HRP920488A2 - Application of peptidoglycan monomers (pgm) its n-acyl derivatives and metal complexes for the preparation of the medicine for the correction of immunosuppressive and hematosuppressive condition of an organism - Google Patents

Description

The invention relates to the use of peptidoglycan monomer (PGM), its N-acyl derivatives and metal complexes for the preparation of drugs for the correction of immunosuppressive and hepatosuppressive effects of anesthetics and operative stress during surgical procedures or other immunosuppressive, immunodeficient and hepatosuppressive conditions with the aim of faster and safer patient recovery .

A large number of clinical and experimental studies published in the last ten years have shown that surgical stress and/or anesthetics applied during surgery lead to transient immunosuppression, which can endanger the patient's life due to a greater tendency for infection, the spread of tumor metastases, poor wound healing and the like. In the pathogenesis of the resulting immunosuppression, anesthetics participate with their toxic effects, operative stress, and the change in neuron-endocrinoimmune relations, caused by the transient paralysis of the central nervous system during anesthesia (Watkins J. /1980/ Br.J.Hosp.Med. 23:583-590., Udović- Širola M., et al /1989/ In: Immune Consequences of Trauma, Schock and Sepsis, p.411-417), and changes in the metabolism at the level of the liver, which occur after the administration of anesthetics and operative stress, also play a certain role. The hepatotoxic effect of anesthetics is explained by the fact that most halogenated narcotics are metabolized in the liver, where toxic intermediate compounds, such as trifluoroacetylhalide or free radicals, can be formed (Satoh H., et al. /1985/ J.Pharm.exp.Therap. 233, 857 ). In predisposed persons, this can result in the development of autoimmune hepatitis (Vergani D., et al. /1980/ N.Engl.J.Med. 303, 66). It should be noted that not only patients operated on under general endotracheal anesthesia are exposed to this immunosuppressive and hepatotoxic effect, but also hospital staff working in operating rooms. In women, chronic exposure to halothane seems to lead to a 1.3-2.0 times higher incidence of cancer (Baden Y.M., et al. /1986/ In: Anesthesia, Eds.Miller R.D. N.Y., p.730). Considering the seriousness of the consequences that can arise during postoperative immunosuppression and long-term exposure to anesthetics, it is not surprising that there are many attempts to act preventively with different therapeutic approaches. This is how the applications of immunoglobulins are described (Nitsche D., et al. /1988/ 1st International Congress on the Immune consequences of trauma, shock and sepsis; OP 52), synthetic 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 of factors and interferon (Josten Ch., et al. /1988/ 1st International Congress on the Immune consequences of trauma, shock and sepsis., OP43; 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 endotoxin (Sagawa T ., et al. /1989/ In: Immune consequences of trauma, shock and sepsis, p.495-507), vasoactive agents (Schonharting M., et al. /1988/ 1st International Congress on the Immune consequences of trauma, with hock and sepsis, OP 57), antagonist of factors that activate platelets (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. al. /1986/ Int.J.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) (general formula - 1) was prepared by biosynthesis (Jug.pat. 35040) and isolated as a chemically defined compound (Klaić B. /1982/ Carbohydr.res. 110:320; Jug.pat. 40472 ; Patentschrift AT 362740), and then N-acyl derivatives of the general formula I were prepared (Jug.pat. application P-626/89; Eur. Pat. Appl. EP 390093), as well as its metal complexes of the general formula Ia and Ib ( Jug. Pat. Application P-1982/86; Eur. Pat. Appl. EP 268271). The isolated substances are well soluble in water and physiological solution, are non-toxic and non-pyrogenic. They show immunostimulating, antimetastatic and antitumor effects.

The subject of this invention is their hitherto unknown immunocorrective and hepatotropic effect in a model of experimentally induced postoperative immunosuppression.

It was found that peptidoglycan monomer (PGM) and its N-acyl derivatives of general formula I

[image]

where X=H, an alkali (Na, K, etc.), an alkaline earth (Ca, Mg, etc.) metal, or a quaternary ammonium salt of an organic base; R=H, acyl=radical of straight-chain C2-18 alkyl carboxylic acid, or branched C5-18 alkyl carboxylic acid, or unsaturated C12-18 alkenyl carboxylic acid, or aromatic C7-12 carboxylic acid, and complexes with bivalent metals of the general formula Ia and Ib ,

[image]

can be used in the preparation of drugs for the correction of humoral and cellular immunosuppressive conditions that arise as a result of the use of various anesthetics and/or operative stress during surgical procedures and other immunosuppressive and immunodeficient conditions caused by sepsis, burns, physical exhaustion, paraneoplastic syndrome, etc., and for the correction of hepatosuppressive conditions of the organism, by canceling changes in hepatic nucleic acids, especially in hepatic proteins caused by anesthesia, and/or operative stress, as well as other conditions related to immunosuppression, and/or liver diseases, intoxication, hepatitis, etc.

Since major surgical procedures in the clinic are performed under general endotracheal anesthesia, which is maintained by the administration of halogenated anesthetics, we developed an experimental model in which, by applying halothane anesthesia, with or without operative stress, we were able to induce immunosuppression similar to that in humans. For this purpose, we placed BALB/c mice, 2.5-3 months old, in hermetically sealed metabolic cages with soda lime in a volume of 1 L, through which, with the help of a small animal respirator, air (flow rate 350 ml/min) mixed with halothane was blown in a concentration of 0.5-1%. The anesthesia was maintained for 1 hour. In the control group, the animals were subjected to the same procedure, but they breathed air without halothane for 1 hour. Some of the mice were exposed only to halothane anesthesia, and some were also subjected to operative stress in the form of laparotomy. This procedure was preceded by putting the animals under halothane anesthesia, and was performed under short-term ether anesthesia.

In order for the control conditions for this group to be identical, the control groups for laparotomy plus halothane were also subjected to short-term ether anesthesia immediately before halothane anesthesia. In order to monitor the process of humoral, i.e. cellular immunity, the animals were then immunized: a) with sheep erythrocytes (OE) and the number of plaques in the spleen was analyzed on the fourth day after sensitization, b) with allogeneic tumor cells and the growth of sarcoma was monitored and ( from A/J mice) and the time of its rejection, and c) paternal splenocytes to analyze the local reaction of donor (BALB/c) cells against recipient (BALB/c x CBA) F1 hybrids. There were 5-8 animals in each group. Statistical analysis was performed with Student's t test.

The results showed that halothane anesthesia per se has an immunosuppressive effect on both humoral and cellular immunity (Figure 1), and that operative stress caused by laparotomy potentiates this immunosuppression. Namely, in mice sensitized with sheep erythrocytes, halothane anesthesia alone blocked the production of plaques (PCF) in the spleen by 48.5%, (p<0.001), and laparotomy by an additional 27% (Figure 1A), while the inhibition of cellular immunity was manifested by the prolongation of carrying of the allogeneic tumor from the 11th to the 14th day (anesthesia per se) or from the 14th to the 16th day (anesthesia + operative stress; p<0.05) (Figure 1B). The inhibition of cellular immunity by halothane anesthesia per se was also confirmed in the model of local reaction of donor cells against the recipient (GVHR - graft versus host reaction), where the anesthesia of the recipient caused a significant reduction of the reaction at the level of the popliteal lymph node on the 7th day after the injection of paternal splenocytes (from a normal value of 8 ,3±1.5 mg to a value of 4.0±1.0, p<0.05).

The reduction of the humoral immune response caused by halothane anesthesia was accompanied by hypoplasia of the bone marrow and spleen, in which it was determined that the proportion of CD4 and CD8+ cells decreases, and the number of cells that do not belong to this phenotype increases (Figure 2).

Simultaneous determination of the content of hepatic proteins and nucleic acids in mice sensitized to OE and anesthetized with halothane showed that anesthesia has a certain hepatosuppressive effect, that is, it significantly reduces the amount and concentration of hepatic proteins, DNA and RNA (Figure 3). Therefore, in the applied experimental model, we succeeded in imitating most of the symptoms that can occur postoperatively in an anesthetized patient, i.e. causing: 1. immunosuppression - a) humoral and b) cellular type and 2. hepatosuppressive effect.

In this state of immune and hepatic suppression, we then tested the effects of PGM and its analogues, and compared them with their effects in intact, non-suppressed mice. The obtained results showed that PGM and its new analogs are very effective precisely in correcting this type of postoperative immunosuppression, while their effects are much weaker in an intact organism.

Preparation of medicines-preparations

PGM and its N-acyl derivatives and complexes with bivalent metals each alone or in a mixture can be administered intravenously, intraperitoneally, intramuscularly and subcutaneously, in a composition with other non-toxic physiologically acceptable substances known in the art. The size and form of the dose depends on the body weight and the individual status of the organism.

PGM, its N-acyl derivatives and metal complexes with bivalent metals are administered at a dose of 5-50 mg/body weight.

The invention is illustrated by the following examples:

Example 1

Application of PGM (Ia) for correction of humoral immunosuppression in anesthetized and operated animals.

Since we found that the greatest suppression of humoral immunity occurs in halothane-anesthetized and laparotomized mice (Fig. 1), we tested the possibility of preventing the occurrence of immunosuppression by applying PGM. For this purpose, we treated the mice once i.p. by injection of PGM dissolved in 0.05 ml of physiological solution immediately after laparotomy and sensitization of the OE, and immediately before halothane anesthesia. The results shown in Table 1 show that PGM (10 mg/kg) in anesthetized mice increased plaque production by 94.3% (PFC/106), and in mice exposed to halothane anesthesia and operative stress, it stimulated PFC production by as much as 206.4 % compared to the control injected only with saline.

Table 2 shows that the best correction of halothane immunosuppression is achieved with a small dose of PGM, and at the same time tests of the effects of PGM in unanesthetized mice show that the effect was obtained only in immunosuppressed mice. The increase in plaque production in anesthetized mice treated with PGM was accompanied by an increase in bone marrow cellularity and marked peripheral leukocytosis.

Example 2

Application of PGM-complexes with divalent metals in the correction of humoral immunosuppression

Metal complexes of PGM with zinc (PGM-Zn) were dissolved in physiological solution and injected in the same dose as in previous experiments (10 mg/kg) into anesthetized and non-anesthetized, OE-treated mice. The experiments were repeated three times and in all studies PGM-Zn showed a better immunocorrective effect than PGM and increased the production of plaques in anesthetized mice by 73.5%, 73.1% and 101.4% compared to the control injected only with physiological solution (table 3). These effects were accompanied by an increase in the cellularity of the spleen and bone marrow.

Example 3

Application of N-acyl derivatives of PGM in the correction of humoral immunosuppression

The sodium salt of N-lauroyl PGM (PGM-L-Na) dissolved in physiological solution and injected into anesthetized and sensitized mice on two occasions caused the best stimulation in the production of plaques in the spleen (an increase of 109.9% and 120.9% compared to the control injected with saline, and this effect was absent in unanesthetized mice.

Example 4

Application of PGM (Ia), complexes of PGM with divalent metals and PGM-N-acyl derivatives in the correction of cell-type immunosuppression

PGM and its analogs were also tested in the local GVHR, where they were administered immediately after the injection of paternal splenocytes into the foot pad of the rear leg of the F1 hybrid, i.e. immediately before halothane anesthesia. It was found that PGM-Zn enhances the reaction at the level of popliteal lymph nodes on the 7th day after injection, and that PGM-L-Na significantly increases the number of large lymph cells in the local lymph node on the 10th day after injection (determined by using a flow cytometer) (table 4 ).

Example 5

Application of PGM in the correction of hepatosuppressive effects during halothane anesthesia

Since we found that halothane anesthesia in the OE of sensitized mice causes a decrease in hepatic proteins and nucleic acids (Fig. 2), we tested the possibility that the application of PGM affects these changes accompanying humoral immunosuppression. The results shown in Table 5 show that a small dose of PGM led to a significant increase in the content of all tested parameters (DNA, RNA and protein) in the liver. Simultaneous testing of the effects of PGM in intact mice showed that the stimulatory effects of PGM are present only in anesthetized animals, i.e. in hepatosuppressed mice.

Example 6

Hepatotropic effects of PGM, complexes of PGM with divalent metals and N-acyl derivatives of PGM in anesthetized and operated mice

We tested the effects of PGM and its analogues in anesthetized and operated mice and found that PGM leads to an increase in the content of DNA, RNA and protein in the liver of these animals. the effects of its N acyl derivative on liver proteins were of the same intensity, while PGM Zn stimulated the accumulation of hepatic proteins, even more intensively than PGM alone (Table 6, Figure 4).

Claims (6)

1. Application of peptidoglycan monomer (PGM), general formula I, and its N-acyl derivatives [image] where X=H, an alkali (Na, K, etc.), an alkaline earth (Ca, Mg, etc.) metal, or a quaternary admonium salt of an organic base; R=H, acyl=radical of straight-chain C2-18 alkyl carboxylic acid, or branched C5-18 alkyl carboxylic acid, or unsaturated C12-18 alkenyl carboxylic acid, or aromatic C7-12 carboxylic acid, and complexes with bivalent metals of the general formula Ia and Ib , [image] for the preparation of drugs to correct the immunosuppressive and hepatosuppressive state of the organism, by canceling the suppression of the humoral and cellular type of immune response and by canceling changes in hepatic nucleic acids and hepatic proteins, which occur as a result of the general state of immunosuppression and immunodeficiency. 2. Application according to claim 1, characterized by the fact that the immunosuppressive state of the organism is a consequence of the use of various anesthetics and/or operative stress, sepsis, extensive burns, physical and/or senile exhaustion, paraneoplastic syndrome, autoimmune diseases, etc. 3. Use according to claim 1, characterized by the fact that the hepatosuppressive state of the organism is a consequence of halothane anesthesia and/or operative stress during surgical procedures, liver diseases such as intoxications and hepatitis. 4. Use according to claims 1, 2 and 3, for the preparation of drugs, which can be administered intravenously, intraperitoneally, intramuscularly and subcutaneously, in a composition with other non-toxic physiologically acceptable substances known in the art. 5. Application according to claim 1-4, characterized in that the size and form of the dose depends on the body weight and the individual status of the organism. 6. Application according to request 1-5. indicated that PGM, its N-acyl derivatives and metal complexes with bivalent metals are given in a dose of 5-50 mg/kg of body weight