WO1988007367A2

WO1988007367A2 - Melatonin-derivatives for psychogenic stress and acute anxiety, and immuno-stimulant drugs

Info

Publication number: WO1988007367A2
Application number: PCT/EP1988/000270
Authority: WO
Inventors: Walter Pierpaoli; Giorgio Maestroni
Original assignee: Cellena (Cell Engineering) A.G.
Priority date: 1987-04-01
Filing date: 1988-04-01
Publication date: 1988-10-06
Also published as: EP0311651A1; WO1988007367A3; AU1494788A

Abstract

The invention relates to the use of melatonin or related active chemical compounds for the production of pharmaceutical compositions for the treatment of psychogenic stress or acute anxiety as well as for the enhancement of immune resistance in a host against viral and parasitical infections.

Description

MELATONIN-DERIVATIVES FOR PSYCHOGENIC STRESS AND ACUTE ANXIETY, AND IMMUNO-STIMULANT DRUGS

The invention relates to compositions applicable to man or animal, containing melatonin or chemical homologues, derived from indolamines for use in the treatment of psychogenic stress or of related acute anxiety conditions created by distressing circumstances, as well as for the production of immune-stimulant drugs against viral and parasitical infection. More particularly the invention relates to compositions containing said melatonin or chemical homologues in association with a pharmaceutical vehicle for the abovesaid uses. Preferred compositions are those suitable for parenteral or oral use.

Psychogenic unescapable stress or acute anxiety stress conditions of the type referred to herein is often caused in animal when the latter is suddently brought in a non-natural environment. This may be the case e.g. for cattle during transportation from one place to another or when gathered on a ground close to the slaughterhouse. It is well known that, particularly in the last mentioned circumstrances, cattle can develop acute stress to an extent such as to induce heavy organic diseases likely to make them unsuitable for later consumption. Similar anxiety conditions can also affect livestock which is raised in non-natural conditions, such as cattle or pigs which are maintained under restraint in stables for the purpose of accelerated growth.

Such acute anxiety can also develop in man. For instance, it is well known that cancer-patients undergoing heavy chemotherapy often suffer or acute anxiety which in itself may accelerate the fatal outcome of the disease, at least seriously impair the chances of recovery of such patients. Similar anxiety can develop in soldiers on the battle field or under less dramatic circumstances, in persons maintained over prolonged period of time in an unusual environment. This may for instance be the case for seamen in submarines remaining under water for prolonged missions.

Thus the invention aims at providing drugs for treating psychological and emotional conditions of most varied nature and origins.

It has been suggested that such psychological factors and stress seem to modify the production in the host concerned of the production of endogenous melatonin (N-acetyl-5-methoxytryptamine) by the pineal gland. The. role of this hormone in vivo is not yet fully understood. Observations have been made that the production of this hormone is under control or circadian cycle. The amount of melatonin which is produced in vivo, is increased in night time or darkness and is known to fall rapidly in day-time or when the host is subjected to light of strong intensity.

The invention is based on the discovery that melatonin, particularly when administrated orally or parenterally, is capable of providing efficient relief in humans or animals afflicted with heavy psyhogenic or acute, apparently unescapable anxiety.

In another field the invention deals with the relief in man or animal of viral and parasitical infections. The ability of viruses to alter immune responses dates back to Von Pirquet who showed an individual with measles with depressed anti-tubercular responses.

Natural resistance to virus infection involves macrophages, lymphocytes and NK cells which interact with one another and in the production of leukokine immuno modulators which alter resistance to infection.

Examples of viral resistance modified by macro phage include flaviviruses, hepatitis viruses, herpe virus and myxoviruses (S.C. Mogensen et al, "Macrophages and genetically determined natural resistance to virus infection. In: Viruses, Immunity and Immunodeficiency". Ed. Szentiuanyi A, Friedman H. Plenum Press, NY, 1986, pp. 13-24).

H. Friedman et al ("Virus interactions with the immune defense system". In: Viruses, Immunity and Immunodeficiency, Ed. Szentiuanyi A, Friedman H. Plenum Press, NY, 1986, pp. 25-39) lists viral infections compromising the immune system : cell mediated or humoral, and the cell type affected : macrophages, B cells, T cells, as it relates to the type of virus infection. In addition, the tabled lists some of the parameters of immune responsiveness suppressed by viral infection (table 2).

The mechanism of immunosuppression can be mediated by direct intracellular viral proliferation or targeted fixation of the virus to the immunocyte or macrophage. Alternatively, it can relate to products produced by the virus, or the host reaction to the virus.

Retrovirus infection from which the AIDS syndrome is derived have been immunologically linked to immunodeficiency in hosts as varied as poultry, mice and man. These agents effect the broad spectrum of immune response including antibody production and responsiveness, immunologic maturation, delayed hypersensitivity, graft rejection and T cell cytotoxic responsiveness.

The invention is also based on the further discovery that melatonin is an "up" regulator of the immune response that stimulates host immunologic compensation for viral or infection induced immunosuppression. It does this by bath a primary effect as a T cell stimulating neurσhormone and as an agent ameliorating the effects of stress secondary to virus action and infection.

Melatonin, because of its action, as an immunostimulator, can modulate and attenuate the severity of retrovirus induced immunosuppression or, alternatively, alleviate the additive or synergistic toxicity of the association cytomegalic herpes and other viruses that accompany the primary decline in resistance induced by the AIDS (HIV) virus.

Of clinical relevance for vaccination procedures is the report here that melatonin is a main regulator of immune resistance in its broadest significance. The level of its plasma concentration during primary against viral or parasitical sensitization provides regulation of the amplitude and duration of the secondary, memory response. It has been found that administration of melatonin in the pre-darkness, evening hours, affects primary and especially secondary (memory) response by potentiating persistently specific immunity to pathogenic viruses and parasites, such as Plasmodium falciparum or related parasites which are responsible for malaria, as well as resistance against said viruses and parasites.

The invention thus relates to the production of pharmaceutical compositions for use in the treatment of psychogenic stress, acute anxiety conditions or both, on the one hand, and of pharmaceutical compositions for use as immunostimulants to protect human or animal hosts against pathogenic viruses or parasites, on the other hand.

The preferred active principle of the compositions of the invention for the above-mentioned uses consists of melatonin itself. The invention also contemplates the use of chemical homologues for the same purpose, there should be mentioned more particularly the class of compounds which can be represented by the general formula :

in which :

- n is 1 or 2,

R₁ and R₂, identical or different from each other, are -H, -NH₂, -COOH, -OH, acyl, -NH-acyl or alcoxy, said acyl or said alcoxy comprising from 1 to 4 carbon atoms

X is -OH or alkoxy comprising from 1 to 4 carbon atoms,

- Y is -H, -OH or -NH₂.

Preferred compounds for use in the compositions of the invention are those in which Y is hydrogen and Xis methoxy. The most preferred compound is melatonin itself, the formula of which is

Other preferred compounds for use in the compositions of the invention are :

- 5-methoxytryptamine,

- 5-methoxytryptophan, - 5-methoxytryptophol,

- 5-methoxyindole-3-acetic acid,

- 6-hydroxy-melatonin.

These compounds can be obtained by synthetic precesses, general methods of manufacture which can be derived from those published by J. SZMUSZKOVTCZ already mentioned, J. SUPNIEWSKI et al, "Synthesis of melatonin (5-methoxy-N-acetyltryptamine", published in Bull. Acad. Polon. Sci. Biol., 8, p. 479-481, 1960 ; or MASHKOVSKY et al, in Farmakol. Toksikol., 26, n·1, 10, 1963, said methods being of course in each case adapted to the particular compound sought.

Further details will appear from the following disclosure of examples which establish the capability of melatonin and related compounds to overcome the effects of stress in vivo. Reference will be made to the drawings in which :

- figure 1 reflects diagrammatically the capability of melatonin to overcome in vivo a major effect of stress i.e. the depression of immune responsiveness to antigenic stimuli which can be observed in a stressed host, figure 2 is a reproduction of photographs of two groups of mice which had received the same sublethal dose of a most pathogenic virus and which underwent a confinment treatment causing acute stressing, yet whereby one of the groups was treated with melatonin and the other was not.

Further details relating to the capability of melatonin. and related compounds to also effectively enhance the immunoresistance of a host against pathological viruses and parasites will also appear from the further disclosure of example 5.

The tests refferred in examples 1 to 4 which follow bring into play the known immunosuppressive effects induced by stress on animals, in other words the capability of otherwise sublethal doses of a live pathogen to overcome the immune response partially immunosuppres sed in the stres sed host. The examples which follow provide evidence of basic experimental and clinical significance that exogenous melatonin can completely abrogate the effects that anxiety stress induced by physical restraint produced on thymus weight, antibody production and on resistance to sublethal inocula of encephalomyocarditis

(EMC) virus in mice.

Example I :

Female, 2-3 months old BALV/cj mice were divided in three groups, i.e. in a group A (15 mice) which was injected subcutaneously (s.c) with 0.5 ml of 0.2 % V/V ethanol in phosphate saline (PBS), a group B (15 mice) which was injected with melatonin (Biosynth AG, Staad, Switzerland, 20 μg/kg body weight, b.w.) dissolved in ethanol and diluted in

PBS to a final concentration of 0.2 % ethanol,

- a group C (14 mice) which was left untreated.

The injections were performed every days at 4 p.m, over a period of four days, approximately 2 hours before onset of darkness. The three groups were kept under a 12 hours light cycle (6 a.m. to 6 p.m.) with free access to food and water. Groups A and B were stressed by restraining them in 50 ml plastic tubes with

10.5 mm-wide ventilation holes. The operation was repeated every day for 4 days from 10 a.m. to 12 a.m.

The restraint produced anxiety but not complete immobilization. Control C group was not stressed.

All mice were injected on the first day at 1 p.m. with 4 x 10⁸ SRBC i.p. (0.2 % V/V ethanol).

Four days after antigen injection, the mice were sacrificed and the primary antibody response to sheep red blood cell's (SRBC) was evaluated by measuring the number of direct (IgM) plaque forming cells (PFC) in the spleen by the conventional haemolytic plaque assay (Jerne test). Briefly, 2 ml of 1.2 % agarose (Difco

Labs, Detroit, USA) in DUBELCCO's PBS was pipetted in 6 cm petri dishes to constitute the bottom layer. 0.8 ml of 0.75 % agarose in Eagle's MEM medium was mixed with

45 μl of 20 % SRBC and 45 μl of a 1:10 dilution of the spleen cell suspensions to be assayed for PFC. Direct,

IgM-mediated plaques were revealed by the addition of

0.4 ml of absorbed Guinea pig complement. p values were obtained by the analysis of variance of melatonin versus

PBS values compared at equal doses.

Thymus and body weight were also measured. The variances were analysed ans the results appear in table

I hereafter, i.e. *^* p < 0.01 A versus B and versus C, * p < 0.05 A versus B and versus C.

TABLE I

GROUP (n) STRESS TREATMENT PFC/SPLEEN mgTHYMUS weight

g BODY weight

A (15) + PBS 99903134420** 1,67±0,57*

B (15) + Melatonin 199157±62888** 2,35±0,75*

C (14) - - 182035±65881 2,39±0,63

The table shows that the severe immunodepression caused by the immobilization stress in group A was completely overcome in the melatonin-treated mice of group B. As a matter of fact the primary antibody responses, as well as the thymus weights, appeared to be of the same order of magnitude in the melatonin-treated animals and in the controls. Example 2 :

This example demonstrates that the evening administration of melatonin reverses the impaired immune resistance of mice stressed acutely by physical restraint and inoculated with a sublethal dose of encephalomyocarditis virus (EMCV) .

The conditions under which this experiment was carried out are stated in the legend under table 2 hereafter.

TABLE 2

(n)^a STRESS^b TREATMENT^d SURVIVAL^e

3 days 6 days 10 days %

13 + PBS only 11 8 2 15

13 + Melatonin 12 11 10 77

13 - Untreated 13 11 11 85

a) Total number from 2 experiments.

Mice were female BALB/cJ, 2-3 months old. b) individual mice of the first and second groups were restrained for 4 days as described in the preceding example. c) 0.2 ml of 10 ^-8 suspension of EMC virus in phosphate saline (PBS) was injected s.c. immediately after the first stressing session. d) Melatonin (40 μg/kg b.w. in 0.2 ml PBS, 0.2 % V/V ethanol) was injected i.p. at 4 p.m. for 10 days from the day of EMC virus inoculation and stressing procedure. Controls were injected with PBS (0.2 % ethanol) only. e) Days after inoculation of EMCV and initiation of restraint stress. The mice were killed a few hours after apearance of irreversible paralysis of the hind legs.

The "surviving mice" did not show any sign of paralysis. It will be appreciated that the rate of survival of the restrained melatonin-treated animals which resisted the sublethal dose of the (EMCV) was close to the rate of survival in unrestrained animals which had been given the same lethal dose. The high survival in these two groups are in sharp contrast with the low survival rate in the restrained animals which did not receive the melatonin treatment.

Example 3 :

This example shows the results of an other assay establishing that the evening administration of melatonin reverses the impaired immune resistance of mice stressed acutely by physical restraint and inoculated with a sublethal dose of encephalimyocarditis virus (EMCV).

Female 2-3 months old BALB/cJ were inoculated s.c. with 0.2 ml of 2 x 10^-8 dilution of EMCV in saline on day 0.

The mice were divided in groups and two of them restrained two hours per day for 4 days as described

(see materials and methods). One of these groups was treated daily for 10 days with 1 μg of melatonin i.p. at

4 p.m.

The remaining stressed group was treated with saline only as control.

The third group was neither stressed nor treated.

Rates of survival in the 3 experiments (each group comprised initially n = 25 mice) are recorded as percentage and reported ± the standard deviation.

Figure 1 illustrates eloquently the results obtained. The rate of survival in stressed PBS-treated after 10 days was very low, in contrast with the rate of survival of stressed melatonin-treated animals, which was of the same order of magnitude than that of non- stressed controls which had received the same amount of EMCV.

Figure 2 also shows the conditions of the mice in the melatonin-treated group and in the PBS-treated group. Whereas the mice of the melatonin-treated group appear in good condition, with a soft fur, the mice that had been stressed showed all signs of a distressed condition including each hair of their fur standing up individually on their backs and installation of paralysis in their hind legs. Example 4 :

This example shows the reversal of the immunosuppression of the response to an antigen caused in mice treated with 100 mg/kg of body weight of cyclo- phosphamide, i.e. a powerful drug used in heavy cancer- chemotherapy.

The results are plotted in table III hereafter

TABLE III

n·of Spleen PFC/10⁶ Group (n) Treatment Cellsx10⁴ Spleen PFC/Spleen cells

A (10) Melatonin

Cyclophosphamide 169 ±30 30±15 6098±1291

Cyclophosphamide B (10) + melatonin 190±37 165±39 31,855±11,379

C (10) PBS(control) 269±37 786 92 199, 170±42, 381

BALB/c inbred female mice, 60 to 90 days old were on a 12-hour light cycle with free access to food and water. Animals were "randomized" into groups (A, B, C). the mice of groups A and B were injected i.p. with 100 mg/kg b.w. of cyclophosphamide on the day before antigen injection. Each evening at 4 p.m. the mice of groups B were injected i.p. with melatonin (2 mg/kg) b.w.). On day 3 the mice were injected i.p. with 4 x 10 ² SRBC and the number of PFC/10⁴ spleen cells was assessed by the conventional PFC assay 4 days later. * p < 0.05 (B versus A) * P > 0.011 (B versus C)

Table III shows that the immunosuppressive effect of the chemotherapeutical drug is antagonized by melatonin at least to a substantial extent, also in this assay model.

Thus these pharmacological new properties of melatonin provide clear evidence of its capability of protecting hosts (man or animals) against psychogenic stress or acute psychological distress in exceptional circumstances or non-natural environment. Because of its well-known innocuity, melatonin can be used at the most efficient dosages to provide for the result sought, i.e. relief of such stress whatever the circumstances which caused it.

The invention provides more particularly a solution of an acute problem in animal husbandry or in animals under the dramatic conditions which have been recalled in the preamble.

Example 5 :

Reference is first made to the materials and methods which have been used in that further example.

Animals . BALB/cJ, C57BL/6J, BALB/c-Bolm and C3H/He inbred female mice, aged 2-3 months, were used. The mice were bred in our laboratory or purchased and were maintained under conventional conditions in rooms at 22 ± 1 C with a dark-light cycle of 12 hours. Great care was taken to avoid environmental stress before and during the course of the experiments (noise, smells, cage crowding and so on).

Melatonin solutions. Melatonin (MEL, N-acetyl-5-methoxytryptamine was purchased from Biosynth Inc., Staad, Switzerland. Solutions were obtained by dissolving MEL in a minimal volume of absolute ethanol and diluting with sterile bidistilled saline to a final 1% alcohol-water dilution. Controls of mel atonin-injected mice were always injected with ethanol-phosphate saline solutions without melatonin.

Circadian inoculation of melatonin When not differently mentioned, a dose of 0.5 or 1 ug of melatonin (about 20 μg and 40 μg/kg body weight, b.w.) prepared as mentioned above was injected s.c. in 0.2 or 0.5 ml volume. The morning injections were done at 8 a.m. and the evening injections at 4 p.m.

Haemcrtytic plaque forming cell (PFC) assay The number of spleen plasma cells producing either direct (igM) or indirect (IgG) plaques after immunization of mice with sheep red blood cells (SRBC) was evaluated by the conventional agar haemolytic PFC assay in petri dishes (Jerne test) IgG producing cells were revealed by the addition of a suitable dilution (1:30) of Fc-specific rabbit anti-mouse IgG followed by exposure to SRBC-absorbed guinea pig complement.

Antiviral cytotoxic T cell response The method used js essentially that described by Zinkernagel and Doherty, Adv.Immunol.,27,51 (1979). Briefly, according to the experiments 1x10⁶ pock forming units (pfu) or 8x10⁶ pfu of Vaccinia virus ( Serum Institute, bern, Switzerland) suspended in 200 ul of phosphate saline (PBS) were injected intravenously (i.v.) through the retroorbital plexus. Target cell lines were L929 (H-2^k) and D2 (H-2^d) and were maintained in IMDM, 2% foetal calf serum (FCS) at 37 C, 5% CO₂. 5x10⁶ cells were pelletted in a 15 ml conical tube and resuspended in 600 ul of complete medium contaning 4x10⁷ pfu of vaccinia virus. These cells were incubated and gently shaked for 1 hour at 37 C. Infected and an equal number of non-infected cells were then incubated with 200 uCi of ⁵¹Cr-sodium chromate (Amersham Int., Amersham, UK) for 1 hr at 37 C. After this labeling procedure, the cells were washed 3 times, counted and diluted to a final concentration of 10⁵ cells /ml. Infected and non-infected 5¹Cr-labeled target cells were mixed in 96-well, U-bottomed microplates (Nunc, Roskilde, denmark) with effector spleen cells at different effector:target ratios (50:1, 25:1, 12:1, 6:1). The final volume was 200 ul./well in triplicate for each sample. After 5 hour incubation at 37 C, 5% CO₂, the microplates were spun at 500g and 75 ul/well were taken and counted in a gamma counter. Specific ⁵¹Cr release was calculated as follows:

where total release is given by infected or non-infected cells lysed with IN HCl .

Preparation of encephalomyocarditis (EMC) virus

0.02 ml of a preparation of EMC virus were injected intracranially into the brain of 20 ether-anesthetized BALB/cJ adult male mice. The brains were removed during the acute, paralysis-myocarditis stage and a homogenate was prepared with an all-glass Potter at 1:10 weight/volume concentration in isotonic saline and stored in 0.5 ml aliquots at -30 C. The same EMC virus preparation was used for all the experiments done.

Immunization-infection with EMC virus When needed, fresh suspensions from 1:10 dilution of EMC virus were prepared in cold isotonic saline and kept in ice until inoculation. The mice were injected s.c. with a standard volume of 0.2 ml.

Primary sensitization to EMC virus (vaccination procedure) For the initial sensitization of the mice against EMC virus a dilution of 5 x 10^-9 was chosen. The large majority of the mice over 20 grams body weight did not contract the infection and were partially protected by a subsequent lethal dose.

Virus neutralization test (VNT) For evaluating the virus-neutralizing properties in the EMC-injected mice, a modification of the method described by E.H. Lennette, N.J. Schmidt, eds., in Diagnostic

Procedures for Viral and Rickettsial Infections, fourth edition (American Public Health Association, New York, 1969), p. 762, was adopted. Blood was obtained from the retroorbital plexus of ether anesthesized mice with a Pasteur pipette. Serum from individual mice was kept separate. The sera were inactivated for 30 minutes at 56°C and storel at -30°C until used. Fresh 1 x 10^-7EMC virus suspensions were prepared from the original 1:10 brain homogenates in sterile isotonic saline. 100 ul of serum from individual mice was mixed with 50 ul of 10^-7 EMC virus. The tubes were shaken and incubated for 30 minutes at 37°C in a water bath. Groups of 3 to 5 normal female BALB/cJ aged 2-3 months were injected i.p. with the virus-antiserum mixtures, 0.15 total volume per mouse. The mice were daily checked for paralysis of the hind legs and mortality. The mice were killed a few hours after the appearance of orreversible paralysis and myocarditis.

The conditions under which the tests were ran follow from the tables hereafter and the legends which accompany them.

Table 1. Administration of melatonin during primary immunizatior enhances the secondary antiviral cytotoxic T cell response to Vaccinia virus in mice.

Effector % of specific ⁵¹Cr release -± S.D. % to P B S MELATONIN VARIATION

(melatonin target (n = 8) (n = 8) vs (ratio) P B S)

50 : 1 40.0 ± 4,6 52,3 ± 4,1* + 30

25 : 1 31,7 ± 6,1 44,8 ± 3,8* + 41 12 : 1 23,5 ± 4,6 32,7 ± 3,0* + 39

6 : 1 14,4 ± 3,8 23,2 ± 3,5* + 64

C3H/He female mice, two months old, were injected i.v. with 1 x 10⁶ PFU of Vaccinia virus (VV) at 12 a.m.. On the following 6 days the mice were injected s.c. with melatonin (40 μg/ kg b.w.) at 4.00 p.m. 2 hours before onset of darkness. Control mice were inoculated s.c. with 0,5 ml of PBS at the same hour. After this treatment the animals were kept undisturbed for 7 weeks with free access to food and water and under a 12 hours light cycle. A booster injection of 8 x 10⁶ PFU of VV i.v. was then performed. Neither melatonin, nor PBS was inoculated during this second immunization. After 4 days the antiviral cytotoxic T cell response was tested by the ⁵¹Cr release assay. Effector spleen cells were diluted with L929 target cells at the following ratios: 50 : 1,

25 :1, 12:1 and 6:1. % of specific ⁵¹Cr release ± standard deviation (S.D.) was calculated according to the formula described in materials and methods.

^*P <0,01

RESULTS

Effect of melatonin on immunization to Vaccinia virus The secondary T cell-mediated cytotoxic antiviral response to a booster injection of Vaccinia virus was evaluated in mice that during primary immunization were treated or not with melatonin. The results of these experiments are shown in table 2. Spleen cells from mice that during primary immunization with 10⁶ pfu of Vaccinia virus were i njected for 7 days with evening melatonin showed a significantly higher

(p<0.01) T cytotoxic antiviral response than spleen cells from control mice that were treated with PBS. In fact, specific ⁵¹Cr-release was consistently higher in melatonin treated mice at all effector to target cell ratios (table 1). Thus, also a most significant antiviral response, i.e. the T-cell -mediated cytotoxic secondary response can be augmented simply by melatonin treatment during primary immunization.

Effect of melatonin on resistance and immunity to encephylomyocarditis (EMC) virus.

A virus which is highly pathogenic for rodents was chosen (EMC, 10) and obtained as described in material and methods. Many series of experiments were performed in 2-3 months -old female BALB/c mice in order to choose a dosage which would not rapidly produce paralysis and death and in which an influence of melatonin may be possibly studied. As known (W.D.

Bresser, in Handbook of Experimental Immunology, D.M. Weir, ed.

Blackwell Scientific Publ., Oxford, 1979, p. 28) EMC virus in very minute concentrations can produce irreversible paralysis and behaves similarly to poliomyelitis virus.

A first immunisation was carried out with a non lethal dose of EMC virus (see materials and methods) in the presence of melatonin.

No antibodies to the EMC virus were measured in the course of primary immunization.

Four to six weeks after infection with the non-lethal dose of

EMC virus and treatment with melatonin, the mice which survived immunization and treatment with melatonin were injected again with a lethal dose (5 x 10^-7) of EMC virus without further treatment with melatonin. As can be seen in table 2, at 3 and 10 days after re-in fection,a larger number df the mice which had been treated with evening melatonin in the course of the primary immunization (vaccination) survived to the lethal dose of EMC virus. When blood was taken from the re-infected mice at 3 days after re-infection and the virus neutralizing capacity of the serum was tested in vivo (see materials and methods), the serum obtained from mice which had been treated with evening-melato nin in the course of primary immunization displayed ahigher virus-neutralizing capacity as measured by the number of surviving mice at 10 days after inoculation of the virus-antiserum mixtures in individual animals (Table 2). Thus definitely, the evening administration of melatonin in the course of primary infection to a vaccinating dose of the EMC virus was able to generate more protection and a higher response when the same mice were injected again with a lethal dose. In this experimental model, protection of melatonin was solely concerning higher production of circulating antibodies in the evening treated mice.

The experiments reported above demonstrate that evening, pre-darkness administration of melatonin in the course of primary sensitization to viruses not only enhances the primary immune response but also remarkably increases the responsiveness of the primed mice to a second inoculation of the same antigens (memory response) eve without further administration of melatonin.When antigen of primary sensitization is a vaccine (small-pox), the mice treated with melatonin during primary immunization display a much higher, H-2 restricted cytotoxic reactivity against virus infected cells. When antigen of the primary immunization is a highly aggressive (EMC ) virus, an higher number of melatonin-treated mice survive to a second, lethal virus infection. Virus specific antibodies are also clearly enhanced in as shown by the virusrneutralizing assay in vivo. These findings are encouraging in respect of the use of melatonin in active immunization or vaccination agsinst viral and parasitic antigens.

Although no information is yet available on the mechanism by which the administration of melatonin during primary immunization to T-dependent antigens produces a subsequent optimalization of the secondary response, it can be hypothesized that it promotes the proliferation or functions of helper T lymphocytes that in turn optimize the formation of "memory" T and B cells. By this way a subsequent infection or antigen inoculation determines maximal amplification of the response. This seems to be proven by sensitization Vaccinia virus and EMC virus in which clearly evening-melatonin treated mice produce more antibodies and a stronger cytotoxic activity than controls or morning-melatonin treated mice.

In the preceding example, melatonin was used only during the primary vaccination. Needless to say that melatonin may also be administered to the host when subjected to the second vaccination and other boostings whenever necessary, depending upon the nature of the vaccinating virus antigen or parasite-antigen.

Melatonin is not able to stimulate the resistance of the host only in vaccination experiments. the immunostimulant activity of melatonin can also be brought to light when the host is afflicted with viral or parasitic infection.

The benificial action of melatonin has been shown in AIDS patients.

Four AIDS patients were injected daily at 5 p.m. with a dose of 20 mg of melatonin over two weeks. The patients underwent a substantial relief.

Serum samples were taken up from these patients. The mononuclear cells, including T-lymphocytes were separated and incubated in the presence of phytohemagglutinine and of tritiated thymidine.

After 3 days of incubation, the amount of tritiated thymidine incorporated in the lymphocytes was measured.

The same experiments were run on mononuclear cells from the same patients and which had been taken up before the begining of the treatment.

On comparing the results, it was established that an increase by 200-300 % of the thymidine incorporation had been obtained in all melatonin-treated patients, which result established an increase of the immune reactivity of their T-lymphocytes although the T8/T4 ratio was not changed. Nevertheless, increased immune reactivity of the lymphocytes considered as a whole accounted for the improvement of the health condition observed in these patients. first The invention thus relates/more particularly to the use of melatonin and its active chemical homologues for the production of pharmaceutical compositions useful in the treatment of psychogenic stress or acute anxiety.

For the ease of language hereafter, the expression "melatonin" is used hereafter to designate both the actual melatonin and the active chemical homologues thereof.

A preferred composition of this invention contains melatonin associated with a pharmaceutically acceptable vehicle, wherein said melatonin is in an amount effective to relieve the host from psychogenic stress or anxiety.

The other components, particularly vehicles of such compositions, can be any of those which can be tolerated by the patients.

When oral use is considered, effective daily dosages range from about 0.01 to about 100 mg/kg a day, preferably from 0.1 to 10 mg/kg a day, the orally administrable compositions being dosed accordingly.

But the amount of active ingredients may be more or less depending on the specific condition to be treated and the degree of treatment needed.

The invention also relates to melatonin in the form of injectable compositions for the same uses. Effective dosages range from 0.01 to 5 mg/kg a day,

preferably from 0.1 to 1 mg/kg a day.

Needless to say that other forms of administrations for the same purpose, e. g. by the rectal route, can be contemplated as well.

There is no need to emphasize that the man skilled in the art will be able to select the most appropriate galenic compositions to achieve the results sought.

Most effective results will be obtained in the host upon administering said melatonin doses at a time between sunset or little earlier, say 4 P. M., and the time at night when the host is to go on sleep.

The duration of treatment will of course depend on the host and the strees-inducing circumstances.

In a human patient the treatment may be furthered over from one to two weeks or more if appropriate. In animal raised in a non-natural environment the dosage may be adjusted bearing in mind that the treatment may have to last longer. Conversely in animals which are to be slaughtered shortly thereupon, the dosages used may be enhanced to have a more rapid effect whenever necessary.

Melatonin and related homologues are also of use for the production of drugs or pharmaceutical compositon for use to combat non-specifically diseases caused by viruses or parasites, of at lest alleviate them.

The uses indicated previously by way of example are not exclusive of other uses bringing into play the immunoregulator properties of melatonin. It is possible also to cite by way of example its reinforcing action at the level of the specific immunization of the host with regard to parasitic antigens, the restoration of the immunocompetence of the host, when the latter is at a lower level than normal, notably when the latter has been damaged by antigens or parasites themselves, or under the effect of chemotherapy, radiotherapy, or of any other treatment which has an immunosuppressive action.

The pharmaceutical compositions containing melatonin, are useful for the treatment or the prevention of infectious diseases of viral or parasitic origin.

Melatonin or its active chemical homologues may be administered to the host-animal or human being - in any suitable manner to obtain the desired effect. When used as an adjuvant, melatonin and the viral or parasitic vaccinating antigen, may be administered simultaneously or separately, in the latter case at suitable staggered time intervals either by similar or different routes of administration (for example parenteral and oral routes respectively or vice versa).

The invention relates more particularly to such suspensions, solutions or liposomes which are suitable for administration by intradermal, intramuscular or subcutaneous injection, or again by scarification.

Melatonin may also be administered by other routes, e.g. by the rectal route, or in the form of aerosols designed to come into contact with the mucous membranes, notably the ocular, nasal, pulmonary or vaginal mucous membranes. Effective use of melatonin or its chemical homologues with a view of reinforcing the immune defenses of the host, comprises administering to the latter an effective dose of said melatonin or homologues, in one of the administration forms which have been mentioned above, preferably from sunset, or a little earlier, say from 4 P.M. up to the time where the host goes to sleep. Examples of daily doses capable of inducing an effect, range from 0.01 to 100 mg, preferably from 0.1 to 10 mg per kg of body weight, when the administration is effected by the oral route, or again from 0.01 to 5 mg, preferably from 0.1 to 1 mg per kg of body weight, when administered by the parenteral route.

The daily administrations are preferably repeated daily over periods of one or two weeks, the first of such administrations coinciding preferably with the administration of the viral or parasitic antigen, although it may precede or follow the administration of the antigen by one or a few days.

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 4 (11) International Publication Number: WO 88/ 073 A61K 31/40, 31/405 A3 (43) International Publication Date: 6 October 1988 (06.10.

(21) International Application Number : PCT/EP88/00270 (74) Agents: GUTMANN, Ernest et al; S.C. Ernest G mann - Yves Plasseraud, 67, boulevard Haussma

(22) International Filing Date: 1 April 1988 (01.04.88) F-75008 Paris (FR).

(31) Priority Application Numbers: 8707792 (81) Designated States: AT (European patent), AU, BE (E 8707793 ropean patent), CH (European patent), DE (Eur pean patent), FR (European patent), GB (Europe

(32) Priority Dates: 1 April 1987 (01.04.87) patent), IT (European patent), JP, LU (European p 1 April 1987 (01.04.87) tent), NL (European patent), SE (European paten US.

(33) Priority Country: GB

Published

(71) Applicant (for all designated States except US): CELLE- With international search report

NA (CELL ENGINEERING) A.G. [CH/CH]; Loh- Before the expiration of the time limit for amending t wisstrasse 50, CH-8123 Ebmatingen (CH). claims and to be republished in the event of the receipt amendments.

(72) Inventors; and

(75) Inventors/Applicants (for US only) : PIERPAOLI, Wal(88) Date of publication of the international search report: ter [IT/CH]; Lohwisstrasse 12, CH-8123 Ebmatingen 26 January 1989 (26.01.8 (CH). MAESTRONI, Giorgio [IT/IT]; Via Madonna Delle Grazie, 16/A, 1-28052 Cannobio (IT).

(54) Title: MELATONIN-DERIVATIVES FOR PSYCHOGENIC STRESS AND ACUTE ANXIETY, AND IMM NO-STIMULANT DRUGS

(57) Abstract

The invention relates to the use of melatonin or related active chemical compounds for the production of pharm ceutical compositions for the treatment of psychogenic stress or acute anxiety as well as for the enhancement of immun resistance in a host against viral and parasitical infections.

FOR THE PURPOSES OF INFORMAHON ONLY

Codes used to identify States party to the PCT on theftontpagesofpampWetepubL-ihinginternationalappli- cations under the PCT.

AT Austria ER France ML Mali

AU Australia GA Gabon MR Mauritania

BB Barbados GB United Kingdom MW Malawi

BE Belgium HU Hungary NL Netherlands

BG Bulgaria π* Italy NO Norway

BJ Benin JP Japan RO Romania

BR Brazil KP Democratic People's Republic SD Sudan

CF Central African Republic ofKorea SE Sweden

CG Congo KR Republic ofKorea SN Senegal

CH Switzerland LI Liechtenstein SU Soviet Union

CM Cameroon LK Sri Lanka TD Chad

DE Germany, Federal Republic of U Luxembourg TG Togo

DK Denmark MG Monaco US United States of America π Finland MG Madagascar

Claims

1. use of a compound hereafter defined for the production of pharmaceutical compositions effective to relieve man or animal of psychogenic stress or acute anxiety or both, wherein said compound has the following formula :

in which

- n is 1 or 2,

- R₁ and R₂, identical or different from each other, are -H, -NH₂ , -COOH, -OH, acyl, -NH-acyl or alcoxy, said acyl or said alcoxy comprising from 1 to 4 carbon atoms,

- Y is H, -OH or -NH₂.

2. The use of claim 1, wherein said compound is melatonin.

3. Use of a compound hereafter defined for the production of pharmaceutical compositions effective as immunostimulant drugs against viral and parasitical infections, wherein said compound has the following formula :

in which n is 1 or 2,

- R₁ and R₂, identical or different from each other, are -H, -NH ₂, -COOH, -OH, acyl, -NH-acyl or alcoxy, said acyl or said alcoxy comprising from 1 to 4 carbon atoms,

- X is -OH or alkoxy comprising from 1 to 4 carbon atoms,

- Y is -H, -OH or -NH₂.

4. The use of claim 3, wherein said compound is melatonin.