Evaluation of a novel macromolecular cascade-polymer contrast medium for dynamic contrast-enhanced MRI monitoring of antiangiogenic bevacizumab therapy in a human melanoma model.

RATIONALE AND OBJECTIVES: To assess the applicability of a novel macromolecular polyethylene glycol (PEG)-core gadolinium contrast agent for monitoring early antiangiogenic effects of bevacizumab using dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI). MATERIALS AND METHODS: Athymic rats (n = 26) implanted with subcutaneous human melanoma xenografts underwent DCE-MRI at 2.0 T using two different macromolecular contrast agents. The PEG core cascade polymer PEG12,000-Gen4-(Gd-DOTA)16, designed for clinical development, was compared to the prototype, animal-only, macromolecular contrast medium (MMCM) albumin-(Gd-DTPA)35. The treatment (n = 13) and control (n = 13) group was imaged at baseline and 24 hours after a single dose of bevacizumab (1 mg) or saline to quantitatively assess the endothelial-surface permeability constant (K(PS), µL⋅min⋅100 cm(3)) and the fractional plasma volume (fPV,%), using a two-compartment kinetic model. RESULTS: Mean K(PS) values, assessed with PEG12,000-Gen4-(Gd-DOTA)16, declined significantly (P < .05) from 29.5 ± 10 µL⋅min⋅100 cm(3) to 10.4 ± 7.8 µL⋅min⋅100 cm(3) by 24 hours after a single dose of bevacizumab. In parallel, K(PS) values quantified using the prototype MMCM albumin-(Gd-DTPA)35 showed an analogous, significant decline (P < .05) in the therapy group. No significant effects were detected on tumor vascularity or on microcirculatory parameters in the control group between the baseline and the follow-up scan at 24 hours. CONCLUSION: DCE-MRI enhanced with the novel MMCM PEG12,000-Gen4-(Gd-DOTA)16 was able to monitor the effects of bevacizumab on melanoma xenografts within 24 hours of a single application, validated by the prototype, animal-only albumin-(Gd-DTPA)35. PEG12,000-Gen4-(Gd-DOTA)16 may be a promising candidate for further clinical development as a macromolecular blood pool contrast MRI agent.

Permeability to macromolecular contrast media quantified by dynamic MRI correlates with tumor tissue assays of vascular endothelial growth factor (VEGF).

PURPOSE: To correlate dynamic MRI assays of macromolecular endothelial permeability with microscopic area-density measurements of vascular endothelial growth factor (VEGF) in tumors. METHODS AND MATERIAL: This study compared tumor xenografts from two different human cancer cell lines, MDA-MB-231 tumors (n=5), and MDA-MB-435 (n=8), reported to express respectively higher and lower levels of VEGF. Dynamic MRI was enhanced by a prototype macromolecular contrast medium (MMCM), albumin-(Gd-DTPA)35. Quantitative estimates of tumor microvascular permeability (K(PS); µl/min × 100 cm(3)), obtained using a two-compartment kinetic model, were correlated with immunohistochemical measurements of VEGF in each tumor. RESULTS: Mean K(PS) was 2.4 times greater in MDA-MB-231 tumors (K(PS)=58 ± 30.9 µl/min × 100 cm(3)) than in MDA-MB-435 tumors (K(PS)=24 ± 8.4 µl/min × 100 cm(3)) (p<0.05). Correspondingly, the area-density of VEGF in MDA-MB-231 tumors was 2.6 times greater (27.3 ± 2.2%, p<0.05) than in MDA-MB-435 cancers (10.5 ± 0.5%, p<0.05). Considering all tumors without regard to cell type, a significant positive correlation (r=0.67, p<0.05) was observed between MRI-estimated endothelial permeability and VEGF immunoreactivity. CONCLUSION: Correlation of MRI assays of endothelial permeability to a MMCM and VEGF immunoreactivity of tumors support the hypothesis that VEGF is a major contributor to increased macromolecular permeability in cancers. When applied clinically, the MMCM-enhanced MRI approach could help to optimize the appropriate application of VEGF-inhibiting therapy on an individual patient basis.

Nephrogenic systemic fibrosis in rats treated with erythropoietin and intravenous iron.

PURPOSE: To use a rat model for nephrogenic systemic fibrosis (NSF) that was administered high-dose gadodiamide to determine whether the co-administration of erythropoietin (Epo) and intravenous iron potentiated development of skin lesions that are thought to be a marker for the development of NSF. MATERIALS AND METHODS: The local committee for animal research approved this study. High-dose gadodiamide was administered, 2.5 mmol per kilogram of body weight for 20 days, or 500 times the U.S. Food and Drug Administration-approved dose, to four groups of Hannover-Wistar rats: group A, gadodiamide only; B, gadodiamide and Epo; C, gadodiamide and intravenous iron; and D, gadodiamide, Epo, and intravenous iron. The animals were sacrificed 7 days after final injection, and the authors examined dermal histologic findings from each animal and measured metal deposition by using inductively coupled plasma mass spectrometry. To compare the effect of metal deposition and cellularity, a linear mixed effects model was used to fit the data within PROC MIXED modeled with rat-specific random effects, and subsequently a Dunnett adjustment was performed. RESULTS: Rats treated with gadodiamide and both Epo and intravenous iron (group D) had significantly worse skin lesions at gross and histologic analysis (P = .004) compared with the rate treated with gadodiamide only (group A). Group D also had increased levels of deposited gadolinium as measured by means of mass spectrometry (P = .012). CONCLUSION: With a rat model similar to those already existing in the literature, skin changes were more marked in animals exposed to gadodiamide, Epo, and intravenous iron, as opposed to those animals exposed to gadodiamide alone; this experiment suggests that great caution may be warranted when prescribing gadolinium-based contrast agents to patients receiving Epo and intravenous iron.

Magnetic resonance imaging for monitoring the effects of thalidomide on experimental human breast cancers.

Thalidomide, which inhibits angiogenesis in certain tumor types, reduced extravasation of a macromolecular contrast medium (MMCM) in a human breast cancer model as assayed by MMCM-enhanced dynamic magnetic resonance imaging (MRI) and fluorescence microscopy in the same tumors. After a 1-week, three-dose course of thalidomide, the mean MRI-assayed endothelial transfer coefficient, K(PS), decreased significantly (p < 0.05) from 19.4 +/- 9.1 to 6.3 +/- 9.1 microl/min.100 cm(3). Correspondingly, microscopic measurements of extravasated MMCM, expressed as fractional area of streptavidin staining, were significantly (p < 0.05) lower in thalidomide-treated tumors (18.6 +/- 11.9%) than in control saline-treated tumors (50.2 +/- 2.3%). On a tumor-by-tumor basis, post-treatment K(PS) values correlated significantly (r(2) = 0.55, p < 0.05) with microscopic measures of MMCM extravasation. However, no significant differences were observed between saline- and thalidomide-treated tumors with respect to rate of growth, vascular richness, or amount of VEGF-containing cells. Because of its sensitivity to the detection of changes in vascular leakage in tumors, this MMCM-enhanced MRI assay could prove useful for monitoring the effects of thalidomide on an individual patient basis. The significant correlation between MRI and fluorescence microscopic measures of MMCM extravasation supports the utility of the non-invasive MRI approach for assessing the action of thalidomide on tumor blood vessels.

Magnetic resonance imaging assays for dimethyl sulfoxide effect on cancer vasculature.

OBJECTIVES: To evaluate the potential of quantitative assays of vascular characteristics based on dynamic contrast-enhanced magnetic resonance imaging (MRI) using a macromolecular contrast medium (MMCM) to search for and measure effects of dimethyl sulfoxide (DMSO) on cancer vasculature with microscopic correlations. MATERIAL AND METHODS: Saline-treated control (n = 8) and DMSO-treated (n = 7) human breast cancer xenografts (MDA-MB-435) in rats were imaged dynamically by MMCM-enhanced MRI using albumin-(Gd-DTPA)27-(biotin)11 (molecular weight approximately 90 kDa), before and after a 1-week, 3-dose treatment course. After the posttreatment MRI examinations, tumors were perfused with lectin and fixative and subsequently stained with RECA-1 and streptavidin for quantitative fluorescent microscopy. Quantitative MRI estimates of cancer microvessel permeability (KPS; microL/min.100 cm3) and fractional plasma volume (fPV; %) were based on a 2-compartment kinetic model. Fluorescent microscopy yielded estimates of MMCM extravasation and vascular density that were compared to the MRI results. RESULTS: DMSO decreased cancer vascular endothelial permeability significantly (P < 0.05) from tumor KPSday0 = 19.3 +/- 8.8 microL/min.100 cm3 to KPSday7 = 0 microL/min.100 cm3). K values in the saline-treated tumors did not change significantly. The amount of extravasated albumin-Gd-(DTPA)27-(biotin)11, as assayed by a fluorescently labeled streptavidin stain that strongly binds to the biotin tag on the MMCM, was significantly (P < 0.05) lower in the DMSO-treated cancers than in the control cancers (57.7% +/- 5.5% vs. 34.2% +/- 4.9%). Tumor vascular richness as reflected by the MRI-assayed fPV and by the RECA-1 and lectin-stained microscopy did not change significantly with DMSO or saline treatment. CONCLUSION: Reductions in cancer microvascular leakiness induced by a 7-day course of DMSO could be detected and measured by dynamic MMCM-enhanced MRI and were confirmed by microscopic measurements of the leaked macromolecular agents in the same cancers. Results support the robustness of an MMCM-enhanced MRI approach to the characterization of cancers and providing first evidence for an in vivo effect of DMSO on cancer blood vessels.

New macromolecular polymeric MRI contrast agents for application in the differentiation of cancer from benign soft tissues.

PURPOSE: To compare three new macromolecular polyethylene glycol (PEG) -core dendrimeric gadolinium(Gd)-based MRI contrast agents for their applicability in quantitative assays of endothelial leakiness and tissue vascular density for the differentiation of cancer from normal soft tissues. MATERIALS AND METHODS: Thirty-two athymic rats with human breast cancer xenografts (MDA-MB-435) were imaged by dynamic MRI following enhancement with one of three new (Gd-DOTA)-conjugated PEG-core dendrimer contrast agents (effective molecular weights 161 to 323 kDa). Results were compared with a prototype macromolecular contrast agent, albumin (Gd-DTPA). Assays of permeabilities (K(PS); microL/min . 100 cm(3)) and tumor fractional plasma volumes (%) based on a two-compartment kinetic model were performed for skeletal muscle and tumors. RESULTS: The largest PEG-core contrast agent, PEG(20,000)-Gen4-(Gd-DOTA), leaked in breast tumors (K(PS) = 50 +/- 23 microL/min . 100 cm(3)), while exhibiting no measurable transendothelial leak (K(PS) = 0 microL/min . 100 cm(3)) in normal soft tissue microvessels allowing successful differentiation (P < 0.05) of cancers from normal muscle. PEG(12,000)-Gen4-(Gd-DOTA) leaked in tumors and in normal muscle (K(PS) = 51 +/- 26 and K(PS) = 21 +/- 18 microL/min . 100 cm(3), respectively). The smallest agent, PEG(12,000)-Gen3-(Gd-DOTA) also showed a measurable leak in both normal and malignant microvessels. CONCLUSION: MRI assays of vascular endothelial leakiness using new PEG-core, (Gd-DOTA)-conjugated macromolecular contrast agents proved applicable for the differentiation of human breast cancer from normal soft tissue. The apparent threshold in effective molecular weight for a clear differentiation of cancer from normal muscle with no measurable leak in the muscle is between 194 and 323 kDa.

Cascade polymeric MRI contrast media derived from poly(ethylene glycol) cores: initial syntheses and characterizations.

Diagnostic contrast media for magnetic resonance imaging (MRI) are often applied to enhance the signal of blood allowing for quantitative definition of vascular functional characteristics including tissue blood volume, flow, and leakiness. Well-tolerated and safe macromolecular formulations are currently being sought that remain in the blood for a relatively long period and that leak selectively from diseased vessels, particularly cancer vessels. We synthesized a new class of macromolecular, water-soluble MRI contrast media by introducing two diverging polylysine cascade amplifiers at each end of a poly(ethylene glycol) (PEG) backbone, followed by substitution of terminal lysine amino groups with Gd-DTPA chelates. Four candidate PEG cascade conjugates are reported here, PEG3400-Gen4-(Gd-DTPA)8, PEG6000-Gen4-(Gd-DTPA)8, PEG12000-Gen4-(Gd-DTPA)8, and PEG3400-Gen5-(Gd-DTPA)13 with descriptions of their basic physical, biological, and kinetic properties, including real and effective molecular sizes, proton T1 relaxivities in water and plasma, partition coefficients, osmolalities, chelate stability, stability in plasma, stability to autoclaving, certain in vivo pharmacokinetics (blood half-life, blood clearance, volume of distribution), and whole body elimination profiles in normal rodents. These candidate PEG-core cascade MRI contrast media showed a range of effective molecular sizes similar to proteins weighing 74-132 kDa, although their actual molecular weights were much smaller, 12-20 kDa. All compounds exhibited a narrow range of size dispersity and relatively high T1 relaxivities (approximately 3 times the value for unconjugated Gd-DTPA at 2 T and 37 degrees C). Representative compounds also showed a high degree of hydrophilicity, stability in solution buffer and plasma, and lack of binding to proteins. The two candidate compounds with the largest effective molecular sizes, PEG12000-Gen4-(Gd-DTPA)8 and PEG3400-Gen5-(Gd-DTPA)13, had longer blood half-lives, 36 and 73 min, respectively (monoexponential kinetics for both), and showed strong, prolonged MRI enhancement of vessels. Results also indicate that in vivo pharmacokinetics and bodily elimination profiles can be adjusted by the selection of molecular size for the PEG core and the selection of the amplification degree of the cascade polylysine clusters. The initially evaluated compounds from this new class of contrast media show acceptable, desirable characteristics in many, but not all, respects. Further efforts are directed toward candidate macromolecules having higher thermodynamic stability, higher degree of substitution by gadolinium chelates, and more rapid bodily elimination.