Background: The α7 nicotinic acetylcholine receptor increasingly has been implicated in normal brain physiology, as well as in neuropsychiatric disorders. The highly cortical distribution of α7 nicotinic acetylcholine receptor suggests a role in cognition. Methods: We expanded the first-in-human PET imaging of α7 nicotinic acetylcholine receptor with [ F]ASEM from 5 to 21 healthy nonsmoking volunteers and added a feasibility study in 6 male patients with schizophrenia. Study aims included: (1) confirmation of test-retest reproducibility of [F]ASEM binding, (2) demonstration of specificity by competition with DMXB-A, an α7 nicotinic acetylcholine receptor partial agonist, (3) estimation of [ F]ASEM binding potentials and α7 nicotinic acetylcholine receptor density in vivo in humans, and (4) demonstrating the feasibility of studying α7 nicotinic acetylcholine receptor as a target for schizophrenia. Received: January 8, 2018; Revised: February 10, 2018; Accepted: March 5, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any 1 medium, provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy021/4924585 by Ed 'DeepDyve' Gillespie user on 08 June 2018 2 | International Journal of Neuropsychopharmacology, 2018 Results: Test-retest PET confirmed reproducibility (>90%) (variability ≤7%) of [ F]ASEM volume of distribution (V) estimates in healthy volunteers. Repeated sessions of PET in 5 healthy subjects included baseline and effect of inhibition after oral administration of 150 mg DMXB-A. From reduction of binding potentials, we estimated the dose-dependent occupancy of α7 nicotinic acetylcholine receptor by DMXB-A at 17% to 49% for plasma concentrations at 60 to 200 nM DMXB-A. In agreement with evidence postmortem,α 7 nicotinic acetylcholine receptor density averaged 0.67 to 0.82 nM and inhibitor affinity constant averaged 170 to 385 nM. Median V in a feasibility study of 6 patients with schizophrenia was lower than in healthy volunteers in cingulate cortex, frontal cortex, and hippocampus (P = 0.02, corrected for multiple comparions, Mann–Whitney test). Conclusions: The current results confirm the reproducibility of [ F]ASEM V estimates and the specificity of the tracer for α7 nicotinic acetylcholine receptor. Preliminary findings from our feasibility study of [ F]ASEM binding in patients with schizophrenia are suggestive and provide guidance for future studies with more subjects. Keywords: PET imaging, nicotinic acetylcholine receptors, schizophrenia Introduction As ligand-gated excitatory cation channels, nicotinic acetyl- hampered noninvasive research of this receptor system. Many choline receptors (nAChRs) in central nervous system (CNS) are radiotracers of interest to α7-nAChR ( C-CHIBA-1001; Toyohara fundamental to physiology (Lukas et al., 1999). The 2 nicotinic et al., 2009), C-NS14492 (Ettrup et al., 2011Ma ; gnussen et al., receptor subtypes, α4β2 and α7, predominate in CNS among 2015), and others have been tested, but most had suboptimal many other nAChR subtypes (Lukas et al., 1999; Gotti and properties in PET of humans (see Gao et al., 2013 for review). Clementi, 2004). Nicotine binds with high affinity to α4β2-nAChR Here, we further tested human subjects with [F]ASEM, the (K ~ 1 nM; Anderson and Arneric, 1994) and with a much lower only α7-nAChR PET tracer available to humans with promis- affinity to α7-nAChR (K ~ 6000–14 000 nM; Davies et al., 1999). ing imaging characteristics (Gao et al., 2013b Horti et ; al., 2014b ; The α7-nAChR subtype is associated with the pathophysi- Horti, 2015; Coughlin et al., 2018Hillmer et ; al., 2017 W ; ong et al., ology of specific CNS diseases, including schizophrenia, drug 2017). Objectives included: (1) confirmation of test-retest repro- addiction, depression, anxiety, and multiple neurodegen- ducibility of [ F]ASEM binding in brain of normal volunteers, (2) erative disorders (Verbois et al., 20002002 , ; Woodruff-Pak and demonstration for the first time of specific binding and target Gould, 2002; Albuquerque et al., 2009; D’Hoedt and Bertrand, engagement/occupancy in human brain of the partial agonist 2009; Philip et al., 2010; Hoffmeister et al., 2011; Ishikawa and DMXB-A (GTS-21), (3) estimation for the first time of [ F]ASEM Hashimoto, 2011; Parri et al., 2011) and therefore is an import- binding potentials and α7-nAChR receptor density (B ) and max ant target of drug discovery. Multiple postmortem studies have inhibitor affinity constant (K) for human brain in vivo, and (4) a shown reduced α7 receptors in brain samples from patients feasibility study of the target role of α7-nAChR in psychosis in 6 with schizophrenia, suggesting a role of α7 in schizophrenia (see patients with schizophrenia. review by Thomsen et al., 2010). In autoradiography of brains from patients with schizophrenia, reductions of α-[I] BTX Methods binding to α7 were demonstrated in hippocampus, anterior cin- gulate cortex, and thalamic reticular nucleus (Freedman et al., Subjects 1995) but not in orbitofrontal or temporal lobes (Court et al., 1999; Marutle et al., 2001). All studies were carried out with the approval of the Johns The α7-nAChR has emerged as a potential therapeutic Hopkins Institutional Review Board, where all subjects provided target for treatment of schizophrenia (Wallace et al., 2011; written informed consent. Participants were recruited via ad-ver AhnAllen, 2012; Geerts, 2012; Wallace and Bertrand, 2013; tisements within the metropolitan Baltimore region. Young and Geyer, 2013a; Freedman, 2014b; Beinat et al., 2015). Twenty-one healthy, nonsmoking control subjects (13 In animals, stimulation of 7 is pr α ocognitive (Young and Geyer, males and 8 females; age range: 18–52 years; means± SD: 2013a; Potasiewicz et al., 2017). In clinical trials with select- 32.6 ± 12.4 years) volunteered for the study. In addition to base- ive α7 agonists, activation of the receptor improved cognitive line PET, 12 subjects had a second baseline PET (test-retest), and performance in patients with schizophrenia. Selected drugs 5 other subjects had a second PET after a single dose of DMXB-A. (DMXB-A, also known as GTS-21, TC-5619, and EVP-6124) show Only 8 of the 12 subjects had successful test-retest PET due to some degree of cognitive improvement in trials of patients with technical problems in either test or retest in 4 subjects. Of the schizophrenia, although none has yet proceeded to late phase subjects, 5 (including 2 with test-retest from a previous publi- or FDA approval. A common trend is the improvement of cog- cation by Wong et al., 2014) were included in this study. Healthy nition with an inverted U-shaped dose–response curve, when volunteers had no psychotropic treatment history, were free of increasing the dose of α7 agonist above a threshold resulted significant medical or neuropsychiatric disorder, and received in loss of drug effect (Wallace and Bertrand, 2015 Le ; wis et al., no current medications. 2017). The latest research has revealed a new generation of To examine the feasibility of measuring α7 nAChR in promising α7-targeting drugs (Beinat et al., 2016). schizophrenia, 6 patients diagnosed with schizophrenia (aged Despite the importance of the nicotinic receptor system, 20–31 years; average 25.0± 4.3 [SD] y; all males; 1 smoker the physiological and pharmacological roles of specific recep- only) were recruited. Volunteers with schizophrenia that met tor subtypes in CNS remain poorly understood (Philip et al., DSM-V criteria, with stable antipsychotic medication other 2010; Ishikawa and Hashimoto, 2011; Marrero et al., 2011P ; arri than clozapine, were referred by collaborating psychiatrists. et al., 2011). Until recently, the lack of radioligands for quanti- Subjects underwent neuropsychiatric characterization includ- tative emission tomography imaging of α7-nAChR in humans ing Brief Psychiatric Rating (BPRS) scores. Demographic data Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy021/4924585 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wong et al. | 3 Table 1A. Group Demographics Age Diagnosis Condition n Males Females (mean/SD) Age (range) Healthy controls Baseline 21 13 8 32.0 (12.4) 18–52 Retest 8 6 2 37.8 (11.7) 20–52 Blocking 5 3 2 22.1 (4.5) 18–28 Patients with schizophrenia Baseline 6 6 0 24.8 (3.9) 20–31 No healthy controls reported current or past smoking history. Table 1B. Patients with Schizophrenia BPRS – Negative BPRS – Positive Subject ID Age Sex Race Ethnicity (M) (M) Smoker Cigarettes/d Medications 01 27 M AA NH 2.25 1.25 No N/A Aripiprazole 02 31 M W NH 3 1 Yes 20 Haloperidol, benztropine, sertraline 03 22 M AA NH 2.75 2.25 No N/A Risperidone 04 25 M AA NH 2.75 1.75 No N/A Quetiapine, haloperidol, benztropine 05 20 M AA NH 1.5 1.5 No N/A Escitalopram, benztropine, aripiprazole, clonezapam 06 – Scan 1 24 M W NH 2.75 3.25 No N/A Risperidone, lorazepam 06 – Scan 2 24 M W NH 1.0 1.0 No N/A Lorazepam for healthy controls and subjects with schizophrenia are listed radiometabolite detection during the 90-min imaging session. in Table 1A, 1B. Participants provided information of history of Arterial plasma samples were drawn as rapidly as possible for tobacco use, including number of cigarettes per day and years the first 2 min and then every minute until 10 min post injection, of tobacco use. every 2 min until 20 min post injection, and at the times 25, 30, All subjects (controls and patients with schizophrenia) had 45, 60, 75, and 90 min post injection. Metabolites were measured negative urinary toxicology tests and provided CO breath sam- for the times of 2, 5, 10, 20, 30, 60, and 90 min post injection. (See ples of <4 ppm on the imaging day to confirm abstinence for at Supplemental Material for HPLC methods.) least 12 h from tobacco smoking for any subjects (see additional Each subject received 13.8 mCi± 0.23 (SEM) [ F]ASEM synthe- inclusion/exclusion criteria in Supplemental Material). sized by the method of Gao et al. (2013) with average specific One patient with schizophrenia was tested with PET [ F] activity of 56 417 Ci/mmol. There were no significant differences ASEM while chronically treated with risperidone and then in injected radioactivity or specific activity between the controls retested with PET without drug for 3 weeks (discontinued for and patients with schizophrenia. Tracer-free fraction in plasma clinical reasons). across subjects averaged 2.79% ± 0.06 (SD) and was not signifi- To address possible effects of antipsychotics administered to cantly different in healthy controls and patients with schizo- patients, we determined biodistribution in mice treated for 5 d phrenia (methods in Supplemental Materials). with cohorts of placebo vs risperidone, aripiprazole, or olanzap- Of the healthy volunteers, 12 were retested with F]ASEM [ ine, the 3 most common drugs in the present patient population, imaging approximately 5 weeks (mean 41.2 d) after the first at doses equivalent to doses used in humans (Reagan-Shaw et al., PET session, including 3 reported by Wong et al. (2014). We care- 2008; Dawe et al., 2010; McOmish et al., 2012; Ning et al., 2017). fully reviewed the radiometabolite HPLC results and elected to Mice were i.v. injected with [ F]ASEM and sacrificed after 60 min exclude 4 subjects for technical reasons (e.g., defective capture (detailed Methods and Figure A in Supplemental Materials). columns during early development of HPLC procedures). To determine the target engagement (occupancy) by a receptor lig- and and to obtain evidence of specific binding of [ F]ASEM to α7- PET nAChR in humans, 5 healthy subjects additionally underwent All subjects underwent PET with the Siemens/CTI High- a second imaging session after administration of oral 150 mg Resolution Research Tomography (HRRT: resolution <2 mm). The of the partial agonist DMXB-A, 40 min before the PET tracer dynamic imaging continued for 90 min with the frame sequences injection. Radial arterial samples for detection of concentra- of 4 x 15 s, 4 x 30 s, 3 x 60 s, 2 x 120 s, 5 x 240 s, and 12 x 300 s. Each tion DMXB-A were obtained during PET imaging at 70, 100, and subject received a radial arterial catheter for collection of approx-130 min post-oral dose of DMXB-A. Concentrations of DMXB-A imately 40 arterial blood plasma samples and subsequent HPLC in subjects’ plasma samples were determined quantitatively by Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy021/4924585 by Ed 'DeepDyve' Gillespie user on 08 June 2018 4 | International Journal of Neuropsychopharmacology, 2018 Figure 1. Estimates of V and test-retest variability for . (A) Re V gional estimates of V for all healthy control subjects (n = 21). (B) Test-retest variability of V in 8 healthy T T T T controls. For both panels, bars show mean ± SD. Abbreviations: Am, amygdala; CN, caudate nucleus; Cb, cerebellum; Cg, cingulate; Fr, frontal lobe; Fs, fusiform gyrus; GP, globus pallidus; Hp, hippocampus; In, insula; LG, lingular gyrus; Oc, occipital lobe; pC, paracentral; PH, parahippocampus; Pa, parietal lobe; PS, postcentral gyrus; Pc, precentral gyrus; Pr, precuneus; Pu, putamen; Tp, temporal lobe; Th, thalamus; vS, ventral striatum. HPLC with a modification of the previously reported method (Kem et al., 2004). (Details in Supplemental Material.) Menstrual phase for the women was determined by a pro- gesterone level using levels exceeding 2 ng/mL, indicating luteal phase. For 2 women (one in luteal phase and one in follicular phase), menstrual phase was determined by self-reported men- strual history, as progesterone samples were not available. PET Analysis Volumes of Interest Volumes of interest (VOI) were selected automatically in indi- vidual subject SPGR MRI volumes using FSL (FMRIB Software Library; Jenkinson et al., 2012) FIRST tool (Patenaude et al., 2011) for subcortical regions and Freesurfer tool (Fischl et al., 2004) for cortical regions. Automated VOI were manually edited to suit the structures of interest, using a locally developed VOI tool (VOILand). Refined VOI were transferred from MRI to PET spaces according to MRI to PET coregistration parameters obtained from the co-registration module of SPM12 (Statistical Parametric Mapping; Ashburner and Friston, 2003). The VOI of PET space were applied to PET frames to obtain time-activity curves of Figure 2. Dose-dependent inhibition with DMXB-A level. The saturation, s, regions. Head-motion correction was performed with the image reflects the fraction of receptor occupied by the blocking agent, DMXB-A. Each point on the curve represents individual measurements of the plasma level of co-registration module of SPM12. DMXB-A, determined by HPLC mass spectroscopy, and the fraction of receptors Total volumes of distribution (V ) and test-retest variability occupied by DMXB-A, estimated by the Extended Inhibition Plot (EIP). The hori- were estimated for all 21 cortical VOI (Figure 1A–B). In addition, zontal error bars represent the SEM from 3 measurements, and the vertical error the full VOI set was used in the calculation of dose-depend- bars reflect the SEM from the linear analysis of the Extended Inhibition plot. ent inhibition (Figure 2) as well as for the averaged estimates These data were fit to the Michaelis-Menten equation, with O assigned to a max of maximum binding capacity (B ; Figure 3) and inhibitor K value of 1; (i.e, 100% occupancy, or a saturation fraction, s = 1). This is typical for max I receptor occupancy studies with PET. However, further studies at higher plasma (Figure 4). For analysis of V differences (in patients vs control concentrations may indicate a lower maximal occupancy, due to the unique subjects, Figure 5) and potential correlation with BPRS score (see nature of the α7 subunit, as stated in the Discussion. Supplemental Material), we chose 3 cortical regions implicated 125 125 in autoradiography with [ I]α-bungarotoxin ([ I]BTX) of brain Kinetic Analysis of patients with schizophrenia postmortem, previously reported with significant reductions in hippocampus (~70%, Freedman Quantification of [ F]ASEM steady-state volumes of distribu- et al., 1995), frontal cortex (40%, Guan et al., 1999), and cingulate tion (V ) proceeded as previously published (Wong et al., 2014). cortex (54%, Marutle et al., 2001). Specifically, the plasma reference graphical analysis was used Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy021/4924585 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wong et al. | 5 Figure 3. Regional binding potentials (BP ). The analysis using the Extended Inhibition Plot (EIP) revealed that the BP decreased significantly in response to compe- ND ND tition with DMXB-A by 31% in average compared with baseline. Each line on the graphs of 6 representative regions of the original 21 regions displays the estimates of BP in individual subjects at baseline and inhibition condition. ND Figure 4. B estimates. Average B across all regions (21 VOIs total) were computed from the known BP estimates, the plasma concentration of DMXB-A and the max max ND receptor affinity, K , which was calculated as 0.35 nM based on known values from I α–bungarotoxin in vitro studies. The analysis reveals that the average acr B oss D max all regions ranged from 0.76 to 0.96 nM in the 5 subjects, and the rK anged from 170 to 385 nM. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy021/4924585 by Ed 'DeepDyve' Gillespie user on 08 June 2018 6 | International Journal of Neuropsychopharmacology, 2018 Figure 5. Distribution of V in healthy patients and controls. Boxplots of the distribution of in Vhealthy controls (HC) and patients with schizophrenia (SCZ) in cingu- T T late cortex (Cg), hippocampus (Hp), and frontal cortex (Fr). The single points above the limits of the boxplot are outliers. to obtain regional V values that served as primary outcome where values of V at baseline and inhibition are different, T ND variable for test-retests and analyses with age, sex, and neuro- denoted V and V at baseline and inhibition, respect- ND (b) ND (b) psychiatric testing as well as serving as secondary outcome ively. The EIP yielded the value of V in the inhibition condi- ND variable for estimates of the reference volume of nondisplace- tion (V ), when the true reference volume at baseline (V ) is ND(i) ND(b) able bound radioligand (V ), competitor saturation of receptors known such that estimates yielded by EIP depend on a measure ND (s), binding potentials (BP ), and B . We completed B and of the magnitude of the reference volume. ND max max affinity (IC , K ) estimates of α7-nAChR in 3 steps in 5 subjects Volumes of distribution (V and V ) yielded BP . We esti- 50 I T ND ND by means of DMXB-A inhibition. mated the BP of 5 subjects with DMXB-A inhibition in the ND We estimated the occupancy of the partial agonist inhibitor by baseline and at inhibition, using the values of V and V in T(b) ND(b) means of estimates of the radioligand’s volume of nondisplace- the baseline condition and the values of V and V at inhib- T(i) ND(i) able distributed tracer in the baseline (V ), obtained from baboon ition obtained by means of Equation (2), in the unblocked and ND brain in vivo (Horti et al., 2014), using the conventional Inhibition DMXB-A inhibited states, with the equation Plot (IP) of Gjedde and Wong (2000). We compared the value of V ND V V obtained from the decline of the magnitude of the total volume of T(b) T(i) BP =-11 andBP =- ND(b) ND(i) , (3) V V V in in the presence of 2 different concentrations of SR180711, of ND(b) ND(i) which we used the value associated with the lower dose to avoid affecting the value of V by changes of protein binding in brain ND where the BP and BP terms symbolize the BP at baseline ND (b) ND(i) ND tissue or circulation or both (see supplemental Figure B illustrat- and inhibition, respectively. ing the V determination in baboon brain). ND The BP and inhibitor concentrations (Ci) yielded the B ND max Applied to regional V, the IP yielded reference volumes (V ) and K (IC ) estimates of the receptor and inhibitor, respect- T ND I 50 and degrees of saturation (s), according to the equation (Phan ively. We completed B and affinity of the competitor (IC , K ) max 50 I et al., 2017), calculation of α7-nAChR in 5 subjects by means of DMXB-A inhibition using the values of BP of baseline and inhibition Vs =- () 1 Vs + V ND T(i) Tb () ND , (1) that led to the novel (never previously published) in vivo esti- mate of the magnitude of the B and K of nAChR in humans, max I where V symbolizes the volume of distribution at baseline T (b) using the equation and V the volume at inhibition in presence of DMXB-A that T (i) competes with the radiotracer for binding to the binding site. max BP = ND (4) æ ö DMXB-A  According to Equation (1), when the regional estimates of in V ÷ T ÷ K ç1+ ç K è ø the inhibition condition were plotted as functions of the esti- I mates in the baseline condition, the solution yielded the refer - ence volume, V , and the fraction of receptors occupied by the where the B is determined relative to the affinity of the radio- ND max competitive drug, s. ligand to the receptor K, , with known values of the inhibitor The initial analysis revealed V estimates before and after concentration [DMXB-A] and the receptor’s affinity for inhibitor, DMXB-A inhibition of similar magnitudes, compensating for K . For this calculation, we used a calculated value of ASEM of K I d values of V that may be different in the inhibition and base- 0.35 nM (established from prior measure of K of 0.4 nM for [ I] ND line conditions. To resolve this issue, we applied the Extended bungarotoxin) (Xiao Y et al., 2009). IP equation (EIP; Phan et al., 2017) that relates the radioli- gand distribution volumes at inhibition and baseline as fol- Schizophrenia lows: As a feasibility study, we compared 6 patients with schizophre- nia to a subset of 15 healthy volunteers matched for age, within é ù ND(i) ê ú Vs =- () 1 Vs + V 3 specific brain regions: hippocampus and cingulate and frontal T(b) ND(i), (2) Ti() ê ú ê ND(b)ú ë û cortices. In this subset, the mean age of healthy volunteers was Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy021/4924585 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wong et al. | 7 23 ± 4 (SD) y, range 18 to 30, and for patients with schizophrenia volunteers precluded analysis of different changes of with V the mean age was 24± 4 (SD) y, range 20–31 y. age in men and women. Results Schizophrenia To evaluate the feasibility of studying patients with schizo- Regional V phrenia, we compared patients with healthy control subjects. Regional estimates of V in selected regions are shown in In the 3 preselected regions (cingulate and frontal cortices, Figure 1A. We tested the test-retest variability of the V estimates T hippocampus) examined for group differences between in 8 healthy subjects, 6 males (average age 41) and 2 females patients and the age-matched control subjects, the median (average age 24 y). Across all cortical and subcortical VOIs, the V values were lower in patients than in age-matched healthy average test-retest variability was 7.8% ± 0.6% (SD , see Figure 1B), volunteers (Figure 5). Potential outliers, one a control sub- ranging from 6.5% to 8.9% (repeatability of >90%). ject and one a patient, were identified (outlier points marked in boxplots of Figure 5). Using the Mann-Whitney U-test, we Receptor Kinetics tested for significant differences in median V values between control subjects and patients, before and after excluding out- The reanalysis of inhibition of [ F]ASEM binding in baboon liers. The results, with corrections for multiple comparisons, (Horti et al., 2014) yielded the V of nondisplaceable tracer accu- are given in Table 2. In the cingulate cortex and hippocampus, mulation at baseline (V ), In the baboon, the V of [ F]ASEM ND(b) T the difference in V was significant after exclusion of outli- declined markedly in response to blocking with the high affin- ers and correction for multiple comparisons ( =P .02 in both ity inhibitor SSR180711. The reference volume V assessed by ND regions). means of the standard IP is presented in Figure 2, with the esti- The correlation of estimates of V with scores of BPRS did 3 T mate of V of 5.4 ml/cm at the lower dose of the SR180711. ND (b) not reach statistical significance, although there were positive We note that the magnitude of V is a property of the tracer ND trends for values in cingulate and frontal cortices in 5 subjects [ F]ASEM that we assume to be representative of mammalian after outlier analyses (see supplementary Figure C for details). brains in general. We used the value of V of 5.4 mL/cm as the value of ND Antipsychotic Medication V in the EIP, required when V values failed to yield evi- ND (b) T dence of inhibition of radioligand binding by DMXB-A in The biodistribution in mice revealed no significant effects on human brain. In 5 subjects, the value of V yielded estimates ND(b) [ F]ASEM binding of aripriprizole, risperidone, or olanzapine (see of saturation () and inhibition r s eference volume (V ), as ND(i) Supplemental Materials). As described in Methods, the patient shown in Figure 2 of the individual estimates of as function s with schizophrenia chronically treated with risperidone had of the concentration of DMXB-A in arterial plasma, confirming repeated PET after a 3-week drug-free period, imposed for clini- the dose-dependent inhibition exerted by DMXB-A. Estimates cal reasons. The V estimates did not differ by more than 7% (i.e., of regional BP declined significantly in 6 of the 21 representa- ND within the test-retest variability of control subjects in any brain tive regions after blocking with DMXB-A, as shown in Figure 3. region for the on- and off–risperidone comparison (Figure 1B) The average values for all 21 regions of B and average values max (supplementary Figure D , points labelled “on” and “off”). of the receptor’s K to DMXB-A ranged from 0.67 to 0.82 nM for B and from 170 to 385 nM for K , as presented in Figure 4. max I Regarding the choice of the V the standard error of the V ND (b), ND (b) Discussion estimate in baboon of 2.7 mL/cm corresponded to ranges of human B estimates of 0.4 to 1.9 nM and DMXB-A K estimates max Subjects of 138 to 363 nM. We extended the first-in-human report of Wong et al. (2014) from 5 to 21 healthy subjects (8 test-retest sessions, 5 sessions with Menstrual Cycle inhibition by DMXB-A of [ F]ASEM binding, and 6 baseline ses- The menstrual cycle phase (luteal vs follicular) was determined sions) and added a preliminary study of 6 patients with schizo- for the 8 healthy control females. Estimates of did not differ V phrenia, with 1 patient receiving test-retest PET in the absence significantly in relation to cycle phase. The lower ages of women and presence of chronic risperidone medication. Table 2. Corrected and Uncorrected P Values from V in Controls vs Patients Results (uncorrected P values and corrected by different methods) of Mann-Whitney tests for V in Healthy Controls vs Patients with Schizophrenia Region Uncorrected P value Adjusted (Horchberg) Adjusted (Bonferroni) Adjusted (Simes) All Data Cg .056 .159 .167 .118 Fr .171 .172 .515 .172 Hp .079 .159 .238 .119 With outliers removed Cg .009 .028 .028 .025 Fr .058 .058 .173 .058 Hp .017 .033 .05 .025 Adjusted P values were obtained by the STATA package qqvalue using 3 different methods of adjustment (Newson, 2010; specific methods are Horcberg, 1998, stand- ard Bonferroni correction and Simes, 1986). Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy021/4924585 by Ed 'DeepDyve' Gillespie user on 08 June 2018 8 | International Journal of Neuropsychopharmacology, 2018 The results demonstrate excellent test-retest repeatability standard IP here, we obtained DMXB-A occupancies in the of [ F]ASEM PET of >90%. In the population of 21 healthy non- human subjects in the range of 17% to 49%. Future studies smoking volunteers, when we plotted tracer distribution vol- with new α7-nAChR drugs will further test the occupancy and ume V against age, we observed an increase with of V with age. the accuracy of the resulting estimates of B and affinity in T T max While our smaller sample and lack of a wide age distribution human brain in vivo. limited our ability to test this relationship formally with regres- With the assumptions and translation from autopsy pro- sion analysis, the trends in our data match the findings from a tein and wet tissue-based B of α7-nAChR, we find remarkable max larger age range in another set of subjects (Coughlin et al., 2018, agreement among the B estimates that we regard as another max which had only 2 subjects in common that we provided to the innovative aspect of the present study. As 150 mg is the highest Coughlin paper previously). The preliminary findings of values single oral dose allowed under the DMXB-A IND based on histor - of V vs age are shown in supplementary Figure D . ical information, we elected not to test lower doses, because of We were unable to determine sex differences because of the the modest occupancy, compared with occupancy of drugs such different age ranges. Of the 8 women, none of them showed a as antipsychotics, and more recently glycine transporter (GlyT1) specific effect of menstrual cycle on estimates of V. ligands (Martin-Facklam et al., 2013 W ; ong et al., 2013). However, the dose dependent occupancy with DMXB-A is evidence of spe- cificity of the radiotracer [ F]ASEM in human brain. The occu- Receptor BP ND pancy is further evidence of the potential usefulness of [ F] Estimates of B are based on determination of BP (Gjedde ASEM for therapeutic occupancy trials, as these are somewhat max ND et al., 2005). Importantly, for the first time, we present evidence complicated due to the inverted-U-shaped dose effect of α7- that competitor occupancy, apparent , and K B of human α7- nAChR partial agonists (Young and Geyer, 2013). I max nAChR can be measured, based on the assumptions presented. The α7-nAChR receptor has 5 binding sites among the 5 α7 We determined regional estimates of [F] ASEM binding as the subunits and is maximally activated when at least 2 binding first step towards complete receptor quantification. In addition sites are occupied by an agonist. At higher doses, agonists bind to regional values of V, the receptor quantification required at up to all 5 binding sites and progressively desensitize the α7. knowledge of the magnitude of V of the tracer in a region of Moreover, α7 partial agonist drugs such as DMXB-A and oth- brain tissue with no specific binding. Previously, no such refer - ers produce their clinical effects at lower concentrations than ence volume has been identified for this tracer with prevalent would be expected from in vitro functional experiments where binding in cerebral cortex. As the first PET study with blockade peak currents are recorded (Prickaerts et al., 2012 Papke ; , 2014; of α7-nAChR in human brain in vivo, this study took advantage Preskorn et al., 2014). The co-agonistic mechanism of in vivo of the original development of the well-documented partial action of α7 partial agonist suggests that DMXB-A, at the clinic- agonist DMXB-A at the University of Florida (Kem, 2000 K ; em ally effective dose, binds at 1 of 5 binding sites of α7, while brisk et al., 2004) and the subsequent successful testing in patients activation of the receptor will, however, occur upon exposure with schizophrenia at the University of Colorado (Olincy et al., to endogenous ACh. Thus, in vivo, a single molecule of DMXB-A 2006; Freedman, 2014). plus 1 molecule of ACh are sufficient to trigger the opening of the α7-nAChR channel (Williams et al., 2011Pric ; kaerts et al., 2012; Papke, 2014; Preskorn et al., 2014). Therefore, in the PET Receptor Occupancy occupancy studies with the DMXB-A-treated humans, the bind- Recent studies of mouse and baboon brains revealed significant ing of [ F]ASEM is expected to be inhibited by as little as 20% to blockade of the α7-nAChR by the high-affinity partial α7-nAChR 40% (one-fifth to two-fifths) instead of 100%. This co-agonistic agonists SSR180711 and DMXB-A in vivo (Horti et al., 2014a; mechanism agrees reasonably with our PET [F]ASEM inhibition Wong et al., 2014), but effective inhibition or competition has not by DMXB-A in humans at 17% to 49% with a maximum clinical yet been confirmed in humans. As a candidate drug, DMXB-A dose of DMXB-A (150 mg). Full blocking of [F]ASEM with α7 ago- was tested in clinical trials of patients with schizophrenia, nists occurs at higher doses, as observed in mice with 20 mg/kg Alzheimer’s disease, or ADHD (Freedman et al., 2008F ; reedman, DMXB-A (Horti et al., 2014a; Wong et al., 2014) and in baboons 2014), and the drug was used here to test target engagement/ administered 5 mg/kg of the more highly potent SSR180711 that occupancy in vivo at the α7-nAChR and to demonstrate specifi- is unavailable in the United States for human use (Horti et al., city of [ F]ASEM binding to these receptors. 2014). Previously, we demonstrated dose-dependent tracer inhib- ition in rodents by DMXB-A with cerebellum as reference region Receptor Density and Tracer Affinity (appropriate in rodents but not human brain) (Wong et al., 2014). Similarly, in the present study, blockade with the highest dose The use of the EIP with the DMXB-A inhibition and the use of of DMXB-A allowed in humans (150 mg orally) exhibited com- the estimate of V from baboon brain (Horti et al., 2014) yielded ND petition with binding of [ F]ASEM. The occupancy of receptors the binding potentials necessary to estimate the average values by DMXB-A displayed a relatively monotonic relationship with across all binding regions of B (0.67–0.82 nM) and K (range max I plasma levels of DMXB-A. 170–385) in human brain. While the value of V in humans cur- ND We used the EIP under the assumption that the apparently rently is unknown, the baboon value of 5.4 mL/cm estimated absent inhibition of receptor binding would be due to factors here with SEM of 2.7 mL/cm corresponded to ranges of B max such as change of free fractions of the radioligand in circulation estimates of 0.75 (range 0.38–1.83) nM and DMXB-A K estimates or brain tissue or both, with the consequence that the value of V of 138 to 363 nM. These values agree favorably with estimates ND would differ between the baseline (V ) and inhibited (V ) postmortem of 0.28 nM B for human cerebral cortex with ND (b) ND (a) max states, as reported by Phan et al. (2017). Using EIP and the α-[ I]bungarotoxin (Davies and Feisullin, 1981) and cortical B max value of V of 5.4 mL/cm obtained from moderate block- values of 0.7 nM in patients with schizophrenia compared with ND(b) ade of the α7-nAChR in baboon brain, reported by Horti et al. 1.5 nM in healthy control subjects (Marutle et al., 2001), assum- (2014) as a simple reduction in slope and reanalyzed by the ing 10% mg protein converted to wet weight. Downloaded from https://academic.oup.com/ijnp/advance-article-abstract/doi/10.1093/ijnp/pyy021/4924585 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Wong et al. | 9 As a future direction, we specifically will recruit partici- Schizophrenia pants for inclusion into cohorts of smokers and nonsmokers of In the feasibility study of 6 patients with schizophrenia, we both healthy controls and patients with schizophrenia to fur - observed reductions of V in 5 of the 6 subjects. The finding of sig- T ther explore smoking effects. The assignments will be verified nificant reduction of regional distribution in 5 of 6 subjects in 2 by smoking history, urine cotinine levels, CO breath measures, of 3 regions after outlier exclusion of a single subject agreed with and assessments of extent of nicotine use and dependence (e.g., previous findings postmortem. However, in the present study, the Fagerstrom, MINI nicotine use, Minnesota Nicotine Scale, and difference between controls and patients is lower than observed Michigan Nicotine Reinforcement Questionnaire). With a larger postmortem. This finding could be due to a number of factors, sample, the schizophrenia group is likely to comprise more 18 125 such as tracer differences ([ F]-ASEM vs [ I]-BTX), in vivo vs in smokers, particularly among older patients. vitro tests, and the heterogeneity of the patient population. Age and Sex Differences Limitations In the group of 21 subjects in the age range of 18 to 50 y, males Antipsychotic Medication and females were not distributed uniformly, ruling out formal determination of sex differences as functions of normal aging. Neuroreceptor studies of patients with schizophrenia in the Also, the small number of 5 subjects with DMXB-A occupancy drug free or naïve state are increasingly logistically challeng- nonetheless yielded a reasonable range of DMXB-A human ing, although such studies have been completed in limited plasma concentrations, presumably due to individual variability populations (Farde et al., 1986W ; ong et al., 1986). In the present in oral absorption and metabolism of the inhibitor. limited population, patients with schizophrenia were tested while on stable medication for more than 3 weeks with antip- Schizophrenia sychotics that are held to have only negligible effect on binding to α7 nAchR and to be nonexclusionary in therapeutic studies In this feasiblity study, the small number of patients with of partial agonists such as DMXB-A, e.g., clozapine (which was schizophrenia is a statistical limitation. In 5 of the 6 subjects, exclusionary). Several precautions were taken to reduce the pos- estimates of V in 2 of 3 regions compared with age-matched sibility of confounding antipsychotic action in the quantifica- control subjects, as well as a formal outlier analysis, suggested tion of binding to α7nAchR. First, the majority of the patients that the high values of V in one subject may have had other were treated with risperidone and haloperidol, which unlike causes leading to the elevation. Thus, expansion of the number clozapine fail to normalize the P50 abnormality (Adler et al., of patients with schizophrenia and detailed documentation of 1998). Second, a rodent biodistribution study found no signifi- the clinical and neuropsychological features of the patients will cant effects on [ F]ASEM binding. Finally, although only 1 sub- help expand the findings of factors that influence the hetero- ject was studied off medications, the repeated PET test on and geneity of α7nAchR findings. off chronic risperidone was suggestive of the conclusion that this commonly used antipsychotic is unlikely to confound the estimates of the V values for nAChR. T α7 Conclusions Here, we confirmed the test-retest reliability of tracer binding in Effects of Smoking healthy human brains, and we demonstrated for the first-time All 21 control subjects and 5 of the 6 patients with schizophrenia specificity of competition from the partial agonist DMXB-A in were nonsmokers. We considered comparing smokers with non- normal healthy volunteers, as previously reported in preclinical smokers but did not recruit a sufficient number of healthy volun- imaging studies. The novel first time presentation of in vivo B max teers with clinically verified smoking status. By design therefore measures agreed favorably with published human postmortem we studied only nonsmoking control subjects in the current values. The effects of antipsychotics in our schizophrenia vol- protocol of test-retest and occupancy studies and comparison unteers may not be a major confound based on our preliminary with the feasibility study of 6 patients with schizophrenia. mice and literature and one subject on and off risperidone. The Despite the relatively high prevalence of smoking in patients preliminary findings in patients with schizophrenia suggest the with schizophrenia, only 1 of the subjects randomly recruited feasibility of imaging this important disorder and generally con- with schizophrenia smoked (20 cigarettes/d). Most subjects were sistent with previous reports of human brains postmortem, but younger (<35 years old), which may be a contributing factor, as it clearly larger samples with a spectrum of clinical severity and has been reported that younger subjects are less likely to smoke comparisons with full cognitive and neuropsychological tests (for an example of this trend, see https://www.hhs.gov/ash/oah/ are needed. adolescent-development/substance-use/drugs/tobacco/trends/ index.html). The patient that happened to be a regular smoker also happened to be the oldest of the patients at 31 y (age range Acknowledgments of patients 20–31 y, mean 24.9 y). The V values did not differ significantly for the smoking subject compared with the other Radiochemistry staff, Johns Hopkins PET Center staff, David Schretlen, PhD, and J. Coughlin, MD for scientific discussions patients with schizophrenia with low value of .V Furthermore, on the day of PET, all control subjects and and the High Resolution Brain Imaging Section faculty, staff, and fellows (including Andrew Crabb and Anil Mathur). Thanks patients with schizophrenia had CO values <4 PPM, indicating successful abstinence (regardless of their self-reports) and likely to Gayane Yenokyan, MD, PhD, Director, Biostatistics Consulting Center, Johns Hopkins School of Public Health for guidance and longer than 12 h, reducing the acute effects of nicotine, if any, on the PET measure of the tracer V . assistance on statistical analyses. 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International Journal of Neuropsychopharmacology – Oxford University Press
Published: Mar 7, 2018
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