In vivo imaging with D-amino acids in animals and in humans
Brain imaging with D-cis-4-[18F]fluoroproline
In 1972 Garweg and Dahnke reported an intensive labelling of the cerebral cortex after intraperitoneal injection of D-[3H]proline in mice, while after injection of L-[3H]proline cortical uptake was negligible (Garweg and Dahnke, 1972). Further experiments demonstrated that radioactivity in the brain after injection of D-Pro was due to incorporation of L-Pro into proteins of the cerebral cortex (Garweg and Dahnke, 1973, 1974). The authors postulated the existence of a proline racemase in the cerebral cortex of the mouse converting D-Pro to L-Pro prior to utilization in protein synthesis.
Stimulated by the observations of preferred uptake of D-[3H]proline in the brain of rodents in relation to L-Pro, Langen and collaborators investigated the differential cerebral uptake of the D- and L-enantiomers of the Positron-Emission-Tomography (PET) tracer cis-4-[18F]fluoroproline and of D-/L-[3H]Pro in rats by dual tracer autoradiography and the uptake of D-cis-4-[18F]fluoroproline in two human subjects by PET (Langen et al., 2005). Analysis of the rat brain tissue after injection of D-cis-4-[18F]fluoroproline (n=3) revealed no radioactivity in the proteins but a relevant part in the form of L-trans-4-[18F]fluoroproline. The PET studies yielded a 4 – 5 fold higher SUV and influx rate constant in the human cortex for D-cis-4-[18F]fluoroproline than for L-cis-4-[18F]fluoroproline. These authors concluded that D-cis-4-[18F]fluoroproline and D-[3H]Pro are preferably transported at the blood-brain barrier compared to their L-enantiomers and isomerized to the L-form within the brain. Thus, D-Pro in the plasma may be a source of intracerebral L-Pro which has been shown to act as a modulator of excitatory neurotransmission. Further experiments investigated the uptake of D-cis-4-[18F]fluoroproline in different brain pathologies (Langen et al., 2007). Focal cortical infarctions were induced in different brain areas of the rat using a photothrombosis model. Ex vivo autoradiography demonstrated increased uptake of D-cis-4-[18F]fluoroproline in the ischemic area but also in brain areas remote from the primary lesion such as the ipsilateral thalamus. Focal D-cis-4-[18F]fluoroproline uptake in thalamic nuclei varied with the site of the cortical infarction corresponding to the thalamocortical projections connecting the nuclei with their specific target regions in the cerebral cortex. Since it is a well-known phenomenon that brain injuries involving cerebral cortex may induce secondary degeneration in thalamic nuclei, uptake of D-cis-4-[18F]fluoroproline appears to be related to the phenomenon of retrograde secondary neurodegeneration (Langen et al., 2007).
Further experiments were performed in rats after implantation of F98 rat gliomas in various brain areas (Geisler et al., 2013). Interestingly, the solid tumor mass of F98 gliomas exhibited no significant uptake of D-cis-4-[18F]fluoroproline but again prominent tracer uptake could be observed in brain areas distant to the primary lesion. Thus, focal uptake of D-cis-4-[18F]fluoroproline was noted in ipsilateral thalamic nuclei according to their thalamocortical projections and additionally in the hippocampal area CA1 in two animals with ipsilateral F98 rat gliomas involving hippocampal subarea CA3 rostral to that area, probably the consequence of a tumor-induced lesioning of corresponding fiber tracts (Geisler et al., 2013). Another observation in that study was a focally increased uptake of cis-4-[18F]fluoro-D-proline in the central necrosis of large F98 tumors while the solid tumor mass was negative. This finding may allow an application of PET using cis-4-[18F]fluoro-D-proline for the differentiation of recurrent brain tumors and radiation necrosis. First PET studies in patients after radiotherapy of brain tumors confirmed high accumulation of cis-4-[18F]fluoro-D-proline in histologically confirmed radionecrosis while brain tumors showed nearly no tracer uptake (Geisler et al., 2014). Furthermore, the evaluation of time activity curves of cis-4-[18F]fluoro-D-proline uptake demonstrated different uptake patterns between tumor and necrotic areas. Brain tumors showed a low tracer uptake with a slowly increasing time activity curve while histologically confirmed radionecrosis exhibited an early peak 5 to 10 min after tracer injection followed by a decrease of the time activity curve. These findings indicate that PET using cis-4-[18F]fluoro-D-proline might be useful to differentiate between recurrent brain tumor and radionecrosis (Geisler et al., 2014).
Preferred stereoselective brain uptake of [3H]-D-serine
Stimulated by the observation of a preferred transport of the D-enantiomer of proline at the blood brain barrier (BBB) the differential uptake of [3H]-D-Ser and [3H]-L-Ser in the rat brain one hour after intravenous injection using quantitative autoradiography was investigated. Owing to its potential role as an agonist of NMDA receptors in the brain the metabolism of D-Ser has been a subject of major interest in recent years. The blood-to-brain transfer of D-Ser is thought to be extremely low and it is assumed that D-serine is generated by isomerisation of L-Ser in the brain. Surprisingly, brain uptake of [3H]-D-Ser was significantly higher than that of [3H]-L-Ser indicating a preferred transport of the D-enantiomer of serine at the BBB. This finding indicates that exogenous D-Ser may have a direct influence on glutamatergic neurotransmission and associated diseases (Bauer et al., 2005).
Preferred transport of O-(2-[18F]fluoroethyl)-D-tyrosine (D-FET) into the porcine brain
Another study investigated the transport of O-(2-[18F]fluoroethyl)-D-tyrosine (D-FET) across the BBB by PET in anesthetized piglets and patients after subtotal resection of brain tumors in comparison with O-(2-[18F]fluoroethyl)-L-tyrosine (L-FET). The initial brain uptake of D-FET in piglets was more than two-fold higher than that of L-FET, whereas the initial brain uptake of D-FET in patients was similar to that of L-FET. The authors concluded that considerable differences of stereoselective amino acid transport at the BBB in different species exist. Therefore, the results from animal experiments concerning BBB amino acid transport may not be transferable to humans.
Other radiolabelled D-amino acid for PET imaging
A study comparing uptake of D-isomers of O-11C-methyl tyrosine and O-18F-fluoromethyl tyrosine to the corresponding L-isomers in tumor bearing mice indicated a low uptake of the D-AAs in inflammation and lower background radioactivity in abdominal organs and faster washout compared with the L-isomers. The authors concluded that D-AAs may be advantageous over L-AAs for tumor imaging with PET (Huang et al., 2015). Another study comparing uptake of the D-enantiomer of a phenylalanine derivative, p-(2-[18F]fluoroethyl)-phenylalanine in comparison to its L-isomer in tumor bearing rats, however, could not detect better imaging properties of the D- compared with the L-enantiomer (Wang et al., 2011).
Bauer D, Hamacher K, Broer S, Pauleit D, Palm C, Zilles K, Coenen HH, Langen KJ (2005) Preferred stereoselective brain uptake of D-serine–a modulator of glutamatergic neurotransmission. Nuclear Med. Biol. 32:793-797.
Garweg G, Dahnke HG (1972) Various use of D-proline and L-proline in the brain of the mouse. Verhandlungen der Anatomischen Gesellschaft 67:297-303.
Garweg G, Dahnke HG (1973) Amino-acid racemase in the organism of mammals. Die Naturwissenschaften 60:201.
Garweg G, Dahnke HG (1974) Is the conversion of D-proline into L-proline in the brain a prerequisite for the incorporation into nerve-cell proteins? (Studies on the murine brain). Verhandlungen der Anatomischen Gesellschaft 68:375-379.
Geisler S, Willuweit A, Schroeter M, Zilles K, Hamacher K, Galldiks N, Shah NJ, Coenen HH, Langen KJ (2013) Detection of remote neuronal reactions in the Thalamus and Hippocampus induced by rat glioma using the PET tracer cis-4-[(1)(8)F]fluoro-D-proline. J. Cerebral Blood Flow Met. 33:724-731.
Geisler S, Ermert J, Stoffels G, Willuweit A, Galldiks N, Filss CP, Shah NJ, Coenen HH, Langen KJ (2014) Isomers of 4-[(18)F]fluoro-proline: Radiosynthesis, biological evaluation and results in humans using PET. Curr. Radiopharm. 7:123-132.
Huang T, Tang G, Wang H, Nie D, Tang X, Liang X, Hu K, Yi C, Yao B, Tang C (2015) Synthesis and preliminary biological evaluation of S-11C-methyl-D-cysteine as a new amino acid PET tracer for cancer imaging. Amino Acids 47:719-727.
Langen KJ, Salber D, Hamacher K, Stoffels G, Reifenberger G, Pauleit D, Coenen HH, Zilles K (2007) Detection of secondary thalamic degeneration after cortical infarction using cis-4-18F-fluoro-D-proline. J. Nuclear Med. 48:1482-1491.
Langen KJ, Hamacher K, Bauer D, Broer S, Pauleit D, Herzog H, Floeth F, Zilles K, Coenen HH (2005) Preferred stereoselective transport of the D-isomer of cis-4-[18F]fluoro-proline at the blood-brain barrier. J. Cereb. Blood Flow Metab. 25:607-616.
Wang L, Lieberman BP, Plossl K, Qu W, Kung HF (2011) Synthesis and comparative biological evaluation of L- and D-isomers of 18F-labeled fluoroalkyl phenylalanine derivatives as tumor imaging agents. Nucl. Med. Biol. 38:301-312.
Karl-Josef Langen, M.D., Institute of Neuroscience and Medicine, INM-4, Forschungszentrum Jülich and Department of Nuclear Medicine, University Clinic of Aachen