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Effect of the Allosteric Hemoglobin Modifier RSR13 on Oxygenation in Murine Tumors

H. Hou1, N. Khan1, J.A. O’Hara1, O.Y. Grinberg1, J.F. Dunn2, M.A. Abajian2, C.M. Wilmot1, E. Demidenko3,
R.P. Steffen4, H.M. Swartz1

1EPR Center for the Study of Biological Systems, Dartmouth Medical School, Hanover, NH, USA;
 2Biomedical NMR Laboratory, Dartmouth Medical School,  Hanover, NH, USA;
 3 Department of Community and Family Medicine, Section of Biostatistics and Epidemiology, Dartmouth Medical School, Hanover, NH, USA;
 4Allos Therapeutics, Inc., Westminster, CO, USA

INTRODUCTION

Several studies have shown that RSR13 can increase the oxygen tension of hypoxic tumors. The techniques that have been employed have produced very valuable data, but they have been limited in their ability to follow changes over time. These limitations include the capability of measuring the acute time course after administration of RSR13 (i.e. seconds and minutes) and the ability to measure effects from repeated measurements over days and weeks. EPR oximetry has the potential to provide repeatedly and relatively non-invasively measurements of intratumoral oxygen tension. BOLD MR imaging is sensitive to the paramagnetic effects of deoxygenated hemoglobin. This imaging method has a high spatial resolution, making it suitable for monitoring and characterizing tumors. The purpose of this study was to measure the time course of tumor oxygenation changes induced by treatment with repeat dosing of RSR13 on a model tumor, the RIF-1 fibrosarcoma in mice.

METHODS

The study was carried out in transplanted RIF-1 tumors in 18 female C3H/HEJ mice. Two aggregates of lithium phthalocyanine (LiPc) were implanted in the tumor when the mean tumor volumes reached about 600 mm3. The distance between the two implants was 6.0 mm. Baseline measurements of tumor pO2 were made for three days. Starting on the fourth day of EPR measurements, after an initial baseline measurement, RSR13 (150 mg/kg) was injected intraperitoneally and measurements of intratumoral oxygen were made for 60 minutes following treatment. The administration of RSR13 was carried out for six consecutive days. In each mouse, every third day BOLD NMR measurements were made instead of EPR oximetry for the 60-minute period following RSR13. The EPR measurements were made with a 1200 GHz in vivo spectrometer developed and built at the EPR Center for the Study of Viable Systems. The BOLD NMR measurements were done at 7T using a multi-echo gradient echo sequence with TR = 1.5s, TE = 0.007s and an interecho spacing of 0.005s. SI vs TE the data were fit to a single exponential function to quantify R2*. After the measurements on the sixth day of administration of RSR13, the animals were sacrificed and studied to determine the histological status of the tumor, including the position of the LiPc within the tumor.

RESULTS

The mean baseline pO2 and mean tumor size at the beginning of the measurements was 5.03 ± 0.48 mm Hg (pooled data from two implants per tumor) and 605.5 ± 62.6 mm3. By the sixth day, the baseline pO2 and mean tumor size were 5.22 ± 0.74 mm Hg and 1288.8 ± 123.3 mm3. As indicated in the table there were statistically significant changes in the magnitude of effect of RSR13 over the 60 minutes time course, with a maximum reached at 30-40 minutes and a lower effect at 50–60 minutes. There was considerable variation in both the pO2 reached after injection of RSR13 (150mg/kg) and the time course of increasing pO2. Some tumors responded more quickly, with the maximum change being within the first 30 minutes, while other tumors showed a slow increase in tumor pO2 within 60 minutes of the study; most of the tumors had a significant increase of tumor pO2 at most time points over the sixty minutes period. The changes in the BOLD effect as measured by DR2* were quite modest, although there were some time-points where there was a small but significant increase in R2*.

 

CONCLUSIONS

      The magnitude of the observed increase of oxygen in the tumor has significant implications for enhancing the effectiveness of radiation therapy. The measured increase from the baseline of tumor pO2 would be expected to lead to a significant increase in the tumor radiosensitivity. These results illustrate a unique and useful capability of EPR oximetry, obtaining repetitive measurements of tissue (tumor) pO2 noninvasively. This provides unambiguous information on the effectiveness of RSR13 to repetitively enhance tumor oxygenation.

 

                                               

                                                      Mean tumor pO2 (mm Hg) after RSR13 at 10 – 60 minutes

 

 

                    Time       Baseline    10 min.         20 min.         30 min.            40 min           50 min.          60 min.

                   (days)       (0 min.)

 

                       1      5.2±0.94       8.1±2.78      11.8±3.15*     13.1±2.80**    11.6±2.64*     10.7±3.42      8.7±2.39

 

                       2      6.7±1.01   10.6±2.30        17.9±4.02*      20.7±4.15**    14.7±3.79*     14.2±3.35*   15.0±5.65

 

                       3      4.6±0.59     5.5±0.90         7.1±1.01*      11.1±1.67**    10.0±2.03**     7.6±1.15*     4.4±0.43

 

                       4      5.0±0.41     4.5±0.38          6.3±0.61        9.1±1.24*      12.8±3.44*    11.4±2.22*      9.3±1.95*

 

                       5      5.9±0.71     7.5±1.18*       10.2±2.19*     12.7±2.71*     10.4±2.97      10.5±2.65*     9.2±2.24

 

                       6      3.8±0.61     4.2±0.89          5.9±1.42        6.6±1.46*        4.3±0.63*       4.9±1.09      3.6±0.46

 

Table. pO2 (mm Hg, mean ± SE) in RIF-1 tumors in mice from two implants after daily treatment with RSR13 (150 mg/kg, i.p.)
The baseline is the average pO2 from 30 min period prior to the injection of RSR13. * p< 0.05; ** p< 0.01, compared with the baseline (two-tailed paired t-test).
 

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