CA2115270A1 - 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 states of the organism - 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

Wo 93/03746 pcr/Eps2/ol8s9 211~270 ., .~.
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PREPARATION OF MEDICAMENTS CONTAINING THE PEPTIDOGLYCAN MONOMER OR ITS - -DERIVATIYES.
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The present invention relates tO the use of the peptidoglycan monomer (PGM), itS ~T-acvl derivatives. c.nd its metal complexes in the preparalion of medicaments for~ the correction of the immunosuppress*e and hepatosuppressive effects of 2s anaesthetics and operative stress in surgical treatment or in other immuno-suppressi~e~ immunodeficient. and hepa~osuppressive states, tO achieve a swift and~saferecoY, ofthepatients.

Numerous pubhcations on clinical and~ experimental research in the last ten vears 30 have shown that surgical stress andlor anaesthetics administered in surgeIy develop a transient immunosuppression which may sepresent a risk for the patient's life due to the augmented suscepn~ilitv to infec~ions, spread of tumour metastases, impairment of wound hea~iIlg, and the like. To the pathogenesis of the generated immunosuppression coIJtn~ute jointly the anaesthetics with their 3s toxic e~ec~, ~he operame stress, and the changes in the rleur~endoc~ino-immuno relationsnip, resl~l~ing from the transient paralysis of the central nervous svste~ during anaesthesia. (Watkins J. 11g~0/ Br.~.Hosp. Med. 23:583-S90., Udovic-~irola M. e~ aL 119~91 Jll: Immune Consequenc~s of Trauma, Shock and .

Wo 93/03746 - Pcr/Eps2/ol8s9 2 11~ 270 2 Sepsis, p. 411~17); whereas, a cer~n cf~ect is caused by the changes in the metabo~ism on the liver level, resul~ing after the administration of the anaesthetics and the operative stress. The hepatotoxic effect of ~aesthetics maybe explained by the fact that the majority of halogenated narcotics are metabolized in the liver, which may yicld to~ac intermediates, such as e.g.
tri~luoroacetyl halides or ~ee radicals (Satoh H., et al. 11985/ J.Pharm.
Exp.Therap. 233, 857). ln predisposed persons this may result in the developmentof autoimmune hepa~itis (Vergani D., et a~ /19801 N.Engl. J. Med. 303, 663. 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 tbe medical staff working in the operating theatre. It seems that the chronic exposure to halothane resu~ts in a 1.3 - 2.0 oftener development of carcinoma inpersons of female sex ~Badcn Y.M., et al. 119861 ln Anesthesia, Eds. MiDer R.D.
N.Y., p. ~130). In view of the senous consequences which may arise aher the post-operative immunosupp. ession and the long-continued cxposition tO anaesthetics, the high number of therapeutic attempts for preveD~ive action is not surprising.Thus, there bas been descnbed the use of: immunoglobulin ~itsche D., et aL
/19881 1st International Congrcss on the lmmunc Conscquenccs of Trauma, Shock and Scpsis; OP 52), synthetical hormoncs (Faist E., et aL /1987/ lnt. J. ain.
Pharm. Res. 7:83-87; Waymack J.P., et al. /1985/ Arch. Surg. 120:43; ~aist E.
/1989/ In Immune Consequcnces of Tsauma, Shock and Sepsis, p. 509-517), transfer factors and mterfcrons (Jostcn C~., et a~ 119881 1st. Intcrnational Congress on the lmmune Consequcnces of Trauma, Shock and Sepsis., OP 43;
Livings~on D.H. et a~ /19891 In: Immun~ Consequences of Trauma, Shock and Sepsis, p. 551-555), ~1-2 receptor b~ockers (Nielsen H. J., et al 11988/ 1st lnternational Congress on thc lmmune Consequences of Trauma, Shock and Sepsis, OP 49), monoc~onal antl~odies against endotoxins (Sagawa T., et al. 119891 ln: Immune Consequences of Trauma, Shock and Sepsis, p. 495-507); vasoactive agents (Schontharting ~1., et aL 1198811st lnternational Congress on the lmmune Consequences of Trauma, Shock and Sepsis, OP 57); trombocite-activating-factors antagonists (~letchcr J.R., et a~ l1988/ 1st lnternational Congress on the lmmune Consequ~nccs of Trauma, Shock and Sepsis, OP 56); and various immunomodulators (Schopf RE., el o~ 119881 1st lnternational Congress on the Immune Consequences of Trauma, Shock and Sepsis; Tsang K.P. et aL 11986/
lnt.J.lmmunopharmacol. 8:437; Hadden J.W. et ~ 119891 ln: 1st International Congress on the lmmune Conscquences of Trauma, Shock and Sepsis, p. 509-517).

SUBSTITUTE SHEET
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- W093/03746 i~ 5 2 7 0 pcr/Ep92/ol859 ~ ~;

The biologically act*e substance peptidoglycan monomer ~PGM) was made ~;
available by biosynthcsis (in accordance with YU Patent 35040) and isolated as a ~ ~;
chernical~y def~ned compound ~according tO Klaic B. Carbohydr. Res. (1982) -~
110:320; YU Patent 40 472; AT Patent Specification 362740~; later, there were s prepared its N-acyl dcrivatives (YU Pat. Appl. P-626/89; Eur. Pa~. Appl. EP
39 00 93), and its metal complexes (YU Pat. Appl. P-1982/86; Eur. Pat. Appl. EP
26~271). The isolated substances a~e well-soluble in water and physioJogical solution, non-toxic, and apyrogenic. They demonstrate immunostimula~ing, ar~timetastatic, 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

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(CEI2)3 c~IcON~2 wherein R stands for hydrogen, Ac stands for a straight (C2 - Cl~ alkyl~ carboxylic acid group, or a branched (Cs - C1g alkyl~ earboxylic acid group, or an 35 unsaturated (C12 - C1g alkenyl) carboxylic acid group, or an aromatic (C7 - C12) carbo3~vlic acid grGup, and X stands for a hydrogen, or an alkaIi metal, or an alkaline earth metal, or ~a quaternary ammonium salt of an organic base, and complexes thereof with bivalent metals of the formulae Ia or Ib SUBSTITUTE SHEET

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wo 93/03746 ~115 2 7 0 pcr/Eps2 in the prepara~ion of medicame~ts for the correction of .he immunosuppress*e states of the humoral and cell-mediated type, induced by the administraaon of various anaesthe~ics and/or operative stress in surgery, and of other immun~
suppressive and immunodeficient states, induced by sepsis, burn injuries, body 5 exhaustion3 paraneoplastic syndrome, alld the )ike, 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 ~y anaesthesia, and/~r operative stress as ~ell as other states, associated with immun~
suppression, and/or hepatic disorders, in~o~ocations, hepatitis, etc.
~0 The hitherto not kno~n activity of PGM, i~s ~-acyl derivatives and their bivalent metal salts ~s il~ustrated by the demons~ration on the model of an experimentally induced post-operative immunosuppression.

1$ Taking into account that the majority of surgical operations is performed in general endotracheal anaesthesia maintained by the administration of halogenated anaesthehcs~ there was designed an E~perimental Model enabling the indu~ement of an immunosuppression ~nilar as in humans, by the application of the halothane anaes~hesia, with or v~thout opera~ive stress. For this 20 reason BAl,B/c micc, agcd ~5-3 months were placed into hermetically closcd 1-L
~etabolic cages contaîniIlg soda lime, into which air (a flow of 350 mLJmin) with added 0.5-1% of halothane was chargcd by means of a respirator for sma~
animals. l~he narcosis was maintained for 1 hour. Animals in the control group were subjeeted to the same procedure, with thc exception of halothane in the air25 for 1 hour. A sub-group of mice was exposed only ~o halothane anacs~hesia, whereas, a sub-group was additionally subjected to operative stress in the form of laparotomy. This opera~ion preceded the cxposure of the animals to halothane anaesthesia, and was performed under short ether narcosis. ln order to maintain identical condi~ions in ~he contro~ for this group, the control groups for 3~ laparstomy ~ halothane were also immediately befoIe halothane anaesthcsia subjected to a shon ether narcosis. For the control of the humoral, and cel)-mediat~d immunity the animals were th~n ilslmunized: a) with she~p erythrocytes (OE), and the number of plaques in the spleen was analyæd on the 4tb day after the sensibilisation; b) with allogeneic tumour ce}ls, and the growth of the sarcoma 35 1 (from A/J mice) and their rejection time; and c) paternal splenocytes for the analysis of the local reaction of tbe donor cells (BALB/c) against the recipient(BALB/c x CBA)Fl hybrid. Each group comprised 5-8 animals. The statistical analysis was performed by ~tudent's t-test .

SUBSTITUTE SHEET

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lhe results demonstrated that the halothane anaesthesia per se exer~ed a;
~munosuppressive e~fect on the humoral a~d cell-media~ed immunity (Fig. 1), and that the operative stress caused by laparotomy potentiated this -immunosuppression. Thus, in mice sensl~ilized with sheep erythrocytes halothane s anaesthesia alone bloc~ced the plaque (PFC) formation in the spleen for 48.5 %
(p < 0.001), and laparotomy for additional 27 % (Fig. lA); whereas, the inhibition of cell-mediated immunity was manifested by the prolongation of the allogeneic tumour-1oearing ~om the 11th to the 14th day (anaesthesiaperse), and ~om the 14th to the 16th day (anaesthesia + operative stress; p < 0.05) (Fig.
o 1B~. 1 he inhibition of cell-mediated immunity by halothane anaesthesia per se was confirmed by the ~Iodel of local response of donor cells against host cells ~GVHR - graft versus host reaction~, in which the anaesthesia of the donor induced a sigruficant response diminishrnent on the popliteal Iymphatic node level on the 7th d~y after the injection o~ paternal lymphocytes (~rom the norma} value :
~ 8.3 + 1.5 rng to the value 4.0 + 1.0; p < 0.05).

The halothane-induced diminishment oî the humoral i~mune response waS
a~compar~ied by hypoplasia of thc bone marrow and thc spleen, and thc assessment of the deereased propornon of CD4 and CD8+ cells, and the increasc 2D of the number pf cells not belonging ~o this phenotype. (Fig. 2). ~:

The simultaneous detcrrDination of ~he hepanc prot~ins and nuclcic acids contents in OE-sénslbilized and halothane-anaesthetized mice demonstrated a certain hepatosuppressive ac~vity of anaesthesia, and its sigIuficant effect on the 2~ decrease of ~he amount and~concentration of hepa~ic proteins, D~A, arJd RNA ~ .
(Fig 3). Accordingly, it was succeeded in the applied Expenmental ~vIodel tO
imitate the majority of ~ptoms, which may anse after the operation in an anaesthetized patient, and cause:
1. immu~osuppression-3~ a) of the humoral, and ~b) ccll-mediate~ type; . ~ -2 a hepatosuppressive e~ect.
Ill this state ~ immuno and hepatosuppression we tested further the e~fects of ~-PG~, and its analogues, and compared their e~ects in i~tact, ~o~-suppressed ~-3S micc. Thc obtained results dcmonstIated that PG~ a~d i~s analogues werc hig~ ~-efficient Lll ~e very corlec~:on of such post~pera~e immunosuppressio~ where~s, its ef~ects were much fcé~ler in a~ ta~t organism~
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, SUB5TITlJT~ S~EET

WO 93~03746 2 ~ I ~ 2 7 ~ PCr/EP92/OlX59 Medical formulations: PGM and itS ~-acyl derivatives and complexes with bivalent metals or mixtures thereof may be administered intravenously, intrapentoneally, intramuscularly, and subcutaneously, in composition with o~hernontoxical, physiologically acceptable substanc~s kno~m in the art T~e ur~it dose 5 size and form depeDd on tbe body weight and the individual st~te of the organism.
PGM and its N-acyl derivatives and complexes with bi~ralent metals may be administered in a dose of 5-50 mg per kg of body weight.
l`he invention is ~lustrated by the following Examples.

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SUBSTITUTE SHEE~T

WO 93/03746 PCI`/EP92/01859 ~115270 8 EXA~LE 1 Correction of humoral type immunosuppression in anaesthetized and operatcd animals with PG~ (la) Since we have found, that the major suppressioD of humoral immuT ity resulted ins halothane-anaesthet~ed and laparotomized tnice (Fig. 1) there was tcstcd the preventioD of immunosuppression by the application of PGM. For this tcasoD
rnice were given one intraperitoneal injection of PGM dissolved in 0.05 mL of physiological solution immediately after laparotomy and OE-sensl~ilization, and imrnediately before ha]othane anacsthesia. The results shown ~ Table 1 .
10 demonstrate that PGM (10 mg/kg) in anaesthetized mice increased the plaque generation for 94.3 % (PFC/106), whereas, in mice subjected to halothanc ~:;
anaesthesia and operative strcss the PFC generation was stimulatcd cven for :
206.4 % with respect to the conirol inJected only with ph~siological solution. .

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TABLE 1~UMOR~LI-~MU~I~AES~ZEDA~DW~ROTOM~ZED ~.
PGM.TIU~I~ ~OE
:: 1- --~ ~Nithh~lolh~De~ ~Correction Orll810tbl~De iDdUCtd j Group immunosuppression with PGM
PFC/10 PFC/Spleen PFC/10PFClSpleen .
; ¦ Làparotom~! + j 30û.6:~42.7-- ~ 629683:tl6551.1- ¦ 206% 124.80~ I .
SRBC~PGM - .
¦lOm~ ¦ : l :~ l~ pnrotom~J~, . 9~15 2~100~79975 ~ ::;
SRB~C+ : ~ : .
Pbysiol.s.
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SRBC+PGM 260~209 50007~ 4043.9 9430% 64.70~ :~

¦10mg/kp ¦ _ _ ¦OE+Ph~S~OI~ ¦ ~4~31.4 30363.5+10194-7 l ¦
St~tisti~ll~ si~nS canl w~th rtspect to control (~ p~O.Ol; ~ p<O.OOl) ,' SUBSTITUTE SHEET

WO 93/03746 ~ 1 1 5 2 7 0 P~/EPg2/01859 Table 2 shows that the best correction of halothane immunosuppression was achieved with a low PGM dose, and the simultaneously perforrned investigation of the PG~f effect in non-anaesthetized mice demonstrated that the effect was achieved only in immunosuppressed mice. The plaque generation increase in 5 anaesthetized PGM-treated mice was accompanied by the bone-marrow cell augmentation and an expressed periphereal leukocy~osis.

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~1 LS270 EX~LE 2 Correction of humoral type immunosuppression with PG~ -complexes with bivalent metals l~e PGM-Zn complex dissolv~d in physiologic~l solution was injected in the 5 same dose as in the fo}egoing Example (10 mg/~g) in anaesthetized and non-anaesthetized OE-treate~ mic~. The experiment was repeated three ~mes, and in all investigations PGM^Zn demonstrated an ~mproved immunocorrectlve . activiry in comparison with PGM. and incTeased the plaque generation in ana~sthetized mice for 73.5 ~c, 73.1 Ci~G, and 101.4 ~c, with respect to ~he control o injected onlv with physio]ogical sc~lution (Tab1e 3 ) .
These e~ects were accornpanied by the cell-augm~ntation of the spleen and the bone-marrow.
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TABLE 3 E~ORAL ~ m ~ zED MlCE
- TREAll~D Wll~ PGM ~ ITS DERIVAI~VES

Group ~lo. otW;th balothaDe CorrecS~on ~n;malspF~o6 (%) PM 1~mg/kg 6 860~66.1 ~9.~0~
PGM Zn 5115~41.1 7~.50%
~-PGM ~a 6:932.5$78.1 4~.40~o Physiol.s. 5 664~1g Control PGM-lOmg/kg 5 295~:423 21.90%
PG~ Zo ~419i:41.0 73.10%
L-PG~ a i 30~ 3g 109.~0 %
Pb~siol.s. ~ 242:~11.1 Control PGM lOmg/kg ~ 1782:~200 68.40 PGM-i~n 62131il64 1û1.4û~
L PGM Na 6~338+184 120.90%
Physiol.s. 6 1058.3i:13~ Co~trol .

~UBSflTU~ SHET

~11527U : ~
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EX~IPLE 3 CoITection of humoral ~pe immunosuppression with ~-acyl derivatives of PG~

The sodium salt of ~I-lauroyl PGM (PGM-L-~la) dissolYed in physiological ~ -s solution and injected in anaesthetized and sens~bi~ized mice caused twice the best stimulation of plaque gelleration in the spleen (augmentation of 109.9 % and 120.9 % with respect to the control injected with physiological solutlon :~
-~Table 3). Thls effect was also absent in ananaesthetized ;mice. ~.
0 EXA~PLE4 Correction o~ cell-mediated type immunosuppression w~th PG~I-complexes with bivalent metals~ and ~I-acyl denvatives of PGM

PG~I and its analogues were tested in local GVHR in which they were ~ ~;
15 administered immediately after the injectjon of paternal splenocytes into the hind leg pad of F1 hybnds, or immediately be~ore halothane anaesthesia. It was found,that PGM-Zn potentiates the response on the popliteal Iymphatic node level on the ?th dav ater the injection, whereas, PGM-L-~a significantly increases then umbe~ of large Iymphatic:cells in the local Iymphatic node on day 10 after the20 : in~ection (assessed by means~of counter-flow cytomete~) (Table 4 ) .

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WO 93/03746 ` i~ ~15 2 7 0 Pcr/Ep92Jol8s9 TABLE 4 GVHR + HALOT~lANE
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Weight difference o~ lymph gland 5th day 7th day 10th d~y PGM 2.2:~1.4 5.2:$0.8 3+13 L-PGM-Na 2.5+1 43:~1.7 2~:~0.7 .
PGM-Zn ~ 1.4 Physiol.s. 1.5*0.2 4tl 1.8i:1,4 Ctll dif~ertnce in lgmph node ~million) 5th d~ 7th dsy 10th d~

PGM ~ 5.66 . 3 62 L-PGM-Na 7.66 1.84 3.57 -P&M-ZD 2,9 ~
- ~. Physioi.s. 238 3.6 . -Number of large cells (thous~nd) 5th d~ 7tb d~y10th d~y PGM 6.034 8.032 7.82 ..
I,P~;M~Na ~ i 6.038 6.462 17.494 PGM-Zo 4.6M
PhysiDI~ 5337 5.3gl :

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21 L5270 `~
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Col~ ectlnn o/ hepatosuppr~ssive ef~ects during halothane anacsthesia with PG~l Since we found ~hat the halo~hane anaesthesia in OE-senslbilized mice caused thes diminishment of the number of hepatic proteins and nucleic acids (Fig 3), we tried to establish whether the application of PG~ influenced these changes whlchaccompany the immunosuppression of the humoral type. The results shoun in Table 5 dernonstrated that a small dose of PG~ induced a significant increase 1nall investiga~ed parameters (D~A, R~ and proteins) in the liver. Thc o simultaneous testing of PGhl effect- in intact mice demonstrated that the . stimulating ef~ects of PG~ were present only in anaesthetized animals, to say in hepatosuppressed mice.

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EX~P~E 6 Hepatotropic e~e~ts of PG~, PG~-complexes with bivalent metals and ~-acyl .
deri~atives of PGM in anaesthetized and operated mic~

s The e~ects of PGM and its analogues were tested in anaesthetized and operated mice and it was estabbshed that PG~ caused an increase of the Dl`~ A, and proteins contents in the livers of said animals. l~e ef~ects of ItS ~^acyl deriva~ives on the liver proteins were of the same intensity, whereas, PG~l Zn stimulated the ~ ~
association of hepa~ic proteins, even ~ore intensive than PGM alone (Tab1e 6 ;;
and F ig . 4 ) .

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Claims (6)

CLAIMS:

1. The use of N-acyl derivatives and metal complexes of the peptido-glycan monomer (PGM) of the formula I

wherein R stands for hydrogen, Ac stands for a straight (C2 - C18 alkyl) carboxylic acid group, or a branched (C5 - C18 alkyl) carboxylic acid group, or an unsaturated (C12 - C18 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 Ia and Ib 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 andimmunodeficient 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 well 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 oh 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.