CA1251141A - Method for minimizing disturbances in circadian rhythms of bodily performance and function - Google Patents

Abstract

ABSTRACT

Disturbances in circadian rhythms of bodily performance and function, as may occur for example in transfer of work patterns from day to night-shift, or rapid crossing of several time zones in an aircraft ("jet lag") are treated by administration of melatonin.
Alleviation or prevention of ill effects associated with disturbance of circadian rhythms is achieved by administration of melatonin is specified ways which are varied to suit particular needs and circumstances.

Description

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METHOD FOR MINIMIZING DISTURBANCES IN

BODILY PERFORMANCE AND FUNCTION

This invention relates to a method oE
minimi~ing disturbances in function and performance rhythms in humans with disturbed circadian rhythms.
All forms of life, from plants, insects, amphibians, reptiles, birds and mammals, including humans, show cyclical patterns of activity of approximately 24 hours periodicity.

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Circadian rhythms in bodily functions are defined as those events with a periodicity of about 24 hours which persist in the absence of external environmental cues. When a rhythm persists in the 5 absence of ex-ternal environmental cues it is said to be free-running. In man, the periodicity of these free-running rhythms is almost invariably greater than 24 hours. In normal life, these rhythms are reset (entrained) every day to about 24 hours by external 10 environmen-tal stimuli (zeitgebers), the light-dar~ cycle being of overriding importance.
~ host of mammalian circadian rhythms have been described, including feeding, drinking, sleep-wake, rapid eye movement (REM) sleep, body`temperature, physical and 15 mental performance, cardiovascular activity, urine flow, electrolyte excretion and mitotic activity.
There are also pronounced daily rhythms in blood hormone concentrates of cortisol, noradrenaline, adrenaline, insulin, growth hormone, adrenocorticotrophic 20 hormone, prolactin, luteinising hormone, testosterone, - melatonin, arginine vasotocin, etc. etc.
In humans, it has been conclusively demonstrated that many of these rhythms free-run, eg.
sleep-wake, REM sleep, body temperature, cortisol, --25 adrenaline, noradrenaline and melatonin and are therefore true circadian rhythms.
Experimentally it has been shown in humans that rhythms normally synchronized with each other in the presence of external environmental cues can free-run with 30 different periodicities in the absence of external environmental cues. This is referred to as 'internal desvnchronization'. For example, in the absence of environmental cues an individual might have a body J~

~) temperature rhythm of 25 hours and a sleep-wake rhythm of 33 hours. On re-introducing an appropriate environmental cue these rhythms would become re-synchronized.
The phenomenon of internal desynchronization 5 has been taken as evidence that there must be more than one central time clock or oscillator, and that in normal life these oscillators are kept in synchrony with one another by the entraining environmental stimuli.~ One would expect persons not experiencing the normal light-10 dark cycle, for example astronauts, submariners and polarexplorers and certain blind individuals to suf~er from internal desynchronization through not receiving the noxmal zeitgeber from the light-dark cycle.
The way in which photoperiod acts as an 15 entraining stimulus for circadian rhythms is highly comple~ and still not completely understood. It is thought that separate neural oscillators are responsive to the daylight changes at dawn and dusk respectively, and that acting in concert these are r'esponsible for the 20 daily entrainment of the circadian ,rhythms~ Thus these rhythms readily adapt to the gradual changes in the light-dark ratio with the progression of the seasons.
Recent research suggests that the suprachiasmatic nuclei of the hypothalamus contain the dawn and dusk oscillators 25 which play an integral role in the synchronization of circadian rythms.
A person subjected to a rapid alteration in the timing of day and night will find his body rhythms to be out of synchrony with the new environment, as in the case 30 of somebody who starts to work on night shift, or'who rapidly crosses several time zones in an aircraft. It may take a week or so for the various rhythms all to become reentrained, some responding faster than others.
This transitory desynchronization is termed 'internal 35 dissociation' and causes the symptoms of "jet lag", eg.

. - 5 -feeling sleepy and tired at times of day inappropriate to the sleep-work schedule of the new location or roster, with disturbed rhythms of appetite, urination, body temperature and performance of physical and mental tasks.
Reference is made to the following publications which have documented some effects experienced by humans subjected to abrupt time changes.
1. Effects of flying and of time changes_on menstrual cycle length and on performance of airline stewardesses. Preston, F.S., Bateman, S.C., Short, R.V. and Wilkinson, R. (1973) Aerospace Medicine 44, 438-4~l3.

2. Effects of "Jet La~" on Hormonal Patterns. I.
Procedures, variations in -total plasma proteins, and disruption of adrenocorticotropin-cortisol periodicity.
Desir, D., VanCauter, E., Fang, V.S., Martino, E., Jadot, C., Spire, J.P., Noel, P., Refetoff, S., Copinschi, G., Goldstein, J., 1981 J. Clin.
Endocrinol Metab. 52:628.~

- 3. Effects of "Jet Lag" on Hormonal Patterns. II.
Adaption of Melatonin Circadian Periodicity.
Fevre-Montange, M., ~anCauter, E., Refetoff, S., Desir, D., Tourniaire, J., Copinschi, G., 1981 J. Clin Endocrinol Metab. 52:642.

~ t is a common experience of airline travellers that flights in a westerly direction (phase delay) are less stressful than flights in an easterly direction (phase advance). This may be because in the latter case 30 some xhythms may phase-advance and some may phase-delay, thereby accentuating the degree of internal dissociation.
Circadian biologis-ts refer to this as entrainment by partition.

25~
. - 6 -It has also been demonstrated that shift workers adapt to phase delays (analagous to westerly flights) better than to phase advances. This is presumably explained by the fact that the free-running 5 rhythms are longer than 24 hours and these have a propensity to delay.
"Jet lag" is considered to be a symptom of internal dissociation.
The recent study by Desir et al (reference 2.
lO above) has confirmed that internal dissociation is associated with trans-meridian -travel. Those researchers have also documented discernible differences between the effects of flights in a westerly direction and ~lights in an easterly direction (also see Fevre-Montange et al, 15 reference 3. above).
In contrast to the "jet lag" symptoms experienced by those individuals suffering from short-term internal dissociation as a results of a trans-meridian flight or an altered work rpster, individuals 20 with long-term internal desynchron-~zation do not suffer - in the same way. Astronauts, submariners, polar explorers and some blind people are likely to fall into this latter category since they are deprived of the normal photoperiodic entraining stimulus, and there is 25 anecdotal evidence to suggest that some of them may have difficulty in synchronizing their sleep-wake times with the imposed work roster.
Volumes have been written on the subject of attempts to prevent or treat "jet lag" within the past 30 decade in the scientific, medical and lay press, and the topic has been exhaustively reviewed.

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SUGGESTIONS IN THE LITERATURE FOR MINIMIZING "~ET LAG"
=

GENERAL
1. Implementation of a travel-time formula which takes into account arrival and departure times and number of time-zones crossed.

PRE-FLIGHT ADAPTATION
1. Simulation of destination clock time.
10 20 Simulation of destination day-night cycle. ) Several days 3. Simulation of destination ) prior to life events eg. meal ) departure.
timing, going to bed and getting-up.

FLIGHT ADAPTATION
1. Avoid alcohol.
2. Avoid heavy meals.
3. Do some e~ercise.
.

1. Keep clock-time the same as that of country of origin.
2. Keep light-dark cycle the same as that of country of origin.
25 ~. Build special hotels with facilities for keeping country of origin light-dark schedules and meal times, (as has been the practice of Russian air crews flying between the U.S.S.R.
and Great Britain).

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4. Schedule important meetings at times of day chosen on basis of country of origin time rather than destination time.

5. Arrive at destination several days in advance of important meetings.

6. Avoid isolation and maximize social interaction to promote synchronization.

7. Induce sleep by moderate exercise, eg. walking.

8. Induce sleep by taking a warm bath.
10 9. Induce sleep by taking hypnotics.
10. ~void hypnotics. ) due to interference 11. Avoid alcohol. ) with REM sleep.
12. Avoid tranquilizers.
13. Manipulate meal timing.
15 14. Manipulate calorific content.
15. Avoid staying indoors and maximize exposure to daylight after arrival.

For further popular discussion of suggested methods to prevent and overcome jet lag see: "The Jet-20 lag Book" Kowet D, Crown Publishers Inc. and "Overcoming Jet lag" Ehret C. Berkley Books.
There is a growing list of substances known to influence (but not entrain) mammalian circadian rhythms.
These include heavy water (D2O), lithium, and the ~5 monoamine oxidase inhibitors, clorgyline and imipramine.
Chronic administration of these compounds to laboratory animals len~thens the unentrained, free-running period, and the monoamine oxidase inhibitors split the rhythm.
As none of these substances actually entrain 30 circadian rhythms none of these substances would be appropriate for treating "jet lag".
It has also been claimed that oestrogen, progesterone, testos-terone and thyroxine, or their removal by gonadectomy or thyroidectomy, may influence ~ 9 ~;~5~

the free-running period in laboratory animals. It has been suggested that in birds the daily rhythm in corticosteroid secre-ion could function as a master oscillator to entrain other circadian rhythms. Recently 5 Alberto Angeli (Glucocorticoid Secretion: A Circadian Synchronizer of The Human Temporal Structure, 1983, J.
Steroid Biochem, Vol. 19, No. 1, pp 545-554) has proposed such a mechanism for man. Since the corticosteroid rhythm is one of the last to reentrain following a time-10 zone change, it is difficult to see how this wouldoperate.
Hypnotics and tranquillizers, eg. diazepam, temazepam, oxazepam, triazolam, botizolam, are frequently used by the travelling public, and although they may 15 induce sleep, its quality is in question since REM sleep is adversely affected. Some have even gone so far as to advise against the use of hypnotics because of their cumulative effects if taken repeatedly.
There is no scientific evidence to suggest that 20 any of these compounds hastens the~reentrainment of circadian rhythms.
A fundarrental problem of the prior art has been the total lack of understanding of the phenomenon known as jet lag. Not surprisingly amidst the welter of 25 contradictory claims there is little consensus.
Indeed there is little reason for the approach of workers in the pxior art to have consensus for it was asserted by Aschoff, one of the pioneers of circadian biology, that the central nervous mechanisms that 30 generate circadian rhythms must be chemicallv and thermally independent in order to ensure the precision of the body's timing mechanisms. Thus the system is rendered immune frorn unintentional manipulation by everyday extraneous influences. The approach of workers in the prior art has been to manipulate just one aspect of a particular circadian rhythm.
In the article "PHY5IOLOGICAL CHANG~.S
5 UNDERLYING JE~ LAG" (British Medical Journal, Vol. 284, January 16, 1982, ppl44-146), Arendt and Marks state at page 146:
"Anticipation of jet lag and its preve~tion by planned manipulation of circadian rhythmicity before departure is possible using particular food-intake protocols, but Eor most people such an approach is impracticable. Pharmacological manipulation of circadian rhythms_poses even greater problems. Compounds including oestradiol, testosterone, theophylline, lithium, and tricyclic anti-depressants affect the length of the period of circadian rhythms in animals, but there have been few, if any, studies of their effects on human rhythms.
Temporary discomfort is s-urely preferable to such medication. ~ecommendations for passengers by the Advisory Group for Aero Space Research and Development include the use of short-acting hypnotics during Transmeridional flights to lessen the effects of jet lag.
~ubsequently, an e~tended night's sleep, together with exposure to local social cues, is essential for rapid resynchronization. Aircrew were recommended to keep sleep deficit to a minimum, remain on home-base time, and return rapidly to base. The practical application of such recommendations clearly presents difficulties, with particular reference to - remaining on home-base time. Various formulae have been devised for calculatins the rest-time necessary to achieve resvnchroniza~io~, depending on the number of time-zones crossed, flight duration, times of depart~lre and arrival, and direction of flight, as well as the individual's age." (emphasis added) Clearly these reviewers consid~r the prior art to be teaching away from the use of pharmacologi~cal manipulation of circadian rhythms in order to relieve the symptoms of "jet lag".
Mela~onin is another hormone with a pronounced 24 hour rhythm, the levels being elevated during the night time in all species. It is secreted by the pineal gland, but pinealectomy in rats is without effect on their free-running circadian rhythms, suggesting that in 15 mammals melatonin is not involved in the generation of such rhythms.
Pinealectomy of rats and hamsters accelerates reentrainment after a phase shift in the light-dark cycle. Thus if the removal of melatonin s~eeds up the 20 readjustment following a phase shift, melatonin administration would be expected to have the opposite effect and postpone reentrainment. From -these findings one would e~pect melatonin to be contraindicated for a person suffering from jet lag.
The first demonstration of chemical entrainment in mammals was published by Redman, J., Armstrong, S., and Ng, K.T., in Science, 1983 Vol. 219, pp 1089-1091.
Contrary to the above predictions, they showed that pharmacological doses of melatonin could entrain the 30 free-running circadian activity rhythm of rats, but only when melatonin was given at the time bf onset of spontaneous activity. But since endogenous melatonin levels in rats are elevated at the time of ma~imal `' ' ~L~S~

activity whereas in humans they are elevated during sleep, the connection bet~Jeen the rat results and the problem of jet lag in humans is not apparent.
A chronobiotic is defined for present purposes 5 as a substance which is capable of therapeutically reentraining short-term dissociated or long-term desynchronized circadian rhythms, or prophylactically preventing their disruption following an environmental insult. The surprising discovery of the present 10 invention is that melatonin is a powerful chronobiotic in humans. This discovery is contr~ry to all expec,ations.
Melatonin is released by the pineal gland in a rhythm generated by the suprachiasmatic nuclei. It was therefore highly surprising to discover that melatonin 15 produced by the "slave" organ was capable of feeding back to regulate some functions of the "master" oscillator, the suprachiasmatic nuclei.
The role played by the suprachiasmatic nuclei in regulating the release of melatonin by the pineal has 20 been reviewed by Winfree (CIRCADIAN TIMING OF SLEEPINESS
- IN MAN AND WOMAN, ~infree A.T., American Journal of Physiology , Vol. 243, 1982, ppl93-204).
It has been long known that pharmacological doses of melatonin administered to human subjects induces 25 drowsiness and sleep as do other hormones like progesterone. (See: ON THE EFFECTS OF MELATONIN ON
SLEEP AND BEHAVIOUR IN MAN, Cramer H, Rudolph J, Consbruch V, Kendel K. 1974, Advances in Biochemical Psychopharmacology, Vol. 11, ppl87-191 Raven Press, NY).
30 The architecture of this melatonin-induced sleep is more natural than that produced by currently available sleeping pills, which suppress REM sleep. However, this cannot be taken as evidence that melatonin is the physio~ogical compound normally responsible for sleep;
35 one can fall asleep during the daytime when melatonin - 13 ~

levels are at their lowest, and melatonin levels can be dissociated from the sleep pattern for several days following an abrupt ti~e-zone change. This is perfectly in keeping with the observation that hypnotics can induce 5 sleep but do not reentrain circadian rhythms. The well documented hypno-tic role of melatonin has not previously been linked with reentrainment of circadian rhythms.
Within the last 10 years it has been shown that physiological doses of melatonin administered by mouth, 10 by implant or by injection are capable of altering the timing of the annual breedinq season in hamsters, sheep, ferrets and deer, and the present inventors have demonstrated similar effects in wallabies. A review o these potential commercial applications of melatonin 15 appeared in Nature on April 28th, 1983 ~Vol. 302 p755), and it is interesting that no mention was made of the possibility that melatonin might regulate events such as "jet lag".
Notwithstanding such work, in 1983 Arendt 20 hypothesised (with no experimental evidence to support her contention) that melatonin might be able to shift circadian rhythms in man and reoryanize the circadian system when it is pathologically disturbed. (BIOLOGICAL
RHYTHMS Arendt J., International Medicine, Vol. 3, Number 25 2, 1983 pp6-9). However that researcher did not identify "jet lag" as internal dissocia-tion nor did she establish that melatonin would in fact shift circadian rhythms.
Arendt did no' specify or suggest a pharmacological procedure involving melatonin that might achieve the 30 hypothesised response.
It is clear from our results that e~o~enous melatonin can act as a chronobiotic in man since it can hasten the reentrainment of the body temperature rhythm following an abrupt time-zone change or a change in work 35 roster such as that e~perienced by night-shift workers.

- 14 - ~25~

It is well known from some of our own and other earlier work that performance in cognitive tasks is closely linked to the body temperature rhythm (see: Colquhoun, W.P., Biological rhythms and human performance, ~cademic 5 Press, 1971), and that the duration and organization of human sleep is dependent on the circadian phase of the body temperature rhythm (Czeisler, C.A., Weitzman, E.D., Moore-Ede, M.C., Zimmerman, J.C., Knaver, R.S.i ~1980 SCIENCE, Vol. 210, pp 1264-1267). We therefore believe 10 that the beneficial effects of melatonin in our experiments are due to its ability to hasten the reentrainment of body temperature rhythms, from which many other rhythms flow. Individuals such as astronauts, submariners, polar explorers or blind people, would 15 benefit from timed daily administration of melatonin.
There is an interesting relationship between time zone changes and psychiatric illness. Depressive illnesses are higher in incidence after a westward flight while mania is higher after an eastward flight. Since a 20 westward flight is a phase delay of- the zeitgeber this leaves, at least initially, the sleep span advanced relative to the new night-time. This fits in exactly with the phase-advanced hypothesis of depression. This states that in endogenous depression, various circadian 25 rhythms are shifted abnormally early (phase advanced) relative to the night-day cycle (Antidepressants and a circadian rhythm phase-advance hypothesis of depression, by T.A. Wher et al, pp 263-274 in Brain Neurottansmitters and Hormones edited by R. Collu et al, Raven Press 30 1982). Therefore, melatonin administered at the appropriate time of day could be used in treatment of all psychotic illnesses which result from abnormal timing of circadian rhythms. The only study in which melatonin has been administered to depressed patients reported an ~.~5~

exacerbation of symptoms including florid psychotic episodes (CARI~AN, J.S. et al ~MERICAN JOURNAL OF
P~YCHIAT~Y 133, pp 1181-1186, 1976).
The prior art is teaching away from the use of 5 melatonin to relieve psychoses, since researchers have made no allowance in the experimental procedur~s for the time of day of administration of melatonin and melatonin's capacity to re-entrain circadian rhy~hms in humans.
The object of this invention is to minimize the disturbances in human bodily performance and function that occurs in the absence of an appropriate photoperiodic zeitgeber or time cue.
By the method of the present invention 15 administration of the hormone melatonin is used as a zeitgeber to rapidly entrain certain circadian rhythms to a new photoperiodic environment, thereby minimizing the period of disturbance consequent upon the disruption of these rhythms.
In one embodiment there is provided a pharmaceutical composition in capsùle form for the re-entrainment of disturbed circadian rhythms in a human subject comprising 1-10 mg melatonin in admixture with a pharmaceutically acceptable diluent~
In another embodiment there is provided a process for the manufacture of a medicament for alleviating the effects of disturbed circadian rhythms on bodily performance and function in a human subject which process is characterized by incorporating melatonin into the 30 medicament as the active ingredient.

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- 15a -It is known that the pineal gland secretes its principal hormone, melatonin (5-methoxy-N-acetyltryptamlne) according to a circadian rhythm generated in the suprachiasmatic nuclei of the hypothalamus and entrained by pnotoperiod. The pineal gland acts as a neuro-endocrine transducer, converting a neural input, in the form of light acting on the retina, into an endocrine output, in the form of melatonin which is released into the systemic circulation to act back on the brain directly. 'rhe pineal gland may also release other regulatory compounds such as arginine vasotocin which act back on the brain.
Since the circadian rhythm of melatonin secretion from the pineal is go~erned by a circadian rhythm generated in the suprachiasmatic nuclei and is entrained by light, our invention is surprising as we are ~ 16 ~5~

using the product of this rhythm, melatonin to act back on the rhythm-generating mechanism within the suprachiasmati^ nuclei to reentrain them. Our understanding of feedback systems would predict that 5 exogenous administration of melatonin would be ineffective since it would merely suppress endogenous release. The surprising discovery is that by applying the method of the present invention -eentrainme~t is hastened.
It is known from measurements of melatonin in blood and of its metabolites in urine that in all species investigated, including the human, melatonin is produced predominantly if not exclusively at night. It has also been observed that this nocturnal secretory pattern takes 15 several days to readjust to a sudden time change.
Application of the method should normally co~ence im~ediately following a time change, when the person would be trying to go to sleep according to the new time schedule. By raising the amount of melatonin in 20 the circulation this will not only induce a sense of drowsiness, helping the person to go to sleep, but it will also improve the quality of the sleep and entrain the circadian rhythms to the new imposed photoperiod.
While the invention has been described with reference to 25 the administration of melatonin, other related indoles or indole derivates may be used. It has been shown that synthetic melatonin analogs do express varying degrees of melatonin agonist activity when tested on fish bioassay (STRUCTURE ACTIVITY RELATIONSHIP OF MELATONIN ANALOGUES
30 Frohn M.A, Seaborn C.J., Johnson D.W., Phillipou G, Seamark R.F., Matthews C.D., Life Science Vol. 27, pp2043-20456, 1980).
Treatment in accordance with the invention may be used by a person until all their bodily rhythms have 35 adjusted to the new environment. It may also be possible t to use the treatment to adjust the circadian rhythms in advance of a time change, thereby anticipating the new environment.
The preferred schedule for treatment requires 5 the subject to take a specified preparation of melatonin at the anticipated time for going to sleep in the new destination or a new activity schedule. The treatment should be taken at least at the commencement of the first sleep period of the new destination. Alternatively 10 treatment may be commenced 1 or more days prior to the commencement of the journey or during the journey at the time anticipated to be the commencement of sleep time in the new time ~one.
The method of -the invention may be achieved by 15 the oral administration of e~ogenous melatonin, the preferred dosage range being from 1 - 10 mg.
The timing of the melatonin administration and the period of time for which the blood level remains elevated are important parameters for-control when 20 performing the method of the invention. Any pharmaceutical preparation containing melatonin in a pharmacologically effective concentration would be suitable for performing the method of the invention.
Maintainins high plasma levels of melatonin throughout 25 the desired sleep period is preferable. Alternatively, it may be necessary to take additional melatonin if the person wakes during the desired sleep period.
While people have administered melatonin before it is not part of the prior art to formulate a dose of 30 melatonin into a slow release form. Our research has shown that it is preferable to maintain appropriate plasma levels of melatonin during the entrainment procedure. Clearly melatonin in a slow release form ~5~

enables the desired plasma levels to be main,ained without the need to take further doses during a particular sleep period.
~hile oral administration is the preferred 5 route of administration of melatonin any other route may be used to achieve the desired plasma levels in accordance with the method of the invention.
I~ view of the Xnown effects of high intensity light in inhibiting endogenous melatonin release in man 10 (A. LE~Y et al Science, Vol. 210 : pp 1257-1268, 19~0) it would be advisable to make use of the phenomenon to bleach out the old endogenous melatonin rhythm following a time zone or work ros-ter change, whilst taking exogenous melatonin to stimula-te the development of the 15 new melatonin rhythm. It is also known that beta blockers such as PROPANOLOL can inhibit endogenous melatonin secretion so these could also be used to eliminate the old endogenous melatonin rhythm following a time zone or work roster change.
It is known that the enzyme N-acetyl - transferase which converts serotonin to N-acetylserotonin provides a rate limiting step in the production of melatonin by the pineal gland. Therefore the method of the invention may also be achieved by administering N-25 acetylserotonin or any of its intermediates in the synthesis of melatonin as the therapeutic agent. Since exogenous L-dopa administration stimulates melatonin synthesis, and presumably release in rats, this method of increasing melatonin bioavailability at an appropriate 30 time of the day is also useful for performing the method of the present invention.
The invention may also include a package containing melatonin in appropriate dosage form, said package including instructions for adminis_-ation ~2~

thereof, consistent with the teachings herein for the alleviation or prevention of ill effects associated with disturbance of circadian rhythms.
In drawings which illustrate embodiments of the invention and which are referred to in the following examples, Figure 1 is a temperature record graph for subject A in Example 4;
Figure 2 is a graph of plasma melatonin versus time for subject A in Example 9;
Figure 3 is a forty~eight hour plot of rectal temperature rhythm for subject F in Example 10 divided into 4 stages; and Figure 4 is the same as Figure 3 except that each lS of the 4 stages has been shown separately.

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This example demonstrates the effectiveness of the present invention at alleviating the distressing effec-ts on circadian rhythms during a west out, east back flight through several time zones.
Subject A was a Professor~f Physiology who had never taken melatonin before.
Flight from Melbourne ~ North Carolina U.S.A. i Melbourne 16.4.83 - 20.4.83 Details of activity schedule, sleeping time, melatonin administration.
Melbourne Time Activity and location Local -time 1205hrs 16.4.83 Left Melbourne by air 1315hrs 17.4.83 Arrived Chapel Hill, 2215 hrs North Carolina. 16.4.83 140Ohrs To bed, took 2.5mg Melatonin, fell asleep 2300 hrs 1730hrs Awoke spontaneously, took 5.Omg Melatonin,- fell 0230 hrs asleep again. - 17.4.83 0030hrs 18.4.83 Awoke spontanéously Felt 0930 hrs fresh, relaxed, not jet-lagged Worked a normal day, chairing Board Meetings 1330hrs To bed, took 5mg Melatonin, fell asleep 2230 hrs 1615hrs Awoke spontaneously took 5.0 mg Melatonin, fell 0115 hrs asleep again 18.4.83 1815hrs Awoke spontaneously, took 5.0 mg Melatonin, fell asleep again 0315 hrs 2245hrs Awoke spontaneously, felt fresh, relaxed 0745 hrs Worked a normal morning. In afternoon caught flight 0875hrs 19.4.83 back to Melbourne, departing at 1657 hrs 2315hrs Hawaii. Took 2.5 mg 0315 hrs Melatonin. fell asleep 19.4.83 ~.2S~

M~lbourne Time Activity and Location Local Time 0600 hrs 20.4.83 Awoke spontaneously, feeling fresh.

Worked on plane 1515 hrs Arrived Mel~ourne Airport .
2330 hrs To bed no Melatonin 0800 hrs Awoke normally.

CO~I~E~T
Although subject A awoke once or -twice during the night in North Carolina, he fell asleep again within 15 minutes of ta~ing additional melatonin on each occasion.
Subject A awoke spontaneously each morning in the U.S.A. feeling in top form, and was able to carry out a hea-~Jy work schedule during the day with no symptoms of jet lag whatsoever.
~ ollowing a similar itinerary, on ~revious occasions, without taking melatonin Subject A had consistently been tired, inattentive, unable to concentrate, - -and felt totally disoriented in time.
- ~n the return flight, Subject A put himself back on Melbourne time and took melatonin in Hawaii to enable him to sleep Melbourne time. As a result, he arrived back in Melbourne in the afternoon feeling fine, and slipped easily into his normal Melbourne routine, going to bed at 11.30 p.m.
and sleeping normally to awa~e at the usual time of 8.00 a.m.
Subject A slept normally for the following three nights, and never felt any ratigue whatsoever from a rather punishing schedule.
Subject A's rapid return to normality on his return to Melbourne probably reflects the fact that the 3 days in the U.S.A. had been insufficient to disturb all o~ his circadian rhvthms, which remained essentially on L~!elbourne time throughout the trip. However, the melatonin treatment in the U.S.A. certainly seemed to entrain his sleep-wake and performance rhythms to U.S.A. time.

~5~

This example demonstrates that the method of the present invention is capable of altering a human's circadian rhythms to avoid the distress associated with a rapid transition from one time zone through at least a second time zone and then return to the first time zone. The most remarkable positive effect in this instance was that Subject A could alter his sleep routine and work routine to suit each time zone visited.
In this example Subject A used a preparation of crystalline melatonin in a gelatine capsule.

. - 23 --Flight from Melbourne ~ Munster, I~. Germany ~ Melbourne.
27.5.83 - 5.6.83 Details of activity schedule, sleeping time, ~elatonin ad~inistration.
elbourne Time Activity and Loca~ion Local T me 1540 hrs 27.5.83 Left Melbourne by air 0140 hrs 29.5.83 Arrived klunster, W. Germany 1740 hrs 2&.5.83 0700 hrs To bed, took 5 mg Melatonin 2300 hrs 1550 hrs Awoke spontaneously 0750 hrs Felt fresh and rested, much less tired. 29.5.83 Worked a normal day, scienti ic meetings 0635 hrs 30.5.83 To bed, too~ 5 mg ~leiatonin 2~35 hrs 1120 hrs A-~oke spontaneously, took 5 mg Melatonin 0320 ~rs fell 2sleep. 30.5.83 I
1415 hrs 4wol-e spor.taneoucly, ieeling eY~cellent 0615 hrs . ~
Worked a normal cay, scien.i.ic meetings -- 0840 hrs 31.5.83 To b ~ took 5 mg Melatonin 0040 hrs 31.5.83 1205 hrs Awoke spontaneously, took 5 mg Melatonin, 0405 hrs fell asleep.

1505 hrs Awoke spontaneously, reeling fine 0705 hrs Worked a normal day, scientific mee~ings 09~6 hrs 1.6.S3 To bed, took 5 ~g Melatonin 0125 hrs 1.6.83 1030 hrs Awoke spontaneously, but fell aslePp 0230 hrs again with no addi~ional Melatonin 24 _ ~L;25~

MQlbourne Time Ac tivitv and Location Local Time .
1400 hrs Awoke sponraneously feeling good. 0600 hrs Flew to Frankfurt, drove to Munich To bed. headache, feeling tired.
05~5 hrs 2.6.83 ~To Melatonin. Fell asleep at once. 2145 hrs 1445 hrs Awoke spontaneously, feeling fine 0645 hrs ~ 2~6.83 Scientific meetings, flew to Frankfurt.

0720 hrs 3,6083 To bed~ no Melatonin, fell asleep at once 2320 hrs.

1430 hrs Awoke spontaneously, feeling very fresh. 0630 hrs 3.6.83 0550 hrs 4.6.83 Cau~ht flight to Melbourne, departing at 2150 hrs 0835 hrs Slept from Rome to Bomhay, going to sleep at Rome time. 0035 hrs 4.6.83 1400 hrs Awoke sponraneously, feeling O.K.
0000 hrs (midnight) Took 5 mg ~lel2tonin, slept deeply.

0610 hrs 5.6.83 Awoken by ~reakfast, felt fresh and rested.

0700 hrs Landed Melbourne Spent day gardenino 2100 hrs To bed. too~ 5 mg Melatonin 0810 hrs 6.6.83 Awoke spontaneously afte- good sleep.
~el~ tired during the day.

2315 hrs To bed, took 5 mg Melatonin, slept very well.

- 25 - ~

Melbourne Time Activitv and Location Local Time 0745 hrs 7.6.83 Awoke spontaneously, felt excellent, busy day in laboratory. ~

2245 hrs To bed, took 5 mg Melatonin, slept well.

0745 hrs 8.6.83 Awoke spo~taneously with a slight hangover.
Discontinued medication.

CO~lENT
The melatonin allowed Subject A to sleep well following both Westerly and Easterly flights with an ~-hour time zone change. Subject A awoke spontaneously each mornin~ at the appropriate (local) time reeling excellent, and experienced no symptoms of jet lag apart from tiredness on the second day after his return to Melbourne.
This example demonstrates that with the method of the present invention it is possible to rapidly entrain sleep routines to suit an interary that crosses several time zones over a period of a week.
As in Example 1, Subject A took melatonin packaged in a gelatine capsule.
In both examples, Subject A spontaneously awoke durins entrainment sleep. Upon taking another capsule he returned to sleep. These experiments show that the preferred way of performing entrainment would be with melatonin made up in a long-acting preparation.

EX~PLE 3.
Subject A Flight from Melbourne ~ Stockholm ~ London~
Melbourne - 25.6.83 - 11.7.83 Details of activity schedule, sleeping times, melatonin administration.
Melbourne Time Ac-tivi-ty and Location Local Time 1300hrs 25.6.83 Left Melbourne by air.
2100hrs 26.6.83 Arrived Stockholm ~ took 1300 hrs bus to Uppsala. 26.6.83 0715hrs 27.5.83 To bed, took 5mg Melatonin 2315 hrs 1222hrs Awoke spontaneously, took 5 mg 0422 hrs Melatonin,fell asleep again 27.6.83 1435hrs Awoke spontaneously, feeling fine. ~ 0635 hrs Attended Acta Congress in Stockholm, evening reception and party. Felt good.
1207hrs 28.6.83 To bed, took 5 mg Melatonin 0407 hrs 28.6.83 1700hrs Awoke spontaneously. Felt good. 0800 hrs Attended Congress and reception and evening party.
1208hrs 29.6.83 To bed, took 5 mg Melatonin 0408 hrs ~ 29.6.83 1630 hrs Awoke spontaneoously feeling good. 0830 hrs Attended Acta Congress, boat trip in evening 0945hrs 30.6.83 To bed, took 5 mg Melatonin 0145 hrs ~ 30.6.83 1540hrs ~woke spontaneously, feeling fine. ~ 0740 hrs Acta Congress all day 1152hrs 1.7.83 To bed, did not take 0352 hrs any Melatonin 1.7.83 1600 hrs Awoke spontaneously feeling fine 0800 hrs Melbourne Time Activity and Location Local time Flew to London an~ train to Cambridge - stayed one night;
Brlstol - stayed 4 nights;
London, stayed one night. No problems sleeping, waking or with feelings of well being. No records kept until return flight.
starting on 9.7.83.
0630hrs 10.7.83 Left London by air. Feeling very tired after a hot day, so slept 2130 hrs on plane 9.7.83 1445hrs Awoke spontaneously.
1700 - 1900hrs Slept 2130 Flight arrived Singapore 1930 hrs (1230 hrs London -time 2345hrs Took 5mg Melatonin, fell asleep 2145 hrs 3/4 hour later (1445 hrs London time`
0500hrs 11.7.83 Awoken by breakfast after a good sleep, bu-t could have slept longer .
0947hrs Arrived Melbourne Airport Went home, shower, shave , went in to lab; for l hour lecture and then discussions with Medical students. Felt Eine.
2334hrs To bed, took 5mcJ Melatonin, slept well.
0800hrs 12.7.83 Awoke to alarm. Feeling fine Normal working day. Felt fine 2247hrs To bed, took 5mg Melatonin, did not go to sleep until 2330 hrs.
0800hrs 13.7.83 Awoke by alarm. Feeling fine.
Normal working day. Felt fine.
2324hrs To bed, took 5mg Melatonin, fell asleep 2400 hrs.
0745hrs 14.7.83 Awoke spontaneously, feeling fine.
Normal working day.
2300hrs To bed, did not take Melatonin 0800hrs 15.7.83 Awoke to alarm, feeling fine Discontinued record.

- 28 ~ ~5 ~
.

COr~lENT
Once again, the melatonin allowed Subject A to sleep well at an ina~propriate time of the day following both Westerly and Easterly flights with an 8-9 hour time change.
It was interes~ing that in Sweden he was quite sleep-deprived, going to bed in the early hours of the morning after much social activity and some alcohol at parties and receptions, nevertheless he always awoke at an appropriate local time feeling refreshed, and did not need to take additional melatonin at night.
Subject A also encountered no problems on his return to Melbourne, and was able to lecture to a class of 170 r1edical students within 4 hours of stepping off the plane. At no time during the day either in Sweden or on his return to Melbourne did he feel jet-lagged.
The example demonstrated the ability of the method of the present invention to raDidly entrain circadian rhy~hms.
Rapid entrainment to periods that vary every 24 hours is clearly demonstrated.

E~,~MPLE 4 ubject A Flight from Melbourne Geneva ~ London~
Chapel Hill, North Carolina ~ Melbourne 18 9.83 - 30.9.83 This e~ample demonstrates the intimate relationship between the human body circ~dian rhythms of temperature and per~ormance.
Durinq this experiment use was made of a portable Vitalog PMS8 mini computer to record the temperature of Subject A. The v~talog PMS8 recorded body temperature at ten minute intervals utilizing a rectal temperature probe.
The temperature record has been displayed in figure 1. A
discussion of the temperature record follows.
During this flight Subject A wore the Vitalog PMS8 mini computer and rectal probe for the continuous recording of his deep body temperature. The equipment malfunctioned after 4 days due to premature battery failure. From 18.9.83 - 23.9.83 Subject A kept a detailed daily log of quality and nature of sleep mood, and alertness according to a technique developed by NASA.
Details of activity schedule, sleeping time, melatonin administration.
Melbourne time Activity and Location Local time 1300hrs 18.9.83 Left Melbourne by air 1610hrs 19.9.83 Arrived Geneva 0810 hrs ~ 19.9.83 Spent morning walking round lake, committee meeting at WHO all afternoon. ~
0535hrs ~0.9.83 To bed, took 5mg Melatonin 2135 hrs fell asleep~at once.
1155hrs Awoke spontaneously, took 5mg Melatonin, fêll asleep after 0355 hrs 30 minutes.~ 20.9.83 1500hrs Awoken by alarm, ~eeling e~cellent. 0700 hrs * Trade Mark ~L~5~

Melbourne Time Activity and Location Local time Worked all day at WHO.
0640hrs 21,9.83 To bed, took 5mg Melatonin. 2240 hrs 1234hrs Awoke feeling drowsy, slight headache, took 5mg Melatonin 0434 hrs Asleep 30 minutes. 21.9.83 1500hrs Awoken by alarm, feeling fine. 0700 hrs Worked all day at WHO.
0626hrs 22.9.83 To bed, took 5mg Melatonin~
Asleep 30 minutes later. 2226 hrs 1145hrs Awoke spontaneously, took 5mg 0345 hrs Melatonin. Did not fall asleep 22.9.83 but lay in bed awake thinking of many resarch ideas. Event-ually fell asleep at 0600 hrs 1450hrs Awoken by alarm, feeling good 0650 hrs J~
Worked all day at WHO
~1, 0807hrs 23.9.83 To bed, took 5mg Melatonin 0007 hrs ~ell asleep at once. 23.9.83 '-- - -- -- 1~ ' 1430hrs Awoke spon-taneously, feeling fine 0630 hrs Worked all day at WHO
0240hrs 24.9.83 Left Geneva by air. 1840 hrs 0700hrs Arrived at hotel in London 2200 hrs 0933hrs To bed, did not Melatonin 0033 hrs 2 ~ . 9 . g 3 1530hrs Awoke spontaneously, felt fine. 0630 hrs 0130hrs 25.9.83 Left London by air 1630 hrs I

~5~

Melbourne Time Activity and Location Local time 1200hrs Arrived Raleigh/Durham,U.S.A. 2200hrs 1335hrs To bed in Chapel Hill, took 5mg Melatonin, fell asleep aftcr 30 minutes. 2335hrs 1532hrs Awoke with proctalgia - ~ook two codeine and Disprin* 0132hrs fell asleep at once. 25.9.83 2050hrs Awoke spontaneously, fel-t fine 0650hrs Worked all day at FHI
chairing meetings.
1355hrs 26.9.83 To bed, took Smg Melatonin, fell asleep at once. 2355hrs 2045hrs ~wo~e spontaneously feeling fine 0645hrs Worked all day at FHI
chaired meetings. 26.9.83 1333hrs 27.9.83 To bed, took 5mq Melatonin, fell asleep at once. 2333hrs 2035hrs Awoke spontaneously, felt fine 0635hrs Worked all day at FHI~
gave seminar in morning.
Flew in afternoon to Washington, D.C. 27.9.83 1127hrs 28.9.83 To bed, took 5mg Melatonin asleep in half an hour. 2127hrs ~.
1615hrs Awoke, worried about catching early morning flight. Doæed 0215hrs fitfully for 2hr 30 minutes 28.9.83 185lhrs Got up. Slight headache, so -took two asprins 0451hrs 211Shrs Flew from Washington National Airport to SanFrancisco via Minneapolis,clock bac~ 3hrs.
Dozed lhr 30min. on flight 0715hrs 0530hrs 29.9.83 Arrived San Francisco 1230hrs * Trade Mark ~.25~

Melbourne Time Activity and Location Local Time Visit Dr. Graeber and Dr.Gander at NASA Aerospace Centre, Manned Vehicle Systems Research Division, Ames. Changed computer battery 1230hrs Departed San Francisco 1930hrs.
1400hrs Arrived Los Angeles, Changed planes 210Ohrs.
1530hrs Flew to Sydney 2230hrs 1800hrs Slept for lhr 30mins until OlOOhrs dinner at 29.9.83 1932hrs Took 5mg Melatonin, asleep at once 0232hrs 2335hrs Awoke. Took 5mg Melatonin, fell asleep at once. 0635hrs.
0323hrs 30.9.83 Awoken by neighbour on plane 0632hrs Landed at Sydney -0745hrs Took off for Melbourne.
O909hrs Landed Melbourne.
Stayed at home all day.
In the evening, felt very tired and sleepy and cold.
2150hrs To bed, took 5mg Melatonin fell asleep in 25 mins.
0152hrs 1.10.83 Awoke to defaecate. Took 5mg Melatonin. Fell asleep at once 0800hrs Awoken by baby. Fel-t very sleepy, but this soon passed.
0815hrs Worked in garden all day.

~ ~25~

Melbourne Time Activity and Location Local Time -2220hrs To bed, tood 5mg Melatonin, slept soundly.
0815hrs 2~10.83 Awoken by baby. Felt fine.
At home all day ~I
2212hrs To bed, took 5mg Melatonin Disturbed once in night by baby, but fell asleep again.
0730hrs 3.10.83 Awoke spontaneously.
Felt fine J~
Normal day at work.
2200hrs To bed, no Mela-tonin.
Asleep by 2230 hrs.
0700hrs 4.10.83 Awoken by baby.
Normal day at work 2330hrs To bed, asleep at once 0600hrs 5.10.83 Awoke spontaneously.
Record discontinued.

COMMENT
On this round-the-world flight in a Westerly direction Melatonin once again enabled Subject A to sleep when he wanted to, and to awake at a normal local time . Each morning Subject A felt fresh and relaxed, and was able to function during the ensuing days with no subjective symptoms of jet-lag.
Figure 1 displays the tempe~ature record of Subject A for four days.
The dark, dotted regions represent dark periods. The time scale is for Melbourne time no matter where Subject A actually was when the recording was made. Each dark period is identified by one of the letters A, B, C, D or E.
Recordings of dark periods B, C and D have been repeated to give continuity to the record.
The black arrows identify tne times o acministration of melatonin.
The Vitalog rectal probe was inserted the day before departure at 1400 hrs on 17.9.83 and the accompanying graph shows the expected nocturnal fall during the first night of sleep (Dark Period A). The second sleep period occurred on the plane en route to Geneva, and again showed a nocturnal fall. The third and fourth sleep periods, assisted by melatonin administration (black a-rot~s) took place after arrival in Geneva, although the record is incomplete du~ to battery failure. The lowest temperatures were now occurring at around mid-day Melbourne time, coincident with the new sleep period, whilst maximal temperatures were occurring at around midnight Melbourne time. This shows that his body temperature rhythm had re-entrained extremely rapidly to Geneva time, under the influence of exogenous melatonin.
It is well known that per~ormance rhythms are closely allied to the body temperature rhythm, pertormance being highest when body temperature is highest. The results of this experiment suggest that the melatonin prevented the subjective feelings of je-t-lag by re-entraining Subject A's .
bodv temperature rhythm, and hence his performance, to the new time-zone. ~ithout the method of the present invention, it would have taken Subject Als temperature rhythm several days to readjust to such a rapid and large -time-zone change.
The temperature record has provided useful date on the temperature circadian rhythm and its response to entrainment by melatonin.

~ ~ .

EXA~PLE 5 Subiect B Fli~ht from i~;elbourne ~ Geneva ~ Rotterda~ ~ `;elbourne 30.1.84 - 19.2.84 This was a double-blind trial. The subject, a Cllnical Professor who had never taken ~lelatonin before. w~s give~ two bottles labelled A and R
containino identical-looking ~elatin capsules, and told to t~ke one capsule on going to bed. and again if awaking in the middle of the nioht, for the 'irst 4 days after arrival in Geneva. ~e -~as told to taXe one capsule B
accordin~ to ;he same scnedule after return to ~lelbourne, and to record the atlality of each ni~ht's sleep and feeli~gs during the dayti~e. Capsule A
cont2ined 5 m~ ~lelatonin, and capsule B contained a pla~ebo (sucrose).
Only after the subject had comPleted h~s written report 4 days after recurnln~ to ~Aelbourne was he told which pills contained ~lela~onin and which contained placebo.
Details of activity schedule, sleeping time, ~el~tonin administration.
~lhourne Time Activit~ and ~ocation Local Time 1345 hrs 30.1.84 Le~t `,~elbourne by air 2130 hrs 31.1.84 Arrived ~.eneva 1130 hrs 31.1.84 0630 hrs 1.2.84 To bed, too'~ Capsule A, fell asleep 2030 hrs 1115 h-s Awoke, took Capsule A, fell asleep a~ain 0115 hrs 1.2.84 1700 hrs ~woken by alarm 0700 hrs Worked all day at I~HO, felt tired in afternoon 1315 hrs 2.2.84 To bed, took Capsule A, fell asleep 0315 hrs 2.2.84 .~
1700 hrs Awoken oy alarm~ havin~ slept well 0700 hrs Worked all day at l~10, felt tired in ~orning oa30 hrs 3.2.~4 To bed, took Capsule A, rell asleep 2330 hrs 1330 hrs Awoke~ took Capsule A, fell asleep a~ain 0330 hrs 3.2.8~i r 1600 h-s Awoke spontaneously havin~ slept ~Jell 0600 hrs . - 36 - ~25~

E~YPLE 5 (continued) ~lelboorne Time Activity and Location Local Ti~e Worked all day at ~HO, felt tired in afternoon 1035 hrs 4. ~4 To bed, too~ Caosule A, fell asleep 0035 hrs - 4.2.84 1745 hrs Awoken by alarm havin~ sleep well ~ 0745 hrs - RETURN JOUR~.lEY
~3~ hrs 19.2.84 ~rrived Melbourne Spent day around the hous e 2250 hrs Took Capsule B, rell asleep 0515 20.2.84 Awo~e after good sleeo. Too~ Capsule B
but could not go to sleep a~aln . - Felt e~tremely tired in the evening 2230 hrs Took Cc~.psule B, Iell asleep 0230 hrs 21.2.84 Awoke, took CapsulP B, fell asleep again Felt e~tre~ely tir d i~ the evenin~ ~

2250 hrs Took Capsule B, fell asleep C640 hrs 2 _.84 Slept well, awoken by alarm ~elt ~remely tired late in the evening ~400 hrs To bed, too'~ Capsule B. Restless sleep 0320 hrs 23.2.84 Awoke, cook Caosule B

0640 hrs Awoken by alarm ~ 37 ~ ~2 COI~lENT
Before being told which was the acti~e substance and which the placebo, the subject concluded that Caps~lle A had had and effect in making him less tired and more alert in the meetings at WHO in Gene~a. In contrast, he feld that Capsule B had had no effect since he was as tired as usual on returnins to ~lelbourne. This was the correct diagnosis. This example shows melatonin's ability to rapidly entrain a subject to a new time zone. Within 2a hours of entering a new time zone Subjec~ B
was able to sleep during a period that coincided with early morning in the zone of departure.

~ 25~

Subject C Flight from Melbourne ~ Los Angeles -~Washington ~ London ~ Vancouver -> Melbourne.
15.2.84 - 27.3.84 The subject, a Professor who had never taken Melatonin before, was given gelatin capsules containing 5 mg Melatonin and instructed to take one capsule on going to bed in the evening (local time) after arrival at his destination, and to take a further capsule if he awoke in the night.
The following is a verbatim account of the subject's impresslons:
"The first use of Melatonin followed an uninter-rupped flight Erom Melbourne to Los Angeles, arriving Los Angeles 9.00am. That night I took one capsule on retiring and a second when I awoke during the night.
The sleep that night appeared normal; it is my normal habit to awake most nights.
The following day I felt very close to normal.
The next night in Los Angeles my sleep was normal without taking any more capsules.
Following a flight from Washington to London which involved a five hours time-zone change I arrived in London mid-morning and felt tired enough that I fell asleep that afternoon. That evening I took one capsule on retiring and on awakening during the night took a second capsule. I awoke next day feeling normal and appeared to be fully adjusted to the London time on that and the following day.
An uninterupted flight from London to Vancouver involved a time-zone change of eight hours. I arrived at Vancouver in the evening. Again I took one capsule on retiring and another when I awo~e during the night.
The next day I felt a little unusual and later around 4.00 pm felt very sleepy, perhaps as if still partly on 5~
- 38a -"London time". The following night I had a normal sleep and on the next day felt fully adjusted to Vancouver time.
Overall, my subjective feeling was that the Melatonin made a great improvement over my normal experience of jet-lag. On flights such as those listed above I
normally feel very peculiar, a kind of unreal feeling.
It usually takes me two or three days to settle into the new sleeping and waking pattern. However the marked improvement in my response to time-zone changes that was brought about by using the Melatonin capsules enabled me to work quite effectively immediately following arrival in the new location. It was such a contrast to previous experiences that I would be anxious to make use oE further Melatonin tablets in this way on any fu-ture trips involving substantial east-west travel."

COMMENT

Subject C found that melatonin prevented the usual, unpleasant symptoms associated with rapid changes in time zones. This Subject undoubtedly gained considerable benefit from entrainment with melatonin and the method of the present invention.

_ 40 - ~ ~5~
.. ~

PL~ 7 Subject D Flight from Melbourne + London ~ Edinburgh -~ London ~ ~elbourne 23.3.84 - 2.4.84 The subject, a Clinical Professor who had never taken Melatonin before. ~as given gelatin capsules containing 5 mg Melatonin and instructured to take one capsule on going to bed ln the evening (local time) after arrival at his destination, and to take a further capsule if he awoke ln ehe night.
The following is a verbatim account of the s~ject's ir~pressionS:
"I am writing lnforr.ally to let you know of my experience using melatonin (capsules, 5 mg) as a hypnotic/regularizer of diurnal rhythms, on a recent (23 March - April 2) trip to and fro~ Edinburgh.
On the out~ard leg, in retrospect, I took ~elatonin at the wrong time. m e flight (Melbourne-Perth-Bo~bay-London) left ~ere at 1925: I worked Melbourne-Perth, slept very soundly without taking anyching Perth-~ombay, and took 5 + 5 mg on the Bombay to London leg. As this ~as morning, Melbourne time, and I had slept so~lndly already~ I slept little if at all on this leg. I was, however, able to work effectively in reviewing a series of manuscripts for journals.
Having arrived in London at lC30 ~T I felt slightly sleepy at around 1600, and slept deeply until approximately 1930 in a day-bed at a fr end~s house. I dined, shared a bottle of wine, talked wlth friends until midnight, took 5 mg of melatonin and slept soundly until 0630 G~T (0730 summer time, the changeover having been effected during the night).
`I took melatonin 5 mg on each of the three succeeding nights, and enjcyed 6-6 1/2 hours excellent sleep. I napped minimally during the talks at the British Endocrine Societies meeting, certainly muc'n- less than when usi~g Mogadon as an hypnotic, and no- more than during a local meeting with no problem of jet lag.-I discontinued melatonin for the last two nights, with no adverse effects,and 6-6 1/2 (normal sleep duration) hours of sleep each night. On the return trip (EdinDurgh-London-~uscat-Singapore-Sydney-~.elbourne) I catnapped on the shuttle between Edinburgh (departure 1940) and London, and ~orked without sleeping London-Muscat-Singapore. After Singapore (approximately 10 a.m. Edinbur~h time, approximately 1.00 a.m. Kelbourne time) I took 5 mg melatonin, and slept for approY~imately 4 hours.
I dozed for 30 minutes as a passenger on a car ride on the day I arrlved home~ in the afternoon; apart fro~ that I have not felt sleepy, even after a couple of glasses of wine. I have taken 5 mg each ni~ht at approximatelY
11.30~ and have slept very satisfyingl; until woken by an alarm at 6.30 a.m. On the return leg~ perhaps due to delaying sleep until the appropriate ti~e in the citv of arrival, I have had absolu~ely no proble2s of jet lag on the outward trip they were present, but much less than either unt eated or when I have used ~gadon in an atte pt to regularize sleep parterns.

Though I realize that this trail was not blind, I am personally convinced of the efficacy of melatonin in easily and painlessly allowing me to adjust to a new time zone, sleeping at the appropriate local time and remaining awake at other times~ This is in marked contrast with my previous behaviour; I would be very grateful if melatonin could continue to be made available to me on furtner long trips.'`

COMMENT
Although this subject undoubtedly gained considerable benefit from administering Melatonin ln accordance with the method of the present inven-tion, it is interesting to no-te that the Melatonin did not induce sleep when taken on the outward journey following a normal sleep period by Melbourne time. This once again suggests that the beneficial effects of Melatonin in the prevention of jet-lag cannot be attributed solely to its weak hypnotic or tranquillising role.
It seems to have a more fundamental action in re-entraining the body's own sleep-wake rhythm. The experiment described in this example also demonstrates the superiority of the use of melatonin as herein described over traditional pharmaoeutical treatments such as the Mogadon.

~Z5~L4~L

EX~IPLE 8 Subject E Flight from 1Ondon-~ Melbourne-~ London 30.12.83 - 16.1.84 The subject, a 13 year old schoolgirl who had never flown across time zones or taken Melatonin before, arrived in Melbourne on the morning of 31.12.83 feeli~g fairly fresh, but had a sleep in the afternoon. On going to bed at 2100 hrs Melbourne time, she took 5 mg Melatonin and awoke at 0700 hrs the following morning after an excellent night's sleep. She appeared alert and active all the following day, with no symptoms of jet-lag, and again took melatonin on going to bed at 2130 hrsl slept well, and awoke at about 0700 hrs the following morning. Once again, she showed no signs of jet-lag on the 3rd day, and the same procedure was repreated on the ~th day.
On the return journey to London, she was given no `~1elatonin to take. Subject E, and a medical practitioner both reported that she had very great,-difficulty in readjusting her sleep-wake rhythm to London time after her return.
She did not feel back to normal for almost a week.

CO~ENT
It is generally accepted that the adverse effects on circadian rhythms caused by Westerly flights are considered to he lesssevere than those caused by Easterly rlights.
It is interesting that in this example Melatonin appeared to prevent all symptoms of jet-lag following a flight in an Easterly direction by a young girl who h~d never before experienced jet-lag. However, the symptoms were abundantly evident both subjectively and objectively after her return Westerly flight, when she did not take any Melatonin.

` _ 43 _ ~25 EX~iPLE 9 Subject A 23.2.84 In order to investigate the rate of 3bsorbtion and the clearance of orally administered melatonin, and the blood levels that are achieved, SubJect ~ had an indwelling catheter inserted in the antecubital vein of his left arm at 0900 hrs and 5 ml blood samples were withdrawn into a heparinised syringe at frequest intervals before and after swallowing 5 mg Melatonin in a gelatin capsule. This was two hours after a light breakfast OL apple juice, cereal and tea. Blood sampling was continued until 1400 hrs. The blood was immediately centr.ifuged, and the plasma frozen until assayed for melatonin using a specific radioimmunoassay developed in the inventors' laboratory as a modification of the procedure originally described by D.3. ~ennaway et al.
(Endocrinology 110, 1766, 1982.) The results are shown in the accompanying Figure 2.
The basal melatonin values in.the first two samples were slightly higher than the expected normal diurnal values of around 50 pg/ml plasma. The plasma concentrations did not start to rise until 20 min after ingestion of the capsule, and the peak concentration, which was 100 times higher than the pre-ingestion basal values was achieved 66 minutes after ingestion. The values then fell, al-though 260 minutes after ingestion they were still 10 times higher than the normal nocturnal values than one might expect to find of around 100 pg/ml plasma. It was interesting that subjective feelings of drowsiness were experienced when the melatonin concentration first exceeded 2 ng/ml, with yawning and sleepiness coincident with the maximal levels.
If pharmacological blood levels of melatonin are necessary for re-entrainment of circadian rhythms and for inducing sleep, there would be an advantage in prolonging the time for which blood levels are eleva~ed above 2 ng/ml.
l~aintaining such levels should prevent the problem of wa.~ing-up 4 hours during melatonin induced sleep as was the experience of most oE the subjects studied.

- - 44 - ~ ~5~
..

This experiment does demonstrate that when ingested melatonin rapidly reaches a high plasma level the peak level is achieved within 60 minutes. However, the peak level is not maintained as it is also rapidly metabolised.
This experiment clearly demonstrates the need for formulating melatonin into a slow release Eorm. By administration of melatonin in a slow release form it will be possible to maintain high plasma levels for the whole sleep period.
The method of the present invention may be enhanced by using melatonin combined with a known slow release pharmaceutical carrier.

- 45 - ~25~

C

~ ~^
~o ~9 }
~ I O

U) ~INO L~l~W ~d~lS~d ~ 6 -25~

EX~`'~LE 10 Subject ~. si.~ulated conditions o. raDid rotating sh~ ~ work roster 15.1. 84 - 8, ?, 8g, The subject was a 38 year old male psychology lecture; wno had never taken rlelatonin beIore. In order to evaluate the efrects oE ~elatonin on adaptation to rotatinq shift work condi_ions the subject undertoo}; three, eight hour phase advances in hls sleeD-wakeJwork-rest schedule at six day intervals. Mela;onin was ao~inistered under douole blind conditions on the first Dhase advance and placebo taken on the second. In marked con,rast to the DlacebO condition -there was good and accelerated entrainment of the body telmDerature rhyth~ due to Melatonin ad~minis.7a~ion.
This entr~inment was not a secondary consecuence OI sleA? induction.
The data can be taken as evidence for Melatonin as an e~fective chronobiotic for adaptation to shirtwork.

The following constitutes the maiil a ?ects o the method and results of tihe study.

The exDeri~ent was designed to inco-porate the salien_ features of a raDidly rotating shifi-work schedule combined witn asDects oE lighting changes encountered on dest~nation arrival after inter-continental jet Elight. The e~periment involved five stages:
pre-baseline (7 days); stage A, baseline (4 days); stage B, first ~ hour advance oE darkness (~ davs); stage C, second ~ hour advance o. da-kness (~ days), and stage D (~ days), a tnird ~ hou~ advance of darkness and hence return to baseline conditions. Tne e~De imental protocol di,rered from that normally found in shift wo~k in that the subject had to spend the dar'; perioc in bed in ar. isolation unit ~ 47 ~
.. ~

irres?ective OT whether sleeDiness was felt or not; where~s, no sleep was allowed during 'he light period no matter how ti-ed the subject _elt. ~uring the ligh~ period _he subjec~ was 'ree eo leave the isolation un t to engage in work or leisure relate~
activities. An example of details of a dailv activity schedule taken rrom a diary is shown in Table 2.

The e~DerL~ent was carried out over the long vacation, the isolated unit being situated hal~ below ground under one Or the residential colleges on La Trobe University Camous. The area was, there~ore, fairly well bu~rered 2sainst external noi;e and a ventilztion e~haust fan w-as continuously running, which aided eY.traneous noise reduction.
Windows located hioh u? on the walls o_ a ~itcr.en alcove a~d snower/toilet rooms we-e blacked out. ~he main slee?in~-living room was illuminated by two iluorescent tubes (40 w2t') while the alcove and ba.hr3cm-are3 had one, 40 watt, fluorescent lich. in each. All lights were conlrolled by a single ti~e clock se' to ~ive an LD 16:8 cycle, lights on at 0780 hours during baseline A bedside læm? was used for rezding durins the light ?eriod. ~o lign.ing W25 allowed during the dark period ~or any reason. There~ore, events sueh as guencning of thirst and urination had to take ?lace ir. total d~rkness.
Tem~eratu-e of the isoiation area was main.ained within the range ~0-22C throughout the e~?eriment. During stage A and pre-baseline breaklast wa5 inaeste~. between 0800 and 0900, lunch between 100 and 1400; and dinner be_ween 1800 and 2000. During s~ages ~ and C meals ar.d in.e~-~eal intervalc ~ere scheduled -or equivalen_ times lrelative to ligh,s on) to t;~ose du-ing staye A.

48 ~

During stages B and C the subjecl was instructrd to taXe one capsule just prior to lights off. If still awake one hour later, the subject was to ta~e a second ca?sule. Similar instructions aDplied to the second and third hours after lights-off but no caDsule was to be ta~en du-ing the last four hours of dar~ness i.e. max,~T~um number of cassules to be taken was four. Capsules were re-ri~erzted and had to be taken in co~plete aarkness once lights were off. A do~le blind procedure was em?loyed with stage B being drug ~lelatonin, Sisma, 5mg/capsule) and stage C being placebo (sucrose Cilled capsule).
No caDsules were ta~en on the Einal dar}: Deriod at the end or each stage i.e. days 10 and 16.

Rect21 te~perzture was sa~.pled every 12 .minules using a ~ritalog ~.~S
Dor,able microcomputer. A diary of life events was ~eD~ . These included details Or s;eep, wor~, leisure, meals, beveragea, urination ana de~ecation.

During baseline a clear sir.usoidal, daily rhythm ir. rec~al temperzture was reco-ded, with the nadi~ of the rhyth~ occu-ring eithe- at the middle or towzrds the end of the dar}: period. The rhythT was oDstu-eà or. the fi-st day on the phase shift (day 5) and missins da.a on day 7, due to battery failure, make it har2 to envisage the 2evelo?ment o~ the entrainment pattern. Neve-theless, bv the third com~lete day after the ~hase shift (day S) the temperatl~re rhyt.hm was re-es,a~lishec znd in ?hase with thr new light-da-.~ cycle. This is re~ar~ably auic~ re-en~rair~ent, esDecially consiQe-ins the fact that (a) the e~ternal cycle OI night and day and (b) social entrai.~ent facto-s are both com~eting with the entrainment c~?~ci~v of arti~icial ~D cvcle The decrease in body te.~era_ure during the czr}~ ?e~iod 5~

w2s not 2 secondary, exoqenous efrect of melatonir-induced slee~
since the s~e temperature waverorm was seen on da,- 10 when no malatonin was ta~en. Fu--hermore fro~. diary desc-i?.ions, it-~as clear that melatonin did not have the same potent sleep induc.ion as ,_ reported by other subjects afte~ intercontinent~l flig~ was only in ret-ospect, after placebo trea,ment, that melatonin's slee?, tranquility ?ro~erties em2rged. Therefore, in staae ~, Mel2tonin phase shi'ted the rectal temperature rhyt~ and aide sleep i~duction.

In s.age B and sæm~ phenomena ~ere not seen fo- place~o ~reat~ent.
The clear wavefor.~ demonstrated for baseline and da~s 8" ~ and 10 o' ~.elatonin contrast mzr.~edly with t~e er~atic pa.tern under placebo conditions. In the placebo con~itions a strong e~ogenous (mas~ing) com~ponent is found. By day 15 the~'oody ,emDerature rhyth~. h~s phas- delaved bac~ to the original bzselir.e ?attern.~ The peak t'ne-efore now coincices with the new dar}: pe-iod. Slee? and bed rest at the peak of the rhythm ~roduces a secor.da-y trou~h thereby obscuring the peak. Tnis phenomenor is ~uite mar~.ec on days 15 and 16. This interDret~.ion is consis~en~ ~ith tne fac_ that on the firs. ~a~v o- tne third ~hzse shi't td_ys 16J17) wner the subject retur~ec to Ihe original baseline light dar~ cycle, tne d2ily t~Der2ture rhythm im~edia'ely re-ent~ained.

These rindings indicate that e~ogenous melztonin a2minis.ration can entrain a subject's body tem?erature rhy'h~ torota'incs~ scne2ules unde- a_t ~'icial ligh~ dzr~. con2itions despite com~e~.ior. .'rom ~ore dominan= er..raining agents such as the natural en~iro.~ment21 cycle o~' nign, and day an2 pe-iodic soci~l 'acto~s. It is ~-e iclec _h~t a _ _ _ _ _ _ . , . _ . _ _ .. ~ _ _ _ _ _ _ _ _ .. .. . . . _ ~ _ _ . _ . _ .. . _ . ..

co~bination of mel.ator.in treatment, taken at dark onset, with beta blockers taken at beginning of ligh. onset or with high artificial`light levels (~ 2,000 Lu~) would enhance melatonin's phase shifting of circadian rhythms.
Lven without these combinations, melatonin will be useful for aiding adjustment of both rotating and permanent night shift workers to their routines.

~5~
, ' ` ' - 51 -TABLE 2.

Daily ~iary of events for (22.1.8~) tne first cc.-.?~e~e d~v a'ter fi-st ?h2ss shift.
Date ~i~e ~ Locztion and Activity 21.1.84 2332 Awoke to radio and got ~? Just zfter lights on. A
" 2350 Urinated (120ml) Made breakfast - cereal, 4 ~ieces Or bread with blac~ber~y jam; mug of tea with 2 sugars.
2q 1.84 0140 Removed rectal probei ~en, to toilet but cons~i~ated; had showe~.
0205 Walked across can~us to of-~ce. h'o-ked at desk.
0235 Black cor-ee with 2 sugdrs.
0330 Went to Behavioural S-iences cuilaing to work on co~?~ater.
04d5 ~'alk~d bac~ to isolatior. unit to coo',: lunch (a little late~ aa 4 sausaaes, 2 ecgs and 2 pieces of bread.
0535 Urinated (9Oml) 0615 DiA crossword 0640 U~inated (~Oml) Did wzsnins in College laundry and reaa book.
07q5 Very ~a~ ~u. ache. ~7ent to to~let. Work-ed in isolatior unit - re.-.~ing ~n~ w_iting.
0915 Urine 80ml, 0925 Drove to Nortncote to visit f-iends.
1030 Coffee with 2 sucars Went for walk 1230 Sleak Sanà~.~ich (big one) for dinner Cor,~ee with 2 sug~rs Drove back to isolation ~Lnit 1335 U-inatec 250ml.
1400 Sat in sun bu~ too hot so cæme back indoors.
14qO Coffee (dec~-finzted) ~ith 2 5ug2-s.
1435 Sittin~ in chair feeling verv sleeDy. Too .ired to read manus~~i~_.
J.R. a-rived to chec'~ .hat all is well.
1515 J.R left. Cleaned m~ teeth. Took ca~sule.
1525 Urinated 100~1. ~en. to ~ed . ~ ~25~

1530 Lignts ou,.
Fell aslee? after a??ro~ 30 minutes.
1754 Awoke. Urinated into con~zine- in the d~rk.
look secon~ capsule from friàse.

2330 Awoke to ~aio alarm one minute be~ore lights on. Had ~een dreamins. Pad "early mornins" erection.
2344 Urinatea 46Gml.
2350 Breakfast.
.

E'igures 3 and 4 display the forty-eight hour plots of rectal temperature rhythm for subject ~ during the 4 stages of the e~periment. The dark dotted regions represent dark periods. The numbers 1 to 23 iden~ifies 24 hour divisions. Divisions 2 to 22 are repeated to give continuity to the record; i.e. the data is plotted over 48 hour scale in order to aid visualization ~f the waveform.
Stage A constitutes baseline; Stage B an 8 hour advance of darkness when melatonin was taken just before dark onset and again hourly if no sleep occured; Stage C is a second 8 hour advance in darkness with placebo administration;
Stage D a third 8 hour advance of darkness to come back in line with Stage A.- Shaded areas indicate the 8 hours of darkness. Figure 4 is the same as figure 3, but with each stage snown separately.

~25~
- 5~ -It will be clearly understood that the invention in its general aspects is not limited to the specific details referred to hereinabove.

Claims (44)

Claims:

1. A process for the manufacture of a medicament for alleviating the effects of disturbed circadian rhythms on bodily performance and function in a human subject which process is characterized by incorporating melatonin into the medicament as the active ingredient.

2. A process for the manufacture of a medicament for entraining circadian rhythms in a human subject to a predetermined cycle having a periodicity of approximately 24 hours which process is characterized by incorporating melatonin into the medicament as the active ingredient.

3. A process for the manufacture of a medicament for alleviating the effects of disturbed circadian rhythms in a human subject due to undergoing a rapid change in time zones which process is characterized by incorporating melatonin into the medicament as the active ingredient.

4. A process for the manufacture of a medicament for alleviating the effects on disturbed circadian rhythms in a human subject of a rapid transition from one time zone to any number of other time zones which process is charac-terized by incorporating melatonin into the medicament as the active ingredient.

5. A process for the manufacture of a medicament for alleviating the effects on circadian rhythms in a human subject of a rapid transition from one time zone to a second time zone which process is characterized by incor-porating melatonin into the medicament as the active ingredient.

6. A process for the manufacture of a medicament for alleviating the effects on circadian rhythms in a human subject of a rapid transition from one time zone through at least a second time zone and a return to the first time zone which process is characterized by incorporating mela-tonin into the medicament as the active ingredient.

7. A process for the manufacture of a medicament for alleviating the effects on circadian rhythms in a human subject of rapidly altering the 24 hour routine which process is characterized by incorporating melatonin into the medicament as the active ingredient.

8. A process for the manufacture of a medicament for alleviating the effects on circadian rhythms in a human subject of living in an environment substantially free of zeitgebers of 24 hour periodicity which process is charac-terized by incorporating melatonin into the medicament as the active ingredient.

9. A process for the manufacture of a medicament for alleviating the effects of a disturbance in the temperature circadian rhythm in a human subject which process is characterized by incorporating melatonin into the medi-cament as the active ingredient.

10. A process for the manufacture of a medicament for entraining a temperature circadian rhythm in a human subject to a predetermined cycle having a periodicity of 24 hours which process is characterized by incorporating melatonin into the medicament as the active ingredient.

11. A process for the manufacture of a medicament for alleviating the effects of a disturbance in the endogenous melatonin circadian rhythm in a human subject which process is characterized by incorporating melatonin into the medicament as the active ingredient.

12. A process for the manufacture of a medicament for entraining the endogenous melatonin circadian rhythm in a human subject to a predetermined cycle having a periodicity of 24 hours wherein the medicament is to be administered at a time which coincides with the upward phase of the pre-determined cycle which process is characterized by incorpo-rating melatonin into the medicament as the active ingredient.

13. A process according to claim 1, 2 or 3 wherein the medicament is adapted to oral administration.

14. A process according to claim 4, 5 or 6 wherein the medicament is adapted to oral administration.

15. A process according to claim 7, 8 or 9 wherein the medicament is adapted to oral administration.

16. A process according to claim 10, 11 or 12 wherein the medicament is adapted to oral administration.

17. A process according to claim 1, 2 or 3 wherein the dose of melatonin is in the range 1-10 mg.

18. A process according to claim 4, 5 or 6 wherein the dose of melatonin is in the range 1-10 mg.

19. A process according to claim 7, 8 or 9 wherein the dose of melatonin is in the range 1-10 mg.

20. A process according to claim 10, 11 or 12 wherein the dose of melatonin is in the range 1-10 mg.

21. A process according to claim 1, 2 or 3 wherein the melatonin is combined with a pharmacologically acceptable carrier.

22. A process according to claim 4, 5 or 6 wherein the melatonin is combined with a pharmacologically acceptable carrier.

23. A process according to claim 7, 8 or 9 wherein the melatonin is combined with a pharmacologically acceptable carrier.

24. A process according to claim 10, 11 or 12 wherein the melatonin is combined with a pharmacologically acceptable carrier.

25. A process according to claim 1, 2 or 3 wherein the melatonin is replaced with melatonin mixed with at least a melatonin agonist so as to achieve a composition that is pharmacologically active for the purposes of the present invention.

26. A process according to claim 4, 5 or 6 wherein the melatonin is replaced with melatonin mixed with at least a melatonin agonist so as to achieve a composition that is pharmacologically active for the purposes of the present invention.

27. A process according to claim 7, 8 or 9 wherein the melatonin is replaced with melatonin mixed with at least a melatonin agonist so as to achieve a composition that is pharmacologically active for the purposes of the present invention.

28. A process according to claim 10, 11 or 12 wherein the melatonin is replaced with melatonin mixed with at least a melatonin agonist so as to achieve a composition that is pharmacologically active for the purposes of the present invention.

29. A process according to claim 1, 2 or 3 wherein the melatonin is replaced with a melatonin agonist.

30. A process according to claim 4, 5 or 6 wherein the melatonin is replaced with a melatonin agonist.

31. A process according to claim 7, 8 or 9 wherein the melatonin is replaced with a melatonin agonist.

32. A process according to claim 10, 11 or 12 wherein the melatonin is replaced with a melatonin agonist.

33. A process according to claim 1, 2 or 3 wherein the melatonin is compounded into a slow release form.

34. A process according to claim 4, 5 or 6 wherein the melatonin is compounded into a slow release form.

35. A process according to claim 7, 8 or 9 wherein the melatonin is compounded into a slow release form.

36. A process according to claim 10, 11 or 12 wherein the melatonin is compounded into a slow release form.

37. A pharmaceutical composition in capsule form for the re-entrainment of disturbed circadian rhythms in a human subject comprising 1-10 mg melatonin in admixture with a pharmaceutically acceptable diluent.

38. A pharmaceutical capsule for the re-entrainment of disturbed circadian rhythms in a human subject, said capsule containing 1-10 mg melatonin and a pharmaceutically acceptable diluent.

39. A pharmaceutical composition in slow release form for the re-entrainment of disturbed circadian rhythms in a human subject comprising 1-10 mg melatonin in admixture with a pharmaceutically acceptable diluent.

40. A preparation for oral administration comprising melatonin and a pharmaceutically acceptable diluent the preparation being adapted to release melatonin over an extended period upon administration to a human.

41. A pharmaceutical capsule for the re-entrainment of disturbed circadian rhythms in a human subject, said capsule containing 1-10 mg melatonin and a pharmaceutically acceptable diluent, said capsule being capable of releasing melatonin over a long period of time upon administration to a human.

42. A preparation for oral administration comprising melatonin mixed with at least a melatonin agonist so as to achieve a composition that is pharmacologically active for the purposes of the present invention.

43. A package containing melatonin in capsule form, 1-10 mg per capsule, said package including instructions for administration thereof for the alleviation or prevention of ill effects associated with disturbance of circadian rhythms in a human subject.

44. A process for preparing a pharmaceutical composi-tion in unit dosage ready-to-use form for the re-entrain-ment of disturbed circadian rhythms in a human subject which process is characterized by incorporating in the pharmaceutical composition as the active ingredient 1-10 mg melatonin.