BR55 Ultrasound Molecular Imaging of Clear Cell Renal Cell Carcinoma Reflects Tumor Vascular Expression of VEGFR-2 in a Patient-Derived Xenograft Model.

Standard imaging cannot reliably predict the nature of renal tumors. Among malignant renal tumors, clear cell renal cell carcinoma (ccRCC) is the most common histological subtype, in which the vascular endothelial growth factor 2 (VEGFR-2) is highly expressed in the vascular endothelium. BR55, a contrast agent for ultrasound imaging, consists of gas-core lipid microbubbles that specifically target and bind to the extracellular portion of the VEGFR-2. The specific information provided by ultrasound molecular imaging (USMI) using BR55 was compared with the vascular tumor expression of the VEGFR-2 by immunohistochemical (IHC) staining in a preclinical model of ccRCC. Patients' ccRCCs were orthotopically grafted onto Nod-Scid-Gamma (NSG) mice to generate patient-derived xenografts (PdX). Mice were divided into four groups to receive either vehicle or axitinib an amount of 2, 7.5 or 15 mg/kg twice daily. Perfusion parameters and the BR55 ultrasound contrast signal on PdX renal tumors were analyzed at D0, D1, D3, D7 and D11, and compared with IHC staining for the VEGFR-2 and CD34. Significant Pearson correlation coefficients were observed between the area under the curve (AUC) and the CD34 (0.84, p < 10-4), and between the VEGFR-2-specific signal obtained by USMI and IHC (0.72, p < 10-4). USMI with BR55 could provide instant, quantitative information on tumor VEGFR-2 expression to characterize renal masses non-invasively.

Nilotinib Enhances Tumor Angiogenesis and Counteracts VEGFR2 Blockade in an Orthotopic Breast Cancer Xenograft Model with Desmoplastic Response.

Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-targeted therapies predominantly affect nascent, immature tumor vessels. Since platelet-derived growth factor receptor (PDGFR) blockade inhibits vessel maturation and thus increases the amount of immature tumor vessels, we evaluated whether the combined PDGFR inhibition by nilotinib and VEGFR2 blockade by DC101 has synergistic therapy effects in a desmoplastic breast cancer xenograft model. In this context, besides immunohistological evaluation, molecular ultrasound imaging with BR55, the clinically used VEGFR2-targeted microbubbles, was applied to monitor VEGFR2-positive vessels noninvasively and to assess the therapy effects on tumor angiogenesis. DC101 treatment alone inhibited tumor angiogenesis, resulting in lower tumor growth and in significantly lower vessel density than in the control group after 14 days of therapy. In contrast, nilotinib inhibited vessel maturation but enhanced VEGFR2 expression, leading to markedly increased tumor volumes and a significantly higher vessel density. The combination of both drugs led to an almost similar tumor growth as in the DC101 treatment group, but VEGFR2 expression and microvessel density were higher and comparable to the controls. Further analyses revealed significantly higher levels of tumor cell-derived VEGF in nilotinib-treated tumors. In line with this, nilotinib, especially in low doses, induced an upregulation of VEGF and IL-6 mRNA in the tumor cells in vitro, thus providing an explanation for the enhanced angiogenesis observed in nilotinib-treated tumors in vivo. These findings suggest that nilotinib inhibits vessel maturation but counteracts the effects of antiangiogenic co-therapy by enhancing VEGF expression by the tumor cells and stimulating tumor angiogenesis.

First-in-Human Ultrasound Molecular Imaging With a VEGFR2-Specific Ultrasound Molecular Contrast Agent (BR55) in Prostate Cancer: A Safety and Feasibility Pilot Study.

OBJECTIVE: BR55, a vascular endothelial growth factor receptor 2 (VEGFR2)-specific ultrasound molecular contrast agent (MCA), has shown promising results in multiple preclinical models regarding cancer imaging. In this first-in-human, phase 0, exploratory study, we investigated the feasibility and safety of the MCA for the detection of prostate cancer (PCa) in men using clinical standard technology. MATERIALS AND METHODS: Imaging with the MCA was performed in 24 patients with biopsy-proven PCa scheduled for radical prostatectomy using a clinical ultrasound scanner at low acoustic power. Safety monitoring was done by physical examination, blood pressure and heart rate measurements, electrocardiogram, and blood sampling. As first-in-human study, MCA dosing and imaging protocol were necessarily fine-tuned along the enrollment to improve visualization. Imaging data were correlated with radical prostatectomy histopathology to analyze the detection rate of ultrasound molecular imaging with the MCA. RESULTS: Imaging with MCA doses of 0.03 and 0.05 mL/kg was adequate to obtain contrast enhancement images up to 30 minutes after administration. No serious adverse events or clinically meaningful changes in safety monitoring data were identified during or after administration. BR55 dosing and imaging were fine-tuned in the first 12 patients leading to 12 subsequent patients with an improved MCA dosing and imaging protocol. Twenty-three patients underwent radical prostatectomy. A total of 52 lesions were determined to be malignant by histopathology with 26 (50%) of them seen during BR55 imaging. In the 11 patients that were scanned with the improved protocol and underwent radical prostatectomy, a total of 28 malignant lesions were determined: 19 (68%) were seen during BR55 ultrasound molecular imaging, whereas 9 (32%) were not identified. CONCLUSIONS: Ultrasound molecular imaging with BR55 is feasible with clinical standard technology and demonstrated a good safety profile. Detectable levels of the MCA can be reached in patients with PCa opening the way for further clinical trials.

In situ validation of VEGFR-2 and α v ß 3 integrin as targets for breast lesion characterization.

Vascular endothelial growth factor receptor 2 (VEGFR-2) and α v ß 3 integrin are the most frequently addressed targets in molecular imaging of tumor angiogenesis. In preclinical studies, molecular imaging of angiogenesis has shown potential to detect and differentiate benign and malignant lesions of the breast. Thus, in this retrospective clinical study employing patient tissues, the diagnostic value of VEGFR-2, α v ß 3 integrin and vascular area fraction for the diagnosis and differentiation of breast neoplasia was evaluated. To this end, tissue sections of breast cancer (n = 40), pre-invasive ductal carcinoma in situ (DCIS; n = 8), fibroadenoma (n = 40), radial scar (n = 6) and normal breast tissue (n = 40) were used to quantify (1) endothelial VEGFR-2, (2) endothelial α v ß 3 integrin and (3) total α v ß 3 integrin expression, as well as (4) the vascular area fraction. Sensitivity and specificity to differentiate benign from malignant lesions were calculated for each marker by receiver operating characteristics (ROC) analyses. Whereas vessel density, as commonly used, did not significantly differ between benign and malignant lesions (AUROC: 0.54), VEGFR-2 and α v ß 3 integrin levels were gradually up-regulated in carcinoma versus fibroadenoma versus healthy tissue. The highest diagnostic accuracy for differentiating carcinoma from fibroadenoma was found for total α v ß 3 integrin expression (AUROC: 0.76), followed by VEGFR-2 (AUROC: 0.71) and endothelial α v ß 3 integrin expression (AUROC: 0.68). In conclusion, total α v ß 3 integrin expression is the best discriminator between breast cancer, fibroadenoma and normal breast tissue. With respect to vascular targeting and molecular imaging of angiogenesis, endothelial VEGFR-2 appeared to be slightly superior to endothelial α v ß 3 for differentiating benign from cancerous lesions.

Liver dysplasia: US molecular imaging with targeted contrast agent enables early assessment.

PURPOSE: To investigate the ability of vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted ultrasonographic (US) microbubbles for the assessment of liver dysplasia in transgenic mice. MATERIALS AND METHODS: Animal experiments were approved by the governmental review committee. Nuclear factor-κB essential modulator knock-out mice with liver dysplasia and wild-type mice underwent liver imaging by using a clinical US system. Two types of contrast agents were investigated: nontargeted, commercially available, second-generation microbubbles (SonoVue) and clinically translatable PEGylated VEGFR2-targeted microbubbles (BR55). Microbubble kinetics was investigated over the course of 4 minutes. Targeted contrast material-enhanced US signal was quantified 5 minutes after injection. Competitive in vivo binding experiments with BR55 were performed in knock-out mice. Immunohistochemical and hematoxylin-eosin staining of liver sections was performed to validate the in vivo US results. Groups were compared by using the Mann-Whitney test. RESULTS: Peak enhancement after injection of SonoVue and BR55 did not differ in healthy and dysplastic livers (SonoVue, P = .46; BR55, P = .43). Accordingly, immunohistochemical findings revealed comparable vessel densities in both groups. The specificity of BR55 to VEGFR2 was proved by in vivo competition (P = .0262). While the SonoVue signal decreased similarly in healthy and dysplastic livers during the 4 minutes, there was an accumulation of BR55 in dysplastic livers compared with healthy ones. Furthermore, targeted contrast-enhanced US signal indicated a significantly higher site-specific binding of BR55 in dysplastic than healthy livers (P = .005). Quantitative immunohistologic findings confirmed significantly higher VEGFR2 levels in dysplastic livers (P = .02). CONCLUSION: BR55 enables the distinction of early stages of liver dysplasia from normal liver.

The high angiogenic activity in very early breast cancer enables reliable imaging with VEGFR2-targeted microbubbles (BR55).

OBJECTIVES: Tumour xenografts of well-discernible sizes can be examined well by molecular ultrasound. Here, we investigated whether very early breast carcinomas express sufficient levels of VEGFR2 for reliable molecular ultrasound imaging with targeted microbubbles. METHODS: MCF-7 breast cancer xenografts were orthotopically implanted in nude mice (n = 26). Tumours measuring from 4 mm(3) (2 mm diameter) up to 65 mm(3) (5 mm diameter) were examined with automated 3D molecular ultrasound using clinically translatable VEGFR2-targeted microbubbles (BR55). Additionally, the relative tumour blood volume was assessed with non-targeted microbubbles (BR38). In vivo ultrasound data were validated by quantitative immunohistochemistry. RESULTS: Very small lesions 2 mm in diameter showed the highest binding of VEGFR2-specific microbubbles. In larger tumours significantly less BR55 accumulated (p = 0.023). Nonetheless, binding of VEGFR2-targeted microbubbles was still high enough for imaging. The relative blood volume was comparable at all tumour sizes. Both findings were confirmed by immunohistochemistry. Additionally, a significantly enhanced number of large and mature vessels were detected with increasing tumour size (p < 0.01), explaining the decrease in VEGFR2 expression during tumour growth. CONCLUSIONS: 3D molecular ultrasound using BR55 is very well suited to depicting the angiogenic activity in very small breast lesions, suggesting its potential for detecting and characterising these lesions.

Ultrasound molecular imaging contrast agent binding to both E- and P-selectin in different species.

OBJECTIVES: Ultrasound molecular imaging is increasingly used in preclinical studies to measure the expression of vascular markers during inflammation process. In this context, a new ultrasound contrast agent functionalized with a recombinant P-selectin glycoprotein ligand-1 analogue (rPSGL-Ig) was developed (MBrPSGL-Ig). This agent was assayed in vitro and in vivo to evaluate its binding performance and potential to image expression of inflammatory markers E- and P-selectin. Performance of this newly developed agent was compared with that of antibody (MBAb) or sialyl Lewis X (MBsLe) containing microbubbles and with control microbubbles (MBC). MATERIALS AND METHODS: The targeted ultrasound contrast agents were prepared by coupling biotin-conjugated ligands onto streptavidin-functionalized microbubbles. First, in vitro experiments were performed to measure the adhesion efficiency of these microbubble constructs under static or flow conditions (114 sec), on cell monolayer (human umbilical vein endothelial cells and bEnd.5), or coatings of E- or P-selectin of various animal species, respectively. Second, molecular imaging studies were performed in a rat inflammatory model 24 hours after intramuscular injection of lipopolysaccharide in the hind limb. Finally, immunohistochemistry staining of rat inflamed muscle tissue was performed to assess expression of E- and P-selectin. RESULTS: Microbubbles functionalized with rPSGL-Ig (MBrPSGL-Ig) displayed firm in vitro binding on the coating of both recombinant E- or P-selectin, with an efficiency similar to microbubbles comprising antibody specific for E-selectin (MBE) or P-selectin (MBP). In contrast, lower binding capacity was measured with MBsLe. At the surface of inflamed endothelial cells, MBrPSGL-Ig were able to interact specifically with E- and P-selectin. Binding specificity was demonstrated by performing blocking experiments with target-specific antibodies, resulting in an 80% to 95% decrease in binding. Ten minutes after microbubble injection, echo signal measured with MBrPSGL-Ig in the inflamed muscles was 20-fold higher compared with MBC. Moreover, the in vivo adhesion of MBrPSGL-Ig was 2- and 7-fold higher compared with P-selectin or E-selectin-specific microbubbles, respectively. Immunohistochemistry revealed a temporal coexpression of E- and P-selectin in the vascular bed of inflamed rat muscle 24 hours after lipopolysaccharide injection. CONCLUSION: The molecular imaging study demonstrates that MBrPSGL-Ig provide imaging signal higher than those measured with antibody or sialyl Lewis X containing microbubbles. These results suggest that MBrPSGL-Ig is a powerful agent to image the expression of both E- and P-selectin in the context of an inflammatory process.

Effects of acoustic radiation force on the binding efficiency of BR55, a VEGFR2-specific ultrasound contrast agent.

This work describes an in vivo study analyzing the effect of acoustic radiation force (ARF) on the binding of BR55 VEGFR2-specific contrast-agent microbubbles in a model of prostatic adenocarcinoma in rat. A commercial ultrasound system was modified by implementing high duty-cycle 3.5-MHz center frequency ARF bursts in a scanning configuration. This enabled comparing the effects of ARF on binding in tumor and healthy tissue effectively in the same field of view. Bubble binding was established by measuring late-phase enhancement in amplitude modulation (AM) contrast-specific imaging mode (4 MHz, 150 kPa) 10 min after agent injection when the unbound bubbles were cleared from the circulation. Optimal experimental conditions, such as agent concentration (0.4 × 10(8)-1.6 × 10(8) bubbles/kg), acoustic pressure amplitude (26-51 kPa) and duty-cycle (20%-95%) of the ARF bursts, were evaluated in their ability to enhance binding in tumor without significantly increasing binding in healthy tissue. Using the optimal conditions (38 kPa peak-negative pressure, 95% duty cycle), ARF-assisted binding of BR55 improved significantly in tumor (by a factor of 7) at a lower agent dose compared with binding without ARF, and it had an insignificant effect on binding in healthy tissue. Thus, the high binding specificity of BR55 microbubbles for targeting VEGFR2 present at sites of active angiogenesis was confirmed by this study. Therefore, it is believed that based on the results obtained in this work, ultrasound molecular imaging using target-specific contrast-agent microbubbles should preferably be performed in combination with ARF.

Use of intravital microscopy to study the microvascular behavior of microbubble-based ultrasound contrast agents.

PURPOSE: The study describes the use of intravital microscopy (IVM) to assess the behavior of ultrasound contrast agents (UCAs), including targeted UCAs, in the microcirculation of rodents. MATERIALS AND METHODS: IVM was performed on various exteriorized organs: hamster cheek pouch, rat mesentery, liver, spinotrapezius muscle, and mouse cremaster muscle. A dorsal skin-fold chamber with MatBIII tumor cells was also implanted in rats. Nontargeted UCAs (SonoVue(®) and BR14) and targeted UCAs (BR55 and P-selectin targeted microbubbles) were tested. IVM was used to measure microbubble size, determine their persistence, and observe their behavior in the blood circulation. RESULTS: Intravenous and intra-arterial injections of high doses of UCAs did not modify the local microvascular hemodynamics. No microbubble coalescence and no increased size were observed. Adhesion of some microbubbles to leukocytes was observed in various microcirculation models. Microbubbles are captured by Kupffer cells in the liver. Targeted microbubbles were shown to adhere specifically to endothelial receptors without compromising local blood flow. CONCLUSION: These results support the safety of both targeted and nontargeted UCAs as no microvascular flow alteration or plugging of microvessels were observed. They confirm that binding observed with targeted microbubbles are due to the binding of these microbubbles to specific endothelial receptors.

Molecular and functional ultrasound imaging in differently aggressive breast cancer xenografts using two novel ultrasound contrast agents (BR55 and BR38).

OBJECTIVES: To characterise clinically translatable long-circulating (BR38) and VEGFR2-targeted (BR55) microbubbles (MB) and to assess their ability to discriminate breast cancer models with different aggressiveness. METHODS: The circulation characteristics of BR38 and BR55 were investigated in healthy mice. The relative blood volume (rBV) of MDA-MB-231 (n = 5) or MCF-7 (n = 6) tumours was determined using BR38. In the same tumours in-vivo binding specificity of BR55 was tested and VEGFR2 expression assessed. Data validation included quantitative immunohistological analysis. RESULTS: BR38 had a longer blood half-life than BR55 (>600 s vs. 218 s). BR38-enhanced ultrasound showed greater vascularisation in MDA-MB-231 tumours (p = 0.022), which was in line with immunohistology (p = 0.033). In-vivo competitive binding experiments proved the specificity of BR55 to VEGFR2 (p = 0.027). Binding of BR55 was significantly higher in MDA-MB-231 than in MCF-7 tumours (p = 0.049), which corresponded with the VEGFR2 levels found histologically (p = 0.015). However, differences became smaller when normalising the levels of BR55 to the rBV. CONCLUSIONS: BR38 and BR55 are well suited to characterising and distinguishing breast cancers with different angiogenesis and aggressiveness. Long-circulating BR38 MB allow extensive 3-dimensional examinations of larger or several organs. BR55 accumulation faithfully reflects the VEGFR2 status in tumours and depicts even small differences in angiogenesis.

Ultrasound molecular imaging of VEGFR2 in a rat prostate tumor model using BR55.

OBJECTIVES: To evaluate BR55, a new VEGFR2-specific ultrasound contrast agent, for imaging prostate tumors in an orthotopic model in the rat. MATERIALS AND METHODS: Rat prostate adenocarcinoma were established by injection of G Dunning R-3327 tumor cells in one lobe of the prostate of Copenhagen rats. Imaging experiments were performed with BR55, SonoVue, and streptavidin-functionalized microbubbles coupled with an anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody using a clinical ultrasound scanner. Contrast enhancement in the tumor and healthy prostate was followed over time by intermittent imaging at low acoustic power. Signal quantification and statistical analysis were performed in the tumor and healthy tissue to compare the behavior of the 3 contrast agents. Immunohistochemistry was performed on the prostate and tumor specimen to determine the expression of VEGFR2. RESULTS: Comparable contrast enhancement was observed in tumors at peak intensity for BR55 and SonoVue. Then, once unbound microbubbles had cleared from the circulation, a strong enhancement of the tumor was obtained with BR55, whereas no significant microbubble accumulation was detected in the healthy prostate tissue. SonoVue microbubbles were rapidly eliminated, and no significant binding was observed in the tumor. The tumor to prostate ratio calculated after signal quantification was about 20 for the 3 doses of BR55 tested. The enhancement obtained with BR55 in the tumor was not significantly different from the one observed with antibody-coupled streptavidin microbubbles. Intense staining for VEGFR2 was detected in the tumor vessels by immunohistochemistry. CONCLUSIONS: This study showed that BR55 binding to prostate tumors resulted in a strong enhancement of the lesions as early as a few minutes after contrast injection, whereas minimal nonspecific accumulation occurred in the healthy part of the gland. BR55, like SonoVue, provide information on tissue perfusion during the early vascular phase, but BR55 binding to the tumoral endothelium allows to gain additional information by highlighting the sites of active angiogenesis. The late phase enhancement of the tumor should be particularly valuable for prostate cancer detection and for biopsy guidance.

BR55: a lipopeptide-based VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis.

OBJECTIVES: BR55, an ultrasound contrast agent functionalized with a heterodimer peptide targeting the vascular endothelial growth factor receptor 2 (VEGFR2), was evaluated in vitro and in vivo, demonstrating its potential for specific tumor detection. MATERIALS AND METHODS: The targeted contrast agent was prepared by incorporation of a biospecific lipopeptide into the microbubble membrane. Experiments were performed in vitro to demonstrate the binding capacities of BR55 microbubbles on immobilized receptor proteins and on various endothelial or transfected cells expressing VEGFR2. The performance of BR55 microbubbles was compared with that of streptavidin-conjugated microbubbles targeted to the same receptor by coupling them to a biotinylated antibody. The specificity of BR55 binding to human and mouse endothelial cells was determined in competition experiments with the free lipopeptide, vascular endothelial growth factor (VEGF), or a VEGFR2-specific antibody. Molecular ultrasound imaging of VEGFR2 was performed in an orthotopic breast tumor model in rats using a nondestructive, contrast-specific imaging mode. RESULTS: BR55 was shown to bind specifically to the immobilized recombinant VEGFR2 under flow (dynamic conditions). BR55 accumulation on the target over time was similar to that of microbubbles bearing a specific antibody. BR55 avidly bound to cells expressing VEGFR2, and the pattern of microbubble distribution was correlated with the pattern of receptor expression determined by immunocytochemistry. The binding of targeted microbubbles on cells was competed off by an excess of free lipopeptide, the natural ligand (VEGF) and by a VEGFR2-specific antibody (P < 0.001). Although selected for the human receptor, the VEGFR2-binding lipopeptide was also shown to recognize the rodent receptor. Tumor perfusion was assessed during the vascular phase of BR55, and then the malignant lesion was highlighted by specific accumulation of the targeted microbubbles on tumoral endothelium. The presence of VEGFR2 was confirmed by immunofluorescence staining of tumor cryosections. CONCLUSIONS: VEGFR2-targeted ultrasound contrast agents such as BR55 will likely prove useful in human for the early detection of tumors as well as for the assessment of response to specific treatments.

Molecular imaging of human thrombus with novel abciximab immunobubbles and ultrasound.

BACKGROUND AND PURPOSE: Molecular imaging of therapeutic interventions with targeted agents that simultaneously carry drugs or genes for local delivery is appealing. We investigated the ability of a novel microbubble carrier (immunobubble) for abciximab, a glycoprotein IIb/IIIa receptor inhibitor, for ultrasonographic molecular imaging of human clots. METHODS: Human thrombi were incubated with immunobubbles conjugated with abciximab. Control clots were incubated in either saline or with immunobubbles conjugated with nonspecific antibody. We evaluated immunobubble suspensions with variable concentrations of encapsulated gas and measured mean acoustic intensity of the incubated clots. In vivo molecular imaging of human thrombi with abciximab immunobubbles was evaluated in a rat model of carotid artery occlusion. RESULTS: Mean acoustic intensity was significantly higher for abciximab immunobubbles as compared with control immunobubbles under all conditions tested with maximum difference in intensity at a gas volume of 0.2 microL (P=0.0013 for mechanical index 0.05, P=0.0001 for mechanical index 0.7). Binding of abciximab immunobubbles to clots in vitro led to enhanced echogenicity dependent on bubble concentration. In vivo ultrasonic detectability of carotid thrombi was significantly higher for clots targeted with abciximab immunobubbles (P<0.05). Quantification of in vivo contrast enhancement displayed a highly significant increment for abciximab immunobubble-targeted clots compared with nonspecific immunobubble-targeted clots (P<0.0001) and to native clots (P<0.0001). CONCLUSIONS: This study demonstrates the feasibility of using a therapeutic agent for selective targeting in vascular imaging. Abciximab immunobubbles improve visualization of human clots both in vitro and in an in vivo model of acute arterial thrombotic occlusion.

Tuftsin binds neuropilin-1 through a sequence similar to that encoded by exon 8 of vascular endothelial growth factor.

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF(165)-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.

Gd-BOPTA transport into rat hepatocytes: pharmacokinetic analysis of dynamic magnetic resonance images using a hollow-fiber bioreactor.

RATIONALE AND OBJECTIVES: To investigate the transport of the hepatobiliary magnetic resonance (MR) imaging contrast agent Gd-BOPTA into rat hepatocytes. MATERIALS AND METHODS: In a MR-compatible hollow-fiber bioreactor containing hepatocytes, MR signal intensity was measured over time during the perfusion of Gd-BOPTA. For comparison, the perfusion of an extracellular contrast agent (Gd-DTPA) was also studied. A compartmental pharmacokinetic model was developed to describe dynamic signal intensity-time curves. RESULTS: The dynamic signal intensity-time curves of the hepatocyte hollow-fiber bioreactor during Gd-BOPTA perfusion were adequately fitted by 2 compartmental models. Modeling permitted to discriminate between the behaviors of the extracellular contrast agent (Gd-DTPA) and the hepatobiliary contrast agent (Gd-BOPTA). It allowed the successfully quantification of the parameters involved in such differences. Gd-BOPTA uptake was saturable at high substrate concentrations. CONCLUSIONS: The transport of Gd-BOPTA into rat hepatocytes was successfully described by compartmental analysis of the signal intensity recorded over time and supported the hypothesis of a transporter-mediated uptake.

Hollow fiber bioreactor: new development for the study of contrast agent transport into hepatocytes by magnetic resonance imaging.

The aim of our study was to develop a magnetic resonance (MR)-compatible in vitro model containing freshly isolated rat hepatocytes to study the transport of hepatobiliary contrast agents (CA) by MR imaging (MRI). We set up a perfusion system including a perfusion circuit, a heating device, an oxygenator, and a hollow fiber bioreactor (HFB). The role of the porosity and surface of the hollow fiber (HF) as well as the perfusate flow rate applied on the diffusion of CAs and O2 was determined. Hepatocytes were isolated and injected in the extracapillary space of the HFB (4 x 10(7) cells/mL). The hepatocyte HFB was perfused with an extracellular CA, gadopentetate dimeglumine (Gd-DTPA), and gadobenate dimeglumine (Gd-BOPTA), which also enters into hepatocytes. The HFB was imaged in the MR room using a dynamic T1-weighed sequence. No adsorption of CAs was detected in the perfusion system without hepatocytes. The use of a membrane with a high porosity (0.5 microm) and surface (420 cm2), and a high flow rate perfusion (100 mL/min) resulted in a rapid filling of the HFB with CAs. The cellular viability of hepatocytes in the HFB was greater than 85% and the O2 consumption was maintained over the experimental period. The kinetics of MR signal intensity (SI) clearly showed the different behavior of Gd-BOPTA that enters into hepatocytes and Gd-DTPA that remains extracellular. Thus, these results show that our newly developed in vitro model is an interesting tool to investigate the transport kinetics of hepatobiliary CAs by measuring the MR SI over time.

Kinetics of gadobenate dimeglumine in isolated perfused rat liver: MR imaging evaluation.

PURPOSE: To compare in the entire liver, the hepatic kinetics of gadobenate dimeglumine (Gd-BOPTA) and gadopentetate dimeglumine (Gd-DTPA) and to evaluate the hepatic transport of Gd-BOPTA. MATERIALS AND METHODS: The authors studied both contrast agents in isolated perfused rat livers by measuring the magnetic resonance (MR) signal intensity (SI) in 12 rats, as well as the gadolinium concentrations in hepatic tissues in 42 rats. The intrahepatic transport of Gd-BOPTA was investigated with pharmacologic antagonism by using bromosulfophthalein. MR imaging was performed at 1.5 T with a fast gradient-echo T1-weighted MR sequence. RESULTS: The hepatic kinetics based on the MR SI measured over time showed a rapid steady state during Gd-DTPA perfusion, while the SI continuously increased during the 30-minute Gd-BOPTA perfusion period. The pharmacokinetic modeling indicated that the half-lives of Gd-DTPA entry and exit were identical (mean, 1.3 minutes +/- 0.9 [standard error of mean]) and shorter than those observed with Gd-BOPTA (P <.001). The uptake of Gd-BOPTA was faster (mean half-life, 4.8 minutes +/- 0.3) than the washout (mean half-life, 17.5 minutes +/- 2.8) (P =.001). The combined perfusion of bromosulfophthalein and Gd-BOPTA decreased the SI enhancement in comparison with the perfusion of Gd-BOPTA alone (mean, 0.56 +/- 0.03 vs 2.54 +/- 0.39, P <.001). The entry and exit kinetic parameters obtained during the perfusion of Gd-BOPTA plus bromosulfophthalein were identical and comparable to those obtained during Gd-DTPA perfusion (P =.95). Acute bile duct ligation did not interfere with the uptake of Gd-BOPTA in hepatocytes, but it slowed down the excretion by approximately 50%. Measurements of gadolinium concentrations in hepatic tissues confirmed these findings. CONCLUSION: In the liver, the hepatospecific contrast agent Gd-BOPTA enters into hepatocytes likely through the organic anion transporting peptide 1.