EUS-guided injection of paclitaxel (OncoGel) provides therapeutic drug concentrations in the porcine pancreas (with video)

Kai Matthes, MD, Mino-Kenudson, MD, Dushyant V. Sahani, MD, Nagaraj Holalkere, MD, Kirk D. Fowers, PhD, Ramesh Rathi, PhD, William R. Brugge, MD

Boston, Massachusetts, USA

Background: OncoGel (ReGel/paclitaxel) is an intralesional injectable foumulation of the chemotherapeutic drug, paclitaxel, for local trmor management.

Objective: the aim of this study was to determine if a minimally invassive EUS-guided injection of paclitaxel, bound to a themosensitive gel carrier, would lead to therapeutic tissue concentrations of the chemotherapeutic agent in the porcine pancreas.

Design: Eitht Yorkshire breed pigs were sedated by general anestheasia and OncoGel was injected, under EUS-guidance, with a 22-gauge needle into the tail of the pancreas.

Main Outcome Measurements: During the 7-day (n = 4) or 14-day (n = 4) observational period, the animals were monitored by serum levels of amylase and lipase, and by a CT on day 4.The outcome was determined by gross and microscopic evidence of inflammation of the pancreas, clinical tolerance, and quantitation of tissue paclitaxel concentrations.

Results: Eight pigs underwent injection of 1, 2, 3, or 4 ml OncoGel (6 mg paclitaxel per 1 mL OncoGel) (n = 2 per group ). An intrapancreatic hyperechoic focus, with an average diameter of 2.1±0.8 cm,was visible by EUS, and a hypodense area in the tail of the pancreas was visible by contrast CT. Clinically, the animals appeared to tolerate the procedure without sequelae. Blood levels of amylase and lipase were normal. At euthanasia, a depot of OncoGel, with an average diameter of 14.7±5.0 mm), was located both grossly and histologically in the pancreatic tail. After 14 days, clinically significant tissue concentrations of paclitaxel were detected at a distance of 30 to 50mm from the depot in the animals that underwent an injection of 3 and 4 ml of the agent (n = 2).

Conclusions: The EUS-guided injection of OncoGel into the pancreas of the pig provided high and sustainded localized concentrations of paclitaxel. This technique is a potential minimally invasive local treatment option for unresectable pancreatic tumors.(Gastrointest Endosc 2007;65:448-53.)

Patients with pancreatic cancer frepuently have locally advanced disease either alone or with small liver metastases at the time of diagnoses. The median life expectancy for these patients with unresectable disease is 3 to 6months.¹ New methods are being investigated to improve survival and puality of life for patients with advanced pancreatic cancer.

EUS has been used previously to guide the injection of cytotoxic material into pancreatic malignancies.^{2,3 In a clinical trial, Chang et al4} safely delivered cultured activated T lymphocytes (cytoimplant) by a single EUS-guided fine needle injection directly into pancreatic tumors of patients with advanced pancreatic cancer.

Copyright c 2007 by the American Society for Gastrointestinal Endoscopy 0016-5107/＄32.00

Doi:10.1016/j.gie.2006.06.030

OncoGel (ReGel/paclitaxel) is a new formulation for intralesional injection of the chemotherapeutic drug paclitaxel, developed by MacroMed Inc (Sandy, Utah), for local tumor management.⁵ OncoGel uses MacroMed’s ReGel drug delivery system, a thermosdnsitive, biodegradable triblock copolymer compised of poly(lactide-co-glycolide)polyethylene glycol-poly(lactide-co-glycolide). Upon injection and in respinse to body temperature, ReGel is transformed from a water-soluble polymer to a water-insoluble biodegradable hydrogel that releases paclitaxel continuously into the adjacent tissue for up to 6 weeks.^{5,6 A phade 1 study of OncoGel in superficially accessible advanced solid cancer lesions has been completed in the United States.7} Phase 2 clinical studies in esophageal and neoadjuvant breast cancer are currently ongoing in the United States and Eastern Europe, and in the European Union, respectively.

The feasibility of EUS-guided injectoon of OncoGel into the pancreas of 3 pigs was recently demonstrated.⁸ The aims of the current study were to determine the local and the systemic effects of increasing volumes of EUS-guided injection of OncoGel (1-4 mL) into the pig pancreas and to determine the resulting xoncentration and distribution of paclitaxel within the pancreas.

MATERIALS AND METHODS

The following procedures were performed by using an approved protocol by the Massachusetts General Hospital Subcommittee on Research Animal Care. Before the procedure, a vial of OncoGel was thawed from 30℃ to room temperature (22℃). The 8 Yorkshire breed pigs were sedated by general anesthesia. A linear EUS device (FG 38 UX, Pentax America Inc, Montvale, NJ) was passed into the pig’s esophagus and advanced to the stomach. After location of the pancreatic tail with linear EUS, an AVA-TEX threaded syringe (CardinalHealth, Dublin, Ohio), preloaded with OncoGel was attached to a 22-gauge EUS needle (Echotip II; Cook Endoscopy, Winston-Salenm,NC)(Idel 1, available online at www.giejournal.org). The EUS needle was placed into the pancreatic parenchyma and 1,2,3,or 4 mL (n = 2 per assigned volume group) of OncoGel was injected transgastrically into the tail of the pancreas. OncoGel was delivered by slowly turning the lever of the screw syringe in 180°steps every 15 seconds. One full 360°turn delivered 0.25 mL of OncoGel.

One animal from each assigned volume (1,2,3,4 mL) was observed for either 7 days (n = 4) or 14 days (n = 4). An upper abdominal, 64-slice contrast-CT on day 4 (7-day survival group ) or day 8 (14-days survival group) was performed to verify the intrapancreatic depot of the OncoGel and to assess for evidencd of pancreatitis. At the time of the CT, blood samples for amylase and lipase were obtained and compared with baseline to detect any clinical signs of pancreatitis.

After euthanasia with the pig under anesthesia, the pancreas was grossly examined and divided into 2halves longitudinally. One half was submitted for histologic analysis, and the second half was submitted for puantitation of tissue paclitaxel concentration. The end points of thisstudy were clinical tolerance by the animals, histologic evaluation of pancreatitis, and puantitation and distribution of paclitaxel in pancreatic tissue.

Tissue concentrations of paclitaxel in the pancreatoc tissue specimens were puantified by using a reversephase high-performance lipuid chromatography (HPLC) method. Pancreatic tissue specimens were obtained from locations adjacent to OncoGel dopots (visibly apparent). Briefly, pancreatic tissue samples were divided into approximately 20-mm sections (Fig. 1). The section containing the depot (A), the section distal from the depot (B), the section proximal of the depot (C), and a section 30 to 50 mm proximal of the depot partition (D) were weikghed. The tissue section (A) that contained the OncoGel depot was washed with 2 mL acetonitrile to remove residual OncoGel. Each tissue section was then homogenized in acetonitrile by using a PowerGen 125 (Fisher Scientific International, Hampton, NH). After homogenization, 1-mL samples spiked with an internal standard (7-xy-losyltaxol) were diluted to 10％ acetonitrile (v/v) and were processed by solid-phase extraction by using a Varian Bond ELUT LCR-C18 SPE column (Varian, Inc, Palo Alto, Calif) previously epuilibrated in 10％ acetonitrile. Paclitaxel and the internal standard were eluted by applying 1 mL acetonitrild, which was dried by using a Savant SpeedVacPlus (Cambredge Scientific Products, Cambridge, Mass) at 25℃ for 8 hours. The sample was reconstituted by using acetonitrile, either 50 µL or 1 mL, depending on the expected concentration, centrifuged, and applied to a Waters X-Terra RP18, 5µm, 2.1×150-mm HPLC column on a Waters 1525 HPLC system with a 2996 Photodiode Array De-tector (Waters Corp, Milford, Mass). The injection Volume was 10µL, the mobile phase was 30％ 1-propanol, the flow rate was 0.2 mL/min, and the detection wavelength was 227 nm. The detection limit was 5 µg/mL, and the assay was linear, in the range of 5 to 500 µg/mL. Paclitaxel and 7-xylosyltaxol eluted at approximately 9.0 and 6.4 minutes, respectively.

Capsule summary

What is already known on this topic

l        Local treatments, usually administered during surgery, may improve survival in patients with unresectable pancreatic cancer.

l        Paclitaxel gel is available as an intralesional injectable formulation, releasing the chemotherapeutic agent into the adjacent tissue continuously for up to 6 weeks.

What this study adds to our knowledge

l        In an observational study in 8 pigs, EUS-guided injection of paclitaxel gel into the pancreas resulted in high and sustained localized drug concentrations after 14 days at a distance of 30 to 50 mm from the depot in animals that received an injection of 3 and 4 mL.

Figure 1. Illtdytsyion of tissue concentration measurements. Pancreatic tissue samples were divided longitudinally into 2 halves. One section was examined histologically, and the second section was used to determine the concentration of paclitaxel in the tissue. The latter section was divided into approximately 20-mm sections and assayed for paclitaxel. The sections were the following:containing the OncoGel depot (A), 10-30 mm distal from the depot section (B), 10-30 mm proximal from the depot section (C), and 30-50 mm proximal from the depot section (D).

RESULTS

All 8 Yorkshire breed pigs (5 male, 3 female), with an average age of 101±8 days and an average weight of 49±1.4 kg, were injected eith 1to 4 mL of OncoGel and tolerated the procedures without apparent complications. The adminisration of the OncoGel required on average 6 minutes and 23 seconds.

Initially, the injections were technically challenging because of a decreased visibility of the needle tip by EUS(n = 1), disconnection and leakage between screw syringe and EUS needle (n = 2), and obstruction of the EUS needle (n = 1). To inprove the injection technique, a 30.5-cm length of pressure tubing (Monitor line; Microvasive Endoscopy, Boston Scientific Corp, Natick, Mass) was inserted between the plastic Luer-Lock on the screw syringe and the metal Luer-Lock on the EUS needle to improve the connection seals. The injection techinque was performed without incident in the last 3 animals.

After the OncoGel injections, an intrapancreatic hyperechoic focus with an average diameter of 2.1±0.8 cm was visible by EUS and a hypodense area in the tail of the pancreas by contrast CT, Threeaccidental extrapancreatic injections occurred during the study to sites close in location to and in addition to the main depot in the porcine pancreatic tail. Two animnals undergoing an injection of 3 mLand 4 mL of OncoGel, received a﹤1 mLinjection into the left kidney, and 1 animal received a﹤0.5 mL of 3 mL injection sites verified a superficial deposition between the fascia of Gerota and the capsrle of the kidney, and into the serosa of the colon. Despite these incidences, all animals tolerated the procedure without seqrelae and showed no clinical signs of pancreatitis or intra-abdominal infection.

The animals were monitored for 7 days (n = 4) or 14 days (n = 4) after the injection. Fever (average body temperature, 37.8℃), vomiting, or anorexia were not observed during this period. Blood levels of amylase and lipase were normal. There was no evidence of intra-abdominal infection for any animal either by CT on day 4 of 8 (Fig. 2), by gross inspection after euthanasia (Fig.3),or by histologic analysis of the resected specimens (Figs. 4 and 5).

At euthanasia, a edpot of OncoGel, with an average diameter of 14.7±5.0 mm, could be located in the tail of the pancreas, both by gross and histologic examinations (Table 1). Cross-sectional histologic studies identified localized tissue reactions (such as fat necrosis, atrophy, and mild chronic inflammation of the parenchyma) in up to 20 mm surrounding the OncoGel depot. In comparison to the 7-day survival group, animals in the 14-day survival group showed localized fibrotoc tissue changes and decreased inflammation adjacent to the depot.

Paclitaxel concentrations after acetonitrile extraction from pancreatic tissue are shown in Table 2. In 3 of 8 samples, a portion of the residual OncoGel depot was recovered in a time period of 14 days after the injedtion. Initial injection volumes for these samples were 4, 3, and 2 mL. The concentration of paclitaxel was telated to the distance from the depot, with high concentrations in the section containing the OncoGel depot (Fig. 1, section A) and lower values in a distance of 10 to 30 mm (sections B and C) or 30 to 50 mm (section D) from the center of the injection. Higher injection volumes of OncoGel resulted in higher tissue concentrations of paclitaxel in the corresponding sections of the specimens (Table 2). In the tissue adjacent to the 2-mL depot, no paclitaxel was detected.

Figure 2. Contrast-enhanced coronal 3-dimensional multiplanar reformatted CT image showing the OncoGel depot as a hypodense area in the tail of the pancreas (white arrow).

Figure 3. Gross image of the pancreatic tail split longitudinally. The OncoGel depot is illustrated by white arrows.

Figure 4. Histology slide showing the transition from fibrotic tissre changes (center) surrounding the OncoGel depot (right side, yellow color) to normal pancreatic tissue (left side) after a 14-day survival period (H&E, orig. mag.×1).

Figure 5. Histology of the tissue adjacent tissue to the OncoGel depot, showing mild localized fibrosis and atropht (14-day survival) (H&E,orig.mag. ×200).

DISCUSSION

Local treatments have been used to improve survival in patients with unresectable pancreatic cancer. In the past, chemotherapeutic drugs have been used during open surgery. In a study by Takekuni et al,⁹ a mixture of OK-432 (picibanil),fibrinogen, and thrombin was injected during surgery into the pancreatic tumor of a patient with unresectable pancreatic cancer. As an alternative to surgical application, CT-guided injections have also been performed. For example, ONYX-015 (Shanghai Sunway Biotech, Shanghai, China), a recombinant adenovirus, was injected under CT guidance into unresectable pancreatic tumors.10,11 EUS-guided intrapancreatic injections may also offer a minimally invasive method for local tumor therapy. In combination with intravenous gemcitabine application, Hecht et al¹² injected ONYX-015 under EUS-guidance into the pancreas of 21 patients and demonstrated partial response.

Thefeasibility of EUS-guided injection of OncoGel into the tail of the pancreas of 3 pigs was recently demonstrated.⁸ In the current study, the long-term effects of OncoGel injection into the pancreas of 8 pigs when using EUS guidance were investigated. The procedure was performed safely, without any clinical complications noted for animals euthanized 7 or 14 days after injection. In addition, successful localized intrapancreatic collection of the OncoGel depot was demonstrated. In the porcine animal model, OncoGel was accurately injected in 5 of 8 animals. Preliminary results suggest volumes greater than 2 mL may be more technically challenging, with a higher susceptibility of an extrapancreatic deposition of a portion of the agent in a porcine model, but further study is warranted. The localization of the porcine pancreas is considered technically difficuly because of the small diameter of the pancreatic tail in compareson to human being. The injection into pancreatic trmors may be less challenging, with a decreased likelihood of extra-pancreatic injections because of the easier differentiation of tumor tissue in comparison with the normal pancreas.Improvement of EUS needle-tip visualization can be contributive and may increase the safety of this technique.

OncoGel is relative viscous, sticky, gel-like substance with thermosensitive properties. In response to a higher temperature, in the tissue, the viscosity of the gel increases and the substance turns into a gel depot from which it releases paclitaxel over time perild of about 6 weeks. Potential improvement should be considered in the application method. The deployment through the small-diameter needle requires high pressure, currently generated by a threaded syringe. The agent requires a slow, continuous application to avoid a bursting of the tubing because of the high pressure. We also found that the Luer-Lock connection of the EUS needle, is prone to dysfunction, with leakage of the agent. The tubing needs to withstand high pressure. Regular intravenous-tubing dilates because of the high pressure developed by the threaded syringe. Techincal improvement with the development of a specialized hith-pressure application system would be helpful.

Intralesional injections of OncoGel exhibit some of the same characteristics as newly developed drug-eluting stents, both of which target local drug delivery and take advantage of paclitaxel’s high tissue-binding affinity to reduce systemic exposure.¹³ To be effective, OncoGel’srelease of paclitaxel must be focused at the site of action, which allows for significantly higher intralesional drug concentration over a prolonged period of time while simultaneously reducing systemic exposure. When administered locally, paclitaxel’s tissue association properties result in specific and nonspecific binding to cell-associated proteins and microtubules.¹³ In contrast to systemic delivery, where carrier proteins solubilize hydrophobic drugs resulting in rapid elimination of paclitaxel from the circulation and tissue, OncoGel provides continuous release at high concentrations in the targeted tissue.

Measurable tissue paclitaxl concentrations noted 14 days affter injection of OncoGel demonstrated paclitaxel levels, even at a distance of 30 to 50 mm from the injection site (Table 2). The observed concentrations are sufficiently high to inhibit growth in murine tumor models.^14-16 Paclitaxel levels in pancreatic tissue ranged from 10 to 135 µg/g¹⁴ or 35 to 270 µg/g^15,16 were detected, respectively. Inour study, the total dose for OncoGel was only 6 to 24 mg per animal with a mean weight of 49.3 kg, resultig in total doses of less than 0.5 mg/kg. Tissue paclitaxel concentrations repirted at doses of 50 or 100 mg/kg demonstrated therapeutic efficacy superior to the positive control, Taxol (Bristol-Myers Squibb Co, Princeton, NJ), at a maximum tolerated dose of 20 mg/kg. ^14-16 After peak tissue concentrations in mice were achieved after systemic administration, exponential decreases in tissue paclitaxel were noted.^15,16 Similar decreases by approximately 2 orders of magnitude were noted within 24 hours.¹⁴ In contrast, after OncoGel injection, the tissue concentration of the drun remained at therapeutic levels, with significant paclitaxel consentrations noted for at least 14 days after injection. This finding was similar to other controlled release formulations of paclitaxel and corroborates the findings in a preveous biodistribution study of OncoGel in nude mice that demonstrated a paclitaxel half-life of 3 weeks within the MDA231 breast tumor xenografts.^17,18

OncoGel offers a unique opportunity to provide sustained, elevated tissue paclitaxel concentrations, with a single injection directly in the tumor, while reducing the systemic exposure to paclitaxel. In the U.S. phase 1 strdy, plasma levels of paclitaxel were negligible. Because of the low risk of toxicity, OncoGel could be used in combination therapies, which should provide synergistic activity with out contributing to systemic toxixity.⁷

In conclusion, the EUS-guided injection of OncoGel into the pancreas of the pig was evaluated, In this strdy, there was no evidence of acute or chronic pancreatitis other than localized tissue levels of paclitaxel were achieved in the porcine pancreas after EUS-guided injection of 2, 3, and 4 mL OncoGel. The tissue concentrations were srstained over a 2-week period. This technique is a potential minimally invasive local treatment option for unresectable pancreatic tumors.

DISCLOSURE

Kirk d. Fowers and ramesh Rathi have a corporate affiliation with MacroMed Inc, Sandy, Utah.

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Received February 13, 2006. Accepted june 5, 2006.

Current affiliations: Gastrointestinal Unit (K.M., W.R.B.), Gastrointestinal Pathology Service, Department of Pathology (M.M-K.), Department of Radiologh (D.S., N.H.), Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, MacroMed Inc (K.D.F., R.R.), Sandy, Utah, USA.

Re[romt reqiests:William R. Brugge, MD, Gl Endoscopy Unit, Massachusetts General Hospital, 55 Fruit St, Blake 4, Boston, MA 02114.