Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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
:
- 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
.
CH20~ CEI20~
_ ~~ ~
~EIAC / ~AC
~3 CON~2 1 ~3 C~3
C~3CHCO-~HC~CC~-NEIlElCE12CH~CO-~THCEICO-N~C~lCO-NEIC~COOX ., ,~
(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
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WO 93/03746 PCI/EP92/01859
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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.
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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
2 7 ~
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~
.
,
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. .
,.".
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)
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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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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.
.
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 : ~
12 :
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
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TABLE 4 GVHR + HALOT~lANE
.
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
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S~E~STITUTE SHEET ~
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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