ANTICANCER RESEARCH 33: 1989-2000 (2013) Evaluation of Melphalan, Oxaliplatin, and Paclitaxel in Colon, Liver, and Gastric Cancer Cell Lines in a Short-term Exposure Model of Chemosaturation Therapy by Percutaneous Hepatic Perfusion RAJNEESH P. UZGARE, TIMOTHY P. SHEETS and DANIEL S. JOHNSTON Pharmaceutical Research and Development, Delcath Systems, Inc., Queensbury, NY, U.S.A. Abstract. Background: The goal of this study was to candidates for use in the CS-PHP system to treat patients determine whether liver, gastric, or colonic cancer may be with gastric and colonic metastases, and primary cancer of suitable targets for chemosaturation therapy with the liver. percutaneous hepatic perfusion (CS-PHP) and to assess the feasibility of utilizing other cytotoxic agents besides Chemotherapeutic molecules exert beneficial clinical effects melphalan in the CS-PHP system. Materials and Methods: by inhibiting cell growth or by inducing cell death via Forty human cell lines were screened against three cytotoxic apoptosis. They can be divided into several categories based chemotherapeutic agents. Specifically, the dose-dependent on their mechanisms of action. The chemotherapeutic agent effect of melphalan, oxaliplatin, and paclitaxel on melphalan hydrochloride, which has been approved by the proliferation and apoptosis in each cell line was evaluated. US Food and Drug Administration and is used in the These agents were also evaluated for their ability to induce treatment of multiple myeloma and ovarian cancer, is a apoptosis in normal primary human hepatocytes. A high- derivative of nitrogen mustard that acts as a bifunctional dose short-term drug exposure protocol was employed to alkylating agent. Melphalan causes the alkylation of DNA at simulate conditions encountered during CS-PHP. Results: the N-7 position of guanine and the N-3 position of adenine The average concentration of melphalan required for (1). Thus, the binding of melphalan to DNA can result in inducing significant apoptosis was 61 μM, or about 3-fold cross-linking between bases, particularly G-G and G-A, on less than the theoretical concentration of 192 μM, achieved complementary strands, which leads to double-stranded DNA in the hepatic artery during CS-PHP dosing with melphalan. breaks and cell death through a caspase-mediated apoptotic Additionally, we found that gastric cancer cell lines were 2- pathway (2-5). Taxanes, such as paclitaxel, exert their effects 5 fold more sensitive to apoptosis than liver cancer cell lines by inhibiting microtubules (6, 7). Disruption of microtubules to all three compounds, suggesting that in addition to colonic during cell division leads to cell-cycle arrest and subsequent and gastric cancer metastases to the liver, primary gastric cell death by apoptosis. Paclitaxel has been used clinically cancer may also be amenable to management by CS-PHP in treatment regimens for a number of cancer types such as using an appropriate therapeutic agent. Significantly, at breast cancer and non–small-cell lung cancer (8). Platinum concentrations that are predicted using the CS-PHP system, agents, such as oxaliplatin, bind to and damage DNA, which these agents caused apoptosis of colonic, gastric, and liver leads to disruption of transcription and replication and to cancer cells but were not toxic to primary human cell-cycle arrest. If this DNA damage is not repaired, hepatocytes. Conclusion: The compounds tested are potential apoptosis will ultimately result (9). These agents comprise part of the treatment regimens for cancer such as colorectal, ovarian, and bladder cancer (10). At the doses required for clinical efficacy, chemotherapeutic Correspondence to: Rajneesh P. Uzgare, Ph.D., Director of agents are often associated with significant side effects. For Pharmaceutical Research, Delcath Systems, Inc., 566 Queensbury example, because these compounds target rapidly dividing Avenue, Queensbury, NY 12804, U.S.A. Tel: +1 5187438892 ext. 349, Fax: +1 5187430139, e-mail: [email protected] cells, they often induce myelosuppression, which contributes significantly to patient morbidity (11). Thus, a system that Key Words: Chemosaturation therapy, percutaneous hepatic enables the delivery of chemotherapeutic compounds perfusion, melphalan, taxanes, platinum agents. specifically to the organ of interest while reducing systemic 0250-7005/2013 $2.00+.40 1989 ANTICANCER RESEARCH 33: 1989-2000 (2013) exposure offers significant advantages. Isolated hepatic cytotoxicity assays, cell lines were grown in Roswell Park Memorial perfusion (IHP) and percutaneous hepatic perfusion (PHP) Institute 1640 medium (RPMI 1640) or Dulbecco’s modified offer this type of delivery system. Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 2 mM L-alanyl-L-glutamine, and 1 mM sodium pyruvate in a humidified The administration of melphalan via IHP and PHP has atmosphere of 5% CO at 37˚C. Cells were seeded into 384-well been studied for the treatment of liver cancer, as well as for 2 plates and incubated in 5% CO2 at 37˚C. metastases to the liver from colorectal cancer and metastatic Human primary hepatocytes (Invitrogen, Carlsbad, CA, USA) melanoma, among others (12-15). IHP permits intensification were thawed in hepatocyte culture medium with supplements and of chemotherapy to the liver, thereby improving efficacy growth factors [hepatocyte basal medium (HBM™) and hepatocyte while limiting extrahepatic toxicity. However, its efficacy is culture medium (HCM™) SingleQuots® from LONZA, restricted because the treatment cannot be repeated since Walkersville, MD, USA], treated with a Percoll (Sigma-Aldrich, St. Louis, MO, USA) separation step, and immediately seeded into 384- post-surgical intra-abdominal adhesions preclude further well collagen I-coated optical plates (BD Biosciences, San Jose, surgery. By contrast, chemosaturation therapy with PHP (CS- CA, USA). Cells were incubated at 37˚C in 5% CO2 for 24 h, after PHP), represented in Figure 1, is a repeatable percutaneous which the medium was exchanged and the cells were treated with procedure for the intra-arterial administration of melphalan test compounds. hydrochloride to the liver with extracorporeal filtration. The CS-PHP device is approved for clinical use in Europe where Cell treatment with test compounds. Melphalan, oxaliplatin, cases are being performed routinely in several countries. The paclitaxel and mitoxantrone were purchased from Sigma-Aldrich or proprietary CS-PHP system delivers melphalan to the liver Calbiochem (EMD Millipore, Billerica, MA, USA). For the via the proper hepatic artery and employs a double-balloon multiplexed cytotoxicity assays, compounds were serially diluted 3.16-fold and assayed over a range of 10 concentrations; the highest aspiration catheter in the retrohepatic inferior vena cava to and lowest concentration for each compound is specified in Tables isolate and collect hepatic venous outflow and direct the I-III. The final dimethyl sulfoxide (DMSO) concentration in the effluent to an extracorporeal circuit containing a proprietary assay did not exceed 0.1%. Compounds were added 24 h after cell hemofiltration system that reduces the concentration of seeding. At the same time, a time-zero untreated cell plate was melphalan in the blood dramatically and returns the filtered generated. The compound treatment period was 50 min. After blood to the systemic circulation through the jugular vein. incubation with a compound, the cells were spun, the supernatant Thus, when combined with an appropriate filtration system, was aspirated to remove the compound, and the medium was exchanged three times and cells incubated for an additional 72 h at this system could potentially be used to deliver other 37˚C. chemotherapeutic agents specifically to the liver. For primary hepatocytes, at 24 h after plating, the medium was In this study, we screened six gastric, seven liver, and 27 exchanged and compounds were added. Each plate of tested colon cancer cell lines for their sensitivity to melphalan, compounds included one reference compound (mitoxantrone) and oxaliplatin, and paclitaxel. We determined whether any of vehicle control. Compounds were serially diluted 3.16-fold and these agents effectively induces cell death of cancer cells in assayed over a range of 10 concentrations (0.008 μM - 250 μM for vitro and, thus, could be used with the CS-PHP device. This mitoxantrone) similar to those described for the cell lines. The final DMSO concentration in the assay did not exceed 0.5%. The compound study is notably relevant to clinical settings because of the treatment period was 1 or 4 h, followed by a wash-out. Fresh medium short time of exposure (50 min) of cells to high doses of the was added to the cells, and the cells were incubated for an additional compound of interest followed by a wash-out step to remove 17 or 14 h, respectively, at 37˚C prior to being assayed for an attached the agent from the cell milieu, simulating CS-PHP. A live count and for caspase-3 activation at 18 h. multiplex system was used to simultaneously measure cell proliferation and apoptosis. Finally, in order to address the Cell fixation and labeling. Following the final incubation step, the safety of these agents in a potential CS-PHP to treat liver cells were fixed and stained with fluorescently labeled antibodies metastases, we performed assays on primary human and nuclear dye to allow visualization of nuclei and apoptotic cells. hepatocytes and measured the relative attached cell count and Cells were fixed with paraformaldehyde