Magnetic Resonance Imaging, Vol. 10, pp. 975-981, Printed in the USA. All rights reserved.

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1992 Copyright

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0730-725x/92 $5.00 + .ca 1992 Pergamon Press Ltd.

Original Contribution NONINVASIVE IN VIVO 13C-NMR SPECTROSCOPY OF A 13C-LABELED XENOBIOTIC IN THE RAT

D. LANENS,* H. J. MULLER,t F. VAN DE VYVER, * TJ. DE COCK-BUNNING,~ M. SPANOGHE,* A. VAN DER LINDEN,* G.J. MULDER,~ R. DOMMISSE,* J. LUGTENBURG~ *Research Group for Biomedical NMR, University of Antwerp, Wilrijk, Belgium, TDivision of Toxicology, Center for Biopharmaceutical Sciences, University of Leiden, the Netherlands, SDepartment for Ethics, Alternatives and History of Animal Experiments, University of Leiden, the Netherlands, §Gorlaeus Laboratories, Department of Organic Chemistry, University of Leiden, the Netherlands

and

This study demonstrates that the xenobiotic product, l-(o-chloropheayl)-l-(p-chlorophenyl)-2,2-dichloro-3-13Cpropane can be monitored in the liver of an intact animal by in vivo 13C surface coil NMR spectroscopy after intraperitoneal administration. The carbon-13 label could be detected after a single dose of only 200 mg/kg of the product. The intrahepatic changes of the signal intensity of the labeled product were monitored as a function of time. No signals corresponding to metabolites could be detected. Keywords: In vivo NMR spectroscopy; 13C NMR; 13C labeling; Xenobiotic.

INTRODUCTION

nol, all in vivo 13C spectroscopy using 13C-labeled compounds dealt with endogenous compounds. In this paper, the feasibility of detecting a 13C-labeled xenobiotic product and the possibility of following the fate of this product in a noninvasive way using in vivo 13C NMR spectroscopy is demonstrated.

In comparison to phosphorous and proton NMR spectroscopy, in vivo carbon NMR spectroscopy presents a number of major disadvantages, including the low sensitivity of the 13C nucleus, the low natural abundance of 13C(l.l%), and the low in vivo concentrations of various interesting metabolites. As a consequence, the number of studies using 13C in vivo NMR spectroscopy is limited. Some of the disadvantages concerning in vivo 13C spectroscopy may be avoided by labeling the molecule of interest with a 13C label, thus drastically increasing the sensitivity of the technique. Moreover, the label can be incorporated at a strategic site with respect to the expected metabolization pathway, without disturbing the structure and chemical properties of the compound. This strategy has been used successfully to follow the metabolic pathways in living animals of several compounds such as glucose,‘-* glycerol,‘*” acetate,” lactate,12 pyruvate, 12,13alanine, 14,15choline,16 ethanol, 13,14and butyrate. l7 With the exception of etha-

MATERIALS AND METHODS

The product used in this study was l-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloropropane, known as DDP (Fig. 1). Synthesis

of 13C Labeled

DDP

For the synthesis of this product an existing procedure” was modified in order to obtain maximal labeling efficiency. In a dry 250 ml, three necked, round bottomed flask in a dry nitrogen atmosphere, lithiumdiisopropylamide (LDA) was prepared at -20°C by addition of 10.3 ml (15 mmol; 1.1 eq; solution in hexane) n-butyllithium (n-Buli; Merck-Schuchardt, Hohenbrunn, Germany) to a solution of 1.56 g (15 mmol; 1.1 eq) diisopropylamide (DIA) in 30 ml tetrahydroPart of this work was presented as an oral presentation at the Eighth Annual Meeting of the Society for Magnetic Resonance Imaging, Abstract no. 129.

RECENED 12/26/91; ACCEPTED4114192. Address correspondence to Dr. R. Dommisse, Research Group for Biomedical NMR, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium. 915

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Table 1. Description of NMR spectra of l-(-o-chlorophenyl)-l-(p-chlorophenyl)2,2-dichloro-3-‘3C-propane 300 MHz ‘H NMR (CDC13) Cl Fig. 1. Chemical structure of 1-( o-chlorophenyl)- 1-(p-chlorophenyi)-2,Zdichioropropane (DDP). *Position of 13C label.

furan (THF) (freshly distilled from lithiumaluminumhydride) and 60 ml sodium dried ether. The mixture was stirred for 15 min at -20°C and then cooled to below -70°C in a liquid nitrogen/acetone bath. Five grams (15 mmol; 1.1 eq) of l-(o-chlorophenyl)-l-(pchlorophenyl)-2,2_dichloroethane (DDD; Lysodren@, Bristol-Meyers, Co., Princeton, NJ, USA) was dissolved in 25 ml ether and 25 ml THF and added dropwise over a period of 45 min to the cooled mixture. Consequently the white slurry was stirred for 1 hr at a temperature below -70°C and then cooled to a temperature below -90°C. Two grams (14 mmol; 1 eq) of 99% 13C-enriched methyliodide (Cambridge Isotope Laboratories, Woburn, MA, USA) dissolved in 25 ml ether was added. A white precipitate of lithiumiodide was formed during 30 min of stirring after raising the temperature to -80°C. Then 100 ml of water was added and the mixture was allowed to warm to room temperature. The two phases were separated and the aqueous layer was extracted twice with ether. The organic fractions were washed with a concentrated sodium chloride solution and dried over magnesium sulfate. Filtration and evaporation yielded 5.6 grams of a yellow oil of 3-13C-DDP. In vitro NMR analysis of this product on a Bruker WM-300 spectrometer showed a purity of 98.5% (see Table 1). Laboratory Animals Healthy female Wistar rats with a body weight of approximateIy 200 g were obtained from inbred colonies at the Department of Biopharmaceutical Sciences, State University of Leiden, The Netherlands. They were fed ad libitum with complete lab chow and had free access to water. The rats were injected intraperitoneally with the desired load of 3-13C-DDP dissolved in a 1: 1 mixture of sesame oil and DMSO. Prior to spectroscopy the animals were anesthetized with a combination of Hypnorm (4 mg/kg IP; Janssen Pharmaceutics, Beerse, Belgium) and diazepam (2.5 mg/kg; subcutaneous; Roche, Base], Switzerland) and the regions of interest of the rats were positioned over the two-turn surface coil described below.

S @pm): 2.20 (d, JC-H 131 Hz, 3H, 13CH3), 5.30 (s, lH, CH), 7.20-7.50 (m, 7H, ArH), 8.10-8.12 (m, lH, ArH). 75

MHz 13CNMR (CDC&)

6 (ppm): 38 (s, CH3); 91.09 (d, Ccl,);

126, 128.2. 128.6, 129.8, 131.8 (s, Arc-H); 133.5, 134.6 (s, quat, Arc-Cl); 136.5, 136.8 (s, quat, Arc-C).

In Vivo NMR Spectroscopy

In vivo measurements were performed on an ORSBiospec (Oxford Research Systems, Oxford, England) equipped with a superconducting magnet with a field strength of 1.9 T and a horizontal free bore of 26 cm. A double tuned coil, resonating at 80 MHz (proton) and 20 MHz (carbon) was built for this study. This transmit/receive coil consisting of 1.5 mm diameter copper wire was a two-turn surface coil with a diameter of 25 mm. Double tuning was achieved using the design proposed by Schnall et al. I9 The resonant circuit was coupled capacitively to both the 13C preamplifier and the ‘H decoupling channel. Accurate matching at both frequencies was possible by means of a three element network as proposed by Murphy-Boesch et al.20 Radiofrequency (RF) leakage from the proton channel into the carbon preamplifier was prevented by using a proton stop filter at the input of the carbon preamplifier and an 80 MHz, high-quality band-pass

filter on the proton transmit channel. The magnetic field homogeneity was optimized by using the high frequency mode of the coil for shimming of the proton signal until the full width at half maximum (FWHM) of the water resonance was below 40 Hz (0.5 ppm). Carbon spectra were obtained using an inversely gated decoupling scheme. The pulse length was calibrated for intrahepatic measurements and a repetition time of 0.5 set was used. For each transient, 1024 data points were registered. The spectral width corresponded to 6000 Hz. Each spectrum was the result of 1024 transients. Proton decoupling was achieved by using the Waltz16 sequence with a 90-degree pulse of 200 hsec with the proton carrier frequency set on the water resonance. By using this irradiation technique, no increase in body temperature of the animals was noticed. A thin Teflon shield separated the coil from the region of interest of the animal.

In vivo 13C-NMR of a labeled xenobiotic 0 D. LANENSET

In Vitro Measurements at 4.7 T Three days after administration of the labeled product, one rat was sacrificed and liver, kidneys, muscle tissue, and sections of the abdominal wall were excised and stored in liquid nitrogen. Prior to the NMR measurements, parts of the excised tissues were placed in a lo-mm diameter NMR tube. Proton decoupled i3C-NMR spectra were obtained with a Jeol FX-200 (4.7 T) spectrometer (Jeol LTD., Akishima, Japan) operating at 50.10 MHz. Spectra were recorded with 8K data points in a spectral width of 10,000 Hz with an acquisition time of 0.4 set, a 30 degree (6 psec) pulse and a repetition time of 0.5 sec. Broadband decoupling was only applied during acquisition of the signal with a power level of approximately 2 W and centered at the frequency of water protons. Each spectrum resulted from 4000 transients. During the experiments, the temperature inside the probe was maintained at 37°C. RESULTS In the reference “C-NMR spectra of the liver of an intact animal, no significant glucose or glycogen signals could be detected, but signals corresponding to choline and ethanolamine are observed (Fig. 2A). The singlet character of the methyl groups (15 ppm) of the fatty acid chains indicates adequate proton decoupling in all spectra. Figures 2B-E show a stacked plot of carbon spectra of the liver of a rat obtained over a period of 12 days after administration of a total amount of 200 mg 3-13C-DDP (dose: 1000 mg/kg). In comparison with the reference spectrum, an additional peak can be observed at 39 ppm. This peak corresponds to the labeled C3 position in the DDP molecule. During the studied period the intensity of the peak at 39 ppm decreased to zero. No additional peaks, corresponding to metabolites of DDP could be detected. In the carbon spectra of the excised liver, kidneys, muscle, and parts of the abdominal wall, obtained 3 days after 3-13C-DDP administration (dose: 1000 mg/kg), a peak at 39 ppm corresponding to the C3 of DDF was observed only in the excised liver and kidneys but not in the abdominal wall and in the muscle tissue (Fig. 3). DISCUSSION This work demonstrates that a 13C-labeled xenobiotic product, 3-13C-DDP, can be monitored noninvasively in the liver of a living animal by means of in vivo surface coil 13C-NMR spectroscopy after IP injection of only 200 mg/kg of the product. Although the excitation pulse, delivered by the surface coil, was opti-

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mized for intrahepatic measurements, signals from the adjacent abdominal wall also contribute to the spectra. Nevertheless, the absence of a detectable signal at 39 ppm in the spectra of the excised abdominal wall and the presence of this signal in the spectra of the excised liver confirm the hepatic origin of the 3-13CDDP signal in the in vivo spectra. The lipid signal of the CH2 moieties in the in vivo spectra was used as an internal reference (30 ppm) to determine the chemical shift of the different peaks in the spectra. This explains the difference in chemical shift (1 ppm) between the C3-DDP signal in vitro (relative to TMS) and in vivo. To eliminate the proton-carbon coupling, high level proton decoupling during the acquisition of the timedomain signal was used. This technique requires the possibility of the simultaneous transmission of pulses at the frequency of proton and carbon during the experiment. The present results demonstrate that adequate decoupling at 1.9 T with the use of a Waltz-16 decoupling scheme in combination with a double tuned surface coil is possible. Due to the lower spectral resolution of in vivo NMR spectra, suboptimal decoupling cannot be resolved. 13C-labeling of a compound is usually a complicated chemical issue because it often requires the exchange of a carbon atom belonging to the backbone of the molecule. Also, one has to take into account the high price of 13C-enriched substrates. The product under study (DDP) represents a potential alternative to l-(o-chlorophenyl)-l-(p-chlorophenyl)-2,2-dichloroethane (DDD), the only FDA-approved adrenocorticolytic agent presently used for the treatment of adrenal cortical carcinomas that metastasize. Both compounds (DDD and DDP) show the ability to cause reversible necrosis of the zona fasiculata and zona reticularis, while sequentially damaging the mitochondria from these areas. However, DDP shows significantly decreased side effects in comparison to DDD, which in the case of DDD, are caused by the massive doses, required for therapeutic effect of the drug and its rapid biotransformation, mainly to l-(o-chlorophenyl)-1-(p-chlorophenyl) acetic acid (DDA). This product was not found in animals treated with DDD. The applied doses in this study were derived from the results of earlier kinetic and metabolic studies in rats. ‘* The 13Clabel was incorporated at the C3 position because metabolites found in the urine of experimental animals, treated with the product, had an altered structure at C2 and C3. l8 However, in the present study, no metabolites could be detected in both in vivo and in vitro liver spectra. This implies that the concentration of the metabolites in the liver is beyond the detection limit of the technique or that metabolization does not take place in the liver.

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Fig. 2. A stacked plot showing in vivo 1.9 T r3C-NMR spectra of the liver of an anesthetized rat after administration of a dose of 200 mg (1000 mg/kg) 3-13C-DDP. (A) Reference spectrum: 15 ppm: methyl group of fatty acyl chains; 23-34 ppm: methylene groups of fatty acyl chains; 40 ppm: ethanolamine; 54 ppm: choline; 62 ppm: Cl,C3 of glycerol; 69 ppm: C2 of glycerol; 128-130 ppm: C spZ of polyunsaturated fatty acids; 171 ppm: carboxyl groups. (B) 1 day after administration. 39 ppm (*): C3-DDP signal. (C) 3 days after administration. (D) 7 days after administration. (E) 12 days after administration.

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Fig. 3. Carbon spectra of excised tissues, obtained three days after 3-13C-DDP administration. (A) Liver; (B) abdominal wall muscle; (C) abdominal wall fat; (D) kidney. A peak at 39 ppm (*) corresponding to the C3 of DDP can only be observed in the excised liver and kidneys but not in the abdominal wall fat and muscle tissue. (Figure continues)

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Finally, it is important to mention that the lowest applied dose (200 mg/kg) is in the clinical range’* of a number of cytostatic agents. Therefore, 13C-NMR spectroscopy opens new vistas for pharmacokinetic studies of those products using in vivo 13C nuclear magnetic resonance spectroscopy. Acknowledgments-This work was supported by grants 3.0096.86 of the Belgian National Fund for Scientific Research in Medicine (FGWO) and 87/92-120 of the Department for the Programming of Scientific Research of the Belgian Government (DPWB).

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of labeled glycerol by cells from euthyroid and hyperthyroid rats. Proc. Natl. Acad. Sci. USA 76: 1603-1607; 1979. 10. Cohen, S.M.; Rognstad, R.; Shulman, R.G.; Katz, J. A comparison of 13C nuclear magnetic resonance and 14C tracer studies of hepatic metabolism. .I. Biol. Chem. 256: 3428-3432; 1981. 11 Baily, I.A.; Gadian, D.G.; Matthews, P.M.; Radda, G.K.; Seeley, P.J. Studies of metabolism in the isolated, perfused rat heart using 13C NMR. FEBS Lett. 123: 315-318; 1981. 12. Sherry, A.D.; Nunnally, R.L.; Peshock, R.M. Metabolic studies of pyruvate- and lactate-perfused guinea pig hearts by “C NMR. J. Bioi. Chem. 260~9272-9279; 1985.

13. Cohen, S.M. Simultaneous ‘% and 31P NMR studies of perfused rat liver. J. Bioi. Chem. 258: 14294-14308; 1983. 14. Cohen, S.M.; Shulman, R.G.; McLaughlin, A.C. Effects of ethanol on alanine metabolism in perfused mouse liver studies by ‘% NMR. Proc. Natl. Acad. Sci. USA 76:4808-4812;

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15. Stromski, M.E.; Arias-Mendoza, F.; Alger, J.R.; Shulman, R.G. Hepatic gluconeogenesis from alanine: 13C nuclear magnetic resonance methodology for in vivo studies. Magn. Reson. Med. 3:24-32; 1986. 16. Tunggal, B.; Hofmann, K.; Stoffel, W. In vivo ‘%Znuclear magnetic resonance investigations of choline metabolism in rabbit brain. Magn. Reson. Med. 1390-102; 1990. 17. Cross, T.A.; Pahl, C.; Oberhansli, R.; Aue, W.P.; Keller, U.; Seelig, J. Ketogenesis in the living rat followed by 13C NMR spectroscopy. Biochemistry 23: 6398-6402;

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18. Jensen, B.L.; Caldwell, M.W.; French, L.G.; Briggs, D.G. Toxicity; ultrastructural effects and metabolic studies with I-(o-chlorophenyl)-I-(p-chlorophenyl)-2;2dichloroethane (o;p’-DDD) and its methyl analog in the guinea pig and rat. Toxic. Appl. Pharmac. 87: l-9; 1987. 19. Schnall, M.D.; Subramanian, V.H.; Leigh, J.S.; Chance, B. A new double tuned probe for concurrent ‘H and “P NMR. J. Mugn. Reson. 65:122-129; 1985. 20. Murphy-Boesch, J.; Thomas, W.; Brown, T.R. Electrically balanced double tuned surface coils for in vivo spectroscopy and imaging at 1.5 Tesla. In: Book of abstracts: Seventh Annual Meeting of the Society of Magnetic Resonance in Medicine. San Francisco: SMRM; 1988: 863.