Access to the CNS: Biomarker Strategies for Dopaminergic Treatments
Access to the CNS: Biomarker Strategies for Dopaminergic Treatments
Willem Johan van den Brink 0
Semra Palic 0
Isabelle Köhler 0
Elizabeth Cunera Maria de Lange 0
0 Division of Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research, Leiden University , Einsteinweg 55, 2333 CC Leiden , The Netherlands
1 Elizabeth Cunera Maria de Lange
Despite substantial research carried out over the last decades, it remains difficult to understand the wide range of pharmacological effects of dopaminergic agents. The dopaminergic system is involved in several neurological disorders, such as Parkinson's disease and schizophrenia. This complex system features multiple pathways implicated in emotion and cognition, psychomotor functions and endocrine control through activation of G protein-coupled dopamine receptors. This review focuses on the system-wide effects of dopaminergic agents on the multiple biochemical and endocrine pathways, in particular the biomarkers (i.e., indicators of a pharmacological process) that reflect these effects. Dopaminergic treatments developed over the last decades were found to be associated with numerous biochemical pathways in the brain, including the norepinephrine and the kynurenine pathway. Additionally, they have shown to affect peripheral systems, for example the hypothalamus-pituitary-adrenal (HPA) axis. Dopaminergic agents thus have a complex and broad pharmacological profile, rendering drug development challenging. Considering the complex system-wide pharmacological profile of dopaminergic agents, this review underlines the needs for systems pharmacology studies that include: i) proteomics and metabolomics analysis; ii) longitudinal data evaluation and mathematical modeling; iii) pharmacokinetics-based interpretation of drug effects; iv) simultaneous biomarker evaluation in the brain, the cerebrospinal fluid (CSF) and plasma; and v) specific attention to condition-dependent (e.g., disease) pharmacology. Such approach is considered essential to increase our understanding of central nervous system (CNS) drug effects and substantially improve CNS drug development.
biomarkers; CNS drug development; dopaminergic agents; systems pharmacology
Over the last decades, the development of therapies targeting
diseases affecting the central nervous system (CNS) has been
facing numerous challenges while the number of people
suffering from CNS disorders has tremendously grown,
exceeding one billion worldwide nowadays (
). The challenges
mostly rely on the insufficient knowledge of biomolecular
mechanisms underlying many CNS-related diseases, as well
as the poor understanding of mechanisms of action of many
CNS drugs. In order to improve drug efficacy, both
pharmaceutical industry and academic community have
fostered the implementation of biomarker-based
approaches for translational pharmacology and dose
decision-making in clinical settings. A biological or
biochemical marker represents a measurable sign with regard to a
pharmacological or pathological process, providing a
clinically meaningful endpoint in predicting the effect of a chosen
). Biological markers are recognized as a
valuable tool in drug development, allowing for further
elucidation of both drug efficacy and side effects. CNS drug discovery
and development faces multiple challenges, including the
large number of drugs that fail in late phases of clinical trials
due to poor understanding of processes underlying the dose
response relation (
). In this context, biomarkers represent an
attractive alternative approach to support identification of
most promising compounds, guide the dosing strategies in
early clinical trials, and help recognizing a patient population
that is most likely to benefit from a specific treatment.
This systematic and exhaustive review presents all
biochemical indicators that have been previously reported
as being related to dopaminergic drug effects, as well as
their potential role in biomarker-driven CNS drug
development, focusing on biomarkers in rodents biofluids, specifically
brain extracellular fluid (brainECF), cerebrospinal fluid (CSF),
plasma and urine.
Anatomy and Physiology of the Dopaminergic System
Dopamine is a neurotransmitter that belongs to the
catecholamine family and is primarily synthesized in the brain and the
kidneys. In the brain, dopamine is produced in the cell bodies
of dopaminergic neurons located in the substantia nigra
(SN), the ventral tegmental area (VTA) and the
hypothalamus. These neurons send projections to multiple brain
areas where dopamine is stored and released, including the
striatum (nigrostriatal pathway), the prefrontal cortex
(PFC) (mesocortical pathway), the nucleus accumbens
(NAc) (mesolimbic pathway) and the pituitary gland
(tuberoinfundibular pathway), as illustrated in Fig. 1. It should
be noted that these pathways do not represent all dopamine
systems in the brain. Other systems, such as the thalamic
dopamine system, are increasingly recognized as important
additional components of the brain dopamine pathways (
The presence of dopamine in the mesolimbic pathway is
related to positive reinforcement, reward and/or pleasure, while
in mesocortical pathway it is involved in cognitive control of
behavior. Furthermore, the role of dopamine in the
nigrostriatal pathway, transmitted from the SN (midbrain) to
the putamen in the dorsal striatum, is to simulate
rewardrelated cognitive processes as well as psychomotor function.
The tuberoinfundibular pathway projects dopaminergic
neurons from the hypothalamus to the pituitary gland to
modulate secretion of hormones, including prolactin. Dopaminergic
pathways also project from the VTA (midbrain) to the
amygdala, the hippocampus, and the cingulate cortex. As such,
dopamine is simultaneously involved in both emotional and
memory processing. Dopaminergic neurons form a tight
network with a number of other neuronal pathways, including
choline, glutamate and gamma-aminobutyric acid (GABA)
systems, showing its possible role in multiple complex
processes. Therefore, any drug targeting the dopaminergic neurons
may influence multiple transduction pathways including both
the dopaminergic and other systems.
Five dopamine receptor subtypes, often referred to as D1–5
receptors, have been reported in the CNS, all being G-protein
coupled receptors that may function independently but of
which the downstream pathways may also interact (
Dopamine receptors are divided into D1- and D2-like receptor
classes, the D1 receptor class including D1 and D5 receptors
while D2 receptor class includes D2, D3, and D4 receptors. D1
receptor and D2 receptor classes have opposing effects with
regard to adenylyl cyclase activity, cAMP concentrations, as
well as phosphorylation of proteins, resulting in either
stimulatory or inhibitory action on voltage-gated and ion channels
in synapses (
). D1 receptor are highly expressed in the
striatum, NAc, SN, frontal cortex and amygdala, while lower
expression of D1 receptor is found in the hippocampus,
thalamus, and cerebellum. D2 receptor are mainly localized in the
striatum, NAc, SN, hypothalamus, cortical areas, amygdala
and hippocampus. Although dopamine receptors are most
densely expressed in the brain, they are also found in the
periphery in different patterns of expression (
the system-wide effects of dopamine that are crucial in
Dopaminergic Agents for Treatment of Neurological
The dopaminergic system has been exploited for treatment
opportunities in a large variety of disorders. Due to its broad
implication in pathophysiology, the current pharmacological
efforts mostly focus on targeting both the dopamine receptors
and subsequent post-receptor mechanisms. Different types of
dopaminergic drugs have been developed so far, primarily
dopamine agonists and dopamine antagonists.
Dopamine agonists have been developed for treating
Parkinson’s disease, a progressive neurodegenerative disorder
presenting both motor and non-motor symptoms. The
pathology of the Parkinson’s disease is characterized with an
extensive loss of dopamine neurons in the SN and accumulation of
the protein α-synuclein in Lewy bodies within nerve cells in
specific brain regions (
). Although the underlying
mechanisms leading to Parkinson’s disease remain poorly
understood, a strong association between low dopamine brain levels
and Parkinson’s disease symptoms has been frequently
). Dopamine receptor agonists, introduced first in 1970
for the treatment of Parkinson’s disease, act directly on
dopamine receptors to mimic endogenous neurotransmission.
Levodopa (L-DOPA), a pro-drug crossing the blood-brain
barrier (BBB), was the first therapeutic option available for
treating Parkinson’s disease. Various other agonists, e.g.,
apomorphine, bromocriptine and pramipexole, have been
later developed and commercialized, showing comparable
While most of the currently available dopamine agonists are
used for Parkinson’s disease, the vast majority of dopamine
antagonists have been developed for the treatment of
schizophrenia. Multiple studies using animal models of
schizophrenia have elucidated a pattern of persistent hyperdopaminergic
state, accompanied with altered stimulus recruits of dopamine
in different brain regions. Cognitive impairments during
psychosis might thus be explained by a rapid release of
dopamine into the mesolimbic and the nigrostriatal regions (
Chlorpromazine was the first and extremely potent antagonist
of D2 receptor discovered, which considerably fostered
antipsychotic drug development. Nevertheless, chlorpromazine
treatment is accompanied with pronounced adverse effects,
including neuroleptic malignant syndrome and
extrapyramidal symptoms (EPS) such as tardive dyskinesia. Other D2
receptor antagonists, e.g., haloperidol, risperidone and
clozapine, have been developed to exhibit comparable or greater
effectiveness with fewer of these side effects, in particular EPS
Many of dopaminergic agents were discovered with
incomplete understanding of their modes of action, often resulting in
unpredictable side effects and/or off-target effects. It is only
after having been introduced to market that studies were
conducted to elucidate their modes of actions, which revealed
multiple pathways affected (
Selectivity of Dopaminergic Drugs
Clozapine is currently the Bgold standard^ for the treatment
). Interestingly, this is one of the least
selective D2 receptor antagonists (
). Indeed, schizophrenia
is a polygenic disease, and therefore a ‘shotgun-approach’
may be more successful than a ‘magic-bullet approach’ (16).
Many D2 receptor antagonists have therefore affinity for more
receptors, including serotoninergic, adrenergic, muscarinic,
and histaminergic receptors (
). Also many D2 receptor
agonists were found non-selective, with affinity for other
dopaminergic, serotonergic, adrenergic and histaminergic
receptors (21). This should be taken into consideration when
evaluating the effects of these agents on the system-wide
This review aims to further improve the understanding of
mechanisms of action by providing an extensive overview of
the pathways that are affected by dopaminergic agents, with
the hope to increase our understanding of system-wide
dopaminergic pharmacology, as well as to provide directions on
how to improve pharmacological biomarker strategies during
early drug development.
A systematic overview of literature over the past 25 years
has been built, focusing on dopaminergic treatment effects
on central and peripheral biomolecular pathways in rats. A
search of the PubMed database was conducted in September
2017 by using the following key words: dopamine antagonists,
dopamine agonists, biogenic amine, amino acid, hormone, cytokine, lipid,
neurotransmitter, cerebrospinal fluid, intracerebral microdialysate, plasma,
urine, rat (see Supplementary Data S1 for the exact search
code), yielding to 1058 articles (English only). Only
studies describing the effects of dopaminergic agents and
elucidating a potential biochemical indicator of drug
action in rats were included. In vitro studies,
experimental studies focusing only on behavioral changes and/or
reactions, studies of cognition patterns or event-related
potentials, and studies that only included
pharmacokinetic information were excluded. Furthermore, studies
including functional imaging techniques or
electroencephalography, investigating dopamine receptor
affinities, functions, and synthesis, exploring the effect of
dopaminergic agents in combination with other
pharmacological agents, under pathological conditions, after
surgical procedures such as adrenalectomy or ovariectomy,
with pregnant or lactating animals, and with animals
under long-term food restriction were excluded as well.
Finally, prolactin, being considered a standard marker
of dopaminergic activity with well-explored functions
and relationship with dopamine (
), has been
excluded. After selection, 260 articles were included.
DOPAMINERGIC TREATMENT EFFECTS
ON ENDOGENOUS METABOLITES LEVELS IN THE CNS
The CNS-wide effects of dopamine receptor agonists and
antagonists reported in the selected studies are shown in Table I
and Fig. 2. Although information was also gathered from
studies involving intracerebral administration, only data after
systemic administration is presented to obtain insights into
clinically relevant effects. Moreover, a distinction is
made between short-term and long-term treatment
effects. Most of the effects reported in the CNS have
been mainly observed in brainECF, using microdialysis,
leading to deeper insights into neurotransmitter
pathways. Overall, the reported literature emphasizes the
CNS-wide effects of dopaminergic agents, including
dopamine pathway but also norepinephrine, cholinergic,
GABA-glutamate, serotonin, kynurenine, nitric oxide
and endocannabinoid pathways.
Several considerations have to be taken into account for the
discovery of easily accessible biomarkers that reflect these
systematic effects, notably (Fig. 3):
detectability in CSF, plasma or/and urine;
simultaneous evaluation together with other markers of
the pathway of interest to understand the dynamics
between the drug and the pathway;
Sufficient understanding of central and peripheral
Identification of distribution rates between brain, CSF,
plasma and urine to understand the temporal relation
between the biomarker peripheral concentration and
effects in the brain.
Effects on the Dopamine Pathway
Metabolism and Signaling of the Dopamine Pathway
The synthesis of dopamine involves the conversion of tyrosine
into L-DOPA, the precursor of dopamine. It is stored into
vesicles in the presynaptic neuron, following uptake via the
vesicular monoamine transporter (VMAT). These vesicles
release dopamine into the synaptic cleft, where it may bind to
pre- or postsynaptic dopamine receptors to pass on neuronal
signals to the post-synaptic neuron. The dopamine present in
the synaptic cleft is eliminated through conversion to its
metabolites homovanillic acid (HVA), 3,4-dihydroxyphenylacetic
acid (DOPAC) or 3-methoxytyramine (3-MT), or by uptake
into the presynaptic neuron via the dopamine transporter. In
the latter case, dopamine is stored into vesicles, or degraded to
HVA or DOPAC.
Effects of Dopaminergic Agents on the Dopamine Pathway
Dopamine receptors are located pre- and postsynaptically,
thereby influencing local concentrations of dopamine and its
metabolites upon the presence of agonists and antagonists
(Table I, Fig. 2). Short-term treatments with D2 receptor
antagonists such as haloperidol, sulpiride, risperidone,
olanzapine and clozapine have shown to stimulate the
dopamine pathway (
), whereas administration of D2 receptor
+ (green): increase; - (red): decrease; +/-, -/0 or +/0 (grey): conflicting results; 0 (grey): no effect.aIn case multiple stubdies were identified for the effects of a
particular drug cclass on a particular marker,donly the 4 most recent publications were reported. Only in striatum; Only observations after intracerebral
administration; Few and/or conflicting data; Measured in the prefrontal cortex
DA dopamine, DOPAC 3,4-dihydroxyphenylacetic acid, HVA homovanillic acid, 3-MT 3-methoxytyramine, NE norepinephrine, E epinephrine, VMA
vanillylmandelic acid, GABA gamma-aminobutyric acid, 5-HT serotonin, brainECF brain extracellular fluid
agonists like quinpirole, quinelorane, 7-OH-DPAT, and
apomorphine inhibit this pathway (
). This has
been observed in brainECF for dopamine as well as for
its major metabolites DOPAC, HVA, 3-MT (Table I).
The influence of D1 receptor agents on the dopamine
pathway remains poorly investigated. Only one study
was identified, showing an increase in dopamine levels
after intraperitoneal treatment with the D1 receptor
antagonist SCH23390 (
), while no studies reported the
effects after systemically injected D1 receptor agonists. The
effects of D2 receptor antagonists and agonists on the
dopamine pathway may be explained by the modulation of
presynaptic D2 autoreceptors that provide a negative feedback
function on dopamine release (
). Moreover, many of these drugs
have affinity for 5-HT receptors (
), which also contribute
to the control of dopamine release (
After long-term treatment with D2 receptor agonists,
the basal dopamine pathway activity is decreased,
similar to the effect observed after short-term treatment
). Interestingly, D2 receptor antagonists inhibit
the dopamine levels after long-term treatment, while
the levels of the dopamine metabolites are increased
). This may, first of all, be explained by the
upregulation of D2 receptor expression after long-term
treatment (94), thereby leading to an enhanced inhibition of
dopamine release via the D2 autoreceptor. Second, the
monoamine oxidase (MAO) and the catechol-O-methyl
transferase (COMT), that metabolize dopamine into
DOPAC, HVA and 3-MT, were upregulated (
providing another explanation, also supporting the
increased concentrations of dopamine metabolites that are
observed with long-term treatment.
Biomarkers for the Dopamine Pathway
Dopamine and its metabolites can be detected in CSF, plasma
and urine (
). In contrast to dopamine, HVA is
able to cross the BBB, providing a way to evaluate
central dopaminergic activity in plasma. The difficult
aspect is to distinguish between the central and the
peripheral effects, since the dopaminergic system is also
peripherally active in, for example, the kidney and the
adrenal glands. The origin of the HVA response in
urine after long-term treatment with haloperidol and
) is therefore not known. Surprisingly,
no further studies were identified that investigated
CSF, plasma or urine biomarkers of the dopamine
pathway after dopaminergic treatment.
Effects on the Norepinephrine Pathway
Metabolism and Signaling of the Norepinephrine Pathway
The largest concentrations of norepinephrine in the brain are
found in neurons in the locus coeruleus. Outside the brain, it is
found in the postganglionic sympathetic adrenal fibers and the
chromaffin cells in the adrenal glands. Within the
norepinephrine neurons, VMAT stores dopamine into synaptic vesicles,
where it is converted to norepinephrine through dopamine
beta-hydroxylase, and released into the synaptic cleft.
Norepinephrine may bind to alpha- or beta-adrenergic
receptors, the former being mostly inhibitory and located
presynaptically, while the latter are stimulatory and located
postsynaptically. From the synaptic cleft, norepinephrine undergoes
reuptake into the presynaptic neuron via the norepinephrine
t r a n s p o r t e r , o r i s m e t a b o l i z e d t o e p i n e p h r i n e ,
dihydroxyphenylglycine and methoxyhydroxyphenylglycol.
In the presynaptic neuron, it may be stored into vesicles, or
degraded into its metabolites.
Effects of Dopaminergic Agents on the Norepinephrine Pathway
Norepinephrine release is stimulated by D2 receptor antagonists
such as clozapine, olanzapine and risperidone, although this has
not been reported for haloperidol (
) (Table I, Fig. 2). While
this may be explained by dopaminergic modulation of
norepinephrine release (97), these drugs also exhibit affinity for the
adrenergic receptors (
). Interestingly, in contrast to
haloperidol, the other D2 receptor antagonists showed affinity for the α2
adrenergic receptor. After long-term treatment, haloperidol
caused a reduction of norepinephrine levels in the striatum
), which may be explained by reduced conversion from
dopamine to norepinephrine, since long-term D2 receptor
antagonist treatment decreased dopamine levels (Table I, Fig. 2).
Plasma norepinephrine concentrations were decreased
after D2 receptor stimulation with the agonist bromocriptine
). This effect was blocked by administration of the D2
receptor antagonist domperidone, which does not cross
the BBB, suggesting the effect to be peripheral (
Furthermore, plasma levels of epinephrine were increased
upon stimulation of D2 receptor, although likely elicited through
direct peripheral action on the adrenal gland and independent
of the effect on norepinephrine (
Biomarkers for the Norepinephrine Pathway
Norepinephrine and its metabolites have been already
analyzed in CSF, plasma and urine (
), indicating that the
latter biofluids can be used to estimate the central
norepinephrine pathway activity. Indeed, reduced levels of the most
downstream norepinephrine metabolite vanillylmandelic acid
were found in urine after long-term treatment with
haloperidol or clozapine (
). However, as discussed in the previous
paragraph, the effect on plasma (and thus also urine)
norepinephrine concentrations are at least partly caused by
peripheral effects. Further understanding of the relative central and
peripheral effects of dopaminergic agents on the plasma or
urine norepinephrine pathway responses is needed to
conclude whether they can be used as biomarker for central
activity. The CSF levels are likely more representative; however,
the evaluation of longitudinal norepinephrine pathway
responses upon dopaminergic treatment is still lacking.
Effects on the Acetylcholine Pathway
Metabolism and Signaling of the Acetylcholine Pathway
Acetylcholine (ACh) is produced from choline in the
presynaptic neurons and stored into vesicles via the vesicular
acetylcholine transporter. These vesicles release ACh into the
synaptic cleft where it binds to the postsynaptic ACh receptors,
which are subclassified into nicotinic receptors that modulate
neuronal activity and muscarinic receptors that elicit
Gprotein dependent signaling. ACh is degraded to choline
and acetate, the former being recycled into the presynaptic
neuron by the sodium-dependent choline transporter.
Interestingly, anticholinergic drugs are typically prescribed
to decrease the EPS accompanying antipsychotic treatments,
suggesting that the dopaminergic and the cholinergic system
are tightly connected. Cholinergic interneurons in the
striatum represent only 1–2% of all neurons, yet they play an
important role in the integration of multiple neurotransmitter
), thereby contributing to the stabilization of
dopaminergic signaling in the psychomotor circuit (also
corticobasal ganglionic system) (
Effects of Dopaminergic Agents on the Acetylcholine Pathway
As listed in Table I and Fig. 2, ACh release from cholinergic
interneurons in the striatum is inversely related to D2 receptor
stimulation or inhibition. On the other hand, choline, the
precursor of ACh, was reduced after D2 receptor antagonist
treatment, probably as a consequence of ACh release, since
the uptake of choline was increased to support ACh
Contrary to their effect in the striatum, D2 receptor
agonists increased ACh levels in the hippocampus and the frontal
). Furthermore, ACh in the PFC and the
hippocampus was increased after treatment with
secondgeneration D2 receptor antagonists, which was not the case
for first-generation D2 receptor antagonists (
ACh levels in the NAc were not affected by D2 receptor
). Overall, this indicates that the relation between
the dopaminergic system and cholinergic signaling is
regionspecific. Indeed, there is evidence for D2 receptor specific
regulation of ACh in the striatum, while for other regions the
results are conflicting. D1 and D2 receptors are certainly
involved, taking into account that several of the D2 receptor
binding drugs discussed here also exhibit affinity for the
muscarinic receptors (
D1 receptor agonists have consistently been reported to lead
to increased ACh levels in several brain regions, including the
), while D1 receptor antagonism led
to decreased ACh concentrations (
), or had no effect (
Cholinergic neurons indeed express the D1, mostly the D5
receptor, increasing excitability after receptor stimulation (
Biomarkers for the Acetylcholine Pathway
Both ACh and choline can be detected in CSF and plasma
with state-of-the-art analytical methods (
Furthermore, the plasma levels of these molecules may reflect
central cholinergic activity, since they both can cross the BBB
(115). However, ACh is an important neurotransmitter of the
PNS, sending signals from neural endfeet to muscle cells. This
might confound the plasma levels as a marker of central
activity. Quantitative understanding of the BBB distribution
relative to the PNS response is essential to be able to interpret the
plasma levels. Moreover, the relation between dopamine
treatment and the cholinergic system appeared brain region
specific, which may limit the usefulness of CSF and plasma for
cholinergic biomarker detection. No studies have investigated
cholinergic CSF and plasma in relation to dopaminergic
treatment so far. Therefore, it is not possible to conclude whether it
is possible to use these biofluids for biomarker evaluation.
Effects on the GABA-glutamate Pathways
Metabolism and Signaling of the GABA-glutamate Pathways
GABA and glutamate are the main inhibitory and excitatory
neurotransmitters, respectively, in the brain. Glutamate is
synthesized from glutamine by the enzyme glutaminase and is
stored in vesicles in glutamatergic neurons via the action of
vesicular glutamate transporters. These vesicles release
glutamate into the synaptic cleft where it binds to the glutamate
receptors, i.e., metabotropic receptor and ionotropic
receptors (NMDA, kainate,and AMPA receptors). From the
synaptic cleft, glutamate distributes into glial cells, using the
glutamate transporter 1 or the glutamate aspartate transporter,
where it is metabolized into glutamine. Glutamine is
subsequently released from the glial cells and recycled into
glutamatergic neurons. Also in GABAergic neurons, glutamate is
produced from glutamine. However, these neurons also
contain the enzyme glutamate decarboxylase that converts
glutamate into GABA. Vesicular GABA transporters store GABA
into vesicles which release it into the synaptic cleft. There, it
binds to the GABA receptors to inhibit the activity of the
postsynaptic neuron. GABA diffuses to the glial cells via the
GABA transporter where it is metabolized to glutamate via
the Krebs cycle, and subsequently converted to glutamine.
Glutamine is recycled into the presynaptic GABAergic
neurons. Although glutamate and GABA have many roles in the
brain and are distinct neurotransmitters, we discuss here their
interconnection in relation to two dopaminergic pathways: the
nigrostriatal pathway and the mesocorticolimbic pathway.
These pathways belong to the so-called circuits that connect
multiple brain regions by neuronal fibers. Concretely, in the
nigrostriatal pathway, activation of the striatal D1 receptor
leads to release of GABA into the internal globus pallidum
(GPi) and the substantia nigra reticula (SNr). This
subsequently reduces the release of GABA into the thalamus. Activation
of the striatal D2 receptor inhibits the release of GABA into
the external globus pallidum (GPe), which then stimulates the
release of GABA into the subthalamic nucleus and the GPi.
This also reduces the release of GABA into the thalamus. As
such, these two pathways, also referred to direct and indirect
pathway, enhance the thalamic release of glutamate into the
PFC. Since cortical glutamatergic neurons project to multiple
regions in the midbrain, amongst which the striatum and the
substantia nigra, many functionalities are stimulated. In the
mesocorticolimbic pathway, activation of D2 receptors in the
VTA stimulates GABAergic neurons in the NAc. This leads to
enhancement of GABA release into the other brain regions
such as VTA and ventral pallidum. Additionally, D2 receptor
activation in the VTA stimulates the release of dopamine into
the PFC. This enhances the activity of the pyramidal neurons
that release glutamate into other brain regions, including NAc
Effects of Dopaminergic Agents on the GABA-glutamate
While these circuits for a large part were unraveled by local
injection of dopaminergic, GABAergic and glutamatergic
), not many studies have been performed
showing the effect of systemically injected dopaminergic
agents (Table I, Fig. 2). Only one D1 receptor agent, an
antagonist, was systemically injected to show no effect on
glutamate levels in the entopeduncular nucleus (EPN) (
cortical GABA levels were increased with systemic injection
of D2 receptor agonists, while glutamate levels in the NAc or
EPN were decreased (
), contrasting the response
expected from the above-described circuits. D2 receptor
antagonists typically did not show an effect on GABA levels in the
ST, the GPe, the PFC and the NAc (
glutamate levels in the ST, EPN, PFC or NAc (
should be noted that the results are not always consistent, since
some studies with D2 receptor antagonists found reduced
GABA levels in the GP, NAc or PFC (
increased GABA concentrations in the GP or the striatum
), or increased glutamate levels in the SN, ST, EPN,
PFC, or NAc (
). These contradictions highlight
the delicate balance of this circuit, which is affected by
multiple factors (e.g., target site exposure, experiment time,
offtarget effects, etc.) that can cause concentration-, time-, or
drug-dependent differences among the studies. Moreover,
with systemic injection, these circuits are perturbed at multiple
regions, rendering its pharmacological interpretation
non-intuitive. Systematic studies that account for these factors, and
that evaluate glutamate, GABA and dopamine in multiple
brain regions simultaneously, are warranted to obtain a
deeper insight into the effects of systemic administration of
dopaminergic agents on such circuits.
Biomarkers for the GABA-glutamate Pathways
Although GABA and glutamate concentrations are well
measurable with modern analytical approaches (
), it is not
known how the levels relate to dopaminergic treatment.
GABA and glutamate responses have shown to be
regiondependent, which may confound the CSF and plasma
response. Further experimental evidence needs to be collected
to evaluate the potential of CSF and plasma to assess the
GABA-glutamate pathway activity in relation to
Effects on the Serotonin Pathway
Metabolism and Signaling of the Serotonin Pathway
Serotonin is produced from the amino acid tryptophan via
5hydroxytryptophan and stored into vesicles by VMAT. When
it is released from these vesicles into the synaptic cleft, it binds
to different classes of 5-HT receptors (5-HT1–5-HT7). It is
recycled into the presynaptic neuron by the serotonin
transporter, where it is stored into vesicles or metabolized to
5hydroxyindoleacetic acid (5-HIAA).
Effects of Dopaminergic Agents on the Serotonin Pathway
In contrast, the modulation of serotonin circuits by dopamine
is mainly restricted to D2 receptor mediated stimulation of
serotonin neuron cell bodies in the dorsal raphe nucleus
(DRN) that control motor activity. This leads to increased
serotonin release in the DRN and other regions such as the
), as identified with systemic administration of D2
receptor agonists (
) (Table I, Fig. 2). No effects of
dopamine agonists were found on the levels of the metabolite
). Additionally, it was suggested that D2
receptor agonists modulate serotonin afferents presynaptically in
the hippocampus (
) or the SN (
). D2 receptor
antagonists did not show an effect on serotonin levels (
except for atypical antipsychotics such as risperidone and
clozapine, likely elicited through presynaptic serotonin receptors
). Moreover, 5-HIAA was found increased
after risperidone in but not all studies (
Biomarkers for the Serotonin Pathway
The serotonin metabolite 5-HIAA, but not serotonin itself, has
been already detected in CSF (
). serotonin, 5-HIAA and the
precursor tryptophan can be also detected in plasma.
Although serotonin cannot pass the BBB, the central serotonin
pathway activity may be inferred from the tryptophan and
5HIAA responses. It is, however, important to realize that the
serotonin pathway is also present in peripheral systems, for
example in platelets. Moreover, tryptophan is provided via
food intake. These factors may confound the plasma
biomarker response to reflect central activity. Experimental evidence is
further needed to investigate the relation between
dopaminergic treatments, central serotonin activity and CSF or plasma
Interactions Among Neurotransmitter Systems
The above-described effects of dopaminergic agents clearly
show that the neurotransmitter systems of dopamine,
norepinephrine, GABA, serotonin, glutamate and ACh are highly
interconnected. Moreover, many of these agents also influence
these neurotransmitter systems via binding to other receptors,
such as serotonineric and adrenergic receptors. Therefore, in
order to understand the effects of these agents,
neurotransmitter responses should be evaluated altogether. Qi et al. (2016)
established a network of the connections between these
neurotransmitters, taking into account the spatial and functional
organization of their neurons and interactions (
) (Fig. 4).
This network was used to understand the neurotransmitter
disbalances in schizophrenia and their normalization upon
antipsychotic treatment. Indeed, disease pathology and drug
action must understood in terms of a disbalance among
multiple signaling pathways, rather than describing pathology and
pharmacology as a single pathway disruption.
Biomarkers that Reflect the Balance
Among the Neurotransmitter Systems
It will become important to identify accessible biomarkers in
CSF, plasma or urine that can reflect the balance among the
neurotransmitter systems. While such approach has been
followed for a glutamate receptor agonist, identifying the
turnover of the dopamine, norepinephrine and serotonin pathway
in CSF (
), there has not been such attempt for dopaminergic
Effects on the Kynurenine Pathway
Metabolism and Signaling of the Kynurenine Pathway
Similar to serotonin, kynurenine is a metabolite of tryptophan.
In fact, about 95% of tryptophan in the brain is metabolized
via the kynurenine pathway, further leading to kynurenic acid,
quinolinic acid and 3-OH-kynurenine (
quinolinic acid is a pro-glutamatergic molecule, kynurenic
acid has several anti-glutamatergic properties, such as the
antagonism of the NMDA receptor and the inhibition of
glutamate release through ACh receptors. 3-OH-kynurenine is
involved in the generation of free radicals, independent of the
glutamate system (133). 3-OH-kynurenine and quinolinic acid
have neurotoxic properties, while kynurenic acid has proven
to be neuroprotective (
). A disbalance in the kynurenine
metabolism was therefore associated with several neurological
disorders, amongst which Parkinson’s disease and
Effects of Dopaminergic Agents on the Kynurenine Pathway
Kynurenic acid was reduced after long-term (1–12 months), but
not after shorter-term (1 week) administration of clozapine,
raclopride and haloperidol (
) (Table I, Fig. 2). D2 receptor
antagonists may potentially interfere with the kynurenine amino
transferase (KAT) enzyme, which converts kynurenine to
kynurenic acid. Indeed, kynurenine and its metabolites other
than kynurenic acid were not altered after treatment with D2
receptor antagonists (
). It is likely that this effect is D2 receptor
specific, given that raclopride is a highly selective D2 receptor
). D2 receptor antagonists thus likely inhibit the
neuroprotective branch of the kynurenine metabolism, which
could be a potential unwanted effect in the long term.
Biomarkers for the Kynurenine Pathway
Kynurenine and kynurenic acid are present in sufficient
concentration in CSF to be quantified (
Moreover, 40% of the kynurenine synthesis occurs in
the brain, while 60% takes place in the blood and is
transported over the BBB. It is thus likely that
kynurenine and kynurenic acid in CSF and plasma
reflect the levels in the brain; however, it is not known to
which extent. CSF and plasma levels changes upon
dopaminergic treatment remain to be investigated.
Effects on the Nitric Oxide Pathway
Metabolism and Signaling of the Nitric Oxide Pathway
Nitric oxide is generated by nitric oxide synthase (NOS)
through the conversion of arginine to citrulline. Nitric oxide
has a short half-life (i.e., few seconds) and is readily oxidized to
nitrite and nitrate, which can then be measured as an
indication of NOS activity. By binding to soluble guanylyl cyclase,
nitric oxide stimulates local postsynaptic excitability via
modulation of voltage-gated ion channels and possibly also
presynaptic neurotransmitter release, thereby modulating synaptic
). Nitric oxide is tightly connected to
glutamatergic signaling. Moreover, it contributes to gonadotrophin
and oxytocin release, circadian and respiratory rhythms,
locomotor and thalamocortical oscillation, as well as learning
process and memory (139). The nitric oxide pathway is
downregulated in Parkinson’s disease and schizophrenia, indicating a
connection with dopamine (
Effects of Dopaminergic Agents on the Nitric Oxide Pathway
Citrulline, nitrite and nitrate have shown to be upregulated
after short-term treatment with D1 receptor and D2 receptor
agonists (Table I, Fig. 2). Only two studies with systemic
administration have been reported (
), while other studies
focused on the effects after intracerebral injections (
possible hypothesis for this upregulation is the stimulation of
NOS activity by dopamine, thereby augmenting the
production of citrulline and nitric oxide (
). The effect on the nitric
oxide pathway was proven to be D2 receptor-specific in the
), while the D1 receptor was involved in the
). Although D2 receptor antagonists blocked
the effect of D2 receptor agonists on nitric oxide
Fig. 4 Mathematical model
containing expressions for the
interactions between the
systems in multiple brain
regions. Rather than looking at
single biomarkers, this model
enables the prediction of
disbalances among the
neurotransmitter systems under
conditions of drug administration.
Adapted from reference (
), they did not exhibit a significant
effect when administered alone (
longterm treatment with haloperidol led to an upregulation
of neuronal NOS in the hypothalamus (94).
Biomarkers for the Nitric Oxide Pathway
Nitrite and nitrate have been measured in the CSF of patients
suffering from neurological disorders (
their potential as easily-accessible biomarkers. Nitrate urine
levels were found increased after intravenous administration
of fenoldopam, a D1 receptor agonist, although this effect
might have been exerted via D1 receptors present in the
kidney, rendering difficult to discriminate between peripheral
and central effects (88).
Effects on the Endocannabinoid System
Metabolism and Signaling of the Endocannabinoid System
The most well-known components of the endocannabinoid
system are anandamide, which is synthesized from
Na r a c h i d o n o y l p h o s p h a t i d y l e t h a n o l a m i n e , a n d 2
arachidonyl glycerol (2-AG), that is produced from
). Anandamide is degraded to
ethanolamine and arachidonic acid by fatty acid amide
hydrolase, while 2-AG is broken down to arachidonic
acid by monoglyceride lipase (
). Arachidonic acid is
the precursor of a wide range of biologically and
clinically important eicosanoids and respective metabolites,
i n c l u d i n g p r o s t a g l a n d i n s a n d l e u k o t r i e n e s . T h e
endocannabinoid system is widely distributed in the
CNS where it reduces synaptic input through retrograde
signaling via cannabinoid receptors, in the brain mainly
the CB1 receptor subclass (
Effects of Dopaminergic Agents on the Endocannabinoid System
Dopamine influences the endocannabinoid system
mainly in the nigrostriatal pathway by upregulation of
endocannabinoid system in the striatum and
downregulation in the GPe in a D2 receptor dependent manner
). Indeed, quinpirole stimulated the release of
anandamide in the striatum (
), an effect that was blocked
by raclopride (Table I, Fig. 2). This provides evidence
for D2 receptor-dependent involvement of the dopaminergic
system in endocannabinoid signaling. Furthermore, although
the D1 receptor agonist SKF38393 did not cause an
effect on anandamide (
), it was found that, with
impaired dopamine release, the striatal D1 receptor may
also affect the endocannabinoid system (
Biomarkers for the Endocannabinoid System
Even though anandamide can be detected and
quantified in the brain, its levels in CSF and plasma are very
), rendering its quantitation challenging. Moreover,
2-AG is chemically unstable in aqueous solution, leading to
the formation of its isomer 1-AG. Nevertheless, ethanolamine
levels can be measured in CSF suggesting this compound as a
potential biomarker candidate to reflect the activity of the
endocannabinoid system (
DOPAMINERGIC TREATMENT EFFECTS
ON THE NEUROENDOCRINE
AND THE ENERGY SYSTEMS
Additional to its role in the CNS, the dopamine system
is widely expressed in peripheral tissues (
the importance of evaluating the peripheral effects of
dopaminergic agents. The CNS is connected to the
periphery via the PNS and the neuroendocrine system,
allowing for the opportunity to capture the consequence
of central drug effects in the periphery, as done for
instance with prolactin (
). A significant influence
on the hypothalamic-pituitary-adrenal (HPA) axis, the
reproductive system, insulin signaling and the lipid
metabolism has been found in this systematic review
(Table II, Fig. 2). With regards to biomarker discovery,
two important aspects can be highlighted (Fig. 3):
Biomarkers need to be evaluated together with other
markers of the pathway of interest to understand its
interaction with the drug;
The connection between brain and target pathway must
be quantitatively understood to allow for estimation on
how the biomarker response reflects the central effect.
Effects on the Hypothalamic-Pituitary-Adrenal (HPA)
Signaling in the HPA Axis
The hypothalamo-pituitary-adrenal (HPA) axis is involved in
the homeostasis of metabolic and cardiovascular systems,
stress response, reproductive system, as well as immune
system. It is a complex system of signals and feedback
mechanisms between the hypothalamus, the pituitary gland and the
adrenal glands. The hypothalamus releases corticotrophin
releasing hormone (CRH) and vasopressin to modulate the
secretion of adenocorticotropin hormone (ACTH) by the
pituitary gland. ACTH subsequently stimulates the release of
glucocorticoids (corticosterone in rodents, cortisol in humans)
and catecholamines, which control CRH and ACTH release
via a negative feedback loop. ACTH is cleaved from the
prohormone pro-opiomelanocortin, which also yields to a
number of different peptides including alpha-melanocyte
stimulating hormone (α-MSH), beta-endorphin and a few
other peptides that are also secreted from the pituitary gland.
Effects of Dopaminergic Agents on the HPA Axis
A wide range of neural systems influence the HPA axis (
including dopaminergic system, both in a D1 and D2 receptor
dependent manner (Table II, Fig. 2) (
). This effect
is mainly observed after short-term treatment with D1 and D2
receptor agonists, while long-term treatment did not show a
significant effect on basal ACTH levels (
Surprisingly, in contrast to haloperidol, the D2 receptor
antagonists eticlopride and remoxipride have been reported
to increase ACTH plasma levels (
remoxipride was 40 times less potent to elicit the ACTH
response than to induce the prolactin response (24), suggesting
that these observations are explained by off-target effects.
Contrary to their conflicting results for ACTH release, D2
receptor antagonists showed a consistent stimulation of
corticosterone plasma levels (Table II, Fig. 2), indicating that
glucocorticoid release is not only mediated via a central
mechanism of ACTH secretion. Additionally, the stimulation of the
PNS was suggested to control the sensitivity of the adrenal
medulla to ACTH, thereby enhancing the release of
corticosterone. It is not certain whether this process is under
dopaminergic control, but catecholamines certainly play a role
). Furthermore, D2 receptor antagonists might directly
modulate the release of corticosterone, given that D2 receptors
have been found on the adrenal cortex (
). It is worth
mentioning that investigations on dopaminergic innervation in the
glucocorticoid release focused on aldosterone release from the
zona glomerula, and not on corticosterone release from the
zona fasciculate and reticularis (
). Whether the effects of
dopaminergic drugs are primarily mediated via dopamine
receptors is not fully elucidated. While the ACTH response to D2
agonist quinpirole was blocked by the D2 antagonist sulpiride,
indicating the involvement of the D2 receptor, the
corticosterone response was not evaluated by such approach (
In addition to ACTH and corticosterone, α-MSH
secretion from the intermediate lobe of the pituitary gland is also
controlled by the dopaminergic system (
). α-MSH levels
+ (green): increase; - (red): decrease; +/-, -/0 or +/0 (grey): conflicting results; 0 (grey): no effect. + (green): increase; - (red): decrease; +/-, -/0 or +/0 (grey):
conflicting results; 0 (grey): noa effect.In case multiple studiesb were identified for the effects of a particular drug class on a particular marker, only the 4 most recent
publications were reported. Few and/or conflicting data; The atypical antipsychotics risperidone and clozapine showed a positive effect, whereas haloperidol
showed a negative effect
ACTH adenocorticotropic hormone, Alpha-MSH alpha-melanocyte stimulating hormone, LH luteinizing hormone, FSH follicle stimulating hormone
were increased after D2 receptor antagonist treatment
) but changed not after D2 receptor agonist treatment
(155), suggesting that α-MSH release is under maximal
inhibitory control of dopamine.
the release of progesterone and estrogens (estrone,
estradiol and estriol) in females, as well as testosterone in
males from the reproductive glands, which act as a negative
feedback on GnRH release.
Biomarkers of the HPA Axis
Although the basal mechanisms of the HPA axis are very well
understood, it remains unclear at which levels dopamine
drugs interfere. The dopamine system is active in the
hypothalamus, the pituitary gland, as well as the adrenal gland.
While α-MSH and ACTH reflect the response in the pituitary
gland upon hypothalamic stimuli, the corticosterone response
is secondary to ACTH, or elicited at the adrenal gland
directly. Therefore, the interpretation of biomarker responses
should rely simultaneous evaluation of α-MSH, ACTH and
corticosterone in a longitudinal manner to enable the
evaluation of dopamine drug effects at the different levels of the HPA
Effects on the Reproductive System
Signaling in the Reproductive System
The reproductive system also involves communication
between the brain and the periphery. It is controlled by the
neuroendocrine system through the release of gonadotropin
releasing hormone (GnRH) from the hypothalamus,
which stimulates the secretion of luteinizing hormone
(LH) and follicle stimulating hormone (FSH) in the
pituitary gland. These hormones subsequently modulate
Effects of Dopaminergic Agents on the Reproductive System
The role of the dopaminergic system in the reproductive system
is supported by a well-known side effect of D2 receptor
antagonists, i.e., sexual dysfunction (
dopamine release in the nigrostriatal, mesolimbic and medial
preoptic area plays a crucial role in mating behavior and
), providing a mechanistic basis for the
involvement of dopamine in sexual function. Other studies have
investigated the dopaminergic drug effects on the sex hormones
testosterone, progesterone and estrogen in plasma (Table II,
Fig. 2). prolactin was excluded from our analysis because of
its well-known relation with dopaminergic agents; however, it
is an important mediator of sexual function, supported by the
higher frequencies of sexual disorders observed with strong
inducers of prolactin (classical antipsychotics and risperidone)
compared to weak inducers (e.g., clozapine and olanzapine)
). The antipsychotic drug-induced disorders are at least
partially mediated via peripheral mechanisms, since the
peripherally acting D2 receptor antagonist domperidone also caused
significant changes in reproductive hormones (
The results observed for testosterone plasma concentrations
were conflicting and mainly associated with high dose levels
). Furthermore, while the D2 receptor antagonists
chlorpromazine and metoclopramide caused a reduction in
progesterone and estrogen levels (
clozapine, risperidone, and haloperidol led to enhanced
). Similarly, LH and FSH were
reduced after long-term chlorpromazine and fluphenazine
), while there was no effect observed after
long-term sulpiride, risperidone and haloperidol treatment
). After short-term haloperidol treatment, however,
increased levels of LH and FSH were observed (162).
Interestingly, the effect of short-term D2 receptor antagonist
treatment was observed in female but not in male rats (
The non-selective characteristics of the abovementioned
D2 receptor antagonists may explain these conflicting results,
particularly since the effects were associated with large dose
). Moreover, sex hormones show a high degree of
intra-individual variability and impact of treatment duration,
the latter being illustrated by the increased testosterone levels
observed after 5 days of domperidone treatment, while it was
reduced after 30 days (194). This dual effect highlights the
importance of longitudinal sampling upon dopaminergic
Finally, in addition to the effects of dopaminergic drugs on
prolactin and the sex hormones, D2 receptor agonists
enhanced oxytocin secretion, likely in a D3R-specific manner
Biomarkers of the Reproductive System
The reproductive system has multiple levels, i.e., the
hypothalamus, the pituitary and the endocrine glands, where further
understanding is required to develop an effective biomarker
strategy. The prolactin response is already difficult to
interpret. Although some studies indicated that it correlates to drug
exposure in the brain (
), another study found plasma
exposure a better predictor (196). A prolactin response has
been also observed with domperidone, which does not cross
the BBB (
). These observations suggest that the prolactin
response is a composite of central and peripheral effects.
Similarly, it is not known to which extent LH and FSH
represent a central or a peripheral effect. Oxytocin, however,
represents a biomarker for central effects only, given that the
release is solely controlled by the hypothalamus. The
testosterone and progesterone responses are secondary to LH and
FSH responses, although they may also have been elicited
through a peripheral mechanism. Overall, similar to the
HPA axis, the longitudinal evaluation of such possible
biomarkers is essential to understand the interaction between
dopamine drugs and the reproductive system.
Effects on the Insulin System
Signaling in the Insulin System
It is well known that many antipsychotics, especially atypical,
increase the risks for complicated disorders such as metabolic
syndrome and type 2 diabetes mellitus (
). Blood glucose
levels are controlled by mainly two hormones; insulin and
glucagon. Upon a rise in glucose levels, insulin is secreted from
pancreatic β-cells, leading to the glucose uptake in the muscles
and storage as glycogen in the liver. As a consequence, the
insulin secretion is reduced. When blood glucose levels fall,
glucagon is released from the pancreatic α-cells, causing
glucose release from the liver.
Effects of Dopaminergic Agents on the Insulin System
Although insulin signaling is under PNS control (
), the role
of dopamine is mainly at the periphery. It is argued that
dopamine and insulin are co-secreted from the pancreatic beta
cells, with dopamine providing a negative feedback on insulin
secretion in a D2-like receptor dependent manner (
However, both insulin and glucagon levels were not influence
by short-term D2 receptor agonist treatment (Table II, Fig. 2)
), highlighting that this mechanism does not play a major
role. In contrast, glucose concentrations were increased after
treatment with the D3 agonist 7-OH-DPAT, which was
antagonized by raclopride. Interestingly, this effect was
confirmed for quinpirole, but not for bromocriptine (
Possibly, off-target mechanisms of bromocriptine normalize
the D3 receptor mediated effect on glucose. Both glucose
and insulin levels were increased with D2 receptor antagonists
(Table II, Fig. 2). Typically, the dose required to elicit a
shortterm glucose response was higher than the one needed for a
corticosterone response (
), indicating that an off-target
effect explains this response.
The results of long-term treatment are conflicting, with in
general no effect on basal fasting glucose or insulin levels
), although for some D2 receptor antagonists
a stimulation of the insulin system has been observed
). Given the large variation in experimental
design (sex, strain, fasting protocol, dose levels), it is difficult to
identify the source of this discrepancy. Moreover, many D2
receptor antagonists were found to share the off-target affinity
for other receptors, such as serotonine, muscarinic and the
histamine receptor, all involved in weight gain which is
associated with insulin resistance and hyperglycemia (
Interestingly, the M3 muscarinic receptor was found to be
crucial in the control of insulin release (
). It is thus likely
that the short- and the long-term effects of D2 receptor
antagonists on the insulin system are mediated via other receptors
than the D2 receptor only.
Biomarkers of the Insulin System
The insulin system has been well described in terms of
biomarkers, including fasting plasma glucose, fasting serum
insulin and glycated hemoglobin. Systematic and well-controlled
studies that longitudinally evaluate these biomarkers in
combination with dopamine treatment are needed to better
understand their potential interaction.
phosphatidylethanolamine as biomarker for antipsychotic
Effects on the Lipid Metabolism
Metabolism and Signaling in the Lipid System
Phospholipid and cholesterol pathways are the main pathways
of lipid metabolism. Both pathways start with acetyl CoA, and
depending on whether the enzyme SREB-1 or SREB-2 is
present, the fatty acid or the cholesterol pathway is activated
). Fatty acids are subsequently converted to triglycerides
or phospholipids, amongst others. Cholesterol and
phospholipids are notably essential to maintain the cell membrane
). A distorted lipid metabolism can lead to the
loss of neural transmission and is involved in brain several
disorders, including schizophrenia (
misbalances in the lipid homeostasis may, for example, cause weight
gain, atherosclerosis and cardiovascular problems. In this
regard, the relation between dopaminergic drugs and the lipid
metabolism is closely related to what is observed with the
insulin system (
Effects of Dopaminergic Agents on the Lipid System
The lipid metabolism has shown to be significantly altered
after long-term treatment, while no studies were identified
for short-term treatment (Table II, Fig. 2). For instance, 2–
3 week treatment with the D2 receptor antagonists risperidone
and olanzapine caused an increase in triacylglycerols and a
decrease in free fatty acids plasma levels, which was not the
case for the partial D2 receptor agonist aripiprazole (
Another study showed that 4 weeks of treatment with
clozapine and risperidone, but not haloperidol, raised the serum
levels of total cholesterol, free fatty acids and triglycerides
via modulation of the pathway that is responsible for their
). The fact that the D2 receptor agonist
ergocryptine, although relatively unselective for this receptor,
has been reported to decrease total cholesterol and
triglycerides concentrations (
), may indicate that these effects are
mediated via D2 receptors. However, given that not all D2
receptor antagonists affect the lipid metabolism, other
receptors than the D2 receptor may be involved. Further
investigations are needed to investigate through which mechanism
dopaminergic agents affect the lipid metabolism.
Biomarkers of the Lipid Metabolism
Cholesterol, free fatty acids, triacylglycerols and
triglycerides can be used as biomarker to evaluate dopamine
treatment effect on the lipid metabolism. Additionally, a
lipidomics-based approach also revealed an increase of
RECOMMENDATIONS FOR BIOCHEMICAL
BIOMARKER STRATEGIES IN CNS DRUG
This review provides an extensive overview into the
effects of dopaminergic agents on multiple biological
pathways in the CNS and the periphery, as well as
the potential of easily accessible biomarkers to reflect
these effects. Overall, there is a strong need for
systematic searches for biomarkers that together can represent
the system-wide effects of dopaminergic agents. Here,
we provide the following recommendations to account
for system-wide effects in early CNS drug development.
Use Proteomics and Metabolomics-Based Biomarkers
Discovery for CNS Drug Effects
We envision a crucial role for proteomics and metabolomics
approach to further elucidate known and unknown pathways
and to identify drug effect-related biomarkers (
Considering the potential lack of insights into the
systemwide effects of a new compound in early drug development,
these methodologies enable preclinical anticipation of wanted
and unwanted effects (
). This information can then be used
to optimize the future dosing strategies. Also, using a targeted
metabolomics approach with monoamines in the brain, it was
shown that risperidone and clozapine are biochemically closer
to the 5-HT2A antagonist M100907 than to haloperidol
). Interestingly, this pattern highly corresponded with
behavioral outcome (116). Indeed, many of the dopaminergic
agents described in this review are non-selective.
Pharmacological effects should be seen as a balance between
multiple components of a network of affected biochemical
pathways (Fig. 4) (
). CNS drug discovery should thus aim
for rational development of non-selective drugs to attack the
polygenic CNS disorders (
). Proteomics and metabolomics
will certainly provide additional and valuable tools for the
investigation of the in vivo pharmacology (
Use Longitudinal Data and Mathematical Modeling
Mathematical modeling to understand CNS drug effects are
further needed. A pharmacological interaction at one or more
receptors will pass on to the neurotransmitter network,
causing the net result on the individual neurotransmitters, as well
as the balance between them, being not so intuitive. A
mathematical evaluation is therefore needed to understand CNS
drug effects (
). In this regard, longitudinal data on
biomarker levels is essential to calibrate these models. Indeed,
the pattern of the response reveals information that cannot be
obtained from single time point measures (
example, it was observed that not only basal levels of dopamine and
norepinephrine were decreased after long-term treatment, but
also the effect size after pharmacological stimulation (
Moreover, it is also difficult to quantify the effect by a single
time point in short-term treatments.
Evaluate CNS Drug Effects in Combination with Pharmacokinetics
This temporal pattern not only depends on the dynamic
interactions within the biological system, but also on the exposure
pattern of the drug and its possible active drug metabolites at the
site of action. It is therefore important to take into account the
pharmacokinetics when evaluating the pharmacodynamics.
Only one study considered the steady state plasma
concentrat i o n s o f c l o z a p i n e a n d i t s a c t i v e m e t a b o l i t e N
desmethylclozapine in combination with a response measure
. The levels of the drug and the metabolite showed high
variability between the animals. Moreover, the ratio between
clozapine and its metabolite was dependent on the sex of the
animal and the dose. Given the fact that the exposure of the drug
and its metabolite drives the response, such variability can have a
significant impact on the biomarker plasma levels. This is
particularly true for CNS drugs, for which the exposure pattern in
the brain is determined by a complex interaction of
pharmacokinetics, BBB transport and distribution through the brain (
Moreover, the drug exposure is likely to be brain region-specific,
which will lead to quantitative differences in drug-receptor
interactions, depending on the brain region (
). Thus, when
pharmacokinetics is taken into account, pharmacodynamics can be
compared between drugs of the same pharmacological class,
excluding the interference of pharmacokinetic differences.
Analyze Brain, Plasma and CSF Biomarkers
Plasma (or urine) samples are typically used for biomarker
identification, while CSF samples are getting more and more
interest in CNS-related diseases. Interestingly, our literature
search did not reveal pharmacological biomarker evaluations
in CSF, even though it has been used for other drug classes
) and discovery of pathological biomarkers (
plasma and CSF have the advantage to be accessible in
humans, biomarker responses in these biofluids may give a
biased view with regard to the actual effects in the brain.
Many biomarkers, for example dopamine, do not cross the
BBB. Even in the case they do (e.g., HVA) or if the biomarker
is measured in CSF, it is difficult to know how is quantitatively
relates to the effects in the brain. The current overview shows
hardly any studies that simultaneously studied biomarker
responses in brainECF and plasma. One study measured plasma
and brain cholesterol levels after long-term treatment with
clozapine or haloperidol, but no significant correlation was
). Another study could positively associate serum
progesterone levels with brain allopregnanolone as a reflection
of GABAA potentiation and anxiolytic effect after short-term
treatment with olanzapine and clozapine (
and simultaneous biomarker evaluations in plasma and brain
are recommended to provide a quantitative relation between
the central effect and the accessible biomarker response.
Investigate the Condition-Dependency of Pharmacological Effects
Dopaminergic effects are highly condition dependent. As an
illustration, dopamine receptors are present on immune cells
to reduce their activation level (
), but no effect of
dopaminergic agents was found on immune markers such as
Creactive protein, interleukin-6 or tumor-necrosis-factor alpha
). On the other hand, haloperidol was found
to have immune-modulatory and anti-inflammatory effects in
an animal disease model of rheumatoid arthritis (
D2 receptor antagonists have been shown to normalize
lipopolysaccharide-induced inflammation (
that only in an activated immune system, D2 receptor
antagonists have an effect on immune markers. Thus, while some
markers may not respond under healthy conditions, these
observations cannot directly be extrapolated to a diseased
condition. Patients or diseased animals need to be evaluated as a
population on its own.
This review highlights that dopaminergic agents, even selective
ones, have a wide array of biochemical effects. Indeed,
dopaminergic drugs may interfere with at least 8 different systems in
the brain, including dopamine signaling, norepinephrine
signaling, ACh signaling, GABA-glutamate circuits, serotonin
signaling, kynurenine metabolism, nitric oxide pathway,
endocannabinoid system, and 4 systems in the periphery, i.e.,
HPA axis, reproductive system, insulin signaling, and lipid
metabolism. Moreover, in line with earlier reviews, many
dopaminergic drugs are non-selective (
). Therefore, although
we refer to ‘dopaminergic drugs’, the biochemical actions of
these drugs may be elicited via non-dopamine receptors. A
systems pharmacology approach is expected to provide deeper
insight into the actions of dopaminergic drugs. With such
approach it will become possible to anticipate unwanted effects,
such as weight gain or sexual disorders. It is stressed that
CNS drug development lacks accessible biomarkers that
represent central effect. Hardly any studies were found
that relate the central effect to an accessible (i.e. CSF,
plasma, urine) biomarker response. Moreover, plasma
samples were mostly obtained at a single time-point,
thereby missing the insight into the longitudinal pattern
of the effect. Overall, given that other neurotransmitter
systems are similarly interconnected as the dopamine
system and also widely expressed, we highlight the need
for longitudinal system-wide biomarker evaluations to
create greater understanding of CNS and to improve
early CNS drug development.
Open Access This article is distributed under the terms of the
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permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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