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EPR Oximetry of Oxygenation in Rat Cerebral 9L Tumor after RSR13 Administering

 

Huagang Hou1, Nadeem Khan1, Julia A. O’Hara1, Oleg Y. Grinberg1, Jeffrey F. Dunn2,
Michelle A. Abajian2, Carmen Wilmot1, Eugene Demidenko3, Shi Lu1, Robert P. Steffen4, and Harold M. Swartz1

 

1EPR Center for the Study of Viable Systems, Dartmouth Medical School, Hanover, N 03755, USA;
 2Biomedical NMR Laboratory, Dartmouth Medical School,  Hanover, NH, USA;
3Section of Biostatistics and Epidemiology, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756 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 over days and weeks. EPR oximetry has the potential to provide repeatedly and relatively
non-invasively measurements of intratumoral oxygen tension. With the new techniques that now are available, it is possible
to use EPR oximetry to measure intratumoral pO2 on intracranial 9L gliosarcoma in rats. The purpose of this study was
to measure the time course of tumor oxygenation changes induced by treatment with repeat dosing of RSR13.

 

METHODS

The study was performed in 9L intracranial tumors in 10 male Fisher 344 rats, which had lithium phthalocyanine (LiPc)
implanted in the tumor and in the normal brain tissue in the opposite hemisphere. Monitoring the cerebral pO2 was started 7-10 days
after the tumor cells were implanted. NMR imaging was used to determine the position and size of tumor in the brain. Baseline EPR
measurements were made first, then for six consecutive days, starting on the second day, after an initial baseline EPR measurement,
RSR13 (150 mg/kg) was injected intravenously for 15 minutes and measurements of tumor and normal brain oxygen were made
alternatively at ten minute intervals for the next 60 minutes.

 

RESULTS

The pO2 in the 9L gliosarcoma  and normal brain tissue in rats were measured while breathing 100% of  oxygen prior
to and after i.v. administration of the RSR13 (150 mg/kg). As indicated in the Table1 and Table 2, under baseline conditions,
the tumor and normal brain tissue from day 1 to day 6 had a range of mean pO2 from 97 ± 9 mm Hg to 113 ± 9 mm Hg
and 64 ± 8 mm Hg to 96 ± 13 mm Hg, respectively. No statistical significant differences between baseline tumor and normal pO2
 were found on day 1, while there were significant differences between baseline tumor and normal brain tissue pO2 from
day 2 to day 6. Both a traditional t-test and more advanced growth curve analysis confirm that statistically significant changes
 in the magnitude of effect of RSR13 over the 60 minutes time course reach a maximum at 53 ± 2 to 61 ± 4 minutes in the tumors
and 54 ± 3 to 63 ± 7 minutes in the normal brain tissues, with pO2 returning to baseline values in 106 ± 5 to 121 ± 9 minutes
and 108 ± 6 to 127 ± 13 minutes post dose. There were no statistically significant changes in the magnitude of effect of RSR13
over the 60 minutes time course between the tumor and normal tissues on day 1 and day 6, while there were statistically significant
changes in the magnitude of effect of RSR13 over the 60 minutes time course between the tumor and normal tissues from day 2 to day 5.
There are no statistically significant changes in the time to reach maximum pO2 and return to baseline between the tumor
and normal tissues from day 1 to day 6.

The extent of the increase in tumor pO2 achieved by RSR13 would be expected to lead to a significant increase
in the effectiveness of radiotherapy of tumors with hypoxic regions. These results illustrate a unique and useful capability
of in vivo EPR oximetry, to obtain repeated measurements of tumor oxygenation and physiology. The information on the dynamics
of tumor pO2 after RSR13 administration should be useful for the design of clinical protocols using allosteric hemoglobin effectors.

 

                                     Tumor Side                                                        Normal Side

                                                  _____________________________________________________________________

Time         pO2 base      pO2 max     T max     T base              pO2  base     pO2 max      T max       T base

(days)       (mm Hg)     (mm Hg)   (minutes) (minutes)          (mm Hg)     (mm Hg)     (minutes)  (minutes)

    

    1            110±12       182±21         58±3        116±6            96±13        136±19        54±3      108±6   

    2            103±7         198±15         57±3        114±6            71±10        110±16        63±7      127±13

    3            103±10       159±16         53±2        106±5            72±11        107±17        58±3      117±6

    4            112±9         164±14         61±4        121±9            70±9          107±14        62±4      123±8

    5             97 ±9         140±13         59±4        119±9            64±8          103±14        55±3      110±5

    6             99 ±9         148±15         60±5        119±10          68±11        111±18        61±5      123±10

 

 

 

 

 

 

 

 

 

 

Table 1. Tumor, normal brain tissue pO2 and time to reach maximum pO2 and taken to return back to baseline after daily treatments
of intracranial 9L gliosarcoma in rats with RSR13 (150 mg/kg, i.v. for 15 min) on 6 consecutive days (mean ± SE).

pO2 base = baseline pO2; pO2 max = maximum pO2; T max = time to reach maximum pO2; T base = time required to come back
to the baseline pO2. The baseline is the average pO2 from 12 min period prior to the injection of RSR13. n = 10 rats.

 

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 Time     Dif. pO2 base   p-value   Dif. pO2 max    p-value   Dif. T max   p-value   Dif. T base    p-value      

(days)         (mm Hg)                       (mm Hg)                        (minutes)                     (minutes)     

     

    1                14.73         0.4108         46.83         0.0957          3.81          0.37             7.61           0.37       

2                32.66         0.0071         87.88         0.0001         -6.39          0.37          -12.78          0.37

    3                31.56         0.0325         51.93         0.0230         -5.46          0.15          -10.94           0.15 

    4                42.49         0.0008         57.03         0.0033         -0.83          0.89            -1.67           0.89

    5                32.92         0.0071         36.68         0.0451          4.40          0.39              8.80           0.39  

    6                31.15         0.0280         37.15         0.1057         -1.69          0.82            -3.37            0.82

 

 

 

 

 

 

 

 

 

 

Table 2. Statistical analysis of absolute differences of tissue pO2 and time to reach maximum pO2 and return to baseline
between 9L gliosarcoma and the normal brain tissue after daily treatments with RSR13 (150 mg/kg, i.v. for 15 min)
on 6 consecutive days in rats.

Dif. pO2 base = difference in baseline pO2 compared with normal brain tissue; Dif. pO2 max = difference in maximum pO2
compared with normal brain tissue; Dif. T max = difference in time to reach maximum pO2 compared with normal brain tissue;
Dif. T base = difference in time to return to baseline pO2 compared with normal brain tissue. n = 10 rats.

 

ACKNOWLEGEMENTS

        This work was supported by Allos Therapeutics Inc., Westminster, CO, and by a NIH (NIBIB) grant PO1 EB002180,
“Measurement of pO2 in Tissues In Vivo and In Vitro,” and used the facilities of the EPR Center for the Study of Viable Systems
supported by NIH (NIBIB) grant P41 EB002032

 

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