Zuxing Kan

Biodistribution of paclitaxel and poly(L-glutamic acid)-paclitaxel conjugate in mice with ovarian OCa-1 tumor

Purpose: Poly(l-glutamic acid)-paclitaxel (PG-TXL) is a water-soluble paclitaxel (TXL) conjugate made by conjugating TXL to poly(l-glutamic acid) via ester bonds. In preclinical studies, PG-TXL has shown significant antitumor activity against a variety of solid tumors. To elucidate the relationship between tissue distribution and antitumor efficacy of PG-TXL, we studied and compared the biodistribution of PG-TXL and TXL. Methods: Female C3Hf/Kam mice bearing syngeneic ovarian OCa-1 tumors were injected with either [3H]TXL or PG-[3H]TXL at an equivalent TXL dose of 20 mg/kg. Mice were killed at various times after drug injection, and samples of blood, spleen, liver, kidney, lung, heart, muscle, brain, fat, and tumor were removed and the radioactivity counted. In addition, concentrations of free [3H]TXL released from PG-[3H]TXL in the spleen, liver, kidney, and tumor were analyzed by using high-performance liquid chromatography (HPLC). Whole-body autoradiographs of mice killed 1 day and 6 days after administration of PG-[3H]TXL were obtained to study the intratumoral distribution of PG-TXL. Results: When [3H]TXL was conjugated to polymer, the biodistribution pattern of PG-[3H]TXL differed from that of [3H]TXL. Based on area under the tissue concentration-time curve (AUC) values, tumor exposure to [3H]TXL was five times greater when administered as PG-TXL than as TXL formulated in Cremophor EL/alcohol vehicle. Furthermore, concentrations of free paclitaxel released from PG-[3H]TXL remained relatively constant in tumor tissue, being 489, 949 and 552 ng/g tumor tissue at 5, 48 and 144 h after dosing, respectively. Autoradiographic images of mice injected with PG-[3H]TXL revealed that radioactivity was primarily located in the periphery of the tumor on day 1 after drug administration and was homogeneously diffused into the center of the tumor by day 6. Over the 144-h study period, [3H]TXL concentrations, predominantly as the inactive conjugate, were higher in tissues with a more abundant reticular endothelial system (i.e. liver, kidney, spleen, lung) than in tissues with less abundant or lacking RE systems (i.e. muscle, fat, brain). Both [3H]TXL and PG-[3H]TXL were excreted primarily through the hepatobiliary route, with a small fraction of each drug (5% and 8.7%, respectively) excreted into the urine within 48 h. Conclusions: This study indicates that the distribution to tumor tissue was enhanced when [3H]TXL was administered as a macromolecular conjugate, and that free TXL was released and maintained within the tumor for a prolonged period. Thus, the antitumor activity of PG-TXL observed in preclinical studies may be attributed in part to enhanced tumor uptake of PG-TXL.

Effect of transcatheter hepatic arterial embolization on angiogenesis in an animal model

Objectives:Transcatheter arterial embolization (TAE) is used for the treatment of patients with malignant liver tumors. However, the proangiogenesis effect of TAE-associated hypoxia has not been adequately studied. The goal of this study was to determine angiogenic activity in tumors subjected to TAE by evaluating the tumor microvessel density (MVD). Materials and Methods:Mammary cancer 13762 NF tumor cells were inoculated into the livers of male Sprague-Dawley rats. TAE was performed 12–14 days after tumor inoculation. Rats were divided into 4 groups on the basis of treatment type. Control group animals (n = 16) were subjected to sham TAE without polyvinyl alcohol (PVA) particles. Animals in the other 3 groups were subjected to TAE with 1 (n = 11), 2 (n = 8), or 3 (n = 10) mg of PVA particles. Rats were killed 3–6 hours or 2 or 3 days after embolization, and the liver tumor tissues were dissected and frozen in liquid nitrogen. Tumor tissue slides were prepared, stained with CD-31, and evaluated for MVD. Blood samples collected just before sacrificing the animals were used to measure serum vascular endothelial growth factor (VEGF) levels. Results:Tumors treated with TAE showed varying degrees of central necrosis with residual viable tumor cells in the periphery. Tumor MVD in animals treated with TAE was significantly higher than that in the control group (23.6 versus 17.5; P = 0.001). Although the MVD in animals treated with TAE using 1 mg of PVA was higher than that in the control group, this difference was not statistically significant. TAE using 2 mg of PVA resulted in a significant increase in tumor MVD (25.9 versus 17.5; P = 0.007). Use of 3 mg of PVA did not result in any further increase in MVD. There was a significant increase in tumor MVD in the animals killed 2 or 3 days after TAE compared with the control group (24.5 versus 17.5; P = 0.002). The animals treated with TAE showed a statistically significant increase in VEGF levels compared with the control group. Conclusions:TAE of hepatic tumors results in the stimulation of angiogenesis in the residual viable tumor, which could have an adverse effect on the therapeutic efficacy of TAE.

Quantification of angiogenesis by functional computed tomography in a matrigel model in rats1

RATIONALE AND OBJECTIVES The aim was to evaluate functional computed tomography (fCT) in the quantification of angiogenesis by comparing the tissue perfusion parameters measured by CT perfusion (CTP) software with histologic vascular parameters in a Matrigel model in rats. It was hypothesized that tissue perfusion parameters and histologic vascular parameters are related. MATERIALS AND METHODS In vivo angiogenesis assays were performed using Matrigel supplemented with escalating doses (0 ng [control group], 250 ng, and 1,000 ng) of recombinant rat vascular endothelial growth factor (VEGF164) subcutaneously injected into the backs of Sprague Dawley rats. On day 7, rats with Matrigel plug underwent fCT following a bolus injection of iodinated contrast medium. Using CTP software, fCT parameters were generated (blood flow [BF], blood volume [BV], mean transit time, and permeability-surface area product) and functional maps on the basis of a distributed parameter tracer kinetic model, the adiabatic approximation to the tissue homogeneity model. The animals were then sacrificed. Matrigel plug was sectioned into slices corresponding to the CT scan plane and stained with CD31 immunohistochemical stain. Histologic vascular parameters, including microvascular density (MVD), vessel number (VN), vascular area, and vascular perimeter, were measured. CTP and histologic parameters were correlated. RESULTS The Matrigel plugs with the 1,000-ng VEGF group exhibited a higher MVD than the 250-ng VEGF and control groups (P < .05). VN differed significantly between the control versus the 250-ng VEGF groups and 250-ng versus 1,000-ng VEGF groups (P < .05), with the highest VN in the 250-ng VEGF group. BF, mean transit time, and permeability-surface area product each differed significantly to VEGF levels. Changes in BF and BV did not correspond with increases in MVD or VN; however, in the 250-ng VEGF group, there was a strong positive correlation (r = 0.9) between BV and VN, vascular area, and vascular perimeter, which was not seen in the control or 1,000-ng VEGF group. All fCT parameters significantly correlated with each other (P < .05), with strong correlations between BF and mean transit time (r = -0.7) and between BF and permeability-surface area product (r = 0.7) and a weak correlation between BF and BV (r = 0.3). CONCLUSION These results validate the VEGF-induced endothelial cell in a rat Matrigel model. In addition, histologic vascular parameter MVD does not correlate with fCT parameters measured by CTP software.

Role of Kupffer cells in iodized oil embolization.

RATIONALE AND OBJECTIVES.Iodized oil is a common oily embolic agent used in chemoembolization for treating hepatic tumors. However, how the iodized oil is cleared from the liver has been an unsettled and controversial issue. In this study, the authors attempt to clarify whether Kupffer cells are involved in the clearance of iodized oil and to evaluate the effect of hepatic arterial injection of iodized oil on the functional status of Kupffer cells. METHODS.Iodized oil was injected into the proper hepatic artery in 42 Fischer 344 rats. In vivo microscopy was performed immediately after and 1,3,7,15,30, and 60 days after injection. Electron microscopy was performed after in vivo microscopy. RESULTS.Kupffer cells actively captured and phagocytosed iodized oil droplets in the hepatic circulation. The number and functional status of Kupffer cells in the liver were significantly increased after the injection of the iodized oil and returned to normal when the liver was cleared of the oil. CONCLUSIONS.Kupffer cells play an important role in clearing iodized oil from the liver. Iodized oil activates the immune defense system in the liver, which may have a synergistic effect in tumor treatment.

Peribiliary plexa--important pathways for shunting of iodized oil and silicon rubber solution from the hepatic artery to the portal vein. An experimental study in rats.

OBJECTIVES.Iodized oil is commonly used in chemoembolization of hepatic tumors, and silicon rubber solution is used for casting studies of hepatic tumor vasculature. Understanding the distribution patterns of iodized oil and silicon rubber solution is of significance in the refinement of iodized oil techniques and proper interpretation of hepatic tumor vascular studies. In this study, the location for iodized oil and silicon rubber solution shunting from the hepatic artery to the portal vein was identified. METHODS.Iodized oil and silicone rubber solution were injected into the hepatic artery in rats. The porta hepatis and the liver periphery were examined using in vivo microscopy. RESULTS.Iodized oil and silicone rubber solution had identical distribution patterns in the hepatic circulation. Both were shunted in large quantities from the hepatic artery into the portal vein through the peribiliary plexa. Other potential shunting sites did not contribute to the shunting. CONCLUSIONS.Though of different chemical natures, iodized oil and silicon rubber solution share similar distribution patterns in the liver. Hepatic arterioportal shunting of these substances occurs via the peribiliary plexa.

Sinusoidal Embolization: Impact of Iodized Oil on Hepatic Microcirculation

PURPOSE To determine the effects of iodized oil, which is used in chemoembolization of liver cancer, on hepatic microcirculation and to measure the time required for recovery of microcirculation. MATERIALS AND METHODS Iodized oil was injected in 0.1-, 0.2-, and 0.4-mL/kg doses into the hepatic artery in three groups of rats (n = 63). In vivo microscopy was performed during and immediately after the procedure and on days 1, 3, 7, 15, 30, and 60. A control group of rats underwent identical microscopy procedures. RESULTS Changes in microcirculation occurred after injections with iodized oil. Oil injected into the hepatic artery entered the portal vein and flowed into the sinusoids to create an incomplete sinusoidal embolization. Recovery of the sinusoidal circulation occurred 3, 7, and 30 days after injections of 0.1, 0.2, and 0.4 mL/kg of oil, respectively. CONCLUSIONS The liver tolerated oil embolization well. Despite the changes in microcirculation and a nonlinear recovery time, the microcirculation completely recovered, even with a 0.4-mL/kg dose.

Biodistribution of Cyclic Carbonate of Ioxilan: A Radiopaque Particulate Macrophage Imaging Agent

RATIONALE AND OBJECTIVES A biodegradable radiopaque particulate contrast agent formulated from cyclic carbonate of ioxilan (IXC), which is a prodrug of nonionic water-solubel contrast ioxilan, recently has been developed. This contrast agent enhances liver attenuation and is cleared from the body as ioxilan. In the current study, we tested whether the biodistribution of IXC particles would be affected by the characteristics of particles. METHODS IXC nanoparticles (average diameter = 290 nm) and IXC microparticles (average diameter = 1.7 mm) were prepared, characterized, and injected intravenously (i.v.; 50 mg I/kg body weight) into rats. Two sensitive, reproducible analytic methods--inductively coupled plasma-mass spectrometry (ICP-MS) and high-performance liquid chromatography (HPLC)- were used to quantify tissue iodine and ioxilan concentrations. RESULTS Both IXC nanoparticles and microparticles were taken up in the liver and spleen. The IXC nanoparticles remained in the liver at high concentrations for 6 hr and were slowly cleared. They also gave a high blood iodine concentration in the first 5 min after i.v. injection, suggesting their potential use as a blood-pool imaging agent. Unlike the nanoparticles, the microparticles had a significantly lower uptake by the kidney. CONCLUSION Because of reduced renal uptake, microparticles are a preferred macrophage imaging agent. Biodegradable radiopaque particles may be used either as blood-pool imaging agents or as macrophage imaging agents depending on their size and distribution characteristics. The ICP-MS and HPLC methods are useful for biodistribution studies of iodinated contrast agents.

Preparation, characterization, and evaluation of ioxilan carbonate particles for computed tomography contrast enhancement of liver

RATIONALE AND OBJECTIVES.To prepare and characterize a new particulate contrast medium, cyclic carbonate of ioxilan (IX-C) particles, as a macrophage imaging agent for computed tomography (CT) enhancement of the liver. METHODS.Cyclic carbonate of ioxilan was synthesized from ioxilan, a nonionic water-soluble contrast agent. The IX-C particles prepared by a solvent extraction-evaporation method were characterized by size distribution, degradability, suspension stability, and median lethal dose. Pharmacokinetics of IXC particles and their effectiveness in enhancing liver attenuation and in detecting hepatic tumors were evaluated using normal and VX2-tumor-bearing rabbits. RESULTS.The IX-C particles were biodegradable, with ioxilan and carbon dioxide as the degradation products. The particles had an average size of 1 to 2 /im and were stable in saline suspension. The median lethal dose determined for IX-C particles was 2.6 and 3.1 g/kg body weight for female and male rabbits, respectively. A dose of 200 mg iodine/kg body weight caused an increase of 38 Hounsfield units in liver attenuation. In rabbits, hepatic clearance of the contrast medium occurred in 2 days. A tumor barely visible in precontrast scans could be detected after contrast injection. CONCLUSIONS.Development of particulate contrast medium from nonionic contrast agents represents a new approach. Ioxilan carbonate particles have suitable physicochemical properties that warrant further studies before clinical evaluation.

Perfusion CT Assessment of Tissue Hemodynamics Following Hepatic Arterial Infusion of Increasing Doses of Angiotensin II in a Rabbit Liver Tumor Model

PURPOSE To investigate the effects of increasing doses of angiotensin II on hepatic hemodynamics in the normal rabbit liver and in hepatic VX2 tumors by using dynamic contrast material-enhanced perfusion computed tomography (CT). MATERIALS AND METHODS This study was approved by the institutional animal care and use committee. Solitary hepatic VX2 tumors were implanted into 12 rabbits. In each animal, perfusion CT of the liver was performed before (at baseline) and after hepatic arterial infusion of varying doses (0.1-50.0 μg/mL) of angiotensin II. Images were acquired continuously for 80 seconds after the start of the intravenous contrast material administration. Blood flow (BF), blood volume (BV), mean transit time (MTT), and capillary permeability-surface area product were calculated for the tumor and the adjacent and distant normal liver tissue. Generalized linear mixed models were used to estimate the effects of angiotensin II dose on outcome measures. RESULTS Angiotensin II infusion increased contrast enhancement of the tumor and distal liver vessels. Tumor BF increased in a dose-dependent manner after administration of 0.5-25.0 μg/mL angiotensin II, but only the 2.5 μg/mL dose induced a significant increase in tumor BF compared with BF in the adjacent (68.0 vs 26.3 mL/min/100 g, P < .0001) and distant (68.0 vs 28.3 mL/min/100 g, P = .02) normal liver tissue. Tumor BV varied with angiotensin II dose but was greater than the BV of the adjacent and distant liver tissue at only the 2.5 μg/mL (4.8 vs 3.5 mL/100 g for adjacent liver [P < .0001], 4.8 vs 3.3 mL/100 g for distant liver [P = .0006]) and 10.0 μg/mL (4.9 vs 4.4 mL/100 g for adjacent liver [P = .007], 4.9 vs 4.3 mL/100 g for distant liver [P = .04]) doses. Tumor MTT was significantly shorter than the adjacent liver tissue MTT at angiotensin II doses of 2.5 μg/mL (9.7 vs 15.8 sec, P = .001) and 10.0 μg/mL (5.1 vs 13.2 sec, P = .007) and significantly shorter than the distant liver tissue MTT at 2.5 μg/mL only (9.7 vs 15.3 sec, P = .0006). The capillary permeability-surface area product for the tumor was higher than that for the adjacent liver tissue at the 2.5 μg/mL angiotensin II dose only (11.5 vs 8.1 mL/min/100 g, P = .01). CONCLUSION Perfusion CT enables a mechanistic understanding of angiotensin II infusion in the liver and derivation of the optimal effective dose. The 2.5 μg/mL angiotensin II dose increases perfusion in hepatic VX2 tumors versus that in adjacent and distant normal liver tissue primarily by constricting normal distal liver vessels and in turn increasing tumor BF and BV.

Hepatic microcirculatory changes induced by hepatic artery embolization in rats: original investigation.

RATIONALE AND OBJECTIVES To evaluate the effects of hepatic artery embolization (HAE), hepatic microcirculatory changes induced by HAE were assessed quantitatively in rats. METHODS Using in vivo microscopy, the blood-flow velocity (BFV) through terminal portal venules (TPVs) and terminal hepatic venules (THVs) was measured during HAE with gelatin sponge powder (GSP), iodized oil (Lipiodol, 0.1, 0.2, and 0.4 mL/kg), or 0.1 mL/kg Lipiodol followed by GSP. RESULTS After HAE with GSP, BFV through TPVs decreased significantly, but BFV through THVs did not decrease. After HAE with Lipiodol (0.2 and 0.4 mL/kg), BFV through TPVs decreased significantly, but BFV through THVs did not. After HAE with Lipiodol followed by GSP, BFV through both TPVs and THVs decreased significantly. CONCLUSIONS Neither GSP nor Lipiodol adversely affects hepatic microcirculation when administered alone; however, HAE with a combination of Lipiodol and GSP does adversely affect hepatic microcirculation.