Determination of an optimal dosing schedule for combining Irinophore C™ and temozolomide in an orthotopic model of glioblastoma.

Our laboratory reported that Irinophore C™ (IrC™; a lipid-based nanoparticulate formulation of irinotecan) is effective against an orthotopic model of glioblastoma (GBM) and that treatment with IrC™ was associated with vascular normalization within the tumor. Here, the therapeutic effects of IrC™ when used in combination with temozolomide (TMZ) in concurrent and sequential treatment schedules were tested. It was anticipated that IrC™ engendered vascular normalization would increase the delivery of TMZ to the tumor and that this would be reflected by improved treatment outcomes. The approach compared equally efficacious doses of irinotecan (IRN; 50 mg/kg) and IrC™ (25 mg/kg) in order to determine if there was a unique advantage achieved when combining TMZ with IrC™. The TMZ sensitive U251MG(O) cell line (null expression of O-6-methylguanine-DNA methyltransferase (MGMT)) modified to express the fluorescent protein mKate2 was inoculated orthotopically into NOD.CB17-SCID mice and treatment was initiated 14 days later. Our results demonstrated that IrC™ and TMZ administered concurrently resulted in optimal treatment outcomes, with 50% long term survivors (>180 days) in comparison to 17% long term survivors in animals treated with IRN and TMZ or TMZ alone. Indeed, the different treatments resulted in a 353%, 222% and 280% increase in median survival time (MST) compared to untreated animals for, respectively, IrC™ combined with TMZ, IRN combined with TMZ, and TMZ alone. When TMZ was administered after completion of IRN or IrC™ dosing, an increase in median survival time of 167-174% was observed compared to untreated animals and of 67% and 74%, respectively, when IRN (50 mg/kg) and IrC™ (25mg/kg) were given as single agents. We confirmed in these studies that after completion of the Q7D×3 dosing of IrC™, but not IRN, the tumor-associated vascular was normalized as compared to untreated tumors. Specifically, reductions in the fraction of collagen IV-free CD31 staining (p<0.05) and reductions in tumor vessel diameter were observed in tumors from IrC™-treated animals when compared to tumors from untreated or IRN treated animals. Analysis by transmission electron microscopy of the ultra-structure of tumors from IrC™-treated and untreated animals revealed that tumor-associated vessels from treated animals were smaller, more organized and exhibited a morphology comparable to normal blood vessels. In conclusion, optimal treatment outcomes were achieved when IrC™ and TMZ were administered concurrently, whereas IrC™ followed by TMZ treatment given sequentially did not confer any therapeutic advantage.

Irinophore C™, a lipid-based nanoparticulate formulation of irinotecan, is more effective than free irinotecan when used to treat an orthotopic glioblastoma model.

Given compelling evidences supporting the therapeutic potential of irinotecan (IRN) for patients with glioblastoma (GBM), the present study evaluated the activity of Irinophore C™ (IrC™), a lipid-based nanopharmaceutical formulation of IRN, in GBM. The levels of IRN and SN-38 were determined in plasma and brain after a single intravenous dose of IRN or IrC™ in tumor-free mice. Treatment with IrC™ significantly increased the plasma AUC(0-24h) of the active (lactone) forms of IRN and SN-38 when compared to free drug (760 and 30-fold increase, respectively). Levels of IRN and SN-38 in brain tissue were also increased significantly (compared to IRN treatment) following IrC™ administration. A tolerability study revealed that IrC™ is better tolerated than IRN. The efficacy of IrC™ and IRN was assessed in an orthotopic model of GBM. The therapeutic efficacy of IrC™ given at 25mg/kg weekly was comparable to the efficacy achieved using twice the dose of IRN. At the maximum tolerated dose, IrC™ (100mg/kg) increased the survival time of tumor-bearing mice of 83% compared to untreated animals. Ki67 immunostaining analysis of IrC™-treated tumors revealed a transient increase in cell proliferation after treatment. The results justify further studies evaluating the use of IrC™ for treating GBM.

Metronomic gemcitabine suppresses tumour growth, improves perfusion, and reduces hypoxia in human pancreatic ductal adenocarcinoma.

BACKGROUND: The current standard of care for pancreatic cancer is weekly gemcitabine administered for 3 of 4 weeks with a 1-week break between treatment cycles. Maximum tolerated dose (MTD)-driven regimens as such are often associated with toxicities. Recent studies demonstrated that frequent dosing of chemotherapeutic drugs at relatively lower doses in metronomic regimens also confers anti-tumour activity but with fewer side effects. METHODS: Herein, we evaluated the anti-tumour efficacy of metronomic vs MTD gemcitabine, and investigated their effects on the tumour microenvironment in two human pancreatic cancer xenografts established from two different patients. RESULTS: Metronomic and MTD gemcitabine significantly reduced tumour volume in both xenografts. However, K(trans) values were higher in metronomic gemcitabine-treated tumours than in their MTD-treated counterparts, suggesting better tissue perfusion in the former. These data were further supported by tumour-mapping studies showing prominent decreases in hypoxia after metronomic gemcitabine treatment. Metronomic gemcitabine also significantly increased apoptosis in cancer-associated fibroblasts and induced greater reductions in the tumour levels of multiple pro-angiogenic factors, including EGF, IL-1alpha, IL-8, ICAM-1, and VCAM-1. CONCLUSION: Metronomic dosing of gemcitabine is active in pancreatic cancer and is accompanied by pronounced changes in the tumour microenvironment.

The phosphoinositide-dependent kinase-1 inhibitor 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012) prevents Y-box binding protein-1 from inducing epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is integral to basal-like and human epidermal growth factor receptor-2 (Her-2)-overexpressing breast cancers. Such tumors are associated with poor prognosis, the majority of which express high levels of EGFR. We reported that EGFR expression is induced by the oncogenic transcription factor Y-box binding protein-1 (YB-1) that occurs in a manner dependent on phosphorylation by Akt. Herein, we questioned whether blocking Akt with 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012), a phosphoinositide-dependent protein kinase-1 (PDK-1) small-molecule inhibitor, could prevent YB-1 from binding to the EGFR promoter. MDA-MB-468 and SUM 149 are basal-like breast cancer (BLBC) cells that were used for our studies because they express high levels of activated PDK-1, YB-1, and EGFR compared with the immortalized breast epithelial cell line 184htrt. In these cell lines, YB-1 preferentially bound to the -1 kilobase of the EGFR promoter, whereas this did not occur in the 184htrt cells based on chromatin immunoprecipitation. When the cells were exposed to OSU-03012 for 6 h, YB-1/EGFR promoter binding was significantly attenuated. To further confirm this observation, gel-shift assays showed that the drug inhibits YB-1/EGFR promoter binding. The inhibitory effect of OSU-03012 on EGFR was also observed at the mRNA and protein levels. OSU-03012 ultimately inhibited the growth of BLBC in monolayer and soft agar coordinate with the induction of apoptosis using an Array-Scan VTI high-content screening system. Furthermore, OSU-03012 inhibited the expression of EGFR by 48% in tumor xenografts derived from MDA-MB-435/Her-2 cells. This correlated with loss of YB-1 binding to the EGFR promoter. Hence, we find that OSU-03012 inhibits YB-1 resulting in a loss of EGFR expression in vitro and in vivo.

Development and assessment of conventional and targeted drug combinations for use in the treatment of aggressive breast cancers.

Combination chemotherapy has been at the forefront of cancer treatment for over 40 years. However, the rationale for selecting drug combinations and the process used to demonstrate clinical effectiveness has primarily followed trial and error methodology. Typically, the selection and assessment of combined drug therapies has been based on the effectiveness of each agent as monotherapy in treating the neoplasm and avoiding overlapping toxicities, followed by clinical trials to establish dose scheduling, toxicity, and efficacy. Unfortunately, this scheme is inefficient in terms of the time required to complete and revise these clinical trials based on the outcome to optimize the drug combination. A more rational approach for the development of combination oncology products should consider (i) in vitro assays for assessing therapeutic effects of drug combinations (antagonistic, additive or synergistic interactions) when added simultaneously; (ii) methods for measuring these interactions in vivo; (iii) the importance of understanding pharmacokinetic and biodistribution parameters when using drug combinations; (iv) the need to assess pathways known to contribute to cancer cell survival as well as metastasis; and (iv) the need to assess the fate of different cell populations (cancer and stroma) contributing to the development of cancer. Therefore, the goal of this article is to provide a road map for the preclinical development of drug combination products that will have improved therapeutic activity and a high likelihood of providing beneficial therapeutic outcomes in patients with aggressive cancers with a specific focus on patients with breast cancer.

Tumor radiosensitization by sustained intratumoral release of bromodeoxyuridine.

We have previously reported that the use of the polymer bis(p-carboxyphenoxy)propane-sebacic acid (20:80) for intratumoral delivery of cis-platinum in a mouse tumor model (RIF-1) potentiated the effects of acute and fractionated radiation. This mode of drug delivery seems particularly applicable to the administration of radiosensitizing drugs because an optimum concentration of radiosensitizer can be maintained in the tumor over the prolonged period required for fractionated radiation treatment. We have now investigated, in the same tumor model, radiosensitization by the thymidine analogue bromodeoxyuridine (BrdUrd). BrdUrd (20%, w/w) was incorporated into bis(p-carboxyphenoxy)propane-sebacic acid (20:80) and polymer rods containing the drug implanted in the RIF-1 tumor. Preliminary in vitro studies of the rate of release of BrdUrd from the polymer showed an initial rapid loss over 24 h, followed by a slower release extending over the next 5 days. In experiments in which tumor cells, which had incorporated BrdUrd in vivo from implanted polymer, were excised and a single cell suspension irradiated in vitro radiosensitization indicative of BrdUrd incorporation was associated mainly with an increase in the alpha constant for the linear quadratic model of cell survival. Radiosensitization was seen for tumor cells harvested between 5 and 10 days after polymer implant, a finding that is consistent with results of experiments in which the percentage of cells that had incorporated BrdUrd were measured by flow cytometry at various times after polymer/BrdUrd implant. The proportion of tumor cells positive for BrdUrd was 40-50% between 3 and 8 days after polymer implant. When tumors were irradiated in situ and response measured in terms of tumor growth delay (TGD), radiosensitization was not seen for an acute dose of 16.5 Gy. In contrast, significant radiosensitization was seen for fractionated treatments when polymer/BrdUrd was implanted 3 days before the first radiation dose. For a dose of 5 x 6 Gy, TGD was increased from 22 days for radiation alone to 27 days for radiation plus polymer implant. For 10 x 6 Gy fractions, TGD increased from 45-77 days for those mice in whom the tumor eventually regrew, whereas for 25% of the mice in this group the tumor volume was reduced to a point where it was no longer detectable and there was no recurrence for at least 120 days after treatment.

Radiosensitization of a mouse tumor model by sustained intra-tumoral release of etanidazole and tirapazamine using a biodegradable polymer implant device.

BACKGROUND AND PURPOSE: Drug toxicities are often a limiting factor in long term treatment regimes used in conjunction with radiotherapy. If the drug could be localized to the tumor site and released slowly, then optimal, intra-tumoral drug concentrations could be achieved without the cumulative toxicity associated with repeated systemic drug dosage. In this paper we describe the use of a biodegradable polymer implant for sustained intra-tumoral release of high concentrations of drugs targeting hypoxic cells. MATERIALS AND METHODS: The RIF-1 tumor was implanted subcutaneously or intramuscularly in C3H mice and irradiated with 60Co gamma rays. The drug delivery device was the co-polymer CPP-SA;20:80 into which the drug was homogeneously incorporated. The hypoxic radiosensitizer Etanidazole or the bioreductive drug Tirapazamine were delivered intra-tumorally by means of implanted polymer rods containing the drugs. Tumor growth delay (TGD) was used as the end point in these experiments. RESULTS: Both Etanidazole and Tirapazamine potentiated the effects of acute and fractionated radiation in the intra-muscular tumors but neither drug was effective in sub-cutaneous tumors. Since both drugs target hypoxic cells we hypothesized that the lack of effect in the subcutaneous tumor was attributable to the smaller size of the hypoxic fraction in this tumor model. This was confirmed using the hypoxia marker EF5. CONCLUSIONS: These results indicate that the biodegradable polymer implant is an effective vehicle for the intra-tumoral delivery of Etanidazole and Tirapazamine and that, in conjunction with radiation, this approach could improve treatment outcome in tumors which contain a sub-population of hypoxic, radioresistant cells.

Cisplatin delivery by biodegradable polymer implant is superior to systemic delivery by osmotic pump or i.p. injection in tumor-bearing mice.

The use of biodegradable polymer implants to deliver cisplatin was compared with delivery by systemic injection and by osmotic pump. Drug levels in the tumor were found to be higher than those in the blood and kidney when the drug was delivered using the polymer implant. In contrast, for the other two delivery methods blood and kidney cisplatin levels were greater than those in the tumor. It has been previously shown that tumor response, in terms of growth delay, was greatest when drug was delivered by polymer implant and least when treatment was by osmotic pump.

The potentiation of the effect of radiation treatment by intratumoral delivery of cisplatin.

PURPOSE: To compare potentiation of the effects of acute or fractionated radiation by cisplatin when the drug was delivered intratumorally by implanted biodegradable polymer, by intraperitoneal injection, or by intraperitoneal osmotic pump. METHODS AND MATERIALS: Radiation was delivered to a mouse tumor (RIF-1) either in a single dose or in a fractionated regime in conjunction with cisplatin delivered either as a bolus injection, with an osmotic pump, or with a biodegradable polymer rod containing cisplatin. The osmotic pump was implanted in the intraperitoneal cavity of the mouse while the polymer implants were placed directly in the tumor. As the polymer degrades, the drug is released at the treatment site leading to high local concentrations. The osmotic pump, in contrast, leads to prolonged systemic exposure to the drug at low concentrations. Tumor growth delay (TGD) was used as an endpoint in these experiments. RESULTS: The most effective treatment protocol, in terms of potentiating the effects of radiation was cisplatin delivered by polymer implanted 2 days before an acute dose of radiation (growth modification factor [DMF] = 2.2). Comparison of single and multifraction regimes where polymer implant was on the same day as the commencement of treatment showed greater potentiation of the effect of fractionated than of acute radiation treatment with the DMF for fractionated treatment remaining relatively constant (1.5-1.9) for 5, 8, and 12 fraction treatments. Cisplatin delivered via the osmotic pump did not deliver a high enough dose of cisplatin to produce therapeutic effect in this mouse tumor model and had little impact on response to treatment. CONCLUSIONS: Our results indicated that cisplatin delivered intratumorally by biodegradable polymer implant was effective in potentiating the effect of both acute and fractionated radiation. For the fractionated treatments the effect was maintained with increasing fraction numbers and treatment time.

Tumor treatment by sustained intratumoral release of cisplatin: effects of drug alone and combined with radiation.

PURPOSE: The effect of intratumoral delivery of cisplatin to a mouse tumor model (RIF-1) by means of a biodegradable polymer implant with and without radiation was studied. METHODS AND MATERIALS: The polymer bis (p-carboxyphenoxy) propane-sebacic acid (CPP:SA; 80:20) and its degradation products have been characterized. Polymer rods (8 x 0.5 mm) containing 17% cisplatin by weight were prepared by extrusion, and the in vitro degradation rate measured. The implants were placed into mouse tumors and their effect (with and without radiation) on tumor growth delay studied. The levels of Pt in the mouse kidney, tumor, and blood plasma at selected intervals after implant were also determined. These results were compared with those obtained when cisplatin was delivered systemically. RESULTS: When cisplatin was delivered by the polymer implants, higher levels were present in the tumor for longer time periods (cf. systemic delivery of the drug). For both nonirradiated and irradiated tumors, those treated with the polymer implants had significantly longer tumor growth delays compared to nonimplanted controls and to systematically treated tumors. CONCLUSIONS: The results show that intratumoral delivery of cisplatin is more efficient than systemic delivery. Using the biodegradable polymer implant, higher doses of cisplatin can be tolerated by the animal as the drug is localized within the tumor, and the high levels of the drug in the tumor can be maintained for an extended period of time. When radiation is given in conjunction with cisplatin, the tumor response is supraadditive for all modes of cisplatin administration but is potentiated to a greater extent when cisplatin is delivered through the polymer implant. The greatest effect is seen for treatment with cisplatin delivered by polymer implant combined with fractionated radiation.