Design, synthesis and evaluation of MCH receptor 1 antagonists--Part III: Discovery of pre-clinical development candidate BI 186908.

Although overweight and obesity are highly prevalent conditions, options to treat them are still very limited. As part of our search for safe and effective MCH-R1 antagonists for the treatment of obesity, two series of pyridones and pyridazinones were evaluated. Optimization was aimed at improving DMPK properties by increasing metabolic stability and improving the safety profile by reducing inhibition of the hERG channel and reducing the potential to induce phospholipidosis. Steric shielding of a labile keto moiety with an ortho-methyl group and fine-tuning of the polarity in several parts of the molecule resulted in BI 186908 (11 g), a potent and selective MCH-R1 antagonist with favorable DMPK and CMC properties. Chronic administration of BI 186908 resulted in significant body weight reduction comparable to sibutramine in a 4 week diet-induced obesity model in rats. Based on its favorable safety profile, BI 186908 was advanced to pre-clinical development.

Design, synthesis and evaluation of MCH receptor 1 antagonists--Part I: Optimization of HTS hits towards an in vivo efficacious tool compound BI 414.

Despite recent approvals of anti-obesity drugs there is still a high therapeutic need for alternative options with higher efficacy in humans. As part of our MCH-R1 antagonist program for the treatment of obesity, a series of biphenylacetamide HTS hits was evaluated. Several issues of the initial lead structures had to be resolved, such as potency, selectivity over related GPCRs and P-gp efflux limiting brain exposure in this series. We could demonstrate that all parameters can be significantly improved by structural modifications resulting in BI 414 as a potent and orally available MCH-R1 antagonist tool compound with acceptable in vivo efficacy in an animal model of obesity.

Design, synthesis and evaluation of MCH receptor 1 antagonists--Part II: Optimization of pyridazines toward reduced phospholipidosis and hERG inhibition.

Despite recent success there remains a high therapeutic need for the development of drugs targeting diseases associated with the metabolic syndrome. As part of our search for safe and effective MCH-R1 antagonists for the treatment of obesity, a series of 3,6-disubstituted pyridazines was evaluated. During optimization several issues of the initial lead structures had to be resolved, such as selectivity over related GPCRs, inhibition of the hERG channel as well as the potential to induce phospholipidosis. Utilizing property-based design, we could demonstrate that all parameters can significantly be improved by consequently increasing the polarity of the compounds. By this strategy, we succeeded in identifying potent and orally available MCH-R1 antagonists with good selectivity over M1 and 5-HT2A and an improved safety profile with respect to hERG inhibition and phospholipidosis.

Phosphoglycerate kinase 1 as a promoter of metastasis in colon cancer.

Oxidative stress due to intratumoral hypoxia in solid cancer has been shown to be associated with increased mortality. Phosphoglycerate kinase 1 (PGK1) is an enzyme of the glycolytic pathway, which is regulated by hypoxia-inducible factor-1α (HIF-1α) and has been described for its role in tumor progression and metastasis in several malignancies. We investigated whether the expression of PGK1 varies between metastatic and non-metastatic colon cancer. We compared PGK1 expression in colon cancer patients either with or without metastasis via polymerase chain reaction (PCR) and immunohistochemistry. Microarray analysis was performed to test altered gene expression after PGK1 silencing, using isolates from HCT116 cell lines. PCR results showed an increased expression of PGK1 in colon cancer tissue from metastatic patients in comparison to patients with no metastasis (fold change 2.6, p<0.001). Immunohistochemical staining of PGK1 showed stronger staining in metastatic tissue in comparison to non-metastatic cancer tissue according to a semi-quantitative evaluation. Microarray and subsequent pathway analysis provided 4 genes of interest (CYR61, FOS, JUN and EGR1) used for pathway proposal. The results indicate that increased expression of PGK1 in colon cancer tissue is associated with metastasis. Furthermore, we propose several genes induced by PGK1 that could account for cell migration, mainly EGR1 and CYR61 together with the transcription factors FOS and JUN.

Transesophageal NOTES--a critical analysis of relevant problems.

The transesophageal access route has not become a principal topic in the discussion about NOTES up to now. Analyzing the problems in this new field of surgery, however, the transesophageal route shows high relevance. Here, all possibilities, limitations, and problems of NOTES become obvious. This paper contains a critical analysis of the literature published to date (nine full papers, five abstracts). Nearly all publications represent experimental studies in living pigs. In most cases a submucosal tunnel technique is performed as access route to mediastinum, pleural cavity, and heart. Interventions and operations include simple mediastinoscopies as well as epicardial operations after exposition of the heart. For access and manipulation, conventional flexible endoscopes and instruments are used. Clips, T-bars, or a combination of both achieve the closure. Some studies rely on spontaneous closure of the incision without any suturing or approximation. In such experimental settings, the following results are presented: Access is achieved in 90% of cases, the aim of the operation is met in 92%, technical success in closure is achieved in 90%, healing of incision assessed as good in two of five studies, satisfactory in three of five studies. Mortality, ranging from 6 to 25%, and complication rates were (surprisingly) high. It has to be stressed that analyzing these papers published to date, no adequate attention is paid to basic facts and problems of general and thoracic surgery (e.g. different forms, prevention, diagnosis and therapy of pneumothorax or differentiated forms of ventilation). Relevant differences in the anatomy and physiology of the esophagus and mediastinum between humans and pigs should additionally be taken into account to choose optimal experimental parameters when transferring results to human settings. Moreover, requirements regarding sterility and hygiene in a structure like the mediastinum, which is at high risk from the point of view of infection biology, have not yet been respected. These factors should be taken into account in further studies--as well as clinically relevant disease patterns in humans--to be able to realize possible advantages of this NOTES access in a clinical setting.

Simultaneous adrenal and extra-adrenal myelolipoma - an uncommon incident: case report and review of the literature.

BACKGROUND: Extra-adrenal myelolipomas are rare benign tumours. Other soft tissue tumours such as well-differentiated liposarcomas appear morphological almost identical. Preoperative imaging and especially biopsy are important tools to diagnose these lesions. CASE PRESENTATION: We report a very seldom case of a simultaneous myelolipoma of the adrenal gland in association with an extra-adrenal myelolipoma in an 75-year-old man. With a review of the literature we describe and discuss the aetiology, differential diagnosis and treatment of patients with respect to adrenal and extra-adrenal lesions. CONCLUSION: The appearance of a simultaneous adrenal and extra-adrenal myelolipoma is a rare incident. We conclude that such lesions should be considered in the differential diagnosis of a fat-containing tumour in the retroperitoneal tissue/compartment.

Minimizing oxidative stress by gene delivery of superoxide dismutase accelerates regeneration after transplantation of reduced-size livers in the rat.

Transplantation of reduced-size livers may lead to a hypermetabolic state and increased production of oxygen radicals. Since oxygen radicals may cause liver injury and impair liver regeneration, we tested the hypothesis that overexpression of superoxide dismutase (SOD) in reduced-size livers (RSL) would accelerate regeneration and reduce injury in a rat model of transplantation of RSL. Donor rats were infected with adenoviruses either expressing SOD1 (Ad.SOD1) or beta-galactosidase (Ad.lacZ). Livers were harvested 72 hours later, reduced to 45% of weight, and transplanted. After transplantation, hepatic SOD activity, graft survival, histopathology, AST/ALT release, and bilirubin were examined. Regeneration was evaluated by BrdU-staining, graft weight, and expression of cyclin D1 and p21. In Ad.SOD1-treated livergrafts, SOD activity increased three-fold compared to controls. Survival was dramatically increased in recipients of Ad.SOD1-RSL (100% vs. 20% in Ad.lacZ-RSL), and peak levels of AST/ALT and bilirubin levels were reduced by 75% and 87.5%, respectively (P < 0.001). In histological sections, hepatocyte necrosis decreased from 24% after Ad.lacZ-treatment to 6% after Ad.SOD1-treatment (P <0.001). Regeneration was also accelerated after Ad.SOD1-treatment as demonstrated by an increase of BrdU-stained cells 24 hours after reperfusion and increased liver weight after 1 week. In conclusion, overexpression of SOD1 in RSL prevents primary non-function of reduced-size liver grafts and accelerates liver regeneration.

Arterial reconstruction in rat liver transplantation--development of a new tubing technique of the common hepatic artery.

Arterialization of liver transplants in rats results in an improved function compared with grafts without artery. Here we compared techniques of reconstruction, focusing on thrombosis, duration of procedure and severity of pancreas damage after dissecting the gastroduodenal artery (GDA). Group 1: tube was inserted into the proper hepatic artery (PHA) of donor and recipient. Group 2: tube was placed into common hepatic artery (CHA) of donor and recipient. Group 3: cuff was placed over the CHA of the recipient and the graft's artery was slipped over the cuff. Tubing in PHA leads to a thrombosis rate of 40% after 6 months. Arteries remain perfused by using a cuff or tube in CHA. Dissection of the GDA does not influence pancreatic perfusion. Reconstruction took 19 s using the large tube, about 30 s for the tube into PHA and 1 min for the cuff. The method of choice is using a tube for the CHA.

C1-inhibitor reduces hepatic leukocyte-endothelial interaction and the expression of VCAM-1 in LPS-induced sepsis in the rat.

INTRODUCTION: Increased leukocyte-endothelial interaction (LEI) leading to hepatic microperfusion disorders is proposed as major contributor for hepatic failure during sepsis. Recently it has been demonstrated that complement inhibition by C1-inhibitor (C1-INH) is an effective treatment against microcirculatory disturbances in various diseases. The purpose of this study was to investigate the influence of C1-INH on microcirculation and LEI in the liver in a rat model of sepsis. MATERIALS AND METHODS: Rats received lipopolysaccharides (LPS) from Escherichia coli intravenously. Controls received Ringer solution only. Ninety minutes after LPS infusion some animals were treated with C1-INH intravenously (LPS + C1-INH). Others (LPS + SC) and controls (Ringer + SC) received sodium chloride (SC). Hepatic LEI and mean erythrocyte velocity (MEV) were quantified by intravital microscopy (IVM) 90 min after LPS or Ringer infusion (0) and 30, 60, 90 and 120 min following treatment. VCAM-1 m-RNA in hepatic tissue, C3a, TNF-alpha and hepatic enzyme liberation in blood was analysed. RESULTS: Leukocyte sticking to the endothelial wall in postsinusoidal venules was significantly reduced in the LPS + C1-INH vs. the LPS + SC group 30, 60, 90 and 120 min after treatment. VCAM-1 m-RNA expression in the hepatic tissue was markedly and C3a levels in plasma were significantly reduced in the LPS + C1-INH vs. the LPS + SC group. No differences in TNF-alpha levels were detected between these two groups. MEV was improved in the LPS + C1-INH vs. the LPS + SC group. CONCLUSIONS: Our results indicate that even upon delayed treatment hepatic adhesion molecule expression and LEI can be reduced by C1-INH. The multifunctional regulator may reduce hepatic microcirculatory disturbances during sepsis under clinical conditions.

Effects of three superoxide dismutase genes delivered with an adenovirus on graft function after transplantation of fatty livers in the rat.

BACKGROUND: Oxygen-derived free radicals play a central role in ischemia/reperfusion injury after organ transplantation and are degraded by endogenous radical scavengers such as superoxide dismutase (SOD). Overexpression of SOD by delivery of the cytosolic SOD gene with an adenovirus (Ad.SOD1) decreases organ injury and increases survival in a rat model of liver transplantation. However, it is unclear which of the three isoforms of SOD provides the most protective effect. The purpose of this study was to identify the isoform with the highest effectiveness against ischemia/reperfusion injury after transplantation of fatty livers, which are particularly susceptible. METHODS: Donor rats were given ethanol by gavage before harvest to induce steatotic livers. Some of the donors were infected with adenoviruses expressing either the gene lacZ encoding bacterial beta-galactosidase (Ad.lacZ), Ad.SOD1, Ad.SOD2 (mitochondrial isoform), or Ad.SOD3 (extracellular isoform). After transplantation, SOD activity in liver, survival, histopathology, transaminases, and activation of nuclear factor (NF)-kappaB, IkappaB kinase, Jun-N-terminal kinase (JNK), and tumor necrosis factor (TNF)-alpha were evaluated. RESULTS: Ad.SOD1 treatment increased survival, blunted transaminase release, and reduced necrosis, whereas Ad.SOD3 had no protective effect. Ad.SOD2 was not as protective as Ad.SOD1. Ad.SOD1 reduced the activation of NF-kappaB, blunted JNK activity, and reduced TNF-alpha activity. Ad.SOD2 treatment resulted in lower kinase, TNF-alpha, and NF-kappaB activities but was not as effective as Ad.SOD1. IkappaB kinase activity was not affected. CONCLUSION: This study demonstrates that cytosolic SOD represents the most effective isoform of SOD to protect transplanted livers from failure; this may be related to lowered NF-kappaB and JNK activities because of reduced oxygen-derived radical production.