SK17994A3 - Usage of a peptidoglycan monomer, its n-acyl derivatives and its metal complexes in the preparation of medicaments - Google Patents

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

Use of peptidoglycan monomer '(PGId), its N-acyl derivatives with its metal complexes in the preparation of drugs for the correction of immunosuppressive e hepa, tosuppressive conditions of the organism

Technical field

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 correcting the immunosuppressive and hepatosuppressive effects of anesthetics and surgical stress in surgery or other immunosuppressive, immunodeficiency and hepatosuppressive. and safe patient recovery.

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The state of the art

Numerous publications of clinical and experimental research over the past decade have shown that surgical stress and / or anesthesia administered in surgery leads to transient immunosuppression, which may pose a risk to patients' lives due to increased susceptibility to infection, spread of tumor metastases, slowed wound healing and the like. Anesthesia with their toxic effects, surgical stress and changes in the neuro-endocrine-immune relationship resulting from transient paralysis of the central nervous system under anesthesia contribute to the pathogenesis of generated immunosuppression ('Vatkins J. (1980): Br. J. Posp. L 23: 5835-590, Udovič-Širola Ivi et al (1989) in Immune Consequence of Trauma (Shock and Sepsis, pp. 411-417), while some effects are due to changes in hepatic metabolism, occurring after administration of anesthesia and surgical stress, the non-pathogenic effect of anesthesia may be explained by the fact that most halogenated narcotics are metebolized in the liver, which can bloom toxic intermediates such as trifluoroacetyl halides or free radicals (Satoh H. et al. J. Pharm. Exp. Thc-rap. 233, 857). In predisposed persons, this may lead to the development of autoimmune hepatitis (Vergani et al. (1980): λ Engl. J. ed. 303, 66). It should be noted that this immunosuppressive hepetotoxic effect is not limited to patients being operated with endotracheal anesthesia, but may also include a medical staff operating in the operating room. Chronic exposure to haloid appears to result in a 1.3 to 2.0 more frequent development of female cancer (Eeden YM et al. (1986) in Anesthesia, Miller B. L. (ed.), NY, 730.). In view of the serious consequences that may occur following post-operative immunosuppression and prolonged exposure to anesthetic agents, the high number of therapeutic attempts for preventive action is not surprising. Thus, the use of: immunoglobulin has been described (Nitsche L. et al. (1988): lst internetionel Congress on the Immune Consequences of Trauma, Shock and Sepsis; OP 52), synthetic hormones (Feist E. et al. (1987): Int. J. Clin Pharm Bes 7, 83-67, Waymack JP et al (1985): Ar Surg 120, 43 Faist E. (1989) in Consequences of Trauma, Shock and Sepsis, 509 to 517), transfer factors and interferons (Josten Ch. et al. (1988) lst International Congress on Immune Consequences of Trauma, Shock and Sepsis, OP 43; Livingston BH et al. (1989) in Immune Consequences of Traume, Shock end Sepsis, pages 551 to 555), H-2 receptor blockers (Nielsen HJ et al 1968): lst International Congress on Immune Consequences of Trauma, Shock and Sepsis, OP 49), monoclonal antibodies against endotoxins (Segawa T (1989) in Immune Consequences of Trauma, Shock End Sepsis, pp. 495-507), vasoactive agents (Schontherting M. et al. (1988): International Congress on Immune Consequences of Trcume, Shock and Sepsis, OP 57), thrombocyte activating factor entagonists (Fletcher J.P. and company. (1988): lst International Congress on Immune Consequences of Trauma, Shock End Sepsis, OP 56) and various immunomodulators (Schopf RE et al. (1988): lst International Congress on Immune Consequences of Trauma, Shock End Sepsis; Tseng KP et al (1986): Int. J. Immunopharmecol., 8, 437; I-adden JW et al (T989) in the International Internet Congress on Immune Consequences of the Treuma, Shock end Sepsis, pp. 509 51 517 ·) .

The biologically active substance peptidoglycane monomer (PGM) is available by biosynthesis (according to Yugoslavian patent 35040) and was isolated as a chemically defined compound (according to Klaic B .; Carbohydr. Res. (1982) 110, 320; Yugoslav patent 40472; Austrian; No. 362740). His N-acylderivatives (South Slovak Patent Application P-626/89, European Patent Application 390093) and his metal complexes (South Slovak Patent Application P-1982/86, European Patent Application 268271) were prepared. The isolated substances are well soluble in water e in physiological solutions, are non-toxic e epyrogenic. they exhibit immunostimulatory, antimetestatic and antitumor activity.

SUMMARY OF THE INVENTION

The present invention provides a new use of the peptidoglycan monomer (PGM) and its K-ecyl derivatives of formula I

CH-, CH-.

I ³ I ³

CH-CHC0 NHCHC0-NHCHCH _{of the} CH-C 0 NHCHC0 NHCHC0-NHCHC00X 3 2 2 ((CH _9).

I ³

R-HNCHCONH ₂ (I) wherein R is hydrogen, Ac is a C 2 -C 18 straight chain alkylcarboxylic acid, a C 5 -C 2 branched chain alkyl carboxylic acid, an unsaturated alkenyl carboxylic acid and X represents a hydrogen atom, an alkali metal or alkaline earth metal atom, or a quaternary ammonium salt of an organic base, and their divalent complexes. with the general formula Ia or Id

N-Ac-Glc-N-Ac-Mur

L-Ala-L-Ala-0

X

M e

2a Co

2b Ni c Zn

2d Cd

N-Ac-Glc-N-Ac-Mur

Me

3a Cu

3b Hg

N-Ac-Glc-N-Ac-Mur

D-Ala-L-Ala-O (Ib) in the presence of drugs for the correction of humoral-type and cell-mediated immunosuppressive conditions induced by administration of anesthetics and / or surgical stress in surgery, other immunosuppressive and immunodeficiency vertebra induced by scsi, burns, body depletion, paraneoplastic syndrome and the like, and for the correction of hepatosuppressive conditions of the body, arresting changes in hepatic nucleic acids, particularly hepatic proteins, induced by anesthesia and / or surgical stress, as well as other conditions associated with immunosungression and / or hepatic disorders, ?.

Hitherto unknown activity of PGM, its N-acyl derivative of their divalent metal salts is illustrated in a model of experimentally induced postoperative immunosuppression.

Taking into account that most surgical operations are performed under general endotracheal anesthesia maintained by the administration of halogenated anesthetics, an experimental model was designed to induce immunosuppression similar to humans by applying halothane anesthesia with or without surgical stress. Therefore, BALB mice / c 2.5 to 3 months old are placed in hermetically sealed 11 metabolic cages containing soda lime (a mixture of calcium oxide and sodium hydroxide) to which air is supplied (by a respirator for small animals) with an addition of 0.5 to 1% halothane (flow 350 ml / min). This anesthesia is maintained for 1 hour. Animals from the control group were subjected to the same procedure except that helothane was not added to the air. A sub-group of mice was only subjected to halothane anesthesia, and the sub-group was further subjected to surgical stress in the form of laparotomy (abdominal wall cut). This operation prevented the animals from being exposed to halothane anesthesia ε was performed under brief ether anesthesia. In order to achieve the same conditions for control of this group, the laparotomy + helothane control groups were subjected to brief ether anesthesia immediately prior to halothane anesthesia. In the humoral and cell-mediated immunity control, the animals were then immunized with: e) sheep erythrocytes (OE) and on the fourth day after sensitization, the number of plaques in the spleen was analyzed, b) allogeneic tumor cells, sarcoma I growth (A / J mice) and c) parental splenocytes for analysis of the local donor cell response (BALB / c) to the (BALB / cx CBA) F ^ hybrid host. Each group comprised 58-8 animals. Statistical analysis was performed by Student's t-test.

The results show that halothane anesthesia per se showed an immunosuppressive effect on humoral and cell-mediated immunity (Fig. 1) and that operative stress caused by 1a, parotomy enhanced this immunosuppression. In mice that are sensitized with sheep erythrocytes, halothane anesthesia alone blocked plaque formation (PFC) in the spleen from 48.5% (p <0.001) and laparotomy of 27% (Fig. 1A); Inhibition of cell-mediated immunity was demonstrated by increasing the time with Biogenic tumor from day 11 to day 14 (all anesthesia) and from day 14 to day 16 (anesthesia + surgical stress; p <0.05) (Fig. 1B). Inhibition of cell-mediated immunity by helothan anesthesia alone was confirmed in a model of local donor cell response to recipient cells (GVHR - graft versus host reaction), where donor anesthesia induced a significant reduction in the knee lymph node size response on day 7 post parental lymphocyte injection 8.3 - 1.5 mg to 4.0 - 1.0; p <0.05).

Halothane-induced diminution of the humoral immune response was accompanied by hpyplasia (imperfect development) of bone marrow and spleen, a decrease in the proportion of CL4 and CL8 ⁺ cells, and an increase in the number of non-phenotype cells (Fig. 2).

Simultaneous determinations of hepatic proteins and nucleic acids in OE-sensitized and halothane-aesthetized mice showed some hepatosuppressive activity of anesthesia and its significant impact on the reduction in the amount and concentration of hepatic proteins, DNA e RNA (Fig. 3). In the experimental model, most of the symptoms that may occur after surgery in an anesthetized patient, and which cause:

1. immunosuppression -

(a) of a humoral type; and

(b) cell-mediated type

2. hepatosuppressive effect.

In this state of immuno and hepatosuppression, we further tested the effects of PGr.i and its analogs and compared their effects on intact mice. The results obtained show that PGi 1 and its analogs are highly effective in correcting such postoperative immunosuppression, while their effects are much weaker in intact organisms.

Medicinal product: PGM and its N-acyl derivatives and double complexes

The heavy metals or mixtures thereof can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, with other non-toxic, physiologically acceptable substances known to those skilled in the art, the unit dosage size and form depends on body weight and individual condition of the organism. PGM and its N-acyl derivatives and divalent metal complexes can be administered at a dose of 5 to 50 mg body weight.

The invention is further illustrated by the following examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Correction of humoral-type immunosuppression in anesthetized and operated animals by PGM (1a)

Since we found that the main suppression of humoral immunity occurs in helothane-anesthetized and laparotomized mice (Fig. 1), we tested the prevention of immunosuppression by application of PGM. For this reason, mice were given a single intraperitoneal injection of PGM dissolved in 0.05 ml of saline immediately after leparotomy and OE-sensitization and immediately before halothane anesthesia. The results presented in Table 1 show that PGM (10 mg / kg) increased plaque formation to 94.3% (PFC / 10 ^) in anesthetized mice, while in mice under helotane anesthesia who were also subjected to surgical stress , PFC formation was even stimulated up to 206.4% relative to the control injected with saline only.

- 8 Table 1

Humoral activity of anesthetized and laparotomized mice treated with PGM

group halothane axle with helothane. . . induced immuno- PFC / 10 suppression by PGM PFC / spleen PFC / 10 PFC / spleen laparotomy + SRBC + PGM 10 mg / kg ** 300.6 to 42.7 62968.3-16551.1 * 206% 124.80% laparotomy + SRBC + physiol. solution 98 to 22.5 2800.2 to 7997.5 SRBC + PGM 10 mg / kg 260.6 to 20.9 50007.8-4043.9 94.30% 64.70% OE + fyziol.r. 134.2 to 31.4 30363.5 to 10194.7 statistically significant appearance control house (* p <0.01; ** p <0.001)

Table 2 shows that the best correction of haemolan immunosuppression is achieved with a low dose of PGM. Simultaneous research into the effect of PGM in unenesthetized mice showed that the effect was only seen in immunosuppressive mice. The increase in plaque formation in anesthetized PGm-treated mice was accompanied by an increase in bone marrow cell counts and peripheral leukocytosis.

- 9 Table 2 (first part)

Humoral immunity in anesthetized and unesthetized mice after treatment with PGM

group without PPC / 10 halothane PFC / mallow OE + PGM 100 mg / kg OE-physiological. solution 225 to 6.9 .60339-5112,6 OE + PGM 50 mg / kg 385.6 to 36.1 111,608.4 to 11,079.3 * OE-physiological. solution 498.7 to 47.1 178262.3 to 25557.8 OE + PGM 10 mg / kg 465.6 to 25.7 87670,5-5552,2 ^m OE-physiological. solution 598.1 36.9 ^ 159847.7 to 10628.2

- 10 Table 2 (second part, completion) halothane group halothane correction

PFC / 10 PFC / spleen induced immunosuppression by PGM

PFC / 10 PFC / spleen

OE + PGM 39.6-6.0 * 100 mg / kg 8129.4 to 845.8 * -67 % -72.80 % OE-physiological. 120-14 29,888 to 6031.5 solution OE + PGM 159.3-39.5 ** 50 mg / kg 36085.2 to 10629.6 * ^Ť -9 % -32.40 % OE-physiological. 175-12 53356.9 to 1864.6 solution OE + PGM 651.2-85.4 * 10 mg / kg 106,092 to 4,812.9 * 66th , 20 í 32.10 % OE-physiological. 391.8 to 7.1 80301.8 to 6369.5

solution statistically significant relative to control (”* p <0.01; * p <0.001)

Example 2

Correction of humoral-type immunosuppression by the action of PGM-bivalent metal complexes

The PGM-Zn complex dissolved in saline is injected at the same dose as in the previous example (10 mg / kg) to anesthetized and non-anesthetized OE-treated mice. This experiment is repeated three times. All research p

PGM-Zn showed improved immunocorecular activity compared to PGM and increased plaque formation in anesthetized mice by 73.5 », ρη

101.4% relative to the control given saline only.

These effects are accompanied by an increase in spleen cells from the bone marrow.

Table 3

Humoral immunity in anesthetized mice treated with PGM with its derivatives

group Count animal with halothane PFC / 10 ⁶ correction (%) PGM-lOmg / kg 6 860-66.1 29.50 PGM-Zn 5 1152 to 41.1 73.50 L-PGM-On 6 932.5 to 78.1 40.40 fvziol rozp. 5 664-51.9 inspection PGM-lOmg / kg 5 295-42.3 21.90 PGM-Zn 5 419-41.0 73.10 L-PGM-On 5 508 to 3.9 109.90 fyziol. Sol. 5 242-11.1 inspection PGM-lOmg / kg 5 1782-200 68,40 PGM-Zn 6 2131-164 101.40 L-PGM-On 6 2338 ^ 184 120.90 fyziol. Sol. 6 1058.3-135 inspection

Example 3

Correction of immunosuppression of humoral type by N-acverivatives PGM

Sodium N-lyuroyl-PGM (PGM-L-Na) is dissolved in saline. This solution is injected into enesthetized and sensitized mice. Thus, there is twice as much a stimulation of plaques in the spleen (an increase of 109.9 ε 120.9 µ) relative to the control given only saline.

-f. ·. ·· .- · -

This effect was not present in non-anesthetized mice.

Example 4

Correction of PGM-mediated immunosuppression with divalent metals and PGM K-ecylderivatives

PGM and its analogs were tested for total GVHP. In this test, they were administered immediately after parental splenocyte injection into the soft hind leg of F1 hybrids or immediately before helothan anesthesia. PGM-Zn has been found to enhance the size of the knee lymph node, 7. day after injection, while PGM-L-Na significantly increases the number of large lymphoid cells in the local lymph node on day 10 post-injection (evaluated by cell flow counter).

Table 4

GVHP + halothane lymphatic gland weight differences

5 day Day 7 10 day PGM 2.2 to 1.4 5.2 to 0.8 3-1,3 L-PGM-On 2,5-1 4.3 to 1.7 2.5 to 0.7 PGM-Zn 6.2 to 1.4 -b fyziol. r oz. 1.5 to 0.2 4-1 1.8-1.4 differences in cells in lymph. glands (in millions) 5 day Day 7 10 day PGM 5.66 3 6.2 L-PGM-On 7.66 1.84 3.57 PGM-Zn 2.9 fyziol ro from. 2.38 3.6 count large cells ( in thousands) Day 5 Day 7 Day 10 PGM 6,034 8,032 7.82 L-PGM-NA 6,038 6,462 17,494 PGM-Zn 4,677 fyziol ro z t. OK 5,337 5,391

Example 5

Correction of hepatosuppressive effects during hypnotic anesthesia by PGM

While we found that halothane anesthesia in OE-sensitized mice caused a decrease in the number of hepatic proteins and nucleic acids (Fig. 2), we tried to determine whether PGM application affected those changes that accompanied immunosuppression of the oumoral type. The results presented in Table 5 show that a small dose of PGM induces a significant increase in all parameters examined (DNA, RNA and proteins) in the liver. Simultaneous testing of the effect of PGM on intact mice showed that the stimulatory effects of PGM were present in the anesthetized animals, i. in mice with suppressed liver function.

Table 5 (first part)

Hepetotropic effect of PGM in anesthetized and non-anesthetized mice DNA RNA group mg / 100b.hm. mg / g mg / 100bm. mg / g halothane + 10.09 * -2

PGM (100 mg / kg) halothane + 6.17-0.6

PGM (50 mg / kg) halothane + 8.74 * -2.72

PGM (10 mg / kg) halothane + 6.88-1.01 saline

1.59 -0.45 from 15.36 to 4.7 2.5 to 0.93 1.2 to 0.09 from 10.11 to 3.04 1.95 to 0.45 1.64 ^ -0.46 ^ 16.73 -4.99 3.15 -0.82 * 1.24 to 0.22 9.34 to 1.78 1.67 to 0.38

- 14 Table 5 (second part, completion)

group LNA RNA mg / lOOb.hm. mg / g mg / lOOb.hm. mg / g The PGM + 50 mg / kg 8.17 to 2.47 1.74 to 0.67 from 12.83 to 2.12 2.57 to 0.59 0+ PGM 10 mg / kg 7.14 to 1.55 1.48 to 0.35 from 12.25 to 1.69 2.54 to 0.38 0-physiological. solution ** 7.91 to 0.97 1.60 ^ 0.45 from 11.74 to 2.08 2.21 * 0.46 pr oteíny mg / lOOb.hm. mg / g halothane + 492.5 to 69.2 78.4 to 12.9

PGM (100 mg / kg) halothane + 379.3-57.3 78.4-12

PGM (50 mg / kg) halothane + 605.5 * 134 134.7 113.9 * -22.3

PGM (10 mg / kg) halothane + saline *

O + PGM (50mg / kg)

O + PGM (10mg / kg)

0-physiological. solution

463.5 to 80.2

491.8- 92.1

** 414.6 - 20.6

529.9- 69.7

83.7 to 15.4

95.9- 10.4

85.9- 3,9

110.7 to 26.8

Statistically significant between groups carrying the same index p <0.0?

- 15 Example 6

Hepatotropic effects of PGM, divalent metal PGM-complexes and PGM N-acylderivatives in anesthetized and operated mice

The effects of PGM and its analogs were tested in anesthetized and operated mice. PGM was found to cause an increase in the DNA, RNA and protein content of the liver of the animals. The effects of N-acyl derivatives on proteins in the liver were equally intense, while PGM-Zn stimulated the association of hepatic proteins, even more intensively than PGM alone.

Table 6 (first part)

Hepatotropic effect of PGM and its analogues in mice that have been anesthetized and surgery

group mg / lOOb.hm. LNA mg / g surgery + 16.7 ^x * -2.23 halothane - * - PGM ^{XÄX (1} ° 2.98 ** - 0.29 surgery - * - 11,39-4,16 halothane + PGM-Zn 10 mg / kg 2.03 to 0.81 surgery - * - 13,37-1,89 halothane - * - L-PGM-Na 10 mg / kg 2.39 to 0.44 surgery * 12,64-2,63 halothane * u v saline solution 2.24 to 0.59 surgery - * - 15,44-2,57 physiological solution * 2.65 to 0.36

- 16 Table 6 (second honor)

group mg / lOOb.hm. RNA mg / g surgery * 23.82 ^M, ** **0.8 halothane * PGM *** (10 mg / kg) 4,26 * '** - 0.23 surgery * 16,45-6,24 helotán * PGM-Zn 10 mg / kg 2.93 to 1.22 surgery * 22,57-4,31 helotán * L-PGM-Na 10 mg / kg 4.03 to 0.81 surgery * 18.14 * ± 2.84 helotán * saline * 3,2OÍO 66 surgery * 22,51-0,92 saline * 3.88 to 0.02

- 17 - Table 6 (third honor, completion) group Proteins mg / lOOb.hm. mg / g ch irurgia * halothane * PGM *** 10 mg / kg 540.2 -61.8 * 96.1 * '**' *** ί _1Ο , 5 surgery * halothane * PGM-Zn 10 mg / kg 667.6 * ^{, m} ' ^± 36.9 11 ₆ “« ^ 5.1 surgery * halothane * L-PGM-Na (10 mg / kg) 616 * '** - 64.3 109.5 * '** - 4.3 surgery * halothane * saline ** 456.7-58 80.2 to 10, 2 surgery * saline * 434.2 6.2 ^ 75-4.2

statistically significant between groups bearing the same index p <0.05.

/ W - 16 -

Claims (5)

PATENTOVÉ. Claims Use of a peptidoglycan monomer (PGM) and its N- -acylderivatives of the formula I ch ₃ chco-nhchco-nhchch ₂ ch ₂ co-nhchco-nhchco-nhchcoox (ch ₉ ) ₇ I ^{2 3} p-nitro ₂ (I) wherein R represents a hydrogen atom, Ac represents a straight chain alkyl group having 2 to 18 carbon atoms, a branched chain alkyl group having 5 to 18 carbon atoms, an unsaturated alkenylcarboxylic acid having 12 up to 18 carbon atoms or an aromatic carboxylic acid having 7 to 12 carbon atoms and X represents a hydrogen atom, an alkali metal or alkaline earth metal atom or a quaternary ammonium salt of an organic base, and its divalent metal complexes of the general formula Ia or Ib (Ia) M e 2a Co 2b Ni 2 C Zn 2d Cd - 19 N-Ac-G1 e-N-Ac-Mu r Me 3a Cu 3b Hg (Ib) characterized in that it is used in combination with other conventional non-toxic physiologically acceptable substances in the preparation of medicaments for correcting immunosuppressive and hepatosuppressive conditions of the body. Use according to claim 1, characterized in that the immunosuppressive conditions comprise a humoral type and a cell-mediated type induced by the administration of various anesthetics and / or surgical stress in surgery, and more particularly immunosuppressive and immunodeficiency conditions induced by sepsis, burns, body exhaustion syndrome and the like. Use according to claim 1, characterized in that the hepetosuppressive state of the body comprises changes in hepatic nucleic acids, in particular hepatic proteins, induced by anesthesia and / or surgical stress, as well as other conditions associated with immunosuppression and / or hepatic disorders, poisoning, hepatitis, etc. Use according to claim 1, characterized in that medicaments are prepared for intravenous, intraperitoneal, intramuscular and subcutaneous administration. Use according to claim 1, characterized by 20 in that the weight of the dose and the formulation depend on the body weight e of the individual condition of the organism. Use according to claim 1, characterized in that PGM, its N-acyl derivatives and bivalent metal complexes are administered at a dose of 5 to 50 mg / kg body weight.