Search

Author(s): Sung Hee Um [1]; Francesca Frigerio [1]; Mitsuhiro Watanabe [2]; Frédéric Picard [2, 4]; Manel Joaquin [1]; Melanie Sticker [1]; Stefano Fumagalli [1]; Peter R. Allegrini [3]; Sara C. [...]

Despite the contribution of changes in pancreatic β-cell mass to the development of all forms of diabetes mellitus, few robust approaches currently exist to monitor these changes prospectively in vivo. Although magnetic-resonance imaging (MRI) provides a potentially useful technique, targeting MRI-active probes to the β cell has proved challenging. Zinc ions are highly concentrated in the secretory granule, but they are relatively less abundant in the exocrine pancreas and in other tissues. We have therefore developed functional dual-modal probes based on transition-metal chelates capable of binding zinc. The first of these, Gd ⋅1, binds ZnII directly by means of an amidoquinoline moiety (AQA), thus causing a large ratiometric Stokes shift in the fluorescence from λem=410 to 500 nm with an increase in relaxivity from r1=4.2 up to 4.9 m M−1 s−1. The probe is efficiently accumulated into secretory granules in β-cell-derived lines and isolated islets, but more poorly by non-endocrine cells, and leads to a reduction in T1 in human islets. In vivo murine studies of Gd ⋅1 have shown accumulation of the probe in the pancreas with increased signal intensity over 140 minutes. [ABSTRACT FROM AUTHOR]

Author(s): Sung Hee Um; Francesca Frigerio; Mitsuhiro Watanabe; Frédéric Picard; Manel Joaquin; Melanie Sticker; Stefano Fumagalli; Peter R. Allegrini; Sara C. Kozma; Johan Auwerx; George Thomas Nature 431, 200-205 (2004).10.1038/nature02866 [...]

Background Effective chemotherapy rapidly reduces the spin--lattice relaxation of water protons (T1) in solid tumours and this change (ΔT1) often precedes and strongly correlates with the eventual change in tumour volume (TVol). To understand the biological nature of ΔT1, we have performed studies in vivo and ex vivo with the allosteric mTOR inhibitor, everolimus. Mice bearing RIF-1 tumours were studied by magnetic resonance imaging (MRI) to determine TVol and T1, and MR spectroscopy (MRS) to determine levels of the proliferation marker choline and levels of lipid apoptosis markers, prior to and 5 days (endpoint) after daily treatment with vehicle or everolimus (10 mg/kg). At the endpoint, tumours were ablated and an entire section analysed for cellular and necrotic quantification and staining for the proliferation antigen Ki67 and cleaved-caspase-3 as a measure of apoptosis. The number of blood-vessels (BV) was evaluated by CD31 staining. Mice bearing B16/BL6 melanoma tumours were studied by MRI to determine T1 under similar everolimus treatment. At the endpoint, cell bioluminescence of the tumours was measured ex vivo. Results Everolimus blocked RIF-1 tumour growth and significantly reduced tumour T1 and total choline (Cho) levels, and increased polyunsaturated fatty-acids which are markers of apoptosis. Immunohistochemistry showed that everolimus reduced the%Ki67+ cells but did not affect caspase-3 apoptosis, necrosis, BV-number or cell density. The change in T1 (ΔT1) correlated strongly with the changes in TVol and Cho and%Ki67+. In B16/BL6 tumours, everolimus also decreased T 1 and this correlated with cell bioluminescence; another marker of cell viability. Receiver-operating-characteristic curves (ROC) for everolimus on RIF-1 tumours showed that ΔT1 had very high levels of sensitivity and specificity (ROCAUC = 0.84) and this was confirmed for the cytotoxic patupilone in the same tumour model (ROCAUC = 0.97). Conclusion These studies suggest that ΔT1 is not a measure of cell density but reflects the decreased number of remaining viable and proliferating tumour cells due to perhaps cell and tissue destruction releasing proteins and/or metals that cause T1 relaxation. ΔT1 is a highly sensitive and specific predictor of response. This MRI method provides the opportunity to stratify a patient population during tumour therapy in the clinic. [ABSTRACT FROM AUTHOR]

Background: Patients with glioblastoma (GBM) inevitably develop recurrent or progressive disease after initial multimodal treatment and have a median survival of 6-9 months from time of progression. To date, there is no accepted standard treatment for GBM relapse or progression. Patupilone (EPO906) is a novel natural microtubule-stabilizing cytotoxic agent that crosses the blood-brain barrier and has been found to have preclinical activity in glioma models. Methods: This is a single-institution, early-phase I/II trial of GBM patients with tumor progression who qualified for second surgery with the goal of evaluating efficacy and safety of the single-agent patupilone (10 mg/m2, every 3 weeks). Patients received patupilone 1 week prior to second surgery and every 3 weeks thereafter until tumor progression or toxicity. Primary end points were progression-free survival (PFS) and overall survival (OS) at 6 months as well as patupilone concentration in tumor tissue. Secondary end points were toxicity, patupilone concentration in plasma and translational analyses for predictive biomarkers. Results: Nine patients with a mean age of 54.6 ± 8.6 years were recruited between June 2008 and April 2010. Median survival and 1-year OS after second surgery were 11 months (95% CI, 5-17 months) and 45% (95% CI, 14-76), respectively. Median PFS was 1.5 months (95% CI, 1.3-1.7 months) and PFS6 was 22% (95% CI, 0-46), with 2 patients remaining recurrence-free at 9.75 and 22 months. At the time of surgery, the concentration of patupilone in tumor tissue was 30 times higher than in the plasma. Tumor response was not predictable by the tested biomarkers. Treatment was generally well tolerated with no hematological, but cumulative, though reversible sensory neuropathy grade ≤3 was seen in 2 patients (22%) at 8 months and grade 4 diarrhea in the 2nd patient (11%). Non-patupilone-related peri-operative complications occurred in 2 patients resulting in discontinuation of patupilone therapy. There were no neurocognitive changes 3 months after surgery compared to baseline. Conclusions: In recurrent GBM, patupilone can be given safely pre- and postoperatively. The drug accumulates in the tumor tissue. The treatment results in long-term PFS in some patients. Patupilone represents a valuable novel compound which deserves further evaluation in combination with radiation therapy in patients with GBM. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Solid tumors have a raised interstitial fluid pressure (IFP) due to high vessel permeability, low lymphatic drainage, poor perfusion, and high cell density around the blood vessels. To investigate tumor IFP as an early-response biomarker, we have tested the effect of seven anticancer chemotherapeutics including cytotoxics and targeted cytostatics in 13 experimental tumor models. IFP was recorded with the wick-in-needle method. Models were either ectopic or orthotopic and included mouse and rat syngeneic as well as human xenografts in nude mice. The mean basal IFP was between 4.4 and 15.2mm Hg; IFP was lowest in human tumor xenografts and highest in rat syngeneic models. Where measured, basal IFP correlated positively with relative tumor blood volume (rTBV) determined by dynamic contrast-enhanced magnetic resonance imaging. Most chemotherapeutics sooner (2 or 3 days) or later (6 or 7 days) lowered tumor IFP significantly, and the cytotoxic patupilone caused the greatest decrease in IFP. In rat mammary orthotopic BN472 tumors, significant drug-induced decreases in IFP and rTBV correlated positively with each other for both patupilone and the cytostatic vatalanib. In the two orthotopic models studied, early decreases in IFP were significantly (P ≤ .005) correlated with late changes in tumor volume. Thus, drug-induced decreases in tumor IFP are an early marker of response to therapy, which could aid clinical development. [ABSTRACT FROM AUTHOR]

Patupilone is a microtubule stabilizer (MTS) currently in clinical development. Here, we evaluate the anti-cancer activity in vitro and in vivo in comparison to paclitaxel and describe the pharmacokinetics (PK) of patupilone in tumor-bearing nude mice and rats. The potency in vitro of patupilone and two other MTS, paclitaxel and ixabepilone, was determined using human colon carcinoma cell lines with low (HCT-116, HT-29, RKO) and high (HCT-15) P-glycoprotein expression (P-gp), as well as two multi-drug resistance (MDR) model cell pairs, MCF7/ADR and KB-8511 cells and their respective drug-sensitive parental counterparts. The PK of patupilone was investigated in nude mice bearing HCT-15 or HT-29 xenografts and in rats bearing s.c. pancreatic CA20498 tumors or A15 glioma tumors. Anti-cancer activity in vivo was compared to that of paclitaxel using three different human tumor colon models. The retention and efficacy of patupilone was compared in small and large HT-29 xenografts whose vascularity was determined by non-invasive magnetic resonance imaging. Patupilone was highly potent in vitro against four different colon carcinoma cell lines including those showing multi-drug-resistance. In contrast, paclitaxel and ixabepilone displayed significantly reduced activity with markedly increased resistance factors. In both rats and mice, a single i.v. bolus injection of patupilone (1.5–4 mg/kg) rapidly distributed from plasma to all tissues and was slowly eliminated from muscle, liver and small intestine, but showed longer retention in tumor and brain with no apparent elimination over 24 h. Patupilone showed significant activity against three human colon tumor models in vivo, unlike paclitaxel, which only had activity against low P-gp expressing tumors. In HT-29 tumors, patupilone activity and retention were independent of tumor size, blood volume and flow. The high potency of patupilone, which is not affected by P-gp expression either in vitro or in vivo, and favorable PK, independent of tumor vascularity, suggest that it should show significant activity in colorectal cancer and in other indications where high P-gp expression may compromise taxane activity. [ABSTRACT FROM AUTHOR]

Assessment of tumour vascularity may characterize malignancy as well as predict responsiveness to anti-angiogenic therapy. Non-invasive measurement of tumour perfusion and blood vessel permeability assessed as the transfer constant, Ktrans, can be provided by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Using the orthotopic murine tumour model B16/BL6 melanoma, the small contrast agent GdDOTA (DOTAREM®; Guerbet, Paris) was applied to assess the vascular transfer constant, Ktrans, and interstitial leakage space, whereas intravascular iron oxide nanoparticles (Endorem®; Guerbet, Paris) were used to detect relative tumour blood volume (rTBV), and in one experiment blood flow index (BFI). No correlations were observed between these four parameters ( r2 always <0.05). The B16/BL6 primary tumour and lymph-node cervical (neck) metastases produced high levels of the permeability/growth factor, VEGF. To probe the model, the novel VEGF receptor (VEGF-R) tyrosine kinase inhibitor, PTK787/ZK222584 (PTK/ZK) was tested for anti-tumour efficacy and its effects on DCE-MRI measured parameters of tumour vascularity. Data from the non-invasive measure of tumour vascularity were compared with a histological measurement of vasculature using the DNA-staining dye H33342. PTK/ZK inhibited growth of the primary and, particularly, cervical tumour metastases following chronic treatment for 2 weeks (50 or 100 mg/kg daily) of 1-week-old tumours, or with 1 week of treatment against more established (2-week-old) tumours. After chronic treatment with PTK/ZK, DCE-MRI detected significant decreases in Ktrans and interstitial leakage space, but not rTBV of both primary tumours and cervical metastases. Histological data at this time-point showed a significant decrease in blood vessel density of the cervical metastases but not the primary tumours. However, in the cervical metastases, the mean blood vessel width was increased by 38%, suggesting overall no marked change in blood volume. After acute (2-4 day) treatment, DCE-MRI of the cervical metastases demonstrated a significant decrease in Ktrans and interstitial leakage space and also in the initial area under the enhancement curve for GdDOTA (IAUC), but no change in the rTBV or BFI. Thus, significant changes could be detected in the DCE-MRI measurement of tumour uptake of a small contrast agent prior to changes in tumour size, which suggests that DCE-MRI could be applied in the clinic as a rapid and sensitive biomarker for the effects of VEGF-R inhibition on tumour blood vessel permeability and thus may provide an early marker for eventual tumour response. Copyright © 2005 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Elucidating the signalling mechanisms by which obesity leads to impaired insulin action is critical in the development of therapeutic strategies for the treatment of diabetes1. Recently, mice deficient for S6 Kinase 1 (S6K1), an effector of the mammalian target of rapamycin (mTOR) that acts to integrate nutrient and insulin signals2, were shown to be hypoinsulinaemic, glucose intolerant and have reduced β-cell mass3. However, S6K1- deficient mice maintain normal glucose levels during fasting, suggesting hypersensitivity to insulin3, raising the question of their metabolic fate as a function of age and diet. Here, we report that S6K1-deficient mice are protected against obesity owing to enhanced β-oxidation. However on a high fat diet, levels of glucose and free fatty acids still rise in S6Kl-deficient mice, resulting in insulin receptor desensitization. Nevertheless, S6K1-deficient mice remain sensitive to insulin owing to the apparent loss of a negative feedback loop from S6K1 to insulin receptor substrate 1 (IRS1), which blunts S307 and S636/S639 phosphorylation; sites involved in insulin resistance4,5. Moreover, wild-type mice on a high fat diet as well as K/KAy and ob/ob (also known as Lep/Lep) mice-two genetic models of obesity—have markedly elevated S6K1 activity and, unlike S6K1-deficient mice, increased phosphorylation of IRS1 S307 and S636/S639. Thus under conditions of nutrient satiation S6K1 negatively regulates insulin signalling. [ABSTRACT FROM AUTHOR]

Objective: The aim was to define: (1) whether bradykinin administration during reperfusion improves postischaemic myocardial recovery; (2) whether high energy phosphate compounds are involved in the protective effects of bradykinin; and (3) whether bradykinin-induced release of prostacyclin and nitric oxide mediate the protective effects of bradykinin. Methods: In the Langendorff rat heart preparation, coronary flow, left ventricular developed pressure, and, using 31P magnetic resonance spectroscopy, the high energy phosphate compounds phosphocreatine and β-ATP were assessed during 15 min of global ischaemia and 30 min of reperfusion. Administration of 10−7 M bradykinin was started before ischaemia and maintained throughout the experiment (BK-pre). This was compared to 10−7 M bradykinin given exclusively with reperfusion (BK-post). Then 10−7 M bradykinin was given simultaneously with 10−4 M Nω-nitro-L-arginine-methyl ester (BK-LNAME) or 10−5 M indomethacin (BK-indo). Results: In comparison to control hearts, BK-pre exerted a significant protective effect on the postischaemic recovery of coronary flow [71(5)% v 43(4)%, P < 0.05], left ventricular pressure [81(8)% v 42(5)%, P < 0.05], phosphocreatine [105(4)% v 67(8)%, P < 0.05], and β-ATP [78(9)% v 48(7)%, P < 0.05]. With BK-post, recovery of coronary flow [71(4)% v 43(4)%, P < 0.05] and left ventricular pressure [78(4)% v 42(5)%, P < 0.05] significantly improved; however the recovery of phosphocreatine [70(4)% v 67(8)%, NS] and β-ATP [58(2)% v 48(7)%, NS] was not different from control. When bradykinin and L-NAME or indomethacin was given the beneficial effects of bradykinin on ischaemic hearts were abolished. Conclusions: (1) Bradykinin improved postischaemic myocardial recovery when given before ischaemia or starting exclusively with reperfusion; (2) this was only partially related to a protective action on the high energy phosphate compounds during ischaemia; (3) the beneficial effects of bradykinin on ischaemic hearts are dependent from an unrestrained action of prostacyclin and nitric oxide. [ABSTRACT FROM PUBLISHER]

Background: Effective chemotherapy rapidly reduces the spin-lattice relaxation of water protons (T1) in solid tumours and this change (ΔT1) often precedes and strongly correlates with the eventual change in tumour volume (TVol). To understand the biological nature of ΔT1, we have performed studies in vivo and ex vivo with the allosteric mTOR inhibitor, everolimus.Methods: Mice bearing RIF-1 tumours were studied by magnetic resonance imaging (MRI) to determine TVol and T1, and MR spectroscopy (MRS) to determine levels of the proliferation marker choline and levels of lipid apoptosis markers, prior to and 5 days (endpoint) after daily treatment with vehicle or everolimus (10 mg/kg). At the endpoint, tumours were ablated and an entire section analysed for cellular and necrotic quantification and staining for the proliferation antigen Ki67 and cleaved-caspase-3 as a measure of apoptosis. The number of blood-vessels (BV) was evaluated by CD31 staining. Mice bearing B16/BL6 melanoma tumours were studied by MRI to determine T1 under similar everolimus treatment. At the endpoint, cell bioluminescence of the tumours was measured ex vivo.Results: Everolimus blocked RIF-1 tumour growth and significantly reduced tumour T1 and total choline (Cho) levels, and increased polyunsaturated fatty-acids which are markers of apoptosis. Immunohistochemistry showed that everolimus reduced the %Ki67+ cells but did not affect caspase-3 apoptosis, necrosis, BV-number or cell density. The change in T1 (ΔT1) correlated strongly with the changes in TVol and Cho and %Ki67+. In B16/BL6 tumours, everolimus also decreased T1 and this correlated with cell bioluminescence; another marker of cell viability. Receiver-operating-characteristic curves (ROC) for everolimus on RIF-1 tumours showed that ΔT1 had very high levels of sensitivity and specificity (ROCAUC = 0.84) and this was confirmed for the cytotoxic patupilone in the same tumour model (ROCAUC = 0.97).Conclusion: These studies suggest that ΔT1 is not a measure of cell density but reflects the decreased number of remaining viable and proliferating tumour cells due to perhaps cell and tissue destruction releasing proteins and/or metals that cause T1 relaxation. ΔT1 is a highly sensitive and specific predictor of response. This MRI method provides the opportunity to stratify a patient population during tumour therapy in the clinic. [ABSTRACT FROM AUTHOR]

Presents a correction to the article "Absence of S6K1 Protects Against Age- and Diet-Induced Obesity While Enhancing Insulin Sensitivity," by Sung Hee Um and colleagues, published in the September 2004 issue of the periodical "Nature".

Despite the contribution of changes in pancreatic β-cell mass to the development of all forms of diabetes mellitus, few robust approaches currently exist to monitor these changes prospectively in vivo. Although magnetic-resonance imaging (MRI) provides a potentially useful technique, targeting MRI-active probes to the β cell has proved challenging. Zinc ions are highly concentrated in the secretory granule, but they are relatively less abundant in the exocrine pancreas and in other tissues. We have therefore developed functional dual-modal probes based on transition-metal chelates capable of binding zinc. The first of these, Gd⋅1, binds Zn(II) directly by means of an amidoquinoline moiety (AQA), thus causing a large ratiometric Stokes shift in the fluorescence from λem =410 to 500 nm with an increase in relaxivity from r1 =4.2 up to 4.9 mM(-1)  s(-1) . The probe is efficiently accumulated into secretory granules in β-cell-derived lines and isolated islets, but more poorly by non-endocrine cells, and leads to a reduction in T1 in human islets. In vivo murine studies of Gd⋅1 have shown accumulation of the probe in the pancreas with increased signal intensity over 140 minutes., (© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[(18)F]fluoro-glucose (FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC(50) values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 microM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.

Purpose: Identification of a generic response biomarker by comparison of chemotherapeutics with different action mechanisms on several noninvasive biomarkers in experimental tumor models., Experimental Design: The spin-lattice relaxation time of water protons (T(1)) was quantified using an inversion recovery-TrueFISP magnetic resonance imaging method in eight different experimental tumor models before and after treatment at several different time points with five different chemotherapeutics. Effects on T(1) were compared with other minimally invasive biomarkers including vascular parameters, apparent diffusion coefficient, and interstitial fluid pressure, and were correlated with efficacy at the endpoint and histologic parameters., Results: In all cases, successful chemotherapy significantly lowered tumor T(1) compared with vehicle and the fractional change in T(1) (DeltaT(1)) correlated with the eventual change in tumor size (range: r(2) = 0.21, P < 0.05 to r(2) = 0.73, P < 0.0001), except for models specifically resistant to that drug. In RIF-1 tumors, interstitial fluid pressure was decreased, but apparent diffusion coefficient and permeability increased in response to the microtubule stabilizer patupilone and 5-fluorouracil. Although DeltaT(1) was small (maximum of -20%), the variability was very low (5%) compared with other magnetic resonance imaging methods (24-48%). Analyses ex vivo showed unchanged necrosis, increased apoptosis, and decreased %Ki67 and total choline, but only Ki67 and choline correlated with DeltaT(1). Correlation of Ki67 and DeltaT(1) were observed in other models using patupilone, paclitaxel, a VEGF-R inhibitor, and the mammalian target of rapamycin inhibitor everolimus., Conclusions: These results suggest that a decrease in tumor T(1) reflects hypocellularity and is a generic marker of response. The speed and robustness of the method should facilitate its use in clinical trials.

Mutations in hemojuvelin (HJV) cause severe juvenile hemochromatosis, characterized by iron loading of the heart, liver, and pancreas. Knockout (KO) mice lacking HJV (Hjv-/-) spontaneously load with dietary iron and, therefore, present a model for hereditary hemochromatosis (HH). In HH, iron chelation may be considered in noncandidates for phlebotomy. We examined the effects of deferasirox, an oral chelator, in Hjv-/- mice. Hepatic, cardiac, splenic, and pancreatic iron were determined by measuring elemental iron and scoring histological sections. Heart and liver iron levels were also determined repeatedly by quantitative R2* magnetic resonance imaging (MRI). The time course of iron loading without intervention was followed from Week 8 of age (study start) to Week 20, when once-daily (5x/week) deferasirox was administered, to Week 28. At 8 weeks, liver iron of KO mice was already markedly elevated versus wild-type mice (P<0.001) and reached a plateau around Week 14. In contrast, Week 8 cardiac and pancreatic iron levels were similar in both KO and wild-type mice and, compared with the liver, showed a delayed but massive iron loading up to Week 20. Contrary to the liver, heart, and pancreas, the KO mice spleen had lower iron content versus wild-type mice. In Hjv-/- mice, liver and heart iron burden was effectively reduced with deferasirox 100 mg/kg (P<0.05). Although deferasirox was less efficacious at this dose in the pancreas, over the observed time period, a clear trend toward reduced organ iron load was noted. There was no noticeable effect of deferasirox upon splenic iron in Hjv-/- mice. Quantitative R2* MRI demonstrated the ability to assess iron concentrations in the liver and myocardial muscle accurately and repetitively. Hepatic (R=0.86; P=3.2*10(-12)) and delayed myocardial (R=0.81; P=2.9*10(-10)) iron accumulation could be followed noninvasively with high agreement to invasive methods.

Purpose: Comparison of the antiangiogenic/vascular properties of the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and the vascular endothelial growth factor receptor (VEGFR) inhibitor vatalanib (PTK/ZK)., Experimental Design: Antiproliferative activity against various tumor histotypes and downstream effects on the mTOR pathway were measured in vitro. In vivo, antitumor activity, plasma, and tumor RAD001 levels were measured. Activity in several different angiogenic/vascular assays in vitro and in vivo was assessed and compared with PTK/ZK., Results: RAD001 inhibited proliferation in vitro (IC50 values<1 nmol/L to >1 micromol/L), and in sensitive and insensitive tumor cells, pS6 kinase and 4E-BP1 were inhibited. Activity in vitro did not correlate with activity in vivo and significant responses were seen in tumors with IC50 values>10-fold higher than tumor RAD001 concentrations. In vitro, RAD001 inhibited the proliferation of VEGF-stimulated and fibroblast growth factor-stimulated human endothelial cells but not dermal fibroblasts and impaired VEGF release from both sensitive and insensitive tumor cells but did not inhibit migration of human endothelial cells. In vivo, in tumor models derived from either sensitive or insensitive cells, RAD001 reduced Tie-2 levels, the amount of mature and immature vessels, total plasma, and tumor VEGF. RAD001 did not affect blood vessel leakiness in normal vasculature acutely exposed to VEGF nor did it affect tumor vascular permeability (Ktrans) as measured by dynamic contrast-enhanced magnetic resonance imaging. However, the pan-VEGFR inhibitor PTK/ZK inhibited endothelial cell migration and vascular permeability but had less effect on mature vessels compared with RAD001., Conclusions: VEGFR and mTOR inhibitors show similar but also distinct effects on tumor vascular biology, which has implications for their clinical activity alone or in combination.

Dysregulated angiogenesis and high tumor vasculature permeability, two vascular endothelial growth factor (VEGF)-mediated processes and hallmarks of human tumors, are in part phosphatidylinositol 3-kinase (PI3K) dependent. NVP-BEZ235, a dual PI3K/mammalian target of rapamycin (mTOR) inhibitor, was found to potently inhibit VEGF-induced cell proliferation and survival in vitro and VEGF-induced angiogenesis in vivo as shown with s.c. VEGF-impregnated agar chambers. Moreover, the compound strongly inhibited microvessel permeability both in normal tissue and in BN472 mammary carcinoma grown orthotopically in syngeneic rats. Similarly, tumor interstitial fluid pressure, a phenomenon that is also dependent of tumor permeability, was significantly reduced by NVP-BEZ235 in a dose-dependent manner on p.o. administration. Because RAD001, a specific mTOR allosteric inhibitor, was ineffective in the preceding experiments, we concluded that the effects observed for NVP-BEZ235 are in part driven by PI3K target modulation. Hence, tumor vasculature reduction was correlated with full blockade of endothelial nitric oxide (NO) synthase, a PI3K/Akt-dependent but mTORC1-independent effector involved in tumor permeability through NO production. In the BN472 tumor model, early reduction of permeability, as detected by K(trans) quantification using the dynamic contrast-enhanced magnetic resonance imaging contrasting agent P792 (Vistarem), was found to be a predictive marker for late-stage antitumor activity by NVP-BEZ235.

Purpose: Evaluation of vascular disruptive activity in orthotopic models as potential surrogate biomarkers of tumor response to the microtubule-stabilizing agent patupilone., Experimental Design: Mice bearing metastatic B16/BL6 melanoma and rats bearing mammary BN472 tumors received vehicle or efficacious patupilone doses (4 and 0.8-1.5 mg/kg i.v., respectively). Tumor vascularity assessment by dynamic contrast-enhanced or dynamic susceptibility contrast magnetic resonance imaging and interstitial fluid pressure (IFP) occurred at baseline, 2 days (mice and rats), and 6 days (rats) after treatment and were compared with histologic measurements and correlated with tumor response., Results: In B16/BL6 metastases, patupilone (4 mg/kg) induced a 21 +/- 5% decrease (P < 0.001) in tumor blood volume and a 32 +/- 15% decrease (P = 0.02) in IFP after 2 days and reduced tumor growth and vessel density (>42%) after 2 weeks (P < or = 0.014). Patupilone dose-dependently inhibited BN472 tumor growth (day 6) and reduced IFP on days 2 and 6 (-21% to -70%), and the percentage change in IFP correlated (P < 0.01) with the change in tumor volume. In both models, histology and vascular casts confirmed decreases in tumor blood volume. One patupilone (0.8 mg/kg) administration decreased (P < 0.01) tumor IFP (54 +/- 4%), tumor blood volume (50 +/- 6%), and vessel diameter (40 +/- 11%) by day 6 but not the apparent diffusion coefficient, whereas histology showed that apoptosis was increased 2.4-fold and necrosis was unchanged. Apoptosis correlated negatively (P < 0.001) with IFP, tumor blood volume, and tumor volume, whereas tumor blood volume and IFP were correlated positively (P = 0.0005)., Conclusions: Vascular disruptive effects of patupilone were detected in situ using dynamic contrast-enhanced or dynamic susceptibility contrast magnetic resonance imaging and IFP. Changes in IFP preceded and correlated with tumor response, suggesting that IFP may be a surrogate biomarker for patupilone efficacy.

Aberrant epidermal growth factor receptor (EGFR) and ErbB2 expression are associated with advanced disease and poor patient prognosis in many tumor types (breast, lung, ovarian, prostate, glioma, gastric, and squamous carcinoma of head and neck). In addition, a constitutively active EGFR type III deletion mutant has been identified in non-small cell lung cancer, glioblastomas, and breast tumors. Hence, members of the EGFR family are viewed as promising therapeutic targets in the fight against cancer. In a similar vein, vascular endothelial growth factor (VEGF) receptor kinases are also promising targets in terms of an antiangiogenic treatment strategy. AEE788, obtained by optimization of the 7H-pyrrolo[2,3-d]pyrimidine lead scaffold, is a potent combined inhibitor of both epidermal growth factor (EGF) and VEGF receptor tyrosine kinase family members on the isolated enzyme level and in cellular systems. At the enzyme level, AEE788 inhibited EGFR and VEGF receptor tyrosine kinases in the nm range (IC(50)s: EGFR 2 nm, ErbB2 6 nm, KDR 77 nm, and Flt-1 59 nm). In cells, growth factor-induced EGFR and ErbB2 phosphorylation was also efficiently inhibited (IC(50)s: 11 and 220 nm, respectively). AEE788 demonstrated antiproliferative activity against a range of EGFR and ErbB2-overexpressing cell lines (including EGFRvIII-dependent lines) and inhibited the proliferation of epidermal growth factor- and VEGF-stimulated human umbilical vein endothelial cells. These properties, combined with a favorable pharmacokinetic profile, were associated with a potent antitumor activity in a number of animal models of cancer, including tumors that overexpress EGFR and or ErbB2. Oral administration of AEE788 to tumor-bearing mice resulted in high and persistent compound levels in tumor tissue. Moreover, AEE788 efficiently inhibited growth factor-induced EGFR and ErbB2 phosphorylation in tumors for >72 h, a phenomenon correlating with the antitumor efficacy of intermittent treatment schedules. Strikingly, AEE788 also inhibited VEGF-induced angiogenesis in a murine implant model. Antiangiogenic activity was also apparent by measurement of tumor vascular permeability and interstitial leakage space using dynamic contrast enhanced magnetic resonance imaging methodology. Taken together, these data indicate that AEE788 has potential as an anticancer agent targeting deregulated tumor cell proliferation as well as angiogenic parameters. Consequently, AEE788 is currently in Phase I clinical trials in oncology.

In this study we investigated the potential of in vivo MRI detection of axonal Mn2+ transport for tracing neuronal projections originating in the sensorimotor cortex in healthy and lesioned rat brains. Special attention was given to the potential of visualizing neuronal sprouting of central nervous system across the midline. After injecting unchelated MnCl2 into the forelimb area of sensorimotor cortex of 18 healthy and 10 lesioned rats corticofugal projections could be traced through the internal capsule to the cerebral peduncle and the pyramidal decussation. Although the neuronal tract was visible as early as 6 h after MnCl2 injection, best contrast was achieved after 24-48 h. Beside the cortico-spinal tract, the cortico-thalamic fibres were also visualized by anterograde Mn2+ transport. Cortico-striatal fibres were partially masked by the very high signal near the MnCl2 injection site but could be discerned as well. Slight, diffuse signal enhancement of cortical tissue contralateral to the MnCl2 injection site in healthy rat brains suggests interhemispheric connections or passive diffusion of Mn2+. However, enhanced fibre tract contrast connecting both hemispheres was visible 16 weeks after onset of focal photothrombotic cortical injury. In conclusion our study has shown that we were able to visualize reproducibly the main descending corticofugal projections and interhemispheric connections by non-invasive MRI after localized injection of MnCl2. The appearance of interhemispheric Mn2+-enhanced fibres after photothrombotic focal injury indicates that the method may bear potential to follow non-invasively gross plastic changes of connectivity in the brain after injury., (Copyright 2001 John Wiley & Sons, Ltd.)

Purpose: To investigate the use of magnetic resonance (MR) imaging enhanced with ultrasmall superparamagnetic particles of iron oxide (USPIO) to identify acute, early chronic, and late chronic abscess formation in an experimental model of soft-tissue abscess., Materials and Methods: Experimental soft-tissue infection in 15 rats was imaged with an MR imaging unit on days 1 and 2 (acute), days 5 and 6 (early chronic), and days 8 and 9 (late chronic) after inoculation of the infectious agent. All animals were imaged without contrast enhancement and immediately and 24 hours after USPIO administration. MR and histopathologic findings were compared. The changes in relative signal intensity (SI) and in the extent and pattern of contrast enhancement (macrophage distribution) between the animal groups were analyzed. Statistical testing was performed with Kruskal-Wallis analysis of variance and the chi2 test., Results: At 24 hours after USPIO administration, the relative SI of the abscess wall and the relative macrophage extent were 0.50 (0.33-0.73) and 1.03 (0.90-1.08), respectively, for acute infection; 0.11 (0.10-0.18) and 0.94 (0.93-1.01) for early chronic infection; and 0.53 (0.44-0.58) and 0.80 (0.77-0.83) for late chronic infection. The changes in enhancement pattern (P <.001), relative SI (P <.001), and relative macrophage extent (P <.05) with time were significant., Conclusion: The macrophage distribution pattern increases the specificity of MR findings in chronic infection and allows differentiation between areas with active inflammation and areas of reparative granulation tissue.