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Novel, non-nitrocatechol COMT inhibitors modulate dopamine neurotransmission in the frontal cortex and improve cognitive

flexibility

Spencer Byers1, Ingrid P. Buchler1, Michael DePasquale1, Helen L. Rowley2, Rajiv S. Kulkarni2, Lucy Pinder2, Anna Kolobova1, Cailian Li1, Vinh Au1, Daniel Akuma1, Gongliang Zhang1, Huijun Wei1,3, Sharon C. Cheetham2, James C. Barrow1,3, and

Gregory V. Carr1,3*,

1Lieber Institute for Brain Development, Johns Hopkins University Medical Campus, Baltimore, MD, USA.

2RenaSci Limited, Nottingham, UK

3Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.

*Correspondence:

Gregory V. Carr Suite 300 855 North Wolfe Street Baltimore, MD 21205 410-955-1000 [email protected]

certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was notthis version posted January 2, 2019. ; https://doi.org/10.1101/510040doi: bioRxiv preprint

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Abstract

Cognitive impairment is a primary feature of many neuropsychiatric disorders and there is a need for new therapeutic options. Catechol-O-methyltransferase (COMT) inhibitors modulate cortical dopaminergic function and have been proposed as potential cognitive enhancers. Unfortunately, currently available COMT inhibitors are not good candidates due to either poor blood-brain barrier penetration or severe toxicity. To address the need for safe, brain-penetrant COMT inhibitors, we tested multiple novel COMT inhibitors in a set of preclinical in vivo efficacy assays to determine their viability as potential clinical candidates. We found that multiple COMT inhibitors, exemplified by LIBD-1 and LIBD-3, significantly modulated dopaminergic function measured as decreases in homovanillic acid (HVA) and increases in 3,4-Dihydroxyphenylacetic acid (DOPAC), two dopamine metabolites, in cerebrospinal fluid (CSF) and the frontal cortex. Additionally, we found the LIBD-1 significantly improved cognitive flexibility in a rat attentional set-shifting assay (ASST), an effect previously seen with the COMT inhibitor tolcapone. These results demonstrate that LIBD-1 is a novel COMT inhibitor with promising in vivo activity and the potential to serve as a new therapy for cognitive impairment.


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Introduction

Cognitive impairment is a core feature of many neurological and psychiatric disorders

and the severity of the impairment is directly correlated with functional outcomes for

patients (1-5). Currently, there are very few therapeutic options for patients with

cognitive impairment. The options that are available have limited efficacy and in some

cases, including psychostimulants in schizophrenia, are contraindicated due to potential

exacerbation of other symptoms (6).

The neuromodulator dopamine (DA) plays a critical role in cognitive function

across species and strategies that augment or refine DA activity in the brain have been

shown to improve cognitive function (7). The enzyme catechol-O-methyltransferase

(COMT) is a key component of the DA metabolic pathway (8) and inhibition of the

enzyme has been proposed as a cognitive enhancement strategy (9). COMT is located

throughout the body with the highest expression in the liver and brain (10). There is a

soluble (S-COMT) and a membrane-bound (MB-COMT) form of the enzyme. S-COMT

has a higher Vmax but lower affinity for DA compared to MB-COMT (11). It is thought

that S-COMT activity predominates in the periphery where it metabolizes many

substrates including catecholestrogens and exogenous catechols (12). In contrast, MB-

COMT accounts for most of the enzyme activity in the brain, especially in the primate

brain, where the major substrate is dopamine, which is present at relatively low

concentrations (13).

The major routes for terminating DA signaling in the brain are reuptake and

metabolic conversion. In the striatum, the dopamine transporter (DAT) is the major

termination mechanism. In the frontal cortex and hippocampus, there are relatively


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small amounts of DAT, most of which is extrasynaptic. There is evidence that the

norepinephrine transporter (NET) can clear DA from the extracellular space in cortex

and hippocampus (14), but NETs also are outside DA synapses and the majority of

functional DA signal termination in these regions appears to depend on COMT activity,

with some contribution from monoamine oxidase (15, 16). COMT directly converts DA

to 3-Methoxytyramine (3-MT) and also converts the DA metabolite 3,4-

Dihydroxyphenylacetic acid (DOPAC) to homovanillic acid (HVA). The fact that COMT

has an outsized role in DA signaling in the cortex and hippocampus compared to the

striatum has led to the hypothesis that selective targeting of COMT activity may

modulate cognitive function without some of the psychostimulant effects (e.g. abuse

liability) seen with other DA modulators (9).

There is a large body of evidence showing that COMT activity has a dynamic

relationship with cognitive function in humans and animal models. There is a highly-

studied functional polymorphism in the COMT gene (Val158Met) that produces a “high”

activity enzyme (Val158) and a “low” activity enzyme (Met158). In general, the Val form

of COMT is associated with lower cognitive function compared to the Met form (17-19).

However, this effect is labile and interactions with other genetic and environmental

factors can modify or even reverse the relationship (20, 21). Administration of COMT

inhibitors also has variable effects on cognitive function based on the individual status

of the Val/Met polymorphism and baseline performance, particularly in studies of

people without a diagnosis of cognitive impairment (22, 23). Overall, there have been a

number of studies of patients across multiple indications that suggest that COMT

inhibition may be a viable strategy for improving cognitive function (24, 25).


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Currently, COMT inhibitors are utilized as an adjunctive therapy alongside L-

DOPA administration in patients with Parkinson’s disease (PD) to improve motor

symptoms. COMT metabolizes L-DOPA to 3-O-Methyldopa (3-OMD) in the periphery

and decreases the amount of L-DOPA available to cross the blood-brain barrier (BBB).

Therefore, COMT inhibition serves to augment the CNS availability and effects of L-

DOPA without having to increase the dosage of L-DOPA (26, 27). Since the target

enzyme is in the periphery, a brain-penetrant COMT inhibitor is not necessary for this

indication. However, for cognitive enhancing effects, there is evidence that a brain-

penetrant or central COMT inhibitor is required. A comparison of the effects of

tolcopone (brain-penetrant) to those of entacapone (peripherally-restricted) in patients

with PD showed that only those taking tolcapone demonstrated improvement in the

cognitive symptoms associated with PD (24).

Tolcapone, however, is not a viable, long-term option as a cognitive enhancer due

to safety concerns. Tolcapone is associated with potentially fatal liver toxicity that makes

the drug a poor choice for a chronic cognitive enhancement therapy (28). Given the

relative safety of the other COMT inhibitors on the market, the toxicity is thought to be

compound-specific as opposed to mechanism-based (29). Therefore, a potent, brain-

penetrant, and safe COMT inhibitor is needed to fully explore this mechanism as a

potential avenue for cognitive enhancement.

Here we describe the in vivo activity of novel COMT inhibitors. Our goal was to

identify a COMT inhibitor with good oral bioavailability and significant effects on

neurochemical biomarkers and behavior in rats. We used tolcapone as our efficacy

benchmark. We identified LIBD-1 as a candidate molecule for future studies. LIBD-1 has


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high selectivity for MB-COMT over S-COMT, which we believe will produce an outsized

effect on central COMT activity while having a smaller impact on liver COMT function.

The evidence presented here indicates that LIBD-1 is a viable lead compound with

favorable properties that support continued development.

Materials and Methods

Animals

Male CD® IGS rats (8-11 weeks old; Charles River Laboratories, Wilmington, MA, USA)

were used for all experiments. Age-matched female CD® IGS rats were also used for

some of the DA metabolite biomarker studies. Rats used for experiments at the Lieber

Institute for Brain Development were housed in a temperature (22°C) and humidity-

controlled facility on a 12h/12h light/dark cycle (lights on at 0600 hours). Rats used for

experiments at RenaSci were housed in a temperature (21 ± 4°C) and humidity-

controlled (55 ± 15%) room on a 12h/12h light/dark cycle (lights on at 0700 hours)

facility. Rats used in behavioral experiments were handled for one minute at least three

times in the week prior to testing. All procedures were approved by the local Animal

Care and Use Committee and were in compliance with the Guide for the Care and Use of

Laboratory Animals.

Drugs

Novel COMT inhibitors (synthesized in-house) and tolcapone (synthesized in-house)

were suspended in vehicle (0.1% Tween80, 0.1% 1510 silicone antifoam, 1%


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methylcellulose in water; Table 1) and administered either orally (po) or through

intraperitoneal (ip) injection (tolcapone). Administration volumes were 10 mL/kg and 5

mL/kg for po and ip dosing, respectively. In the microdialysis experiment, a dose

volume of 2 mL/kg was used for tolcapone administration.

DA metabolite biomarker protocol

On the day of testing, rats were transferred to a holding room and weighed. After an

hour acclimation period, rats received either vehicle or a COMT inhibitor. At the

appropriate time point (1 or 4 hours), rats were moved to a separate procedure room

where they were anaesthetized via isoflurane. Once the rats were determined to be

unresponsive, their heads were shaved using electric clippers. The rats were positioned

in a stereotaxic frame, with their heads pointed down at a 45-degree angle. To collect

cerebrospinal fluid (CSF), previously published protocols (30, 31) were adapted. Briefly,

a 23 gauge needle, connected via PE50 tubing to a collection syringe, was used to access

the cisterna magna. Slight negative pressure was used to ensure the CSF flowed evenly.

The CSF was collected in previously chilled (dry ice) Eppendorf tubes containing 0.05M

perchloric acid (4:1 CSF:perchloric acid ratio). The tubes were put back on dry ice until

the end of the procedure. CSF samples with visible blood contamination were not used

in subsequent bioanalytical analyses. Next, the chest cavity was opened, and blood was

collected by cardiac puncture. The blood was collected in Lithium-Heparin 1.3mL

microtubes (Sarstadt, Numbrecht, Germany) and stored on ice. The rat was then

transcardially perfused with ice-cold phosphate buffered saline (PBS) via a peristaltic

pump set to a flow rate of 20 mL/min. Following perfusion, the brains were collected in


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5mL Thermo Scientific Nunc tubes (Thermo Fisher Scientific, Waltham, MA, USA).

Blood was then centrifuged at 2000 g at 4°C for 10 minutes to separate the plasma.

Plasma was then transferred into Thermo Scientific Matrix tubes (Thermo Fisher

Scientific, Waltham, MA, USA) for storage. CSF, plasma, and brains were stored at -

80°C until analysis. DA metabolite and COMT inhibitor concentrations were measured

by LC-MS/MS as previously described (32).

Frontal cortex microdialysis

Surgery

Rats were anaesthetised with isoflurane (5% to induce, 2% to maintain) in O2 (1 L/min)

delivered via an anaesthetic unit (Burtons Medical Equipment Ltd, UK). A concentric

microdialysis probe (CMA 12 Elite, with an exposed polyarylethersulphone (PAES)

membrane tip; Linton Instrumentation, Norfolk, UK) was stereotaxically implanted into

the frontal cortex (2 mm tip, coordinates: AP: +3.2 mm; L: +/-2.5 mm relative to

bregma; V: –4.0 mm relative to the skull surface). Coordinates were taken from the

stereotaxic atlas of Paxinos and Watson (1986). The upper incisor bar was set at 3.3 mm

below the interaural line so that the skull surface between bregma and lambda was

horizontal. Additional burr holes were made for skull screws (stainless steel) and the

probes were secured using dental cement. Carprofen (Carprieve, Norbrook Laboratories

(GB) Ltd, Newry, UK) was administered for pain relief at least 30 min prior to animals

regaining consciousness following surgery (5 mg/kg sc). Following surgery, animals

were individually housed in the dialysis bowls (245 mm internal diameter at base of

bowl, 360 mm wall height, Harvard Apparatus, Cambridge, UK), with the microdialysis


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probes connected to swivels and a counter-balanced arm to allow unrestricted

movement. Rats were allowed a recovery period of at least 16 h with standard rodent

chow and filtered water available ad libitum. During this time, the probes were

continuously perfused at a flow rate of 1.2 μL/min with an artificial cerebrospinal fluid

(aCSF; Harvard Apparatus, UK) of the following electrolyte composition (in mM):

sodium 150; potassium 3.0; magnesium 0.8; calcium 1.4; phosphate 1.0; chloride 155.0,

pH 7.2.

Dialysate sample collection

Dialysate samples were collected at 20 min intervals for a baseline period of 80 min

prior to the onset of administration of vehicle, tolcapone, LIBD-1, or LIBD-3. Sampling

continued for an additional 2 h. Samples were collected into Eppendorf vials (300 μl,

Microbiotech, Stockholm, Sweden) containing 5.0 μL of 0.1 M perchloric acid to prevent

oxidation of DA and its metabolites. Samples were stored at -80°C until assayed by

HPLC with electrochemical detection.

Additional details on bioanalytical analysis and microdialysis probe location

verification are provided in Supplemental Information.

Attentional set-shifting task (ASST)

This version of the ASST was adapted from previously described protocols (33, 34), and

was performed over a four-day period. Rats were food restricted to 90% of free-feeding


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weight during the week before testing. A full description of the methods is provided in

the Supplemental Material.

Data analyses

In our CSF neurochemical experiments, we compared drug-treated rats to vehicle-rats

using either one-way ANOVAs or t-tests. For ANOVAs significant main effects were

followed up with post hoc Tukey’s tests. Data are represented by the mean value as a

percentage of the vehicle-treated group. For the microdialysis experiment, data were log

transformed and analyzed by ANCOVA with log(baseline) as a covariate. Baseline was

defined as the geometric mean of the four pretreatment samples. Comparisons to the

vehicle-treated group were by the multiple t-test (Supplemental Tables 2-4). Data are

presented as the mean (% of vehicle-treated) ± SEM. Some extreme values were

removed from the analysis. These are listed in Supplemental Table 5. The ASST data

were analyzed using two-way ANOVA with repeated measures. Drug treatment was the

between-subjects variable and ASST stage was the within-subjects variable. Significant

main effects were further probed with post hoc Tukey’s tests to determine significant

pairwise comparisons. ASST data are presented as the mean ± SEM.

Results

COMT inhibitors modulate HVA and DOPAC concentrations in CSF


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To determine if our novel COMT inhibitors (Table 1) prevent COMT activity in vivo, we

measured the concentrations of the dopamine metabolites HVA and DOPAC in

cerebrospinal fluid (CSF). Previous research has shown that potent brain-penetrant

COMT inhibitors reliably decrease HVA concentrations while increasing DOPAC

concentrations in CSF (35). Here, we benchmarked our COMT inhibitors to the

prototypical COMT inhibitor tolcapone (Table 2). Tolcapone (15 mg/kg) significantly

decreased HVA (1 h: t9 = 8.730, p < 0.0001; 4 h: t13 = 13.090, p < 0.0001) and increased

DOPAC (1 h: t9 = 8.528, p < 0.0001; 4 h: t13 = 9.866, p < 0.0001) at both time points

sampled (1 and 4 h). All doses of our MB-COMT selective inhibitors LIBD-1 and LIBD-3

also significantly decreased HVA (LIBD-1 1 h: F3,30 = 26.450, p < 0.0001; 4 h: F3,33 =

29.470, p < 0.0001; LIBD-3 1 h: F3,32 = 42.700, p < 0.0001; 4 h: F2,19 = 126.200, p <

0.0001) and increased DOPAC (LIBD-1 1 h: F3,30 = 46.850, p < 0.0001; 4 h: F3,33 =

42.670, p < 0.0001; LIBD-3 1 h: F3,32 = 32.730, p < 0.0001; 4 h: F2,19 = 41.510, p <

0.0001) at both time points tested. The highest doses of LIBD-1 and LIBD-3 were

unable to modulate the DA metabolites to the same degree as tolcapone despite reaching

concentrations that would be expected to completely block MB-COMT, suggesting some

role for S-COMT in regulating DA metabolism in rats. Previous research has shown

sexually dimorphic effects related to COMT activity (36, 37), so we tested both tolcapone

(HVA: t10 = 9.505, p < 0.0001; DOPAC: t10 = 6.307, p < 0.0001) and LIBD-1 (HVA: t11 =

6.696, p < 0.0001; DOPAC: t11 = 5.478, p = 0.0003) in female rats and found similar

changes in DA metabolites. In contrast, LIBD-2, a potent, isoform nonselective COMT

inhibitor, with extremely low brain penetrance, does not significantly alter CSF DA

metabolites (HVA: t13 = 0.1482, p = 0.8845; DOPAC: t13 = 1.793, p = 0.0962), indicating

that our biomarker assay is a sensitive measure of in vivo target engagement.


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COMT inhibitors modulate HVA and DOPAC concentrations in the frontal cortex

The CSF biomarker assay is a good measure of target engagement, but we also wanted to

confirm the activity of our compounds in the putative brain subregion, the cerebral

cortex, thought to be the neural substrate for the effects of COMT inhibition on cognitive

function. Therefore, we also tested the effects of our novel COMT inhibitors on DA,

HVA, and DOPAC concentrations in extracellular fluid (ECF) sampled from the cortex

by microdialysis (Figure 1). As previously reported, none of the COMT inhibitors altered

extracellular DA concentrations (38-40). Here, both LIBD-1 and LIBD-3 significantly

decrease HVA and increase DOPAC as expected (Figure 1b-c). As seen in the CSF

experiment, LIBD-1 effects do not quite reach the same magnitude as our positive

control tolcapone. In contrast, the effects of LIBD-3 were not significantly different from

tolcapone. These microdialysis results support the effects we see in CSF and further

demonstate that our MB-selective compounds may have similar utility as compared to

tolcapone.

LIBD-1 improves cognitive flexibility in the set shifting assay

Both LIBD-1 and LIBD-3 demonstrate good in vivo COMT inhibitor activity. The next

step was to test whether LIBD-1 and LIBD-3 would be able to modulate cognitive

function, measured as performance in the ASST. The rat digging version of the ASST is a

validated assay for measuring cognitive flexibility and the task is dependent on an intact

frontal cortex (33). Moreover, COMT inhibition has been shown to improve


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performance during the extradimensional shift (ED) stage of the task (38), analogous to

the task in monkeys that has been shown to be a dopamine mediated prefrontal

cognitive task (41). To establish the validity of our in-house protocol, we first measured

the effects of tolcapone (15 mg/kg) as a positive control. As expected, there were

significant differences in the number of trials to criterion between the stages (F6,126 =

4.729, p = 0.0002) and tolcapone improved performance (F1,21 = 5.075, p = 0.0351);

Figure 2a). Post hoc analyses revealed that the ED stage required more trials to criterion

than the compound discrimination stage (CD; p = 0.0104) and the intradimensional

shift stage (ID; p = 0.0003) in vehicle-treated rats. Tolcapone improved ED stage

performance (p = 0.0177) without significantly altering performance on any of the other

stages.

Next, we tested the effects of LIBD-1 (30 and 100 mg/kg) in the ASST. Again,

there was a significant difference in the number of trials to criterion between stages

(F6,300 = 31.8, p < 0.0001). In the vehicle-treated rats, there was a significant difference

in trials to criterion between the ED stage and the CD (p < 0.0001) and ID (p < 0.0001)

stages. Like tolcapone, LIBD-1 improved performance in the ASST compared to vehicle

(F2,50 = 7.52, p = 0.0014; Figure 2b). LIBD-1 (100 mg/kg) significantly improved

performance in the ED stage of the task (p = 0.0087). Additionally, the 30 mg/kg group

performed worse than the vehicle group on the compound discrimination reversal (CDr;

p = 0.0123) and worse than both the vehicle and 100 mg/kg groups on the

intradimensional shift reversal stage (IDr; p = 0.0296 and p = 0.0142, respectively).

These data indicate that LIBD-1 modulates cognitive function and the 100 mg/kg dose

produces effects similar to the FDA-approved COMT inhibitor tolcapone.


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Finally, we tested the effects of LIBD-3 in the ASST (Figure 2c). There was a

significant difference in trials to criterion between the stages (F6,252 = 12.83, p =

0.0001). The ED stage required more trials than the CD and ID stages, although only the

differences with the CD stage reached the significance threshold (p = 0.0019 and p =

0.0674. respectively). Unlike both tolcapone and LIBD-1, LIBD-3 did not affect

performance (F2,42 = 0.4392, p = 0.6475).

Discussion

Research suggests that COMT inhibition is a viable strategy for improving executive

function, with potential utility across many clinical indications (42, 43). Data from

patients with Parkinson’s disease indicates that the cognitive benefits of COMT

inhibition require a brain-penetrant compound (24). Of the FDA-approved COMT

inhibitors, tolcapone has marginal brain penetration. Unfortunately, long-term use of

tolcapone is associated with potentially fatal liver toxicity in some patients, therefore,

novel, safe, and brain-penetrant COMT inhibitors are needed to explore the utility of the

mechanism for cognitive enhancement. Here we describe the in vivo activity of novel,

non-nitrocatechol, brain-penetrant COMT inhibitors in rats and identify our current

lead drug candidate.

In the CNS, COMT inhibitors are known to alter the concentrations of the DA

metabolites HVA and DOPAC. Specifically, COMT inhibition decreases HVA and

increases DOPAC concentrations. Previous research has used post-administration

measurement of CSF HVA and DOPAC as in vivo markers of COMT inhibition (35). We


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found that two of our novel compounds, LIBD-1 and LIBD-3, produced sustained

modulation of HVA and DOPAC concentrations in CSF. Additionally, we showed that

the changes in CSF HVA and DOPAC were due to central COMT inhibition by testing the

effects of a third compound, LIBD-2. LIBD-2 is extremely potent in vitro, but its brain-

penetrance is negligible and it did not significantly alter central DA metabolism.

While CSF DA metabolites are translational biomarkers for central COMT

inhibition, we believe the ultimate target for the use of COMT inhibitors as cognitive

enhancers is the frontal cortex. To determine if our novel COMT inhibitors could

modulate COMT function directly within our proposed site of action, we conducted

microdialysis experiments with probes sampling from the frontal cortex. We found that

both LIBD-1 and LIBD-3 significantly altered HVA and DOPAC concentrations within

the cortex similar to the effect produced by tolcapone. Our results confirmed previously

reported findings for tolcapone and show that non-nitrocatechol COMT inhibitors

produce relevant changes in DA metabolism in the cortex.

The experiments discussed so far showed that our novel COMT inhibitors

produce qualitatively similar effects compared to the COMT inhibitor tolcapone. We

also confirmed that central measures of COMT inhibition require a brain-penetrant

inhibitor. The final characteristic we wanted in a potential lead compound was the

ability to modulate behavior in a cognition assay. In the next series of experiments, we

showed that LIBD-1 produced behavioral changes at relevant central exposure levels.

LIBD-1 improves cognitive flexibility in the ED phase of the task comparable to the

effects of tolcapone. Interestingly, the lower dose of LIBD-1 increased the number of

trials required to complete the CDr and ID stages. Although it is not clear what caused


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this change in performance, one hypothesis generated from the clinical literature

suggests that tolcapone can impair performance by increasing cognitive control at times

when more automatic responding is advantageous (22). This may be a particular

problem for “normal” subjects, like the rats in our experiments, compared to those with

cognitive impairment. Taken together, these results indicate that our novel non-

nitrocatechol COMT inhibitor possesses many of the in vivo properties required to test

its utility as a cognitive enhancer in a clinical trial.

LIBD-3 produced similar, and arguably superior, results in the neurochemical

assays compared to LIBD-1, but did not significantly improve cognitive flexibility in the

ASST. At this time, it is not clear what is responsible for the discrepancy in performance.

In a cell-based assay, LIBD-3 produces significant cellular toxicity and maximum

changes in DOPAC concentrations much lower than LIBD-1 and tolcapone (G. Zhang,

personal communication). These data suggest that there may be compound-specific

effects that could impede performance in the ASST, but are not evident given the limited

temporal and spatial resolution of our in vivo neurochemical measures.

A large body of evidence suggests that COMT inhibition improves cognitive

function across multiple clinical domains (24, 25, 44). Although there are multiple FDA-

approved COMT inhibitors, none of them are suitable for long-term use as cognitive

enhancers. Entacapone and opicapone (approved in Europe) do not cross the blood-

brain barrier. Tolcapone, which is brain-penetrant, has a black-box warning due to

potentially fatal liver toxicity. The toxicity associated with tolcapone appears to be

compound-specific, as no other marketed COMT inhibitor shares this liability (29). The


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lack of an approved, brain-penetrant, and safe COMT inhibitor has prevented the full

exploration of the utility of the mechanism for cognitive disorders.

In contrast with tolcapone, which is a nonselective inhibitor, our novel COMT

inhibitors are selective for MB-COMT compared with S-COMT. In the rat brain, MB-

COMT and S-COMT protein levels are approximately equal (45). We believe the lack of

S-COMT activity of our inhibitors contributes to the lower in vivo potency in the rat

when compared to tolcapone. In clinical usage, MB-COMT selectivity may actually be an

advantage for our compounds. In humans, and nonhuman primates, roughly 85% of

total brain COMT is the MB-COMT form (46). Moreover, given the enzyme kinetics and

endogenous dopamine concentrations in the brain, MB-COMT is most likely responsible

for more than 85% of total brain COMT activity in humans (11, 47).

A critical outstanding question concerning the utility of COMT inhibitors is which

clinical indication is most likely to be improved by treatment. Small, short-duration

pilot studies have suggested potential efficacy in multiple disorders (24, 25, 44). In

contrast, other studies have shown that in certain circumstances COMT inhibition has

no effect on cognitive function and can actually impair cognition (22, 23). However,

there is a convergence of data suggesting that disorders characterized by impaired

executive function and/or impulse control, including traumatic brain injury (TBI),

behavioral variant frontotemporal dementia (bvFTD), and ADHD may be good

candidates for a COMT inhibitor clinical trial. To be clear, we do not believe that a

COMT inhibitor strategy would be disease-modifying in these indications. However,

there are currently no treatments available to significantly improve the chronic

symptoms of TBI or bvFTD and there is a still a need for effective ADHD treatments


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without stimulant effects and abuse liability (48-50). LIBD-1 has shown significant

efficacy in our preclinical models to suggest it may be able to serve as the COMT

inhibitor to test these hypotheses.


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Funding and Disclosure

This work was funded by US National Institutes of Health grant R01MH107126 and The Lieber Institute for Brain Development. IPB and JCB are inventors on patents that include the novel COMT inhibitors (WO2016123576 and WO2017091818). HLR, RSK, LP, and SCC are employees of RenaSci Ltd. The remaining authors have nothing to disclose.


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Acknowledgments

The authors thank Michael Poslusney and Noelle White for technical assistance, Richard Brammer for statistical analysis of the microdialysis data, Elizabeth Tunbridge for advice on the ASST assay, and Daniel R. Weinberger for constructive comments on a draft of this manuscript.


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Figure legends

Figure 1. Effects of COMT inhibitors on DA and metabolites in the frontal cortex. a DA concentration. b HVA concentration. c DOPAC concentration. Results are adjusted means; n=8 for all groups except tolcapone (n = 7). Vertical arrow indicates time of drug administration (t = 0). Tolcapone, LIBD-1, and LIBD-3 all decreased HVA concentrations and increased DOPAC concentrations without significantly altering DA concentrations compared to vehicle treatment. Statistical results, including timepoints with significant differences between groups, are presented in Supplemental Tables 1-3.

Figure 2. Effects of COMT inhibitors on behavior in the ASST. a Tolcapone (n = 12/vehicle and n = 11/tolcapone b LIBD-1 (n = 24/vehicle, n = 15/LIBD-1 30 mg/kg group, and n = 14/LIBD-1 100 mg/kg group). c LIBD-3 (n = 21/vehicle, n = 14/LIBD-3 30 mg/kg group, and n = 10/ LIBD-3 100 mg/kg group). d Comparison of performance in the ED stage across the three experiments. *p <0.05 and **p <0.01 compared to the vehicle-treated group in the stage.


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Compound Human MB‐COMT IC50 (nM) Human S‐COMT IC50 (nM) Rat MB‐COMT IC50 (nM) Rat S‐COMT IC50 (nM)Tolcapone < 1 < 1 < 1 < 1

LIBD‐1 5.8 1906 < 1 3750

LIBD‐2 7.7 3.8 < 1 236

LIBD‐3 1.5 > 5000 2.2 > 5000

All COMT potency values are for recombinant enzyme

LIBD‐1 and LIBD‐2 are 8‐Hydroxyquinolines from the family of compounds described in reference 32

LIBD‐3 is a pyrido[1,2‐a ]pyrazine‐8‐one described in patent WO2017091818


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Compound Dose Timepoin

t (h) Free Plasma

(nM) Free Brain

(nM) CSF (nM)

CSF HVA (%vehicle) CSF DOPAC (%vehicle)

Tolcapone 15 mg/kg 1 35 BLQ BLQ 7 291

15 mg/kg 4 4 BLQ BLQ 28 304

15 mg/kg (IP); females 4 10 BLQ BLQ 31 233

LIBD‐1 10 mg/kg 1 69 62 132 63 234

30 mg/kg 1 98 BLQ 352 49 229

100 mg/kg 1 211 191 1220 36 273

10 mg/kg 4 35 35 157 72 140

30 mg/kg 4 64 BLQ 176 66 210

100 mg/kg 4 175 67 423 49 251

100 mg/kg; females 4 142 128 349 42 238

LIBD‐2 100 mg/kg 4 41 BLQ BLQ 101 119

LIBD‐3 10 mg/kg 1 N/A N/A N/A 59 201

30 mg/kg 1 145 156 84 39 197

100 mg/kg 1 496 674 328 35 276

30 mg/kg 4 46 39 15 48 182

100 mg/kg 4 290 430 78 22 207

BLQ = below limit of quantitation

Limits of quantitation: Tolcapone (74 nM in CSF and 144 nM in brain). LIBD‐1 (66 nM in brain and CSF). LIBD‐2 (58 nM in CSF and 233 nM in brain).

N/A = no values


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Figure 1a b

c


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Figure 2

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*

*

a b

c d


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