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Measurement of Upregulation of Inducible Nitric Oxide Synthase in the Experimental Autoimmune Encephalomyelitis Model Using a Positron Emitting Radiopharmaceutical
NITRIC OXIDE: Biology and Chemistry Vol. 1, No. 3, June, pp. 263–267 (1997) Article No. NO970120
BRIEF COMMUNICATION Measurement of Upregulation of Inducible Nitric Oxide Synthase in the Experimental Autoimmune Encephalomyelitis Model Using a Positron Emitting Radiopharmaceutical Jian Zhang,*,† Anne H. Cross,‡ Timothy J. McCarthy,* and Michael J. Welch*,†,1 *Mallinckrodt Institute of Radiology, †Department of Chemistry, and ‡Department of Neurology and Neurological Surgery, Washington University School of Medicine, St. Louis, Missouri 63110
Received December 16, 1996, and in revised form February 24, 1997
Excess nitric oxide has been implicated in the pathogenosis of experimental autoimmune encephalomyelitis (EAE) which is an animal model for multiple sclerosis. Positron emission tomography (PET) is an imaging technique that has shown utility for studying enzyme systems in vivo. A positron-labeled inducible nitric oxide synthetase (iNOS) inhibitor has been studied in EAE-affected mice as well as controls. Greater uptake of the radiolabeled inhibitor was observed in the spinal cord of the affected mice than of control mice. Increased uptake was also observed in other organs not previously implicated in this experimental model. The increased uptake of the radiopharmaceutical in this model suggests that this tracer may have the potential for measuring increased levels of iNOS in humans by PET. q 1997 Academic Press Key Words: positron emission tomography (PET); enzyme systems; nitric oxide synthase; radiopharmaceutical; encephalomyelitis; multiple sclerosis.
1 To whom correspondence should be addressed at Division of Radiological Sciences, Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 South Kingshighway Blvd., St. Louis, MO 63110. Fax: (314) 362-8399. E-mail: [email protected].
Positron emission tomography (PET)2 has demonstrated utility for studying enzyme systems in vivo and providing information about enzyme activities in both normal and diseased states, as well as the effects of novel pharmaceuticals on these enzymatic systems (1). Excess production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in a number of pathologic states such as chronic inflammation, septic shock, diabetic vascular dysfunction, and transplant rejection (2–4). An iNOS PET radiopharmaceutical, which noninvasively detects iNOS levels, would have many potential applications and would also provide a new approach to elucidating the role of excess NO generated via iNOS in vivo. Previously, we have synthesized two positronlabeled iNOS-selective inhibitors, S-[11C]methylisothiourea ([11C]MITU) and S-2-[18F]fluoroethylisothiourea ([18F]FEITU) and performed preliminary evaluations with in vitro and in vivo models (5). Increased uptake of both agents by cells stimulated to 2 Abbreviations used: PET, positron emission tomography; iNOS, inducible nitric oxide synthase; [11C]MITU, S-[11C]methylisothiourea; [18F]FEITU, S-2-[18F]fluoroethylisothiourea; LPS, lipopolysaccharide; EAE, experimental autoimmune encephalomyelitis; CNS, central nervous system; MS, multiple sclerosis.
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express iNOS and efficient blocking of iNOS under the controlled in vitro conditions were observed. In preliminary biodistribution studies using Sprague– Dawley rats, enhanced uptake of both of these labeled iNOS inhibitors (compared to control) was detected in some organs reported to express iNOS in rats pretreated with LPS (6, 7). These results suggested that both of these labeled inhibitors have potential for monitoring increased levels of iNOS in vivo. Experimental autoimmune encephalomyelitis (EAE) is an inflammatory central nervous system (CNS) demyelinating disorder which serves as a prime animal model for multiple sclerosis (MS) (8, 9). Recent evidence has suggested a possible role of excess NO in the pathogenesis of EAE. iNOS mRNA has been detected in the spinal cord and brain of the EAE mouse model (10–12). Levels of iNOS expression as well as enzyme activity were increased in spinal cords and CNS of mice with acute EAE while increased iNOS levels have not been observed in other organs of EAE-affected mice (13). Consequently, the spinal cord and other areas of the CNS might serve as target tissue for iNOS radiopharmaceutical evaluation in this mouse model. In the present work, the EAE model was used to assess whether increased iNOS activity could be detected in vivo. Enhanced uptake of the radiopharmaceutical in the EAE (13) mouse model would suggest that these agents could be used to detect iNOS activity in diseases such as multiple sclerosis. Furthermore, these radiopharmaceuticals might be used to study iNOS activity with disease progression and also allow the assessment of drug dose and effects in therapy. An initial step in the evaluation of any PET radiopharmaceutical is to examine the biodistribution of the tracer in a carefully controlled and relatively inexpensive animal model. When a suitable candidate has been identified, it is then taken on to larger animal models suitable for PET imaging. Because of the longer half-life of fluorine-18 (t1/2 Å 109.8 min) compared to carbon-11 (t1/2 Å 20.4 min), and the greater selectivity as well as potency of FEITU over MITU, [18F]FEITU was chosen as the tracer for initial evaluation. The EAE mouse model was induced as described previously in 6- to 8-week-old female SJL/J mice (13). CNS myelin (purified from guinea pig spinal
cords, purchased from Rockland, Gilbertsville, PA) was emulsified in 1:1 mixture of sterile saline and complete Freund’s adjuvant (Difco Laboratories Inc., Detroit, MI). The emulsion was injected subcutaneously at two sites on the back (base of tail and dorsal neck) to induce EAE. Each mouse received 0.6 mg of whole myelin in 0.1 ml of emulsion subcutaneously on Day 0 and 100 ng of pertussis toxin (List Laboratories, Campbell, CA) dissolved in 100 ml of sterile PBS intravenously on Days 0, 2, and 7 postimmunization. Clinical disease was scored from 0 to 5 in a blind fashion according to accepted criteria (14): 0, no clinical sign; 1, limp tail; 2, limp tail and hind limb weakness; 3, moderate hind limb paralysis; 4, severe hind limb paralysis, 5, moribund. [18F]FEITU was synthesized as described previously (5). The synthetic precursor, [18F]fluoroethyl triflate, was prepared as reported (15). [18F]Fluoride was produced by an 18O(p,n) 18F reaction on an enriched oxygen-18 water target. [18F]Fluoride from the water target was added to a solution of Kryptofix [2.2.2] (6.0 mg, 16.2 mM) and K2CO3 (1.5 mg, 10.9 mM) in a 5-mL Vacutainer. Water was azeotropically evaporated using HPLC grade acetonitrile (3 1 0.5 mL) in a 1107C oil bath under a stream of nitrogen. The resultant anhydrous [18F]fluoride was resolubilized into 300 mL anhydrous acetonitrile and transferred to a 1-mL Reactivial precharged with ethylene glycol bistriflate (2 mg, 6.2 mmol) in 100 mL anhydrous acetonitrile. The container was capped tightly and the mixture was heated at 1107C for 2 min and then cooled to 07C in an ice bath. Triflic anhydride (5 mL) was added to the reaction solution and this mixture was allowed to incubate for 3 min at room temperature. To remove unreacted [18F]fluoride, the solution was passed through a short alumina plug (1 cm in a Pasteur pipet), which was eluted with 2 1 300 mL anhydrous acetonitrile. The eluate (containing [18F]fluoroethyl triflate) was transferred to a 1 mL Reactivial containing 2 mg of thiourea in 100 mL of DMF. The resulting mixture was capped and heated at 1107C. After 30 min, the reaction mixture was removed from the oil bath and the organic solvent (acetonitrile) was evaporated under a stream of nitrogen. The residue was taken up in 2 mL HPLC solvent and purified by HPLC (SCX semiprep column eluted isocratically with 2.5% ethanol in 0.05 N saline at flow of 5 mL/min). The desired product
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[18F]FEITU, retention time 14.5 min, was collected for further study. The total reaction and purification time was 110 min, decay corrected radiochemical yield was 4.5–10% (ú98% purity, sp act Å 120–300 Ci/mmol at the end of synthesis). Biodistribution studies were performed using the murine EAE model (13). Radiolabeled tracer, [18F]FEITU in saline solution, was administered to the mice under Metafane anesthesia, via tail vein injection. The animals were allowed free access to water and food. Biodistribution was examined at 10 min postinjection. After the mice were sacrificed, the organs and tissues of interest were removed and weighed. The radioactivity levels in the samples were determined using an automatic well-type gamma counter, Beckman 8000 (Fullerton, CA). The percentage injected dose per gram of tissue (%ID/ g) was calculated by comparison to a weighed and counted standard solution. The EAE-affected mice (n Å 4) were employed in the biodistribution study and naive female SJL/J mice of the same age were used as controls. At the time of death, blood was removed by cardiac puncture, and then cold, sterile PBS was perfused through the left cardiac ventricle to remove blood from tissues, particularly the CNS. The intact spinal cord, lung, heart, brain, kidney, and samples of liver, muscle, and blood were collected for counting. The clinical course of EAE is characterized by weight loss and a progressive paralysis, which commonly leads to complete hind limb paralysis. By Day
TABLE I
Weight and Ratio of Control and EAE Micea Control group (n Å 4) Mouse weight (g) Lung weight (g) Liver weight (g) Kidney weight (g) Heart weight (g) Brain weight (g) Spinal cord weight (g) Spinal cord wet/dry wt ratio
20.74 0.24 0.88 0.11 0.10 0.35 0.12 3.68
{ { { { { { { {
1.31 0.03 0.09 0.01 0.01 0.02 0.01 0.05
EAE group (n Å 9) 14.79 0.23 0.79 0.12 0.10 0.36 0.14 3.83
{ { { { { { { {
1.29* 0.03 0.09 0.01 0.01 0.01 0.01** 0.09***
a
Data are presented as means { SD (standard deviation). * P õ 1.0 1 10019, versus control. ** P õ 0.04, versus control. *** P õ 0.002, versus control.
TABLE II
Biodistribution of [18F]FEITU in EAE Mouse Model at 10 Min Postinjection % Injected dose/g { SDa
Tissue and organs
Control group (n Å 4)
Blood Lung Liver Kidney Muscle Heart Brain Bone Spinal cord Spinal cord/muscle Spinal cord/blood
2.27 1.90 4.56 13.0 2.60 1.48 0.79 2.68 0.89 0.34 0.39
{ { { { { { { { { { {
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2.99 2.88 4.93 27.0 3.26 1.86 1.17 7.28 1.51 0.46 0.51
{ { { { { { { { { { {
0.02* 0.34* 0.80* 6.7* 0.45 0.35 0.16* 2.69* 0.16* 0.04** 0.06***
a
SD is standard deviation. * P õ 0.01 versus control. ** P õ 0.02 versus control. *** P õ 0.04 versus control.
15, the treated mice developed EAE with a clinical score of 3.2 { 1.1. Although the EAE-affected mice weighed 30% less than the control, there was no difference in the weight of sampled internal organs such as lung, liver, kidney, heart, and brain between each group (Table I). The weight difference was thus attributed mainly to water and muscle weight loss. The spinal cords of the EAE-affected mice (wet weight) were slightly heavier than that of control mice (P õ 0.04), perhaps due to the effects of inflammation. After water removal via lyophilization, the spinal cord dry weight was determined and the values of the two groups were not statistically different. EAE mice demonstrated significantly higher spinal cord wet-to-dry weight ratios than the control (P õ 0.002). Uptake of [18F]FEITU (% ID/g) in the lung, liver, kidney, brain, bone, and spinal cord in the EAEaffected mice was significantly higher compared to the control (Table II). Similar results were observed with total organ uptake (% injected dose/organ) except that the higher uptake found in the bone of the EAE-affected mice was not significantly different from control mice (Table III). [18F]FEITU-associated activity in the spinal cord, kidney, and bone of EAE mice was increased twofold compared with control mice (Tables II and III). In addition, the uptake (%
Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.
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0.12 0.22 1.47 4.2 0.35 0.19 0.05 0.10 0.16 0.06 0.05
EAE group (n Å 4)
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Biodistribution of [18F]FEITU in EAE Mouse Model at 10 Min Postinjection % Injected dose/organ { SDa Control group (n Å 4)
Tissue and organs Blood Lung Liver Kidney Muscle Heart Brain Bone Spinal cord
3.30 0.44 3.92 1.41 7.53 0.14 0.28 6.06 0.11
{ { { { { { { { {
0.29 0.03 1.08 0.36 0.75 0.02 0.02 0.49 0.02
EAE group (n Å 4) 3.10 0.69 4.10 3.19 6.76 0.17 0.42 12.0 0.20
{ { { { { { { { {
0.37 0.05* 0.25 0.87* 1.12 0.03 0.07* 5.3 0.00**
a SD is standard deviation. * P õ 0.01 versus control. ** P õ 0.00005 versus control.
ID/g) ratio of spinal cord/muscle as well as spinal cord/blood was significantly increased in the EAEaffected mice compared to the normal control group. Although the muscle mass was reduced in EAE-affected animals, it can be regarded as control tissue for iNOS levels since muscle is unaffected histologically by EAE. Previous results from our laboratory in LPStreated rats indicated that increased uptake of [18F]FEITU was seen in several organs with known iNOS induction at 10 min but not at 30 min (5). This is probably due to the rapid metabolism of [18F]FEITU; as a result, the biodistribution studies in the present investigation were carried out at 10 min. The time points for evaluation on the EAE mouse model were chosen based on previous results (16). Both NO and iNOS enzyme have been shown in the spinal cord of mice with EAE (12, 16), the highest frequency of iNOS-positive cells were observed at the peak of EAE rather than in the early phase, based on immunohistochemical examination of spinal cord with iNOS-specific antibody (12). Therefore, the mice were studied during the acute EAE period, 15 days postimmunization. It is known that paralytic episodes coincide with an acute perivascular inflammatory response in the CNS and the spinal cord is the areas of greatest pathology (9, 17). This inflammation is composed predominantly of infiltrating macrophages and T
cells (9). Our observation of increased tissue water content agrees with the edema formation associated with this process. The increased wet weight of the spinal cord can be attributed to the increased water content. Since the body weight of the EAE-affected mice were nearly 30% less than the control group, the observed differences in uptake (%ID/g) between EAE mice and normal mice may partially be due to mass differences. Therefore, the spinal cord/muscle ratio (used as a target tissue to non-target tissue ratio) was used to provide a better indication of tracerspecific uptake in our animal model. We observed significant increases (by nearly 30%) in the spinal cord/muscle ratio of the biodistribution of [18F]FEITU in EAE mice compared to normal mice. An increase in the uptake of [18F]FEITU is observed in the spinal cord (the target of autoimmune attack in EAE) when the data are expressed either as %ID/g or %ID/organ. As discussed above, the spinal cords in the EAE mice contain increased amounts of water associated with the inflammation response. The protein-bound [18F]FEITU is even greater in the EAE mice, since water should not affect uptake and only add to the weight. Increased [18F]FEITU uptake is not only observed in the spinal cord but also in other organs (kidney, brain, bone). Based on the data obtained in the well controlled in vitro system (5), our data suggest that upregulation of iNOS may be occurring in these other organs which were believed to be unaffected by EAE. In these experiments each mouse was injected with 20 mCi of [18F]FEITU. Based on the specific activity of the tracer we have calculated the FEITU concentration in the upregulated tissues, for example, 1 nM in the spinal cord and 20 nM in the kidney. These values are approximately 1 and 20%, respectively, of the calculated iNOS levels observed during upregulation (5, 16). During any inflammatory process many systems are affected; however, since FEITU is known to be a specific tracer for iNOS, we believe that increased uptake in these regions is indeed an indication of iNOS upregulation. Preliminary experiments in which marrow was separated from the bone suggest the bone activity to be in the marrow. Due to its rapid metabolism [18F]FEITU appears to not be an ideal tracer; however, we have recently determined (McCarthy and Welch, unpub-
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iNOS MEASUREMENT BY POSITRON EMISSION TOMOGRAPHY
lished data) that this tracer is metabolized at a much slower rate in nonhuman primate blood both in vitro and in vivo. This observation combined with the data showing increased levels of uptake in several tissues of the murine EAE model suggests that it may be useful for determining upregulation of iNOS by PET in humans.
ACKNOWLEDGMENTS This work was supported by NIH (HL13851) to M.J.W. and RG2681 from the National Mutiple Sclerosis Society to A.H.C. The authors thank Bill Margenau and David Ficke for isotope production and Manuel San and Elizabeth Sherman for assistance with the animal model.
REFERENCES 1. Fowler, J. S., Volkow, N. D., and Wolf, A. P. (1993). in Nuclear Imaging in Drug Discovery, Development, and Approval (Burns, H. D., Gibson, R. E., Dannals, R. F., and Siegl, P. K. S., Eds.), pp. 179–200, Birkha¨user, Boston. 2. Kerwin, J. F. K., Jr., and Heller, M. (1994). The argininenitric oxide pathway: A target for new drugs. Med. Res. Rev. 14, 23–74. 3. Kerwin, J. F. K., Jr., Lancaster, J. R., Jr., and Feldman, P. L. (1995). Nitric oxide: A new paradigm for second messengers. J. Med. Chem. 38, 4343–4362. 4. Moncada, S., and Higgs, A. (1993). The L-arginine-nitric oxide pathway. N. Engl. J. Med. 329, 2002–2012. 5. Zhang, J., McCarthy, T. J., Moore, W. M., Currie, M. G., and Welch, M. J. (1996). Synthesis and evaluation of two positron labeled nitric oxide synthase inhibitors, S-[11C]methylisothiourea and S-(2-[18F]fluoroethyl)isothiourea, as potential positron emission tomography tracers. J. Med. Chem. 39, 5110– 5118. 6. Evans, T., Carpenter, A., and Cohen, J. (1992). Purification of a distinctive form of endotoxin-induced nitric oxide synthase from rat liver. Proc. Natl. Acad. Sci. USA 89, 5361–5365. 7. Knowles, R. G., Merrett, M., Salter, M., and Moncada, S. (1990). Differential induction of brain, lung and liver nitric oxide synthase by endotoxin in the rat. Biochem. J. 270, 833– 836.
8. Alvord, E. D., Kies, M. W., and Suckling, A. J. (1984). Experimental allergic encephalomyelitis: A useful model for multiple sclerosis. Prog. Clin. Biol. Res. 146, 1–554. 9. Raine, C. S. (1984). Biology of disease: The analysis of autoimmune demyelination: Its impact on multiple sclerosis. Lab. Invest. 50, 608–635. 10. Hooper, D. C., Ohnishi, S. T., Kean, R., Numagami, Y., Dietzschold, B., and Koprowski, H. (1995). Local nitric oxide production in viral and autoimmune diseases of the central nervous system. Proc. Natl. Acad. Sci. USA 92, 5312–5316. 11. Koprowski, H., Zheng, Y. M., Heber-Katz, E., Fraser, N., Rorke, L., Fu, Z. F., Hanlon, C., and Dietzschold, B. (1993). In vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases. Proc. Natl. Acad. Sci. USA 90, 3024–3027. 12. Okuda, Y., Nakatsuji, Y., Fujimura, H., Esumi, H., Ogura, T., Yanagihara, T., and Sakoda, S. (1995). Expression of the inducible isoform of nitric oxide synthase in the central nervous system of mice correlates with the severity of actively induced experimental allergic encephalomyelitis. J. Neuroimmunol. 62, 103–112. 13. Cross, A. H., Girard, T. J., Giacoletto, K. S., Evans, R. J., Keeling, R. M., Lin, R. F., Trotter, J. L., and Karr, R. W. (1995). Long-term inhibition of murine experimental autoimmune encephalomyelitis using CTLA-4-Fc supports a key role for CD28 costimulation. J. Clin. Invest. 95, 2783–2789. 14. Pettinelli, C. B., and McFarlin, D. E. (1981). Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vitro activation of lymphnode cells by myelin basic protein: requirement for Lyt1/2/ T lymphocytes. J. Immunol. 127, 1420–1423. 15. Kiesewetter, D. O., Bru¨cke, T., and Finn, R. D. (1989). Radiochemical synthesis of [18F]fluororaclopride. Appl. Radiat. Isotopes 40, 455–460. 16. Cross, A. H., Keeling, R. M., Goorha, S., San, M., Rodi, C., Wyatt, P. S., Manning, P. T., and Misko, T. P. (1996). Inducible nitric oxide synthase gene expression and enzyme activity correlate with disease activity in murine experimental autoimmune encephalomyelitis. J. Neuroimmunol. 71, 145– 153. 17. Waldburger, K. E., Hastings, R. C., Schaub, R. G., Goldman, S. J., and Leonrad, J. P. (1996). Adoptive transfer of experimental allergic encephalomyelitis after in vitro treatment with recombinant murine interleukin-12: Preferential expansion of interferon-g-producing cells and increased expression of macrophage-associated inducible nitric oxide synthase as immunomodulatory mechanisms. Am. J. Physiol. 148, 375– 382.
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BRIEF COMMUNICATION Measurement of Upregulation of Inducible Nitric Oxide Synthase in the Experimental Autoimmune Encephalomyelitis Model Using a Positron Emitting Radiopharmaceutical Jian Zhang,*,† Anne H. Cross,‡ Timothy J. McCarthy,* and Michael J. Welch*,†,1 *Mallinckrodt Institute of Radiology, †Department of Chemistry, and ‡Department of Neurology and Neurological Surgery, Washington University School of Medicine, St. Louis, Missouri 63110
Received December 16, 1996, and in revised form February 24, 1997
Excess nitric oxide has been implicated in the pathogenosis of experimental autoimmune encephalomyelitis (EAE) which is an animal model for multiple sclerosis. Positron emission tomography (PET) is an imaging technique that has shown utility for studying enzyme systems in vivo. A positron-labeled inducible nitric oxide synthetase (iNOS) inhibitor has been studied in EAE-affected mice as well as controls. Greater uptake of the radiolabeled inhibitor was observed in the spinal cord of the affected mice than of control mice. Increased uptake was also observed in other organs not previously implicated in this experimental model. The increased uptake of the radiopharmaceutical in this model suggests that this tracer may have the potential for measuring increased levels of iNOS in humans by PET. q 1997 Academic Press Key Words: positron emission tomography (PET); enzyme systems; nitric oxide synthase; radiopharmaceutical; encephalomyelitis; multiple sclerosis.
1 To whom correspondence should be addressed at Division of Radiological Sciences, Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 South Kingshighway Blvd., St. Louis, MO 63110. Fax: (314) 362-8399. E-mail: [email protected].
Positron emission tomography (PET)2 has demonstrated utility for studying enzyme systems in vivo and providing information about enzyme activities in both normal and diseased states, as well as the effects of novel pharmaceuticals on these enzymatic systems (1). Excess production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in a number of pathologic states such as chronic inflammation, septic shock, diabetic vascular dysfunction, and transplant rejection (2–4). An iNOS PET radiopharmaceutical, which noninvasively detects iNOS levels, would have many potential applications and would also provide a new approach to elucidating the role of excess NO generated via iNOS in vivo. Previously, we have synthesized two positronlabeled iNOS-selective inhibitors, S-[11C]methylisothiourea ([11C]MITU) and S-2-[18F]fluoroethylisothiourea ([18F]FEITU) and performed preliminary evaluations with in vitro and in vivo models (5). Increased uptake of both agents by cells stimulated to 2 Abbreviations used: PET, positron emission tomography; iNOS, inducible nitric oxide synthase; [11C]MITU, S-[11C]methylisothiourea; [18F]FEITU, S-2-[18F]fluoroethylisothiourea; LPS, lipopolysaccharide; EAE, experimental autoimmune encephalomyelitis; CNS, central nervous system; MS, multiple sclerosis.
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express iNOS and efficient blocking of iNOS under the controlled in vitro conditions were observed. In preliminary biodistribution studies using Sprague– Dawley rats, enhanced uptake of both of these labeled iNOS inhibitors (compared to control) was detected in some organs reported to express iNOS in rats pretreated with LPS (6, 7). These results suggested that both of these labeled inhibitors have potential for monitoring increased levels of iNOS in vivo. Experimental autoimmune encephalomyelitis (EAE) is an inflammatory central nervous system (CNS) demyelinating disorder which serves as a prime animal model for multiple sclerosis (MS) (8, 9). Recent evidence has suggested a possible role of excess NO in the pathogenesis of EAE. iNOS mRNA has been detected in the spinal cord and brain of the EAE mouse model (10–12). Levels of iNOS expression as well as enzyme activity were increased in spinal cords and CNS of mice with acute EAE while increased iNOS levels have not been observed in other organs of EAE-affected mice (13). Consequently, the spinal cord and other areas of the CNS might serve as target tissue for iNOS radiopharmaceutical evaluation in this mouse model. In the present work, the EAE model was used to assess whether increased iNOS activity could be detected in vivo. Enhanced uptake of the radiopharmaceutical in the EAE (13) mouse model would suggest that these agents could be used to detect iNOS activity in diseases such as multiple sclerosis. Furthermore, these radiopharmaceuticals might be used to study iNOS activity with disease progression and also allow the assessment of drug dose and effects in therapy. An initial step in the evaluation of any PET radiopharmaceutical is to examine the biodistribution of the tracer in a carefully controlled and relatively inexpensive animal model. When a suitable candidate has been identified, it is then taken on to larger animal models suitable for PET imaging. Because of the longer half-life of fluorine-18 (t1/2 Å 109.8 min) compared to carbon-11 (t1/2 Å 20.4 min), and the greater selectivity as well as potency of FEITU over MITU, [18F]FEITU was chosen as the tracer for initial evaluation. The EAE mouse model was induced as described previously in 6- to 8-week-old female SJL/J mice (13). CNS myelin (purified from guinea pig spinal
cords, purchased from Rockland, Gilbertsville, PA) was emulsified in 1:1 mixture of sterile saline and complete Freund’s adjuvant (Difco Laboratories Inc., Detroit, MI). The emulsion was injected subcutaneously at two sites on the back (base of tail and dorsal neck) to induce EAE. Each mouse received 0.6 mg of whole myelin in 0.1 ml of emulsion subcutaneously on Day 0 and 100 ng of pertussis toxin (List Laboratories, Campbell, CA) dissolved in 100 ml of sterile PBS intravenously on Days 0, 2, and 7 postimmunization. Clinical disease was scored from 0 to 5 in a blind fashion according to accepted criteria (14): 0, no clinical sign; 1, limp tail; 2, limp tail and hind limb weakness; 3, moderate hind limb paralysis; 4, severe hind limb paralysis, 5, moribund. [18F]FEITU was synthesized as described previously (5). The synthetic precursor, [18F]fluoroethyl triflate, was prepared as reported (15). [18F]Fluoride was produced by an 18O(p,n) 18F reaction on an enriched oxygen-18 water target. [18F]Fluoride from the water target was added to a solution of Kryptofix [2.2.2] (6.0 mg, 16.2 mM) and K2CO3 (1.5 mg, 10.9 mM) in a 5-mL Vacutainer. Water was azeotropically evaporated using HPLC grade acetonitrile (3 1 0.5 mL) in a 1107C oil bath under a stream of nitrogen. The resultant anhydrous [18F]fluoride was resolubilized into 300 mL anhydrous acetonitrile and transferred to a 1-mL Reactivial precharged with ethylene glycol bistriflate (2 mg, 6.2 mmol) in 100 mL anhydrous acetonitrile. The container was capped tightly and the mixture was heated at 1107C for 2 min and then cooled to 07C in an ice bath. Triflic anhydride (5 mL) was added to the reaction solution and this mixture was allowed to incubate for 3 min at room temperature. To remove unreacted [18F]fluoride, the solution was passed through a short alumina plug (1 cm in a Pasteur pipet), which was eluted with 2 1 300 mL anhydrous acetonitrile. The eluate (containing [18F]fluoroethyl triflate) was transferred to a 1 mL Reactivial containing 2 mg of thiourea in 100 mL of DMF. The resulting mixture was capped and heated at 1107C. After 30 min, the reaction mixture was removed from the oil bath and the organic solvent (acetonitrile) was evaporated under a stream of nitrogen. The residue was taken up in 2 mL HPLC solvent and purified by HPLC (SCX semiprep column eluted isocratically with 2.5% ethanol in 0.05 N saline at flow of 5 mL/min). The desired product
Copyright q 1997 by Academic Press. All rights of reproduction in any form reserved.
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[18F]FEITU, retention time 14.5 min, was collected for further study. The total reaction and purification time was 110 min, decay corrected radiochemical yield was 4.5–10% (ú98% purity, sp act Å 120–300 Ci/mmol at the end of synthesis). Biodistribution studies were performed using the murine EAE model (13). Radiolabeled tracer, [18F]FEITU in saline solution, was administered to the mice under Metafane anesthesia, via tail vein injection. The animals were allowed free access to water and food. Biodistribution was examined at 10 min postinjection. After the mice were sacrificed, the organs and tissues of interest were removed and weighed. The radioactivity levels in the samples were determined using an automatic well-type gamma counter, Beckman 8000 (Fullerton, CA). The percentage injected dose per gram of tissue (%ID/ g) was calculated by comparison to a weighed and counted standard solution. The EAE-affected mice (n Å 4) were employed in the biodistribution study and naive female SJL/J mice of the same age were used as controls. At the time of death, blood was removed by cardiac puncture, and then cold, sterile PBS was perfused through the left cardiac ventricle to remove blood from tissues, particularly the CNS. The intact spinal cord, lung, heart, brain, kidney, and samples of liver, muscle, and blood were collected for counting. The clinical course of EAE is characterized by weight loss and a progressive paralysis, which commonly leads to complete hind limb paralysis. By Day
TABLE I
Weight and Ratio of Control and EAE Micea Control group (n Å 4) Mouse weight (g) Lung weight (g) Liver weight (g) Kidney weight (g) Heart weight (g) Brain weight (g) Spinal cord weight (g) Spinal cord wet/dry wt ratio
20.74 0.24 0.88 0.11 0.10 0.35 0.12 3.68
{ { { { { { { {
1.31 0.03 0.09 0.01 0.01 0.02 0.01 0.05
EAE group (n Å 9) 14.79 0.23 0.79 0.12 0.10 0.36 0.14 3.83
{ { { { { { { {
1.29* 0.03 0.09 0.01 0.01 0.01 0.01** 0.09***
a
Data are presented as means { SD (standard deviation). * P õ 1.0 1 10019, versus control. ** P õ 0.04, versus control. *** P õ 0.002, versus control.
TABLE II
Biodistribution of [18F]FEITU in EAE Mouse Model at 10 Min Postinjection % Injected dose/g { SDa
Tissue and organs
Control group (n Å 4)
Blood Lung Liver Kidney Muscle Heart Brain Bone Spinal cord Spinal cord/muscle Spinal cord/blood
2.27 1.90 4.56 13.0 2.60 1.48 0.79 2.68 0.89 0.34 0.39
{ { { { { { { { { { {
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2.99 2.88 4.93 27.0 3.26 1.86 1.17 7.28 1.51 0.46 0.51
{ { { { { { { { { { {
0.02* 0.34* 0.80* 6.7* 0.45 0.35 0.16* 2.69* 0.16* 0.04** 0.06***
a
SD is standard deviation. * P õ 0.01 versus control. ** P õ 0.02 versus control. *** P õ 0.04 versus control.
15, the treated mice developed EAE with a clinical score of 3.2 { 1.1. Although the EAE-affected mice weighed 30% less than the control, there was no difference in the weight of sampled internal organs such as lung, liver, kidney, heart, and brain between each group (Table I). The weight difference was thus attributed mainly to water and muscle weight loss. The spinal cords of the EAE-affected mice (wet weight) were slightly heavier than that of control mice (P õ 0.04), perhaps due to the effects of inflammation. After water removal via lyophilization, the spinal cord dry weight was determined and the values of the two groups were not statistically different. EAE mice demonstrated significantly higher spinal cord wet-to-dry weight ratios than the control (P õ 0.002). Uptake of [18F]FEITU (% ID/g) in the lung, liver, kidney, brain, bone, and spinal cord in the EAEaffected mice was significantly higher compared to the control (Table II). Similar results were observed with total organ uptake (% injected dose/organ) except that the higher uptake found in the bone of the EAE-affected mice was not significantly different from control mice (Table III). [18F]FEITU-associated activity in the spinal cord, kidney, and bone of EAE mice was increased twofold compared with control mice (Tables II and III). In addition, the uptake (%
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0.12 0.22 1.47 4.2 0.35 0.19 0.05 0.10 0.16 0.06 0.05
EAE group (n Å 4)
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ZHANG ET AL. TABLE III
Biodistribution of [18F]FEITU in EAE Mouse Model at 10 Min Postinjection % Injected dose/organ { SDa Control group (n Å 4)
Tissue and organs Blood Lung Liver Kidney Muscle Heart Brain Bone Spinal cord
3.30 0.44 3.92 1.41 7.53 0.14 0.28 6.06 0.11
{ { { { { { { { {
0.29 0.03 1.08 0.36 0.75 0.02 0.02 0.49 0.02
EAE group (n Å 4) 3.10 0.69 4.10 3.19 6.76 0.17 0.42 12.0 0.20
{ { { { { { { { {
0.37 0.05* 0.25 0.87* 1.12 0.03 0.07* 5.3 0.00**
a SD is standard deviation. * P õ 0.01 versus control. ** P õ 0.00005 versus control.
ID/g) ratio of spinal cord/muscle as well as spinal cord/blood was significantly increased in the EAEaffected mice compared to the normal control group. Although the muscle mass was reduced in EAE-affected animals, it can be regarded as control tissue for iNOS levels since muscle is unaffected histologically by EAE. Previous results from our laboratory in LPStreated rats indicated that increased uptake of [18F]FEITU was seen in several organs with known iNOS induction at 10 min but not at 30 min (5). This is probably due to the rapid metabolism of [18F]FEITU; as a result, the biodistribution studies in the present investigation were carried out at 10 min. The time points for evaluation on the EAE mouse model were chosen based on previous results (16). Both NO and iNOS enzyme have been shown in the spinal cord of mice with EAE (12, 16), the highest frequency of iNOS-positive cells were observed at the peak of EAE rather than in the early phase, based on immunohistochemical examination of spinal cord with iNOS-specific antibody (12). Therefore, the mice were studied during the acute EAE period, 15 days postimmunization. It is known that paralytic episodes coincide with an acute perivascular inflammatory response in the CNS and the spinal cord is the areas of greatest pathology (9, 17). This inflammation is composed predominantly of infiltrating macrophages and T
cells (9). Our observation of increased tissue water content agrees with the edema formation associated with this process. The increased wet weight of the spinal cord can be attributed to the increased water content. Since the body weight of the EAE-affected mice were nearly 30% less than the control group, the observed differences in uptake (%ID/g) between EAE mice and normal mice may partially be due to mass differences. Therefore, the spinal cord/muscle ratio (used as a target tissue to non-target tissue ratio) was used to provide a better indication of tracerspecific uptake in our animal model. We observed significant increases (by nearly 30%) in the spinal cord/muscle ratio of the biodistribution of [18F]FEITU in EAE mice compared to normal mice. An increase in the uptake of [18F]FEITU is observed in the spinal cord (the target of autoimmune attack in EAE) when the data are expressed either as %ID/g or %ID/organ. As discussed above, the spinal cords in the EAE mice contain increased amounts of water associated with the inflammation response. The protein-bound [18F]FEITU is even greater in the EAE mice, since water should not affect uptake and only add to the weight. Increased [18F]FEITU uptake is not only observed in the spinal cord but also in other organs (kidney, brain, bone). Based on the data obtained in the well controlled in vitro system (5), our data suggest that upregulation of iNOS may be occurring in these other organs which were believed to be unaffected by EAE. In these experiments each mouse was injected with 20 mCi of [18F]FEITU. Based on the specific activity of the tracer we have calculated the FEITU concentration in the upregulated tissues, for example, 1 nM in the spinal cord and 20 nM in the kidney. These values are approximately 1 and 20%, respectively, of the calculated iNOS levels observed during upregulation (5, 16). During any inflammatory process many systems are affected; however, since FEITU is known to be a specific tracer for iNOS, we believe that increased uptake in these regions is indeed an indication of iNOS upregulation. Preliminary experiments in which marrow was separated from the bone suggest the bone activity to be in the marrow. Due to its rapid metabolism [18F]FEITU appears to not be an ideal tracer; however, we have recently determined (McCarthy and Welch, unpub-
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iNOS MEASUREMENT BY POSITRON EMISSION TOMOGRAPHY
lished data) that this tracer is metabolized at a much slower rate in nonhuman primate blood both in vitro and in vivo. This observation combined with the data showing increased levels of uptake in several tissues of the murine EAE model suggests that it may be useful for determining upregulation of iNOS by PET in humans.
ACKNOWLEDGMENTS This work was supported by NIH (HL13851) to M.J.W. and RG2681 from the National Mutiple Sclerosis Society to A.H.C. The authors thank Bill Margenau and David Ficke for isotope production and Manuel San and Elizabeth Sherman for assistance with the animal model.
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8. Alvord, E. D., Kies, M. W., and Suckling, A. J. (1984). Experimental allergic encephalomyelitis: A useful model for multiple sclerosis. Prog. Clin. Biol. Res. 146, 1–554. 9. Raine, C. S. (1984). Biology of disease: The analysis of autoimmune demyelination: Its impact on multiple sclerosis. Lab. Invest. 50, 608–635. 10. Hooper, D. C., Ohnishi, S. T., Kean, R., Numagami, Y., Dietzschold, B., and Koprowski, H. (1995). Local nitric oxide production in viral and autoimmune diseases of the central nervous system. Proc. Natl. Acad. Sci. USA 92, 5312–5316. 11. Koprowski, H., Zheng, Y. M., Heber-Katz, E., Fraser, N., Rorke, L., Fu, Z. F., Hanlon, C., and Dietzschold, B. (1993). In vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases. Proc. Natl. Acad. Sci. USA 90, 3024–3027. 12. Okuda, Y., Nakatsuji, Y., Fujimura, H., Esumi, H., Ogura, T., Yanagihara, T., and Sakoda, S. (1995). Expression of the inducible isoform of nitric oxide synthase in the central nervous system of mice correlates with the severity of actively induced experimental allergic encephalomyelitis. J. Neuroimmunol. 62, 103–112. 13. Cross, A. H., Girard, T. J., Giacoletto, K. S., Evans, R. J., Keeling, R. M., Lin, R. F., Trotter, J. L., and Karr, R. W. (1995). Long-term inhibition of murine experimental autoimmune encephalomyelitis using CTLA-4-Fc supports a key role for CD28 costimulation. J. Clin. Invest. 95, 2783–2789. 14. Pettinelli, C. B., and McFarlin, D. E. (1981). Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vitro activation of lymphnode cells by myelin basic protein: requirement for Lyt1/2/ T lymphocytes. J. Immunol. 127, 1420–1423. 15. Kiesewetter, D. O., Bru¨cke, T., and Finn, R. D. (1989). Radiochemical synthesis of [18F]fluororaclopride. Appl. Radiat. Isotopes 40, 455–460. 16. Cross, A. H., Keeling, R. M., Goorha, S., San, M., Rodi, C., Wyatt, P. S., Manning, P. T., and Misko, T. P. (1996). Inducible nitric oxide synthase gene expression and enzyme activity correlate with disease activity in murine experimental autoimmune encephalomyelitis. J. Neuroimmunol. 71, 145– 153. 17. Waldburger, K. E., Hastings, R. C., Schaub, R. G., Goldman, S. J., and Leonrad, J. P. (1996). Adoptive transfer of experimental allergic encephalomyelitis after in vitro treatment with recombinant murine interleukin-12: Preferential expansion of interferon-g-producing cells and increased expression of macrophage-associated inducible nitric oxide synthase as immunomodulatory mechanisms. Am. J. Physiol. 148, 375– 382.
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