Antitarget Selectivity and Tolerability of Novel Pyrrolo[2,3-d]pyrimidine RET Inhibitors.

The selective inhibition of RET kinase as a treatment for relevant cancer types including lung adenocarcinoma has garnered considerable interest in recent years and prompted a variety of efforts toward the discovery of small-molecule therapeutics. Hits uncovered via the analysis of archival kinase data ultimately led to the identification of a promising pyrrolo[2,3-d]pyrimidine scaffold. The optimization of this pyrrolo[2,3-d]pyrimidine core resulted in compound 1, which demonstrated potent in vitro RET kinase inhibition and robust in vivo efficacy in RET-driven tumor xenografts upon multiday dosing in mice. The administration of 1 was well-tolerated at established efficacious doses (10 and 30 mg/kg, po, qd), and plasma exposure levels indicated a minimal risk of KDR or hERG inhibition in vivo, as evaluated by Miles assay and free plasma concentrations, respectively.

A small molecule UPR modulator for diabetes identified by high throughput screening.

Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and in vivo disease models. Using asialoglycoprotein receptor 1 (ASGR) fused with Cypridina luciferase (CLuc) as reporter assay for folding capacity, we have screened a million small molecule library and identified APC655 as a potent activator of protein folding, that appears to act by promoting chaperone expression. Furthermore, APC655 improved pancreatic ß cell viability and insulin secretion under ER stress conditions induced by thapsigargin or cytokines. APC655 was also effective in preserving ß cell function and decreasing lipid accumulation in the liver of the leptin-deficient (ob/ob) mouse model. These results demonstrate a successful uHTS campaign that identified a modulator of UPR, which can provide a novel candidate for potential therapeutic development for a host of metabolic diseases.

Efficacy and Tolerability of Pyrazolo[1,5-a]pyrimidine RET Kinase Inhibitors for the Treatment of Lung Adenocarcinoma.

RET (REarranged during Transfection) kinase gain-of-function aberrancies have been identified as potential oncogenic drivers in lung adenocarcinoma, along with several other cancer types, prompting the discovery and assessment of selective inhibitors. Internal mining and analysis of relevant kinase data informed the decision to investigate a pyrazolo[1,5-a]pyrimidine scaffold, where subsequent optimization led to the identification of compound WF-47-JS03 (1), a potent RET kinase inhibitor with >500-fold selectivity against KDR (Kinase insert Domain Receptor) in cellular assays. In subsequent mouse in vivo studies, compound 1 demonstrated effective brain penetration and was found to induce strong regression of RET-driven tumor xenografts at a well-tolerated dose (10 mg/kg, po, qd). Higher doses of 1, however, were poorly tolerated in mice, similar to other pyrazolo[1,5-a]pyrimidine compounds at or near the efficacious dose, and indicative of the narrow therapeutic windows seen with this scaffold.

Cell-specific discrimination of desmosterol and desmosterol mimetics confers selective regulation of LXR and SREBP in macrophages.

Activation of liver X receptors (LXRs) with synthetic agonists promotes reverse cholesterol transport and protects against atherosclerosis in mouse models. Most synthetic LXR agonists also cause marked hypertriglyceridemia by inducing the expression of sterol regulatory element-binding protein (SREBP)1c and downstream genes that drive fatty acid biosynthesis. Recent studies demonstrated that desmosterol, an intermediate in the cholesterol biosynthetic pathway that suppresses SREBP processing by binding to SCAP, also binds and activates LXRs and is the most abundant LXR ligand in macrophage foam cells. Here we explore the potential of increasing endogenous desmosterol production or mimicking its activity as a means of inducing LXR activity while simultaneously suppressing SREBP1c-induced hypertriglyceridemia. Unexpectedly, while desmosterol strongly activated LXR target genes and suppressed SREBP pathways in mouse and human macrophages, it had almost no activity in mouse or human hepatocytes in vitro. We further demonstrate that sterol-based selective modulators of LXRs have biochemical and transcriptional properties predicted of desmosterol mimetics and selectively regulate LXR function in macrophages in vitro and in vivo. These studies thereby reveal cell-specific discrimination of endogenous and synthetic regulators of LXRs and SREBPs, providing a molecular basis for dissociation of LXR functions in macrophages from those in the liver that lead to hypertriglyceridemia.

Targeting Plasmodium PI(4)K to eliminate malaria.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery.

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Laparoscopic Roux-en-Y gastric bypass in patients with body mass index >70 kg/m2.

BACKGROUND: Sparse published data support the optimal surgical management of megaobesity (body mass index >70 kg/m(2)). The purpose of the present study was to compare laparoscopic Roux-en-Y gastric bypass (LRYGB) and open Roux-en-Y gastric bypass (ORYGB) in megaobese patients. METHODS: We conducted a retrospective review of 89 consecutive patients with a body mass index >70 kg/m(2) who underwent LRYGB or ORYGB from January 2003 to May 2007 at the Ohio State University Medical Center. RESULTS: LRYGB was performed in 37 patients, with 3 conversions to open surgery, and 52 underwent ORYGB. No statistically significant demographic or preoperative co-morbidity differences were discerned. The mean intraoperative blood loss was lower in the LRYGB group (54 mL versus 211 mL; P < .0001). The median length of stay for both LRYGB and ORYGB groups was 4 days. One patient in the open group died. The postoperative complications were statistically equivalent between the 2 groups. The hernia rate for the LRYGB group was 3% and was 19% in the ORYGB group (P = .02). The patients who underwent LRYGB had greater excess body weight loss at 3 (22.7% versus 17.5%, P = .02) and 6 (37.5% versus 30.5%, P = .03) months. However, the average excess body weight loss at 12 and 24 months was similar (48% and 60%, respectively). CONCLUSION: LRYGB is a technically feasible and safe surgical approach in the megaobese. The intraoperative blood loss was less with LRYGB than with ORYGB. The overall mortality and complications were not different, with the exception of hernia frequency, which was significantly greater after ORYGB. The percentage of excess body weight loss at 3 and 6 months was better for the LRYGB group. In both groups of patients, the 12- and 24-month excess body weight loss were similar.

Do gastrotomies require repair after endoscopic transgastric peritoneoscopy? A controlled study.

BACKGROUND: The optimal method for closing gastrotomies after transgastric instrumentation has yet to be determined. OBJECTIVE: To compare gastrotomy closure with endoscopically delivered bioabsorbable plugs with no closure. DESIGN: Prospective, controlled study. SETTING: Animal laboratory. SUBJECTS: Twenty-three dogs undergoing endoscopic transgastric peritoneoscopy between July and August 2007. INTERVENTIONS: Endoscopic anterior wall gastrotomies were performed with balloon dilation to allow passage of the endoscope into the peritoneal cavity. The plug group (n = 12) underwent endoscopic placement of a 4 x 6-cm bioabsorbable mesh plug in the perforation, whereas the no-treatment group (n = 11) did not. Animals underwent necropsy 2 weeks after the procedure. MAIN OUTCOME MEASUREMENTS: Complications related to gastrotomy closure, gastric burst pressures, relationship of burst perforation to gastrotomy, and the degree of adhesions and inflammation at the gastrotomy site. RESULTS: After the gastrotomy, all dogs survived without any complications. At necropsy, burst pressures were 77 +/- 11 mm Hg and 76 +/- 15 mm Hg (P = .9) in the plug group and no-treatment group, respectively. Perforations occurred at the site of the gastrotomy in 2 of 12 animals in the plug group and in none of the 11 dogs in the no-treatment group (P = .5). Finally, there were minimal adhesions in all dogs (11/11) in the no-treatment group and minimal adhesions in 3 and moderate adhesions or inflammatory masses in 9 of the 12 animals in the plug group (P = .004). LIMITATIONS: Small number of subjects, animal model, no randomization. Gastrotomy trauma during short peritoneoscopy may not be applicable to longer procedures. CONCLUSIONS: After endoscopic gastrotomy, animals that were left untreated did not show any clinical ill effects and demonstrated adequate healing, with fewer adhesions and less inflammation compared with those treated with a bioabsorbable plug.

Transgastric endoscopic peritoneoscopy does not require decontamination of the stomach in humans.

INTRODUCTION: Natural orifice translumenal endoscopic surgery (NOTES) is a rapidly evolving field that provides endoscopic access to the peritoneum via a natural orifice. One important requirement of this technique is the need to minimize the risk of clinically significant peritoneal contamination. We report the bacterial load and contamination of the peritoneal cavity in ten patients who underwent diagnostic transgastric endoscopic peritoneoscopy. METHODS: Patients participating in this trial were scheduled to undergo diagnostic laparoscopy for evaluation of presumed pancreatic cancer. Findings at diagnostic laparoscopy were compared with those of diagnostic transgastric endoscopic peritoneoscopy, using an orally placed gastroscope, blinding the endoscopist to the laparoscopic findings. We performed no gastric decontamination. Diagnostic findings, operative times, and clinical course were recorded. Gastroscope and peritoneal fluid aspirates were obtained prior to and after the gastrotomy. Each sample was sent for bacterial colony counts, culture, and identification of species. RESULTS: Ten patients, with an average age of 63.7 years, have completed the protocol. All patients underwent diagnostic laparoscopy followed by successful transgastric access and diagnostic peritoneoscopy. The average time for laparoscopy was 7.2 min, compared with 18 min for transgastric instrumentation. Bacterial sampling was obtained in all ten patients. The average number of colony-forming units (CFU) in the gastroscope aspirate was 132.1 CFU/ml, peritoneal aspirates prior to creation of a gastrotomy showed 160.4 CFU/ml, and peritoneal sampling after gastrotomy had an average of 642.1 CFU/ml. There was no contamination of the peritoneal cavity with species isolated from the gastroscope aspirate. No infectious complications or leaks were noted at 30-day follow-up. CONCLUSIONS: There was no clinically significant contamination of the peritoneal cavity from the gastroscope after transgastric endoscopic instrumentation in humans. Transgastric instrumentation does contaminate the abdominal cavity but, the pathogens do not mount a clinically significant response in terms of either the species or the bacterial load.

Targeted parathyroidectomy: effectiveness and intraoperative rapid-parathormone dynamics.

OBJECTIVE: The objective of this study is to assess the effectiveness of selective parathyroid exploration, using preoperative image localization and intraoperative rapid-parathormone (rPTH) assay. The kinetics of intraoperative rPTH in parathyroid adenoma vs. multiglandular disease is assessed. DESIGN: This is a prospective noncontrolled study of a cohort of 100 patients with primary hyperparathyroidism, at a single academic institution. The patients underwent selective parathyroidectomy after preoperative localization, including sestamibi scan and ultrasonography. Intraoperative rPTH assay was used to determine the extent and success of parathyroidectomy. Frozen sections were used as additional confirmation. Follow-up serum calcium levels were used to assess the effectiveness of selective parathyroidedcomy. RESULTS: Mean preoperative serum calcium (Ca) and baseline intact-parathormone were 11.6 mg/dL and 136, respectively. Data were available in 96 cases: 87 single-gland adenoma with two in ectopic mediastinal position, two double adenoma and seven cases of hyperplasia. Ten percent of patients with adenoma needed bilateral exploration for nonlocalizing or false negative imaging, or for intraoperative rPTH failure to decay. All of the patients undergoing unilateral targeted exploration were normocalcemic on follow up. There were only one failed exploration and two cases of recurrent mild hypercalcemia, all three in bilateral exploration cases. Intraoperative rPTH reduction by standard curves was predictive of successful excision of all of the abnormal glands, as confirmed by postoperative serum calcium levels. More than one postexcision rPTH measurement was useful by showing failure of a decaying slope in multiglandular disease. CONCLUSION: Targeted parathyroidectomy, when appropriately selected and carried out, is an effective treatment of primary hyperparathyroidism in most cases. Intraoperative rPTH can correctly guide removal of hyperfunctioning glands. Targeted parathyroidectomy offers the advantage of less invasive surgery with less tissue dissection confined to one side and avoids surgically disturbing the remainder of the neck. This should reduce postoperative complications and allow for easier and safer re-exploration in the few cases with persistent or recurrent disease.

Endovascular management of a splenic artery aneurysm.

Splenic artery aneurysms represent the third most common aneurysm in the abdomen. The majority are asymptomatic and discovered incidentally by abdominal imaging. The overall rupture rate is low but the associated mortality rate is high, especially in pregnant women and patients with portal hypertension. Traditionally, open surgical modalities represented the only viable treatments. More recently, laparoscopic and endovascular techniques have been applied to these vascular lesions. We report a case of a giant splenic artery aneurysm that was diagnosed incidentally and managed successfully by percutaneous splenic artery embolization. The case is presented and general considerations regarding the presentation, diagnosis, and management of splenic artery aneurysms are reviewed.

Synthesis and single-molecule studies of a well-defined biomimetic modular multidomain polymer using a peptidomimetic beta-sheet module.

In the pursuit of advanced biomaterials with combined strength, toughness, and elasticity, a new class of well-defined modular polymers has been synthesized, and their nanomechanical properties have been studied using atomic force microscopy. These polymers are based on a peptidomimetic beta-sheet-based double-closed loop (DCL) module, which was designed to overcome the limitation of the modular polymers we reported previously (J. Am. Chem. Soc. 2004, 126, 2059). Single-molecule force-extension experiments revealed the sequential unfolding of these modules as the polymer is stretched, resulting in more regular sawtooth-patterned curves similar to those seen in titin and other biopolymers. The single-molecule data agreed well with computer modeling, which suggested that hydrogen bonding and pi-stacking are both involved in the formation of small DCL clusters along the polymer chain.