International Journal of Applied Radiation and Isotopes. Vol. 29. pp. 443--447 © Pergamon Press Ltd.. 1978. Printed in Great Britain
0020.708X/78/0701-0443502.00/0
Synthesis and Preliminary Scintigraphic Evaluation of in vivo Distribution of 11C-Hydroxyurea/Isohydroxyurea and C-Cyanate M E L D R U M B. W I N S T E A D , C H U E N - I N G C H E R N , T Z - H O N G L I N , A R C H I E K H E N T I G A N , J A M E S F. L A M B a n d H. S. W l N C H E L L Bucknell University, Lewisburg, PA 17837, U.S.A. and Medi-Physics, Inc., Emeryville, CA 94608, U.S.A. (Received 27 June 1977; in revised form 16 August 1977)
HHCN, produced directly from proton bombardment of 99% N2-1~o H2 [14N(p, a) ~~C], was oxidized by KMnO4 to l~C-cyanate which was added to hydroxylamine at - 5 to 0°C to yield a mixture of hydroxyurea and isohydroxyurea. Scintigraphie evaluation of in vivo distribution in a dog following administration of KO~CN showed diffuse whole-body distribution, apparently including brain, with greater activity seen in regions of the heart, liver, and kidneys. Similar evaluation of a dog given t ~C-hydroxyurea/isohydroxyurea showed early localization of activity in the region of the heart which appeared to be due to activity in the myocardium. However, tissue activity measured in rats after administration of this material failed to demonstrate concentration in the myocardium.
camera fitted with a pinhole collimator. Tissue distribution of ~~C activity was studied at necropsy 10 and 25 min after i.v. administration of ~tC-hydroxyurea/ isohydroxyurea to 175-200g female Sprague--Dawley rats.
INTRODUCTION HYDROXYUREA has been employed as a chemotherapeutic agent in treatment of certain lymphomas and leukemias/~ However, there appears to be little pharmacokinetics data available tracing the distribution of the agent through the body as a function of time. Consequently, as part of our ongoing scintigraphic evaluation of the in vivo distribution of l t C compounds, ~2~ we synthesized and studied t tC-labeled hydroxyurea/isohydroxyurea.
RESULTS
METHODS H ~~CN was produced directly on bombardment of 99% N2 and 1% H2 with 22MeV protons as described previously.~s) K ~1CN was formed by exchange between HI~CN and 0.5 or 10m-mole of carrierKCN in aqueous solution. K ~ C N was oxidized by KMnO~ and Cu(OH)2 catalyst employing the method of HALEY and LAMBODY.~6~This method claims quantitative conversion of KCN to KOCN. We did not verify the quantitative aspects of this conversion, but did validate the method by demonstrating that most, if not all, of the product of the reaction was KOCN. KO ~tCN and hydroxylamine-HC1 were reacted to form a mixture of ~C-labeled hydroxyurea and isohydroxyurea using the method of KOFOD.~7~ Using melting point and infrared analysis of the crystalline product materials, we verified that the bulk of the product was a mixture of hydroxyurea and isohydroxyurea. The in vivo distribution of the intravenously administered ~~C compounds was determined scintigraphically using a Searle Radiographics HP scintillation A.e..L 29:7--t"
11C-Hydrogen cyanide was generated directly by 22 MeV proton bombardment of 99% N z - I % H2 using the target system previously described/5~ The ~C-hydrogen cyanide was continuously flushed from the target and carried in Teflon tubing to a reaction vessel containing carrier-KCN located in a shielded enclosure approximately 30ft from the cyclotron target. In the first study, 821mCi of t~C-hydrogen cyanide was collected in the trap at EOB following a 1-hr bombardment at 36/~A, while in the second study, 684 mCi (EOB) was collected in the trap following a l-hr bombardment at 35pA. In the first study, a 78.9% radiochemieal yield of ~C-cyanate was obtained following a processing time of 37 rain, and in the second study, a 92.6% radiochemical yield of ~C-cyanate was obtained following a processing time of 32.5 rain. In vivo distribution in the dog was studied following i.v. administration of 75 mCi of ~lC-cyanate contained in 0.25 m-mole carrier. The scintigraphic images obtained are shown in Fig. 1. Activity was diffusely distributed throughout the body, including the region of the brain, with greater activity noted in the region of the heart, liver and kidneys. ~lC-Hydroxyurea/isohydroxym'ea was synthesized from ~1C-cyanide in a total synthesis time of 124 rain
443
Meldrum B. Winstead et al.
444
TABLE !. Tissue activity in rats (average of 4 rats per point) following i.v. administration of 11C. hydroxyurea/isohydroxyurea Tissue or organ Adrenals Blood Brain Carcass Heart Kidneys Liver Lungs Muscle Pancreas Bladder/Urine
% Dose 10 rain 25 rain 0.08
0.12
0.68 78.21 0.50 5.46 9.01 1.01
0.53 79.96 0.38 5.90 5.76 0.52
0.72 2.56
0.47 6.56
% Dose/g 10 rain 25 rain 1.04 1.16 0.47 0.57 0.98 3.47 1.25 0.96 0.70 1.01
1.50 0.66 0.37 0.57 0.65 3.78 0.78 0.53 0.73 0.59
in a 72% radiochemical yield. 3.9mCi of t lChydroxyurea/isohydroxyurea was administered i.v. to a dog. The scintigraphic images obtained are shown in Fig. 2. Activity is seen to initially accumulate in the region of the heart and kidneys. In the scintiphoto of the chest taken at 25 rain following administration of the agent, a 50 ml blood sample in a syringe placed beneath the chest failed to show appreciable counts relative to the counts accumulated in the region of the heart. By 47 min following administration, activity in the region of the heart had diminished .to background levels. Following the resynthesis of ~ C hydroxyurea/isohydroxyurea and its i.v. administration to rats, tissue distributions of activity at necropsy at 10 and 25 rain were obtained as shown in Table 1. Concentration of activity in the myocardium was not seen in the rat at these time periods and liver activity exceeded that in either blood or myocardium. DISCUSSION ~tC-Cyanate was formed by the catalyzed oxidation of K~ICN and subsequently converted to 11C-labeled hydroxyurea (A) and isohydroxyurea (B). 3K~tCN + 2KMnO4 + H 2 0 c,~om~, 3 K O : t C N + 2MnO2 + 2KOH HONH2" HCI + KO 11CN ---* H2N 1: C O N H O H (A) + H2N t 1COONH2 (B). Since cyanate has been employed in treatment of sickle eell anemia, ~8~ it was of some interest to us to study the in vivo distribution of the O : t C N - produced as a precursor material in our synthesis of t~C-hydroxyurea/isohydroxyurea. The appearance of l~C activity in the region of the brain following administration of ~~C-cyanate suggests that the "bloodbrain barrier" is permeable to cyanate or its carboncontaining metabolites. The relatively higher concentration of t i c label in heart, liver and kidneys may be related to the relatively high perfusion of these organs in the anesthetized dog. The lack of evidence for rapid urinary excretion suggested by the minimal accumulation of activity in the bladder at 20 min
postinjection speaks for a prolonged retention of at least the carbon moiety of cyanate in the body. : t C Activity was seen in both the region of the heart and the kidneys following administration of ~lC-hydroxyurea/isohydroxyurea to the dog. Accumulation of activity in the kidneys was expected since this is the organ affecting its excretion from the body. We were surprised to see activity in the region of the heart greater than that which could be ascribed to blood activity. But then we were also surprised when :3NH3 ~9~and 13N-asparagine~a°~ were found to accumulate in the myocardium. Interestingly, the amide portion of asparagine resembles that of the amide portion of hydroxyurea/isohydroxyurea. We initially speculated that this terminal amide region is somehow responsible for accumulation of these agents in the myocardium. Tissue distribution of activity at necropsy of rats given this agent differed substantially from the distribution of activity in dogs evaluated scintigraphically. In rats, the concentration of activity in the liver exceeded that in both heart and blood and at 10 and 25 min the heart failed to show myocardial activity substantially different from that in blood. Consequently, scintiphotos of the rat showed liver activity substantially greater than that seen in the region of the heart. The opposite was seen in the dog. Whether myocardial localization of this material occurs in man remains to be determined.
CONCLUSIONS 11C-Cyanate was obtained from oxidation of tiC-cyanide using KMnO4 and Cu(OH)z catalyst in aqueous solution. Total synthesis time from E.O.B. was approximately 35 rain. Following i.v. administration to a dog, activity was diffusely distributed throughout the body, including the region of the brain, with greater activity noted in the region of the heart, liver, and kidneys. 11C.Hydroxyurea/isohyd. roxyurea was obtained by reacting 11C-cyanate with hydroxylamine hydrochloride. Total synthesis time from 11C-cyanate was approximately 90 rain. Following i.v. administration to a dog, activity accumulated in the regions of the heart and the kidneys. Activity in the region of the heart did not appear to be due to activity in the heart blood pool. Tissue activity in rats 10 and 25 min after administration of this agent was dissimilar to that seen in dogs and no evidence of myocardial concentration of activity was noted.
Acknowledgements--The authors thank ROBERT BOWMAN, MICHAELGPa~RETT,and I~NNtS LUM of Medi-Physics, Inc., for their contributions to this study. This work was supported by Atomic Energy Commission Contract AT (04-3)-849 to Medi-Physics and by grants from the Camille and Henry Dreyfus Foundation, Inc., and the Merck Foundation to Bucknell University.
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IN VIVO
DISTRIBUTION OF 11C-LABELED HYDROXYUREA, H2N.11CO_NHOH LEFT LATERAL IMAGES OF CHEST F"IEART
SAMPLE 10 MIN
25 MIN
47 MIN
LEFT LATERAL IMAGES
15 MIN
32 MIN
ABDOMEN
HEAD & NECK
FIG. 2. In vivo distribution of 1tC activity following i.v. administration of t tC-hydroxyurea/isohydroxyurea in a dog.
Synthesis and preliminary scintioraphic
REFERENCES 1. KRAFOFF I. H., SAVEL H. and MURPHY M. L. Cancer Chemotherapy Rep. 40, 53 (1964). 2. W,NSTEAD M. B., LAMa J. F. and WINCt~Lt. H. S. J. nucl. Med. 14, 747 (1973). 3. WINSTEAD M. B. et al. J. nucl. Med. 16, 1049 (1975). 4. WINSTEAD, M. B. et al. J. Med. Chem. 19, 279 (1976). 5. LAMe J. F., JAmS R. W. and WINCHELL H. S. Int. J. appl. Radiat. Isotopes 22, 475 (1971).
447
6. HALEr E. E. and LAblnODV J. P. J. Am. chem. Soc. 76, 2926 (1954). % KOFOD H. Acta Chem. Scand. 7, 938 (1953). 8. CERAMI A. and MANNING J. M. Proc. hate. Acad. Sci. 68, 1180 (1971). 9. HUNTER W. W., Jr. and MONAHAN W. G. J. nucl. Med. 12, 368 (1971). 10. GELaARD A. S., CLARKE L. P. and LAUGHLIN J. S. J. nucl. Med. 15, 1223 (1974).
0020.708X/78/0701-0443502.00/0
Synthesis and Preliminary Scintigraphic Evaluation of in vivo Distribution of 11C-Hydroxyurea/Isohydroxyurea and C-Cyanate M E L D R U M B. W I N S T E A D , C H U E N - I N G C H E R N , T Z - H O N G L I N , A R C H I E K H E N T I G A N , J A M E S F. L A M B a n d H. S. W l N C H E L L Bucknell University, Lewisburg, PA 17837, U.S.A. and Medi-Physics, Inc., Emeryville, CA 94608, U.S.A. (Received 27 June 1977; in revised form 16 August 1977)
HHCN, produced directly from proton bombardment of 99% N2-1~o H2 [14N(p, a) ~~C], was oxidized by KMnO4 to l~C-cyanate which was added to hydroxylamine at - 5 to 0°C to yield a mixture of hydroxyurea and isohydroxyurea. Scintigraphie evaluation of in vivo distribution in a dog following administration of KO~CN showed diffuse whole-body distribution, apparently including brain, with greater activity seen in regions of the heart, liver, and kidneys. Similar evaluation of a dog given t ~C-hydroxyurea/isohydroxyurea showed early localization of activity in the region of the heart which appeared to be due to activity in the myocardium. However, tissue activity measured in rats after administration of this material failed to demonstrate concentration in the myocardium.
camera fitted with a pinhole collimator. Tissue distribution of ~~C activity was studied at necropsy 10 and 25 min after i.v. administration of ~tC-hydroxyurea/ isohydroxyurea to 175-200g female Sprague--Dawley rats.
INTRODUCTION HYDROXYUREA has been employed as a chemotherapeutic agent in treatment of certain lymphomas and leukemias/~ However, there appears to be little pharmacokinetics data available tracing the distribution of the agent through the body as a function of time. Consequently, as part of our ongoing scintigraphic evaluation of the in vivo distribution of l t C compounds, ~2~ we synthesized and studied t tC-labeled hydroxyurea/isohydroxyurea.
RESULTS
METHODS H ~~CN was produced directly on bombardment of 99% N2 and 1% H2 with 22MeV protons as described previously.~s) K ~1CN was formed by exchange between HI~CN and 0.5 or 10m-mole of carrierKCN in aqueous solution. K ~ C N was oxidized by KMnO~ and Cu(OH)2 catalyst employing the method of HALEY and LAMBODY.~6~This method claims quantitative conversion of KCN to KOCN. We did not verify the quantitative aspects of this conversion, but did validate the method by demonstrating that most, if not all, of the product of the reaction was KOCN. KO ~tCN and hydroxylamine-HC1 were reacted to form a mixture of ~C-labeled hydroxyurea and isohydroxyurea using the method of KOFOD.~7~ Using melting point and infrared analysis of the crystalline product materials, we verified that the bulk of the product was a mixture of hydroxyurea and isohydroxyurea. The in vivo distribution of the intravenously administered ~~C compounds was determined scintigraphically using a Searle Radiographics HP scintillation A.e..L 29:7--t"
11C-Hydrogen cyanide was generated directly by 22 MeV proton bombardment of 99% N z - I % H2 using the target system previously described/5~ The ~C-hydrogen cyanide was continuously flushed from the target and carried in Teflon tubing to a reaction vessel containing carrier-KCN located in a shielded enclosure approximately 30ft from the cyclotron target. In the first study, 821mCi of t~C-hydrogen cyanide was collected in the trap at EOB following a 1-hr bombardment at 36/~A, while in the second study, 684 mCi (EOB) was collected in the trap following a l-hr bombardment at 35pA. In the first study, a 78.9% radiochemieal yield of ~C-cyanate was obtained following a processing time of 37 rain, and in the second study, a 92.6% radiochemical yield of ~C-cyanate was obtained following a processing time of 32.5 rain. In vivo distribution in the dog was studied following i.v. administration of 75 mCi of ~lC-cyanate contained in 0.25 m-mole carrier. The scintigraphic images obtained are shown in Fig. 1. Activity was diffusely distributed throughout the body, including the region of the brain, with greater activity noted in the region of the heart, liver and kidneys. ~lC-Hydroxyurea/isohydroxym'ea was synthesized from ~1C-cyanide in a total synthesis time of 124 rain
443
Meldrum B. Winstead et al.
444
TABLE !. Tissue activity in rats (average of 4 rats per point) following i.v. administration of 11C. hydroxyurea/isohydroxyurea Tissue or organ Adrenals Blood Brain Carcass Heart Kidneys Liver Lungs Muscle Pancreas Bladder/Urine
% Dose 10 rain 25 rain 0.08
0.12
0.68 78.21 0.50 5.46 9.01 1.01
0.53 79.96 0.38 5.90 5.76 0.52
0.72 2.56
0.47 6.56
% Dose/g 10 rain 25 rain 1.04 1.16 0.47 0.57 0.98 3.47 1.25 0.96 0.70 1.01
1.50 0.66 0.37 0.57 0.65 3.78 0.78 0.53 0.73 0.59
in a 72% radiochemical yield. 3.9mCi of t lChydroxyurea/isohydroxyurea was administered i.v. to a dog. The scintigraphic images obtained are shown in Fig. 2. Activity is seen to initially accumulate in the region of the heart and kidneys. In the scintiphoto of the chest taken at 25 rain following administration of the agent, a 50 ml blood sample in a syringe placed beneath the chest failed to show appreciable counts relative to the counts accumulated in the region of the heart. By 47 min following administration, activity in the region of the heart had diminished .to background levels. Following the resynthesis of ~ C hydroxyurea/isohydroxyurea and its i.v. administration to rats, tissue distributions of activity at necropsy at 10 and 25 rain were obtained as shown in Table 1. Concentration of activity in the myocardium was not seen in the rat at these time periods and liver activity exceeded that in either blood or myocardium. DISCUSSION ~tC-Cyanate was formed by the catalyzed oxidation of K~ICN and subsequently converted to 11C-labeled hydroxyurea (A) and isohydroxyurea (B). 3K~tCN + 2KMnO4 + H 2 0 c,~om~, 3 K O : t C N + 2MnO2 + 2KOH HONH2" HCI + KO 11CN ---* H2N 1: C O N H O H (A) + H2N t 1COONH2 (B). Since cyanate has been employed in treatment of sickle eell anemia, ~8~ it was of some interest to us to study the in vivo distribution of the O : t C N - produced as a precursor material in our synthesis of t~C-hydroxyurea/isohydroxyurea. The appearance of l~C activity in the region of the brain following administration of ~~C-cyanate suggests that the "bloodbrain barrier" is permeable to cyanate or its carboncontaining metabolites. The relatively higher concentration of t i c label in heart, liver and kidneys may be related to the relatively high perfusion of these organs in the anesthetized dog. The lack of evidence for rapid urinary excretion suggested by the minimal accumulation of activity in the bladder at 20 min
postinjection speaks for a prolonged retention of at least the carbon moiety of cyanate in the body. : t C Activity was seen in both the region of the heart and the kidneys following administration of ~lC-hydroxyurea/isohydroxyurea to the dog. Accumulation of activity in the kidneys was expected since this is the organ affecting its excretion from the body. We were surprised to see activity in the region of the heart greater than that which could be ascribed to blood activity. But then we were also surprised when :3NH3 ~9~and 13N-asparagine~a°~ were found to accumulate in the myocardium. Interestingly, the amide portion of asparagine resembles that of the amide portion of hydroxyurea/isohydroxyurea. We initially speculated that this terminal amide region is somehow responsible for accumulation of these agents in the myocardium. Tissue distribution of activity at necropsy of rats given this agent differed substantially from the distribution of activity in dogs evaluated scintigraphically. In rats, the concentration of activity in the liver exceeded that in both heart and blood and at 10 and 25 min the heart failed to show myocardial activity substantially different from that in blood. Consequently, scintiphotos of the rat showed liver activity substantially greater than that seen in the region of the heart. The opposite was seen in the dog. Whether myocardial localization of this material occurs in man remains to be determined.
CONCLUSIONS 11C-Cyanate was obtained from oxidation of tiC-cyanide using KMnO4 and Cu(OH)z catalyst in aqueous solution. Total synthesis time from E.O.B. was approximately 35 rain. Following i.v. administration to a dog, activity was diffusely distributed throughout the body, including the region of the brain, with greater activity noted in the region of the heart, liver, and kidneys. 11C.Hydroxyurea/isohyd. roxyurea was obtained by reacting 11C-cyanate with hydroxylamine hydrochloride. Total synthesis time from 11C-cyanate was approximately 90 rain. Following i.v. administration to a dog, activity accumulated in the regions of the heart and the kidneys. Activity in the region of the heart did not appear to be due to activity in the heart blood pool. Tissue activity in rats 10 and 25 min after administration of this agent was dissimilar to that seen in dogs and no evidence of myocardial concentration of activity was noted.
Acknowledgements--The authors thank ROBERT BOWMAN, MICHAELGPa~RETT,and I~NNtS LUM of Medi-Physics, Inc., for their contributions to this study. This work was supported by Atomic Energy Commission Contract AT (04-3)-849 to Medi-Physics and by grants from the Camille and Henry Dreyfus Foundation, Inc., and the Merck Foundation to Bucknell University.
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IN VIVO
DISTRIBUTION OF 11C-LABELED HYDROXYUREA, H2N.11CO_NHOH LEFT LATERAL IMAGES OF CHEST F"IEART
SAMPLE 10 MIN
25 MIN
47 MIN
LEFT LATERAL IMAGES
15 MIN
32 MIN
ABDOMEN
HEAD & NECK
FIG. 2. In vivo distribution of 1tC activity following i.v. administration of t tC-hydroxyurea/isohydroxyurea in a dog.
Synthesis and preliminary scintioraphic
REFERENCES 1. KRAFOFF I. H., SAVEL H. and MURPHY M. L. Cancer Chemotherapy Rep. 40, 53 (1964). 2. W,NSTEAD M. B., LAMa J. F. and WINCt~Lt. H. S. J. nucl. Med. 14, 747 (1973). 3. WINSTEAD M. B. et al. J. nucl. Med. 16, 1049 (1975). 4. WINSTEAD, M. B. et al. J. Med. Chem. 19, 279 (1976). 5. LAMe J. F., JAmS R. W. and WINCHELL H. S. Int. J. appl. Radiat. Isotopes 22, 475 (1971).
447
6. HALEr E. E. and LAblnODV J. P. J. Am. chem. Soc. 76, 2926 (1954). % KOFOD H. Acta Chem. Scand. 7, 938 (1953). 8. CERAMI A. and MANNING J. M. Proc. hate. Acad. Sci. 68, 1180 (1971). 9. HUNTER W. W., Jr. and MONAHAN W. G. J. nucl. Med. 12, 368 (1971). 10. GELaARD A. S., CLARKE L. P. and LAUGHLIN J. S. J. nucl. Med. 15, 1223 (1974).
