Supplementary Materials for

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1 Supplementary Materials for Local iontophoretic administration of cytotoxic therapies to solid tumors James D. Byrne,* Mohammad R. N. Jajja,* Adrian T. O Neill, Lissett R. Bickford, Amanda W. Keeler, Nabeel Hyder, Kyle Wagner, Allison Deal, Ryan E. Little, Richard A. Moffitt, Colleen Stack, Meredith Nelson, Christopher R. Brooks, William Lee, J. Chris Luft, Mary E. Napier, David Darr, Carey K. Anders, Richard Stack, Joel E. Tepper, Andrew Z. Wang, William C. Zamboni, Jen Jen Yeh,* Joseph M. DeSimone* *Corresponding author. jen_jen_yeh@med.unc.edu (J.J.Y); desimone@unc.edu (J.M.D.) The PDF file includes: Published 4 February 2015, Sci. Transl. Med. 7, 273ra14 (2015) DOI: /scitranslmed Materials and Methods Fig. S1. Gemcitabine transport into PDX tumors after a single device treatment. Fig. S2. Drug transport through freshly excised murine skin. Fig. S3. Role of gemcitabine concentration in drug transport in vitro. Fig. S4. PK of cisplatin delivered transdermally by iontophoretic devices. Fig. S5. Body weight changes in response to chemotherapy. Fig. S6. Change in laboratory values after the device gemcitabine treatment schedule. Fig. S7. Short-term renal toxicity after a single cisplatin treatment in mice.

2 Supplementary Materials Materials and Methods PK studies Pancreatic cancer patient-derived xenografts. Devices were surgically implanted onto the pancreatic tumors when they reached a median volume of 260 mm 3. The device tubing was thread from the abdominal cavity to the dorsum of the neck, exiting through a skin button (SAI infusion). Treatment was started one week post-implantation in order for epithelialization around the device. The gemcitabine solutions used for IV and device delivery were formulated by diluting gemcitabine hydrochloride powder in normal saline. A constant direct current (DC) power supply was used to provide the electric potential gradient. The positive lead was connected to the device wire, and the negative lead was connected to a silver chloride electrode placed on the mouse's skin with electrolyte gel. A syringe pump was used to circulate the gemcitabine solution through the device at a flow rate of 50 µl/min over a period of 10 minutes. When the constant voltage was applied, drug was driven from the device reservoir into the tumor tissue. Upon completion of the treatment, the device was emptied of drug solution. Terminal bleeds and tissues were removed at their designated times. An inhibitor of cytidine deaminase, tetrahydrouridine (THU) (Calbiochem), was added to the blood. For the IV treatment arm of the study, mice were given a single dose of gemcitabine at 80 mg/kg. SUM149 orthotopic xenograft model. Five million SUM149 cells resuspended in Matrigel (50:50) were injected into the left inguinal mammary fat pad of 4-8 week old athymic nude mice. Devices were transdermal adhered onto the skin (3M Vetbond) above the orthotopic tumors

3 when they reached a single dimension of 5-7 mm. The cisplatin solutions used for device delivery were formulated at ph 5.9, and the cisplatin used for IV delivery was administered directly from the clinical formulation. A constant DC power supply was used to provide power to the device. The positive lead was connected to the device wire, and the negative lead was connected to a silver chloride electrode placed on the mouse's skin with electrolyte gel. A syringe pump was used to circulate the cisplatin solution through the device at a flow rate of 50 µl/min over a period of 25 minutes. When the constant voltage was applied, drug was driven from the device reservoir into the tumor tissue. Upon completion of the treatment, the device was emptied of drug solution. Terminal bleeds were performed. The mice were treated with a single transdermal iontophoretic dose of Cisplatin or 5 mg/kg bolus of Cisplatin via tail vein. Terminal bleeds and tissues were removed at their designated times. Dogs. The canine model was housed at Synecor LLC and treated according to Synecor IACUC protocol. The dogs weighed between kilograms and were between 1-2 years old. The dogs were anesthetized, and the abdomen was shaven and cleaned prior to surgery. A laparotomy was performed, and the right lobe of the canine pancreas was exposed. The devices were sutured on the pancreas, ensuring good contact with the tissue. A 5 x7 electrocautery patch placed on the back of the animal was used as the cathode. A constant DC power supply was used to provide power to the device. A 1-gram vial of gemcitabine hydrochloride powder was solubilized in 25 ml of saline, and the 25 ml was diluted to 100 ml (ph between 4 and 5). Using an IV infusion pump, the solution was pumped through the device at a flow rate of 1.5 ml/min over a period of 60 minutes. Upon drug flow through the device, a constant current of 10 ma (current density 1.4 ma/cm 2 ) was applied for 60 minutes. Ten ml of blood was sampled at 15-minute increments prior to and during therapy and collected into heparinized tubes with 40

4 µl of a 10 mg/ml THU solution. Upon completion of the 60 minutes, the device was emptied of drug solution. The dogs were euthanized with potassium chloride solution, and the blood and tissue/device were extracted and snap frozen with liquid nitrogen and stored at -80 C. Quantification of gemcitabine by distance of drug transport The frozen tissues or 2 wt% agarose gels formed in 8-well tissue culture dishes were sectioned at 50 µm increments by a Leica cryostat microtome at -20 C. Frozen tissue sections were combined to reduce the sample number. To the specimens, the internal standard, 2 dc, was added at a concentration of 20 µg/ml. To the specimens, 0.08 mg/ml THU, 0.4 M perchloric acid solution was added at a liquid to mass ratio of 3:1 (v/w). The samples were vortexed and sonicated in an ice bath for 10 minutes. The samples were centrifuged at 4000 rpm for 10 minutes, the pellet was washed with 0.4 M perchloric acid and centrifuged again at 4000 rpm for 10 minutes), and the supernatants were combined. Potassium hydroxide was added to bring the ph to 3 and potassium perchlorate was removed by centrifugation. The supernatant was analyzed by UV-HPLC. To the plasma samples, the internal standard was added for a concentration of 20 µg/ml. 2.0 M perchloric acid was added to the sample at 10% v/v of the sample. The samples were vortexed, incubated on ice for 15 minutes, and centrifuged at 16,000 rpm at 4 C for 10 minutes. Potassium hydroxide was added to bring the ph to 3 and the insoluble salt, potassium perchlorate, was discarded. A portion of the solution was analyzed by UV-HPLC.

5 SUPPLEMENTARY FIGURES Fig. S1. Gemcitabine transport into PDX tumors after a single device treatment. Three different concentrations of gemcitabine were tested compared with 80 mg/kg intravenously. Data are means ± SD (n = 4). P values were determined by unpaired t test.

6 Fig. S2. Drug transport through freshly excised murine skin. Cisplatin (1 mg/ml) iontophoretic transport into the through the skin was evaluated using a modified Franz diffusion cell. Transport was quantified after iontophoretic (25 minutes at 1 ma) or passive diffusion conditions (0 ma). Data are means ± SD (n = 4). P values were determined by unpaired t test.

7 Fig. S3. Role of gemcitabine concentration on drug transport in vitro. Gemcitabine transport through 2 wt% agarose gel was evaluated by applying 2 ma for 10 minutes with 3 different inflow gemcitabine concentrations and comparing drug transport into the agarose gels. Data are means ± SD (n = 5). P values were determined by unpaired t test.

8 Fig. S4. PK of cisplatin delivered transdermally by iontophoretic devices. Mice were administered a single treatment of cisplatin (1 mg/ml) using the transdermal device that was placed on the skin directly above the orthotopic tumor. Skin, kidney, right inguinal mammary gland, and left inguinal lymph node were collected from each animal at various times, and total cisplatin concentrations were analyzed (µg/g mass of tissue). Data are means ± SD (n = 5).

9 Fig. S5. Body weight changes in response to chemotherapy. Mice with patient-derived pancreatic tumor xenografts (PDX) were given saline or gemcitabine (80 mg/kg via IV or 20 mg/ml concentration via device). Mice with syngeneic T11 orthotopic tumors and human SUM149 orthotopic xenograft tumors were given saline, cisplatin (5 mg/kg via IV and/or 1 mg/ml concentration via device), or radiation (10 Gy). Data are mean body weight changes as a percentage of initial weight ± SEM (n = 5-7 for the PDX study; n = 8 for T11 radiation study; n = 8-9 for SUM149 study; n = 9 for T11 study). P values were determined by ANOVA tests.

10 Fig. S6. Change in laboratory values after the device gemcitabine treatment schedule. Evaluation of pertinent laboratory values for gemcitabine treatment in an orthotopic mouse model of human pancreatic cancer. Mice with patient-derived pancreatic tumor xenografts were given saline or gemcitabine (80 mg/kg IV or 20 mg/ml via device). Pre-treatment labs were collected 10 days before the treatments were started. Post-treatment labs were collected 3 days after the last treatment. Data are means ± SD (n = 7). P values determined by unpaired t test.

11 Fig. S7. Short-term renal toxicity after a single cisplatin treatment in mice. Mice with a SUM149 human breast cancer xenografts were treated with cisplatin delivered IV (5 mg/kg) or by iontophoretic device (1 mg/ml). Kidneys were collected from animals at 6 and 24 hours after treatment and stained for platinum-dna adducts and γh2ax. Data are individual animals and horizontal line is the mean value (n = 5). P values determined by unpaired t test.