Analysis of [11C]flumazenil PET studies
[11C]Flumazenil ([11C]FMZ, [11C]Ro15-1788) is a "benzodiazepine receptor" antagonist that binds reversibly to GABAA receptors.
[11C]Flumazenil has the properties of a good brain PET ligand:
- the metabolites formed in the liver are hydrophilic molecules that do not cross the blood-brain barrier (Shinotoh et al., 1986; Debruyene et al., 1991),
- it is not metabolized in the brain (Shinotoh et al., 1986),
- its binding in the brain is reversible with fast kinetics, and
- its non-specific binding in brain is low (Abadie et al., 1991; Hansen et al., 1991).
After intravenous injection, [11C]flumazenil is initially distributed according to cerebral blood flow (Shinotoh et al., 1986). However, it should be noted that the uptake of [11C]flumazenil is affected by the blood-brain barrier (BBB) efflux transporter P-glycoprotein, at least in rodents (Froklage et al., 2012). In humans, [11C]flumazenil is still useful for measuring the GABAA receptor density (Froklage et al., 2017).
fP and fND
In blood plasma, the fraction of [11C]FMZ bound to plasma proteins is about 0.4 (Klotz et al., 1984), i.e. free fraction fP is about 0.6. Lassen et al (1995) reported an average fP=0.64 ± 0.03 from five subjects, and Price et al (1993) estimated fP=0.50 ± 0.03 also in five subjects.
For [11C]flumazenil, it seems that in the four-compartmental model (three tissue compartmental model)
In an inhibition study with five healthy subjects, Price et al (1993) estimated that average grey matter K1/k2 is 0.68 ± 0.08 in a two-tissue compartmental model, which does not consider nonspecific binding separately; also for white matter K1/k2 was similar, 0.68 ± 0.17. Millet et al (1995) estimated using a multi-injection protocol and five-parameter model that K1/k2 = 0.555 ± 0.056 in healthy control subjects.
Calculating K1/k2 using the ratio of the water contents of the brain and plasma (77.4/94) divided by (1+(1-fP)) gives an estimate 0.51. Lassen et al (1995) estimated VF+VNS=0.79 using cold flumazenil infusion. If we assume that
and that fP and 1-fND are about 0.6 and 0.28, respectively, we can calculate that VF+VNS=0.8. This is close to the estimate by Lassen et al (1995). Magata et al (2003) estimated with Logan plot that VT in brain stem is 0.84 ± 0.33, suggesting that specific binding is very low in white matter.
Two-compartmental model (one-tissue compartmental model)
Compartmental model analysis using arterial plasma input function (Koeppe et al., 1991; Holthoff et al., 1991) have been validated for the analysis of [11C]FMZ PET studies. The estimation of receptor density and dissociation constant requires measurements under different concentrations of bound ligand, which is usually obtained using at least two injections of the ligand with different specific radioactivity (Blomqvist et al., 1990) or using different multi-injection protocols (Millet et al., 1995).
A single tissue compartmental (2-compartmental model) has been found sufficient to describe the tissue dynamics of [11C]FMZ in several studies (Koeppe et al., 1991; Lassen et al., 1995; Millet et al., 1995 and 2002; Klumpers et al., 2008). Altered ligand delivery does not affect distribution volume of [11C]flumazenil even with this simple model (Holthoff et al., 1991).
It must be noticed that iomazenil, which is widely used in SPET if labelled with 123I, has different kinetics, and Buck et al (1996) have recommended three-compartment model for [11C]iomazenil and fitting simultaneously multiple brain regions with coupled k4 or k4 and K1/k’2.
Logan plot can be used to estimate VT without assumptions on compartmental model setting (Miederer et al., 2009). Richardson et al (1997) and Koepp et al (1997) have applied spectral analysis (SA) to produce VT images to be used in SPM, and since then SA has been widely used.
One analysis method used by Lassen et al (1995) was to estimate distribution volumes using integrals of both the metabolite corrected plasma curves and the tissue curves. Both curves were extrapolated using a single exponential obtained from a fit over the interval from 40 to 120 min.
Arterial plasma input
Plasma protein binding is relatively low, about 40%, and blood-to-plasma ratio approaches unity (Klotz et al., 1984; Lassen et al., 1995), with somewhat higher concentration in plasma than in whole blood (Ishiwata et al., 1998). Red blood cells do not contain labelled metabolites.
Sanabria-Bohórquez et al (2000) have suggested omitting the measurement of plasma metabolite fractions, but instead using a mathematical metabolite correction by fitting several regions simultaneously, assuming that [11C]flumazenil fraction in plasma can be modelled by a mono-exponential function plus a constant. The method was validated in a three-injection imaging protocol, but for single-injection protocol the validation was based on simulated data only (Sanabria-Bohórquez et al., 2000).
Okazawa et al (2004) proposed the use of exponential function with fixed parameters to replace individual metabolite analysis; using this method provided valid BPND estimates (calculated as distribution volume ratio - 1, where distribution volumes were estimated using Logan analysis). Using fixed metabolite correction will lead to bias in distribution volume, but since this bias probably is similar in all brain regions, the bias cancels out in calculation of distribution volume ratio (DVR) and BPND. Therefore, if fixed metabolite correction is used, only DVR or BPND should be reported, not VT; this should be kept in mind when interpreting VT results from for example Pascual et al (2012).
Image-derived input function
If appropriate partial volume correction method is used when PET images are reconstructed, then the input function can be derived noninvasively from carotid arteries in the PET image (Mourik et al., 2008).
Reference tissue input
Reference tissue input methods have been proposed to avoid the invasive arterial cannulation and error-prone plasma metabolite analysis also in [11C]FMZ studies.
Logan plot, SRTM and MRTM2 using pons as reference tissue have been shown to provide robust BPND estimates and parametric images (Klumpers et al., 2008; Miederer et al., 2009), although reproducibility and reliability are lower than when using arterial plasma input methods (Salmi et al., 2008). Normandin et al (2012) proposed that the noise in reference region TAC should be included in calculation of weighting factors.
Pons as reference region
In [11C]FMZ studies, pons is most often used as reference region, because it provides reasonable binding estimates with acceptable coefficients of variation and is easier to define anatomically than hemispheric white matter (Abadie et al., 1991 and 1992).
In displacement studies the binding in pons was not changed but the binding in other regions was reduced to the level of pons (Persson et al., 1985). Yet, pons may contain a significant amount of benzodiazepine receptors (Braestrup et al. 1977; Alkire & Haier, 2001; Millet et al., 2002). Hall et al (1992) estimated that pons region has about 2% benzodiazepine receptors compared with frontal cortex. Delforge et al (1995, 1997) estimated that B’max in pons is 5-7 % of cortical values, and the binding potential derived from their results is about 1.1. In vitro benzodiazepine binding densities in cortical regions are about 3.5-5 times higher than in pons (Alkire & Haier, 2001).
Neuroinflammation in rat brain does not prevent usage of pons as reference region (Parente et al., 2017).
Delforge et al (1996, 1997) have corrected the pons curve for bound tracer using previously estimated (Delforge et al., 1995) model parameter values and average plasma curve. They estimated that the percent of bound ligand in pons is 52% with very small injected mass of flumazenil. However, because the receptor sites also affect the free concentration, the correction was simplified by subtracting the pons curve with the bound concentration estimated at 50 min after injection (Delforge et al., 1996).
White matter as reference region
As an alternative, Magata et al (2003) have used white matter, when calculating regional binding potential from VT values derived from plasma-input Logan plots. Their estimate of VT in white matter (brain stem) was 0.84 ± 0.33.
Hammers et al (2003) have found differences also in white matter VT in certain forms of epilepsy.
Klumpers et al (2008) have used white matter and pons as reference tissue with simplified reference tissue model (SRTM), and since white matter led to lower BPND and some rejected fits, recommended using pons.
Benzodiazepine binding in white matter (corpus callosum) may be about 70% of pontial values (Alkire & Haier, 2001).
Previously, Lassen et al (1995) have considered white matter not suitable as a reference region, because it cannot a priori be assumed to have the same non-specific distribution volume (in this case VF + VNS) of the tracer as the grey matter. They estimated that BP in white matter is about 0.2 (Lassen et al., 1995).
Methods for producing parametric maps for SPM analysis were compared by Klumpers et al (2012), and recommended using Logan plot with metabolite corrected plasma, or SRTM, SRTM2 with basis function method or MRTM2 when using pons as reference tissue input.
Arterial input function
- Compartmental models
- Reference region input compartmental models
- Bmax and KD
- Binding potential
- Tissue-to-reference ratio
- Partial saturation approach
Abadie P, Baron JC, Bisserbe JC, Boulenger JP, Rioux P, Travère JM, Barré L, Petit-Taboué MC, Zarifian E. Central benzodiazepine receptors in human brain: estimation of regional Bmax and Kd values with positron emission tomography. Eur J Pharmacol 1992; 213: 107-115. doi: 10.1016/0014-2999(92)90239-z.
Blomqvist G, Pauli S, Farde L, Eriksson L, Persson A, Halldin C. Maps of receptor binding parameters in the human brain - a kinetic analysis of PET measurements. Eur J Nucl Med. 1990;16:257-265. doi: 10.1007/bf00842777.
Debruyene D, Abadie P, Barre L, Albessard F, Moulin M, Zariflan E, Baron J. Plasma pharmacokinetics and metabolism of the benzodiazepine antagonist [ 11 C] Ro 17-88 (flumazenil) in baboon and human during positron emission tomography studies. Eur J Drug Metab Pharmacokinet. 1991; 16: 141-152. doi: 10.1007/BF03189951.
Delforge J, Pappata S, Millet P, Samson Y, Bendriem B, Jobert A, Crouzel C, Syrota A. Quantification of benzodiazepine receptors in human brain using PET, [11C]flumazenil, and a single-experiment protocol. J Cereb Blood Flow Metab. 1995; 15: 284-300. doi: 10.1038/jcbfm.1995.34.
Delforge J, Spelle L, Bendriem B, Samson Y, Bottlaender M, Papageorgiou S, Syrota A. Quantitation of benzodiazepine receptors in human brain using the partial saturation method. J Nucl Med 1996; 37(1): 5-11. PMID: 8544001.
Delforge J, Spelle L, Bedriem B, Samson Y, Syrota A. Parametric images of benzodiazepine receptor concentration using a partial-saturation injection. J Cereb Blood Flow Metab. 1997; 17: 343-355. doi: 10.1097/00004647-199703000-00011.
Friston KJ, Malizia AL, Wilson S, Cunningham VJ, Jones T, Nutt DJ. Analysis of dynamic radioligand displacement or "activation" studies. J Cereb Blood Flow Metab. 1997; 17: 80-93. doi: 10.1097/00004647-199701000-00011
Froklage FE, Postnov A, Yaqub MM, Bakker E, Boellaard R, Hendrikse NH, Comans EFI, Schuit RC, Schober P, Velis DN, Zwemmer J, Heimans JJ, Lammertsma AA, Voskuyl RA, Reijneveld JC. Altered GABAA receptor density and unaltered blood-brain barrier [11C]flumazenil transport in drug-resistant epilepsy patients with mesial temporal sclerosis. J Cereb Blood Flow Metab. 2017; 37(1): 97-105. doi: 10.1177/0271678X15618219.
Gyulai F, Mintun MA, Firestone LL. Dose-dependent enhancement of in vivo GABAA-benzodiazepine receptor binding by isoflurane. Anesthesiology 2001; 95: 585-593. doi: 10.1097/00000542-200109000-00008.
Hansen TD, Warner DS, Todd MM, Baker MT, Jensen NF. The influence of inhalational anesthetics on in vivo and in vitro benzodiazepine receptor binding in the rat cerebral cortex. Anesthesiology 1991; 74: 97-104. doi: 10.1097/00000542-199101000-00016.
Holthoff VA, Koeppe RA, Frey KA, Paradise AH, Kuhl DE. Differentiation of radioligand delivery and binding in the brain: validation of a two-compartment model for [11C]flumazenil. J Cereb Blood Flow Metab. 1991; 11: 745-752. doi: 10.1038/jcbfm.1991.131.
Ihara M, Tomimoto H, Ishizu K, Mukai T, Yoshida H, Sawamoto N, Inoue M, Doi T, Hashikawa K, Konishi J, Shibasaki H, Fukuyama H. Decrease in cortical benzodiazepine receptors in symptomatic patients with leukoaraiosis. a positron emission tomography study. Stroke 2004; 35(4): 942-947. doi: 10.1161/01.str.0000122624.32167.e0.
Ishiwata K, Itou T, Ohyama M, Yamada T, Mishina M, Ishii K, Nariai T, Sasaki T, Oda K, Toyama H, Senda M. Metabolite analysis of [11C]flumazenil in human plasma: assessment as the standardized value for quantitative PET studies. Ann Nucl Med. 1998; 12(1): 55-59. doi: 10.1007/bf03165418.
Iyo M, Itoh T, Yamasaki T, Fukuda H, Inoue O, Shinotoh H, Suzuki K, Fukui S, Tateno Y. Quantitative in vivo analysis of benzodiazepine binding sites in the human brain using positron emission tomography. Neuropharmacology 1991; 30(3): 207-215. doi: 10.1016/0028-3908(91)90147-4.
Klotz U, Ziegler G, Reimann IW. Pharmacokinetics of the selective benzodiazepine antagonist Ro 15-1788 in man. Eur J Clin Pharmacol. 1984; 27: 115-117.21. PMID: 6436030.
Klumpers UM, Veltman DJ, Boellaard R, Comans EF, Zuketto C, Yaqub M, Mourik JE, Lubberink M, Hoogendijk WJ, Lammertsma AA. Comparison of plasma input and reference tissue models for analysing [11C]flumazenil studies. J Cereb Blood Flow Metab. 2008; 28(3): 579-587. doi: 10.1038/sj.jcbfm.9600554.
Klumpers UM, Boellaard R, Veltman DJ, Kloet RW, Hoogendijk WJ, Lammertsma AA. Parametric [11C]flumazenil images. Nucl Med Commun. 2012; 33:422–430. doi: 10.1097/MNM.0b013e3283505f7b.
Koeppe RA, Holthoff A, Frey KA, Kilbourn MR, Kuhl DH. Compartmental analysis of [11C]flumazenil kinetics for the estimation of ligand receptor distribution using positron emission tomography. J Cereb Blood Flow Metab. 1991; 11: 735-744. doi: 10.1038/jcbfm.1991.130.
Laruelle M. Imaging synaptic neurotransmission with in vivo binding competition techniques: a critical review. J Cereb Blood Flow Metab. 2000; 20: 423-451. doi: 10.1097/00004647-200003000-00001.
Lassen NA, Bartenstein PA, Lammertsma AA, Prevett MC, Turton DR, Luthra SK, Osman S, Bloomfield PM, Jones T, Patsalos PN, O’Connell MT, Duncan JS, Andersen JV. Benzodiazepine receptor quantification in vivo in humans using [11C]flumazenil and PET: application of the steady-state principle. J Cereb Blood Flow Metab. 1995; 15: 152-165. doi: 10.1038/jcbfm.1995.17.
Litton JE, Neiman J, Pauli S, Farde L, Hindmarsh T, Halldin C, Sedvall G. PET analysis of [11C]flumazenil binding to benzodiazepine receptors in chronic alcohol-dependent men and healthy controls. Psychiatry Res. 1993; 50(1): 1-13. doi: 10.1016/0925-4927(93)90019-e.
Lucignani G, Panzacchi A, Bosio L, Moresco RM, Ravasi L, Coppa I, Chiumello G, Frey K, Koeppe R, Fazio F. GABAA receptor abnormalities in Prader-Willi syndrome assessed with positron emission tomography and [11C]flumazenil. Neuroimage 2004; 22: 22-28. doi: 10.1016/j.neuroimage.2003.10.050.
Magata Y, Mukai T, Ihara M, Nishizawa S, Kitano H, Ishizu K, Saji H, Konishi J. Simple analytic method of 11C-flumazenil metabolite in blood. J Nucl Med. 2003; 44(3): 417-421. PMID: 12621009.
Malizia AL, Gunn RN, Wilson SJ, Waters SH, Bloomfield PM, Cunningham VJ, Nutt DJ. Benzodiazepine site pharmacokinetic/pharmacodynamic quantification in man: direct measurement of drug occupancy and effects on the human brain in vivo. Neuropharmacology 1996; 35: 1483-1491. doi: 10.1016/s0028-3908(96)00072-x.
Miederer I, Ziegler SI, Liedtke C, Spilker ME, Miederer M, Sprenger T, Wagner KJ, Drzezga A, Boecker H. Kinetic modelling of [11C]flumazenil using data-driven methods. Eur J Nucl Med Mol Imaging 2009; 36: 659-670. doi: 10.1007/s00259-008-0990-z.
Millet P, Delforge J, Mauguiere F, Pappata S, Cinotti L, Frouin V, Samson Y, Bendriem B, Syrota A. Parameter and index images of benzodiazepine receptor concentration in the brain. J Nucl Med. 1995; 36: 1462-1471. PMID: 7629596.
Millet P, Graf C, Buck A, Walder B, Ibáñez V. Evaluation of the reference tissue models for PET and SPECT benzodiazepine binding parameters. Neuroimage 2002; 17: 928-942. doi: 10.1006/nimg.2002.1233.
Mourik JE, Lubberink M, Klumpers UM, Comans EF, Lammertsma AA, Boellaard R. Partial volume corrected image derived input functions for dynamic PET brain studies: methodology and validation for [11C]flumazenil. Neuroimage. 2008; 39(3): 1041-1050. doi: 10.1016/j.neuroimage.2007.10.022.
Nagy F, Chugani DC, Juhász C, da Silva EA, Muzik O, Kupsky W, Canady A, Watson C, Shah J, Chugani HT. Altered in vitro and in vivo flumazenil binding in human epileptogenic neocortex. J Cereb Blood Flow Metab. 1999; 19: 939-947. doi: 10.1097/00004647-199909000-00001.
Normandin MD, Koeppe RA, Morris ED. Selection of weighting factors for quantification of PET radioligand binding using simplified reference tissue models with noisy input functions. Phys Med Biol. 2012; 57: 609-629. doi: 10.1088/0031-9155/57/3/609.
Okazawa H, Yamauchi H, Sugimoto K, Magata Y, Kudo T, Yonekura Y. Effects of metabolite correction for arterial input function on quantitative receptor images with 11C-flumazenil in clinical positron emission tomography studies. J Comput Assist Tomogr. 2004; 28: 428–435. doi: 10.1097/00004728-200405000-00021.
Persson A, Ehrin E, Eriksson L, Farde L, Hedström CG, Litton JE, Mindus P, Sedvall G. Imaging of [11C]-labelled Ro 15-1788 binding to benzodiazepine receptors in the human brain by positron emission tomography. J Psychiatr Res. 1985; 19(4): 609-622. doi: 10.1016/0022-3956(85)90080-9.
Price JC, Mayberg HS, Dannals RF, Wilson AA, Ravert HT, Sadzot B, Rattner Z, Kimball A, Feldman MA, Frost JJ. Measurement of benzodiazepine receptor number and affinity in humans using tracer kinetic modeling, positron emission tomography, and [11C]flumazenil. J Cereb Blood Flow Metab. 1993; 13: 656-667. doi: 10.1038/jcbfm.1993.84.
Salmi E, Aalto S, Hirvonen J, Långsjö JW, Maksimow AT, Oikonen V, Metsähonkala L, Virkkala J, Någren K, Scheinin H. Measurement of GABAA receptor binding in vivo with [11C]flumazenil: a test-retest study in healthy subjects. Neuroimage 2008; 41(2): 260-269. doi: 10.1016/j.neuroimage.2008.02.035.
Salmi E, Kaike KK, Metsähonkala L, Oikonen V, Aalto S, Någren K, Hinkka S, Hietala J, Korpi ER, Scheinin H. Sevoflurane and propofol increase 11C-flumazenil binding to gamma-aminobutyric acidA receptors in humans. Anesth Analg. 2004; 99: 1420-1426. doi: 10.1213/01.ane.0000135409.81842.31.
Samson Y, Bernuau J, Pappata S, Chavoix C, Baron JC, Maziere MA. Cerebral uptake of benzodiazepine measured by positron emission tomography in hepatic encephalopathy. New Engl J Med. 1987;316:414-415. doi: 10.1056/nejm198702123160716.
Sanabria-Bohórquez SM, Labar D, Levêque P, Bol A, De Volder AG, Michel C, Veraart C. [11C]Flumazenil metabolite measurement in plasma is not necessary for accurate brain benzodiazepine receptor quantification. Eur J Nucl Med. 2000; 27(11):1674-1683. doi: 10.1007/s002590000336.
Shinotoh H, Yamasaki T, Inoue O, Itoh T, Suzuki K, Hashimoto K, Tateno Y, Ikehira H. Visualization of specific binding sites of benzodiazepine in human brain. J Nucl Med. 1986; 27: 1593-1599. PMID: 3020192.
Updated at: 2020-01-01
Created at: 2013-06-18
Written by: Vesa Oikonen