Direct delivery and liposomal encapsulation of CPT-11 have been shown to decrease local toxicity and allow much higher concentrations of drug to be achieved at the target site compared with either systemic or free drug delivery.39,45
The efficacy of liposomal topoisomerase inhibitors delivered by CED in rodent glioma models has been previously documented,39,45–47
as has our ability to monitor CED infusions by co-infusion of liposomes containing gadolinium as a surrogate marker.37,48
We have also previously demonstrated that CED of liposomally encapsulated CPT-11 and gadoteridol results in negligible clinical and histopathological adverse effects in normal experimental dogs,40
and the current study suggests that this is also true for dogs with intra-axial gliomas. Although most animals exhibited mild lymphocytic pleocytosis in CSF analyzed after infusions, only 2 animals showed evidence of either histopathological or MRI changes consistent with an adverse reaction to the infusion. Both animals exhibited a deterioration in neurological status and both were rapidly responsive to corticosteroids (Fig. ; Table ); although marked atrophy/malacia involving both grey and white matter was seen in the most severely affected animal (Dog 3). While marked leakage of infusate into nontargeted peritumoral tissues was documented in this case, T2W hyperintensity in the second dog (Dog 8) occurred after infusions that were essentially limited to the tumor volume, and leakage of infusate in other animals was not associated with apparent adverse effects. The likelihood of adverse effects secondary to inappropriate targeting of liposomes to normal brain or CSF were minimized, since real-time imaging allowed for the termination of infusions, alteration of infusion rates, and/or redirection of infusion cannulae when leakage, distribution to normal brain, or static volumes of distribution were documented during the infusion process.
Although assessment of therapeutic efficacy was not a defined endpoint for the study, evidence for efficacy of intratumoral liposomal CPT-11 in these spontaneous tumors and indications of its potential mechanism of action were apparent in the study animals. The natural biology of canine spontaneous gliomas is poorly documented, as is response to conventional therapy such as radiation and chemotherapy. The endpoint for most animals is euthanasia rather than overall survival, and published “survival” data range from several days to several months for all treatment options. As such, any efficacy of direct intratumoral infusion of liposomal CPT-11 could only be assessed subjectively for individual cases on the basis of MRI evidence of static or decreasing tumor volumes and histopathology after euthanasia or death. Liposomal encapsulation has been shown to improve the pharmacokinetic properties and tissue residence time of CPT-11, which has been detected in rodent glioma models up to 2 months after a single CED administration.39,45
Consistent with this experimental data, the efficacy of treatment in dogs receiving multiple infusions appeared to be present over several weeks, based on serial MRI (Fig. ).
Five tumors decreased in volume ranging from 40% to 88%, and 1 dog with grade II astrocytoma (Dog 4) had apparently stable disease (Fig. ). The small area of malacia present within this tumor was probably because of expansion of the CT-biopsy cavity since pooling of infusate was noted in this area during treatments. Malacia secondary to toxic effects in infused neuropil was considered less likely because this was not a finding in other cases or in previous experimental dogs.40
Two tumors, both anaplastic oligodendrogliomas, had rapid growth progression and had no apparent response based either on MRI or histopathology. Importantly, the lack of response in these tumors was associated with successful intratumoral infusions, as defined by real-time imaging (Fig. ). The percent coverage of tumor determined by real-time imaging varied from 32% to 72%, a degree of coverage associated with apparent positive responses in other tumors. These cases, therefore, suggested that there was a true failure of the therapeutic drug rather than a failure of the delivery system. In contrast, the lack of response in Dog 2 (grade III astrocytoma) after the initial 2 infusions was clearly associated with poor Vd secondary to leakage and tumor coverage of <10% (Figs and ). Subsequent delivery of liposomal CPT-11 covering >50% of this tumor resulted in a marked decrease in tumor volume. Similar findings were apparent in Patient 1 (grade III astrocytoma) where limited infusions covering approximately 10% of tumor volume had only slight clinical effect compared with subsequent infusions where tumor coverage was 25% or greater (Fig. ). Although the number of dogs in the study was small and tumor phenotype varied, the critical value of real-time imaging in defining Vd of infusions and subsequent assessment of therapeutic efficacy of a novel therapeutic delivered by CED is clearly demonstrated, as are the potential pitfalls of assessing therapeutic value in the absence of imaging.
The mechanism for the decreased sensitivity and lack of response in the 2 anaplastic oligodendrogliomas is unclear. CPT-11, (7-ethyl-10-(4-[1-piperidinio]-1-piperidinio) carbonyloxycamptothecin) is a water-soluble derivative of the potent alkaloid anticancer agent camptothecin and acts as a specific inhibitor of topoisomerase I.34
CPT-11 is converted into a more active metabolite, SN-38 (7-ethyl-10-hydroxycamptothecin), by carboxylesterase (CES) activity, predominantly in the liver and serum.49
The precise contribution of SN-38 to the antineoplastic effect of CPT-11 compared with Irinotecan hydrochloride is unclear, as is the necessity for systemic metabolism. Some data suggest that intratumoral or neuropil activation of Irinotecan might be as important as systemic/hepatic activation and is an important factor when considering direct intratumoral infusion.50–60
One of the largest decreases in tumor volume (~90%) following therapy was seen in the third anaplastic oligodendroglioma, suggesting, not surprisingly, that factors influencing therapeutic efficacy are not consistent across individual tumors of similar histological type and grade. Further investigation of factors such as carboxylesterase and topoisomerase expression in individual treated tumors may be more informative regarding the sensitivity when tumor infusion has been documented by MRI.
A major advantage of the canine spontaneous tumor model is the availability of necropsy (all nonsurviving animals in this study), providing valuable correlative data often unavailable from human clinical trials. Two major responses were noted in tumors examined histopathologically: necrosis and a modulation of the tumor phenotype. Consistent with the predictive value of real-time imaging in relation to clinical response, histological findings after necropsy were also closely correlated with real-time infusion images. Overlay of MR infusion images and histological sections showed areas of tumor necrosis and/or modulation of tumor phenotype in areas of infused tumor, with residual/new tumor present beyond the margins of infusion exhibiting a more aggressive phenotype (Fig. ). Modulation of tumor phenotype was seen in 4 of 5 necropsied animals that displayed a decreased tumor volume or static disease. Modulation was characterized by more benign, less anaplastic cellular characteristics, and a decrease in the MIB-1 proliferative index (Fig. ; Table ). The mechanism for this altered phenotype following exposure to CPT-11 is unclear, but may involve selective targeting of a more proliferative compartment of tumor cells, leaving terminally differentiated tumor cells as a residual population. The authors have also seen this tumor modulation after CED of liposomal CPT-11 in orthotopic murine tumor models with a canine glioma cell line. The presence of essentially nondividing, yet apparently viable, tumor tissue after therapy has important implications for both the prognosis and assessment of efficacy simply measured in terms of reduction in tumor volume. Routine MRI did not discriminate between untreated tumor and tumor with modified phenotype, and additional imaging approaches such as diffusion-weighted imaging and spectroscopy may be beneficial in defining this modified compartment of the tumor.
In summary, we have shown that canine spontaneous gliomas may provide a valuable large animal, spontaneous tumor model for the investigation of novel delivery, and novel therapeutic strategies for intracranial tumors. It is hoped that retrospective analysis of MRI parameters and infusion data in this more clinically realistic spontaneous model may help to further optimize cannula and infusion parameters. CED of a chemotherapeutic agent (liposomal CPT-11) directly into spontaneous gliomas in a large volume brain is feasible and well tolerated clinically. On the basis of responses in canine patients, intratumoral liposomal CPT-11 should be considered as a potential therapeutic option, particularly in nonresectable or recurrent tumors. As was the case with these spontaneous gliomas in dogs, assessment and optimization of any therapy delivered by CED in future human glioma clinical trials will depend critically on the ability to accurately define infusions in real time. In addition to providing an appropriate mechanistic and biological environment to assess CED parameters more realistically, the canine model has provided an equally realistic system to assess potential adverse effects associated with the infusion procedure, the delivery system, therapeutic agent, and surrogate marker in the context of a large immunocompetent mammal with ongoing intracranial disease.
Conflict of interest statement. D.B.K., C.O.N., and D.C.D. both hold stock and stock options in Merrimack Pharmaceuticals, the company working to develop this novel drug formulation. D.B.K. was previously the Vice President of Pharmaceutical Research and Development for Hermes Biosciences, a company working to develop the product that was recently merged with Merrimack Pharmaceuticals. D.B.K. and D.C.P. are the inventors on a patent application that describes the highly stabilized nanoliposome formulation of CPT-11 used in this manuscript. D.C.P. is the Senior Director of Research for Merrimack Pharmaceuticals. J.W.P. is a Board member, officer in Hermes Biosciences, Inc. and also has equity in the same company. The University of California–San Francisco has a patent related to this work.