A double-blind, randomized, placebo controlled, single oral dose, two way cross-over trial to investigate the effects of an orally administered 5-HTP challenge test on hypothalamic fMRS activation in healthy male volunteers.

Major depressive disorder is a severely disabling medical illness that is
characterized by anhedonia, depressed mood, anxiety, cognitive dysfunction,
neurovegetative disturbances, psychomotor retardation or agitation and
increased suicide risk. The depressive disorders may be conceived of as being a
dysregulation of the physiological response to stress. Investigation of altered
regulation of the hypothalamus-pituitary-adrenal (HPA) axis is therefore one of
the main research strategies in depressive disorders.

Pharmacological agents are frequently utilized in challenge tests to
investigate the HPA-axis in both healthy volunteers and depressed patients.
Such challenges could be helpful in delineating HPA-axis abnormalities
associated with psychiatric disorders and in innovative central nervous system
drug development. One of the more frequently used serotonergic challenge tests
consists of a single dose of L-5-Hydroxytryptophane (5-HTP), the immediate
precursor of serotonin (5-HT). Its decarboxylation into serotonin in raphe
neurons with hypothalamic projections induces increases in
adreno-corticotrophic hormone (ACTH), cortisol and prolactin in peripheral
blood, which are frequently used neuroendocrine endpoints of serotonergic
challenges. Recently a 100mg 5-HTP combined with CBD 100mg + 50mg
(5-HTP100/CBD100+50) challenge test was investigated and found to produce a
reliable dose-dependent release of cortisol. Its pharmacokinetics were
predictable and variability was acceptable, but its applicability was limited
due to the frequent occurrence of nausea and vomiting. Therefore, maximal
stimulation of the serotonergic system was precluded. Since suboptimal
stimulation of central serotonergic pathways may lead to minimilization of
differences between treatment effects (eg. healthy volunteers vs. patients)
this challenge was combined with an anti-emetic. 200mg 5-HTP combined with CBD
100mg + 50mg (5-HTP200/CBD100+50) challenge test was combined with 2 mg
granisetron and found to be effective in curbing its side-effects with
maintenance of a predictable serum cortisol response and pharmacokinetics.

Although this serotonergic challenge seems to be reliable and reproducible,
endpoints are mostly restricted to measurement of peripheral neuro-endocrine
effects. It is assumed that these effects ensue from stimulation of central
serotonergic nuclei, which indirectly activate CRH-production in the
hypothalamus. Using the BOLD fMRI imaging technique, the 5-HT2c agonist
m-chlorophenylpiperazine (mCPP) increased the BOLD signal in human brain areas
believed to be particularly rich in 5-HT2c receptors: medial hypothalamus,
hippocampus, PVN, caudate, pallidum, amygdala and pyriform cortex, anterior
cingulated gyrus and choriod plexus (Anderson et al 2002). In rats, mCPP
significantly increased BOLD signals in brain regions in the amygdala, dorsal
hippocampus and medial hypothalamus. These BOLD increases were blocked by
pre-treatment with the 5-HT2c receptor antagonist SB 242084.
Pharmacologically-induced, stimulus-specific functional changes may uncover
different functions of the hypothalamus, which might be helpful in identifying
functional anatomical areas involved in HPA-axis regulation. Furthermore, other
functional areas which are assumed to be involved in 5-HT-CRH activation (eg.
limbic system, periventricular nucleus, raphe nucleus) may be confirmed and
unknown areas leading to potentially unwanted effects may be identified. Other
central nervous system (CNS)-systems that do not involve the HPA-axis may also
be stimulated by serotonergic challenges. Therefore, non-invasive methods are
needed to investigate the effects of serotonergic stimulation of different
CNS-areas.

Another non-invasive method to investigate the effects of the 5-HTP on central
serotonergic pathways is functional magnetic spectroscopy (fMRS). fMRS records
protons from tissue chemicals other than water, intrinsic phosphorous
containing metabolites, sodium, potassium, carbon, nitrogen and fluorine. A
number of neuronal markers containing some of the above-mentioned elements have
been identified in the past and are often used as surrogate endpoints for
neuronal activity. These include aspartate, creatine, choline and glutamate.
fMRS is therefore an imaging technique with the potential to record human
biochemistry in either resting state or during pharmacological stimulation.
Thus, fMRS can provide basic information on the effects of a CNS-active drug.

Concentrating on the target response rather than on the physiological response
(in healthy volunteers) and eventually the pathophysiological response (in
depressed patients) may give more insight into the more proximal biological
pathways involved in CNS-functions including the HPA-axis in health and
disease.

The main objective of the current study is to detect 5-HTP induced hypothalamic
fMRS-effects and to relate these to the known neuroendocrine effects using a
reliable oral 5-HTP challenge test.

Primary Objectives:
- To investigate the effects of a single dose (plasma concentration) 5-HTP
challenge test (5-HTP200mg/CBD100+50mg/granisetron2mg) on hypothalamic fMRS
activation patterns in healthy male volunteers.

Secondary Objectives:
- To assess the feasibility of PK/PD-analyses for 5-HTP-induced hypothalamic
fMRS-activation patterns;
- To relate the hypothalamic fMRS-activation patterns associated with the 5-HTP
challenge test with plasma ACTH, cortisol and prolactin response.
- To relate the hypothalamic fMRS activation patterns, associated with the
5-HTP challenge test, with saliva cortisol response.

Double-blind, randomized, placebo controlled, single oral dose, two way
cross-over trial.

5-HTP challenge test

7.0 Tesla MRI scanner (fMRS)
At the moment the scientific community agrees that 7.0 or 8.0 Tesla MRI is safe
for human use. LUMC approved protocol: 7 Tesla MRI protocol development,
protocol no: P07.096. Some subjects may become dizzy or nauseous when
introduced quickly into the magnetic field of the fMRI scanner. This is
probably caused by interference with the balance organs of the inner ear.
Subjects will therefore be introduced slowly into the scanner to prevent these
symptoms. Dizziness and nausea caused by the MRI scanner are not dangerous and
of transitory nature.

5-HTP
Single-dose oral administration of 5-HTP can result in short-lasting nausea,
vomiting, palpitations and lightheadedness.

Carbidopa
There are no expected side effects of short-term use of carbidopa, although it
can intensify the side-effects of 5-HTP.

Granisetron
Granisetron can cause headache, constipation, diarrhoea and sedation.

Drug-drug interaction effects
No pharmacokinetic or pharmacodynamic drug-drug interaction effects for the
combinations 5-HTP; CBD; granisetron are known.

Rare adverse events during the study
As with any medication, rare side effects cannot be excluded beforehand.
Occasional reports of the following adverse events have been made:
- 5-HTP may cause dullness and depressed mood;
- Granisetron may rarely cause allergic reactions (varying from rash to
anaphylactic reactions) or transient elevation of serum transaminases.

Antidotes
Adverse events will be treated symptomatically if necessary (paracetamol for
headaches, 2mg granisetron p.o. additionally for breakthrough nausea or
vomiting). For 5-HTP and granisetron no specific antidotes exist. Furthermore a
crash-car will be positioned near the fMRI scanner for safety purposes.

Burden
- no coffee/tea/chocolate on studydays;
- no smoking on studydays;
- no alcohol 24 hours prior to and on studydays;
- no excessive physical activitity 48 hours prior to and on studydays;
- tryptophane-deficient diet on studydays, fluid intake restricted to water and
no fruit allowed;
- 6 times 10 minutes fMRS scans on each studyday.
- no brushing of teeth on the study days