SLC28A3 genotype and gemcitabine rate of infusion affect dFdCTP metabolite disposition in patients with solid tumours.

BACKGROUND: Gemcitabine is used for the treatment of several solid tumours and exhibits high inter-individual pharmacokinetic variability. In this study, we explore possible predictive covariates on drug and metabolite disposition. METHODS: Forty patients were enrolled. Gemcitabine and dFdU concentrations in the plasma and dFdCTP concentrations in peripheral blood mononuclear cell were measured to 72 h post infusion, and pharmacokinetic parameters were estimated by nonlinear mixed-effects modelling. Patient-specific covariates were tested in model development. RESULTS: The pharmacokinetics of gemcitabine was best described by a two-compartment model with body surface area, age and NT5C2 genotype as significant covariates. The pharmacokinetics of dFdU and dFdCTP were adequately described by three-compartment models. Creatinine clearance and cytidine deaminase genotype were significant covariates for dFdU pharmacokinetics. Rate of infusion of <25 mg m(-2) min(-1) and the presence of homozygous major allele for SLC28A3 (CC genotype) were each associated with an almost two-fold increase in the formation clearance of dFdCTP. CONCLUSION: Prolonged dFdCTP systemic exposures (≥72 h) were commonly observed. Infusion rate <25 mg m(-2) min(-1) and carriers for SLC28A3 variant were each associated with about two-fold higher dFdCTP formation clearance. The impacts of these covariates on treatment-related toxicity in more selected patient populations (that is, first-line treatment, single disease state and so on) are not yet clear.

Interleukin 2 in cancer therapy.

Cancer immunotherapy with interleukin-2 (IL-2) has demonstrated long term disease control in metastatic renal cell carcinoma and malignant melanoma. With introduction of novel kinase inhibitors, immunomodulatory molecules, cytokines, and vaccines for treatment of cancer there is an increased interest in combining these therapeutic strategies with IL-2. Here we discuss toxicity and established activity of IL-2 in the management of advanced malignancies, and speculate on future use of this cytokine for treatment of cancer.

Blood outgrowth endothelial cell-based systemic delivery of antiangiogenic gene therapy for solid tumors.

Endothelial cells and endothelial cell precursors encoding a therapeutic gene have induced antitumor responses in preclinical models. Culture of peripheral blood provides a rich supply of autologous, highly proliferative endothelial cells, also referred to as blood outgrowth endothelial cells (BOECs). The aim of this study was to evaluate a novel antiangiogenic strategy using BOECs expressing fms-like tyrosine kinase-1 (sFlt1) and/or angiostatin-endostatin (AE) fusion protein. Conditioned medium from BOECs expressing sFlt1 or AE suppressed in vitro growth of pulmonary vein endothelial cells by 70% compared with conditioned medium from non-transduced BOEC controls. Reverse transcriptase-PCR analysis indicated that systemically administered BOECs proliferated in tumor tissue relative to other organs in C3(1)SV40 TAG transgenic (C3TAG) mice with spontaneous mammary tumors. Tumor volume was reduced by half in C3TAG mice and in mice bearing established lung or pancreatic tumors in response to the treatment with sFlt1-BOECs, AE-BOECs or their combination. Studies of tumor vascular density confirmed that angiogenic inhibition contributed to slowed tumor growth. In an orthotopic model of glioma, the median survival of mice treated with sFlt1-BOECs was double that of mice receiving no BOEC treatment (P=0.0130). These results indicate that further research is warranted to develop BOECs for clinical application.

Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity.

BACKGROUND: Potency, immunogenicity, and toxicity are three problems that limit the use of targeted toxins in solid tumour therapy. METHODS: To address potency, we used genetic engineering to develop a novel bispecific ligand-directed toxin (BLT) called EGF4KDEL, a novel recombinant anti-mesothelioma agent created by linking human epidermal growth factor (EGF) and interleukin-4 (IL-4) to truncated pseudomonas exotoxin (PE38) on the same single-chain molecule. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGF4KDEL 7Mut. RESULTS: In vitro, bispecific EGF4KDEL showed superior anti-mesothelioma activity compared with its monospecific counterparts. Toxicity in mice was diminished by having both ligands on the same molecule, allowing administration of a 10-fold greater dose of BLT than a mixture of monomeric IL4KDEL and EGFKDEL. EGF4KDEL 7Mut, retained all of its functional activity and induced about 87% fewer anti-toxin antibodies than mice given the parental, non-mutated form. In vivo, intraperitoneal (IP) injection of the BLT showed significant (P<0.01) and impressive effects against two aggressive, malignant IP mesothelioma models when treatment was begun 14-16 days post tumour innoculation. CONCLUSION: These data show that EGF4KDEL 7Mut is a promising new anti-mesothelioma agent that was developed to specifically address the obstacles facing clinical utility of targeted toxins.

Phase I study of bortezomib and cetuximab in patients with solid tumours expressing epidermal growth factor receptor.

Bortezomib inhibits nuclear factor-kappaB (NF-kappaB). Cetuximab is a chimeric mouse-human antibody targeted against epidermal growth factor receptor (EGFR). We hypothesised that concomitant blockade of NF-kappaB and EGFR signalling would overcome EGFR-mediated resistance to single-agent bortezomib and induce apoptosis through two molecular pathways. The aim of this phase I trial was to establish the maximum tolerated dose (MTD) for bortezomib plus cetuximab in patients with EGFR-expressing epithelial tumours. The 21-day treatment cycle consisted of bortezomib administered on days 1 and 8 through dose escalation (1.3-2 mg m(-2)). Cetuximab was delivered at a dose of 250 mg m(-2) on days 1, 8 and 15 (400 mg m(-2) day 1 cycle 1). A total of 37 patients were enroled and given a total 91 cycles. No grade > or =3 haematological toxicity was noted. Non-hematological grade > or =3 toxicities included fatigue (22% of patients), dyspnoea (16%) and infection (11%). The MTD was not reached at the highest tested bortezomib dose (2.0 mg m(-2)). Efficacy outcomes included disease progression in 21 patients (56.7%) and stable disease (SD) at 6 weeks in 16 patients (43.3%). Five of the six patients with SD at 12 weeks were diagnosed with cancers of the lungs or head and neck. This combination therapy was moderately effective in extensively pretreated patients with non-small cell lung or head and neck cancers and warrants further investigation.

Systemic inhibition of tumour angiogenesis by endothelial cell-based gene therapy.

Angiogenesis and post-natal vasculogenesis are two processes involved in the formation of new vessels, and both are essential for tumour growth and metastases. We isolated endothelial cells from human blood mononuclear cells by selective culture. These blood outgrowth cells expressed endothelial cell markers and responded correctly to functional assays. To evaluate the potential of blood outgrowth endothelial cells (BOECs) to construct functional vessels in vivo, NOD-SCID mice were implanted with Lewis lung carcinoma cells subcutaneously (s.c.). Blood outgrowth endothelial cells were then injected through the tail vein. Initial distribution of these cells occurred throughout the lung, liver, spleen, and tumour vessels, but they were only found in the spleen, liver, and tumour tissue 48 h after injection. By day 24, they were mainly found in the tumour vasculature. Tumour vessel counts were also increased in mice receiving BOEC injections as compared to saline injections. We engineered BOECs to deliver an angiogenic inhibitor directly to tumour endothelium by transducing them with the gene for human endostatin. These cells maintained an endothelial phenotype and decreased tumour vascularisation and tumour volume in mice. We conclude that BOECs have the potential for tumour-specific delivery of cancer gene therapy.

Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins.

We have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell "niches" in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1alpha or PF4 (LTC-IC maintenance without GAGs, 32 +/- 2%; with GAGs, 95 +/- 7%; P <.001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1alpha, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 +/- 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1alpha. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1alpha, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association and dissociation rates of heparin for PF4, were higher than those of O-S-heparin. We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)

Structurally specific heparan sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche.

Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P <.005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34(+)/HLA-DR- cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34(+) cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.

Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C. A potential mechanism for enhancing generation of activated protein C.

Platelet factor 4 (PF4) is an abundant platelet alpha-granule heparin-binding protein. We have previously shown that PF4 accelerates up to 25-fold the proteolytic conversion of protein C to activated protein C by the thrombin.thrombomodulin complex by increasing its affinity for protein C 30-fold. This stimulatory effect requires presence of the gamma-carboxyglutamic acid (Gla) domain in protein C and is enhanced by the presence of a chondroitin sulfate glycosaminoglycan (GAG) domain on thrombomodulin. We hypothesized that cationic PF4 binds to both protein C and thrombomodulin through these anionic domains. Qualitative SDS-polyacrylamide gel electrophoresis analysis of avidin extracts of solutions containing biotinylated PF4 and candidate ligands shows that PF4 binds to GAG+ but not GAG- forms of thrombomodulin and native but not Gla-domainless protein C. Quantitative analysis using the surface plasmon resonance-based BIAcoreTM biosensor system confirms the extremely high affinity of PF4 for heparin (KD = 4 nM) and shows that PF4 binds to GAG+ thrombomodulin with a KD of 31 nM and to protein C with a KD of 0.37 microM. In contrast, PF4 had no measurable interaction with GAG- thrombomodulin or Gla-domainless protein C. Western blot analysis of normal human plasma extracted with biotinylated PF4 demonstrates PF4 binding to protein C in a physiologic context. Thus, PF4 binds with relative specificity and high affinity to the GAG- domain of thrombomodulin and the Gla domain of protein C. These interactions may enhance the affinity of the thrombin.thrombomodulin complex for protein C and thereby promote the generation of activated protein C.