A phase I study of the combination of ro4929097 and cediranib in patients with advanced solid tumours (PJC-004/NCI 8503).

BACKGROUND: The Notch signalling pathway has been implicated in tumour initiation, progression, angiogenesis and development of resistance to vascular endothelial growth factor (VEGF) targeting, providing a rationale for the combination of RO4929097, a γ-secretase inhibitor, and cediranib, a VEGF receptor tyrosine kinase inhibitor. METHODS: Patients received escalating doses of RO4929097 (on a 3 days-on and 4 days-off schedule) in combination with cediranib (once daily). Cycle 1 was 42 days long with RO4929097 given alone for the first 3 weeks followed by the co-administration of both RO4929097 and cediranib starting from day 22. Cycle 2 and onwards were 21 days long. Soluble markers of angiogenesis were measured in plasma samples. Archival tumour specimens were assessed for expression of three different components of Notch signalling pathway and genotyping. RESULTS: In total, 20 patients were treated in three dose levels (DLs). The recommended phase II dose was defined as 20 mg for RO4929097 on 3 days-on and 4 days-off schedule and 30 mg daily for cediranib. The most frequent treatment-related adverse events (AEs) were diarrhoea, hypertension, fatigue and nausea. Eleven patients had a best response of stable disease and one patient achieved partial response. We did not detect any correlation between tested biomarkers of angiogenesis or the Notch pathway and treatment effect. There was no correlation between mutational status and time to treatment failure. CONCLUSION: RO4929097 in combination with cediranib is generally well tolerated at the DLs tested. Preliminary evidence of antitumour efficacy with prolonged disease stabilisation in some patients with progressive malignancies warrants further clinical investigation of this treatment strategy.

Microvascular preconditioning is not detectable by corrosion casting in the isolated perfused rat heart after 30 minutes of ischaemia.

OBJECTIVE: Ischaemic preconditioning protects the myocardium from ischaemic injury and may also protect the vascular endothelium from the deleterious effects of ischaemia and reperfusion. We examined the possibility that ischaemic preconditioning might preserve the integrity of the coronary microcirculation following ischaemia and reperfusion. METHODS: Isolated rat hearts were perfused in Langendorff mode for 30 minutes and then subjected to 30 minutes of global ischaemia with or without ischaemic preconditioning (threexthree minute cycles). Some hearts underwent an additional 60 minutes of reperfusion. At the end of each protocol, microvascular corrosion casts were made by methylmethacrylate injection. RESULTS: Median left ventricular capillary density [interquartile range] after ischaemia was slightly but not significantly better with preconditioning at 6.8 [4.0-14.7]x10(-2) mm3.mg(-1) vs. 5.2 [2.6-7.1]x10(-2) mm3.mg(-1) (p=0.13). After 60 min of reperfusion, capillary density in preconditioned left ventricles was 20.7 [10.7-22.8]x10(-2) mm3.mg(-1) vs. 16.0 [10.2-23.0]x10(-2) mm3.mg(-1) for untreated ventricles (p=0.47). Coronary blood flow and heart rate were unchanged from before ischaemia. CONCLUSIONS: Ischaemia for 30 minutes induced global left ventricular capillary loss which was unmodified by preconditioning. We did not demonstrate vascular preconditioning using this model.

Platelet aggregation and high-intensity transient signals (HITS) in a sheep model of mitral valve replacement.

BACKGROUND AND AIMS OF THE STUDY: The composition of microemboli detected as high-intensity transient signals (HITS) by Doppler ultrasound in patients with prosthetic heart valves is still debated. Here, platelet aggregation and HITS were investigated in a sheep model. METHODS: Insonation of the carotid artery was performed in 20 sheep with either a mechanical or a biological mitral valve prosthesis in place. The effect of ICI 170809, a 5HT2a antagonist, on the frequency of HITS and on platelet aggregates, counted in arterial blood smears per nine high-power fields, was assessed at three and six months after valve implantation. The mitral transvalvular gradient was measured by transthoracic echocardiography at three and six months. RESULTS: Data are expressed as median and interquartile range. At three months, there were 36 (20-114) HITS/h in the mechanical group, and 0 (0-15) HITS/h in the biological group. At six months, there were 21 (0-82) and 0 (0-2) HITS/h, respectively. The occurrence of HITS was unaffected by either ICI 170809, or by duration of implant in either group. Platelet aggregate counts were higher with the mechanical than with the biological valve at three months, but not at six months. ICI 170809 reduced platelet aggregate counts in both valve types; the reduction was not significant in the bioprosthetic valve group. The pressure gradient across the bioprosthesis increased during the study from 2 (2-3) mmHg to 7.5 (6-10) mmHg, but was unchanged in the mechanical valve. CONCLUSIONS: (i) It was confirmed that the frequency of HITS is higher with the mechanical prosthesis than the bioprosthesis; (ii) circulating platelet aggregates in the bioprosthetic valve group tended to increase as structural valve deterioration occurred; (iii) the frequency of HITS was not influenced by either an increase or a decrease in circulating platelet aggregates; and (iv) HITS detected in patients with prosthetic valves are unlikely to be due to circulating platelet aggregates.

Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli.

The herpes simplex virus type 1 (HSV-1) gene encoding the ribonucleotide reductase (RR) small subunit (R2) was cloned as an unfused and intact open reading frame into a T7 RNA polymerase expression system in Escherichia coli. The expressed product was recovered from bacteria in soluble form and constituted 7% of the soluble protein. Protein purification yielded 3.5 mg of 95% pure R2 per litre of bacterial culture. The correct composition of the purified protein was verified by amino acid analysis and N-terminal sequencing. The isoelectric point of the protein was 5.3. Atomic emission spectroscopy indicated that the iron content of the E. coli-expressed R2 was 0.2 to 0.5 atoms of iron per R2 protomer as compared with a theoretical maximum value of 2. The E. coli-expressed HSV-1 R2 existed as a combination of a stable dimer and monomer. Combination of the E. coli-expressed R2 with the E. coli-expressed large subunit (R1) gave an active holoenzyme. Thus, the T7 expression system provides a rich source of enzymically active HSV-1 RR.

Bone-derived growth factors modulate collagenase and TIMP (tissue inhibitor of metalloproteinases) activity and type I collagen degradation by mouse calvarial osteoblasts.

The finding that osteoblasts synthesize collagenase has led to the hypothesis that bone cells play a major role in bone resorption by degrading the surface osteoid layer, thereby preparing the underlying mineralized bone for osteoclastic action. To further understand the mechanisms regulating osteoid removal, mouse calvarial osteoblasts were cultured on 14C-labelled type I collagen films and the abilities of (i) bovine bone matrix extracts and (ii) purified or recombinant human growth factors, to modify their collagenolytic behaviour were investigated. EDTA/Tris-HCl extracts of bone matrix containing growth factor activity, exerted a dose-dependent inhibition of type I collagenolysis by osteoblasts stimulated with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, 10 ng/ml). Inhibition was accompanied by a reduction in collagenase activity and an increase in free TIMP (tissue inhibitor of metalloproteinases) in the culture medium. Transforming growth factor-beta, epidermal growth factor, platelet-derived growth factor and the acidic and basic fibroblast growth factors all mimicked these effects. In contrast, insulin-like growth factors-I and -II did not inhibit type I collagenolysis, only partially inhibited collagenase activity, and did not stimulate TIMP production by either 1,25(OH)2D3-treated or untreated cells. These findings provide additional evidence for the tight control exerted on the proteolytic activity of osteoblasts and the importance of TIMP in its regulation. They suggest strongly that the conversion (coupling) of the initial resorptive phase of the bone remodelling cycle to one of deposition, may be mediated by polypeptide growth factors either produced locally by osteoblasts, or released by proteolysis from the bone matrix.

Type I collagen degradation by mouse calvarial osteoblasts stimulated with 1,25-dihydroxyvitamin D-3: evidence for a plasminogen-plasmin-metalloproteinase activation cascade.

To understand the mechanisms regulating osteoid removal by osteoblasts, mouse calvarial osteoblasts were grown on 14C-labelled type I collagen films and stimulated with 1,25-dihydroxyvitamin D-3 (2.5.10(-8) M) for 48-72 h. In the presence of 5% non-inhibitory rabbit serum this resulted in a 2-3-fold increase in collagen degradation and a dramatic change in osteoblast morphology, when compared with untreated osteoblasts. Collagenolysis was accompanied by increased synthesis and release of latent collagenase, gelatinase and stromelysin and a concomitant decrease in their specific inhibitor, TIMP (tissue inhibitor of metalloproteinases). In serum-free medium, osteoblasts failed to degrade collagen, but their ability to lyse collagen could be restored by adding plasminogen (5 micrograms/ml) to the cultures. Plasminogen-dependent collagenolysis was inhibited by human recombinant TIMP (5 units/ml), demonstrating that plasmin, derived from plasminogen, activated latent collagenase and did not itself degrade collagen. Plasminogen activator production was confirmed by culturing osteoblasts on 125I-labelled fibrin plates. Comparison with urokinase-type and tissue-type plasminogen activator standards suggested that osteoblast plasminogen activator was predominantly cell-associated and likely to be of the urokinase type. Immunocytochemistry indicated that osteoblasts also constitutively produce plasminogen activator inhibitor-1. These findings provide evidence for the involvement of a plasminogen-plasmin-latent metalloproteinase activation cascade in type I collagen degradation by osteoblasts, and for its regulation by TIMP and plasminogen activator inhibitor-1.

Gelatinase (type IV collagenase) immunolocalization in cells and tissues: use of an antiserum to rabbit bone gelatinase that identifies high and low Mr forms.

An antiserum was raised to rabbit bone gelatinase (type IV collagenase). It was shown by immunoblotting to detect both the low Mr (72,000) enzyme produced by connective tissue cells from rabbit, pig, human and mouse, as well as the high Mr (94,000-97,000) enzymes secreted by macrophages and polymorphonuclear leucocytes from these species, and by rabbit chondrocytes and endothelial cells. Crossed immunoblotting, antibody inhibition and deglycosylation studies indicated that the high and low Mr forms of gelatinase are immunologically distinct gene products, although their substrate specificity profiles are identical. The anti-gelatinase antiserum was used to immunolocalize the enzyme. Gelatinase was most efficiently detected in rabbit monocytes and connective tissue cells, but cells derived from the human and pig gave poor immunostaining, although mouse gelatinase stained well. The anti-gelatinase antiserum stained cells of the synovial tissue of rabbits at 14 days after induction of an antigen-induced arthritis, demonstrating its usefulness as a tool to assess the role of this enzyme in degradative events.