The difference in risk for the proportion of patients with serious adverse events was 2.4% (- 0.8% to 5.6%)., Conclusions: Perioperative metoprolol did not significantly affect mortality and cardiac morbidity in these patients with diabetes. Confidence intervals, however, were wide, and the issue needs reassessment., Trial Registration: Current Controlled Trials ISRCTN58485613.

Exenatide is a 39 amino acid incretin mimetic for the treatment of type 2 diabetes, with glucoregulatory activity similar to glucagon-like peptide-1 (GLP-1). Exenatide is a poor substrate for the major route of GLP-1 degradation by dipeptidyl peptidase-IV, and displays enhanced pharmacokinetics and in vivo potency in rats relative to GLP-1. The kidney appears to be the major route of exenatide elimination in the rat. We further investigated the putative sites of exenatide degradation and excretion, and identified primary degradants. Plasma exenatide concentrations were elevated and sustained in renal-ligated rats, when compared to sham-operated controls. By contrast, exenatide elimination and degradation was not affected in rat models of hepatic dysfunction. In vitro, four primary cleavage sites after amino acids (AA)-15, -21, -22 and -34 were identified when exenatide was degraded by mouse kidney membranes. The primary cleavage sites of exenatide degradation by rat kidney membranes were after AA-14, -15, -21, and -22. In rabbit, monkey, and human, the primary cleavage sites were after AA-21 and -22. Exenatide was almost completely degraded into peptide fragments <3 AA by the kidney membranes of the species tested. The rates of exenatide degradation by rabbit, monkey and human kidney membranes in vitro were at least 15-fold slower than mouse and rat membranes. Exenatide (1-14), (1-15), (1-22), and (23-39) were not active as either agonists or antagonists to exenatide in vitro. Exenatide (15-39) and (16-39) had moderate-to-weak antagonist activity compared with the known antagonist, exenatide (9-39). In conclusion, the kidney appears to be the primary route of elimination and degradation of exenatide.

Context: First-phase insulin secretion (within 10 min after a sudden rise in plasma glucose) is reduced in type 2 diabetes mellitus (DM2). The incretin mimetic exenatide has glucoregulatory activities in DM2, including glucose-dependent enhancement of insulin secretion., Objective: The objective of the study was to determine whether exenatide can restore a more normal pattern of insulin secretion in subjects with DM2., Design: Fasted subjects received iv insulin infusion to reach plasma glucose 4.4-5.6 mmol/liter. Subjects received iv exenatide (DM2) or saline (DM2 and healthy volunteers), followed by iv glucose challenge., Patients: Thirteen evaluable DM2 subjects were included in the study: 11 males, two females; age, 56 +/- 7 yr; body mass index, 31.7 +/- 2.4 kg/m2; hemoglobin A1c, 6.6 +/- 0.7% (mean +/- sd) treated with diet/exercise (n = 1), metformin (n = 10), or acarbose (n = 2). Controls included 12 healthy, weight-matched subjects with normal glucose tolerance: nine males, three females; age, 57 +/- 9 yr; and body mass index, 32.0 +/- 3.0 kg/m2., Setting: The study was conducted at an academic hospital., Main Outcome Measures: Plasma insulin, plasma C-peptide, insulin secretion rate (derived by deconvolution), and plasma glucagon were the main outcome measures., Results: DM2 subjects administered saline had diminished first-phase insulin secretion, compared with healthy control subjects. Exenatide-treated DM2 subjects had an insulin secretory pattern similar to healthy subjects in both first (0-10 min) and second (10-180 min) phases after glucose challenge, in contrast to saline-treated DM2 subjects. In exenatide-treated DM2 subjects, the most common adverse event was moderate nausea (two of 13 subjects)., Conclusions: Short-term exposure to exenatide can restore the insulin secretory pattern in response to acute rises in glucose concentrations in DM2 patients who, in the absence of exenatide, do not display a first phase of insulin secretion. Loss of first-phase insulin secretion in DM2 patients may be restored by treatment with exenatide.

Exenatide (exendin-4) is an incretin mimetic with potential antidiabetic activity. This study examined the effects of a continuous subcutaneous (SC) infusion of exenatide (0.2, 0.4, 0.6, or 0.8 microg/kg/day) or placebo (PBO) on glycemic control over 23 h intervals. Twelve subjects with type 2 diabetes treated with metformin and/or diet received 10 infusions (4 exenatide, 6 PBO) on consecutive days. Exenatide was given in a dose-increasing design with at least one placebo infusion between each exenatide infusion, and with meals and a snack provided during the first 14 h of infusion. Plasma exenatide concentrations were dose-proportional. Plasma glucose (4-23 h) was lower in all exenatide arms compared to placebo (p<0.0001). The change in insulin/glucagon ratio and amylin concentrations from pre-infusion to post-infusion was increased (p<0.005, p<0.05, respectively) in the combined exenatide arms, but remained unchanged in the placebo groups. Nausea and vomiting were the most common treatment emergent adverse events. Exenatide infusion also appeared to have positive effects on beta-cell and alpha-cell function as measured by proinsulin/insulin ratios and mean glucagon concentrations. In summary, exenatide lowered plasma glucose during both prandial and fasting states when delivered as a continuous SC infusion over twenty-three hours, suggesting that exenatide can provide day-long glycemic control in patients with type 2 diabetes.

A novel two-step protein refolding strategy has been developed, where continuous renaturation-bydilution is followed by direct capture on an expanded bed adsorption (EBA) column. The performance of the overall process was tested on a N-terminally tagged version of human beta2-microglobulin (HAT-hbeta2m) both at analytical, small, and preparative scale. In a single scalable operation, extracted and denatured inclusion body proteins from Escherichia coli were continuously diluted into refolding buffer, using a short pipe reactor, allowing for a defined retention and refolding time, and then fed directly to an EBA column, where the protein was captured, washed, and finally eluted as soluble folded protein. Not only was the eluted protein in a correctly folded state, the purity of the HAThbeta2m was increased from 34% to 94%, and the product was concentrated sevenfold. The yield of the overall process was 45%, and the product loss was primarily a consequence of the refolding reaction rather than the EBA step. Full biological activity of HAT-hbeta2m was demonstrated after removal of the HAT-tag. In contrast to batch refolding, a continuous refolding strategy allows the conditions to be controlled and maintained throughout the process, irrespective of the batch size; i.e., it is readily scalable. Furthermore, the procedure is fast and tolerant toward aggregate formation, a common complication of in vitro protein refolding. In conclusion, this system represents a novel approach to small and preparative scale protein refolding, which should be applicable to many other proteins.

Aims/hypothesis: Exenatide (exendin-4) injected subcutaneously twice daily reduces glycaemic deterioration in diabetic fatty Zucker (ZDF) rats and reduces HbA1c in humans with type 2 diabetes. Because tachyphylaxis may develop with continuous peptide exposure, we examined the activity of a long-acting-release (LAR) formulation of exenatide on HbA1c, insulin sensitivity and beta cell secretion in ZDF rats., Methods: Single subcutaneous injections of a poly-lactide-glycolide microsphere suspension (3% peptide) containing 0, 1, 10, 100, 1,000, 3,000 or 9,000 mug exenatide were administered to 9-week-old ZDF rats with matched initial HbA1c values (n=7 rats/group)., Results: In contrast to the progressive 3.22+/-0.42% increase in HbA1c in control ZDF rats observed over 28 days, single exenatide-LAR injections dose-proportionally prevented such glycaemic deterioration (median effective dose 74 microg+/-0.1 log per rat; median effective concentration 52 pmol/l+/-0.06 log). Hyperinsulinaemic-euglycaemic clamp procedures incorporating an intraclamp glucose challenge performed 28 days after treatment revealed increases in beta cell response to the glucose challenge at lower exenatide-LAR doses, and up to a 2.1-fold increase in insulin sensitivity at higher exenatide-LAR doses., Conclusions/interpretation: The finding that a single dose of exenatide-LAR enhanced glucose control for 28 days in the ZDF rat model of type 2 diabetes suggests that tachyphylaxis is unlikely to be a feature of exenatide-LAR preparations, and supports further clinical exploration.

Incretin mimetics are a new class of pharmacological agents with multiple antihyperglycemic actions that mimic the actions of incretin hormones such as glucagon-like peptide (GLP)-1. Dipeptidyl peptidase (DPP-IV) inhibitors suppress the degradation of many peptides, including GLP-1, thereby extending their bioactivity. Several incretin mimetics and DPP-IV inhibitors are undergoing late-stage clinical trials for the treatment of type 2 diabetes. These agents appear to have multiple mechanisms of action, including some or all of the following: enhancement of glucose-dependent insulin secretion; suppression of inappropriately elevated glucagon secretion; slowing of gastric emptying; and decreased food intake (i.e. appetite suppression). Based on preliminary clinical data, incretin mimetics and DPP-IV inhibitors show potential for treating type 2 diabetes.

The effects of the incretin mimetic exenatide (exendin-4) on metabolic parameters, insulin sensitivity, and beta-cell mass were examined in nondiabetic, insulin-resistant obese fa/fa Zucker rats. After 6 wk of treatment, ad libitum-fed exenatide-treated (EX) and pair-fed vehicle control (PF) rats had comparable food intake, body weight, hemoglobin A(1c) (HbA(1c)), and fasting plasma concentrations of glucose, insulin, and lipids. Concurrent decreases in food intake and weight gain were observed in EX and PF rats, compared with ad libitum-fed vehicle control (CON) rats (P < 0.001). The increases in HbA(1c) and fasting plasma insulin concentrations that occur during the normal progression of this disease model were significantly reduced in EX and PF rats, compared with CON rats (P < 0.001). The insulin sensitivity index (ISI; glucose infusion rate to plasma insulin concentration) measured during a hyperinsulinemic euglycemic clamp was 224% higher in EX rats than CON rats (P < 0.001) and 61% higher in EX rats than PF rats (P < 0.004). The latter difference was despite comparable HbA(1c), fasting glucose, fasting insulin, total cholesterol, high-density lipoprotein, and daily food consumption between EX and PF animals. In the absence of exenatide, beta-cell mass was hyperbolically related to ISI (beta-cell mass * ISI was constant). Analogous to the disposition index, the beta-cell mass * ISI product was 63% greater in EX than PF rats (P < 0.05). Thus, exenatide increased beta-cell mass to a greater extent than would be expected in animals of comparable insulin resistance, suggesting a direct trophic effect on islet neogenesis in obese fa/fa rats independent of body weight and glycemia.

Purpose: The pharmacology and tolerability of exenatide in patients with type 2 diabetes mellitus were studied., Methods: Two randomized, single-blind, placebo-controlled studies were conducted. Treatment with oral antidiabetic agents was stopped 14 days before study initiation. In the first study (study A), eight subjects received placebo, 0.1-, 0.2-, 0.3-, and either 0.4-microg/kg exenatide or placebo five minutes before a meal combined with liquid acetaminophen (to assess the rate of gastric emptying) on days 1, 3, 5, 7, and 9. In the second study (study B), subjects received a single s.c. dose of exenatide or placebo on consecutive days. Part 1 of study B used exenatide doses of 0.01 and 0.1 microg/ kg; 0.02-, 0.05-, and 0.1-microg/kg doses were given in part 2. After an overnight fast, the study drug was injected 15 minutes before a meal (part 1) and before a meal and acetaminophen (part 2). Parts 1 and 2 of study B enrolled six and eight patients, respectively., Results: In both studies, plasma exenatide pharmacokinetic profiles appeared dose proportional. Exenatide doses of 0.02-0.2 microg/kg dose-dependently lowered postprandial glucose excursions. Exenatide suppressed postprandial plasma glucagon and slowed gastric emptying. There were no serious adverse events and no patient withdrawals related to treatment. Nausea and vomiting were the most common adverse events and were mild to moderate in severity at doses ranging from 0.02 to 0.2 microg/kg., Conclusion: Administration of preprandial exenatide by s.c. injection resulted in dose-proportional exenatide pharmacokinetics and antidiabetic pharmacodynamic activity. At doses ranging from 0.02 to 0.2 microg/kg, exenatide dose-dependently reduced postprandial plasma glucose excursion by insulinotropism, suppression of plasma glucagon, and slowing of gastric emptying.

Exenatide (synthetic exendin-4), glucagon-like peptide-1 (GLP-1), and GLP-1 analogues have actions with the potential to significantly improve glycemic control in patients with diabetes. Evidence suggests that these agents use a combination of mechanisms which may include glucose-dependent stimulation of insulin secretion, suppression of glucagon secretion, enhancement of beta-cell mass, slowing of gastric emptying, inhibition of food intake, and modulation of glucose trafficking in peripheral tissues. The short in vivo half-life of GLP-1 has proven a significant barrier to continued clinical development, and the focus of current clinical studies has shifted to agents with longer and more potent in vivo activity. This review examines recent exendin-4 pharmacology in the context of several known mechanisms of action, and contrasts exendin-4 actions with those of GLP-1 and a GLP-1 analogue. One of the most provocative areas of recent research is the finding that exendin-4 enhances beta-cell mass, thereby impeding or even reversing disease progression. Therefore, a major focus of this is article an examination of the data supporting the concept that exendin-4 and GLP-1 may increase beta-cell mass via stimulation of beta-cell neogenesis, stimulation of beta-cell proliferation, and suppression of beta-cell apoptosis.

New therapies for the long-term treatment of type 2 diabetes are needed to ameliorate declining pancreatic beta-cell function. Ideally, these therapies should lower fasting and post-prandial blood glucose, produce no hypoglycemia or weight gain, cause no other limiting side effects, and reduce cardiovascular complications. Exenatide (synthetic exendin-4) is a potential therapeutic which may fulfill these criteria. Dose-ranging studies have identified an optimal dose of 0.05 to 0.2 microgram/kg administered subcutaneously twice daily. Pharmacokinetic data support a pivotal study design which mitigates the transient nausea observed in early studies by including a dose initiation period of 1 month at 5 micrograms twice daily, followed by maintenance therapy at 10 micrograms twice daily. Ongoing studies suggest exenatide improves glycemic control through a combination of mechanisms discussed in this review.

The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1). correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2). optimal in vitro conditions for disulfide bond formation ( approximately pH 8) and peptide binding ( approximately pH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules with correct disulfide bonding are formed under non-reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond-assisted folding in the presence of beta(2)-microglobulin and a specific peptide. Under conditions optimized for peptide binding, refolding and simultaneous peptide binding of the correctly oxidized heavy chain was much more efficient than that of the fully reduced molecule.

Survivin is an inhibitor of apoptosis protein, which is over-expressed in most tumors. Aberrant expression of survivin and loss of wild-type p53 in many tumors prompted us to investigate a possible link between these two events. Here we show that wild-type p53 represses survivin expression at both mRNA and protein levels. Transient transfection analyses revealed that the expression of wild-type p53, but not mutant p53, was associated with strong repression of the survivin promoter in various cell types. The over-expression of exogenous survivin protein rescues cells from p53-induced apoptosis in a dose-dependent manner, suggesting that loss of survivin mediates, at least, in part the p53-dependent apoptotic pathway. In spite of the presence of two putative p53-binding sites in the survivin promoter, deletion and mutation analyses suggested that neither site is required for transcriptional repression of survivin expression. This was confirmed by chromatin immunoprecipitation assays. Further analyses suggested that the modification of chromatin within the survivin promoter could be a molecular explanation for silencing of survivin gene transcription by p53.

Progressive growth and metastasis of solid tumors require angiogenesis, or the formation of new blood vessels. Endostatin is a 20-kDa carboxy-terminal fragment of collagen XVIII that has been shown to inhibit endothelial cell proliferation and tumor angiogenesis. Replication-deficient recombinant adenovirus (rAd) vectors were constructed, which encoded secreted forms of human and mouse endostatin (HECB and MECB, respectively), and, as a control, human alkaline phosphatase (APCB). Accumulation of endostatin was demonstrated in supernatants of cultured cells infected with the endostatin rAds. These supernatants disrupted tubule formation, inhibited migration and proliferation, and induced apoptosis in human dermal vascular endothelial cells or human vascular endothelial cells. Endostatin-containing supernatants had no effect on the proliferation of MidT2-1 mouse mammary tumor cells in vitro. A pharmacokinetic study of MECB in immunocompetent FVB mice demonstrated a 10-fold increase of serum endostatin concentrations 3 days after intravenous administration of 1x10(10) particles of this rAd (215-257 ng/mL compared to 12-38 ng/mL in control rAd-treated mice). Intravenous administration of MECB reduced b-FGF stimulated angiogenesis into Matrigel plugs by 38%. Intratumoral MECB inhibited growth of MidT2-1 syngeneic mammary tumors in FVB mice, but had minimal impact on the growth of MDA-MB-231 human breast tumors in SCID mice. Intravenous therapy with MECB also initially inhibited growth of MidT2-1 tumors, but this activity was subsequently blocked by induced anti-rAd antibodies. In summary, endostatin gene therapy effectively suppressed angiogenic processes in vitro and in vivo in several model systems.

Adenovirus-mediated gene therapy is a promising new approach for treatment of ovarian cancer. In animal models, complete elimination of cancer cells is often achieved, although the therapeutic gene has not been delivered to all these cells. This is referred to as a bystander effect, because tumor cells near those that receive the therapeutic gene are also eliminated. Several mechanisms have been proposed for the bystander effect, including intercellular communication within the tumor via gap junctions, apoptosis, antiangiogenesis, cytokines or other soluble mediators, and immunological mechanisms. There are two well-documented antitumor effector cell populations in athymic nude mice: macrophages and natural killer (NK) cells. We hypothesize that peritoneal populations of NK cells in nude mice treated with adenoviruses are involved in the observed bystander effect in this in vivo model. We investigated the role of NK cells as immunological mediators for the bystander effect using the p53 tumor suppressor as the therapeutic anticancer gene. Most ovarian cancer cell lines tested were sensitive to lysis by NK cells, although different ovarian cancer cell lines exhibited different sensitivities to NK cell-mediated lysis. To determine the importance of NK cells in the overall efficacy and in the bystander effect of gene therapy, NK cells were depleted in mice by administration of anti-NK1.1 monoclonal antibodies. To study the efficacy of NK depletion, C57BL/6 (nu/nu) mice were given injections i.v. by a single tail vein injection or i.p. with increasing doses of anti-NK1.1 IgG. All doses of anti-NK1.1 antibody, from 100-500 micrograms, essentially eliminated cytotoxic NK activity. To assess the duration of depletion after a single dose of anti-NK1.1 IgG, a time-course experiment was performed. NK 1.1 antibody was effective in completely depleting cytotoxic NK cell activity in the mice for up to 7 days, whether given as 500 micrograms (i.p.) or 200 micrograms (i.v.). Flow cytometric analysis performed on peritoneal cell populations confirmed depletion of NK cells by approximately 80%. Finally, a survival study was performed, in which animals were depleted of NK cells. In this experiment, NK cell-depleted mice were injected with anti-NK1.1 IgG, and control mice were mice were treated with normal saline. Two days later, all mice were inoculated with a lethal i.p. dose of NIH:OVCAR-3 ovarian cancer cells. After 3 days, the mice were divided into two treatment groups; one treatment group received three consecutive daily i.p. injections of Ad-CMV-p53 (SCH58500), and the second treatment group received three consecutive daily i.p. injections of control adenovirus construct, rAd-null. All of the NK cell-depleted animals, whether treated with rAd-null or with Ad-CMV-p53 (SCH58500) were dead of disease by 116 and 138 days, respectively, after initiation of adenovirus treatment, and no statistically significant difference in survival was observed (P = 0.349). A significant survival advantage was seen in control (NK-competent) mice treated with rAd-null (P = 0.04), although all were dead of disease by day 184. Importantly, control NK-competent mice treated with Ad-CMV-p53 (SCH58500) showed no tumor growth or ascites production, and all animals survived. These results indicate that immunological mechanisms involving natural killer cells play an important role in the bystander effect involving adenovirus-p53 gene therapy for ovarian cancer.

The expression of major histocompatibility class I (MHC-I) crucially depends upon the binding of appropriate peptides. MHC-I from natural sources are therefore always preoccupied with peptides complicating their purification and analysis. Here, we present an efficient solution to this problem. Recombinant MHC-I heavy chains were produced in Escherichia coli and subsequently purified under denaturing conditions. In contrast to common practice, the molecules were not reduced during the purification. The oxidized MHC-I heavy chain isoforms were highly active with respect to peptide binding. This suggests that de novo folding of denatured MHC-I molecules proceed efficiently if directed by preformed disulfide bond(s). Importantly, these molecules express serological epitopes and stain specific T cells; and they bind peptides specifically. Several denatured MHC-I heavy chains were analyzed and shown to be of a quality, which allowed quantitative analysis of peptide binding. The analysis of the specificity of the several hundred human MHC haplotypes, should benefit considerably from the availability of pre-oxidized recombinant MHC-I.

TP53 is the most commonly altered tumor-suppressor gene in cancer and is currently being tested in Phase II/III gene replacement trials. Many tumors contain wild-type TP53 sequence with elevated MDM2 protein levels, targeting p53 for degradation. These tumors are more refractory to treatment with exogenous wild-type p53. Here we generate a recombinant adenovirus expressing a p53 variant, rAd-p53 (d 13-19), that is deleted for the amino acid sequence necessary for MDM2 binding (amino acids 13-19). We compared the apoptotic activity of rAd-p53 (d 13-19) with that of a recombinant adenovirus expressing wild-type p53 (rAd-p53) in cell lines that differ in endogenous p53 status. rAd-p53 (d 13-19) caused higher levels of apoptosis in p53 wild-type tumor lines compared with wild-type p53 treatment, as measured by annexin V-FITC staining. In p53-altered tumor lines, rAd-p53 (d 13-19) showed apoptotic activity similar to that seen with wild-type p53 treatment. In normal cells, no increase in cytopathicity was detected with rAd-p53 (d 13-19) compared with wild-type p53 treatment. This variant protein displayed synergy with chemotherapeutic agents to inhibit proliferation of ovarian and breast cell lines. The p53 variant showed greater antitumor activity in an established p53 wild-type tumor compared with treatment with wild-type p53. The p53 variant represents a means of expanding TP53 gene therapy to tumors that are resistant to p53 treatment due to the cellular responses to wild-type p53.

The in vivo evaluation process described here was instrumental in the identification of SCH 66336 as a clinical candidate. Our lead FTI, SCH 66336, and several other FTIs are being evaluated in early-phase clinical trials to establish proof-of-principle for farnesyl transferase inhibition in human patients. The preclinical studies described here suggest that FTIs may have utility against a wide array of human cancers as a single agent and may, at least in some cases, lead to tumor regression. In addition, the results to date in combination with cytotoxic chemotherapeutic agents in animal models indicate that these combinations may enhance the clinical efficacy of FPT inhibitors. Further preclinical studies should help to guide the clinical development of this class of novel antitumor agents.