Design of potent and selective GPR119 agonists for type II diabetes.

Screening of the Merck sample collection identified compound 1 as a weakly potent GPR119 agonist (hEC(50)=3600 nM). Dual termini optimization of 1 led to compound 36 having improved potency, selectivity, and formulation profile, however, modest physical properties (PP) hindered its utility. Design of a new core containing a cyclopropyl restriction yielded further PP improvements and when combined with the termini SAR optimizations yielded a potent and highly selective agonist suitable for further preclinical development (58).

Identification of a potent synthetic FXR agonist with an unexpected mode of binding and activation.

The farnesoid X receptor (FXR), a member of the nuclear hormone receptor family, plays important roles in the regulation of bile acid and cholesterol homeostasis, glucose metabolism, and insulin sensitivity. There is intense interest in understanding the mechanisms of FXR regulation and in developing pharmaceutically suitable synthetic FXR ligands that might be used to treat metabolic syndrome. We report here the identification of a potent FXR agonist (MFA-1) and the elucidation of the structure of this ligand in ternary complex with the human receptor and a coactivator peptide fragment using x-ray crystallography at 1.9-A resolution. The steroid ring system of MFA-1 binds with its D ring-facing helix 12 (AF-2) in a manner reminiscent of hormone binding to classical steroid hormone receptors and the reverse of the pose adopted by naturally occurring bile acids when bound to FXR. This binding mode appears to be driven by the presence of a carboxylate on MFA-1 that is situated to make a salt-bridge interaction with an arginine residue in the FXR-binding pocket that is normally used to neutralize bound bile acids. Receptor activation by MFA-1 differs from that by bile acids in that it relies on direct interactions between the ligand and residues in helices 11 and 12 and only indirectly involves a protonated histidine that is part of the activation trigger. The structure of the FXR:MFA-1 complex differs significantly from that of the complex with a structurally distinct agonist, fexaramine, highlighting the inherent plasticity of the receptor.

Peroxisome proliferator-activated receptor (PPAR)-alpha agonism prevents the onset of type 2 diabetes in Zucker diabetic fatty rats: A comparison with PPAR gamma agonism.

Peroxisome proliferator-activated receptor (PPAR)-gamma agonists are insulin sensitizers, whereas PPAR alpha agonists are lipid-lowering agents in humans. Chronic treatment with PPAR gamma agonists has been shown to prevent the onset of diabetes in young Zucker diabetic fatty (ZDF) rats; however, the effects of PPAR alpha agonists have not been well characterized in this model. Here we investigated chronic efficacy of PPAR alpha and nonthiazolidinedione (nTZD) PPAR gamma agonists on the onset of diabetes in 6-wk-old male ZDF rats. Whereas treatment with the nTZD PPAR gamma agonist completely prevented development of hyperglycemia, PPAR alpha activation was associated with lowering of food intake and body weight and reductions in fed and fasting hyperglycemia, with prevention of the hyperinsulinemic peak preceding the development of hyperglycemia in ZDF rats. Both compounds improved glucose tolerance during an oral glucose tolerance test with concomitant increases in insulin response. Such improvements of insulin secretion were associated with increased islet to total pancreatic area ratio and pancreatic insulin contents. Hyperinsulinemic-euglycemic clamp studies demonstrated that nTZD PPAR gamma reduced basal endogenous glucose production and increased insulin-stimulated glucose disposal, consistent with an improved insulin action as a cause of the improved glucose homeostasis. In contrast, activation of PPAR alpha did not significantly improve glucose metabolism during the hyperinsulinemic-euglycemic clamp. In conclusion, chronic treatment of ZDF rats with a PPAR gamma agonist completely prevented the onset of diabetes by improving both insulin action and secretion, whereas PPAR alpha agonism was partially effective, primarily by improving the pancreatic islet insulin response. Unlike the PPAR gamma agonist, the PPAR alpha agonist demonstrated efficacy without inducing body weight gain and cardiomegaly. This study suggests a possible role for PPAR alpha agonists in the prevention of type 2 diabetes mellitus.

Different roles of liver X receptor alpha and beta in lipid metabolism: effects of an alpha-selective and a dual agonist in mice deficient in each subtype.

Liver X receptor (LXR) alpha and LXRbeta are closely related nuclear receptors that respond to elevated levels of intracellular cholesterol by enhancing transcription of genes that control cholesterol efflux and fatty acid biosynthesis. The consequences of inactivation of either LXR isoform have been thoroughly studied, as have the effects of simultaneous activation of both LXRalpha and LXRbeta by synthetic compounds. We here describe the effects of selective activation of LXRalpha or LXRbeta on lipid metabolism. This was accomplished by treating mice genetically deficient in either LXRalpha or LXRbeta with an agonist with equal potency for both isoforms (Compound B) or a synthetic agonist selective for LXRalpha (Compound A). We also determined the effect of these agonists on gene expression and cholesterol efflux in peritoneal macrophages derived from wild-type and knockout mice. Both compounds raised HDL-cholesterol and increased liver triglycerides in wild-type mice; in contrast, in mice deficient in LXRalpha, Compound B increased HDL-cholesterol but did not cause hepatic steatosis. Compound B induced ATP-binding cassette transporter (ABC) A1 expression and stimulated cholesterol efflux in macrophages from both LXRalpha and LXRbeta-deficient mice. Our data lend further experimental support to the hypothesis that LXRbeta-selective agonists may raise HDL-cholesterol and stimulate macrophage cholesterol efflux without causing liver triglyceride accumulation.

Manual examination of the spine: a systematic critical literature review of reproducibility.

OBJECTIVE: Poor reproducibility of spinal palpation has been reported in previously published literature, and authors of recent reviews have posted criticism on study quality. This article critically analyzes the literature pertaining to the inter- and intraobserver reproducibility of spinal palpation to investigate the consistency of study results and assess the level of evidence for reproducibility. METHODS: Systematic review and meta-analysis were performed on relevant literature published from 1965 to 2005, identified using the electronic databases MEDLINE, MANTIS, and CINAHL and checking of reference lists. Descriptive data from included articles were extracted independently by 2 reviewers. A 6-point scale was constructed to assess the methodological quality of original studies. A meta-analysis was conducted among the high-quality studies to investigate the consistency of data, separately on motion palpation, static palpation, osseous pain, soft tissue pain, soft tissue changes, and global assessment. A standardized method was used to determine the level of evidence. RESULTS: The quality score of 48 included studies ranged from 0% to 100%. There was strong evidence that the interobserver reproducibility of osseous and soft tissue pain is clinically acceptable (kappa > or = 0.4) and that intraobserver reproducibility of soft tissue pain and global assessment are clinically acceptable. Other spinal procedures are either not reproducible or the evidence is conflicting or preliminary.

Distinct properties and advantages of a novel peroxisome proliferator-activated protein [gamma] selective modulator.

Antidiabetic thiazolidinediones (TZDs) and non-TZD compounds have been shown to serve as agonists of the peroxisome proliferator-activated receptor gamma (PPARgamma). Here, we report the identification and characterization of a novel non-TZD selective PPARgamma modulator (nTZDpa). nTZDpa bound potently to PPARgamma with high selectivity vs. PPARalpha or PPARdelta. In cell-based assays for transcriptional activation, nTZDpa served as a selective, potent PPARgamma partial agonist and was able to antagonize the activity of PPARgamma full agonists. nTZDpa also displayed partial agonist effects when its ability to promote adipogenesis in 3T3-L1 cells was evaluated. Assessment of protein conformation using protease protection or solution nuclear magnetic resonance spectroscopy methods showed that nTZDpa produced altered PPARgamma conformational stability vs. full agonists, thereby establishing a physical basis for its observed partial agonism. DNA microarray analysis of RNA from 3T3-L1 adipocytes treated with nTZDpa or several structurally diverse PPARgamma full agonists demonstrated qualitative differences in the affected gene expression profile for nTZDpa. Chronic treatment of fat-fed, C57BL/6J mice with nTZDpa or a TZD full agonist ameliorated hyperglycemia and hyperinsulinemia. However, unlike the TZD, nTZDpa caused reductions in weight gain and adipose depot size. Feed efficiency was also substantially diminished. Unlike TZDs, nTZDpa did not cause cardiac hypertrophy in mice. When a panel of PPARgamma target genes was examined in white adipose tissue, nTZDpa produced a different in vivo expression pattern vs. the full agonist. These findings establish that novel selective PPARgamma modulators can produce altered receptor conformational stability leading to distinctive gene expression profiles, reduced adipogenic cellular effects, and potentially improved in vivo biological responses. Such compounds may lead to preferred therapies for diabetes, obesity, or metabolic syndrome.

Miniaturization of cell-based beta-lactamase-dependent FRET assays to ultra-high throughput formats to identify agonists of human liver X receptors.

Activation of liver X receptors (LXRs) induces reverse cholesterol transport and increases high-density lipoprotein cholesterol in vivo. Here, we describe novel, functional, homogeneous cell-based fluorescence resonance energy transfer assays for identifying agonists of LXRs using beta-lactamase as the reporter gene. Stable Chinese hamster ovary cell lines expressing LXRalpha-GAL4 or LXRbeta-GAL4 fusion proteins that regulate beta-lactamase transcription from upstream 7 x UAS GAL4 DNA binding sequences were generated and characterized. Synthetic and natural ligands of LXR dose-dependently activated the expression of beta-lactamase in a subtype-specific manner. These assays were used to demonstrate that a 1-pyridyl hydantoin small molecule LXR synthetic ligand specifically activates LXRalpha receptors. The beta-lactamase assays were optimized for cell density, dimethyl sulfoxide sensitivity, and time of agonist stimulation. Clonal LXRbeta-GAL4-beta-lactamase cells were miniaturized into an ultra high throughput (3456-well nanoplates) screening format.

Effects of treatment protocols and subcutaneous implantation on bovine pericardium: a Raman spectroscopy study.

Using Raman microspectroscopy, we have studied mineral deposition on bovine pericardia, fixed according to three different protocols and either implanted subcutaneously or not implanted (controls). A lightly carbonated apatitic phosphate mineral, similar to that found in bone tissue, was deposited on the surface of a glutaraldehyde-fixed, implanted pericardium. Implanted pericardia fixed in glutaraldehyde followed by treatment in either an 80% ethanol or a 5% octanol/40% ethanol solution did not mineralize on implantation. Collagen secondary structure changes were observed on glutaraldehyde fixation by monitoring the center of gravity of the amide I envelope. It is proposed that the decrease in the amide I center of gravity frequency for the glutaraldehyde-fixed tissue compared to the nonfixed tissue is due to an increase in nonreducible collagen cross-links (1660 cm(-1)) and a decrease in reducible cross-links (1690 cm(-1)). The amide I center of gravity in the glutaraldehyde/ethanol-fixed pericardium was higher than the glutaraldehyde-fixed tissue center of gravity. This increase in center of gravity could possibly be due to a decrease in hydrogen bonding within the collagen fibrils following the ethanol pretreatment. In addition, we found a secondary structure change to the pericardial collagen after implantation: an increase in the frequency of the center of gravity of amide I is indicative of an increase in cross-links.

Treatment of bioprosthetic heart valve tissue with long chain alcohol solution to lower calcification potential.

The use of glutaraldehyde-treated biological tissue in heart valve substitutes is an important option in the treatment of heart valve disease. These devices have limited durability, in part, because of tissue calcification and subsequent tearing of the valve leaflets. Components thought to induce calcification include lipids, cell remnants, and residual glutaraldehyde. We hypothesized that treatment of glutaraldehyde-treated bioprosthetic heart valve material using a short and long chain alcohol (LCA) combination, composed of 5% 1,2-octanediol in an ethanolic buffered solution, would reduce phospholipid content and subsequently lower the propensity of these tissues to calcify in vivo. Phospholipid content of glutaraldehyde-treated porcine valve leaflets and bovine pericardium was found to be 10.1 +/- 4.3 (n = 7) and 3.9 +/- 0.48 (n = 2) microg/mg dry tissue, respectively, which was reduced to 0.041 +/- 0.06 (n = 7) and 0.21 +/- 0.05 (n = 4) microg/mg dry tissue, respectively, after LCA treatment. Calcification potential of the treated tissues was assessed using a rat subcutaneous implant model. After 60 days of implantation, calcium levels were found to be 171 +/- 32 (n = 11) and 83 +/- 70 (n = 12) mg/g dry weight for glutaraldehyde-treated porcine leaflets and bovine pericardium, respectively, whereas prior LCA treatment resulted in reduced calcium levels of 1.1 +/- 0.6 (n = 12) and 0.82 +/- 0.1 (n = 12) mg/g dry weight, respectively. These data, taken together, support the notion that treatment of glutaraldehyde-treated tissue with a short and long chain alcohol combination will reduce both extractable phospholipids and the propensity for in vivo calcification.

Content validity of manual spinal palpatory exams - A systematic review.

BACKGROUND: Many health care professionals use spinal palpatory exams as a primary and well-accepted part of the evaluation of spinal pathology. However, few studies have explored the validity of spinal palpatory exams. To evaluate the status of the current scientific evidence, we conducted a systematic review to assess the content validity of spinal palpatory tests used to identify spinal neuro-musculoskeletal dysfunction. METHODS: Review of eleven databases and a hand search of peer-reviewed literature, published between 1965-2002, was undertaken. Two blinded reviewers abstracted pertinent data from the retrieved papers, using a specially developed quality-scoring instrument. Five papers met the inclusion/exclusion criteria. RESULTS: Three of the five papers included in the review explored the content validity of motion tests. Two of these papers focused on identifying the level of fixation (decreased mobility) and one focused on range of motion. All three studies used a mechanical model as a reference standard. Two of the five papers included in the review explored the validity of pain assessment using the visual analogue scale or the subjects' own report as reference standards. Overall the sensitivity of studies looking at range of motion tests and pain varied greatly. Poor sensitivity was reported for range of motion studies regardless of the examiner's experience. A slightly better sensitivity (82%) was reported in one study that examined cervical pain. CONCLUSIONS: The lack of acceptable reference standards may have contributed to the weak sensitivity findings. Given the importance of spinal palpatory tests as part of the spinal evaluation and treatment plan, effort is required by all involved disciplines to create well-designed and implemented studies in this area.

Searching biomedical databases on complementary medicine: the use of controlled vocabulary among authors, indexers and investigators.

BACKGROUND: The optimal retrieval of a literature search in biomedicine depends on the appropriate use of Medical Subject Headings (MeSH), descriptors and keywords among authors and indexers. We hypothesized that authors, investigators and indexers in four biomedical databases are not consistent in their use of terminology in Complementary and Alternative Medicine (CAM). METHODS: Based on a research question addressing the validity of spinal palpation for the diagnosis of neuromuscular dysfunction, we developed four search concepts with their respective controlled vocabulary and key terms. We calculated the frequency of MeSH, descriptors, and keywords used by authors in titles and abstracts in comparison to standard practices in semantic and analytic indexing in MEDLINE, MANTIS, CINAHL, and Web of Science. RESULTS: Multiple searches resulted in the final selection of 38 relevant studies that were indexed at least in one of the four selected databases. Of the four search concepts, validity showed the greatest inconsistency in terminology among authors, indexers and investigators. The use of spinal terms showed the greatest consistency. Of the 22 neuromuscular dysfunction terms provided by the investigators, 11 were not contained in the controlled vocabulary and six were never used by authors or indexers. Most authors did not seem familiar with the controlled vocabulary for validity in the area of neuromuscular dysfunction. Recently, standard glossaries have been developed to assist in the research development of manual medicine. CONCLUSIONS: Searching biomedical databases for CAM is challenging due to inconsistent use of controlled vocabulary and indexing procedures in different databases. A standard terminology should be used by investigators in conducting their search strategies and authors when writing titles, abstracts and submitting keywords for publications.

Writing narrative literature reviews for peer-reviewed journals: secrets of the trade.

OBJECTIVE: To describe and discuss the process used to write a narrative review of the literature for publication in a peer-reviewed journal. Publication of narrative overviews of the literature should be standardized to increase their objectivity. BACKGROUND: In the past decade numerous changes in research methodology pertaining to reviews of the literature have occurred. These changes necessitate authors of review articles to be familiar with current standards in the publication process. METHODS: Narrative overview of the literature synthesizing the findings of literature retrieved from searches of computerized databases, hand searches, and authoritative texts. DISCUSSION: An overview of the use of three types of reviews of the literature is presented. Step by step instructions for how to conduct and write a narrative overview utilizing a 'best-evidence synthesis' approach are discussed, starting with appropriate preparatory work and ending with how to create proper illustrations. Several resources for creating reviews of the literature are presented and a narrative overview critical appraisal worksheet is included. A bibliography of other useful reading is presented in an appendix. CONCLUSION: Narrative overviews can be a valuable contribution to the literature if prepared properly. New and experienced authors wishing to write a narrative overview should find this article useful in constructing such a paper and carrying out the research process. It is hoped that this article will stimulate scholarly dialog amongst colleagues about this research design and other complex literature review methods.

SAR studies: designing potent and selective LXR agonists.

Counterscreening compounds from a Merck PPAR program discovered lead 1, as a nanomolar LXR/PPAR dual agonist. SAR optimization developed a series of heterocyclic LXR agonists having excellent selectivity over all PPAR isoforms and possessing high LXR affinity and strong in vivo potency.

A randomized trial of medical care with and without physical therapy and chiropractic care with and without physical modalities for patients with low back pain: 6-month follow-up outcomes from the UCLA low back pain study.

STUDY DESIGN: A randomized clinical trial. OBJECTIVES: To compare the effectiveness of medical and chiropractic care for low back pain patients in managed care; to assess the effectiveness of physical therapy among medical patients; and to assess the effectiveness of physical modalities among chiropractic patients. SUMMARY OF BACKGROUND DATA: Despite the burden that low back pain places on patients, providers, and society, the relative effectiveness of common treatment strategies offered in managed care is unknown. METHODS: Low back pain patients presenting to a large managed care facility from October 30, 1995, through November 9, 1998, were randomly assigned in a balanced design to medical care with and without physical therapy and to chiropractic care with and without physical modalities. The primary outcome variables are average and most severe low back pain intensity in the past week, assessed with 0 to 10 numerical rating scales, and low back-related disability, assessed with the 24-item Roland-Morris Disability Questionnaire. RESULTS: Of 1,469 eligible patients, 681 were enrolled; 95.7% were followed through 6 months. The mean changes in low back pain intensity and disability of participants in the medical and chiropractic care-only groups were similar at each follow-up assessment (adjusted mean differences at 6 months for most severe pain, 0.27, 95% confidence interval, -0.32-0.86; average pain, 0.22, -0.25-0.69; and disability, 0.75, -0.29-1.79). Physical therapy yielded somewhat better 6-month disability outcomes than did medical care alone (1.26, 0.20-2.32). CONCLUSIONS: After 6 months of follow-up, chiropractic care and medical care for low back pain were comparable in their effectiveness. Physical therapy may be marginally more effective than medical care alone for reducing disability in some patients, but the possible benefit is small.

A randomized trial of chiropractic manipulation and mobilization for patients with neck pain: clinical outcomes from the UCLA neck-pain study.

OBJECTIVES: This study compared the relative effectiveness of cervical spine manipulation and mobilization for neck pain. METHODS: Neck-pain patients were randomized to the following conditions: manipulation with or without heat, manipulation with or without electrical muscle stimulation, mobilization with or without heat, and mobilization with or without electrical muscle stimulation. RESULTS: Of 960 eligible patients, 336 enrolled in the study. Mean reductions in pain and disability were similar in the manipulation and mobilization groups through 6 months. CONCLUSIONS: Cervical spine manipulation and mobilization yield comparable clinical outcomes.

Patients using chiropractors in North America: who are they, and why are they in chiropractic care?

SUMMARY OF BACKGROUND DATA AND OBJECTIVES: Alternative health care was used by an estimated 42% of the U.S. population in 1997, and chiropractors accounted for 31% of the total estimated number of visits. Despite this high level of use, there is little empirical information about who uses chiropractic care or why. METHODS: The authors surveyed randomly sampled chiropractors (n = 131) at six study sites and systematically sampled chiropractic patients seeking care from participating chiropractors on 1 day (n = 1275). Surveys collected data about the patient's reason for seeking chiropractic care, health status, health attitude and beliefs, and satisfaction. In addition to descriptive statistics, the authors compared data between patients and chiropractors, and between patients and previously published data on health status from other populations, corrected for the clustering of patients within chiropractors. RESULTS: More than 70% of patients specified back and neck problems as their health problem for which they sought chiropractic care. Chiropractic patients had significantly worse health status on all SF-36 scales than an age- and gender-matched general population sample. Compared with medical back pain patients, chiropractic back pain patients had significantly worse mental health (6-8 point decrement). Roland-Morris scores for chiropractic back pain patients were similar to values reported for medical back pain patients. The health attitudes and beliefs of chiropractors and their patients were similar. Patients were very satisfied with their care. CONCLUSION: These data support the theory that patients seek chiropractic care almost exclusively for musculoskeletal symptoms and that chiropractors and their patients share a similar belief system.

Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity.

Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.

Effectiveness of four conservative treatments for subacute low back pain: a randomized clinical trial.

STUDY DESIGN: A randomized, assessor-blinded clinical trial was conducted. OBJECTIVE: To investigate the relative effectiveness of three manual treatments and back school for patients with subacute low back pain. SUMMARY OF BACKGROUND DATA: Literature comparing the relative effectiveness of specific therapies for low back pain is limited. METHODS: Among the 5925 inquiries, 206 patients met the specific admission criteria, and 200 patients randomly received one of four treatments for 3 weeks: back school, joint manipulation, myofascial therapy, and combined joint manipulation and myofascial therapy. These patients received assessments at baseline, after 3 weeks of therapy, and 6 months after the completion of therapy. The primary outcomes were evaluated using visual analog pain scales and Roland-Morris activity scales. RESULTS: All four groups showed significant improvement in pain and activity scores after 3 weeks of care, but did not show further significant improvement at the 6-month follow-up assessment. No statistically significant between-group differences were found either at the 3-week or 6-month reassessments. CONCLUSIONS: For subacute low back pain, combined joint manipulation and myofascial therapy was as effective as joint manipulation or myofascial therapy alone. Additionally, back school was as effective as three manual treatments.

Amphipathic 3-phenyl-7-propylbenzisoxazoles; human pPaR gamma, delta and alpha agonists.

A series of amphipathic 3-phenylbenzisoxazoles were found to be potent agonists of human PPARalpha, gamma and delta. The optimization of acid proximal structure for in vitro and in vivo potency is described. Results of po dosed efficacy studies in the db/db mouse model of type 2 diabetes showed efficacy equal or superior to Rosiglitazone in correcting hyperglycemia and hypertriglyceridemia. Good functional receptor selectivity for PPARalpha and gamma over PPARdelta can be obtained.