Laboratory Investigations

The Effect of Benzamide Riboside on the VX2 Model of Liver Cancer in Rabbits

Gordon McLennan, MD, Erik N.K. Cressman, MD, Yonghua Xu, MD, Dianbo Zhang, MD, Mandar R. Jagtap, BS, and Hiremagular N. Jayaram, PhD

PURPOSE: Benzamide riboside (BR) causes apoptosis in multiple tumor cell lines by its inhibition of guanylate biosynthesis. The purpose of this study was to determine the feasibility of the use of BR as a therapeutic agent for hepatic artery infusional cancer therapy in a rabbit VX2 papilloma tumor model.

MATERIALS AND METHODS: VX2 tumor was implanted into the left lobe of the liver of each of 14 New Zealand White rabbits and allowed to grow for 19 days � 3. The proper hepatic artery was selected with a 3-F catheter via right femoral cutdown. The animals were treated with one infusion of 0.9% saline solution (n � 2), 1 mg/kg BR (n � 4), 5 mg/kg BR (n � 4), or 10 mg/kg BR (n � 4). One animal treated with 5 mg/kg BR did not develop tumor. Livers were explanted after 24 hours and sectioned through the tumor. Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) staining was performed on the slides and they were imaged at a magnification of 40 to detect apoptotic cells. Four random fields were obtained from each slide and the percentage of apoptotic cells was calculated by dividing the number of TUNEL-positive cells by the total number of cells. Sections of liver not involved with tumor were seen in five animals: two that received 1 mg/kg BR, one that received 5 mg/kg, and two that received 10 mg/kg.

RESULTS: The mean tumor apoptosis rates were 1.3% with saline solution treatment, 44.8% with 1 mg/kg BR, 52.7% with 5 mg/kg BR, and 70.7% with 10 mg/kg BR. The mean tumor apoptosis in treated animals was significantly greater than in control animals (P � .003) and mean tumor apoptosis was significantly greater with 10 mg/kg BR than with 1 mg/kg BR (P � .03). There were no apoptotic cells in normal liver treated with 1 mg/kg BR or 10 mg/kg BR. The animal that received 5 mg/kg BR exhibited 10.5% apoptotic cells in the field examined (eight of 76 cells). In the animal treated with 5 mg/kg BR but in which tumor did not grow, only one of 76 cells (0.65%) was apoptotic in the area of the injection scar.

CONCLUSION: BR induces apoptosis in VX2 tumor in the rabbit model with minimal apoptosis in normal liver.
J Vasc Interv Radiol 2005; 16:1499–1504

Abbreviations: BR � benzamide riboside, TUNEL � terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling

HEPATOCELLULAR carcinoma (1–3) than 5% (4). Key factors are alcoholic suitable candidates (4 – 6). Although and liver metastases from colon cancer cirrhosis and Hepatitis B and Hepatitis systemic therapeutic agents such as are a significant and increasing prob-C viruses. Colorectal carcinoma is the irinotecan and oxaliplatin have lem in North America. Primary liver third most common cancer, killing achieved a 2–3-month improvement in cancer and intrahepatic biliary ductal 56,000 people per year, most with met-survival for patients with colorectal carcinoma have an annual incidence of astatic disease to the liver. Surgical re-metastases (7,8), the same has not been

5.25 per 100,000 persons and the section can be curative, but fewer than seen with hepatocellular carcinoma

5-year survival rate is dismal at less 20% of patients with liver disease are (9). Therefore, there is a large group of patients with liver cancer with no satisfactory treatment available.

Arterial chemoembolization with

From the Department of Radiology (G.M., E.C., Y.X., D.Z., M.J.), Indiana University Medical Center; and Department of Biochemistry (H.N.J.), Indiana University/Purdue University at Indianapolis, Indianapolis, Indiana. Received November 12, 2004; accepted August 27, 2005. From the SIR 2005 Annual Meeting. Address correspondence to G.M., Department of Radiology, University Hospital Room 0279, 550 N. University Blvd., Indianapolis, IN 46202-5253; E-mail: gmclenna@iupui.edu

This research project was funded by a pilot grant-from the Society of Interventional Radiology Foundation. G.M. has received grant support from Arrow International, Bard Inc., and Genentech, Inc. H.N.J. is the patent holder of the use of BR to induce apoptosis (U.S. Patent No. 5,902,792) and US Patent Application No. 20030148966 (10/347990). None of the other authors have identified a conflict of interest.

© SIR, 2005

DOI: 10.1097/01.RVI.0000185416.08458.01

doxorubicin, cisplatin, and/or mitomycin C results in destruction of healthy tissue along with tumor (10,11). Yttrium 90 microspheres are currently being investigated (12,13), but distal embolization in the pulmonary vascular bed and the attendant costs and hazards of radioisotopes must be considered. A new approach involves targeting of tumor cell mitochondria in light of the high glycolytic rate of many neoplasms, but the active compound is somewhat unstable chemically as a result of the reactive nature of the alkylating agent (14). Treatment is often undertaken in the setting of substantial underlying liver disease, so preservation of noncancerous tissue is important. Local infusion with a compound that is selective for tumor cells and preferentially induces apoptosis would therefore represent an improvement over current or experimental therapies.

Benzamide riboside (BR) is an oncolytic agent that inhibits guanylate synthesis through its inhibition of inosine monophosphate dehydrogenase. The resulting depletion of guanylates prevents normal DNA replication and causes apoptosis in a variety of cancer cell lines (15,16). Because BR is metabolized in normal hepatocytes by carboxylation to an inactive benzene carboxylic acid riboside (17), we hypothesize that BR will have selective toxicity in liver tumors while preserving normal hepatocytes.

The purpose of this study was to evaluate the feasibility of inducing tumor apoptosis within 24 hours of administration of BR into the hepatic artery in a VX2 tumor model of liver cancer. Apoptosis in nontumorous liver tissue was also evaluated to look for evidence of hepatic toxicity.

MATERIALS AND METHODS

All procedures were approved by the institutional animal care and use committee. All animal procedures complied with the Guide for the Care and Use of Laboratory Animals. Animals were sedated with 44 mg of ketamine and 6 mg of xylazine hydrochloride (Phoenix Medical, St. Joseph, MO) injected intramuscularly. Anesthesia was induced with 0.15 mL/kg of intramuscular acepromazine and butorphanol tartrate (Amvet, Lexington, KY) and maintained with 1%–3% isoflurane (Halocarbon Laboratories, River Edge, NJ) gas anesthesia. During the procedure, animals were monitored with electrocardiography. Respiratory rate was recorded every 10 minutes and pupils were checked every 10 minutes to assess depth of anesthesia. Before recovery from anesthesia, all animals received 0.02–0.04 mg/kg of subcutaneous buprenorphine (Reckitt Benckiser, Richmond, VA) for analgesia in the postoperative period. Animals were housed in the laboratory animal resource center after recovery to the sternal position overnight and euthanized the next day with an overdose of sodium pentothal. Death was assured with bilateral pneumothorax.

Experimental Design

VX2 tumor (provided by John Hilton, PhD, of Johns Hopkins University, Baltimore, MD) was implanted in the thighs of four New Zealand White rabbits. When the tumors were approximately 2 cm in diameter, they were harvested, homogenized, and implanted with an 18-gauge Angiocath (Becton Dickinson, Sandy, UT) into the exposed left lobes of the livers of 14 additional female New Zealand White rabbits (weight 4.7 kg � 0.2) and allowed to grow for 19 days � 3. The right common femoral artery was then exposed via cutdown and a 3-F angled catheter (Cook, Bloomington, IN) was used to selectively catheterize the hepatic artery with use of a 0.025inch Glidewire (Boston Scientific, Natick, MA). Hepatic arteriography was performed (Fig 1) and the animals were treated once with an infusion of 0.9% saline solution (n � 2), 1 mg/kg BR (n � 4), 5 mg/kg BR (n � 4), or 10 mg/kg BR (n � 4) into the proper hepatic artery. The drug was prepared in a 0.5-mL volume and administered by hand injection over 60 seconds. One animal in the group treated with 5 mg/kg BR was found to have no tumor at the time of euthanasia. The livers were explanted after 24 hours. A gross image of the tumor (Fig 2) was obtained and an approximate measurement of the tumor diameter was made with the ruler on a scalpel handle. Explanted livers were fixed in a 70% ethanol solution for 24 hours and then transferred to a 30% sucrose solution for 3 days. Tissue was then mounted in OTC freezing medium (Fisher Scientific, Fair Lawn, NJ) and frozen sections were obtained with a cryotome set for 10-�m sections. Sections were mounted on glass slides and stored at �20°C. Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) staining (Pro-mega, Madison, WI) was performed on the slides according to the manufacturer’s instructions (18) and imaged on a Zeiss LSM 510 two-photon confocal microscope (Zeiss, Oberkochen, Germany) equipped with UV, argon, and helium lasers at a magnification of 40 to detect apoptotic cells (Fig 3). The cells were stained with DAPI (4,6-dia

=

midino-2-phenylindole; Sigma Aldrich, St. Louis, MO), to allow identification of all cells. Four random fields were obtained from each slide and the percentage of apoptotic cells was calculated by dividing the number of TUNEL-positive cells by the total number of cells stained with DAPI. Sections of liver not involved with tumor were sectioned and stained for TUNEL in five animals: two that received 1 mg/kg BR, 1 that received 5 mg/kg, and two that received 10 mg/ kg.

Study Endpoints

The study was designed to determine the apoptosis induced in liver cancer 24 hours after treatment. The primary endpoint was therefore the percentage of apoptosis in tumor as determined by TUNEL assay. The percentage of apoptosis in normal livers was calculated as a secondary control and is reported here as descriptive data.

Statistical Analysis

The percentage of apoptosis was compared among dosages with use of one-way analysis of variance. Differences between groups were analyzed with the Fisher least significant difference test. The mean tumor volume and the percentage of apoptosis in normal liver specimens are reported for descriptive purposes only.

RESULTS

There were no adverse effects of the procedure or drug administration in the 24-hour follow-up period. Mean tumor volume was 14.8 mm3 � 20.8, with tumors ranging from 0.5 mm to 5 mm in diameter.

The mean tumor apoptosis measurements were 1.3% in the saline solution group, 44.8% with 1 mg/kg BR, 52.7% with 5 mg/kg BR, and 70.7% with 10 mg/kg BR (Fig 4). There were no apoptotic cells in sections of normal liver treated with 1 mg/kg BR and 10 mg/kg BR. The normal liver of the animal that received 5 mg/kg BR demonstrated 10.5% apoptotic cells in the field examined (eight of 76 cells). In the animal treated with 5 mg/kg BR that did not develop tumor, only one of 76 cells (0.65%) were apoptotic in the area of the injection scar. This result was not included in the mean tumor apoptosis measurements reported earlier.

Results from a one-way analysis of variance model showed strong evidence of dose effect. In particular, the Fisher least significant difference post hoc test showed that the mean tumor apoptosis measurements in treated animals were significantly greater than in animals treated with saline solution (P � .003) and the mean tumor apoptosis was greater with 10 mg/kg BR than with 1 mg/kg BR (P � .03).

DISCUSSION

Transcatheter therapy and ablative techniques have become an important component in the treatment of hepatocellular carcinoma and liver metastases, with prospective randomized trials demonstrating improved survival with transarterial chemoembolization (19,20). The drugs used for transarterial chemoembolization have largely been chosen on the basis of what was available from systemic oncology practice (10,11). As our understanding of tumor biology has advanced, new pharmaceutical agents have been developed that provide fewer side effects and greater tumor specificity.

The major advantage of transarterial therapy is that higher doses of chemotherapy agents can be delivered to the vasculature that supplies the tumor. This is particularly helpful when the drug has significant side effects. When the toxicity profile is better, higher systemic doses can be delivered, but even in this case, a higher concentration of drug can, in theory, be delivered to the artery supplying tumor. This could lead to more rapid cell kill and may allow curative treatment of the cancer.

Inhibitors of inosine monophosphate dehydrogenase, such as BR and tiazofurin, are promising candidates for therapy. These compounds block the rate-limiting step in guanylate biosynthesis, depleting pools of guanosine triphosphate and thereby inhibiting DNA synthesis and cell replication (15,16). They also induce apoptosis in human myelogenous leukemia K562 cells (21), promyelocytic leukemia HL-60 cells (22), and N.1 ovarian carcinoma cells (22) at micromolar concentrations in culture when metabolized to the dinucleotide analogue of nicotinamide adenine dinucleotide (23). Selenazofurin, thiophenfurin, and imidazofurin have been synthesized, but limited in vitro data and no animal data are available for these three analogues. BR has activity against other solid tumors such as HCT-15 colon cancer cells, several central nervous system tumor lines, RXF

Figure 3. TUNEL assay of liver tumors from animals treated with saline solution (a), 1 mg/kg BR (b), 5 mg/kg BR (c), and 10 mg/kg BR (d). Microscopy was performed at a magnification of 40X. The green cells are positive for the TUNEL assay and indicate that the cells are apoptotic. All cells are stained with the blue DAPI stain.

393 renal carcinoma, and M14 mela-tiazofurin (24), which is currently in BR is primarily metabolized in nor-noma. It has shown three-fold greater phase III clinical trials. In vivo antitu-mal liver cells to inactive metabolites inhibitory activity against inosine mor and curative activity of BR (17). Therefore, the potential therapeumonophosphate dehydrogenase and against murine leukemia L1210 lines tic effect of delivering the drug into increased apoptosis compared with has been demonstrated as well (25). the hepatic artery is that the tumor cells would be affected and normal hepatocytes would be spared. The primary toxicity is hindlimb paralysis related to depletion of nicotinamide adenine dinucleotide and is seen at doses of 20 mg/kg in mice (26). In this study, we evaluated the intraarterial administration of doses of 10 mg/kg and lower.

The results of this study clearly show increased apoptosis with each of the doses used compared with controls (Figs 3,4). Sham treatment with saline solution demonstrated only minimal tumor apoptosis. Although the sample size was not large enough to demonstrate a clear dose response, there was a significantly higher percentage of apoptosis with 10 mg/kg BR compared with 1 mg/kg and there was an average of increased apoptosis with increased doses.

Specimens without tumor demonstrated less apoptosis than specimens with tumor, with the highest percentage seen in one specimen treated with 5 mg/kg BR. In this animal, the normal liver had 10.5% apoptosis whereas the tumor had 52.1% apoptosis. The two animals treated with 1 mg/kg BR and the two animals treated with 10 mg/kg BR in which the normal liver was examined exhibited no apoptosis in normal liver sections. These findings suggest minimal effects on the normal liver.

Because this study was a pilot study to assess the feasibility of the use of BR as an intraarterial therapeutic agent, the sample size was small and the focus was on the intraarterial use of the drug. The small sample size limited our ability to demonstrate a dose–response ratio, even though there was clearly an increase in mean apoptosis with increasing doses and tumor apoptosis with 1 mg/kg BR was significantly less than with 10 mg/kg. Also, the study did not include a control for systemic administration of BR or a control for standard chemoembolization techniques, embolization, or administration of BR with embolization. In addition, when we looked at normal liver, we found apoptosis in the liver of only one animal that received 5 mg/kg of BR and no apoptosis with 1 mg/kg or 10 mg/kg BR. It is unclear given the limited sample size whether this is a result of sampling error or processing error with the specimen treated with 5 mg/kg BR. Multiple sections of normal liver will need to be obtained in future studies to determine if the drug has any apoptotic effect on normal liver. Finally, the study was limited to the acute effects of BR in the first 24 hours after treatment. Although each of these limitations will be important to address in subsequent studies, they were beyond the scope of this study. Further study will need to include larger sample sizes based on the variability defined in this study. In addition, we will need to include control groups to look at systemic therapy and the effect of embolization. When these issues are dealt with, it will be necessary to examine the long-term effects of therapy to see if a single treatment can cause tumor regression or if multiple treatments are needed.

Despite the limited scope of this study, we clearly demonstrated that BR, at doses as high as 10 mg/kg, induces apoptosis in intrahepatic VX2 tumors with relative preservation of normal liver.

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