Comparison of the binding of the irreversible monoamine oxidase tracers, [11C]clorgyline and [11C]l-deprenyl in brain and peripheral organs in humans

Nuclear Medicine and Biology 31 (2004) 313–319

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Comparison of the binding of the irreversible monoamine oxidase tracers, [11C]clorgyline and [11C]l-deprenyl in brain and peripheral organs in humans Joanna S. Fowlera,*, Jean Logana, Gene-Jack Wanga, Nora D. Volkowa, Frank Telanga, Yu-Shin Dinga, Colleen Sheaa, Victor Garzaa, Youwen Xua, Zizhong Lia, David Alexoffa, Paul Vaskaa, Richard Ferrieria, David Schlyera, Wei Zhub, S. John Gatleya a

b

Brookhaven National Laboratory, Chemistry Department, Bldg 555, Upton, NY 11973, USA Department of Applied Mathematics and Statistics, State University of New York at Stony Brook, Stony Brook, NY 11794, USA Received 26 August 2003; accepted 3 October 2003

Abstract The monoamine oxidase A and B (MAO A and B) radiotracers [11C]clorgyline (CLG) and [11C]L-deprenyl (DEP) and their deuterium labeled counterparts (CLG-D and DEP-D) were compared to determine whether their distribution and kinetics in humans are consistent with their physical, chemical and pharmacological properties and the reported ratios of MAO A:MAO B in post-mortem human tissues. Irreversible binding was consistently higher for DEP in brain, heart, kidneys and spleen but not lung where CLG ⬎DEP and not in thyroid where there is no DEP binding. The generally higher DEP binding is consistent with its higher enzyme affinity and larger free fraction in plasma while differences in regional distribution for CLG and DEP in brain, heart, thyroid and lungs are consistent with different relative ratios of MAO A and B in humans. Published by Elsevier Inc. Keywords: Monoamine oxidase A and B; Brain; Peripheral organs

1. Introduction Monoamine oxidase (MAO) is a key regulatory and protective enzyme because its substrates include many physiologically active amines including neurotransmitters, drugs and dietary amines. It occurs in two different subtypes, MAO A and MAO B which are different gene products and have different substrate and inhibitor specificities [reviewed in 1]. MAO A preferentially oxidizes norephinephrine and serotonin and is selectively inhibited by clorgyline [2] while MAO B preferentially breaks down benzylamine and phenylethylamine and is selectively inhibited by L-deprenyl [3]. The relative ratios of MAO A and B are both organ and species dependent [4]. In addition, MAO can be affected by a number of variables including age, drugs, tobacco smoking and genetics [1,5]. We have developed the irreversibly binding suicide en* Corresponding author. Tel.: ⫹1-631-344-4365; fax: ⫹1-631-3225815. E-mail address: [email protected] (J.S. Fowler). 0969-8051/04/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.nucmedbio.2003.10.003

zyme inactivators [11C]clorgyline (CLG) and [11C]L-deprenyl (DEP) and their deuterium substituted isotopomers (CLG-D and DEP-D) to measure MAO A and B activity respectively in the human brain and in certain peripheral organs [6,7,8,9,10] (Fig. 1 for structures). In these PET studies we have observed a generally much more robust binding of DEP than CLG even though the mechanisms of irreversible covalent binding of these tracers to their respective MAO subtype are basically the same. This stimulated us to directly compare their kinetics and distribution in brain and peripheral organs and to investigate factors which might account for the more robust binding of DEP. Here we compared CLG and DEP with respect to plasma clearance, magnitude of the isotope effect, plasma to tissue transport, irreversible binding to MAO and relative organ distribution in healthy human subjects studied previously [7,8,9,10]. In addition, we studied two subjects who received both tracer pairs and one who had a whole body scan with both CLG and DEP for direct within-individual comparison. We measured lipophilicity (Log P) and plasma protein binding for CLG and DEP in order to determine whether the similarities

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Fig. 1. Structures of [11 C]clorgyline (CLG) and [11 C]clorgyline-D2 (CLG-D) and [11 C]L-deprenyl (DEP) and [11 C]L-deprenyl-D2 (DEP-D). The arrows indicate the position of the bond cleaved by MAO.

and differences in these properties are consistent with our observations. We discuss the results in terms of the physical, chemical and pharmacological properties of these two molecules as well as the different regional concentration of MAO A and B.

2. Materials and methods 2.1. PET studies These studies were approved by the Institutional Review Board at Brookhaven National Laboratory and written informed consent was obtained from each subject after the procedures had been explained. We compared data from PET studies of the brain and peripheral organs with CLG and CLG-D and DEP and DEP-D in a retrospective analysis. Subject data and original references are summarized in Table 1. Arterial blood samples were taken over the entire scanning period. Plasma was separated and counted and selected plasma samples were analyzed for the presence of parent tracer [11]. Time-activity curves for parent tracer in arterial plasma were integrated over the experimental period for comparison. Regions of interest for the brain (global region only) and the peripheral organs were determined as described (Table 1). We also scanned the torso area of two of the subjects with both CLG/CLG-D and DEP/DEP-D using the dynamic protocol [9,10] (Siemens/CTI ECAT HR⫹, with 4.6 x 4.6 x 4.2 mm resolution at the center of the field of view) and one of the subjects had two whole body scans, one with CLG (7.23 mCi) and one with DEP (7.66 mCi)) from pelvis to brain using a standard clinical whole body protocol provided by the PET camera manufacturer (CTI/Siemens). For

the whole body scans, scanning was initiated 25 minutes after radiotracer injection (the time at which tracer accumulation plateaued). Data was acquired using 8 bed positions of 10 minutes each. For each bed position transmission data was acquired for 4 minutes and emission data was collected for 6 minutes with septa extended (2D mode). This whole body data was processed using segmented transmission attenuation correction and iterative image reconstruction. Urine was collected and counted at 84-87 minutes post injection for six of the subjects for CLG and four of the subjects for CLG-D. Similarly urine was collected from 3 subjects who received DEP and 1 subject who received DEP-D. Model terms for the brain and the peripheral organs were estimated as described previously (Table 1 references). Briefly, a three-compartment model was applied to timeactivity data from different organs and the arterial input function to calculate K1, the plasma to organ transfer constant, which is related to blood flow, k2 which is related to the back-transfer of tracer from organ to plasma and also to blood flow and k3, the kinetic constant describing the rate of binding to MAO. MAO concentration is not determined directly but inferred from the model term k3. However, since estimates of k2 and k3 are highly correlated, a bias can occur in the estimation of k3. This is particularly problematic in regions of high MAO concentration where k2 may be underestimated leading to an underestimation of k3 and of the inferred relative enzyme concentration. By using the combination model parameter ␭k3 as an index of MAO activity, the correlation problem is reduced since ␭k3 depends upon the ratio k3/k2. Reproducibility in test/retest studies is improved by using ␭k3 rather than k3 [12]. The combination parameter, ␭k3, is used as an index of MAO. ␭ is defined as K1/k2 and is independent of blood flow [13].

Table 1 Subject and scanner information for comparative analysis of CLG and DEP binding in brain and peripheral organs Radiotracers

Number of subjects

Region

Average age (gender)

Scanner

Reference

CLG/CLG-D CLG/CLG-D DEP/DEP-D DEP/DEP-D

5 11 5 9

brain torso* brain torso

40 ⫾ 4.3 (5 M) 37 ⫾ 8 (10 M/1F) 52 ⫾ 8 (4M/1F) 37 ⫾ 7 (7 M/2F)

EXACTHR⫹ EXACTHR⫹ CTI-931 EXACTHR⫹

[7] [9] [8] [10]

* eight subjects received torso scans (heart to kidney field of view) and three subjects received scans of their throat (thyroid) area

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Fig. 2. (top row) Time-activity curves for CLG (closed circles) and CLG-D (open circles) and (bottom row) DEP (closed squares) and DEP-D (open squares) in heart, lungs and kidneys. Data for the kidney and lungs is from the same subject and data from the heart is from a different subject so that for each organ, the time activity curves are directly comparable for all four tracers. [Positioning difficulties prevented us from having the heart, lungs and kidneys in the same field of view for all four tracers in a single subject].

2.2. Log P measurements A modification of a literature procedure [14] was used. Briefly, an aliquot of CLG or DEP solution was added to a mixture of 1-octanol (0.1 mL) and PBS buffer (0.9 mL; pH: 7.4). The mixture was vortexed at room temperature for 2 min and then centrifuged at 7000 rpm for 2 min. and the aqueous layer separated very carefully by a syringe. The two phases were counted, the aqueous layer was discarded and PBS buffer (0.9 mL) added to the octanol phase. The procedure was repeated until a constant ratio of decaycorrected counts in the two phases was obtained, and Log P was then derived. 2.3. Plasma protein binding An aliquot of labeled CLG or DEP was counted and added to 500 ␮l of human plasma and this is incubated for 10 min at room temperature. Precisely measured volumes (20-40 ␮l) of the incubated spike plasma are counted. 200400 ␮l of the incubation mixture was placed in the upper level of a Centrifree tube (Amicon Inc, Beverly, Mass) and this was centrifuged for 10 minutes. After centrifuging, the top portion of Centrifree tube containing the bound portion

was removed and discarded and precisely measured aliquots (20-40 ␮l) of the liquid in the cup (unbound fraction) were counted. The free fraction is the ratio of the decay-corrected counts the unbound aliquots to the decay-corrected counts of the unspun aliquots. 2.4. Statistical analysis The integrated value of the plasma input function (from 0-52.5 min) and the model terms K1, ␭k3 and k3/k2 and the isotope reduction factors (ratio of ␭k3(H)␭k3 (D)) for brain, heart lungs, kidneys, spleen and thyroid for CLG and DEP were compared using unpaired samples t-tests. The integrals of the time-activity curves for the radiotraces in the arterial plasma were compared using paired t-tests (2-tail).

3. Results Both CLG and DEP show rapid distribution and uptake with peak concentration occurring at ⬃3 minutes and plateau occurring by 20 minutes in the heart, lungs, kidneys and spleen with a reduction in uptake with deuterium substitution. These data are shown in Fig. 2 which presents the

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Fig. 3. (left panel) Time-activity curves for CLG and CLG-D (closed and open circles) and DEP and DEP-D (closed and open squares) for the liver for the same subject.

time-activity curves for the lungs and kidneys for one of the subjects who receive scans with both CLG and CLG-D and DEP and DEP-D and for the heart for another subject who received both tracer pairs. [These plots provide a direct comparison of measured time-activity data for peripheral organs for CLG and DEP without the confounds of individual variability.] CLG also accumulated in the thyroid and uptake was reduced with deuterium substitution (data not shown). There was a similar pattern in the brain for CLG and DEP [7,8] though we did not obtain brain scans with both tracer pairs in the same subject. In the liver C-11 peaks at ⬃15 min and plateaus thereafter for both CLG and DEP and shows no isotope effect indicating that liver MAO is probably serving as a pathway for the excretion of labeled metabolites (Fig. 3 for comparison of CLG/CLG-D and DEP/DEP-D in the same subject). Both CLG and DEP have low excretion into the urine (1-3% over 90 min). Contrasting with the similarity of the shapes of the timeactivity curves, the absolute uptake of CLG and DEP differed both in magnitude and in relative regional distribution. DEP clearly shows overall higher uptake and this is most strikingly illustrated in whole body images of the same subject scanned with approximately the same dose of CLG and DEP (7.23 vs 7.66 mCi; Fig. 4) with scanning beginning at 25 minutes post radiotracer injection. The higher uptake of DEP over CLG occurred in brain, kidneys, heart and liver. The time-activity curves in Fig. 2 show the greater binding of DEP vs CLG in heart and kidneys and greater binding of CLG vs DEP in the lungs in subjects scanned with both tracers pairs. This high specific binding of CLG and relatively low binding of DEP in the lungs as well as the opposite pattern for the heart is also quite evident in the whole body images shown in Fig. 4 which also shows thyroid binding for CLG but not for DEP.

There is a significantly greater plasma clearance rate for DEP relative to CLG. This is also true for the CLG-D and DEP-D (Fig. 5). More specifically, the integral of the timeactivity data for the arterial plasma for unchanged tracer over the 52.5 minute experimental period scaled to a 1 mCi dose showed values of 344⫾42 and 227⫾40 nCi/ml x min for CLG and DEP respectively (p⫽0.0001, unpaired t test) and 423⫾45 and 311⫾43 nCi/ml x min for CLG-D and DEP-D respectively. In each case the deuterium substituted counterpart had a significantly higher value for the integral than the parent tracer (p⫽0.0001, paired t test). For the sake of simplicity, we only compare the model terms for CLG and DEP and not CLG-D and DEP-D since

Fig. 4. (left panel) Whole body images of one of the subjects who was injected with both CLG (7.23 mCi) and DEP (7.66 mCi) on different days. Imaging was started 25 minutes after the radiotracer injections when radiotracer tracer accumulation plateaus. Scanning was carried out from pelvis to head.

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Fig. 5. Values of the integral of the plasma radiotracer concentration over a 52.5 minute experimental period for CLG and CLG-D and for DEP and DEP-D scaled to a 1 mCi dose. Differences between CLG and DEP and between the H and D isotopomers were significant at the P⫽0.0001 level.

the results for CLG-D and DEP-D show the same patterns as CLG and DEP. The model term K1 which is the plasma to tissue transfer constant and which is a function of blood flow and the permeability surface product in the organ is significantly greater for DEP than CLG in the heart, lungs and kidneys with a trend for a greater value for the spleen (Fig. 6A). The model term ␭k3 which is proportional to MAO concentration is larger for DEP than for CLG except for the lung (Fig. 6B). Both CLG and DEP showed a robust isotope effect (2.6-5.7; p⬍0.001) on ␭k3 in the brain, heart, lungs, kidneys, spleen and thyroid (CLG only). For the peripheral organs, the magnitude of the isotope effect differed for different organs but not for CLG vs DEP (Table 1). In contrast, in the brain (global) there was a significantly lower isotope effect for CLG than for DEP. The ratio of k3/k2 which is an index of the potential influence of blood flow on the binding of the radiotracer to MAO was significantly greater for DEP than for CLG in the heart and brain with a trend (p⫽0.07) for DEP⬎CLG for the kidney (Fig. 6C). This high rate of binding has been particularly problematic for [11C]L-deprenyl in the brain and, in fact, to improve quantification, we now routinely use [11C]L-deprenyl-D2 for brain imaging because its forward binding rate is slower [15]. The measured log P in pH 7.4 buffer was 2.6 for both CLG and DEP. The free fractions in plasma were 0.6% and 6.0% for CLG and DEP respectively.

4. Discussion A major challenge in radiotracer development is to improve our ability to predict and control the behavior of

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labeled compounds in the human body. In this regard, human PET data presents an opportunity to investigate whether radiotracer distribution, specificity and kinetics are consistent with the physical, chemical and pharmacological properties of the parent compound. In this study we sought to gain a better understanding of the factors underlying the generally more robust binding of DEP relative to CLG in brain and in peripheral organs in humans. Both CLG and DEP are suicide enzyme inactivators for MAO with high affinity and high selectivity for their respective enzyme subtypes [2,3]. However, DEP has a higher affinity for MAO than CLG (6.8 vs 39 nM for DEP vs CLG for MAO B and MAO A in rat brain [16,17]). Both inhibit MAO by irreversible covalent attachment after enzyme catalyzed cleavage of the C-H bond on the methylene carbon of the propargyl group in the rate limiting (or rate-contributing) step (Fig. 1 for structures). Bond cleavage occurs within the enzyme substrate complex and results in an activated intermediate which covalently binds to the enzyme, irreversibly deactivating it. When CLG and DEP are labeled with C-11, the enzyme becomes labeled [6]. The significant isotope reduction factor for CLG and DEP in brain and in certain peripheral organs provides compelling evidence for this mechanism (Table 2). A number of factors may account for similarities and differences between CLG and DEP. Irreversible labeling of MAO in vivo is consistent with the similar shape of the time-activity curves. Assuming that the relative values for affinity can be extrapolated to humans and that affinities and concentrations are similar for different organs, one could predict a more avid binding to MAO in the case of DEP. More robust organ uptake is consistent with the more rapid plasma clearance for DEP relative to CLG and may reflect the higher free fraction in plasma for DEP relative to CLG (6% and 0.6% for DEP and CLG respectively). The higher free fraction is also reflected in the high values of the model term K1 for DEP for the heart and the kidney which represents the transfer of tracer from plasma to tissue although we realize that the rate at which the radiotracers dissociates from the plasma protein may also contribute to differences between CLG and DEP. It is unlikely that lipophilicity plays a role in the observed differences since the measured log P value for both CLG and DEP is 2.6. MAO A and MAO B have been reported to have different relative regional distributions in the human body as has been determined using autoradiography with the reversible, mechanism based selective MAO A and B inhibitors, [3H]Ro 41 1049 and [3H]Ro19 6327 respectively [18]. High lung uptake and high values for ␭k3 in the lung for CLG vs DEP are consistent with the known high MAO A:MAO B ratio in the lung [18] which probably overwhelms other factors such as DEP’s high affinity and high plasma free fraction relative to CLG. Expressed in another way, the heart/lung ratios of ␭k3 are 0.63⫾0.38 and 3.3⫾0.83 for CLG and DEP respectively (p⫽0.0001) which is similar to the ratios obtained from the post mortem report (⬃0.9 vs

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Fig. 6. (A) Comparison of K1 (plasma to tissue transfer in units of ml/min/g) for CLG and DEP in heart, lungs, kidneys, spleen and brain; (B) comparison of ␭k3 (model term proportional to MAO activity in units of ccbrain (mlplasma)-1 min-1) for CLG and DEP; (C) comparison of k3/k2 for CLG and DEP. * pⱕ0.03; **pⱕ0.003.

3.1; [18]). We note that the role of lung MAO A in the oxidation of circulating vasoactive amines such as norepinephrine and serotonin which are both MAO A substrates has long been appreciated [19]. The values of ␭k3 in the heart for DEP vs CLG are also consistent with the higher levels of MAO B in the human heart [18]. The exclusive appearance of the MAO A subtype in the human thyroid has been reported in the analysis of human thyroid post mortem [20]. This is apparent in the whole body images in one of the subjects in whom thyroid uptake is seen for CLG but not DEP (Fig. 4). Furthermore, binding is specific as shown by a robust deuterium isotope effect. Thyroidal MAO A appears to have an indirect effect on iodide transport mediated by the accumulation of monoamines in neuroendocrine areas involved in thyrotrophin regulation [21,22]. Similar to results in peripheral organs, we also note that values of K1 and ␭k3 in brain were significantly larger for DEP than for CLG [7,8]. This is shown in Fig. 6 for CLG and DEP. We also note that the ratio of k3/k2 is significantly higher for DEP than for CLG (Fig. 6C) making DEP-D a far more sensitive radiotracer than DEP for brain MAO B studies. Deuterium substitution also reduces the k3/k2 ratio for CLG in the brain but at the expense of revealing nonMAO A binding in white matter. In fact the significantly Table 2 Isotope reduction factors (equal to the ratio ␭k3(H)/ ␭k3(D). There were no significant differences between the degree to which deuterium substitution reduced the binding of [11C]clorgyline and [11C]L-deprenyl* except for the brain. There is no MAO B in the thyroid [20]. Organ

MAO A

MAO B

Brain (global) Heart Lungs Kidneys Spleen Thyroid

2.57 ⫾ 0.17 3.4 ⫾ 0.4 3.7 ⫾ 0.9 4.7 ⫾ 1.0 2.44 ⫾ 1.65 3.49 ⫾ 0.42

4.30 ⫾ 0.56* 3.7 ⫾ 0.62 3.3 ⫾ 0.9 5.7 ⫾ 1.5 2.6 ⫾ 0.85

* p⫽0.0002 (paired t, ⫺6.7)

lower isotope reduction factor for CLG vs DEP in the global brain region is due to high non-specific white matter binding which comprises a large area of the global region [7]. Again for the brain as in the peripheral organs, values of K1 probably reflect the larger free fraction for DEP in plasma whereas high values of ␭k3 probably reflect the higher affinity of DEP vs CLG and the higher MAO B:MAO A ratio in the human brain [23]. While heart/lung ratios of ␭k3 for CLG and DEP are similar to the reported post-mortem values, the heart:kidney ratios are not consistent between the PET and the autoradiographic methods. For example, while heart:kidney ratios are not significantly different for ␭k3 for CLG and DEP (0.59⫾0.09 vs 0.55⫾0.15), post mortem ratios show similar levels of MAO A in the heart and kidney and greater levels of MAO B in heart vs kidney. Though we do not know the reasons for this discrepancy, we note that the age of the subjects in the post-mortem analysis was 72⫾1.7 years [18] which is considerably older than the volunteers (average age 39.2⫾8.7 yrs) used in the PET study. In summary, the binding patterns of CLG and DEP in the human brain and peripheral organs have been compared and are consistent with similarities in their mechanism of action. Differences including more rapid plasma clearance and generally more robust binding of DEP relative to CLG are consistent with its higher enzyme affinity and higher plasma free fraction. However, differences in human MAO subtype concentration overwhelms these other factors in the lung in which high MAO A:MAO B ratio underlies correspondingly higher CLG binding and in the thyroid where the absence of MAO B in the human thyroid is reflected by the absence of DEP binding.

Acknowledgments This work was carried out at Brookhaven National Laboratory under contract DE-AC02-98CH10886 with the U. S. Department of Energy and supported by its Office of Bio-

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logical and Environmental Research and by the National Institute for Biomedical Imaging and Bioengineering (EB002630), the National Institute on Drug Abuse (DA 7092-01& DA00280) and the Office of National Drug Control Policy. We are grateful to Michael Schueller for cyclotron operations and to Karen Apelskog for protocol coordination. We are also grateful to the people who volunteered for these studies.

[11]

[12]

[13]

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