University of Groningen Melatonin on-line Drijfhout ... · as the light/dark (LD) cycle. In the...

University of Groningen

Melatonin on-lineDrijfhout, Willem Jan

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Publication date:1996

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Citation for published version (APA):Drijfhout, W. J. (1996). Melatonin on-line: Development of trans pineal microdialysis and its application inpharmacological and chronobiological studies. Groningen: s.n.

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C h a p t e r 7

A new entrainment modelto test melatonin agonists

One of the important properties of new melatonin agonists, presentlyunder development as potential new drugs, is their ability to interact withthe circadian system. Well known is the entraining effect of exogenousmelatonin on circadian rhythms. Many data in this field of chronobiologyhave been gathered from behavioural experiments, because physiologicalparameters are often difficult to monitor with sufficient time resolution.Therefore, the trans pineal microdialysis technique, as described in thisthesis, may open new opportunities for such chronobiological studies. Thischapter describes the use of microdialysis in entrainment studies, and itsapplication as a test model for newly developed melatonin agonists.

The circadian rhythm of melatonin production was studied with veryhigh time resolution. To take advantage of this, three new phase-markersof the rhythm were introduced to describe the circadian profile. Time ofincrease (IT50), time of decrease (DT50) and amplitude of the rhythm werecalculated from the data of each individual experiment. The possible effectswere tested and evaluated mainly by these phase markers.

Placing animals in constant darkness (DD) conditions for 2 and 4 weeksresulted in marked phase shifts of both IT50 and DT50, which wereproportional to the duration in DD. Amplitude was not affected. Entrain-

7

7. A new entrainment model to test melatonin agonists 127

ing the animals under these free running conditions with 15 min lightpulses daily at the beginning of the subjective day (circadian time (CT) 0)for 2 weeks, entrained the IT50, while the DT50 was phase advanced by 1h. Again, amplitude was unchanged.

Daily melatonin injections (0.5 mg/kg s.c.) at the beginning of thesubjective night (CT12) for 2 and 4 weeks, entrained both IT50 and DT50.A small phase delay of DT50 compared to full entrainment indicated apartial uncoupling of IT50 and DT50. Unexpectedly, the amplitude de-creased following melatonin treatment, which was proportional to theduration of treatment. After 2 weeks melatonin treatment, injected at CT8,no entrainment of either IT50 or DT50 was achieved, but the amplitude wasdecreased, similar to melatonin treatment at CT12.

Several non-indolic melatonin agonists were tested in this entrainmentmodel by injecting animals daily at CT12, for periods of either 2 or 4 weeks.The (in)ability to entrain the circadian system could mostly be related totheir in vitro pharmacological activity. In one case however, the compound(GR196429b) did not entrain either IT50 or DT50 in doses up to 5 mg/kg.This was in contrast to its potency in several other test models, includingthe reversal of the direction of reentrainment after a phase shift. Interest-ingly, all non-indolic melatonin agonists, including GR196429b, enhancedthe amplitude, an effect that was significant in almost all cases. Thispotential to increase amplitude was not related to entraining capability.The increases were substantial, up to a factor of two. Possible mechanismsthat mediate the amplitude effects independently from the entrainingeffects are discussed.

The data presented here provide us with new information about thenature of entrainment by melatonin and include not only the time of onsetand offset of production (phase), but also the amount of melatoninproduced (amplitude). Since the present development of melatonergicagents for clinical use focuses on their role in the circadian system, effectsof these compounds on amplitude of circadian rhythms needs to beaddressed. In vivo microdialysis appeared to be a good technique for that.

The data presented in this chapter are published in the following papers:Drijfhout WJ, Homan EJ, Brons HF, Oakley NR, Skingle M, Grol CJ and Westerink BHC (1996)

Exogenous melatonin entrains rhythm and reduces amplitude of endogenous melatonin: anin vivo microdialysis study. J. Pineal Res., in press.

Drijfhout WJ, De Vries JB, Homan EJ, Brons HF, Copinga S, Gruppen G, Hagan RM, BeresfordIJM, Grol CJ and Westerink BHC (1996) Novel non-indolic melatonin agonists differentiallyentrain endogenous melatonin rhythm and increase its amplitude. Submitted.

128 7. A new entrainment model to test melatonin agonists

7.1 Introduction

Melatonin has been reported to be a regulatory factor in a variety of physiological,immunological and behavioural processes in the body. It’s production is mainly triggeredby signals originating from the suprachiasmatic nuclei (SCN). These nuclei innervate thegland through a multisynaptic pathway, finally consisting of sympathetic nerve fibersoriginating from the superior cervical ganglion. The SCN contain a pacemaker which isconsidered to be the driving force behind rhythmicity in general, including the circadianrelease of melatonin. The interaction between SCN and pineal gland is not a one-wayprocess. Melatonin is reported to have a feedback on the SCN, most clearly demonstratedby its effects on circadian rhythms.

Under normal conditions, the SCN are synchronized by environmental factors, suchas the light/dark (LD) cycle. In the absence of such synchronizing factors, for examplein constant darkness (DD), rhythms start to free-run. Once in free-running, the clockcan be synchronized by several exogenous “Zeitgebers” other than LD cycles, such asrestricted food and water availability41,224 and melatonin administration,129,272,366 a processcalled entrainment. This entraining effect of melatonin, possibly by a feedback on theSCN is an important mechanism which has led to the development of melatonin agonists.Entrainment of circadian rhythms either in constant darkness, or to new LD cycles canhave valuable clinical implications, such as improvement of complaints related to dis-turbed circadian rhythms such as jet-lag, disturbed sleep-wake cycles, shift-work syn-drome etc. Reinforcement of the circadian rhythms in such cases would most importantlyimprove the sleep/wake cycle. At present melatonergic compounds are in (clinical)development for the treatment of sleep disorders15,368 and they might become a newfamily of drugs acting on the biological clock.

The entraining effect of melatonin is only restricted to specific administration timesin the circadian cycle. Only when melatonin administration coincides with the onset ofthe subjective night, entrainment is achieved.272 For the investigation of entrainment,several approaches are reported. Many studies describe the measurement of locomotoractivity,275,373,401 body temperature26,223 or hormone production366 as a parameter of theclock activity. Although the circadian rhythms of these parameters are clear, they can bedisturbed by masking conditions such as stress, activity or feeding.186,291 Pineal metabo-lism, however, shows a remarkable robust diurnal rhythm which seems not to be affectedby such conditions. Therefore, it is becoming increasingly popular in circadian research.

Pineal metabolism can be monitored in several ways, including measurement ofN-acetyltransferase activity,134,352 6-sulphatoxy-melatonin excretion36,155 and plasmaconcentrations of melatonin,126,262 the principal hormone of the pineal. Since these areall indirect parameters of actual pineal melatonin production, information on amplitudeof the rhythms must be interpreted with caution. The microdialysis technique, as de-scribed in this thesis, may be a good alternative. It has a high time resolution, uses lownumbers of animals and yields good quantification of pineal melatonin production. Themethod allows quantification of the circadian rhythm of pineal melatonin productionwith three parameters, i.e. onset, offset and amplitude. Therefore this technique may besuitable to characterize the properties of the biological clock.


7.2 Experimental setup

Animals were used as described on page 58. In all cases, surgery as described on page 59took place one day before the measurements. From each animal a melatonin profile wasrecorded by 24 h microdialysis and on-line assay of melatonin as described on page 63.

The experiments were performed on groups of 8-10 rats. The realization of theexperiments can be divided into two periods, one in which the entrainment with lightand melatonin was investigated and one in which the entrainment model was used to testmelatonin agonists. In each period one group of rats was measured under LD conditionsand served as a control group.

ProtocolUpon arrival animals were adjusted to a new LD cycle (lights on from 01.00 h until 13.00h) for at least two weeks and were housed in groups of 4-5 rats. Then they weretransferred to individual accommodations and released into DD. The next day animalsreceived their first injection (s.c.) with either saline, melatonin or melatonin agonist.Injections took place daily around CT12 (between 1245 h and 1315 h), except for oneexperiment in which melatonin was injected around CT8 (between 0845 h and 0915 h).Duration of the treatment period was either 2 or 4 weeks. In one experiment light wasused as an entraining agent. In this case lights were turned on daily from 0100 until 0115h for a period of two weeks. At the end of the treatment period (light or injections)animals underwent surgery and microdialysis the subsequent day. In order not to letexogenously administered melatonin interfere with endogenously released melatonin,animals received no injection on the day of the microdialysis. Because the animals couldonly be operated and measured on subsequent days, treatment of rats from the samegroup was started on subsequent days, so that each individual rat had received treatmentfor exactly two or four weeks.

Drug treatmentThe following treatments were given: 1% ethanolic saline at CT12 for 2 weeks (Sal-2);saline at CT12 for 4 weeks (Sal-4); melatonin (0.5 mg/kg) at CT12 for 2 weeks (Mel-2);melatonin (0.5 mg/kg) at CT 12 for 4 weeks; melatonin (0.5 mg/kg) at CT8 for 2 weeks(8 Mel); AH-001 (10 mg/kg) at CT12 for 2 weeks; AH-017 (2 mg/kg) at CT12 for 2weeks; GG-012 (5 mg/kg) at CT12 for 2 weeks; GR196429b (0.5 mg/kg) at CT12 for4 weeks (GR 0.5); GR196429b (5 mg/kg) at CT12 for 4 weeks (GR 5). The effects of asingle dose of melatonin (0.5 mg/kg) on pineal melatonin levels during daytime wasrecorded in a separate set of experiments.

Figure 7.1 Chemical structures of the compounds tested in the entrainment model.


AH-001 (2-acetamido-8-methoxytetralin) and AH-017 (2-chlooracetamido-8-methoxytetralin) were synthesized by Dr. S. Copinga;64 GG-012 (4-methoxy-2-methylene-propionamide-indane) was synthesized by G. Gruppen. GR196429b (1-[2-(N-acetyl)aminoethyl]-7,8-dihydrofuro-[2,3-γ]-2,3-dihydroindole.HCl) was providedby Glaxo Wellcome Research Ltd, Stevenage, Hertfordshire, U.K. Chemical structuresof these compounds are provided in Fig. 7.1.

Melatonin solutions were prepared by dissolving melatonin in ethanol and dilutingwith saline, resulting in a final melatonin concentration of 0.25 mg/ml (1% ethanolicsaline). AH-001 was dissolved in equal amounts ethanol/polyethyleneglycol and dilutedwith saline to a final concentration of 5 mg/ml (20% ethanol, 20% polyethyleneglycol,60% saline). AH-017 and GG-012 were dissolved in Solutol® (BASF, Switzerland) anddiluted with saline to a final concentration of 1 and 2.5 mg/ml respectively (40% Solutol®,60% saline). GR196429b was dissolved in saline in concentrations of 0.288 and 2.88mg/ml, resulting in free base concentrations of 0.25 and 2.5 mg/ml respectively. Animalsreceived 2 ml injection fluid per kg body weight. Generally solutions were prepared oncein sufficient amount for the experiments. Solutions of GR196429b were prepared freshlyevery 3-4 days. All solutions were kept refrigerated (4 °C).

Data analysisStart of the experiment was synchronized between individual animals, resulting in equalcircadian time points of sampling. This allowed data on the melatonin production fromdifferent animals to be averaged. Resulting profiles are presented in the figures. Fromeach individual experiment the three phase markers IT50, DT50 and amplitude werecalculated, as explained on page 70. These data were subject to statistical analysis.

Figure 7.2 Melatonin profile in a normal 12/12 LD cycle. This curve is indicated ascontrol curve in the first set of data. Melatonin is expressed as percentage of averagedaytime levels and presented as mean ± S.E.M. (n = 6).


7.3 Characterization of entrainment

Table 7.1, page 146 summarizes the numerical values of the three phase markers (IT50,DT50 and amplitude) of all experiments. A graphical representation of the first set of datais provided by a set of bar graphs (Fig. 7.10, page 137). When the treatment did not fullyentrain, data from the few animals that entrained on the injection procedure were notincluded in the calculations of the phase markers. Sometimes the number of experimentsincluded in the IT50 and DT50 data is larger than the number of experiments included inthe presentation of the melatonin profile. In those cases few animals showed undetectablebasal melatonin production. In all cases, data are expressed as percentage of average basalmelatonin production. Therefore undetectable basal levels resulted in omission from thegraphic and amplitude calculations. Calculation of IT50 and DT50 however was stillpossible. In table 7.2, page 147 the number of entrained and not entrained animals inall experimental groups of this chapter is indicated.

Circadian profile of melatonin releaseIn Fig. 7.2, the diurnal release of melatonin is shown in a 12/12 LD cycle. Melatoninlevels started to increase approximately 2 h after the onset of darkness, resulting in anIT50 of 2.9 ± 0.5 h. Before the lights were turned on, levels declined, resulting in a DT50of -1.0 ± 0.2 h. Slopes were different for both phase markers. While the increaseextended over a period of about 3 h, the decline was completed in about 30 minutes.After reaching the maximal production, a plateau level of melatonin remained ratherconstant throughout the night. The amplitude measured was 1460 ± 57 %.

Free-runningSaline treatment for 2 and 4 weeks resulted in significant phase shifts that were propor-tional to the duration of treatment. After 2 weeks treatment (Fig. 7.3) the phase delaywas approximately 2.5 h (IT50 = 5.6 ± 0.5 h; DT50 = 3.2 ± 0.5 h). Four weeks salinetreatment (Fig. 7.4) resulted in a phase delay of approximately 5 h (IT50 = 8.0 ± 0.4 h;DT50 = 5.2 ± 0.2 h). The amplitudes after 2 weeks saline (1446 ± 31 %) and after 4weeks saline (1338 ± 54 %) were not significantly different from control conditions. Inboth groups, a small number of animals were entrained to the injection procedure. Oneanimal in the 2 weeks saline group was extremely phase delayed up to about 8 h (IT50 =11.1 h; DT50 = 7.3 h).

Entrainment by melatoninA single dose of melatonin (0.5 mg/kg s.c.) resulted in an increase of the daytime levelsin the dialysates to 2627 ± 311 % (Fig. 7.5). Clearance of melatonin was fast and within2 h less than 10% of the maximum levels was left. Because this increase was in the sameorder as day/night differences, the dose of melatonin used can be considered as aphysiological dose at the level of the pineal. The blood concentrations were not measured,but were presumably in the pharmacological range, as is generally agreed when using thisdose.

Animals injected with melatonin for 2 (Fig. 7.6) and 4 weeks (Fig. 7.7) at CT12 wereall entrained and IT50 values did not significantly deviate from control conditions (2.6 ±0.2 h and 3.3 ± 0.3 h respectively). However, DT50 values increased with the duration


Figure 7.3 The effect of saline on free running conditions. Saline was injected daily atCT12 (t = 0 h) for a period of 2 weeks. The dotted curve represents the control curveunder LD conditions as presented on page 131. Data are presented as the mean ± S.E.M.(n = 5).

Figure 7.4 The effect of saline on free running conditions. Saline was injected daily atCT12 (t = 0 h) for a period of 4 weeks. The dotted curve represents the control curveunder LD conditions as presented on page 131. Data are presented as the mean ± S.E.M.(n = 5).


of melatonin treatment. After 2 weeks melatonin, the DT50 value was slightly increasedto -0.2 ± 0.3 h, while the DT50 value was 1.2 ± 0.1 h in the group that received 4 weeksmelatonin, which was a significant phase delay. Melatonin injected at CT8 was not ableto entrain the circadian rhythm (Fig. 7.8). Both IT50 (6.7 ± 0.8 h) and DT50 (2.7 ± 0.3h) were significantly increased compared to control. The amplitude in all melatonintreated animals was decreased, an affect that appeared to be proportional to the durationof treatment. After 2 weeks treatment the amplitude was decreased to 1269 ± 62 %,whereas 4 weeks treatment resulted in an amplitude of 1018 ± 42 %. The decrease didnot seem to be dependent on the time of injection, because also the group that receivedmelatonin at CT8 showed a significantly reduced amplitude (1094 ± 48 %)

Entrainment by lightIn Fig. 7.9 the results are shown of 2 weeks entrainment by daily light pulses of 15 minat CT0. All animals entrained on this treatment. The IT50 value (2.8 ± 0.2 h) was notdifferent from controls, but the DT50 value was significantly phase advanced by morethan 1 h (-2.3 ± 0.2 h). The amplitude (1302 ± 62 %) was not affected by the treatmentand did not show significant differences from control.

Figure 7.5 The effect of exogenous melatonin on daytime melatonin levels. Melatoninwas injected subcutaneously in a dose of 0.5 mg/kg at t = 0 h. Melatonin levels areexpressed both as percentage of average daytime level (left axis) and as absolute outputin fmol/sample (right axis). Data are presented as the mean ± S.E.M. (n = 8).


Figure 7.6 The effect of melatonin on free running conditions. Melatonin was injecteddaily at CT12 (t = 0 h) for a period of 2 weeks in a dose of 0.5 mg/kg (s.c.). The dottedcurve represents the control curve under LD conditions as presented on page 131. Dataare presented as the mean ± S.E.M. (n = 5).




Figure 7.9 The effect of light on free running conditions. Light was applied in pulses of15 min, daily at CT0 (t = 12 h) for a period of 2 weeks. The dotted curve represents thecontrol curve under LD conditions as presented on page 131. Data are presented as themean ± S.E.M. (n = 5).


Ctrl Sal-2 Sal-4 Mel-2 Mel-4 8 Mel Light

Figure 7.10 Graphical representation of IT50 (upper panel), DT50 (middle panel) andamplitude (lower panel) data from the first set of experiments. The columns representcontrol conditions (Ctrl.) and entrainment with saline for 2 (Sal-2) and 4 (Sal-4) weeks,melatonin for 2 (Mel-2) and 4 (Mel-4) weeks, melatonin at CT8 for 2 weeks (8 Mel) andlight at CT0 for 2 weeks (Light). Asterisks (*) indicate statistical significance (p < 0.05).


7.4 Agonists tested in entrainment model

As was the case with saline treated animals, in all experiments where the agonists wereineffective in entraining the melatonin profile, some animals entrained to the injectionprocedure. Mostly this percentage was in the range of 20-30%. In contrast, when a certaintreatment did entrain the melatonin profile, no single animal ever showed a phase shift.This leads to the conclusion that, whenever an animal in a certain experimental group isnot entraining to the treatment, chances are that the compound is not an entrainingagent. When the percentage of entrained animals drops below 50%, one could concludewith certainty that the treatment is ineffective in entraining the melatonin profile. Intable 7.2, page 147 the number of entrained and not entrained animals in all experimentalgroups of this chapter is indicated.

Absolute daytime values in all experimental groups were not significantly different.Therefore amplitude changes of relative amounts are not caused by changes in absolutebasal daytime output, but by increased night-time melatonin production.

Because of the repeated injections each day and the sometimes high concentrationsof organic solvents in the vehicle, sometimes irritation of the skin was noticed. Especiallyin the vehicle consisting of ethanol and polyethyleneglycol (AH-001), some necrosis ofthe skin occurred on the site of injection. Similarly, a rather high concentrations ofSolutol® (AH-017 and GG-012), caused an oedema on the site of injection. However,

Figure 7.11 Melatonin profile in a normal 12/12 LD cycle. This curve is indicated ascontrol curve in the second set of data. Melatonin is expressed as percentage of averagedaytime levels and presented as mean ± S.E.M. (n = 7).


these effects did not seem to affect the behaviour of the rat to a great extent and apparentlydid not influence the entraining process.

In table 7.3, page 149, an overview is presented on the in vitro pharmacological dataof the melatonin agonists tested in this chapter. Table 7.1, page 146 summarizes thenumerical values of the three phase markers (IT50, DT50 and amplitude) of the entrain-ment experiments with the melatonin agonists. A graphical representation of the data isprovided by a set of bar graphs (Fig. 7.18, page 144).

Control curvesIn this set of experiments, two control curves were included, the melatonin profile undernormal LD conditions, and entrainment in DD by 4 weeks of melatonin treatment. InFig. 7.11, the control curve in a normal LD cycle is presented. The IT50 (3.2 ± 0.1 h)and DT50 (-1.2 ± 0.1 h) appeared to be similar to the phase markers of the control curvein the previous set of experiments (page 131). Also the amplitude (1563 ± 85 %) wasexactly in the same range and in a qualitative way, the profile overlapped the one fromthe previous set, with a rather constant high plateau level.

Melatonin treatment (0.5 mg/kg) for 4 weeks in DD (Fig. 7.12) entrained the mela-tonin rhythm in a similar way as in the previous set of experiments. Both IT50 (2.4 ± 0.2h) and DT50 (0.5 ± 0.2 h) were in the same range as reported earlier and again, the DT50was phase delayed significantly, while the IT50 was entrained. The significant decrease inamplitude (1090 ± 40 %) appeared to be reproducible as well.

Figure 7.12 The effect of melatonin on free running conditions. Melatonin was injecteddaily at CT12 (t = 0 h) for a period of 4 weeks in a dose of 0.5 mg/kg (s.c.). Thisexperiment is intended as a control experiment. Data from a similar experiment arepresented on page 135. The dotted curve represents the control curve under LDconditions as presented on page 138. Data are presented as the mean ± S.E.M. (n = 7).


Figure 7.13 The effect of AH-001 on free running conditions. AH-001 was injecteddaily at CT12 (t = 0 h) for a period of 2 weeks in a dose of 10 mg/kg (s.c.). The dottedcurve represents the control curve under LD conditions as presented on page 138. Dataare presented as the mean ± S.E.M. (n = 5).

Figure 7.14 The effect of AH-017 on free running conditions. AH-017 was injecteddaily at CT12 (t = 0 h) for a period of 2 weeks in a dose of 2 mg/kg (s.c.). The dottedcurve represents the control curve under LD conditions as presented on page 138. Dataare presented as the mean ± S.E.M. (n = 6).


AH-001The amidotetralin analogue of melatonin, AH-001, appeared not to entrain the mela-tonin rhythm in a dose of 10 mg/kg (Fig. 7.13). In a large number of animals, themelatonin profile was clearly phase delayed, resulting in an IT50 of 5.1 ± 0.4 h and aDT50 of 2.5 ± 0.3 h. An unexpected finding in this group was a tendency of the amplitudeto be somewhat increased (1811 ± 70 %). Although this effect did not reach the level ofsignificance, the finding was nevertheless intriguing, especially because melatonin itselfcaused a reduction in amplitude.

AH-017The most potent analogue from the amidotetralin-based series of melatonin agonists waseffective in entraining the endogenous melatonin profile in DD (Fig. 7.14). Both IT50(2.7 ± 0.3 h) and DT50 (-0.4 ± 0.2 h) were similar to control LD conditions. The small,not significant phase delay of DT50 compared to control LD conditions was similar tothe melatonin induced entrainment. As might be suggested from the data with AH-001,the amplitude of the rhythm was increased (2471 ± 76 %), a highly significant effectfollowing AH-017 treatment.

GG-012A lead structure of the amidoindane-based series of melatonin agonists did not entrainthe endogenous melatonin profile in a concentration of 5 mg/kg (Fig. 7.15). A significantphase delay was obtained after 2 weeks daily dosing, resulting in an IT50 of 8.0 ± 1.1 h

Figure 7.15 The effect of GG-012 on free running conditions. GG-012 was injecteddaily at CT12 (t = 0 h) for a period of 2 weeks in a dose of 5 mg/kg (s.c.). The dottedcurve represents the control curve under LD conditions as presented on page 138. Dataare presented as the mean ± S.E.M. (n = 7).


and a DT50 of 4.9 ± 1.0 h. Remarkably, this shift was larger than was seen in saline treatedanimals, or in AH-001 treated animals. Again, the amplitude was significantly increased,actually the increase was the largest from all melatonin agonists investigated (3089 ± 99%).

GR196429bThe selective full agonist GR196429b was tested for 4 weeks in concentrations of 0.5mg/kg (Fig. 7.16) and 5 mg/kg (Fig. 7.17) In both concentrations no full entrainment ofthe melatonin profile occurred. In the lower dose, the phase shift in IT50 (8.5 ± 0.8 h)and DT50 (6.0 ± 0.9 h) were significantly different from control and in the same rangeas saline treatment for 4 weeks. Also in the higher dose, the phase shift was significant,but IT50 (13.4 ± 0.9 h) and DT50 (8.9 ± 1.1 h) were approximately 3-5 h larger than inthe lower dose. Apparently the higher dose resulted in a larger phase delay of themelatonin profile. In both doses, a small percentage of the animals entrained to theinjection procedure. In both doses, the curves look a little sloppy, which was caused byaveraging individual curves that were shifted to a different extent. In the higher dose thiseffect is enhanced by the relatively low number of successful experiments.

Amplitude was significantly increased in both dosages. The increase was even larger inthe low dose (2868 ± 66 %) than it was in the high dose (2367 ± 66 %), indicating thatthe amplitude effect was independent from entraining properties.

Figure 7.16 The effect of GR196429b on free running conditions. GR196429b wasinjected daily at CT12 (t = 0 h) for a period of 4 weeks in a dose of 0.5 mg/kg (s.c.). Thedotted curve represents the control curve under LD conditions as presented on page 138.Data are presented as the mean ± S.E.M. (n = 6).


7.5 Discussion

Circadian profile of melatonin productionThe present data clearly show the pronounced diurnal rhythm of melatonin secretion inrat pineal glands. The high time resolution in fact, inherent to microdialysis studies,reveals detailed information about the phase markers of the rhythm. The rise in melatoninis relatively slow. It is associated with activation and induction of N-acetyltransferase,followed by increase in melatonin biosynthesis and subsequent release. The decline, onthe contrary, is a fast process. It’s completion about 1 h before the onset of light indicatesthe loss of an endogenous stimulation, after which the half-life of melatonin determinesthe slope of the curve. This half-life is in rats reported to be about 20 min,6,339 sufficientto explain the rapid decrease. The amplitude of about 15-fold increase during the darkperiod is consistent with data reported on pineal total tissue contents and plasmaconcentrations.

Characteristics of the new entrainment modelIn most entrainment studies concerned with locomotor activity, drinking behaviour etc.,free-running circumstances are established before experiments in DD are carried out.Generally it takes several days before the rhythms begin to free-run. In these situationsanimals are on-line connected to the recording system and measurements can continuefor weeks. In our setup, there is no continuous monitoring of the rhythmicity and animalsare only examined for one day during the complete experiment. In order to be sure that

Figure 7.17 The effect of GR196429b on free running conditions. GR196429b wasinjected daily at CT12 (t = 0 h) for a period of 4 weeks in a dose of 5 mg/kg (s.c.). Thedotted curve represents the control curve under LD conditions as presented on page 138.Data are presented as the mean ± S.E.M. (n = 3).


Figure 7.18 Graphical representation of IT50 (upper panel), DT50 (middle panel) andamplitude (lower panel) data from the second set of experiments. The columns representcontrol conditions (Ctrl) and entrainment with melatonin for 4 weeks (Mel-4), AH-001,AH-017 and GG-012 for 2 weeks and GR196429b for 4 weeks in doses of 0.5 mg/kg(GR 0.5) and 5 mg/kg (GR 5). Asterisks (*) indicate statistical significance (p < 0.05).


injections are given at the correct CT, injections have to start as soon as animals arereleased into DD. This will enhance the sensitivity of entrainment to all kinds of external“Zeitgebers” such as injections. It is in this respect that entrainment in 20-30 % of theanimals after treatment with non-entraining agents has to be explained. The relativeconstant percentage of entrained animals in non-entraining treatments indicates that thiseffect is highly reproducible. The marked sphase shifts of the other animals, shows thatthis method is capable of inducing free-running circumstances.

It is well recognized that melatonin is capable of entraining its own release, as long asit is administered at the onset of the subjective night.129,272 Clearly our data as shown inFig. 7.6 and Fig. 7.7 are in support of this. In most cases less attention is paid to theother phase marker, the decline in melatonin. The present study describes in entrainedanimals a phase delay of the offset, increasing with the duration of the experiment, whilethe onset is entrained. Entrainment with light shows a similar but opposite effect. Lightis known to phase advance circadian rhythms when applied at the end of the subjectivenight.39,150,222 In the present experiments, such light treatment resulted in entrainmentof the onset of melatonin production and a phase advance in the offset (Fig. 7.9). This“uncoupling” of onset and offset of the melatonin rhythm supports the theory of acomplex two-component pacemaker system controlling the pineal melatonin produc-tion. For an extensive description of such a two-oscillator pacemaker, see chapter 8. Inshort, the theory260 was based on the occurrence of “splitting” of free-running activityrhythms of rodents into two distinct components. Two mutually interacting oscillatorswere suggested with different intrinsic periods: an evening component (E) controllingthe onset of activity and a morning component (M) controlling onset of the quiescentperiod. Illnerova and Vanecek135 extended this model to the rhythm in pineal N-acetyl-transferase activity, which appeared to be differential affected by lightly affected by lightpulses applied at different circadian times in constant darkness. The phase-relationbetween E and M, ΨEM, is measured as the time distance between the phase-referencefor E (IT50) and M (DT50). Under conditions of constant darkness, ΨEM is uncompressed,resulting in less interaction between E and M. This makes it possible that phase delaysand advances in DT50 are recorded when IT50 is entrained.

The entraining effect of melatonin is only effective at one specific time, i.e. whenadministration of melatonin coincides with the onset of activity. When looking at a singledose injection of melatonin (Fig. 7.5), it appeared that within two hours after injection,melatonin has returned approximately to basal levels. Taking these facts together, it isnot surprising that melatonin, injected at CT8 was not effective as an entraining agent(Fig. 7.8). If melatonergic agents are to be developed as entraining drugs in order torestore sleeping patterns, effectiveness of such a drug could be greatly enhanced com-pared to melatonin if it entrained not only at CT12, but also at CT8.

Entrainment with melatonin agonistsWhile melatonin is effective as an entraining agent in relatively low doses, entrainmentwith synthetic melatonin agonists seems to be more difficult. AH-001 has a low relativepotency compared to melatonin. Therefore, a much higher dose was used in the experi-ments. However, the poor solubility of the compound limited the maximal dose to be10 mg/kg. The low solubility may also have resulted in precipitation of the compound


the moment it was injected into the tissue. If so, it would have been taken up in thecirculation over a long period of time, following a release pattern similar to slow releasepreparations. Because the window of entrainment sensitivity in terms of the circadiantime point of injection is very narrow, it seems unlikely that a compound that is releasedin such a way will be effective as an entraining agent. Taken together, the dose may havebeen too low to be effective and thus caused the failing of this compound to entrain. Asimilar reason may be applicable to the failure of GG-012 to entrain melatonin levels.Considering GG-012 an alternative explanation could be that although its concentrationused was better corrected for the lower potency, from in vitro pharmacological data itappeared that this compound may be a partial agonist instead of a full agonist.

In the case of AH-017, the situation was quite different. Although its potency is about25% of that of melatonin, the dose used was fourfold higher than melatonin, resultingin a complete compensation for the lower potency. Its effectiveness in entraining themelatonin profile may therefore not be surprising. One of the main problems withAH-017 might be the fact that it contains a chloride atom in the acetyl-group. Generally,it is assumed that activated halogens in the side chain increase the chance of irreversiblebinding to the receptor and can be mutagenic. The potential irreversible binding ofN-bromoacetyl 5-methoxytryptamine to the melatonin receptor was investigated.57 Noindications of such a mechanism were obtained. Although this does not exclude potentialirreversible binding of AH-017, it may at least be less likely. Nevertheless, one mightprobably be inclined not to initiate further clinical investigations, unless this subject iscompletely cleared.

The compound GR196429b was developed as a highly potent melatonin agonist. Inthe in vitro pharmacology, the compound was as potent as melatonin in inhibitingdopamine release from rabbit retina. Therefore, initially the compound was tested in a

IT50 DT50 Amplitude

h ± S.E.M. h ± S.E.M. h ± S.E.M.Ctrl 1 2.9 0.5 -1.0 0.2 1460 57

Sal-2 5.6* 0.5 3.2* 0.5 1446 31

Sal-4 8.0* 0.4 5.2* 0.2 1338 54

Mel-2 2.6 0.2 -0.2 0.3 1269* 62

Mel-4 3.3 0.3 1.2* 0.1 1018* 42

8-Mel 6.7* 0.8 2.7* 0.3 1094* 48

Light 2.8 0.2 -2.3* 0.2 1302 62

Ctrl 2 3.2 0.1 -1.2 0.1 1563 85

Mel-4 (2) 2.4 0.2 0.5 0.2 1090* 40

AH-001 5.1* 0.4 2.5* 0.3 1811 70

AH-017 2.7 0.3 -0.4 0.2 2471* 76

GG-012 8.0* 1.1 4.9* 1.0 3089* 99

GR 0.5 8.5* 0.8 6.0* 0.9 2868* 66

GR 5 13.4* 0.9 8.9* 1.1 2367* 66

Table 7.1 Numerical values of the three phase markers (IT50, DT50 and amplitude) of allexperiments from this chapter. Asterisks (*) indicate statistical significance (p < 0.05).


dose of 0.5 mg/kg. When the drug did not entrain in this dose, a higher dose was used,but lack of entrainment in this higher dose makes it seem unlikely that the dosing wasthe problem. Most remarkably, the compound appeared to be very effective in thephase-shift paradigm (experiments performed at Glaxo Wellcome Research Ltd.). Oneof the properties of melatonin, often used in the characterization of melatonin agonists,is its ability to reverse the direction of reentrainment after an 8h phase advance shift.275,274

Normally animals respond by a phase delay to the new LD cycle. When melatonin isadministered daily at the new dark onset, animals respond by a phase advance to the newsituation, instead of a phase delay. From other experiments it appeared that in a dose of5 mg/kg five out of six animals phase advanced to the new LD cycle, whereas with vehicleinjections all six animals phase delayed. At first glance these data seem to be in contra-diction. However, there are important differences in the models used.

In the phase-shift paradigm, animals remain in LD conditions. Because light is animportant external “Zeitgeber”, it may have an additional effect to the effect of thecompound itself. Because the entrainment experiments are carried out in DD, such anadditional effect is lacking, which may result in poor entrainment. Another difference isthe duration of the experiment. Generally, in the phase-shift experiments the compoundis dosed for several days, after which full reentrainment has occurred. In the presentexperiments, a chronic dosing was applied for four weeks. During such long periods,additional effects may be expected, such as desensitization of the system. Another effectcould be the plasma concentrations to reach steady-state levels, a process highly depend-ent on specific pharmacokinetic parameters. Also differences in rat strains used in thetwo laboratories could account for differences in the outcome of experiments. Finally,the basic principle of the two test systems is different. In the phase-shift paradigm,reentrainment is a fact, primarily because the animals remain in LD. Only its directionis subject of the study. Such an effect may be achieved shortly after the phase-shift, maybeeven during the first day. In the entrainment studies, entrainment is not a fact, but mustbe imposed by the agonist for the duration of the experiment. When the compound

Total # # Entrained # not Entrained % Entrained

Ctrl 1 6 - - -

Sal-2 8 2 6 25

Sal-4 8 2 6 25

Mel-2 6 6 0 100

Mel-4 4 4 0 100

8-Mel 5 0 5 0

Light 5 5 0 100

Ctrl 2 6 - - -

Mel-4 (2) 8 8 0 100

AH-001 7 2 5 28

AH-017 6 6 0 100

GG-012 8 1 7 12

GR 0.5 10 3 7 30

GR 5 7 3 4 43

Table 7.2 Overview of entrained and not entrained animals in each experimental group.


would have an effect for only a couple of days, possibly by desensitization of the system,this would imply ineffectiveness of the compound in the test system.

Although GR196429b did not entrain the melatonin profile, the higher dose resultedin a much larger phase shift than the lower dose, suggesting that the compound indeedinteracted with the circadian system. That this interaction did not result in entrainment,could possibly be caused by an increase in the tau (τ) period. A similar effect was seenwith GG-012, which resulted in a phase shift in two weeks that was similar to the phaseshift after saline treatment in four weeks.

Clearly, the entrainment model described here provides us with interesting informa-tion on the entraining properties of melatonin agonists. The predictable effects ofmelatonin itself appear to be reproducible, and also melatonin agonists can be effectiveas entraining agents. The highly reproducible and low number of false positive resultsincreases the power of the test, especially when compared to similar test systems whichuse behavioural parameters such as locomotor activity as indicators of circadian rhythms.The sometimes poor correlation between the present data and in vitro pharmacologicaldata indicates that in vitro pharmacology must not be the only screening test whendeveloping new ligands. Generally additional experiments using in vivo models providecrucial information.

Amplitude variationsData on changes in amplitude of circadian rhythms are scarce. Locomotor activityprovides only poor information about amplitude. N-acetyltransferase activity is relatedto pineal melatonin production, but a quantitative correlation has not been proved yetand has even been called into question.74 Plasma concentrations and excreted 6-sulpha-toxy-melatonin show substantial variability and the possibility of interfering extra-pineal,not rhythmic, production of melatonin is still a matter of debate.423 Based on the datapresented here, clear changes in amplitude can be monitored. All melatonin treatedanimals show a decrease in amplitude, while melatonin agonists generally increase theamplitude.

These amplitude effects do not seem to be limited to one specific circadian time point,because also melatonin injected at CT8 showed a significant decrease in amplitude.Although the agonists were not tested at other circadian time points, similar data may beobtained when not injected at CT12.

When considering the effects on amplitude, two important questions arise. The firstone is about the mechanism behind the amplitude changes. The second one is thedifference between melatonin on one hand and merely all melatonin agonists on the otherhand. All agonists show an increase in amplitude, varying from almost significant, to highlysignificant, while melatonin reduced amplitude, an effect that appeared to be highlyreproducible. Generally, less specific endogenous compounds elicit a wider variety ofeffects than more specific agonists. In this respect it could be speculated that melatoninexerts both an inhibitory and excitatory effect, from which the inhibitory dominates,whereas in the case of the agonists the inhibitory effect is either not present, or at leastless pronounced, resulting in increased amplitudes. In this section, both the possiblemechanisms behind amplitude regulation and the differences between agonists andmelatonin will be discussed.


Melatonin is suggested to entrain rhythms by a direct action on the SCN. One of theexplanations for its amplitude effects could be a negative effect on amplitude of SCNrhythmicity, apart from an entraining effect on its phase. In this case, the differencesbetween melatonin and the agonists could be based on differences in specificity forreceptor subtypes. The existence of various subtypes of the melatonin receptor has notbeen proven yet, but there are clear indications that they exist.88 Existence of bothsubtypes in the SCN, although not impossible, has never been suggested. A more seriousproblem with direct action on the SCN arises from the nature of signal transductionbetween SCN and pineal gland. When the noradrenaline profile is monitored in thepineal gland, this has an on/off character.82 It was proposed that a relay station existsbetween SCN and pineal, which would pass through the signals from the SCN when acertain threshold level was reached. In such a system, it would be difficult to propose amechanism that would cause changes in amplitude, other than at the level of, or down-stream from, that relay station. These arguments make an amplitude effect caused by adirect interaction with SCN rhythmicity unlikely.

When the amplitude effects are receptor mediated, a different interaction site is muchmore likely. In the pituitary, especially the pars tuberalis, a high receptor density isreported in almost all mammals.337 Because this area plays a major role in the endocrinesystem, interactions at this site could result in manipulation of plasma levels of a varietyof hormones. Such a regulatory role in the endocrine system has been described repeat-edly, including the stimulation of growth hormone production,332,382 the suppression offollicle stimulating hormone (FSH) and luteinizing hormone (LH),391,422 the inhibitionof vasopressin and oxytocin425 and a delay of the seasonal rise in prolactin.152,302 To explainthe effects on the amplitude, not only an effect on plasma levels of various hormones isnecessary, also an effect of these hormones on pineal metabolism is a prerequisite. Verylittle is known on this subject, but recently a gonadotropin-releasing hormone im-munoreactive protein has been characterized in rat pineal gland.245 Interactions of theendocrine system with pineal melatonin production may be very interesting and needfurther research.

Melatonin also plays an important role in the immune system.203 It is known thatmelatonin receptors are present on blood lymphocytes.198,204 Also, interferon-γ canstimulate pineal melatonin production.418,419 Because interferon-γ is produced by T-helper lymphocytes, this may be an interesting neuroimmunological loop that could be

Ki (nM)(chicken retina)

IC50 (nM)(rabbit retina)

relativepotency

(partial)agonist

Melatonin 0.57 0.017 1.0 agonist

AH-001 46.3 1.4 82 agonist

AH-017 3.75 0.063 3.7 agonist

GG-012 9.5 0.48 28 partial agonist

GR196429b 0.72 0.04 2.4 agonist

Table 7.3 Data from in vitro pharmacology studies of the melatonin agonists tested.


involved in regulating the amplitude of melatonin production. Again, the differencesbetween melatonin and its agonists are difficult to explain.

Perhaps the amplitude effects are not directly receptor mediated, or at least notmelatonin receptor mediated. Intrapineal effects could explain the results found. Directeffects on the enzymes involved in the biosynthesis of melatonin, such as N-acetyltrans-ferase, or hydroxy-indole-O-methyltransferase (HIOMT) may occur. From the chroma-tograms obtained during the studies with GR196429b, also the amount ofN-acetylserotonin could be estimated. The resulting ratio between N-acetylserotonin andmelatonin could give an indication on the activity of HIOMT. However, no differencesin this ratio were noticed between melatonin and GR196429b treated animals. There-fore, if an intrapineal effect of melatonin would exist, N-acetyltransferase seems to be amore likely candidate as the target site.

Affinity for other receptors could also result in differences in effect between melatoninand melatonin agonists. From the 8-methoxy-2-amidotetralin series of compounds, it isknown that some have α-adrenergic receptor affinity. Because α2-adrenergic receptorsplay a regulatory role in melatonin production, affinity for these receptors could influencethe amount of melatonin produced. This regulatory role for α2-adrenergic receptoragonists only occurs when the melatonin production is activated by β-adrenergic stimu-lation. At CT12, when the compounds were injected, the release of noradrenaline is stillvery low, so the compound should stay in the circulation for at least several hours. Thisrequirement and the improbability that all agonists have α2-adrenergic affinity make thisexplanation less likely.

In humans, the amplitude of melatonin production was reported to increase followingtreatment with fluvoxamine, a serotonin re-uptake inhibitor.329 Such a re-uptake inhibi-tor enlarges the serotonin availability, a situation which could result in enhanced mela-tonin production. Based on structural similarities between serotonin and melatonin, aneffect on the re-uptake mechanism for serotonin should not be excluded. However, therole of serotonin availability in melatonin production is still a matter of debate.

Monoamine oxidase (MAO)-A inhibitors are reported to have a stimulatory effect onmelatonin production in rats.240,289 Although most of these experiments have beenperformed under LD, generally also a phase-advance was reported. The lack of anyphase-advancing effect of the agonists makes it unlikely that they have MAO-A inhibitoryproperties.

In all explanations which involve receptor mediated processes, or interactions withenzyme systems, it is difficult to understand that all agonists react differently frommelatonin. Especially this observation may lead to explanations that are based on specialproperties of melatonin, instead of the agonists. One item is the metabolism of melatonin,which is probably different from the metabolism of melatonin agonists. What if melatonininjections result in the introduction of an active metabolite? The well known metabolitesof melatonin are 6-hydroxy-melatonin and N-acetyl-5-methoxy-kynurenamine, but alsothe production of N-acetylserotonin as a metabolite of exogenously administered mela-tonin is described.179 Because N-acetylserotonin is an endogenous compound, which isreleased from the pineal similar to melatonin, target sites of N-acetylserotonin may beexpected. Administration of melatonin would activate such target sites, which are notactivated by melatonin agonists. Also, the speed of metabolism may be crucial. Melatonin


has a short half-life, where probably the half-lives of the agonists are at least larger. Thiswould result in different pharmacokinetic parameters of the agonists, resulting in adifferent concentration profile throughout the circadian cycle.

Because many possible mechanisms could also result in acute effects, in a pilotexperiment GR196429b was injected in a concentration of 0.5 mg/kg at CT18. Nochanges in melatonin production were measured, indicating that amplitude effects of themelatonin agonists only occur after chronic dosing. Because of this, many explanationsmentioned above remain possible, but are probably more difficult to prove.

Therapeutical implicationsDespite the difficulties in explaining the amplitude effects of the melatonin agonists, itis a feature that may well be interesting from a therapeutical point of view. Many clinicaldevelopments are directed towards the use of these agonists in elderly people withsleeping problems. In elderly people it is known that the endogenous production ofmelatonin is greatly reduced compared to young individuals. An increase in the amplitudeof the melatonin profile might therefore be as beneficial, if not better, as an entrainmentof the phase.

Several studies are undertaken to replace endogenous melatonin with controlledrelease of exogenous melatonin.115,163,175 Special slow-release preparations are thereforeused to mimic the normal profile. Based on the present data, a melatonin agonist maybe able to have similar effects.

In chronobiology, the attention is often directed towards the phase of rhythms.Because many times the circadian system itself is subject of research, this will result inappropriate data. For the development of melatonergic agents, mere circadian data arevery important, but must be accompanied by data on physiological effects, such as theamplitude of melatonin production. The trans pineal microdialysis technique offersunique possibilities to study both aspects of the new class of “circadian drugs” simulta-neously.


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