Pharmacodynamic and efficacy profile of TGN 255, a novel direct thrombin inhibitor, in canine cardiopulmonary bypass and simulated mitral valve repair.

Heparin-induced thrombocytopenia can be a life-threatening sequel to conventional use of unfractionated heparin in cardiopulmonary bypass (CPB). This study evaluated the pharmacokinetic/pharmacodynamic (PK/PD) and efficacy profile of a novel direct thrombin inhibitor, TGN 255, during cardiac surgery in dogs. Point-of-care coagulation monitoring was also compared against the plasma concentrations of TRI 50c, the active metabolite of TGN 255. The study was conducted in three phases using 10 animals: phase 1 was a dose-ranging study in conscious animals (n = 6), phase 2 was a similar but terminal dose-ranging study in dogs undergoing CPB (n = 6), and phase 3 was with animals undergoing simulated mitral valve repair (terminal) using optimal TGN 255 dose regimens derived from phases I and II (n = 4). During the study, PD markers and drug plasma levels were determined. In addition, determinations of hematologic markers and blood loss were undertaken. Phase 1 studies showed that a high-dose regimen of a 5-mg/kg bolus and infusion of 20 mg/kg/h elevated PD markers in conscious animals, at which time there were no measured effects on platelet or red blood cell counts, and the mean plasma concentration of TRI 50C was 20.6 microg/mL. In the phase 2 CPB dose-ranging study, this dosing regimen significantly elevated all the PD markers and produced hemorrhagic and paradoxical thrombogenic effects. In the phase 3 surgical study, a lower TGN 255 dose regimen of a 2.5-mg/kg bolus plus 10 mg/kg/h produced anticoagulation, elevated PD markers, and produced minimal post-operative blood loss in the animals. Plasma levels of TRI 50C trended well with the conventional point-of-care coagulation monitoring. TGN 255 provided effective anticoagulation in a canine CPB procedure, enabling successful completion with minimal blood loss. These findings support further evaluation of TGN 255 as an anticoagulant for CPB.

Directed osteogenic differentiation of human mesenchymal stem/precursor cells on silicate substituted calcium phosphate.

Insufficient, underactive, or inappropriate osteoblast function results in serious clinical conditions such as osteoporosis, osteogenesis imperfecta and fracture nonunion and therefore the control of osteogenesis is a medical priority. In vitro mesenchymal stem cells (MSCs) can be directed to form osteoblasts through the addition of soluble factors such as ß-glycerophosphate, ascorbic acid, and dexamethasone; however this is unlikely to be practical in the clinical setting. An alternative approach would be to use a scaffold or matrix engineered to provide cues for differentiation without the need for soluble factors. Here we describe studies using Silicate-substituted calcium phosphate (Si-CaP) and unmodified hydroxyapatite (HA) to test whether these materials are capable of promoting osteogenic differentiation of MSCs in the absence of soluble factors. Si-CaP supported attachment and proliferation of MSCs and induced osteogenesis to a greater extent than HA, as evidenced through upregulation of the osteoblast-related genes: Runx2 (1.2 fold), Col1a1 (2 fold), Pth1r (1.5 fold), and Bglap (1.7 fold) Dmp1 (1.1 fold), respectively. Osteogenic-associated proteins, alkaline phosphatase (1.4 fold), RUNX2, COL1A1, and BGLAP, were also upregulated and there was an increased production of mineralized bone matrix (1.75 fold), as detected by the Von Kossa Assay. These data indicate that inorganic substrates are capable of directing the differentiation programme of stem cells in the absence of known chemical drivers and therefore may provide the basis for bone repair in the clinical setting.

Increasing strut porosity in silicate-substituted calcium-phosphate bone graft substitutes enhances osteogenesis.

Synthetic, porous silicate-substituted calcium phosphate bone graft matrices (SiCaP; 0.8 wt % Si) with varying strut porosity were applied to ovine critical-sized defect sites as either 1-2 mm microgranules (SiCaP-23G, SiCaP-32G, and SiCaP-46G) or 1-2 mm microgranules in an aqueous poloxamer carrier (SiCaP-23P, SiCaP-32P, and SiCaP-46P). Defect sites treated with SiCaP-23G or SiCaP-23P showed evidence of bone formation at 8 and 12 weeks in central zones. More advanced neovascularization and increased bone contact was observed for graft materials with higher strut porosities. At 12 weeks, graft materials with higher strut porosities (32% and 46%) had statistically significantly higher absolute bone volumes (p < 0.05) versus those with a strut porosity of 23%. Absolute bone volume in defects treated with grafts of matched strut porosities as microgranules, or microgranules with poloxamer carrier, were similar at 12 weeks. Absolute graft volume for SiCaP-46 reduced over 12 weeks (not statistically significant). In conclusion, bone formation patterns in critically-sized defects confirm strut porosity to be a clinically relevant property of porous silicate-substituted calcium phosphate bone grafts in promoting osteogenesis. Increasing graft matrix strut porosity encouraged earlier neovascularization and increased the absolute equilibrium volume of bone growth within the graft without compromising graft stability.

Studies of benzothiophene template as potent factor IXa (FIXa) inhibitors in thrombosis.

FIXa is a serine protease enzyme involved in the intrinsic pathway of the coagulation cascade. The upstream intervention of the coagulation cascade in selectively inhibiting FIXa would leave hemostasis intact via the extrinsic pathway, leading to an optimum combination of efficacy and safety with low incidence of bleeding. We have identified 2-amindinobenzothiophene template as a lead scaffold for FIXa inhibiton based on its homology with urokinase plasminogen activator (uPA). Subsequent SAR work on the template revealed a number of highly potent FIXa inhibitors, though with moderate selectivity against FXa. X-ray study with one of the analogues demonstrated active site binding interaction with the induced opening of the S1 beta pocket and a secondary binding at the S2-S4 sites, which is in direct contrast with the previous finding.

Structure based drug design: development of potent and selective factor IXa (FIXa) inhibitors.

On the basis of our understanding on the binding interactions of the benzothiophene template within the FIXa active site by X-ray crystallography and molecular modeling studies, we developed our SAR strategy by targeting the 4-position of the template to access the S1 beta and S2-S4 sites. A number of highly selective and potent factor Xa (FXa) and FIXa inhibitors were identified by simple switch of functional groups with conformational changes toward the S2-S4 sites.