MRI in drug discovery

MRI is in widespread use in clinical diagnosis as well as in research areas such as preclinical drug discovery. MRI allows non-invasive acquisition of tomographic images of animal soft tissue with high resolution and contrast. Furthermore its ability to assess organ function in a broad sense renders this technique to a versatile tool to answer specific scientific questions on the drug actions in disease models. Primarily imaging of patho-physiological mechanisms and molecular processes are in the focus of MRI in drug research. Finally MRI is translational and thus has the potential to bridge the gap between preclinical research on one hand and clinical development or therapy monitoring on the other

Quantitative dynamic contrast-enhanced MRI in tumor-bearing rats and mice with inversion recovery TrueFISP and two contrast agents at 4.7 T.

PURPOSE: To characterize tumor vascularization by dynamic-contrast enhanced (DCE) MRI using low and medium molecular weight paramagnetic contrast agents (CA) and inversion recovery (IR) true fast imaging with steady state precession (TrueFISP) in tumor-bearing rats and mice. MATERIALS AND METHODS: T(1) mapping was performed using IR True FISP in phantoms and in vivo at 4.7 T and validated with a segmented IR gradient-echo (IR GE) method. CA concentration in DCE-MRI studies in vivo was calculated from time-series T(1) maps using the CAs GdDOTA and P792 (low and medium molecular weight, respectively). Standard vascular input functions (VIFs) were measured in the jugular veins and were used for modeling of the CA kinetics with a two-compartment model. In rat breast tumors, vascular permeability (transfer constant K(trans)), fractional plasma volume v(p), and fractional leakage space v(e) were quantified and parametric maps were generated. RESULTS: The IR TrueFISP T(1) was slightly underestimated in phantoms and overestimated in vivo (10%) with respect to IR GE. VIFs showed only small interindividual variation. Mean K(trans) values were 0.062 +/- 0.017 min(-1) for GdDOTA and 0.015 +/- 0.005 min(-1) for P792 (N = 12). Mean v(e) and v(p) values were 0.15 +/- 0.04 (0.09 +/- 0.03) and 0.04 +/- 0.01 (0.03 +/- 0.01) for GdDOTA (P792). CONCLUSION: DCE-MRI with IR TrueFISP provided absolute values for K(trans), v(p), and v(e). Direct comparison between GdDOTA and P792 revealed significant differences in the VIF, model-fit-quality, permeability, leakage space, and plasma volume. The larger molecular weight CA P792 appears to be better for measuring tumor vascular parameters

Tumour T1 changes in vivo are highly predictive of response to chemotherapy and reflect the number of viable tumour cells

We have shown that chemotherapy rapidly reduces the spin-lattice relaxation of water protons (T1) in solid tumours and this change (T1) often precedes and strongly correlates with the eventual change in tumour volume [McSheehy et al 2010]. To understand the biological nature of T1, we have performed further 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 analyzed to permit full cellular and necrotic quantification as well staining for the proliferation antigen Ki67 and cleaved-caspase-3 as a measure of apoptosis. The number of blood-vessels per slice was evaluated by haematoxyline-eosin staining. Everolimus blocked tumour growth and significantly reduced the tumour T1 and total choline (Cho) levels, and increased polyunsaturated fattyacids (PUFA) which are markers of apoptosis. Histology and IHC showed that everolimus reduced the %Ki67+ cells and number of blood vessels but did not affect caspase-3 apoptosis, necrosis or cell density. The change in T1 (T1) strongly correlated with the TVol and also the Cho and the endpoint Ki67. In murine B16/BL6 melanoma tumours, everolimus also decreased T1 and this correlated with cell bioluminescence; another marker of cell viability. These studies suggest that T1 is not a measure of cell density but reflects the number of viable and proliferating tumour cells because cell and tissue destruction release factors that cause T1 relaxation. Receiver-operator-curves for everolimus on RIF-1 tumours showed that T1 had very high levels of sensitivity and specificity (AUC=0.91) and this was confirmed for the cytotoxic patupilone in the same tumour model (AUC=0.95)

Patupilone induced vascular disruption in orthotopic rodent tumor models detected by magnetic resonance imaging and interstitial fluid pressure.

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 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

AEE788: a dual family epidermal growth factor receptor/ErbB2 and vascular endothelial growth factor receptor tyrosine kinase inhibitor with antitumor and antiangiogenic activity.

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

mTOR inhibitor RAD001 (everolimus) has antiangiogenic/vascular properties distinct from a VEGFR tyrosine kinase inhibitor

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 values1 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

Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET1

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-[18F]fluoro-glucose (FDG) and 3′-deoxy-3′-[18F]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 IC50 values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 µM, 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

A novel potent oral series of VEGFR2 inhibitors abrogate tumor growth by inhibiting angiogenesis

This paper describes the identification of 6-(pyrimidin-4-yloxy)-naphthalene-1-carboxamides as a new class of potent and selective human vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitors. In biochemical and cellular assays the compounds exhibit single digit nanomolar potency toward VEGFR2. Compounds of this series show good exposure in rodents when dosed orally. They potently inhibit VEGF-driven angiogenesis in a chamber model and rodent tumor models at daily doses of less than 3 mg/kg by targeting the vasculature as demonstrated by ELISA for TIE-2 in lysates, or by immunohistochemical analysis. This novel series of compounds shows potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role

Oxamyl dipeptide caspase inhibitors developed for the treatment of stroke

Structural modifications were made to a previously described acyl dipeptide caspase inhibitor, leading to the oxamyl dipeptide series. Subsequent SAR studies directed toward the warhead, P2, and P4 regions of this novel peptidomimetic are described herein