PET technology for drug development in psychiatry

Abstract Positron emission tomography (PET) is a non‐invasive imaging method to measure the molecule in vivo. PET imaging can evaluate the central nervous system drugs as target engagement in the human brain. For antipsychotic drugs, adequate dopamine D2 receptor occupancy (“therapeutic window”) is reported to be from 65%‐70% to 80% to achieve the antipsychotic effect without extrapyramidal symptoms. For antidepressants, the clinical threshold of serotonin transporter (5‐HTT) occupancy is reported to be 70%‐80% although the relation between the side effect and 5‐HTT occupancy has not yet been established. Evaluation of norepinephrine transporter (NET) occupancy for antidepressant is ongoing as adequate PET radioligands for NET were developed recently. Measurement of the target occupancy has been a key element to evaluate the in vivo target engagement of the drugs. In order to evaluate new drug targets for disease conditions such as negative symptoms/cognitive impairment of schizophrenia and treatment‐resistant depression, new PET radioligands need to be developed concurrently with the drug development.

Consequently, the degree of targeted molecule of each PET radioligand can be evaluated quantitatively. For example, [ 18 F]fluorodeoxyglucose ([ 18 F]FDG), which can measure the glucose metabolism, accumulates in tissues of high glucose consumption such as tumor and inflammation, and produces the large amount of gamma radiations there. By detecting the radiation, the location of the tumor and inflammation is identified. In this section, as an example, we explain 2TCM as follows ( Figure 1). In brief, 4 rate constants, K 1 -k 4 , are calculated mathematically from the time-activity curves of radioactivity in both blood and brain. Usually, binding potential (BP ND ), which is calculated by k 3 /k 4 , is estimated as main outcome measures. BP ND is defined as B avail /K d , as B avail is the density of target available to bind PET radioligand in vivo and K d is the dissociation constant of PET radioligand. 3 Consequently, BP ND is a proportional value of the density of target molecule. The total distribution volume (V T ), which is calculated by K 1 /k 2 × (k 3 /k 4 + 1), is also used. This value represents the ratio of radioactivity between brain and plasma.

| Kinetic modeling
Non-compartment models are also used as alternative approach, for example, Logan graphical analysis (GA) 4 and Ichise multilinear analysis (MA1) 5 , which allow estimating V T . GA shows a robust estimation of V T because of linear regression method, but the values decrease with increasing noise. MA1 is a modified method of GA, which aims to improve the bias.

| Reference tissue model
Estimation of the kinetic parameter requires the arterial blood sampling to obtain the radioactivity in the blood. However, it is invasive and high burden for the participants. If there is the region without target molecule, BP ND can be calculated, without arterial blood sampling, that is, using only the radioactivity in the brain. Simply, the relative difference of target and reference regions represents the BP ND (Figure 2). For example, we can use the cerebellum as reference region for the evaluation of dopamine and serotonin system, and the caudate for norepinephrine system. The PET radioligands which are mentioned in this review are fit to the reference tissue models, meaning no requirement of the arterial blood sampling.
Several methods have been developed as reference tissue models, for example, simplified reference tissue model (SRTM) 6 , multilinear reference tissue model (MRTM) 7 , and reference Logan 8 . SRTM is based on the two assumptions: (1) V ND (distribution volume of nondisplaceable compartment) is the same for the target and reference tissues and (2) the kinetics in the target tissue can be fitted by a 1TCM.

| TARG E T ENG AG EMENT (O CCUPAN C Y )
In the drug evaluation, it is important to estimate its binding property of test drugs to the target molecule such as receptors or trans-

| PET radioligands for dopamine D2 receptor
Main mechanism of action of antipsychotics has been the inhibition of dopamine D2 receptors. [ 11 C]raclopride is the PET radioligand, which selectively binds to dopamine D2 receptors. 9 This radioligand has ideal properties as PET radioligand such as brain uptake, affinity, time course, and mostly used for the brain PET studies. [ 11 C] raclopride can measure dopamine D2 receptors in the striatum in the brain due to moderate affinity to the receptor. For the evaluation of extrastriatum, where the density of dopamine D2 receptor is quite low, [ 11 C]FLB457 10 and [ 18 F]fallypride 11 , which have high affinity to the dopamine D2 receptor, are used. It would not be optimal to evaluate the striatum with these radioligands because the uptake in the striatum does not reach equilibrium during PET measurements. [ 11 C]PHNO has the affinity for dopamine D3 receptor as well as dopamine D2 receptor. 12 The binding of [ 11 C]PHNO reflects the dopamine D3 receptors in the regions which has relatively higher density of dopamine D3 receptors than dopamine D2 receptors such as globus pallidum.  16 They concluded that 65% was the threshold of the antipsychotic effect, and over 78% occupancy was the risk of EPS. Ziprusky et al 17 also reported the relation between dopamine D2 receptor occupancy and antipsychotic effect. They concluded that 70% occupancy is the threshold of antipsychotic effect. Taken together, the adequate occupancy ("therapeutic window") is thought to be from 65%-70% to 80% ( Figure 4). This concept has been widely accepted and applied to confirmation of the dose setting of so-called second-generation antipsychotics, such as risperidone 18-20 and olanzapine 21,22 . When first-generation antipsychotics were re-evaluated, it was reported that optimal dose setting of some antipsychotics might not have been made in the target occupancy's point of view. 23

| PET radioligands for 5-HT2A receptor
Many second-generation antipsychotics have the affinity for 5-HT2A receptors in addition to the dopamine D2 receptors. Initially, [ 11 C]NMSP had been used for the quantification of 5-HT2A receptors. 24 However, this radioligand also has the affinity for dopamine

| Relationship between the clinical efficacy and 5-HT2A receptor
Compared to the dopamine D2 receptor occupancy, there has not been clear evidence of the relationship between 5-HT2A receptor occupancy and clinical efficacy. For example, 5mg/day of olanzapine, which is considered as lower dose than having clinical effectiveness, already showed over 90% 5-HT2A receptor occupancy. 22 One possible mechanism for the low risk of EPS about second-generation antipsychotics is thought as the blockade of 5-HT2A receptors.
However, the likelihood of EPS depends on the dopamine D2 receptor occupancy, regardless of 5-HT2A receptor occupancy.
Paliperidone ER is the extended release formulation of paliperidone, which is available in Japan from 2011. The dopamine D2 receptor occupancy of paliperidone ER was estimated in a part of phase II clinical trial of this drug. 28 This is the first study in Japan using PET and occupancy data in the phase II clinical trial. Thirteen patients with schizophrenia, who were stable in the antipsychotic treatment, were involved in this study. Three doses of 3, 9, and 15 mg/day were tested.
Based on 70%-80% of striatal and extrastriatal dopamine D2 receptor occupancy, the optimal dosage was estimated to be 6-9 mg/d. After the clinical trials, the approved dosage of paliperidone ER in Japan is 6-12 mg/d.

| Blonanserin
Blonanserin is an antipsychotic drug highly selective to dopamine

| Aripiprazole
Aripiprazole, which is available in Japan form 2006, has lower affinity for 5-HT2 receptors than dopamine D2 receptors, different from many other second-generation antipsychotics. Dopamine D2 receptor occupancy of aripiprazole showed over 80% at a low dose of 10 mg/d although the risk of EPS is quite low. [31][32][33] As aripiprazole is a partial agonist for the dopamine D2 receptors, it is assumed that its intrinsic activity can avoid the excessive antagonism even at nearly full saturated target occupancy.   MeNER, which was previously developed with less optimal brain kinetics. 46

| Relationship between clinical efficacy and NET occupancy
Because the development of the appropriate PET radioligand for NET was relatively new, the investigation of NET occupancy by antidepressants has not been widely performed yet. Therefore, the relationship between clinical efficacy and NET occupancy has not been reported so far.

| Duloxetine
Duloxetine is a SNRI (in vitro affinity: 0.8 nmol/L for 5-HTT, 7.5 nmol/L for NET), approved in Japan from 2010 ( relationship of combination of 5-HTT and NET occupancy has not been fully established.

| Nortriptyline
Nortriptyline is a classical antidepressant. It has relatively high selectiv- in patients with depression. They concluded that the required NET occupancy for clinical effect is considered as over 50% using the reported value of effective plasma concentration of nortriptyline.

| Tramadol
Tramadol is an analgesic drug, which has opiate agonism as well as inhibitory effect for 5-HTT and NET. Ogawa et al reported that 100 mg of tramadol showed 50% 5-HTT occupancy in human brain using healthy subjects. 53 The approved dose of tramadol in Japan is up to 400 mg for pain, which might induce 80% 5-HTT occupancy.
This result indicated that the high end of clinical doses of tramadol could block similar level of 5-HTT to other antidepressants.

| Quetiapine
Quetiapine is approved as an antipsychotic drug, but anti-depressive effect has been also reported. A possible explanation for anti-depressive effect is that norquetiapine, a metabolite of quetiapine, has high affinity for NET. Nyberg et al reported that 300 mg of quetiapine XR blocked NET at 35% occupancy in healthy subjects. 54 Yatham et al 55 also reported that 300 mg of quetiapine showed clinical effect for depressive symptoms and it induced 40% NET occupancy. Both studies support that NET inhibition by norquetiapine contributes to the antidepressant effect.

| CON S IDER ATI ON FAC TOR S FOR PE T APPLI C ATI ON
As described above, carbon-11 and fluorine-18 radionuclides are used for the PET radioligands. Due to the short half-life (around 20 minutes) of carbon-11, the site production using a cyclotron and synthesis apparatus is required for the carbon-11 PET radioligands.
In case of fluorine-18 PET radioligands (around 110 minutes halflife), the delivery might be available. When multiple PET measurements in one day are considered to perform such as one baseline PET measurement and occupancy PET measurement with a drug, carbon-11 PET radioligands are feasible due to the shorter half-life.
When the PET measurements are performed, radiation exposure from the radionuclides is needed to consider. Typical radiation dose from PET measurements is at similar level to that from computed tomography (CT) scans for the clinical usage or natural background radiation for a few years.
Duration of PET measurement is relatively long, for example, 1 to 2 hours, and the subjects have to stay still during the measurement, which may make the participants feel uncomfortable especially in the patients with psychiatric/ neurodegenerative disorders.
Simplified methods which can make the measurement time shorter should be considered if possible.

| CON CLUS ION
PET imaging has been widely used for the evaluation of CNS drugs.
Especially, the estimations of dopamine D2 receptor occupancy