Sensitization of Human Cancer Cells to Gemcitabine by the Chk1 Inhibitor MK-8776: Cell Cycle Perturbation and Impact of Administration Schedule in Vitro and in Vivo

Chk1 inhibitors have emerged as promising anticancer therapeutic agents particularly when combined with antimetabolites such as gemcitabine, cytarabine or hydroxyurea. Here, we address the importance of appropriate drug scheduling when gemcitabine is combined with the Chk1 inhibitor MK-8776, and the mechanisms involved in the schedule dependence

Synergistic Combination of Hyperoxygenation and Radiotherapy by Repeated Assessments of Tumor pO2 with EPR Oximetry

The effect of hyperoxygenation with carbogen (95% O2 + 5% CO2) inhalation on RIF-1 tumor pO2and its consequence on growth inhibition with fractionated radiotherapy is reported. The temporal changes in the tumor pO2 were assessed by in vivo Electron Paramagnetic Resonance (EPR) oximetry in mice breathing 30% O2 or carbogen and the tumors were irradiated with 4 Gy/day for 5 consecutive days; a protocol that emulates the clinical application of carbogen. The RIF-1 tumors were hypoxic with a tissue pO2 of 5–9 mmHg. Carbogen (CB) breathing significantly increased tumor pO2, with a maximum increase at 22.9–31.2 min on days 1–5, however, the magnitude of increase in pO2 declined on day 5. Radiotherapy during carbogen inhalation (CB/RT) resulted in a significant tumor growth inhibition from day 3 to day 6 as compared to 30%O2/RT and carbogen (CB/Sham RT) groups. The results provide unambiguous quantitative information on the effect of carbogen inhalation on tumor pO2 over the course of 5 days. Tumor growth inhibition in the CB/RT group confirms that the tumor oxygenation with carbogen was radiobiologically significant. Repeated tumor pO2 measurements by EPR oximetry can provide temporal information that could be used to improve therapeutic outcomes by scheduling doses at times of improved tumor oxygenation

First-In-Human Study in Cancer Patients Establishing the Feasibility of Oxygen Measurements in Tumors Using Electron Paramagnetic Resonance With the OxyChip

Objective: The overall objective of this clinical study was to validate an implantable oxygen sensor, called the ‘OxyChip’, as a clinically feasible technology that would allow individualized tumor-oxygen assessments in cancer patients prior to and during hypoxia-modification interventions such as hyperoxygen breathing. Methods: Patients with any solid tumor at ≤3-cm depth from the skin-surface scheduled to undergo surgical resection (with or without neoadjuvant therapy) were considered eligible for the study. The OxyChip was implanted in the tumor and subsequently removed during standard-of-care surgery. Partial pressure of oxygen (pO2) at the implant location was assessed using electron paramagnetic resonance (EPR) oximetry. Results: Twenty-three cancer patients underwent OxyChip implantation in their tumors. Six patients received neoadjuvant therapy while the OxyChip was implanted. Median implant duration was 30 days (range 4–128 days). Forty-five successful oxygen measurements were made in 15 patients. Baseline pO2 values were variable with overall median 15.7 mmHg (range 0.6–73.1 mmHg); 33% of the values were below 10 mmHg. After hyperoxygenation, the overall median pO2 was 31.8 mmHg (range 1.5–144.6 mmHg). In 83% of the measurements, there was a statistically significant (p ≤ 0.05) response to hyperoxygenation. Conclusions: Measurement of baseline pO2 and response to hyperoxygenation using EPR oximetry with the OxyChip is clinically feasible in a variety of tumor types. Tumor oxygen at baseline differed significantly among patients. Although most tumors responded to a hyperoxygenation intervention, some were non-responders. These data demonstrated the need for individualized assessment of tumor oxygenation in the context of planned hyperoxygenation interventions to optimize clinical outcomes

Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration schedule in vitro and in vivo

Background: Chk1 inhibitors have emerged as promising anticancer therapeutic agents particularly when combined with antimetabolites such as gemcitabine, cytarabine or hydroxyurea. Here, we address the importance of appropriate drug scheduling when gemcitabine is combined with the Chk1 inhibitor MK-8776, and the mechanisms involved in the schedule dependence. Methods: Growth inhibition induced by gemcitabine plus MK-8776 was assessed across multiple cancer cell lines. Experiments used clinically relevant bolus administration of both drugs rather than continuous drug exposures. We assessed the effect of different treatment schedules on cell cycle perturbation and tumor cell growth in vitro and in xenograft tumor models. Results: MK-8776 induced an average 7-fold sensitization to gemcitabine in 16 cancer cell lines. The time of MK-8776 administration significantly affected the response of tumor cells to gemcitabine. Although gemcitabine induced rapid cell cycle arrest, the stalled replication forks were not initially dependent on Chk1 for stability. By 18 h, RAD51 was loaded onto DNA indicative of homologous recombination. Inhibition of Chk1 at 18 h rapidly dissociated RAD51 leading to the collapse of replication forks and cell death. Addition of MK-8776 from 18-24 h after a 6-h incubation with gemcitabine induced much greater sensitization than if the two drugs were incubated concurrently for 6 h. The ability of this short incubation with MK-8776 to sensitize cells is critical because of the short half-life of MK-8776 in patients\u27 plasma. Cell cycle perturbation was also assessed in human pancreas tumor xenografts in mice. There was a dramatic accumulation of cells in S/G(2) phase 18 h after gemcitabine administration, but cells had started to recover by 42 h. Administration of MK-8776 18 h after gemcitabine caused significantly delayed tumor growth compared to either drug alone, or when the two drugs were administered with only a 30 min interval. Conclusions: There are two reasons why delayed addition of MK-8776 enhances sensitivity to gemcitabine: first, there is an increased number of cells arrested in S phase; and second, the arrested cells have adequate time to initiate recombination and thereby become Chk1 dependent. These results have important implications for the design of clinical trials using this drug combination

Dynamic changes in oxygenation of intracranial tumor and contralateral brain during tumor growth and carbogen breathing:a multisite EPR oximetry with implantable resonators

INTRODUCTION: Several techniques currently exist for measuring tissue oxygen; however technical difficulties have limited their usefulness and general application. We report a recently developed electron paramagnetic resonance (EPR) oximetry approach with multiple probe implantable resonators (IRs) that allow repeated measurements of oxygen in tissue at depths of greater than 10 mm. METHODS: The EPR signal to noise (S/N) ratio of two probe IRs was compared with that of LiPc deposits. The feasibility of intracranial tissue pO(2) measurements by EPR oximetry using IRs was tested in normal rats and rats bearing intracerebral F98 tumors. The dynamic changes in the tissue pO(2) were assessed during repeated hyperoxia with carbogen breathing. RESULTS: A 6–10 times increase in the S/N ratio was observed with IRs as compared to LiPc deposits. The mean brain pO(2) of normal rats was stable and increased significantly during carbogen inhalation in experiments repeated for 3 months. The pO(2) of F98 glioma declined gradually, while the pO(2) of contralateral brain essentially remained the same. Although a significant increase in the glioma pO(2) was observed during carbogen inhalation, this effect declined in experiments repeated over days. CONCLUSION: EPR oximetry with IRs provides a significant increase in S/N ratio. The ability to repeatedly assess orthotopic glioma pO(2) is likely to play a vital role in understanding the dynamics of tissue pO(2) during tumor growth and therapies designed to modulate tumor hypoxia. This information could then be used to optimize chemoradiation by scheduling treatments at times of increased glioma oxygenation