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

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-STIMULANTDRUGS The invention relates to compositions applicable to man or animal, containing melatonin or chemical homo- logues, 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 _Ln 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 inten¬ sity. 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 infec¬ tions. 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 wit one another and in the production of leukokine im uno 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 Immuno¬ deficiency, 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 immuno¬ deficiency 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 dis¬ covery 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 immuno- stimulator, can modulate and attenuate the severity of retrσvirus induced immunosuppression or, alternatively, aJ.l_s.ari.ate the additive or synergistic toxicity of the association cytomegalic herpes and other viruses that ac o pany the primary decline in resistance induced by the &iπ£ (HIV) virus. Of clinical relevance for vaccination procedures rεε 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 espe¬ cially 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 pha_αιra_ceutical compositions for use in the treatment of ps^cϋogen c stress, acute anxiety conditions or both, on the. one hand, and of pharmaceutical compositions for use a_E xrαmunostimulants 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, R 1 and R-,, 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 -NH-, . Preferred compounds for use in the compositions of the invention are those in which Y is hydrogen and X is methoxy. The most preferred compound is melatonin itself, the formula of which is Other preferred compounds for use in the composi¬ tions 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. SZMUSZKOV CZ already mentioned, J. SUPNIE SKI et al r "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 coπpounds 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 l to 4 which follow bring into play the known im unosuppressive 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 immunosuppressed in the stressed 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 10 8 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 GR O UP (n) S TRESS TREATMENT PFC/SPLEEN ^THYMUS we i ght g BODY weight A (15) + PBS 99903134420** 1,67+0,57* B (15) + Melatonin 199157+62888** 2,35±O r 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 13 TREATMENT' 1 SURVIVAL 6 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 —8 s.c. wi Λth 0.2 ml of 2 x 10 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 I 1 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 immuno¬ suppression 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. TABLE III n'of Spleen PFC/10 4 Group (n) Treatment CellsxIO 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 4 the number of PFC/10 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 labo¬ ratory or purchased and were maintained under conventional condi¬ tions in rooms at 22 +_ 1 C with a dark-light cycle of 12 hours. Great care was taken to avoid environmental stress before and du¬ ring 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 bidi ' stilled saline to a final 1% alcohoT-water dilution. Controls of mel atonin-i njected 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 describ by Zinkernagel and Doherty, Adv.Immunol. ,27,51 (1979). Briefly, according to the experiments 1x10 pock forming units (pfu) or 8x10 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 ) and were maintained in IMDM, 2% foetal calf serum (FCS) at 37 C, 5% CO... 5x10 cells were pelletted in a 15 ml coni¬ cal tube and resuspended in 600 ul of complete medium contaninq 7 4x10 pfu of vaccinia virus. These cells were incubated and gently shaked for 1 hour at 37 C. Infected and an equal number of non-in¬ fected cells were then incubated with 200 uCi of Cr-sodium chro- mate (Amersham Int., Amersha , UK) for 1 hr at 37 C. After this labe¬ ling procedure, the cells were washed 3 times, counted and diluted ttoo a final concentration of 10 cells /ml. Infected and non-infected 51, Cr-labeled target cells were mixed in 96-well, U-bottomed micro- plates (Nunc, Roskilde, den ark) with effector spleen cells at different effectorrtarget 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 * CO , the microplates were spun at 500g and 75 ul/well were taken and counted in a gamma counter. Specific Cr release was calculated as follows: Experimental release - Spontaneous release % specific rel ease = Total release where total release is given by infected or non-infected cells lysed with IN HC1. Preparation of encephalomyocarditis (EMC) virus 0.02 ml of a preparation of EMC virus were injected intra- cranially 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 -g 5 x 10 was chosen. The large majority of the mice over 20 grams body weight did not contract the infection and were partially protec¬ ted by a subsequent lethal dose. Virus neutralization test (VNT) For evaluating the virus-neutrali¬ zing properties in the EMC-in.iected 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 " EMC 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 1 7 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 an myocarditis. The conditions under which the tests were ran follow from the tabl hereafter and the legends which accompany them. Table-1.'. Administration of melatonin during priiiiaiy i munizritiop enhances the secondary antiviral cytotoxic T cell response to Vaccinia virus in mice. Effector £ of specific Cr release -±.S.D. to P B S MELATONIN VARIATION (melatonin target (n = 8) (n = 8) vs . (ratio) P B S) ■ 5D: : I 40.0 ± 4,6 52,3 ± 4,1* + 30 25 -.: 1 . 31,7 ± 6,1 44,8 t 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 CSSl/He female mice, two months old, were injected i.v. with 1 x 10 : 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 dark¬ ness. 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 in- jj-Etixwr- of; 8: x TXT PFU of VV i.v. was then performed. Neither melatonin, nor FES? as. iτroα.Xa_fcex£ during this second immunization. After 4 days the antiviral cytotoxic T celt response was tested by the Cr release assay. Effector sple¬ en; cell S- were- dn uted. with L929 target cells at the following ratios: 50 : 1, Z5B::i , l rJI antfc 6=1. 5ETt 3T of- " specif rfc 'Dr release ± standard deviation (S.D.) was calculated according to ~ the_formuT.a_ cfescrfbed in materials and methods. P <0,01 SUBSTITUTE SHEET Table 2. A n on letal dose of encep aloinyocardltls (EMC) virus combined with drcadian, evening administration of melatonin (vaccination procedure) potentiates the resistance when the mice are re-Infected with a lethal dose of the virus. EMC virus dilutions Survival rate after Virus neutralization test (VHT) Origin of mice (n) First Injection Second Injection re-Infection ( % ) Survival rate (15 mice) (vaccination table 4) 3 days 10 days 3 days 6 days 10 days 5 x 10 22(67%) 1 (3%) 10 Ex-EMC only (33) 5 x 10 Ex-EMC + ω Melatonin (28) 5 x 10 5 x 10 -7 20(71%) 2 (4%) c morning CD Ex-EMC + _. H Melatonin (38) 5 x 10 5 x 10 30 (79%) 12 (32%) 13 C evening_ . H m ω z Virus neutralization test (VNT). Blood was obtained from the retroorbltal plexus of ether-anestheslzed mice with a Pasteu m m pipette at three days after re-1nfect1on with 0,2 ml of EMC virus diluted to 5 x 10 " with isotonic saline and injected s. H The serum of Individual mice was Inactivated for 20 min at 57°C and stored frozen. Fresh 1 x 10 EMC virus suspensions we prepared each time from the frozen original allquots of 1:10 brain homogenates of EMC-lnfected mice. The dilution medium w sterile isotonic saline. 100 ul of serum from Individual mice was mixed, with 50 l of 1 x 10 EMC virus. The tubes were s ken and incubated for 30 min 1n a water bath. Groups of normal recipient BΛLB/c female mice were injected i.p. with the vi antlserum mixtures, 0,15 ml total volume per mouse. The mice were checked daily for paralysis of the hind legs and mortali The number of Individual mice Injected with the EMC virus and antlserum from individual donors were 3 to 5 per group. The sults of VNTs are plotted together. 0,2 ml s.c. The mice were killed a few hours after the appearance of irreversible paralysis of the hind legs. after i .p. injection of the virus-antl serum mixtures 20 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 10 pfu of Vaccinia virus were injected for 7 days with evening melatonin showed a significantly higher (p<0.01) T cytotoxic antiviral response than spleen cells from 51 control mice that were treated with PBS. In fact, specific Cr-release was consistently higher in melatonin treated mice at all effector to target cell ratios (table LJ. Thus, also a most ~ .^significant anti¬ viral response, i.e. the T-cell -mediated cytotoxic secondary response can be augmented simply by melatonin treatment during primary immuniza¬ tion. 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 experimen 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 whi an influence of melatonin may be possibly studied. As known ( .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 iπuπunization. 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~ ) 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 dfthe mice which had been treated with evening melatonin in the course of the primary immunization (vaccination) sur¬ vived to the lethal dose of EMC virus. When_blood was taken frnm hp re-infected mice at 3 days after re-iπfection and the virus neutralizin 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-neu¬ tralizing 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 mela¬ tonin 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 alethal dose. In this expe¬ rimental model, protection of melatonin was solely concerning higher production of circulating antibodies in the eveningtreated 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 immu¬ ne response . but also remarkably increases the responsiveness of th primed mice to a second inoculation of the same antigens (memory respons without further administration of melatoni_n.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 cyto¬ toxic 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 infec¬ tion. 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 sg^i-st 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 secon¬ dary response, it can be hypothesized that it promotes the proliferatio or functions of helper T lymphocytes that in turn optimize the formatio of "memory" T and B cells. By this way a subsequent infection or antige inoculation determines maximal amplification of the response. This seem 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 phytohemagglu- tinine 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 o administrations for the same purpose, e. g. by th rectal route, can be contemplated as well. There is no need to emphasize that the ma skilled in the art will be able to select the mos appropriate galenic compositions to achieve the result sought. Most effective results will be obtained in th host upon administering said melatonin doses at a tim between sunset or little earlier, say 4 P. M. , and th time at night when the host is to go on sleep. The duration of treatment will of course depen on the host and the strees-inducing circumstances. In a human patient the treatment may b furthered over from one to two weeks or more if appropriate. In animal raised in a non-natura 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 us for the production of drugs or pharmaceutical composito for use to combat non-specifically diseases caused b viruses or parasites, o at lest alleviate them. The uses indicated previously by way of exampl are not exclusive of other uses bringing into play th immunoregulator properties of melatonin. It is possibl also to cite by way of example its reinforcing action a the level of the specific immunization of the host wit regard to parasitic -antigens, the restoration of th immunocompetence of the host, when the latter is at lower level than normal, notably when the latter has bee damaged by antigens or parasites themselves, or under th effect of chemotherapy, radiotherapy, or of any othe treatment which has an immunosuppressive action. The pharmaceutical compositions containin melatonin, are useful for the treatment or the preventio of infectious diseases of viral or parasitic origin. Melatonin or its active chemical homologues may b administered to the host-animal or human being - in an suitable manner to obtain the desired effect. When used a an adjuvant, melatonin and the viral or parasiti vaccinating antigen, may be administered simultaneously o separately, in the latter case at suitable staggered tim intervals either by similar or different routes o administration (for example parenteral and oral route respectively or vice versa) . The invention relates more particularly to suc suspensions, solutions or liposomes which are suitable fo administration by intrader al, intramuscular o subcutaneous injection, or again by scarification. Melatonin may also be administered by othe routes, e.g. by the rectal route, or in the form o aerosols designed to come into contact with the mucou membranes, notably the ocular, nasal, pulmonary or vagina mucous membranes. Effective use of melatonin or its chemi homologues with a view of reinforcing the immune defen of the host, comprises administering to the latter effective dose of said melatonin or homologues, in one the administration forms which have been mentioned abo preferably from sunset, or a little earlier, say fro P.M. up to the time where the host goes to sleep. Examp of daily doses capable of inducing an effect, range f 0.01 to 100 mg, preferably from 0.1 to 10 mg per kg body weight, when the administration is effected by oral route, or again from 0.01 to 5 mg, preferably f 0.1 to 1 mg per kg of body weight, when administered the parenteral route. The daily administrations are preferably repea daily over periods of one or two weeks, the first of su administrations coinciding preferably with t administration of the viral or parasitic antigen, althou it may precede or follow the administration of the antig by one or a few days.

Claims: CLAIMS 1. use of a compound hereafter defined for th production of pharmaceutical compositions effective t relieve man or animal of psychogenic stress or acut anxiety or both, wherein said compound has the followin formula : V in which - n is 1 or 2, - R and R~ , identical or different from eac other, are -H, -NH 2 ' -COOH, -OH, acyl, -NH-acyl or alcoxy said acyl or said alcoxy comprising from 1 to 4 carbo atoms, - Y is H, -OH or -NH,. 2. The use of claim 1 , wherein said compound i melatonin. 3. Use of a compound hereafter defined for th production of pharmaceutical compositions effective a immunostimulant drugs against viral and parasitica infections, wherein said compound has the followin formula : in which n is 1 or 2, - R_ and 2» i-_d-!e<_:n--t_.i_.c_.a<_.l._ or different from eac other, are -H, -NH 2 , -COOH, -OH, acyl, -NH-acyl or alcoxy said acyl or said alcoxy comprising from 1 to 4 carbo atoms, - X is -OH or alkoxy comprising from 1 to 4 car atoms, - Y is -H, -OH or -NH-,. 4. The use of claim 3, wherein said compound melatonin.

Inventors: MAESTRONI GIORGIO, PIERPAOLI WALTER, MAESTRONI, GIORGIO, PIERPAOLI, WALTER
Applicants: CELL ENG AG, CELLENA (CELL ENGINEERING) A.G.
Application year: 1988
Main classification: A61K31
IPC codes: A61K31