PREPARATION OF MEDICAMENTS CONTAINING THE PEPTIDOGLYCAN MONOMER OR ITS DERIVATIVES

Abstract: The invention relates to the use of the peptidoglycan monomer (PGM), its N-acyl derivatives of formula (I), and its metal complexes of formulae (Ia) or (Ib) in the preparation of medicaments for the correction of the immunosuppressive and hepatosuppressive effects of anaesthetics and operative stress in surgical treatment or in other immunosuppressive, immunodeficient, and hepatosuppressive states, to achieve a swift and safe recovery of the patients.

Description: PREPARATION OF MEDICAMENTS CONTAINING THE PEPTIDOGLYCAN MONOMER OR ITS DERIVATIVES. The present invention relates to the use of the peptidoglycan monomer (PGM), its N-acyl derivatives, and its metal complexes in the preparation of medicaments for the correction of the immunosuppressive and hepatosuppressive effects of anaesthetics and operative stress in surgical treatment or in other immuno¬ suppressive, immunodeficient, and hepatosuppressive states, to achieve a swift and safe recovery of the patients. Numerous publications on clinical and experimental research in the last ten years have shown that surgical stress and/or anaesthetics administered in surgery develop a transient immunosuppression which may represent a risk for the patient's life due to the augmented susceptibility to infections, spread of tumour metastases, impairment of wound healing, and the like. To the pathogenesis of the generated immunosuppression contribute jointly the- anaesthetics with their toxic effects, the operative stress, and the changes in the neuro-endocrino- i muno relationship, resulting from the transient paralysis of the central nervous system during anaesthesia. ( atkins J. /1980/ Br.J.Hosp. Med. 23:583-590., Udovic-Sirola M. et al /1989/ In: Immune Consequences of Trauma, Shock and Sepsis, p. 411-417); whereas, a certain effect is caused by the changes in the metabolism on the liver level, resulting after the administration of the anaesthetics and the operative stress. The hepatotoxic effect of anaesthetics may be explained by the fact that the majority of halogenated narcotics are metabolized in the liver, which may yield toxic intermediates, such as e.g. trifluoroacetyl halides or free radicals (Satoh H., et al. /1985/ J.Pharm. Exp.Therap. 233, 857). In predisposed persons this may result in the development of autoimmune hepatitis (Vergani D., et al /1980/ N.Engl. J. Med. 303, 66). It has to be emphasized that this immunosuppressive and hepatotoxic action is not limited to the patients operated in endotracheal anaesthesia, but may involve also the medical staff working in the operating theatre. It seems that the chronic exposure to halothane results in a 1.3 - 2.0 oftener development of carcinoma in persons of female sex (Baden Y.M., et al. /1986/ In Anesthesia, Eds. Miller R.D. N.Y., p. 730). In view of the serious consequences which may arise after the post- operative immunosuppression and the long-continued exposition to anaesthetics, the high number of therapeutic attempts for preventive action is not surprising. Thus, there has been described the use of: immunoglobulin (Nitsche D., et al /1988/ 1st International Congress on the Immune Consequences of Trauma, Shock and Sepsis; OP 52), synthetical hormones (Faist E., et al /1987/ Int. J. Clin. Pharm. Res. 7:83-87; Waymack J.P., et al. /1985/ Arch. Surg. 120:43; Faist E. /1989/ In Immune Consequences of Trauma, Shock and Sepsis, p. 509-517), transfer factors and interferons (Josten Ch., et al /1988/ 1st. International Congress on the Immune Consequences of Trauma, Shock and Sepsis., OP 43; Livingston D.H. et al /1989/ In: Immune Consequences of Trauma, Shock and Sepsis, p. 551-555), H-2 receptor blockers (Nielsen H. J., et al /1988/ 1st International Congress on the Immune Consequences of Trauma, Shock and Sepsis, OP 49), monoclonal antibodies against endotoxins (Sagawa T., et al. /1989/ In: Immune Consequences of Trauma, Shock and Sepsis, p. 495-507); vasoactive agents (Schontharting M., et al /1988/ 1st International Congress on the Immune Consequences of Trauma, Shock and Sepsis, OP 57); trombocite-activating- factors antagonists (Fletcher J.R., et al /1988/ 1st International Congress on the Immune Consequences of Trauma, Shock and Sepsis, OP 56); and various immunomodulators (Schopf R.E., et al /1988/ 1st International Congress on the Immune Consequences of Trauma, Shock and Sepsis; Tsang K.P. et al. /1986/ IntJ.Immunopharmacol. 8:437; Hadden J.W. et al /1989/ In: 1st International Congress on the Immune Consequences of Trauma, Shock and Sepsis, p. 509- 517). The biologically active substance peptidoglycan monomer (PGM) was made available by biosynthesis (in accordance with YU Patent 35040) and isolated as a chemically defined compound (according to Klaic B. Carbohydr. Res. (1982) 110:320; YU Patent 40 472; AT Patent Specification 362740); later, there were prepared its N-acyl derivatives (YU Pat. Appl. P-626 89; Eur. Pat. Appl. EP 39 00 93), and its metal complexes (YU Pat Appl. P-1982/86; Eur. Pat. Appl. EP 268271). The isolated substances are well-soluble in water and physiological solution, non-toxic, and apyrogenic. They demonstrate immunostimulating, antimetastatic, and antitumour activity. The object of the present invention is the novel use of the peptidoglycan monomer (PGM), and its N-acyl derivatives of the formula I wherein R stands for hydrogen, Ac stands for a straight (C2 - C^g alkyl) carboxylic acid group, or a branched (C5 - Cjg alkyl) carboxylic acid group, or an unsaturated (C12 - jg alkenyl) carboxylic acid group, or an aromatic (C7 - C12) carboxylic acid group, and X stands for a hydrogen, or an alkali metal, or an alkaline earth metal, or a quaternary ammonium salt of an organic base, and complexes thereof with bivalent metals of the formulae la or lb SUBSTITUTE SHEET N - Ac-Gle-N- Ac-Mur lb in the preparation of medicaments for the correction of the immunosuppressive states of the humoral and cell-mediated type, induced by the administration of various anaesthetics and/or operative stress in surgery, and of other immuno¬ suppressive and immunodeficient states, induced by sepsis, burn injuries, body exhaustion, paraneoplastic syndrome, and the like, and for the correction of the hepatosuppressive state of the organism, the cessation of the changes in the hepatic nucleic acids, especially in hepatic proteins, induced by anaesthesia, and/or operative stress as well as other states, associated with immuno¬ suppression, and/or hepatic disorders, intoxications, hepatitis, etc. The hitherto not known activity of PGM, its N-acyl derivatives and their bivalent metal salts is illustrated by the demonstration on the model of an experimentally induced post-operative immunosuppression. Taking into account that the majority of surgical operations is performed in general endotracheal anaesthesia maintained by the administration of halogenated anaesthetics, there was designed an Experimental Model enabling the inducement of an immunosuppression similar as in humans, by the application of the halothane anaesthesia, with or without operative stress. For this reason BALB/c mice, aged 2.5-3 months were placed into hermetically closed 1-L metabolic cages containing soda lime, into which air (a flow of 350 mL/min) with added 0.5-1% of halothane was charged by means of a respirator for small animals. The narcosis was maintained for 1 hour. Animals in the control group were subjected to the same procedure, with the exception of halothane in the air for 1 hour. A sub-group of mice was exposed only to halothane anaesthesia, whereas, a sub-group was additionally subjected to operative stress in the form of laparotomy. This operation preceded the exposure of the animals to halothane anaesthesia, and was performed under short ether narcosis. In order to maintain identical conditions in the control for this group, the control groups for laparotomy + halothane were also immediately before halothane anaesthesia subjected to a short ether narcosis. For the control of the humoral, and ceD- mediated immunity the animals were then immunized: a) with sheep erythrocytes (OE), and the number of plaques in the spleen was analyzed on the 4th day after the sensibilisation; b) with allogeneic tumour cells, and the growth of the sarcoma I (from A/J mice) and their rejection time; and c) paternal splenocytes for the analysis of the local reaction of the donor cells (BALB/c) against the recipient (BALB/c x CBA)Fι hybrid. Each group comprised 5-8 animals. The statistical analysis was performed by Student's /-test . The results demonstrated that the halothane anaesthesia per se exerted an immunosuppressive effect on the humoral and cell-mediated immunity (Fig. 1), and that the operative stress caused by laparotomy potentiated this immunosuppression. Thus, in mice sensibilized with sheep erythrocytes halothane anaesthesia alone blocked the plaque (PFC) formation in the spleen for 48.5 % (p < 0.001), and laparotomy for additional 27 % (Fig. 1A); whereas, the inhibition of cell-mediated immunity was manifested by the prolongation of the 'allogeneic tumour-bearing from the 11th to the 14th day (anaesthesia perse), and from the 14th to the 16th day (anaesthesia + operative stress ; p < 0.05) (Fig. IB). The inhibition of cell-mediated immunity by halothane anaesthesia per se was confirmed by the Model of local response of donor cells against receiver cells (GVHR - graft versus host reaction), in which the anaesthesia of the donor induced a significant response diminishment on the popliteal lymphatic node level on the 7th day after the injection of paternal lymphocytes (from the normal value 8.3 +. 1.5 mg to the value 4.0 ± 1.0; p < 0.05). The halothane-induced diminishment of the humoral immune response was accompanied by hypoplasia of the bone marrow and the spleen, and the assessment of the decreased proportion of CD4 and CD8 + cells, and the increase of the number of cells not belonging to this phenotype. (Fig.2). The simultaneous determination of the hepatic proteins and nucleic acids contents in OE-sensibilized and halothane-anaesthetized mice demonstrated a certain hepatosuppressive activity of anaesthesia, and its significant effect on the decrease of the amount and concentration of hepatic proteins, DNA, and RNA (Fig. 3). Accordingly, it was succeeded in the applied Experimental Model to imitate the majority of symptoms, which may arise after the operation in an anaesthetized patient, and cause: 1. immunosuppression - a) of the humoral, and b) cell-mediated type; 2. a hepatosuppressive effect In this state of immuno and hepatosuppression we tested further the effects of PGM, and its analogues, and compared their effects in intact, non-suppressed mice. The obtained results demonstrated that PGM and its analogues were highly efficient in the very correction of such post-operative immunosuppression, whereas, its effects were much feebler in an intact organism. Medical formulations: PGM and its N-acyl derivatives and complexes with bivalent metals or mixtures thereof may be administered intravenously, intraperitoneally, intramuscularly, and subcutaneously, in composition with other nontoxical, physiologically acceptable substances known in the art The unit dose size and form depend on the body weight and the individual state of the organism. PGM and its N-acyl derivatives and complexes with bivalent metals may be administered in a dose of 5-50 mg per kg of body weight The invention is illustrated by the following Examples. EXAMPLE 1 Correction of humoral type immunosuppression in anaesthetized and operated animals with PGM (la) Since we have found, that the major suppression of humoral i mumty resulted in halothane-anaesthetized and laparotomized mice (Fig. 1) there was tested the prevention of immunosuppression by the application of PGM. For this reason mice were given one intraperitoneal injection of PGM dissolved in 0.05 mL of physiological solution immediately after laparotomy and OE-sensibilization, and immediately before halothane anaesthesia. The results shown in Table 1 demonstrate that PGM (10 mg kg) in anaesthetized mice increased the plaque generation for 94.3 % (PFC/10^), whereas, in mice subjected to halothane anaesthesia and operative stress the PFC generation was stimulated even for 206.4 % with respect to the control injected only with physiological solution. TABLE 1 HtMORAL IMMUNITY IN PGM-TREATED MICE Statistically significant with respect to control (* p<0.01; ** p<0.001) Table 2 shows that the best correction of halothane immunosuppression was achieved with a low PGM dose, and the simultaneously performed investigation of the PGM effect in non-anaesthetized mice demonstrated that the effect was achieved only in immunosuppressed mice. The plaque generation increase in anaesthetized PGM-treated mice was accompanied by the bone-marrow cell augmentation and an expressed periphereal leukocytosis. SUBSTITUTE SHEET TABLE 2 HUMORAL IMMUNITY IN ANAESTHETIZED AND NON-ANAES HETIZED MICE AFTER PGM TREATMENT CO c DO ω H c H m ω x m =3 Slnlisllcnlly significant with respect lo con l iol ( ♦ *p<0.0I ; *p<0.001) EXAMPLE 2 π Correction of humoral type immunosuppression with PGM -complexes with bivalent metals The PGM-Zn complex dissolved in physiological solution was injected in the same dose as in the foregoing Example (10 mg/kg) in anaesthetized and non-anaesthetized OE-treated mice. The experiment was repeated three times, and in all investigations PGM-Zn demonstrated an improved immunocorrective activity in comparison with PGM. and increased the plaque generation in anaesthetized mice for 73.5 , 73.1 %, and 101.4 %, with respect to the control injected only with physiological solution. These effects were accompanied by the cell-augmentation of the spleen and the bone-marrow. SUBSTITUTE SHEET ISA/EP EXAMPLE 3 Correction of humoral type immunosuppression with N-acyl derivatives of PGM The sodium salt of N-lauroyl PGM (PGM-L-Na) dissolved in physiological solution and injected in anaesthetized and sensibilized mice caused twice the best stimulation of plaque generation in the spleen (augmentation of 109.9 % and 120.9 % with respect to the control injected with physiological solution. This effect was also absent in unanaesthetized mice. EXAMPLE 4 Correction of cell-mediated type immunosuppression with PGM-complexes with bivalent metals, and N-acyl derivatives of PGM PGM and its analogues were tested in local GVHR in which they were administered immediately after the injection of paternal splenocytes into the hind leg pad of Fl hybrids, or immediately before halothane anaesthesia. It was found, that PGM-Zn potentiates the response on the popliteal lymphatic node level on the 7th day after the injection, whereas, PGM-L-Na significantly increases the number of large lymphatic cells in the local lymphatic node on day 10 after the injection (assessed by means of counter-flow cytometer). 13 TABLE 4 GVHR + HALOTHANE Weight difference of lymph gland 5th day 7th day 10th day Cell difference in lymph node (million) 5th day 7th day 10th day Number of large cells (thousand) 5th day 7th day 10th day UBSTITUTE SHEET EXAMPLE 5 14 Correction of hepatosuppressive effects during halothane anaesthesia with PGM Since we found that the halothane anaesthesia in OE-sensibilized mice caused the diminishment of the number of hepatic proteins and nucleic acids (Fig. 2), we tried to establish whether the application of PGM influenced these changes which accompany the immunosuppression of the humoral type. The results shown in Table 5 demonstrated that a small dose of PGM induced a significant increase in all investigated parameters (DNA, RNA, and proteins) in the liver. The simultaneous testing of PGM effect in intact mice demonstrated that the stimulating effects of PGM were present only in anaesthetized animals, to say in hepatosuppressed mice. SUBSTITUTE SHEET ISA/EP TABLE 5 HEPATOTROPIC EFFECT OF PGM IN ANAESTHETIZED AND NON-ANAESTHETIZED MICE Statistically significant between groups bearing the same superscript p < 0.05. EXAMPLE 6 Hepatotropic effects of PGM, PGM-complexes with bivalent metals and N-acyl derivatives of PGM in anaesthetized and operated mice The effects of PGM and its analogues were tested in anaesthetized and operated mice and it was established that PGM caused an increase of the DNA, RNA, and proteins contents in the livers of said animals. The effects of its N-acyl derivatives on the liver proteins were of the same intensity, whereas, PGM-Zn stimulated the association of hepatic proteins, even more intensive than PGM alone. TABLE 6 HEPATOTROPIC EFFECT OF PGM AND ITS ANALOGUES IN MICE SUBJECTED TO ANAESTHESIA AND SURGERY c CD ω c H m o X m Statistically sigπincant between groups bearing the same superscript p < 0.05.

Claims: CLAIMS: 18 alkaline earth metal, or a quaternary ammonium salt of an organic base, lb in combination with other conventional nontoxic, physiologically acceptable substances, in the preparation of medicaments for the correction of the immunosuppressive and hepatosuppressive states of the organism. 2. The use as claimed in claim 1, wherein the immunosuppressive states comprise the humoral and the cell-mediated types, induced by the administration of various anaesthetics and/or operative stress in surgery, and other immunosuppressive and immunodeficient states, induced by sepsis, burn injuries, body exhaustion, paraneoplastic syndrome, and the like. 3. The use as claimed in claim 1, wherein the hepatosuppressive state of the organism comprises the changes in the hepatic nucleic acids, especially in hepatic proteins, induced by anaesthesia, and/or operative stress as weD as other states, associated with immunosuppression, and/or hepatic disorders, intoxications, hepatitis, etc. 4. The use as claimed in claim 1, in the preparation of medicaments for intravenous, intraperitoneal, intramuscular and subcutaneous administration. 5. The use as claimed in claim 1, wherein the dose weight and formulation depend on the body weight and individual state of the organism. 6. The use as claimed in claim 1, wherein PGM, its N-acyl derivatives and metal complexes with bivalent metals are administered in a dose of 5-50 mg kg body weight. AMENDED CLAIMS [received by the International Bureau on 15 February 1993 (15.02.93 ) ; original claim 1 amended; other claims unchanged (1 page ) ] 1. The use °f N-acyl derivatives and metal complexes of the peptido¬ glycan monomer (PGM) of the formula I (CH 2 ) 3 -HNCHCONH 2 wherein R stands for hydrogen, Ac stands for a straight (C - Cjg alkyl) carboxylic acid group, or a branched (C5 - Cjg alkyl) carboxylic acid group, or an unsaturated (Cγi • ig alkenyl) carboxylic acid group, or an aromatic (C7 - C12) carboxylic acid group; and X stands for a hydrogen, or an alkali metal, or an alkaline earth metal, or a quaternary ammonium salt of an organic base, and its metal complexes with bivalent metals of the formulae la and lb

Inventors: NAUMSKI RADMILA, RADOSEVI-STASIC BISERKA, RUKAVINA DANIJEL, SUSKOVIC BOZIDAR, NAUMSKI, RADMILA, RADOSEVI-STASIC, BISERKA, RUKAVINA, DANIJEL, SUSKOVIC, BOZIDAR
Applicants: PLIVA HANDELS GMBH, PLIVA PHARM & CHEM WORKS, PLIVA FARMACEUTSKA, KEMIJSKA, PREHRAMBENA I KOZMET
Application year: 1992
Main classification: A61K37
IPC codes: A61K38, A61P1, A61P3, A61P17, A61P25, A61P29, A61P37, C07K9