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Ann Thorac Surg 2011;92:2007-2014. doi:10.1016/j.athoracsur.2011.04.106
© 2011 The Society of Thoracic Surgeons
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Original Articles: General Thoracic

Paclitaxel-Loaded Expansile Nanoparticles in a Multimodal Treatment Model of Malignant Mesothelioma

Morgan D. Schulz, MD, Kimberly Ann V. Zubris, PhD, Jacqueline E. Wade, BS, Robert F. Padera, MD, PhD, Xiaoyin Xu, PhD, Mark W. Grinstaff, PhD, Yolonda L. Colson, MD, PhD*

Division of Thoracic Surgery, Departments of Surgery, Pathology, and Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston; and Departments of Chemistry and Biomedical Engineering, Boston University, Boston, Massachusetts

Accepted for publication April 7, 2011.

* Address correspondence to Dr Colson, Division of Thoracic Surgery, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (Email: ycolson{at}partners.org).

Presented at the Forty-seventh Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 31–Feb 2, 2011.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Discussion
 Acknowledgments
 References
 
Background: Malignant mesothelioma has a poor prognosis even when treated aggressively with multimodal therapy. Traditional murine tumor models can be used to evaluate drug efficacy and toxicity in malignant mesothelioma, but not to assess the effect of a multimodal approach that includes the surgical resection of tumor. We therefore developed a murine model of multimodal therapy in which we evaluated paclitaxel-loaded expansile nanoparticles (Pax-eNP) for delivering intracavitary chemotherapy in malignant mesothelioma.

Methods: Paclitaxel-loaded expansile nanoparticles (Pax-eNP) of 100 nm, designed to release drug at an endosomal pH below 5, were synthesized. Xenografts of human malignant mesothelioma were established intraperitoneally in nude mice, followed by cytoreductive surgery (CRS) via laparotomy, and with omentectomy and resection of abdominal fat pads done 14 days later. At fascial closure, 10 mg/kg paclitaxel was delivered as traditional paclitaxel/paclitaxel Cremophor-EL (Pax-CE) or Pax-eNP. Morbidity and survival were assessed over a period of 90 days.

Results: Cytoreductive surgery in mice was feasible and reproducible, and incurred less than 5% operative mortality. By itself, CRS did not significantly prolong survival; however, the addition of intraoperative Pax-CE or Pax-eNP significantly increased survival as compared with that of mice with untreated disease. In the case of Pax-eNP, the increase in survival was also statistically significant as compared with that following resection alone.

Conclusions: A murine model of CRS for malignant mesothelioma allows the in vivo assessment of multimodal therapy, including nanoparticle delivery. Combination therapy was superior to no treatment or CRS alone in prolonging survival. Treatment with Pax-eNP improved overall survival in the setting of CRS, suggesting that Pax-eNP merits further evaluation for intracavitary drug delivery following the surgical resection of malignant mesothelioma.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Discussion
 Acknowledgments
 References
 
Median survival in malignant pleural mesothelioma without treatment ranged from 4 to 18 months, but even with surgical resection and adjuvant therapy the overall 5-year survival is only 10% to 15% [1, 2]. Five-year disease-free survival rates approaching 50% have been reported in some series following the multimodal treatment of malignant mesothelioma with radical cytoreductive surgery (CRS) and adjuvant chemoradiotherapy in highly selected subsets of patients with early-stage disease and favorable tumor histology [3, 4]. However, morbidity associated with this approach is 40%, perioperative mortality ranges from 4% to 6%, and disease recurs in more than half of treated patients despite macroscopically complete cytoreduction [3]. Clinical trials of intraoperative intracavitary hyperthermic chemotherapy have been conducted with the rationale of optimizing the local control of disease at resection. Although early trials show feasibility and potential survival benefit with this approach, morbidity and mortality in the treated series of patients approach 50% and 5% to 11%, respectively [5, 6]. Given these outcomes, there is a significant need for more effective and less toxic treatments for malignant pleural mesothelioma.

Preclinical testing of therapeutic agents for use in multimodal treatment regimens for malignant mesothelioma is challenging because traditional models evaluate drugs in a setting of physiologic homeostasis and do not take into account the impact of major surgery on disease progression or drug efficacy and toxicity profiles. Studies of multimodal treatment regimens have been conducted in surgical models using tumor-bearing rats [7, 8], but these models have been largely limited to allogeneic cell lines and have not been used to evaluate the effect of interventions on the in vivo growth of human tumor implants after surgery. We describe here a murine model of surgically resected malignant mesothelioma xenografts for study of the multimodal treatment of this disease, in which we have evaluated in vivo the efficacy following surgery of pH-responsive expansile nanoparticles (eNP) designed for prolonged intracellular drug release.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Discussion
 Acknowledgments
 References
 
Animals and Cell Line
Female athymic nude mice (age, 4 to 6 weeks; Harlan Laboratories, South Easton, MA) were housed under sterile conditions. Animal care and procedures were approved by the Animal Care and Use Committee of the Dana-Farber Cancer Institute, in strict compliance with federal and institutional guidelines. The human malignant mesothelioma cell line MSTO-211H/CMMPnlacZ/LucNeo[5/11/04T] (MSTO-211H/luc), expressing both firefly luciferase and β-galactosidase, was provided by Dr James Rheinwald (Harvard Medical School, Boston, MA) and maintained in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum, streptomycin (100 μg/mL), and penicillin (100 U/mL) at 37°C in 5% CO2.

Nanoparticle Synthesis
Paclitaxel-loaded expansile nanoparticles (Pax-eNP) were prepared as previously described, using a miniemulsion polymerization technique with the addition of 5% (wt/wt) paclitaxel (MP Biomedicals, Solon OH) before emulsification [9]. The resultant nanoparticle suspensions were stirred overnight and dialyzed to remove solvents and synthetic byproducts before dilution in saline for use in vivo.

Model of Cytoreductive Surgery
Intraperitoneal (IP) xenografts were established in female athymic nude mice through the injection of 5 x 106 MSTO-211H/luc cells on day 0. On day 14 the mice underwent general anesthesia and midline laparotomy. Following incision, a 500-μL bolus of saline was instilled to maintain normovolemia. The animals' lower abdominal fat pads were resected, with care taken not to injure the urinary bladder. The omentum was clamped proximally using curved microdissection forceps, and was divided, with care taken to ensure hemostasis. Residual omental macroscopic tumor was carefully resected. Immediately before fascial closure with 5-0 Vicryl suture (Ethicon, Somerville NJ), the experimental drug or control was normalized to a total volume of 500 μL in warm physiologic saline and administered IP. The skin was closed in an interrupted fashion. Viable tumor burden was assessed with bioluminescent imaging, using a Xenogen IVIS 100 bioluminescence imaging station (Xenogen, Alameda, CA) with a 2-second exposure time following the subcutaneous administration of 150 mg/kg firefly luciferin.

Experimental Design
Experimental mice were divided into five groups. Group 1 did not undergo CRS. Group 2 underwent CRS with 500 uL saline administered at fascial closure. Group 3 underwent CRS with 200 mg/kg expansile nanoparticles without drug (unloaded eNP). This dose of unloaded eNP is the polymer equivalent of Pax-eNP delivering the 10 mg/kg dose of paclitaxel used in group 5. Unloaded eNP have previously been shown to be noncytotoxic to tumor cells in vitro and to have no measurable effect on tumor progression in an in vivo model of microscopic residual disease [10]. Group 4 underwent CRS with 10 mg/kg paclitaxel suspended at 6 mg/mL in 1:1 (v/v) polyethoxylated castor oil (Cremophor EL, Sigma-Aldrich, St. Louis, MO)/ethanol mixture (Pax-CE). Group 5 underwent CRS with 10 mg/kg paclitaxel formulated as Pax-eNP.

Study Endpoints and Analysis
Animals were monitored twice daily for 72 hours following surgery, and then daily for the remainder of the study, with euthanasia at evidence of morbid disease progression. Necropsy was performed with in situ X-Gal (Sigma–Aldrich, St. Louis, MO) staining of the peritoneal and thoracic cavities to assess the extent and distribution of tumor burden through adaptation of the technique of Murphy and colleagues [11]. Briefly, 100 μL paraformaldehyde/glutaraldehyde fixative was instilled into each cavity and aspirated after a 15-minute incubation, and the cavities were washed with 100 μL PBS x 6 followed by the introduction of 100 uL X-Gal staining solution (1 mg/mL in X-Gal buffer prepared according to the manufacturer's instructions) and a 2.5-hour incubation period. The stain was then removed and the peritoneal and thoracic cavities were opened for inspection to assess the extent and distribution of tumor. Kaplan-Meier estimates for overall survival were compared through the log-rank test, with p < 0.05 considered statistically significant. All statistical analyses were done with GraphPad Prism 5.0 software (La Jolla, CA).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Discussion
 Acknowledgments
 References
 
Establishment of Murine CRS Model and Cytoreduction With CRS
Cytoreductive surgery was performed 2 weeks after the establishment of MSTO-211H/luc xenografts. The distribution and extent of disease at surgery were predictable, with a tumor-establishment rate of 100%. The bulk of macroscopic tumor burden was distributed within the omentum and lower abdominal fat pads (Fig 1 ). Cytoreductive surgery was technically successful and reproducible, with macroscopically complete cytoreduction in more than 90% of animals. Imaging of representative animals demonstrated a 10- to 100-fold reduction in luciferin-mediated bioluminescence, which was consistent with the intraoperative assessment of reduction of tumor burden (Fig 2 ). One intraoperative death occurred in an animal with an atypical pattern of primary tumor burden concentrated at the porta hepatis, with ascites and venous engorgement consistent with portal hypertension. These results confirmed the feasibility and low operative mortality of a murine model of CRS performed for intraperitoneal disease.


Figure 1
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  		Fig 1. Cytoreductive surgery with intraoperative intracavitary chemotherapy in a mouse model of malignant mesothelioma. At 14 days post-xenograft implantation, the bulk of solid tumor burden (A) is within the omentum (dashed circle) and the lower abdominal fat pads (arrows indicate tumor nodules). The abdominal fat pads are ligated and divided (B), and omental tumor (C) is resected following clamp hemostasis (D). Following removal of all resectable solid tumor (E), a 24G catheter is used to instill drug or control solution (F) just prior to completing fascial closure.

 

Figure 2
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  		Fig 2. Cytoreductive surgery achieves significant reduction of viable tumor burden. Bioluminescent imaging of representative animals at 14 days of tumor growth before cytoreductive surgery (A) and 24 hours after cytoreductive surgery (B), done with a Xenogen IVIS 100 bioluminescence imaging station with a 2-second exposure time after the administration of 150 mg/kg firefly luciferin, shows significant reduction of viable tumor burden after cytoreductive surgery.

 
Overall Survival and Pattern of Disease Progression After CRS Alone
Survival and disease progression in untreated mice were compared with those in mice successfully debulked with CRS. Animals that did not have CRS (n = 11) demonstrated a median survival of 35 days (range, 32 to 48 days). No difference in median survival was detectable for animals with untreated disease (n = 8, median survival, 35 days) and those that underwent sham laparotomy without tumor resection (n = 3; median survival, 36 days), and these animals were therefore pooled for statistical analysis. Animals that underwent CRS with the administration of saline control (n = 8) survived for a median of 42 days (range, 34 to 65 days, p = 0.137; Fig 3 ). Necropsy demonstrated differences in the pattern of tumor distribution and associated morbidity in animals with untreated disease and those with recurrent disease after CRS. Untreated disease resulted in bulky tumor deposits confined to the peritoneal cavity, with extensive tumor burden in the omentum and lower abdominal fat pads. Bowel obstruction, tumor bulk limiting ambulation, or both were the major observed morbidities. In contrast, recurrent disease following CRS was characterized by tumor burden distributed along the peritoneal reflections of the uterine horns and within the gastrosplenic ligament. Large-volume epigastric tumors and innumerable mesenteric tumor nodules were noted in these animals. Morbidity was primarily secondary to cachexia and proximal bowel or biliary obstruction.


Figure 3
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  		Fig 3. Overall survival after cytoreductive surgery (CRS). Mice that underwent surgical debulking without adjuvant chemotherapy at 14 days after establishment of mesothelioma xenograft (n = 8) and animals with untreated disease (n = 11) were monitored until morbid disease progression necessitated euthanasia. The median survival was 35 days in untreated animals and 42 days among animals treated with cytoreductive surgery without adjuvant intraoperative chemotherapy (p = 0.137).

 
Survival Benefit of CRS and Intraoperative Adjuvant Treatment With Pax-eNP
At physiologic pH, Pax-eNP are hydrophobic, drug-encapsulating polymeric particles of approximately 100 nm diameter. At an endosomal pH of 5 or lower, the side chains of the NP polymer are cleaved, resulting in its expansion to a hydrophilic hydrogel of approximately 1 μm, with subsequent intracellular release of the encapsulated drug over a period of 24 hours [12]. Although mesothelioma is often considered resistant to taxanes, Pax-eNP have been shown to significantly decrease the tumor burden in peritoneal mesothelioma and to prolong survival when coinjected with tumor cells in an orthotopic murine model [10]. We therefore hypothesized that intraoperative treatment with Pax-eNP at the time of CRS might significantly prolong both the time to disease recurrence and overall survival. There was no difference in median survival in CRS control animals treated with saline or unloaded eNP (42 vs 44 days, p = 0.864). However, the adjuvant use of Pax-CE with CRS significantly increased median survival to 52 days (range, 29 to 66 days) as compared with that of untreated animals (p = 0.0039), but the improvement in survival was not statistically significant compared to CRS alone (p = 0.259). In contrast, addition of Pax-eNP resulted in a statistically significant increase in survival relative to both that of mice with untreated disease and CRS controls (54 days; range, 49 to 104 days; p < 0.0001 vs untreated disease, p = 0.0003 vs controls with unloaded eNP), though not relative to treatment with Pax-CE (Figure 4 ).


Figure 4
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  		Fig 4. Adjuvant treatment with paclitaxel-loaded expansile nanoparticles (eNP) improves survival as compared with surgery alone. Overall survival from time of establishment of mesothelioma xenograft was compared among untreated animals (n = 11), animals that underwent cytoreductive surgery (CRS) with intraoperative intracavitary chemotherapy (10 mg/kg paclitaxel as paclitaxel–Cremophor EL [Pax-CE] or paclitaxel-loaded expansile nanoparticles [Pax-eNP], n = 9/group), and animals in which cytoreductive surgery was performed with a volume-equivalent vehicle control consisting of unloaded expansile nanoparticle polymer (n = 8) at fascial closure. Median survival was prolonged to 53 days among animals treated with either paclitaxel formulation (p < 0.0001 vs no cytoreductive surgery; p = 0.001 vs cytoreductive surgery alone). Median overall survival of animals treated with paclitaxel–Cremophor EL was 52 days (range, 29 to 66 days, p = 0.0039 vs no cytoreductive surgery; p = 0.2810 vs saline). Treatment with paclitaxel-loaded expansile nanoparticles resulted in a statistically significant increase in survival relative to both untreated disease and cytoreductive surgery without adjuvant paclitaxel (54 days; range, 49 to 104 days; p < 0.0001 vs no cytoreductive surgery, p = 0.0003 vs unloaded expansile nanoparticles).

 
The distribution of recurrent tumor following surgery with adjuvant treatment was similar to that described clinically, with occasional evidence of invasive retroperitoneal extension of tumor as manifested by animals treated with Pax-eNP. Although no significant difference in survival was noted among animals treated with Pax-CE and those treated with Pax-eNP, in situ thoracic staining of available animals with X-gal demonstrated pleural or pericardial implants or both in 6 of 7 (86%) animals treated with Pax-CE versus 1 of 5 (20%) animals treated with Pax-eNP. Several of the Pax-CE–treated animals underwent euthanasia for clinically evident respiratory distress. Tumor growth was present at the site of the laparotomy incision at the time of euthanasia in all of the animals that underwent CRS with or without adjuvant treatment, and tumor nodules were frequently observed in all of the groups of animals at the tumor injection site in the left lower quadrant. No macroscopic deposits of residual polymer were seen among animals that received nanoparticles, nor were significant intra-abdominal adhesions observed at necropsy.


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Discussion
 Acknowledgments
 References
 
The results of the present study demonstrate a technically straightforward murine surgical model for the assessment of multimodal therapy in malignant mesothelioma, and an innovative approach for preventing local disease recurrence following surgical resection. The improvement in overall survival observed with CRS and intraoperative adjuvant therapy as opposed to that with untreated disease, as well as the distinct patterns of disease progression in primary versus recurrent disease following resection, highlight important aspects of multimodal therapy seen clinically and reflected in the current animal model. Cytoreductive surgery plus intracavitary chemotherapy in animals with gross tumor results in survival equivalent to that achieved previously in the setting of microscopic disease [10], which accords with the effective surgical downstaging of established disease. The reproducibility and low mortality demonstrated in this study suggest that it is technically feasible to further develop the surgical aspect of the model to include a more aggressive "peritonectomy" component, allowing for pharmacokinetic evaluation more closely paralleling that in the clinical setting.

The low incidence of distant metastasis in mesothelioma argues that effective local control is potentially curative, since even advanced disease is generally "contained" within the pleural or peritoneal space. In practice, however, the diffuse distribution of tumor burden frequently precludes complete resection, and even in the setting of "optimal debulking" or "macroscopically complete cytoreduction," microscopic residual disease ultimately results in disease recurrence in the majority of patients. Single-modality therapy with CRS, systemic chemotherapy, or radiotherapy may provide effective palliation in selected patients, but has not been shown to reproducibly confer a survival benefit beyond the 9- to 18-month median survival reported with supportive care alone [2, 13, 14] This clinical observation is reflected in the current animal model, in which, despite the achievement of aggressive tumor debulking (Fig 2), median survival was prolonged by only 7 days, which was not statistically significant as compared with the survival of untreated controls (Fig 3).

Intracavitary chemotherapy increases local drug concentrations by as much as 1,000-fold relative to serum drug levels, with much slower clearance than with systemic drug administration, thereby increasing tumor cell toxicity through prolonged drug exposure [15]. Intraperitoneal chemotherapy with a platinum-containing drug and a taxane has been shown to significantly improve survival in patients with optimally debulked ovarian cancer, and is currently regarded as the standard of care for appropriately selected patients [16, 17]. Multimodal treatment of peritoneal mesothelioma in which aggressive peritonectomy to achieve macroscopic cytoreduction is combined with intraoperative hyperthermic intracavitary chemotherapy have increased median survival from 12 months to 34 to 92 months, with mortality rates of 0% to 8% and overall morbidity rates of 25% to 40% [18]. Given the 60- to 90-minute "dwell time" of intra-operative chemotherapy, hyperthermia and cell-cycle–independent agents (mitomycin C, cisplatin, doxorubicin) are used in treating peritoneal mesothelioma, with the addition of early postoperative IP regimens using paclitaxel with or without 5-fluorouracil to achieve cell-cycle specific cytotoxicity [19]. Nevertheless, disease recurs in nearly 50% of patients, despite being delayed in the setting of macroscopically complete cytoreduction. In contrast to the diffuse peritoneal distribution with relative sparing of the small bowel noted in primary peritoneal mesothelioma, recurrent disease commonly involves the small bowel serosa and mesentery and the epigastric region, areas in which complete cytoreduction is technically difficult to achieve [20]. Results with intraperitoneal paclitaxel following CRS in the animal model used in the current study reflect these clinical outcomes and limitations. Survival was improved in animals treated with CRS and Pax-CE as compared to with that in untreated animals (p = 0.004), but was not significantly improved as compared with that of animals treated with CRS alone (plus saline or unloaded eNP controls). These findings suggest an additive treatment effect with surgery plus chemotherapy as opposed to resection alone, as has also been clinically demonstrated.

Similar attempts to improve local control of disease and subsequent survival in pleural mesothelioma have led to the development of complex multimodal treatment regimens, including aggressive CRS via extrapleural pneumonectomy or pleurectomy and intraoperative intracavitary hyperthermic cisplatin, with postoperative radiotherapy commonly included because of the continued high incidence of local disease recurrence. Improved outcomes, with a median survival exceeding 50 months, have been reported in a select subset of patients with node-negative, early-stage epithelial disease [3, 6]; however, it has not been possible to extend this benefit to all patients with pleural mesothelioma. Moreover, morbidity remains high, at nearly 50%, and more than 40% of patients experience locoregional recurrence [5, 21]. Prolonged survival following intracavitary chemotherapy in peritoneal mesothelioma, which has been less successful in pleural mesothelioma, may reflect effects of various multidrug regimens or differing pharmacokinetics in peritoneal versus pleural intracavitary therapy. Animal models such as ours, together with orthotopic pleural models such as that described by Kelly and colleagues [22], offer the opportunity to conduct preclinical studies that can lead to a better understanding of how to leverage the potential benefit of this approach in the treatment of pleural mesothelioma.

Local and systemic toxicities, together with high local disease recurrence rates because of a limited depth of tumor penetration and short exposure times, have limited the clinical application of intracavitary chemotherapy in nearly all peritoneal malignancies, focusing the standard of care for patients with these diseases on macroscopically complete cytoreduction. Therefore, novel chemotherapeutic agents, immunotherapy, and drug-delivery systems designed for prolonged or targeted intracavitary cytotoxicity or both have been evaluated in preclinical and early clinical studies [23–26]. Controlled-release formulations of current chemotherapeutic agents are particularly attractive because they offer the advantage of well-characterized safety and efficacy profiles while allowing extension of the duration of treatment and preventing catheter-related complications common in current regimens [27]. Despite significant promise and the absence of dose-limiting toxicity in a phase I clinical trial of a paclitaxel nanocarrier in recurrent ovarian cancer [24], neither this nor any of the novel approaches described here has so far been evaluated for efficacy in the setting of first-line intracavitary therapy given intraoperatively at the time of radical debulking procedures, for malignant mesothelioma.

Paclitaxel-loaded expansile nanoparticles were designed for the selective intracellular delivery of chemotherapeutic agents with the goal of intraoperative administration and prolonged local release of cytotoxic drugs for eradicating residual disease following CRS. Prolonged local release appears to be particularly important for efficacy with cell-cycle–specific agents such as paclitaxel. Previous in vitro studies show that Pax-eNP enter and remain within tumor cells of mesothelioma for a prolonged period in vitro [10], and these results suggest that prolonged local delivery of paclitaxel takes place in vivo. Following the demonstrated efficacy of Pax-eNP in a heterotopic tumor model of non-small–cell lung cancer [12] and in an orthotopic model of primary malignant peritoneal mesothelioma [10], the present study describes an orthotopic model of surgical debulking used to evaluate Pax-eNP for the intracavitary treatment of mesothelioma after surgical debulking, for maximal clinical relevance. In this study, survival was statistically significantly increased over that with CRS alone only with the addition of paclitaxel delivered via eNP, although there was a trend toward prolonged survival with paclitaxel. Interestingly, although the pattern of recurrence in our animal model was similar to the clinical findings described after CRS [20], there was a preponderance of extracavitary (thoracic) extension of tumor noted in Pax-CE–treated animals. It is not possible to draw conclusions about differences in drug delivery in this setting, but these observations suggest differences in the mechanism, if not in the degree, of locoregional disease control that will be investigated in future studies. Taken together, these findings highlight the influence of dose, duration of exposure, and intracellular access in drug sensitivity and effective locoregional treatment of disease. Future studies will evaluate whether increased or multiple dosing further improves survival, and studies of alternate and combination drug regimens delivered via eNP are also planned in this model, in view of studies demonstrating clinical resistance to monotherapy with paclitaxel [28, 29] and systemic toxicity following the local administration of platinum agents in the perioperative setting.

This study highlights important aspects of CRS and intracavitary chemotherapy in the treatment of mesothelioma. The surgical technique used in the study is straightforward and adaptable for use in preclinical studies of other peritoneal malignancies for which representative cell lines are available, or against xenografts of individual patient tumors, thereby encompassing the goal of including the risks and benefits of CRS in the in vivo assessment of multimodal therapies. As proof of principle in demonstrating the utility of this model for the in vivo testing of emerging treatment modalities, we have demonstrated the efficacy, without observed morbidity, of a polymeric nanoparticle designed to optimize the treatment of residual malignant disease following surgical debulking.


   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Discussion
Acknowledgments
 References
 
DR THOMAS K. WADDELL (Toronto, Ontario, Canada): Yolonda, I'd like to ask you about the tumor targeting. You mentioned that you were going to get back to it, and maybe I drifted away, but it certainly seems to me that there are many cells in the postoperative period that could take up these particles and generate intracellular organelles with a pH of less than 5, the most obvious one being the peritoneal or pleural macrophage.

So is that a bad thing? Is that potentially detrimental? One could certainly imagine difficulty with the clearance of intraoperative bacterial contamination, and just in general, it may not be a very selective strategy. Is there a good rationale for why you used a pH-based approach to drug release?

DR COLSON: The pH isn't necessarily the way it's targeting. It's more a trigger for release. So I see these as two separate issues. One is how do we get nanoparticles to where we want drug to be, and the second is how do we then tell them to release the drug when they get there? What's happening with a large number of other polymer-type particles is that they release the drug so fast that it's all gone in less than an hour, with the result that by the time you actually get it injected and get it done, the drug is all released, and cleared rather quickly.

So one of the strategies here is that it actually has to get inside and then has to release, and it takes almost 24 hours for that pH change to do that, so that we get a longer delay.

What we don't understand yet is that when we've actually taken nanoparticles and color-coded them, so to speak, with different fluorescent molecules, and when we then inject them and go back even up to 2 weeks later, what we find is that the only areas that have any gross uptake of these nanoparticles are the sites of tumor.

When we've looked at this with the bioluminescent tumor, the first thing we've done is to document that when we've debulked it, it's gone. But the second thing with the bioluminescence is that if we inject nanoparticles when we know there's tumor, they're drawn to that site and they associate with it. We don't know the mechanism for that. Is it tumor-associated macrophages, as you brought up? But it doesn't seem to get into other macrophages.

So we don't know if there's a specific mechanism or what that is, and so that's the thing we're working on.

DR ROSS M. BREMNER (Phoenix, Arizona): Yolonda, thank you. That's really fantastic work that you're doing.

I just want to ask a short question. I see that you're going to increase the dose at the time of your initial injection. Have you got plans for doing repeated intraperitoneal injections, because it seems like you might have it hanging around for a little bit, but the actual drug response is going to go away shortly?

DR COLSON: We've evaluated this with established disease for a week or two and then treated but never resected it. We've done repeated doses, and we've been successful at the fourth dose, seeing, for the most part, that disease is obliterated, with some long-term survivors, and it's significantly different. And that work is presented elsewhere.

But our thought, at least in thinking that we'll just go to a dose of 40 mg/kg here, is that we'd like drug delivery to occur at the time of surgery. You could do your treatment and close up the surgical wound, and not have to give multiple drug doses all the time, because one of the major complications with patients who've got ovarian carcinomatosis is that you have to have a catheter in place, and with a catheter they develop more complications.

So we thought we would start with just trying to give a single large dose because even the 40 mg/kg dose isn't a toxic dose at all; we have a large safety margin, and by having it delivered over a longer period of time, maybe we can get the same effect.

DR BREMNER: Thanks very much.

DR RASHINDRA M. REDDY (Ann Arbor, Michigan): I was just wondering about all the mice that ended up dying. Did they all die from bulky intra-abdominal disease, then?

DR COLSON: Yes. They ended up getting recurrent disease. There are differences in that if they're only given the drug alone after debulking, they actually tend to also get disease in the thoracic cavity. So there are some differences in disease spread.

What we see here is that after you've debulked and given nanoparticles, the animals tend to get more diseases deep within the mesentery, so there are some differences in how the recurrent disease presents itself. But eventually they will end up with recurrence, usually in the abdomen.

DR MALCOLM M. D E CAMP (Chicago, Illinois): You and your colleagues at Brigham have done a lot of work on the pharmacokinetics of intracavitary chemotherapy. The approach you're discussing today has to do with delivering a drug intracellularly.

How do you do safety and pharmacokinetic studies as you start to escalate your dose of paclitaxel-loaded nanoparticles? Are you able to detect paclitaxel in the serum? How are you going to figure out what the maximum tolerated dose is when you begin dose-finding studies?

DR COLSON: There will be two ways. One is going to be to monitor the drug level in the peritoneum, which we're going to have to sample, there's no way around that, and the other is looking at it in the serum.

To date we haven't seen a lot of drug in the serum at all. If we inject it intravenously, we do. However, when we've given different kinds of delayed local polymer formulations, we get minimal levels in the blood stream and a thousand times more drug locally. But monitoring both local and systemic levels is what we really have to do.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Discussion
 Acknowledgments
References
 
The authors express their appreciation to James Rheinwald, PhD, and the Brigham and Women's Cell Culture Core Facility, who kindly provided the luciferase-transfected human mesothelioma cell line used for our bioluminescent imaging studies; and the expertise of Catherine Sypher, LATG, and Jessica Felch, LATG, which was invaluable in developing the operative model described in this report. This work was supported by National Science Foundation grant DMR-1006601 to Dr Mark W. Grinstaff. Dr Morgan D. Schulz's contribution to this work is dedicated to the memory of Prof William D. Schulz.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Discussion
 Acknowledgments
 References
 

  1. Herndon JE, Chahinian AP, Corson JM, Suzuki Y, Vogelzang NJ. Factors predictive of survival among 337 patients with mesothelioma treated between 1984 and 1994 by the Cancer and Leukemia Group B Chest 1998;113:723-731.[Medline]
  2. Law MR, Gregor A, Hodson ME, Bloom HJ, Turner-Warwick M. Malignant mesothelioma of the pleura: a study of 52 treated and 64 untreated patients Thorax 1984;39:255-259.[Abstract/Free Full Text]
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