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Supplementary Data from Pharmacokinetic Analysis and Phase 1 Study of MRX-1024 in Patients Treated with Radiation Therapy with or without Cisplatinum for Head and Neck Cancer

Purpose: A previous study reported radiation protection from mucosal injury with D-methionine (D-met) in preclinical evaluation; therefore, the pharmacokinetics, safety, and utility of D-met were evaluated clinically.Experimental Design: The pharmacokinetics of D-met following oral administration of a bioavailable formulation (MRX-1024) was evaluated in normal volunteers. Subsequently, 25 patients were enrolled on a phase 1 study of MRX-1024 concurrent with radiation therapy (RT) with or without weekly cisplatinum. Toxicity and mucosal events were evaluated weekly.Results: Oral MRX-1024 resulted in rapid and dose-dependent increases in plasma D-met concentrations with a half-life of 3 hours. When administered concurrent with RT without chemotherapy, it was associated with a modest increase in grade 2 (two of six patients) and grade 3 (one of six patients) emesis. In those treated with MRX-1024 along with RT and weekly cisplatinum, there was no appreciable increase in emesis. Overall, five patients withdrew from the study due to emesis (four grade 2 and one grade 3). Only one incidence of dose-limiting toxicity (grade 3 emesis) was identified in 25 patients (4%). Finally, in 18 evaluable patients treated with MRX-1024 at 100 mg/kg twice daily (BID), the incidence of severe (grade 3) oral mucositis was 6% (1 of 18) with no grade 4 mucositis.Conclusions: There is a dose-dependent increase in D-met exposure following MRX-1024 administration at 50 and 100 mg/kg, and MRX-1024 is safe for use concurrent with combined radiation and chemotherapy. The observed rate of mucositis seems less than that for similar treatment regimens within the published literature. Clin Cancer Res; 16(9); 2666–76. ©2010 AACR.

Purpose: Oral mucositis is a common acute morbidity associated with radiation and/or chemotherapy treatment for cancer. d-Methionine (d-Met), the dextro-isomer of the common amino acid l-methionine, has been documented to protect normal tissues from a diverse array of oxidative insults.Experimental Design: We evaluated if d-Met could selectively prevent radiation-induced oral mucositis using in vitro cell culture models as well as an in vivo model of radiation injury to the oral mucosa in C3H mice.Results: Unlike free-radical scavengers, which protected both normal and transformed tumor cells in vitro from radiation-induced cell death, treatment with d-Met in culture protected nontransformed primary human cells from radiation-induced cell death (protective factor between 1.2 and 1.6; P < 0.05) whereas it did not confer a similar protection on transformed tumor cells. d-Met treatment also provided significant protection to normal human fibroblasts, but not to tumor cell lines, from radiation-induced loss of clonogenicity (protection factor, 1.6 ± 0.15). d-Met treatment did not alter DNA damage (as measured by histone phosphorylation) following irradiation but seemed to selectively mitigate the loss of mitochondrial membrane potential in nontransformed cells, whereas it did not provide a similar protection to tumor cells. Tumor control of implanted xenografts treated with radiation or concurrent cisplatin and radiation was not altered by d-Met treatment. Pharmacokinetics following administration of a liquid suspension of d-Met in rats showed 68% bioavailability relative to i.v. administration. Finally, in a murine model of mucositis, a dose-dependent increase in protection was observed with the protective factor increasing from 1.6 to 2.6 over a range of oral d-Met doses between 200 and 500 mg/kg (P < 0.0003).Conclusions:d-Met protected normal tissues, but not tumor cells, in culture from radiation-induced cell death; it also protected normal cells from radiation-induced mucosal injury in a murine model but did not alter tumor response to therapy. Further studies on the use of d-Met to protect from oral mucositis are warranted.

Supplementary Data from Inhibition of mTOR Radiosensitizes Soft Tissue Sarcoma and Tumor Vasculature

Supplementary Data from Evaluation of d-Methionine as a Novel Oral Radiation Protector for Prevention of Mucositis

Purpose: Currently, radiologic response of brain tumors is assessed according to the Macdonald criteria 10 weeks from the start of therapy. There exists a critical need to identify nonresponding patients early in the course of their therapy for consideration of alternative treatment strategies. Our study assessed the effectiveness of the parametric response map (PRM) imaging biomarker to provide for an earlier measure of patient survival prediction. Experimental Design: Forty-five high-grade glioma patients received concurrent chemoradiation. Quantitative MRI including apparent diffusion coefficient (ADC) and relative cerebral blood volume (rCBV) maps were acquired pretreatment and 3 weeks midtreatment on a prospective institutional-approved study. PRM, a voxel-by-voxel image analysis method, was evaluated as an early prognostic biomarker of overall survival. Clinical and conventional MR parameters were also evaluated. Results: Multivariate analysis showed that PRMADC+ in combination with PRMrCBV− obtained at week 3 had a stronger correlation to 1-year and overall survival rates than any baseline clinical or treatment response imaging metric. The composite biomarker identified three distinct patient groups, nonresponders [median survival (MS) of 5.5 months, 95% CI: 4.4–6.6 months], partial responders (MS of 16 months, 95% CI: 8.6–23.4 months), and responders (MS has not yet been reached). Conclusions: Inclusion of PRMADC+ and PRMrCBV− into a single imaging biomarker metric provided early identification of patients resistant to standard chemoradiation. In comparison to the current standard of assessment of response at 10 weeks (Macdonald criteria), the composite PRM biomarker potentially provides a useful opportunity for clinicians to identify patients who may benefit from alternative treatment strategies. Clin Cancer Res; 17(14); 4751–60. ©2011 AACR.

Purpose: The PI3K/Akt/mTOR prosurvival pathway is frequently up-regulated in soft tissue sarcoma. Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin, have recently shown clinical benefit in soft tissue sarcoma, and mTOR inhibition has also been associated with radiosensitization of carcinoma and endothelial cells. This study tested the hypothesis that rapamycin radiosensitizes soft tissue sarcoma and endothelial cells in vitro and in vivo through the inhibition of mTOR. Experimental Design: Colony formation assays were done to determine the radiosensitizing properties of rapamycin on three human soft tissue sarcoma cell lines (SK-LMS-1, SW-872, and HT-1080) and human dermal microvascular endothelial cells (HDMEC). The functional effects of rapamycin and radiation on the endothelial compartment were evaluated with microvascular sprouting assays. The in vivo radiosensitizing activity of rapamycin was assessed with s.c. SK-LMS-1 nude mice xenografts treated with concurrent daily rapamycin, radiation, or both for three weeks. Results: In vitro radiosensitization was shown in all three soft tissue sarcoma cell lines with minimally cytotoxic doses of rapamycin. SK-LMS-1 xenografts displayed significant tumor growth delay with rapamycin and radiation compared with either treatment alone. Radiation resulted in transient increased mTOR function, whereas rapamycin abolished this signaling in irradiated and unirradiated samples. In HDMEC, rapamycin and radiation reduced microvessel sprouting, but did not alter colony formation. Conclusions: Minimally cytotoxic concentrations of rapamycin inhibited the mTOR cascade in culture and in vivo while radiosensitizing soft tissue sarcoma, and produced synergistic effects with radiation on HDMEC microvessel formation. By targeting both tumor and endothelial compartments, rapamycin produced potent radiosensitization of soft tissue sarcoma xenografts. Clinical trials combining rapamycin and radiotherapy in soft tissue sarcoma are warranted.

Purpose: In a previous report, a recombinant luciferase reporter, activated during apoptosis via caspase-3 cleavage, was developed for imaging of apoptosis using bioluminescence. The ability to noninvasively image apoptosis in vivo could dramatically benefit the preclinical development of therapeutics targeting the apoptotic pathway. In this study, we examined the use of 5-fluorouracil (5-FU) for sensitizing D54 tumors to tumor necrosis factor α–related apoptosis–inducing ligand (TRAIL) therapy by monitoring apoptotic activity in vivo using bioluminescence imaging. Experimental Design: Using our apoptosis imaging platform and diffusion magnetic resonance imaging (MRI), we monitored the antitumor effects of 5-FU, TRAIL, and 5-FU + TRAIL using D54 xenografts. Additionally, volumetric and histologic analyses were done for correlation with findings from bioluminescence imaging and diffusion MRI. Results: Bioluminescence imaging showed that therapy with TRAIL alone produced an initial 400% increase in apoptotic activity that rapidly diminished during the 10-day treatment period despite continued therapy. In contrast, concomitant 5-FU and TRAIL therapy elicited an apoptotic response that was sustained throughout the entire therapeutic course. Using diffusion MRI, an enhanced tumor response was detected when concomitant therapy was given versus TRAIL-alone therapy. Last, concomitant therapy resulted in a prolonged growth delay (∼9 days) compared with TRAIL alone (∼4 days). Conclusion: We showed that concomitant 5-FU and TRAIL therapy indeed enhanced apoptotic activity in vivo, which translated into greater tumor control. Moreover, this technique sheds light on the synergy of 5-FU and TRAIL as evidenced by differences in the temporal activation of caspase-3 resulting from the different therapeutic regimens.

We assessed whether a new method of quantifying therapeutic-associated hemodynamic alterations may help to distinguish pseudoprogression from true progression in patients with high grade glioma. Patients from a prospective IRB-approved trial with high grade glioma received concurrent chemoradiation. Relative cerebral blood volume (rCBV) and blood flow (rCBF) maps were acquired prior and at week 3 mid-treatment. Pseudoprogression was defined as imaging changes 1-3 months after chemoradiation that mimic tumor progression but stabilized or improved without change in treatment or for which resection revealed radiation effects only. Clinical and conventional MR parameters, including average percent change of rCBV and CBF, were evaluated as potential predictors of pseudoprogression. Parametric response map (PRM), an innovative, voxel-by-voxel method of image analysis, was also performed. Median radiation dose was 72 Gy (range: 60-81). Of 27 patients, stable disease/partial response was noted in 13 and apparent progression in 14. Adjuvant temozolomide was continued in all patients. Pseudoprogression occurred in 6 patients. Based upon PRM analysis, a significant portion of the tumor volume had reduced rCBV (PRMrCBV) at week 3 in patients with progressive disease compared to those with pseudoprogression (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2698.

We assessed whether quantification of therapeutic-associated tumor cellularity and hemodynamic alterations may help in the early assessment of treatment resistance in patients with high grade glioma. Patients with high grade glioma (n=45) received concurrent chemoradiation. Apparent diffusion coefficient (ADC) and relative cerebral blood volume (rCBV) maps were acquired prior to chemoradiation and at week 3 during treatment on a prospective IRB-approved study. Clinical and conventional MR parameters, including average baseline ADC and rCBV and radiological response, were evaluated as prognostic indicators of one-year survival. Parametric response map (PRM), an innovative, voxel-by-voxel method of image analysis, was also performed. Of 45 patients, median survival was found to be 14.9 months with 44% of patients living shorter than one year (median survival of 6.6 months). Baseline ADC was the only single model not found to be predictive of survival at one year, whereas in a multivariate analysis PRMADC+ and PRMrCBV- had a stronger correlation to survival than baseline rCBV. Overall, a composite model of PRMADC+ and PRMrCBV- was found to have the strongest correlation with one-year survival. This study demonstrates the potential of a composite parametric model sensitive to both tumor cellularity and hemodynamic alterations following first line therapy for providing an early assessment of patient resistant to chemoradiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3754.

Introduction: Standard assessment of radiologic response in patients with malignant glioma is traditionally carried out using T1 gadolinium-enhanced MRI at approximately 10 weeks following treatment initiation. Diffusion-weighted MRI of gliomas, analyzed by the functional diffusion map (fDM), may provide an early measure of response during treatment to predict patient survival (OS). Tumor cells restrict the random diffusion of water molecules within a tumor; therefore, diffusion will increase during successful therapy. The fDM imaging biomarker is a voxel-wise method for analysis of DW-MRI data providing for quantification of treatment-associated spatial alterations in tumor diffusion values earlier than radiologic response. Methods: A prospective single-institution trial of early assessment of brain tumor response by MRI was performed early during radiation therapy. Sixty patients with high grade glioma undergoing radiation therapy were enrolled in an IRB-approved study of intra-treatment MRI 1, 3, and 10 weeks after the initiation of treatment. Receiver operating characteristic curve analysis was used to evaluate imaging parameters as a function of patient survival at one-year. Log Rank and Cox Proportional Hazards models were utilized to assess OS. Results: Measurable changes in diffusion MRI were observed as early as one week into treatment. The volume of tumor with increased diffusion as assessed by fDM 3 weeks after the start of therapy was the strongest predictor of patient survival at one year, with larger fDM predicting longer median survival [52.6 vs. 10.9 months, log-rank, P Conclusions: Compared to conventional neuro-imaging, fDM provided an earlier assessment of equal predictive value during the course of treatment, providing the opportunity to individualize therapy. The combination of fDM and RR provided a more accurate prediction of patient survival than either metric alone.