- Email: [email protected]
Application of ZK 150 471, a fluorinated pH probe for 19F-magnetic resonance spectroscopy, to in vivo pH measurement after hyperthermic treatment of tumors in mice
S e s s i o n 11 Application of ZK 150 471, a Fluorinated pH Probe for 19F-Magnetic Resonance Spectroscopy, to In Vivo pH Measurement After Hyperthermic Treatment of Tumors in Mice Tomoaki Miyazawa, PhD 1, Yoshizumi Aoki, MD 2, Kiyoshi Akagi, MD 2, Masaya Takahashi, PhD 1, Bernhard Fritz-Zieroth, PhD 1, Thomas Frenzel, PhD 3, HannsoJoachim Weinmann, PhD 3
I ntmtumor pH is one factor that defines the so-called metabolic microenvironment, and, in some conditions, the tumor pH correlates with the therapeutic response of malignant tumors [1]. T u m o r pH can be measured by the microelectrode and by 3tp-magnetic resonance (MR) spectroscopy, but these nlethods have several limitations with respect to invasiveness, applicability, and sensitivity. Recently, in vivo pH measurement by MR spectroscopy using a fluorinated c o m p o u n d became possible [2]. Compared with existing methods, 19F-MR spectroscopy is advantageous because it is more sensitive and less invasive. This new method was used to assess the effectiveness of hyperthermic treatment of experimental tumor in mice. MATERIALS AND METHODS
From the 1 Diagnostics 2 Laboratory, Nihon Schering KK, Osaka, Japan; Department of Radiology, Kansai Medical University,. Osaka, Japan; and 3MRI Contrast Media Research, Schering AG, Berlin, Germany. Address reprint requests to T. Miyazawa, PhD, 6-64, Nishimiyahara 2-chome, Yodogawa-ku, Osaka 532, Japan. ; Acad Radio11996;3:S363-S364 9 1996, Association of University Radiologists
FM3A tumor cells derived from mammary carcinoma in mice were grown in an ascites culture, and 0.1 ml of tile diluted ascites containing 1 x 105 tumor cells was injected into the hindlimb of C3H/He mice a g e d / ~ 5 weeks. Animals with a tumor of 8-9 m m in diameter 1 week after implantation were selected for the experiments. This study was conducted in accordance with in-house guidelines for use and care of laboratory animals. Mice were anesthetized with pentobarbital, and the hindlimb with the ttnnor was immersed in a water bath at 44~ for 10, 15, or 20 rain (n = 5). In the other experiments, hyperthermic treatment was done with and without administration of hydmlazine, a vasoactive agent that is k n o w n to potentiate the effect of the hyperthernlic treatment [3]. Hydralazine (Sigma, St. Louis, MO) was injected intravenously at a dose of 5 m g / k g 20 min before the hyperthermia for 15 rain (n = 4). To measure in vivo pH, mice were injected intravenously with 3-(N-[4fluoro-2-trifluoronmthylphenyl]-sulfamoyl)-propionic acid (Schering AG, Berlin, Germany), which works as a pH p r o b e for ]9F-MR spectroscopy [2], at a dose of 1 m m o l / k g 10 rain after the hyperthermic treatment; two sequential 19F spectra were obtained using a h a n d - m a d e surface coil with a
$363
MIYAZAWA ET AL.
Vol. 3, suppl. 2, August 1996
4.7-T MR imaging/spectroscopy system (General Electric Medical Systems, Fremont, CA). The first and the second spectra were obtained between 10 and 30 min, and between 30 and 50 min, respectively, after the hyperthermic treatment. The chemical-shift difference between the two fluorine signals, one of which is pHdependent and the other pH-independent, was converted to a pH value using the calibration curve reported by Frenzel et al. [2]. By plotting the doubling time (i.e., the time the tumor requires to doub!e in diameter) against pH values in the groups with and without hyperthermic treatment in the absence of hydralazine, we determined whether a change in pH would correlate with the growth inhibition of the tumor.
FIGURE 2. Correlation between pH and the doubling time of tumorwith and without hyperthermic treatment (HT). Values are the mean + standard deviation (n = 5).
RESULTS TumoripH measured by 19F-MR spectroscopy was 6.9 or 7.0 before the hyperthermic treatment in any group, and the pH decreased after the treatment at 44~ in the absence o f hydralazine (Fig. 1). The first spectrum, which was obtained between 10 and 30 min after the treatment, showed q decrease in pH (mean _+ standard deviation) from an initial value of 6.97 -+ 0.06 to 6.72 _+ 0.36 (group undergoing 10-min treatment), from 6.94 +_ 0.09 to 6.78 + 0.17 (group undergoing 15-min treatment, p < .05 versus initial value), and from 6.93 -+ 0.09 to 6.21 + 0.18 (group undergoing 20-min treatment, p < .01 versus initial value). The second spectrum obtained
between 30 and 50 min after the treatment showed a further decrease in pH. After the hyperthermic treatment in the presence of hydralazine, pH decreased from an initial value of 6.92 _+ 0.10 to 6.46 + 0.29 (p < .01 versus initial value), whereas the decrease was only marginal without a significant difference in the absence of the drug (data not shown). Tumor pH in the groups with and without hyperthermic treatment s h o w e d a good correlation with the doubling time of the tumor (Fig. 2), suggesting in vivo pH 9as a useful parameter for assessing the effectiveness of the treatment.
I"~" ~ .
5" i
4"
1
I
O before treatment 3'
2
9 HT l O m i n @ liT 20mln
5.6
-
&
6.4
pH
6.6
E.6
i.o
7.2
CONCLUSIONS
6.8"
*
~O.7.0 6.6"~
.HT 10mln I ~
~~
L
,
~-
~~ .
*
6.4' 6.2 6,0
~
9L
HT 15mli'~
HT a0rn~
before treatment
~
[
10-30mln
I
~"------..4...~0 30-50mln
FIGURE 1. Change in intratumor pH before and after hyperthermic treatment (HT).Values are the mean + standard deviation (n = 5). * = p < .05, ** = p < .01 versus before treatment.
S364
Tumor pH decreased in response to the conditions of treatment, which correlated well with the growth inhibition of the tumor. In vivo pH measurement by 19F-MR spectroscopy using the fluorinated c o m p o u n d could be useful for assessing the effectiveness of hyperthermic treatment of tumors. REFERENCES 1. Vaupel PW. Blood flow, oxygenation, tissue pH distribution, and bioenergetic status of tumors. Berlin: Ernst Schering Research Foundation, 1994: 50-65. 2. Frenzel T, KoBler S, Bauer H, NiedbaIla U, Weinmann HJ. Noninvasive in vivo pH measurement using a fluorinated pH probe and fluorine-19 magnetic resonance spectroscopy. Invest Radio11994;29[suppl]:S220-S222. 3. Aoki Y, Akagi K, Kitada N, Tanaka Y, Fritz-Zieroth B. Study of anti-tumor effects by hyperthermia combined with hydralazine on experimental tumor. Nippon Acta Radio11995;55:987-992:
I ntmtumor pH is one factor that defines the so-called metabolic microenvironment, and, in some conditions, the tumor pH correlates with the therapeutic response of malignant tumors [1]. T u m o r pH can be measured by the microelectrode and by 3tp-magnetic resonance (MR) spectroscopy, but these nlethods have several limitations with respect to invasiveness, applicability, and sensitivity. Recently, in vivo pH measurement by MR spectroscopy using a fluorinated c o m p o u n d became possible [2]. Compared with existing methods, 19F-MR spectroscopy is advantageous because it is more sensitive and less invasive. This new method was used to assess the effectiveness of hyperthermic treatment of experimental tumor in mice. MATERIALS AND METHODS
From the 1 Diagnostics 2 Laboratory, Nihon Schering KK, Osaka, Japan; Department of Radiology, Kansai Medical University,. Osaka, Japan; and 3MRI Contrast Media Research, Schering AG, Berlin, Germany. Address reprint requests to T. Miyazawa, PhD, 6-64, Nishimiyahara 2-chome, Yodogawa-ku, Osaka 532, Japan. ; Acad Radio11996;3:S363-S364 9 1996, Association of University Radiologists
FM3A tumor cells derived from mammary carcinoma in mice were grown in an ascites culture, and 0.1 ml of tile diluted ascites containing 1 x 105 tumor cells was injected into the hindlimb of C3H/He mice a g e d / ~ 5 weeks. Animals with a tumor of 8-9 m m in diameter 1 week after implantation were selected for the experiments. This study was conducted in accordance with in-house guidelines for use and care of laboratory animals. Mice were anesthetized with pentobarbital, and the hindlimb with the ttnnor was immersed in a water bath at 44~ for 10, 15, or 20 rain (n = 5). In the other experiments, hyperthermic treatment was done with and without administration of hydmlazine, a vasoactive agent that is k n o w n to potentiate the effect of the hyperthernlic treatment [3]. Hydralazine (Sigma, St. Louis, MO) was injected intravenously at a dose of 5 m g / k g 20 min before the hyperthermia for 15 rain (n = 4). To measure in vivo pH, mice were injected intravenously with 3-(N-[4fluoro-2-trifluoronmthylphenyl]-sulfamoyl)-propionic acid (Schering AG, Berlin, Germany), which works as a pH p r o b e for ]9F-MR spectroscopy [2], at a dose of 1 m m o l / k g 10 rain after the hyperthermic treatment; two sequential 19F spectra were obtained using a h a n d - m a d e surface coil with a
$363
MIYAZAWA ET AL.
Vol. 3, suppl. 2, August 1996
4.7-T MR imaging/spectroscopy system (General Electric Medical Systems, Fremont, CA). The first and the second spectra were obtained between 10 and 30 min, and between 30 and 50 min, respectively, after the hyperthermic treatment. The chemical-shift difference between the two fluorine signals, one of which is pHdependent and the other pH-independent, was converted to a pH value using the calibration curve reported by Frenzel et al. [2]. By plotting the doubling time (i.e., the time the tumor requires to doub!e in diameter) against pH values in the groups with and without hyperthermic treatment in the absence of hydralazine, we determined whether a change in pH would correlate with the growth inhibition of the tumor.
FIGURE 2. Correlation between pH and the doubling time of tumorwith and without hyperthermic treatment (HT). Values are the mean + standard deviation (n = 5).
RESULTS TumoripH measured by 19F-MR spectroscopy was 6.9 or 7.0 before the hyperthermic treatment in any group, and the pH decreased after the treatment at 44~ in the absence o f hydralazine (Fig. 1). The first spectrum, which was obtained between 10 and 30 min after the treatment, showed q decrease in pH (mean _+ standard deviation) from an initial value of 6.97 -+ 0.06 to 6.72 _+ 0.36 (group undergoing 10-min treatment), from 6.94 +_ 0.09 to 6.78 + 0.17 (group undergoing 15-min treatment, p < .05 versus initial value), and from 6.93 -+ 0.09 to 6.21 + 0.18 (group undergoing 20-min treatment, p < .01 versus initial value). The second spectrum obtained
between 30 and 50 min after the treatment showed a further decrease in pH. After the hyperthermic treatment in the presence of hydralazine, pH decreased from an initial value of 6.92 _+ 0.10 to 6.46 + 0.29 (p < .01 versus initial value), whereas the decrease was only marginal without a significant difference in the absence of the drug (data not shown). Tumor pH in the groups with and without hyperthermic treatment s h o w e d a good correlation with the doubling time of the tumor (Fig. 2), suggesting in vivo pH 9as a useful parameter for assessing the effectiveness of the treatment.
I"~" ~ .
5" i
4"
1
I
O before treatment 3'
2
9 HT l O m i n @ liT 20mln
5.6
-
&
6.4
pH
6.6
E.6
i.o
7.2
CONCLUSIONS
6.8"
*
~O.7.0 6.6"~
.HT 10mln I ~
~~
L
,
~-
~~ .
*
6.4' 6.2 6,0
~
9L
HT 15mli'~
HT a0rn~
before treatment
~
[
10-30mln
I
~"------..4...~0 30-50mln
FIGURE 1. Change in intratumor pH before and after hyperthermic treatment (HT).Values are the mean + standard deviation (n = 5). * = p < .05, ** = p < .01 versus before treatment.
S364
Tumor pH decreased in response to the conditions of treatment, which correlated well with the growth inhibition of the tumor. In vivo pH measurement by 19F-MR spectroscopy using the fluorinated c o m p o u n d could be useful for assessing the effectiveness of hyperthermic treatment of tumors. REFERENCES 1. Vaupel PW. Blood flow, oxygenation, tissue pH distribution, and bioenergetic status of tumors. Berlin: Ernst Schering Research Foundation, 1994: 50-65. 2. Frenzel T, KoBler S, Bauer H, NiedbaIla U, Weinmann HJ. Noninvasive in vivo pH measurement using a fluorinated pH probe and fluorine-19 magnetic resonance spectroscopy. Invest Radio11994;29[suppl]:S220-S222. 3. Aoki Y, Akagi K, Kitada N, Tanaka Y, Fritz-Zieroth B. Study of anti-tumor effects by hyperthermia combined with hydralazine on experimental tumor. Nippon Acta Radio11995;55:987-992: