Nucl. Med. Bid. Vol. 18, No. 3, pp. 357-362, 1991 ht. J. Rod&at..&I. Instrum.Part B Printed in Great Britain. All rights reserved

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PET and Plasma Pharmacokinetic Studies after Bolus Intravenous Administration of [ “C]Melatonin in Humans D. LE BARS’**, P. THIVOLLE2, P. A. VITTE’, C. BOJKOWSK14, G. CHAZOT3, J. ARENDP, R.S.J. FRACKOWIAK’ and B. CLAUSTRAT’*t ‘MRC Cycloton Unit, Hammersmith Hospital, Ducane Road, London W12 OHS, England %Zentre de Mkdecine Nuckaire, Service de Radiopharmacie et Radioanalyse, Hop&al Neurocardiologique 69394 Lyon, France, %rvice de Neurologie, Hop&al de l’Antiquaille, 69005 Lyon, France and ‘University of Surrey, Department of Clinical Chemistry, Guildford GU2 5XH, England (Received 16 November 1989; in revisedform 13 June 1990)

A human PET study was performed with carbon-11 labelled melatonin in a healthy volunteer. Plasma pharmacokinetics of melatonin and 6-sulfatoxymelatonin were simultaneously determined using radioimmunoassay. Analysis of tracer kinetics showed maximum activity in the brain 8.5min following injection, which was different from the curve observed for the plasma radioactivity (maximum at 3.5 rnin). The results continned that melatonin readily crosses the blood-brain barrier and that Csulfatoxymelatonin is the main plasma metabolite. The distribution of tracer as a function of time in this study failed to reveal any specific binding.

Introduction The pineal gland plays a central role in the circadian organization of biological rhythms in lower vertebrates and a modulatory role in mammals, including man. The main compound secreted by this gland is melatonin, which can be considered a good marker of cyclic pineal activity. Blood concentrations of the hormone, a consequence of an exclusive production by the pineal gland in humans (Kopp et al., 1980), rise- markedly during the night and show seasonal variations. This indolic hormone is a very lipophilic compound and readily crosses the blood-brain barrier (Pardridge and Mietus, 1980). Previous [ %-Ilmelatonin-binding studies on membrane preparations from whole animal brains have indicated the presence of melatonin bindingsites (Vacas and Cardinali, 1979). These results have been recently confirmed using the melatonin analogue 2-[‘251]iodo-melatonin (Laudon and Zisapel, 1986).

We report here the first, preliminary, Positron Emission Tomography (PET) experiment with melatonin labelled with carbon-11 in humans, coupled with radioimmunoassays of carrier melatonin and its major plasma metabolite, 6-sulfatoxymelatonin. This approach aims at determining precisely the brain and plasma pharmacokinetics of melatonin, a natural compound known to show a psychopharmacological activity in man (Arendt et al., 1984).

Materials sod Methods (1) Preparation of [“C]labelled melatonin for injection CHS Br + Mg + CH, MgBr + “COz + CH:’ COOMgBr(?CH:lCOCl (i) Phthaloyl dichloride (ii) Di-tbutylpyridine

Melatonin

S-Methoxytryptamine *Present address: CERMEP Cyclotron Biomedical, 59 Bd Pinel, 69003 Lyon, France. TAuthor for correspondence.

[“C]Melatonin was obtained in high specific activity by acetylation of S-methoxytryptamine with [“Clacetyl chloride; this labelling agent was prepared 357

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by the carbonation of methylmagnesium bromide with cyclotron produced carbon-11 carbon dioxide, followed by reaction with phthaloyl dichloride. The [ “C]melatonin so produced was purified by HPLC on a preparative silica column and formulated for i.v. injection in saline containing 2% ethanol. The preparation takes 31 min from the end of radionuclide production (EOB) and gives [“Clmelatonin in 25% radiochemical yield, i.e. 126 mCi from 500 mCi of radioactive carbon dioxide, with a specific activity of 700 mCi/pmol (results corrected to EOB, carrier 42pg). Chemical and radiochemical purities were checked by HPLC, TLC/autoradiography; the validity of the synthesis was further confirmed by parallel synthesis of [“C]melatonin from [ “C]carbon dioxide and examination of the product by i3C NMR spectroscopy and mass spectrometry. This synthesis is described in detail elsewhere (Le Bars et al., 1987, 1988). (2) PET and kinetic studies The subject was a 38 year old volunteer (P.T.) without disease or recent medication. At 4 p.m., the tracer was injected as a bolus via the right antecubital vein. The radial artery of the left arm was cannulated for blood sampling. Blood was collected onto heparinized tubes and centrifuged at 4°C for 20 min (2000 g). Melatonin and 6-sulfatoxymelatonin concentrations were determined on plasma samples by radioimmunoassay (Arendt ef al., 1975, 1985); crossreactivities for other indole compounds are very low ( < 2% for N-acetylserotonin, < 0.001% for other indoles). From plasma melatonin and 6-sulfatoxymelatonin kinetic data, a deconvolution (matricial method) led to the impulse response of the biological system. The radioactivity measurement from brain areas were performed using a positron emission tomograph (CT1 931/12/8); fifteen planes were simultaneously recorded, from the orbitomeatal line (plane 15) to the top of the head (plane 1). After positioning the subject centra!ly in the PET camera, transmission data were recorded using an external germanium-68 ring source. This was used to correct for tissue attenuation (Frackowiak et al., 1980). A C”O blood volume (CBV) scan was performed with a 4 min inhalation, 2 min for equilibration and 6 min scan period; after a 30 min pause for decay of residual IsO, 13 mCi of [“Clmelatonin, specific activity 77mCi/pmol with 42pg of carrier were injected intravenously. Acquisition of data was performed dynamically over 75 min; a total of 23 samples of arterial blood were taken and plasma was counted. Finally, a steady-state C”Oz study was performed over 15 min to obtain cerebral blood flow (CBF). The brain areas of interest were chosen by reference to autoradiographic and pharmacokinetic studies with [14C]melatonin in the rat (Vitte et al., 1988). The areas of high melatonin binding site density are

mainly thalamus, striatum, pineal gland, hypothalamus and colliculi, for which time-activity curves were obtained over 75 min.

Results (1) Tolerance No adverse side effects were recorded; subjective feelings appeared in the second minute after injection and were described as “transitory happiness, followed by pulses of intermittent sensations of loss of attention, diminishing over ten minutes or so”.

(2) Plasma and brain kinetics Plasma radioactivity, which is made up of [“Clmelatonin plus its radiolabelled metabolites peaked at 3.5 min [Fig. l(a)]. Immunoreactive plasma melatonin and 6-sulfatoxymelatonin peaked at 3.5 and 20 min, respectively [Fig. l(b) and (c)]. The plasma radioactivity curve was superimposable on that obtained by summation of melatonin and 6sulfatoxymelatonin molar concentrations [Fig. l(d), proving that 6-sulfatoxymelatonin is the major metabolite. Deconvolution of curve lc (plasma 6-sulfatoxymelatonin, output function) by curve 1b (plasma melatonin, input function) yielded the impulse response of the organism [Fig. 2(a)], which showed a maximum located at 8.5 min [Fig. 2(b)]. The kinetics of activity in brain structures was prolonged with respect to the kinetics of plasma radioactivity. Nevertheless all regions showed rapid ingress and wash-out with peak activity around 5-10 min after injection. In comparison, the temporal muscle gave a response similar to the plasma activity. In the pineal gland area, the activity was found to be low. The PET images of plane 8 (Fig. 3) represent the time-activity distribution in the orbito-meatal + 5.1 cm plane over the time course of the experiment, cutting through cerebellum and deep grey nuclei. Images showed maximum radioactivity at 3.5 min in the occipital area and in the sylvian and anterior cerebral areas, corresponding to CBV and CBF activities. At later times, a largely homogenous distribution was observed without accumulation in regions expected to contain specific binding-sites.

Discussion As far as were are aware, this is the first study describing Positron Emission Tomography and radioimmunoassays for a hormone and its main metabolite. From a technical point of view, the excellent superimposition of the plasma radioactivity and immunoreactivity profiles confirms that 6-sulfatoxymelatonin is the major plasma metabolite of melatonin (Jones er al., 1969) and affords a supplementary

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Fig. 3. PET images obtained from a 7 mm thick slice of the transaxial plane, passing through the region of the caudate nuclei (Plane 8, Oh4 + 5.1 cm) over the time course of the experiment; the cerebral blood flow (CBF) and cerebral blood volume (CBV) for the same plane are also illustrated.

361

PET and plasma phannacokinetic studies

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Fig. 1. Plasma activity against time (a), plasma melatonin kinetics (b) plasma 6-sulfatoxymelatonin kinetics (c); concentrations are normalized to their maximum. (d) A reconstruction of molar concentration of melatonin plus &ulfatoxymelatonin is closely superimposable upon curve a. validation of radioimmunoassays. The uniqueness of this metabolite enables an easy determination of the impulse response of the system, knowing the melatonin input function (arterial blood sampling, conlirmed a posteriori by radioimmunology) and the unique metabolite (RIA). Our results concerning plasma kinetics are similar in interpretation to previous results obtained with cold melatonin (Iguchi et al., 1982), showing a biphasic pattern for the disappearance of serum melatonin, with two half-lives (t, l/2 = 5.6 and t2 l/2 = 43.6 mm). Similarly, we observed rapid melatonin clearance

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from brain, from the eighth minute. These findings can be related to parameters governing hormonal uptake, metabolism and clearance, e.g. low plasma protein binding (Cardinali et al., 1972; Laud and Smith, 1979), presence of tissue binding sites of high affinity (Vacas and Cardinali, 1979; Laudon and Zisapel, 1986), relatively low apparent volume of distribution of less than one liter per kg (Claustrat et al., 1988). All suggest rapid diffusion and turnover of the hormone. Under physiological conditions, these phenomena are balanced by the melatonin secretion which con-

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Fig. 2. Deconvolution of the output function (plasma sulfatoxymelatonin) by the input function @lasma melatonin) [Fig. l(a)] gives the impulse response H (1) function of the system. (b) represents this activity A(t) as a function of time showing a maximum at 8.5 min.

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D. LE BARSet al.

sists of a nocturnal infusion of several hours, episodically reinforced by secretory bursts. The brain kinetics reported in Figs 2 and 3 confirm that melatonin readily crosses the blood-brain barrier at a low but significant level and has access to areas such as hypothalamus, striatum and collicuh, which are reported to have specific binding (Cardinali et al., 1979; Niles et al., 1979; Vanecek et al., 1987) or which are labelled with [14C]melatonin in ex vivo autoradiographic studies in animals (Vitte et al., 1988). Our data showing a rapid brain turnover and the existence of a sustained nocturnal secretion for melatonin are two major criteria that will contribute to the development of a sustained-release pharmaceutical form for this natural hormone. Finally, we were not able to image specific binding sites in the human brain, although they do exist (Reppert et al., 1988). This may easily be due to the physico-chemical and metabolic properties of melatonin (high lipophilicity, very short apparent halflife). The possibility exists that there is a specific binding with short off-time such that wash-out of the substance is relatively long in comparison. There is therefore little or no hold-up of the labelled hormone relative to the non-specific pool. It is also possible that the tracer was produced with insufficient specific activity at time of injection (this parameter could be increased by at least an order of magnitude in further studies). The development of agonists or antagonists for melatonin or the use of analogues such as &Auorinated melatonin (Frohn et al., 1980; Le Bars et al., 1988) which possess a longer biological half-life may be of interest for further studies. Acknowledgrment-The authors gratefully acknowledge the support of the Research Commission of the EEC for a grant to D.L.B. This work was presented in abstract form at the Satellite Symposium of the 8th International Congress of Endocrinology Melatonin and the Pineal Gland, Hong Kong, 25-27 July 1988.

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Pardridge W. H. and Mietus L. J. (1980) Transport of albumin-bound melatonin through the blood-brain barrier. J. Neurochem. 34, 1761-1763. Reppert S. M., Weaver D. R., Rivkees S. A. and Stopa E. G. (1988) Putative melatonin receptors in human biological clock. Science 242, 78-81. Vacas M. I. and Cardinali D. P. (1979) Diurnal changes in melatonin binding sites of hamster and rats brains. Correlation with neuroendocrine responsiveness to melatonin. Neurosci. Lea. 15, 259-263. Vanecek J., Pavlik A. and Illnerova H. (1987) Hypothalamic melatonin receotor site revealed by autoradiography. - _ . Bruin Rex 435,. 359-362.

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