Optimisation of pharmacokinetic properties to afford an orally bioavailable and selective V1A receptor antagonist.

The previously described lead compound 5 is a potent and selective V(1A) antagonist with affinity at both the rat and human receptor, but displays poor oral bioavailability and moderate clearance. We report herein the successful optimisation of the pharmacokinetic (PK) properties to afford the potent, selective, orally bioavailable and CNS penetrant compound 15f. A custom optimisation approach was required which demonstrated the value of using early, rapid in vivo PK studies to show improvements in oral exposure. Such assays may be of particular value where low oral bioavailability is anticipated to be multifactorial (e.g., permeability, gut wall metabolism and/or transport) where satisfactory modelling of in vitro data is likely to be difficult within a drug discovery context.

Synthesis and SAR studies of novel 2-(6-aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) antagonists are described. 2-(6-Aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.

Identification and optimisation of novel sulfonamide, selective vasopressin V1B receptor antagonists.

The synthesis and preliminary structure-activity relationships (SAR) of a novel class of vasopressin V(1B) receptor antagonists are described. Hit compound 5, identified via high throughput screening of the corporate collection, showed good activity in a V(1B) binding assay (K(i) 63 nM) but did not possess the lead-like physicochemical properties typically required in a hit compound. A 'deletion approach' on the HTS hit 5 was performed, with the focus on improvement of physicochemical properties, yielding the selective V(1B) antagonist 9f (K(i) 190 nM), with improved druglike characteristics.

The discovery of novel 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamides as vasopressin V1A receptor antagonists.

The discovery of a novel series of 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamide antagonists of the vasopressin V(1A) receptor is disclosed. Compounds 47 and 48 were found to be high affinity, selective vasopressin V(1A) antagonists.

Synthesis and SAR studies of novel 2-(4-oxo-2-aryl-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) (V(3)) antagonists are described. 2-(4-Oxo-2-aryl-quinazolin-3(4H)-yl)acetamides have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and oxytocin (OT). Optimised compound 12j demonstrates a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.

The identification, and optimisation of hERG selectivity, of a mixed NET/SERT re-uptake inhibitor for the treatment of pain.

Hit compound 1, a selective noradrenaline re-uptake transporter (NET) inhibitor was optimised to build in potency at the serotonin re-uptake transporter (SERT) whilst maintaining selectivity against the dopamine re-uptake transporter (DAT). During the optimisation of 1 it became clear that selectivity against the Kv11.1 potassium ion channel (hERG) was also a parameter for optimisation within the series. Discrete structural changes to the molecule as well as a lowering of global cLogP successfully increased the hERG selectivity to afford compound 11 m, which was efficacious in a mouse model of inflammatory pain, complete Freund's adjuvant (CFA) induced thermal hyperalgesia and a rat model of neuropathic pain, spinal nerve ligation (SNL) induced mechanical allodynia.

Identification and optimization of novel 2-(4-oxo-2-aryl-quinazolin-3(4H)-yl)acetamide vasopressin V3 (V1b) receptor antagonists.

The discovery, synthesis, and preliminary structure-activity relationship (SAR) of a novel class of vasopressin V3 (V1b) receptor antagonists is described. Compound 1, identified by high throughput screening of a diverse, three million-member compound collection, prepared using ECLiPS technology, had good activity in a V3 binding assay (IC50=0.20 microM), but less than desirable physicochemical properties. Optimization of compound 1 yielded potent analogs 19 (IC50=0.31 microM) and 24 (IC50=0.12 microM) with improved drug-like characteristics.

Selectin ligand expression regulates the initial vascular interactions of colon carcinoma cells: the roles of CD44v and alternative sialofucosylated selectin ligands.

Selectin-mediated binding of tumor cells to platelets, leukocytes, and vascular endothelium may regulate their hematogenous spread in the microvasculature. We recently reported that CD44 variant isoforms (CD44v) on LS174T colon carcinoma cells possess selectin binding activity. Here we extended those findings by showing that T84 and Colo205 colon carcinoma cells bind selectins via sialidase-sensitive O-linked glycans presented on CD44v, independent of heparan and chondroitin sulfate. To assess the functional role of CD44v in selectin-mediated binding, we quantified the adhesion to selectins of T84 cell subpopulations sorted based on their CD44 expression levels and stable LS174T cell lines generated using CD44 short hairpin RNA. High versus low CD44-expressing T84 cells tethered more efficiently to P- and L-selectin, but not E-selectin, and rolled more slowly on P- and E-selectin. Knocking down CD44 expression on LS174T cells inhibited binding to P-selectin and increased rolling velocities over P- and L-selectin relative to control-transfected cells, without affecting tethering and rolling on E-selectin, however. Blot rolling analysis revealed the presence of alternative sialylated glycoproteins with molecular masses of approximately 170 and approximately 130 kDa, which can mediate selectin binding in CD44-knockdown cells. Heparin diminishes the avidity of colon carcinoma cells for P- and L-selectin, which may compromise integrin-mediated firm adhesion to host cells and mitigate metastasis. Our finding that CD44v is a functional P-selectin ligand on colon carcinoma provides a novel perspective on the enhanced metastatic potential associated with tumor CD44v overexpression and the role of selectins in metastasis.

Variant isoforms of CD44 are P- and L-selectin ligands on colon carcinoma cells.

The initial selectin-dependent events that mediate tumor cell tethering to platelets, leukocytes, and vascular endothelium can regulate the extravasation and colonization of metastatic cells into distant tissues. Little is known, however, about the identity of selectin counter-receptors on tumor cells, which facilitate the metastatic process. To address this issue, we performed SDS-PAGE analysis of membrane proteins, metabolic inhibition studies, blot rolling assays, and cell-free flow-based adhesion experiments using microbeads coated with CD44 immunoprecipitated from carcinomas and purified selectins as substrate. Here, we demonstrate that variant isoforms of CD44 (CD44v) on LS174T colon carcinoma cells possess P-/L-/E-selectin binding activity, in contrast to the standard isoform of CD44 (CD44s) on hematopoietic-progenitor cells (HPCs), which is primarily an L-/E-selectin ligand. Moreover, the selectin-binding determinants on CD44v from LS174T cells are sialofucosylated structures displayed on O-linked glycans, akin to those on P-selectin glycoprotein ligand-1, but distinct from the HECA-452-reactive N-glycans on CD44s expressed on HPCs. Using flow-based adhesion assays, we systematically characterize shear-dependent LS174T CD44 vs. HL60 CD44s adhesion to E-/P-/L-selectin. The novel finding that CD44v are selectin ligands offers a unifying perspective on the apparent enhanced metastatic potential associated with tumor cell CD44v overexpression and the critical role of selectins in metastasis.

Preferential binding of platelets to monocytes over neutrophils under flow.

This study was undertaken to systematically investigate the binding kinetics of platelet recruitment by monocytes relative to neutrophils in bulk suspensions subjected to shear as well as the molecular requirements of leukocyte-platelet binding. Hydrodynamic shear-induced collisions augment the proportion of monocytes with adherent platelets more drastically than that of neutrophils with bound platelets. These heterotypic interactions are further potentiated by platelet activation with thrombin or to a lesser extent by monocyte stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Monocyte-platelet heteroaggregation increases with increasing shear rate and shear exposure time. Platelet P-selectin binding to monocyte P-selectin-glycoprotein-ligand-1 is solely responsible for maximal platelet adhesion to unstimulated monocytes in shear flow. However, the enhanced platelet binding to fMLP-treated monocytes involves a sequential two-step process, wherein P-selectin-PSGL-1 interactions are stabilized by CD18-integrin involvement. Blocking platelet alpha(IIb)beta(3) or monocyte beta(1)-integrin function had no effect. This study underscores the preferential recruitment of platelets by monocytes relative to neutrophils in shear flow, and demonstrates that the shear environment of the vasculature coupled to the state of cell activation modulates the dynamics and molecular constituents mediating monocyte-platelet adhesion.

Systemic lupus erythematosus genome scan: support for linkage at 1q23, 2q33, 16q12-13, and 17q21-23 and novel evidence at 3p24, 10q23-24, 13q32, and 18q22-23.

OBJECTIVE: To identify chromosome regions likely to harbor genes that predispose to the development of systemic lupus erythematosus (SLE) by analyzing a full genome scan in nuclear families ascertained for siblings with SLE. METHODS: Approximately 400 multiallelic markers spaced an average of 10 cM apart were genotyped in a multiethnic panel of 238 individuals from 62 multiplex SLE families having 88 affected sibling pairs and 456 total sibling pairs. Findings were analyzed by 2 model-free statistical linkage procedures. RESULTS: Evidence supporting linkage to 4 previously reported (1q23, 2q33, 16q12-13, and 17q21-23) and 4 novel (3p24, 10q23-24, 13q32, and 18q22-23) chromosome regions was revealed. Stratification by family ethnicity indicated that linkage to 3 regions, 2q33, 10q23-24, and 18q22-23, was derived primarily from the Caucasian families, while linkage to 17q21-23 was seen primarily in the non-Caucasian families. CONCLUSION: Linkage to the same chromosome regions in independent cohorts is a critical step in finding the genes that predispose to a complex disorder such as SLE. Four linked regions also seen in independent SLE cohorts lend credibility to the 4 novel regions identified by these analyses. Substantial linkage information was gleaned by genotyping and analyzing the unaffected siblings. These results provide additional evidence that the SLE clinical phenotype is genetically complex, multigenic, and heterogeneous.

Quantitative trait loci for apolipoprotein B, cholesterol, and triglycerides in familial combined hyperlipidemia pedigrees.

OBJECTIVE: Familial combined hyperlipidemia (FCHL) is a genetically complex lipid disorder that is diagnosed in families by combinations of increased cholesterol, triglycerides, and/or apolipoprotein B (apoB) levels in patients and their first-degree relatives. Identifying the predisposing genes promises to reveal the primary risk factors and susceptibility pathways and suggest methods of prevention and treatment. As with most genetically complex disorders, a clinical definition of disease may not be the most useful phenotype for finding the complement of predisposing genes, and the quantitative traits used to define the disorder can provide important information. This is a report of a quantitative trait loci (QTL) analysis of FCHL. METHODS AND RESULTS: A full genome scan of 377 multi-allelic markers genotyped at approximately 10 centimorgan (cM) intervals was conducted in 150 sibling pairs from 22 nuclear families in FCHL pedigrees. These data were analyzed by 2 multipoint QTL linkage methods using the nonparametric and Haseman-Elston procedures of the Genehunter software. Using a criterion of P<0.001 by the nonparametric analysis, we found evidence of 2 apoB QTL at 1p21-31 (P<0.000009) and 17p11-q21 (P<0.000009), a total serum cholesterol QTL at 12p13 (P<0.0001), and a serum triglycerides QTL at 4p15-16 (P<0.0002). Using the criterion of P<0.03 for at least 2 traits at the same locus, additional evidence for cholesterol (P<0.01) and a triglycerides P<0.02) was observed at 17p11-21, as well as suggestive evidence for apoB (P<0.02) and triglycerides (P<0.01) at 4q34-35, and cholesterol (P<0.01) and triglycerides (P<0.02) and a binary FCHL trait (lod=1.5) at 16p12-13. CONCLUSIONS: QTL analyses of the traits that define FCHL are effective for localizing disease-predisposing genes.