Purpose: Trabectedin (Yondelis, ET-743) is a novel anticancer drug with potent activity against various tumors. However, dose-limiting hepatotoxicity was observed during clinical trials. Because recent reports have suggested that cytochrome P450 3A (CYP3A), as well as the drug transporters ABCB1, ABCC2, and ABCC3 might protect against trabectedin-mediated hepatotoxicity, we investigated the individual and combined roles of these detoxifying systems. Experimental Design: Madin-Darby canine kidney cells expressing ABCC2 and ABCC3 were used to study in vitro trabectedin transport. We investigated the hepatotoxicity of trabectedin, and the plasma and liver levels of this drug and its metabolites in mice deficient for CYP3A, Abcb1a/1b, Abcc2, and/or Abcc3 after i.v. trabectedin administration. Results: Trabectedin was transported by ABCC2 but only modestly by ABCC3. Contrary to our expectation, absence of CYP3A resulted in only a marginal increase in hepatotoxicity. Some hepatotoxicity was observed in Abcc2 -/-mice, but very little in Abcb1a/ 1b -/-and Abcc3 -/-mice. Strikingly, severe hepatotoxicity was found in Abcb1a/1b/ Abcc2 -/-and Abcc2/Abcc3 -/-mice. However, hepatotoxicity was drastically decreased in Cyp3a/Abcb1a/1b/Abcc2 -/-compared with Abcb1a/1b/Abcc2 -/-mice. This suggests that the formation of CYP3A-specific metabolites is an important prerequisite for trabectedin-mediated hepatotoxicity. Further studies revealed that there is increased accumulation of metabolites of trabectedin, but not of trabectedin itself, in the livers of mice that lack Abcc2 but are CYP3A proficient. Conclusions: Our data show that ABCB1, ABCC2, and ABCC3 have a profound and partially redundant function in protection from trabectedin-mediated hepatotoxicity, presumably by clearing the liver from hepatotoxic trabectedin metabolites that are primarily formed by CYP3A. (Clin Cancer Res 2009;15(24):7616-23) Trabectedin is an anticancer drug isolated from a marine tunicate that has recently been approved for the treatment of soft tissue carcinoma. In addition, several phases II and III clinical trials of trabectedin for treatment of other cancer types have been published or are still ongoing, including trials for breast (1), ovarian (2, 3), and prostate cancer (4). Although trabectedin has impressive antitumor activity, dose-limiting hepatotoxicity became apparent during several clinical trials (reviewed in ref. 5).Mass balance studies have revealed that trabectedin is extensively metabolized, and virtually all trabectedin is excreted in its metabolite form into bile. The chemical structure of trabectedin provides numerous sites for metabolic conversion. Indeed, a large number of (unknown) metabolites, without clearly predominating metabolites, could be observed in radiochromatograms after i.v. administration of ( 14 C)trabectedin to patients (6). No metabolites in plasma have been structurally identified so far and only a few metabolites from feces (6). The low dose and high volume of distribution, and long terminal half-l...

The use of growth factors in osteogenic constructs to promote recruitment of bone forming endogenous cells is not clear, while the advantage of circumventing cell seeding techniques before implantation is highly recognized. Therefore, the additive effect of the chemokine stromal cell-derived factor-1α (SDF-1α) on endogenous cell recruitment and vascularization was investigated in a hybrid construct, consisting of a ceramic biomaterial, hydrogel, and SDF-1α, in an ectopic mouse model. We demonstrated in vivo that local presence of low concentrations of SDF-1α resulted in a significant increase in recruited endogenous cells, which remained present for several weeks. SDF-1α stimulated vascularization in these hybrid constructs, as shown by the enhanced formation of erythrocyte-filled vessels. The presence of CD31-positive capillaries/small vessels after 6 weeks in vivo substantiated this finding. The SDF-1α treatment showed increased number of cells that could differentiate to the osteogenic lineage after 6 weeks of implantation, demonstrated by expression of collagen I and osteocalcin. Altogether, we show here the beneficial effects of the local application of a single growth factor in a hybrid construct on angiogenesis and osteogenic differentiation, which might contribute to the development of cell-free bone substitutes.

Vascularization is crucial for the induction of bone formation. In this study, we investigated the application of two subtypes of peripheral blood-derived endothelial progenitor cells (EPCs) to stimulate vessel formation in ectopic bone constructs. Early and late outgrowth EPCs (E-EPC and L-EPC, respectively) were characterized for their ability to form network structures in vitro and perfused vessels subcutaneously in mice. Only L-EPCs showed the formation of fully connected networks on Matrigel two-dimensional (2D) angiogenesis assays. The presence of multipotent stromal cells (MSCs) inhibited network formation in 2D assays, but stimulated network formation in three-dimensional plugs. In vivo studies revealed that at 2 weeks, the highest incidence of formed perfused vessels was reached by implanted E-EPC/MSC constructs and this could be attributed to the presence of E-EPCs. L-EPCs displayed a significantly lower frequency of blood vessel formation than E-EPCs and this was accompanied by a lowering of total luminal area densities. Nevertheless, combined E-EPC/L-EPC application somewhat increased the percentage incidence of perfused vessels. After 6 weeks, differences in vascularization were still obvious as all three EPC-based constructs contained higher numbers of perfused vessels than constructs containing MSCs alone. Bone was formed in all constructs at an incidence that coincided with high density of perfused vessels after 2 weeks. Altogether, our findings suggest the differential establishment of vascular networks by E-EPCs and L-EPCs and suggest the importance of early vasculogenesis in ectopic bone formation.

Bone defect healing is highly dependent on the simultaneous stimulation of osteogenesis and vascularization. In bone regenerative strategies, combined seeding of multipotent stromal cells (MSCs) and endothelial progenitor cells (EPCs) proves their mutual stimulatory effects. Here, we investigated whether stromal-cell-derived factor1a (SDF-1a) stimulates vascularization by EPCs and whether SDF-1a could replace seeded cells in ectopic bone formation. Late EPCs of goat origin were characterized for their endothelial phenotype and showed to be responsive to SDF-1a in in vitro migration assays. Subsequently, subcutaneous implantation of Matrigel plugs that contained both EPCs and SDF-1a showed more tubule formation than constructs containing either EPCs or SDF-1a. Addition of either EPCs or SDF-1a to MSC-based constructs showed even more elaborate vascular networks after 1 week in vivo, with SDF-1a/MSC-laden groups showing more prominent interconnected networks than EPC/MSC-laden groups. The presence of abundant mouse-specific CD31/PECAM expression in these constructs confirmed ingrowth of murine vessels and discriminated between angiogenesis and vessel networks formed by seeded goat cells. Importantly, implantation of EPC/MSC or SDF-1a/MSC constructs resulted in indistinguishable ectopic bone formation. In both groups, bone onset was apparent at week 3 of implantation. Taken together, we demonstrated that SDF-1a stimulated the migration of EPCs in vitro and vascularization in vivo. Further, SDF-1a addition was as effective as EPCs in inducing the formation of vascularized ectopic bone based on MSC-seeded constructs, suggesting a cell-replacement role for SDF-1a. These results hold promise for the design of larger centimeter-scale, cell-free vascular bone grafts.

For the bone implant market, a large number of surface modifications are available on different types of (dental and orthopedic) bone implants. As the implant surface provides the interface at which the biomaterial interacts with the surrounding (bone) tissue, it is of utmost importance to know what surface modification has optimal osteophilic properties. In contrast to numerous earlier studies on bone implant surface modifications with limited number of comparison surfaces, the manuscript by van Oirschot et al. describes the data of in vivo experiments using a large animal model that allows for direct and simultaneous comparison of a large variety of surface modifications, which included both commercially available and experimental surface modifications for bone implants. These data clearly show the superiority of plasma-sprayed hydroxyapatite coatings regarding bone-to-implant contact, bone amount, and bone height.