19F magnetic resonance imaging and spectroscopy of a fluorinated neuroleptic ligand: In vivo and in vitro studies

Psychiatry Research, 25, 73-79

73

Elsevier

19 F

Magnetic Resonance Imaging and Spectroscopy of a Fluorinated Neuroleptic Ligand : In Vivo and In Vitro Studies

David C . Arndt, Adam V . Ratner, Kym F . Fault, Jack D . Barchas, and Stuart W . Young Reerired hithruar. r 19, 1988 ; revised version retched April l, 1988; act zpled June /0, 1988 .

Abstract . The bulk biodistributton of a trill uorinated neuroleptic (fl uphenazine) was studied using 19 l- magnetic resonance imaging (M RI) . Fifteen male SpragueDawlcy' rats were injected with Iluphenazine (120 mgt kg) and scanned in a Q .E . CSI 2 .0 tests MR I system . I he rats were killed lollowing scanning and the brains were removed . '1 - he excised brains were then scanned using 1 14 and "F MR techniques . l he fluorinated neuroleptic was imaged at the injection site, speetroscopically detected in vivo in the head, and spectroscopically localized in the whole brain . These data suggest that in vivo'yl- M RI of fluorinated agents is possible and could have clinical and research applications to the neurosciences . Key Words . Magnetic resonance imaging and spectroscopy, fluorine, neuroleptics, dopamine, ncuroreceptors, imaging . Functional disturbances of the central dopamine neuronal systems have been implicated in a large number of diseases, including Parkinson's disease (Lecnders et al ., 1986 ; Janowski ct al ., 1987) . Huntington's chorea (Klawans, 1971 ; Spokes, 1979), narcolepsy (Bowersox et al ., 1987), and schizophrenia (Carlsson and Llndgvist, 1963 ; Matthysse and Kety, 1985) . The capacity to evaluate, in vivo, agents that interact with dopaminetgic ncuroreceptors would permit characterization of receptor distribution and densities, increase understandingof the metabolic pathwaysol neurotransmitters, and facilitate in vivo pharmacokinetic studies of neuroleptic drugs . Previous attempts to visualize ncuroreceptors have used positron emission tomography (PET) scanning of radiolabeled neuroreceptor ligands (Garnets et al ., 1983 ; Arnettetal ., 1984 ; Wagner,etal ., 1984 ; Wongetal ., 1984 ;Fardeetal ., 1986;Sedvallet a1 . .. 1986 ; Buchshaum, 1987) . While PET scanning is a powerful technique and has been successfully used in receptor distribution (Sedvall et al ., 1986) and metabolite uptake (Farde et al ., 1986) studies, there arc constraints on the use of the method . PET

David C. Arndt . M .S ., was a research assistant in the Nancy Pritzker Laboratory of Behavioral Neurochemistry at Stanford University Medical School . and he is currently a medical student al Harvard Medical School. Adam V Ratner, M . D ., is Felix Block Fellow, and Stuart W . Young . M .D ., is Associate Professor, Department of Diagnostic Radiology and Nuclear Medicine, Stanford University Medical Center . Kym F . Fault . Ph .11, a Head ol'the Pasarow Analytical Neurochemistry lacuity of the Nancy Pritzker Laboratory . and lack D . Barchas, M .D ., is Nancy Friend Pritzker Professor of Psychiatry, Nancy Pritzker Laboratory of Behavioral Neurochemistry and Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford . CA . (Reprint requests to Or . J .D . Barchas, Dept . of Psychiatry and Behavioral Sciences, Stanford University Medical Center, Stanford, CA 94305-5227, USA .) 0165-1781/88/$03 .50 ® 1988 Elsevier Scientific Publishers Ireland Ltd .

74 scanning requires a costly particle accelerator for isotope production in addition to a positron camera . A sophisticated on-site radiochemistry facility is usually required for the production of specialized ligands . Because the halflives of these positron-emitting ligands are short ("C= 20 min ; 19 F= l20 min), and since several hours are required to reach brain-binding equilibrium, observations during equilibrium are difficult_ Only a few observations can be made within the usable halflives of the radioligands, and radiation exposure restricts the number of administrations . Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (M RS) are well-established modalities in basic clinical investigation and are not limited by the same constraints imposed by the use of PET scanning . M R techniques can be used to obtain information from any region of the body : as a result, it should be possible to follow compounds of interest from the injection site to the brain . Furthermore, M R I and MRS equipment is more widely disseminated than are PET scanners . Fluorinated probes are appealing for MR1 ; MRS studies . 19F is the only naturally occurring isotope of fluorine : it has a spin of one-half, a gyromagnetic ratio close to that of hydrogen, and an MRI sensitivity second only to 'H . '9F MR has been successfully used in studies with blood substitutes (Joseph et al ., 1985), chemotherapeutic drugs (Wolf et al ., 1987), fluorinated anesthetics (Chew et al ., 1987), and metabolic markers (Nakada et al ., 1986) . Attempts have been made to use 19 F MRS to determine the concentration of a fluorinated psychopharmacological agent (Iiuphenazine) in vivo (Bartels et al ., 1986) .Inthisstudy,weused fluphenazine(Fig .I)asafluorinated probe to study, in vivo, the bulk biodistribution of a neuroleptic . This study was directed at developing methods of MRS and MRI to evaluate the pharmacokinetics of a fluorinated neuroleptic compound . We obtained "F images and spectra from the depot injection site and in vivo and in vitro 1'F spectra from the brain . Fig . 1 . Chemical structure of fiuphenazine $

r1

(CH 2 ) 3 ~vN-~CH 2 y 2 "OH Fluphenazine is a widely used trif luorinated neuroleptic that bi nds preferentially to dopami ne neu roreceptors in the brain . Methods Animal Preparation . Fifteen male, adult, Sprague-0awley rats (body weight 200-250 g) were injected both intraperituneally with fiuphenazine HO (1_0 ml, 2 .5 mg . ml, E .R. Squibb &Sons, Inc .) and intramuscularly with fiuphenazine deeanoate (1 .0 ml, 25 .0 mg ; ml, E .R . Squibb & Sons, Inc.) 24 hours before scanning . In addition, I hour before scanning, tiuphenazine HCI was injected intravenously through the tail vein (1 .0 ml, 2.5 mg ; ml) . The animals were anesthetized with intraperitoneal pentobarbital (50 mg ; kg) . The hindquarters (site of the depot injection) were scanned ; then the rats were positioned so that only their heads were within the RE "birdcage" coil, and the heads were scanned . After scanning, the rats were killed via cervical dislocation, and the brain was removed from the skull and surrounding tissues immediately . The excised whole brain and the skull ; surrounding tissues were then scanned separately .



75 MRS and MRI . MRS and MRI were performed using a CSI-2T broadband spectrometer imaging system with a bore size of 45 .0 cm . A 5 .2 cM inner-diameter "birdcage" resonator coil (for whole animal work) and a L0 cm inner diameter to-turn solenoidal coil (for excised brain studies) were used for RF excitation and detection . These coils were tunable to both 19 F (80.6 MHz) and ' H (85 .7 MHz) resonance frequencies . ' H and ' 5 F M R I spectra were obtained using a one-pulse sequence . ' H images were obtained using a spin-echo sequence . 19 F images were obtained using a chemical shift selective, driven equilibrium, projection-imaging sequence (Freeman et al ., 1988) . Homogeneity over each sample was optimized by shimming on the sample's 'H signal . Preliminary studies were done with 10 .0 ml phantoms of both fluphenazine HCl (2 .5 mg/ mL) and nuphenazine decanoate (25 .0 mg ; ml) . A 1 YF spectrum obtained from the Iluphenazine HCI phantom is shown in Fig . 2 (the 19 F spectrum obtained from the decanoate phantom was, as would be expected, identical) . The fluphenazine HCI phantom was also used to measure Ti's using an inversion recovery pulse sequence and T2's using a Hahn spin-echo approach . 19 F i l and T2 values of 355 ms and 115 ms, respectively, were obtained .

Fig . 2 . 19F spectrum of a fluphenazine HCl standard (10 .0 ml, 2 .5 mg/mI)

60

40

20

0

-20

-40

The lYF spectrum was obtained using a 1-pulse sequence with a spectral width approximately 10 min .

-so

vPr, Acquisition time was

at 5 kHz .

Results IH images, 1 9 F spectra, and 19 F images were obtained from the hindquarters of all I S rats (examples are shown, respectively, in Figs . 3a, 3b, 3e) . In all of the rats, a I H image and a' F spectrum were obtained from the head and neck region (examples are shown, respectively, in Figs . 4a and 4b) . In all of the rats, a 1 9 F spectrum was obtained from the excised whole brain (Fig . 5) . Inadequate signal-to-noise precluded obtaining a 19F image from the intact heads or excised whole brains .

Discussion The M R resonance techniques used in this study can detect and map the biodistribution of fluorinated agents in vivo . The fluorinated ligand Huphenazine was imaged at the injection site, spectroscopically detected in vivo in the head, and spectroscopically localized in the excised brain in all IS rats . The fluphenazine in the



76 Fig . 3a . A spin-echo 1H coronet projection image of hindquarters (white arrows) of a live supine rat

Fig . 3b . Corresponding 19F image to the 1H image in Fig . 3a

The animal has had 0 .5 nil of 25 mg ; ml fluphenazine decanoate injected mtramuscularlymtoeachlhign24 hours before scanning . Depot injections of tluphenazine in oil are seen bllaterally in the thighs black arrows . The high signal intensity is due to the short T1 of the oil, however, some iH T1 shortening due to the OF of the fluphenazine may also contribute to the high signal . This IH image was acquired using a TA 400 ms . TE 14 ms . and a 256 R 512 matrix': the field of view was 100 mm, and the acquisition tune was approximately 15 min .

1 ne orientation is the same . The 'eF signal is clearly seen in both thighs .arrows . This IaF Image was acquired using a TA of 1,500 ms and a 120 256 matrix ; the field of view was 150 mm . and the acquisition time was approximately 90 min .

Fig . 3c . Corresponding 1gF spectrum to the 1H and 19F images in Figs . 3a and 3b

60

40

20

-20

-40

-60

PPM

The spectrum was obtained using al-pulse sequence with a spectral width of 5 kHz . To enhance signal-to-noise ratio and spectral resolution, line broadening 5 Hz was applied in the data processing . Acquisition time was approximately 10 min . F



77 Fig . 4a . Spin-echo 1 H corona) projection image of a live rat positioned prone, such that only its head was within the coil

Fig . 4b . Corresponding 19F spectrum to the 1H image in Fig, 4a

the orientation of the animal is identical. Trio ICE spectrum was obtained using a 1-pulse sequence with The location of the brain is clearly visible arrowss a spectral width of 10 kHz . To enhance signal-to-noise This 1H image was acquired using aTR 400 ms and a ratio and spectral resolution, line oroadenmg .5 Hz 256'512mabix ;meheld ofview was100mrri .andthe was applied in the data processing Acquisition time acquisition Vme was approximately 15 mini was approximately 60 mm .

Fig . 5 . 9F spectrum obtained from an excised rat brain

Y 100

50

-50

-100

PPM

I he spectrum was obtained using a I-pulse sequence with a spectral width of 10 kHz To enhance signal-tonoise rlio and spectral resolution, line broadening 5 Hz, was applied in the data processing . Acquisition time was approximately 300 min .

78 rat brain is distributed in three distinct pools consisting of receptor bound, unbound, and nonspecifically bound neuroleptic (Crewe et al., 1983) . The ' 9 F spectra that were obtained from the excised rat brains reflect the total concentration of fluphenazine in the brain-not just fluphenazine bound to dopamine receptors . [he finding of '"F in the excised rat brains confirmed that some portion of the in vivo "F signal obtained from the head and neck region arose from the brain itself . To be able to detect spectroscopically, or to image, receptor-bound ligand alone (which, while not currently possible, might permit characterization of receptor density and distribution), the sensitivity of MRS and MRI would need to he increased significantly . This increased sensitivity might possibly be achieved, in part, by using of shielded gradients (Macovski, 1985), increasing B t,, and using postprocessing techniques, e .g ., estimation theory technique (Macovski and Spielman, 1986) . Although a dose of approximately 100 times the standard clinical dose in humans20 .0 mgt kg fluphenazine HCI, as opposed to the standard clinical dose of 0 .25 mg/ kg (Shader, 1978 ; Mason and Granacher, 1980) - was used to obtain a "'F spectrum from the rat hrains, it was possible to image a standard humanclinicaldose of fluphenazine deeanoate- LO ml of 25 mg ml (Shader, 1978 : Mason and Granacher, 1980)-as an intramuscular bolus . The capacity to image and to localize spectroscopically bulk biodistribution of fluorinated neuroreceptor ligands may prove to be a valuable, inexpensive, and noninvasive technique for monitoring the kinetics of neuroleptic uptake from the intramuscular injection site . It is currently possible to use M R for in vivospeetroscopic localizationand imaging of fluorinated neuroleptic compounds . Future technological developments that increase the sensitivity of M R should dramatically extend its potential applications to the basic and clinical neuroseiences . Acknowledgments . The authors thank Dr . Mike Moseley for his technical assistance and support . this work was supported by NIMH grants MH-40153 and MH 23861 .

References Arnett, C .D ., Fowler, JS ., Wolf, A .P ., Logan, J ., and MacGregor, R .R . Mapping brain neuroleptic receptors in the live baboon . Biological Psrchiairr, 19, 1365 (1984) . Bartels, M ., Albert, A ., Kruppa, G ., Mann, K ., Sehroth, G ., labarelli, S ., and label, M . Fluorinated psychopharmacological agents : Noninvasive observation by fluorine-19 nuclear magnetic resonance . Psrehiatrr Research . I8, 197 (198(i) . Bowersox, S.S ., Kilduff, IS ., Faull, KR, Zellcr-deAmicis . L ., Dement, W .C, and Ciaranello, RI) . Brain dopamine receptor levels elevated in canine narcolepsy . Brain Research, 402, 44 (1987) . Buchsbaum, M .S . Positron emission tomography in schizophrenia . In : Me1Ver, H .Y ., ed . Psrchopharnmrologr: The Third Generation of Progress Raven Press, New York (1987) . Carlsson, A ., and Lindgvist, M . Effect of chlorpromazine or haloperidol on formation of 3-methoxytyramineand nurmetanephrine in mouse brain . Aria Pharmacologiru et Toxicologira . 20, 140 (1963) . Chew, W .M ., Moseley, ME ., Mills, PA . . Sessler, D ., Gonzalez-Mendez, R ., James, 1 .1 .,, and Litt, L . Spin-echo fluorine magnetic resonance imagingat 2T : In vivo spatial distrihution of hatothane in the rabbit head . Magnetic Resonance Imaging, 5, 51 (1987) . Crewe, L, Sibley, DR ., Hamblin, MW ., and Leff, S .E . The classification of dopamine receptors : Relationship to radioligand binding . Annual Review ul Neuroscience, 6,43 (1983) .

79 Farde, L ., Hall, H ., Ehrin, E ., and Sedvall, (i, Quantitative analysis of D2 dopamine receptor binding in the living human brain by PET . Science, 231, 258 (1986), Freeman, ll .M . . Mutler, H .H . . Hurd, RE ., and Young . S .W . 19F magnetic resonance imaging of periluorooetyl bromide in vivo :LJagneric Resonance Imaging, 5, 61 (1988) . Garnets, E. S„ Fintau . G ., and Nahmias, C . Dopamine visualized in the basal ganglia of living man . Nature, 305, 137 (1983) . Janowski, A„ Vocci . F„ Bergcr, P ., Angel, 1„ Zelnik, N_ Klcinman, J .E . . Skolnick, P ., and Paul, S .M . ["H]GBR-12935 binding to the dopamine transporter is decreased in the caudate nucleus in Parkinson's disease, Journal of Neurochenrisn .y, 49, 617 (1987) . Joseph, P .M ., Vuasa, Y„ Kundel, H .1,,, Mukherji, B„ and Sloviter, H .A . Magnetic resonance imaging of fluorite in rats infused with artificial blood . /m •estigarive Radiology, 20, 504 (1985) . Klawans, H,L ., Jr . Cerebrospinal fluid homovanillic acid in Huntington'schorea . Journal of Neurological Sciences, 13, 277 (1971), Leenders, K .L ., Palmer, A .J ., Quinn, N ., Clark, i C, Firnau, G ., Garnctt, ES . . Nahmias, C ., Jones, T ., and Marsden, C.D . Brain dopamine metabolism in patients with Parkinson'sdisease measured with positron emission tomography. Journal of Neurology, Neurosurgery and Ps,ychiatrr, 49, 853 (1986) . Macovski, A . Volumetric NMR imaging with time-varying gradients . .Magnetic Resonance in Medicine, 2, 29 (1985) . Macovski . A ., and Spietman, D, In vivo spectroscopic magnetic resonance imaging using estimation theory . Magnetic Resonance in Medicine, 3,97 (1986) . Mason, AS ., and Granacher, R,1 ' . Clinical Handbook at .4nupsarhuiic Drug Therapy, Brunner Mazel, New York (1980) . Matthysse, S_ W ., and Kety, S .S ., eds, Catecholanunes and Schizophrenia : Proceedings ojthe Third International Symposium on Catecholantines (Strasbourg, Prance, May- 1983) . Pergamon Press, Oxford (1985) . Nakada, T., Kwee, IL ., and Conboy, CB. Noninvasive in vivo demonstration of the 2-fluoro-2-deoxy-o-glucose metabolism beyond the hexokinase reaction in rat brain by wF nuclear magnetic resonance spectroscopy, Journal of Neurochemistry, 46, 198 (1986) . Sedvall, G ., Farde, L ., Person, A ., and Wiescl, F .A . Imaging of neurotransmitter receptors in the living human brain . Archives of General Psychiatry, 43, 995 (1986) . Shader, R .1 . Manual of Psychiatric Therapeutics . Little, Brown and Company, Boston (1978) . Spokes, E .G .S . Dopamine in Huntingtori s disease : A study of postmortem brain tissue . In : Chase, T .N ., Wexler, N .S ., and Barbeau, A ., eds . Advances in .Neurology : Huntingdon's Disease Vol .23 . Raven Press, New York, p, 481 (1979) . Wagner, H .N ., Burns, HI) . . Dannals, RE ., Wong, D,F ., Langstrom, B„ Dueller, T ., Frost, ii ., Raven, H .T ., Links, J,M„ Rosenbloom, S .B ., Lukas . SE ., Kramer, A .V ., and Kuhar, M .J . Assessment of dopamine receptor densities in the human brain with carbon-I l-labeled N-methylspiperone. Annals of Neurology, 15 (Suppl), S79 (1984), Wolf, W ., Atbright, M .J ., Silver, MS . . Weber, H ., Reichardt, U ., and Saner, R . Fluorine-19 NMR speetroscopicstudies of the metabolism of 5-Buorouracil in the liner of patients undergoing chemotherapy . Magnetic Resonance Imaging, 5, 165 (1987) . Wong, D,F ., Wagner, H .N ., Dannals, RE ., Links, J .M ., Frost, Ii ., Ravert, UT ., Wilson, A .A ., Rosenhaum, A .E ., Gjedde, A ., Douglas, K .H„ Petranis, .,JDFolstein, ME . . Toung, .LK .T ., Burns, H,D, and Kuhar, M .J . E(leels of age on dopamine and serotonin receptors measured by positron tomography in the living human brain . Science, 226, 1393 (1984) .