EMPLOI DE PEPTIDES DU TYPE MURAMYLPEPTIDES POUR LE TRAITEMENT DE TROUBLES DE MYELOSUPPRESSION OU D'AUTRES ETATS AFFECTANT L'IMMUNITE

THE USE OF MURAMYLPEPTIDES IN THE TREATMENT OF MYELOSUPPRESSED OR OTHERWISE IMMUNOCOMPROMISED STATES

Abrégé français

L'invention porte sur l'emploi de composés de peptides de type muramyle pour la préparation d'un agent atténuant l'immunosuppression.

Abrégé anglais

The use of a muramyl peptide compound in the preparation of an agent for the
alleviation of immunosuppression is disclosed.

Revendications

30
CLAIMS:
1. The use of a muramyl peptide compound of general
formula I:
<IMG>
wherein:
R represents a residue of an amino acid or a linear
peptide built of from 2 to 6 amino acid residues, at
least one of the residues being optionally
substituted with a lipophilic group; and
n is 1 or 2;
in the preparation of an agent for the treatment of
myelosuppressed or otherwise immunocompromised states.
2. The use as claimed in claim 1 wherein the agent is
adapted for oral administration and comprises 0.5-50mg of
the muramyl peptide.
3. The use as claimed in claim 1 or claim 2 wherein n
is 1.

31
4. The use as claimed in any one of claims 1 to 3
wherein the proximal amino acid residue is a residue of
an L-amino acid.
5. The use as claimed in claim 4, wherein the proximal
amino acid residue (or the only amino acid residue, if
there is only one) is a residue of L-alanine.
6. The use as claimed in any one of claims 1 to 5,
wherein the second amino acid residue from the proximal
end of the peptide, if present, is of the D-configuration.
7. The use as claimed in claim 6, wherein the said
second amino acid residue is of D-glutamic or D-aspartic
acid or a mono-, di- or mixed C1-C22 (preferably C1-C6)
alkyl ester, amide or C1-C4 alkyl amide thereof.
8. The use as claimed in any one of claims 1 to 7,
wherein the said second amino acid residue is
D-isoglutaminyl or D-glutamyl.
9. The use as claimed in any one of claims 1 to 8,
wherein the third amino acid residue from the proximal
end of the peptide, if present, is in the
L-configuration.
10. The use as claimed in claim 9, wherein the third
amino acid residue is L-alanyl or L-lysyl.
11. The use as claimed in any one of claims 1 to 10
wherein the amino acid residue or linear peptide is
optionally substituted with at least one lipophilic
group.

32
12. The use as claimed in claim 1 or claim 2, wherein
the compound is N-acetyl-glucosaminyl-N-acetyl-muramyl-L-
alanyl-D-isoglutamine (GMDP).
13. The use as claimed in claim 1 or claim 2, wherein
the compound is:
N-acetyl-glucosaminyl-N-acetyl-muramyl-L-alanyl-D-
glutamic acid (GMDP-A);
N-acetyl-D-glucosaminyl-(.beta.1-4)-N-acetylmuramyl-L-
alanyl-D-glutamine n-butyl ester (GMDP-OBu);
N-[Na-Acetyl-D-glucosaminyl-(.beta.1-4)-N-acetylmuramyl-
L-alanyl-D-isoglutaminyl]-N.epsilon.-stearoyl-L-lysine
(GMDP-Lys (St));
N.alpha.-[N-Acetyl-D-glucosaminyl- (.beta.--4) -N-acetyl-muramyl-
L-alanyl-.gamma.-D-glutamyl]-N.epsilon.-stearoyl-L-lysine (GMDPA-
Lys(St));
N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-L-
alanyl-D-glutamic acid dibenzyl ester (GMDPA(OBz1)2);
N-Acetyl-D-glucosaminyl-(.beta.--4)-N-acetylmuramyl-N-
methyl-L-alanyl-D-isoglutamine (Me-GMDP);
N-Acetyl-D-glucosaminyl-(.beta.--4)-N-acetylmuramyl-(.beta.1--4)
-N-acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-
bis-(L-alanyl-D-isoglutamine) ((GMDP)2);
N-Acetyl-D-glucosaminyl-(.beta.--4)-N-acetylmuramyl-(.beta.1--4)
-N-acetyl-D-glucosaminyl-(.beta.--4)-N-acetylmuramyl-
bis-(L-alanyl-D-glutamic acid) ((GMDPA)2);

33
N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-(.beta.1--4)
-N-acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-
bis-(L-alanyl-D-isoglutaminyl-L-lysine) ((GMDP
Lys)2) ;
N-acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-(.beta.1--4)
-N-acetyl-D-glucosaminyl-((.beta.1--4)-N-acetylmuramyl-
bis-[L-alanyl-D-isoglutaminyl-N.epsilon.-stearoyl-L-lysine]
([GMDP-Lys(St)]2);
N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-L-
alanyl-D-isoglutamine 1-adamantyl ester (GMDP-Ad);
L-Threonyl-N.epsilon.-[N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-
acetyl-muramyl-L-alanyl-.gamma.-D-isoglutaminyl]-L-lysyl-
L-prolyl-L-arginine (GMDP-tuftsin E);
N-Acetyl-D-glucosaminyl-((.beta.--4)-N-acetyl-muramyl-L-
alanyl-.gamma.-D-isoglutaminyl-L-threonyl-L-lysyl-L-
prolyl-L-arginine (GMDP-tuftsin A);
N-Acetyl-D-glucosaminyl-((.beta.1--4)-N-acetylmuramyl-L-
alanyl-.alpha.-D-glutamyl-L-lysyl-L-threonyl-N.epsilon.-stearoyl-L-
lysyl-L-prolyl-L-arginine (GMDPA-tuftsin
lipophilic);
N.epsilon.-[N-Acetyl-D-glucosaminyl-((.beta.1--4)-N-acetyl-muramyl-
L-alanyl-.gamma.-D-isoglutaminyl]-L-lysyl-L-histidyl-L-
glycine amide (GMDPA-bursin);
N-Acetyl-D-glucosaminyl-((.beta.1--4)-N-acetylmuramyl-L-
alanyl-D-isoglutaminyl-L-glutamyl-L-tryptophan
(GMDP-thymogen I);

34
N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-L-
alanyl-D-isoglutaminyl-.epsilon.-aminohexanoyl-L-glutamyl-L-
tryptophan (GMDP-thymogen II);
N.alpha.-[N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetyl-muramyl-
L-alanyl-D-isoglutaminyl]-N.epsilon.-stearoyl-L-lysyl-L-
glutamyl-L-tryptophan (GMDP-thymogen III);
N-acetylmuramyl-L-threonyl-D-isoglutamine (Thr-MDP);
or
N-acetylmuramyl-L-alanyl-D-glutamine n-butyl ester
(Murabutide).
14. The use as claimed in any one of claims 1 to 13
wherein the agent comprises 1-5mg of the muramyl peptide.
15. The use as claimed in claim 14 wherein the agent
comprises 5mg of the muramyl peptide.
16. The use as claimed in any one of claims 1 to 15
wherein the myelosuppression or immunocompromised state
results from radiation treatment or chemotherapy, eg when
used for cancer therapy.
17. The use as claimed in any one of claims 1 to 15
wherein the myelosuppression or immmunocompromised state
results from myeloablative therapy followed by
transplantation of marrow or other progenitor material.
18. A method for the treatment of myelosuppressed or
otherwise immunocompromised states in a subject which
comprises orally administering to the subject 0.5-50mg of
a muramyl peptide compound as defined in any one of
claims 1 to 13.

35
19. A method as claimed in claim 18 wherein 1-5mg of
muramyl peptide is administered.
20. A method as claimed in claim 19 wherein 5mg of
muramyl peptide is administered.
21. A method as claimed in any one of claims 18 to 20
wherein the immunosuppression is caused by radiation
treatment or chemotherapy.

Description

CA 02265890 1999-03-04wo 93/09989 PCT/GB97/024221THE USE OF MURAMYLPEPTIDES IN THE TREATMENT OF MYELOSUPPRESSED OR OTHER-WISE IMMUNOCOMPROMISED STATESThe present invention relates to the use of Muramylpeptides in the preparation of an agent for the5 alleviation of myelosuppression. In particular, itrelates to such use in the preparation of an agent foralleviating myelosuppression following treatment ofcancer patients by radiation or chemotherapy.10 The muramyl peptides are a class of glycopeptidecompounds which in nature constitute part of bacterialcell walls (Ellouz et al, Biochem Biophys Res Comm,59:1317—l325, (1974)). This term has also come toinclude synthetic variants of naturally occurring forms.15 Muramyl peptides were discovered by virtue of theirability to act as adjuvants to injected vaccines (Adam,Synthetic adjuvants, In: Modern concepts in Immunology,Vol 1, (1985)). As studies progressed, it was also foundthat these molecules also possessed other immunostimulant20 properties, and had the ability to enhance nonspecificimmunity to infectious organisms (Chedid et al, PNAS USA,7422089, (1977)) possibly by activating specific celltypes of the immune system, causing them for example toingest foreign particles with greater efficiency25 (Andronova and Ivanov, SoV' Med Review D. Immunology4:1—63, (1991)).More recently, it has been found that under someconditions, certain muramyl dipeptides can also have30 anti—inflammatory or immunosuppressive properties, whichis contrary to their usual immunostimulant effect(Adeleye et al, APMIS, 1o2:145-52, (1994)).Thus, the utility of this class of molecule in theWO 98/09989l015202530CA 02265890 1999-03-04PCT/GB97/02422treatment of myelosuppression has been very limited asevidenced by the lack of commercial products.The smallest functional muramyl peptide, N—acetylmuramyl—L—alanyl—D-isoglutamine (MDP) and its close analoguescontain one sugar residue, N—acetylmuramic acid. Anotherclass of muramyl peptide contains a disaccharide, namelyN—acetylglucosaminyl—N—acetylmuramic acid. The simplestof thesecompounds is N—acetylglucosaminyl—N—acetylmuramyl—L—alanyl—D—isoglutamine, abbreviated asGMDP. These forms are exemplified by the range ofcompounds described in US patent 4,395,399. Some studieshave shown that GMDP has properties distinct from MDP.For example, GMDP is less pyrogenic (fever inducing) thanMDP, and in some circumstances has more distinctanti~inflammatory properties (Adeleye et al, (1994),supra).The development of chemotherapeutic drugs has greatlyincreased the medical profession’s ability tosuccessfully treat numerous types of cancer. Similarly,irradiation of the human body is used for certaincancers. The principal of these treatments is to killrapidly dividing cells, such. as the rapidly‘ dividingcells of a tumour. Unfortunately, certain healthy cellsof the body also divide rapidly, and their damage by thecancer treatment methods results in unwanted side effectsof the treatment. For example, skin and hair bulb cells,and the cells lining the gut are rapidly dividing, anddisturbances in these tissues are well known in patientsundergoing cancer therapy. Of particular concern is("bloodtissues, the consequence of which is adamage caused toso—called hematopoieticforming”)reduction in the number of circulating cells of theWO 981099891015202530CA 02265890 1999-03-04PCT/GB97/02422immune system. While this is not harmful per se, thereduced immune function renders these patients moresusceptible to life threatening infections.The seriousness of this so called myelosuppressiveside~effect is evidenced by the fact that repeated cyclesof anti—cancer treatment are often reduced in intensityor delayed in time, in order for immune function tobecome restored to an acceptable level. Therapeuticagents have therefore been developed which can counteractthe myelosuppressed state. The best known and currentlymost widely" used of these are the so—called "colonystimulating factors", which cause precursors of theimmune cells to differentiate and multiply, and thereforereplenish the cells killed by the radiation orchemotherapy. These factors are proteinaceous, expensiveto produce, and have to be administered by repeatinjection or infusion (Gabrilove et al, New England JMed, 318:l4l4-22, (1988)).The ability of MDPs and GMDP to counteract states ofmyelosuppression arising from the use of radiationtherapy and chemotherapy has been illustrated in animalmodels (Azuma and Otani, Medicinal Res Reviews,l4:401-14, (1994); Adrianova et al, Radiobiologia,32:566—70, (1992)) and, in the case of the hydrophobicanalogue MDP—stearoyl—L—lysine, has even been used forspecific therapeutic advantage in cancer patients(Tsubura et al, Arzneim. ~Forsch/Drug Res, 38:lO70—74,W089/01778). In all these cases, the drug has beenadministered to patients by injection, and in doseseffective for human use has been associated with theoccurrence of side effects such as fever and localreactions at the site of injection (Tsubura et al,1015202530WO 98/09989CA 02265890 1999-03-04PCT/GB97/02422supra).The most effective agents for the restoration ofneutrophils and protection from neutropenia are thecolony stimulating factors (CSF) described above, ofwhich granulocyte - CSF (G—CSF) is the most commonly usedin the clinic at the present time. These agentsspecifically target precursor cells, thereby inducingformation of neutrophils, and it might be anticipatedthat as such they would be more effective than a muramylpeptide, including GMDP, which does not in itself havethe ability to induce precursor cells, but needs to causerelease of messengers which in turn induce precursorcells. The limited ability of MDPs to increaseneutrophil numbers is highlighted even by marketedproducts such as Romurtide, which perform less well thanG-CSF and has never been a major commercial success.Our studies (see Example 1 below) confirm this suspicion,and demonstrate a lower neutrophil peak in mice renderedneutropenic with cyclophosphamide and subsequentlytreated with GMDP than was obtained with G—CSF. In fact,in this model where mice rendered neutropenic withcyclophosphamide (CYA) were challenged with an infectionof Candida, GMDP did. not increase neutrophil numbersabove the CYA. controls. However, most surprisingly,although the effect on neutrophil number was lower, whenthese same animals were challenged with a potentiallylethal dose of the micro—organism Candida, GMDP gave avery effective protection against lethality, compared tothe high mortality seen in animals receiving CYA alone.Surprisingly, it would therefore appear that neutrophilinduction alone is not a good indicator of overallefficacy against "neutropenia", in which the most_ WO 98/099891015202530CA 02265890 1999-03-04PCT/GB97/02422important feature is protection from the clinicalconsequences of immune suppression, not just the cellcount per se. Clearly, "immunostimulation" is not asingle activity, or G—CSF would have been more effectivethan GMQ§.immunostimulator is not sufficient to predict that itTherefore the knowledge that GMDP is anwould be so effective at treating chemotherapy-inducedneutropenic immunosuppression. Vast numbers ofimmunostimulators have been identified over the yearshowever only a very small handful have found utility inthe market place. The correct balance of immune cellactivation appears to be critical for the identificationof drugs with clinical utility.As has been discussed, muramyl peptides are able toincrease neutrophil numbers inanimals renderedimmunosuppressed by chemotherapy, when delivered byinjection. However, such an effect has never beenobserved following delivery by the oral route. InExample 2 we have shown that in a mouse model, mostsurprisingly, a strong‘ effect on neutrophils is seenafter oral administration of GMDP as well as afterinjection of GMDP. The significance of this observationneeds to be considered in the light of human clinicaldata. Thus, in human clinical Phase I studies (example3), it was found that an intramuscular injection of aslittle as 250 micrograms of GMDP induced fever(temperatures increased by l.SoC) and flu like symptoms.When temperatures were raised, white blood cell countswere elevated, but clearly the side effects wouldpreclude the use of this route of administration as atreatment for neutropenia.In other Phase I trials, GMDP was administered as oralWO 981099891015202530CA 02265890 1999-03-04PCT/GB97/02422tablets. Doses of up to 50 gm were given with nosignificant induction of fever or side effects (Example4). From animal studies, it is known that whenadministered by the oral route typically about 10% ofGMDP enters the systemic circulation. However, the humanoral dose cited above (50mg), is 200 times greater thanthe 250 micrograms which causes fever by injection. Thisabsence of side effects from oral administration istherefore most surprising, meaning that it has thepotential to provide a useful treatment for neutropenia,particularly in situations where patients are alreadysuffering from infections i.e. febrile neutropenia. Theabsence of pyrexia, despite the substantial dose of GMDPin the circulation following oral administration, isparadoxical and as yet no specific mechanistic accountfor it exists. It is possible however that the oralroute limits exposure of endothelial cells to the drugwhilst increasing availability‘ to organs such as theliver.Effective treatments for post—chemotherapeuticneutropenia are typically expected to induce neutrophils.We have demonstrated that this can be done with oral GMDPin Example 5, in which the intensity and period of post-chemotherapeuticneutropenia was reduced by GMDPtreatment. Moreover, this occurred in the absence ofsignificant side effects. A surprising finding relatedto this is disclosed in Example 6: Thus, as expected,neutrophil numbers are increased by treatment of patientswith oral GMDP. However, in addition the number ofmonocytes is also increased. It is well known thatmonocytes are important in phagocytosing infectiousagents, and their increase in number is thereforerelevant to protection from infection. Eosinophils andl015202530WO 98/09989CA 02265890 1999-03-04PCT/GB97/02422basophils did not increase, indicating that this is notsimply a generalised effect of GMDP on all blood celltypes. Killion. et al, (Oncology'.Research, 6:357—364,(1994)) have shown that the lipophilic muramyl peptideanalogue MTP—PE is capable of increasing monocyte numbersfollowing cytotoxic therapy in a mouse model, but in thisinstance no beneficial effect on neutrophils was seen.In fact, a slight but insignificant decrease in totalwhite blood cells occurred. Also, analogues such asMTP—PE are specifically designed to be targeted tomonocytes by" addition of a hydrophobic tail. Thisincreases their effectiveness, but also increases theside effect of fever that they induce. As furtherillustration of this novelty, the MDP analogue MDP lysylstearate has been shown to induce thrombocytes (US5,037,804), whereas GMDP had. no beneficial effect onthese cells.Finally, although it is known that GMDP is effective atprotecting from infection in general, and this has beendisclosed in Khaitov et al, (In Immunology of Infections,Ed Masihi pp205—2l5 Pub Dekker,occurring(1994)), infectionduring the immunosuppressedpost—chemotherapeutic state can take several forms, andthe highest occurrence is of pulmonary infection orpneumonia. To our surprise, we have found in clinicaltrials (Example 7) that GMDP is particularly effectiveat preventing such infections. Thepulmonaryaccumulation of this evidence is that (1) GMDP shows asurprising degree of efficacy in the treatment offunctional consequences of neutropenia (i.e.susceptibility to infection) compared to agents such asG—CSF which are more potent in the induction ofneutrophils, (2) it is active orally for this indicationWO 98/099891015202530CA 02265890 1999-03-04PCT/GB97l02422at doses which would be potentially lethal when appliedto other MDPs, (3) this activity occurs in the absence ofsignificant side effects which are nevertheless seen whenGMDP is injected and (4) it is particularly effectiveagainst pulmonary infections, which are a characteristicconsequence of chemotherapy induced neutropenia. Withoutwishing to be bound by any particular theory, it appearsthat these surprising findings are based on the hithertounrecognised discovery that administration by the oralroute presents GMDP to different tissues of the body andwith a different kinetic profile than after injection,provoking a therapeutically‘ advantageous induction ofsubsets of immune cells and resulting in less effect onthose tissues such as endothelial cells which couldmediate harmful or otherwise unwanted side effects.Muramyl peptides show a diverse range of properties andit is impossible to predict in which clinicalcircumstance a particular biological property can beexploited as a useful treatment. This fact isparticularly highlighted by the observation that althoughMDPs were discovered over 20 years ago, to this day theyhave only found limited clinical utility. The specificuse of GMDP as an effective method for the treatment ofneutropenia therefore represents a non—obvious and novelinvention over the existing state of the art.Thus, in a first aspect, the present invention providesthe use of a muramyl peptide compound of general formulal0l520253035WO 98/09989CA 02265890 1999-03-04PCTIGB97/024229I :HOCH24——o 00000000 000,,O\HOCH2 O \\SHAC/ O H3CCHCORH . . . . . . _ _ . O OHNHAC nIwherein:R represents a residue of an amino acid or a linearpeptide built of from 2 to 6 amino acid residues, atleast one of the residues being optionallysubstituted with a lipophilic group; andn is 1 or 2;in the preparation. of an agent for the treatment ofmyelosuppressed or otherwise immunocompromised states,wherein the agent is adapted for oral administration andcomprises O.5—50mg of the muramyl peptide.In the context of the present invention, the term"alleviation of myelosuppression" will be clear to theskilled. man. In general it will mean that the agentcomprising the muramyl peptide compound will be capableof restoring immune function in patients who willpotentially be myelocompromised, for instance followingradiation treatment or chemotherapy. Moreover, thisshould be associated with an increased resistance to theclinical consequence of myelosuppression, ie infection.R preferably represents a mono—, di— or tri—peptide. Theproximal peptide residue (or the only peptide residue, ifthere is only one) is preferably that of an L—amino acid.Examples include:CA 02265890 1999-03-04WO 98109989 PCT/GB97/024221 OL-alanyl L—tryptophanylL-valyl L—lysylL-leucyl L-ornithylL—isoleucyl L—arginyl5 L—a—aminobutyryl L—histidylL—seryl L—glutamylL—threonyl L—glutaminylL—methionyl L—aspartylL—cysteinyl L—asparaginyl10 L—phenylalanyl L—prolyl152O2530L-tyrosyl L—hydroxyprolylL—alanyl is preferred, as is L—threonyl.It is particularly preferred that the peptide Rcorrespond to the peptide in. prototype MDP (L-Ala—D—isoGln). Alternatively, in another preferred embodiment,R may represent L—Ala—D-Glu.The preferred Value for n is 1.The next amino acid from the proximal end of the peptideis preferably of the D—configuration. It is preferablyacidic and may be D—glutamic or D—aspartic acid or amono—, di- or mixed Cl~Cn (preferably C5-C5) alkyl ester,amide or C1-C4 alkyl amide thereof. (The expression"mixed" is illustrated when one carboxyl group isamidated and the other esterified. D—isoglutamine and D-glutamate are preferred.A third amino acid residue from the proximal end of thechain, if there is one, is preferably of the L-configuration, as indicated above in relation. to theproximal amino acid residue. L—alanyl and L—lysyl are‘WO 98/09989l0l5202530CA 02265890 1999-03-04PCT/GB97/02422llpreferred.The amino acid residue or linear peptide is optionallysubstituted with at least one lipophilic group. Thelipophilip group may’ be a Cm—Cn acyl group such asstearoyl or a di—(Cm~Cm acyl)-sn—glycero—3'—hydroxy-phospheryloxy—group wherein for example each of the Cm-Cgacyl groups can be a palmitoyl group. The lipophilicgroup may alternatively (or in addition, as more than onesubstitution may be present) be a C1—Cm ester group, suchas a C2—C6 ester group: a butyl ester is an example.Compounds of general formula I are disclosed in US—A-4395399 and the preferences set out in that document areequallypreferred in the invention.presentAdditionally, in this invention, the group R may besubstituted lipophilically as described above.One of the most preferred compounds for use in thepresent invention falls within general formula I and isN—acetyl—D—glucosaminyl—(B1—4)-N—acetylmuramyl-L—alanyl—D—isoglutamine (GMDP), the structure of which is:HOCH3OHO\nNHAHOCH2 O C}*—Olo CHCO-L—~Alo—D-—isoC|nHO H 'CH3NH/-\CGMDPOther preferred compounds within the scope of generalformula I include:WO 98/099891015202530CA 02265890 1999-03-04PCT/GB97/02422l2N—acetyl-D-glucosaminyl-(51-4)-N~acetylmuramyl~L-alanyl—D—glutamic acid (GMDP-A) which has the structure:HOCH2 OOHOHHOCH2 O NHAC,{—OOH ('ZHCO—L—A|o—D-GluHOCH3NHACGMDP-AN—acetyl—D—glucosaminyl-(Bl-4)—N acetylmuramyl—L-alanyl~L—isoglutamine (GMDP—LL) which has the structure:HOCH2 OOHO\/0| H O CI2HCO—L—A|o-L—isoGlnHOCH3NHACGMDP—LLN—acetyl—D-glucosaminyl—(81-4)-N acetylmuramyl—L—alanyl—D-glutamine n—butyl ester (GMDP—OBu) which has thestructure:HOCH2OOHOHOCH2 O NHAC/ O CHCO-L—A|o—D-Gm-OBU“OOH &H3NHACGMDP—OBuCA 02265890 1999-03-04wo 98/09989 9 PCTIGB97/0242213N—acetyl—D-glucosaminyl-(fil-4)—N acetylmuramyl—L—alany1—D-isoglutaminyl—L-lysine (GMDP—Lys) which has thestructure:HOCH25 OOH0HOCH2 O NHAc/I O CHCO—L—No~D—boGn—L-Lfi“OOH tH3NHAc10GMDP~Lys15 Mk[N—acetyl—D—glucosaminyl—(81-4)—N—acetylmuramyl—L—alanyl—D—isoglutaminyl]—N‘-stearoyl-L—lysine (GMDP—Lys(St)) which has the structure:HOCH202 0 O OHHOCH2 O NHAC/ O CHCO-L~—A|0-D-isoGln—L-Lys-COC17H35_N<:+H0 0“ CIIH3NHAc25GMDP—Lys(St)30 Other useful compounds include:N“-[N-Acetyl—D—glucosaminyl—(Bl——4)—N—acety1-muramyl—L—alanyl-y—D—g1utamyl]-N‘—stearoyl~L—lysine which has thestructure:35CA 02265890 1999-03-04W0 98/09989 PCT/GB97/0242214HO CH2 0‘OH0/ O CHCO—L—Alo—D—G|u—L—Lys—COC1 7H35‘No+“OOH (‘EH3NHAc10 GMDPA—Lys(St)N~Acetyl—D-glucosaminyl—(Bl--4)—N—acetylmuramyl-L—alanyl-D—g1utamic acid dibenzyl ester which has the structure:1520HOCH2OOHO NHAcHOCH2OCHCO-L-AIO-D-C|U-’(OBZl)2HO OH ICH3NHACGMDPA(OBzl)225N—Acetyl-D—glucosaminyl—(B1——4)—N—acetylmuramyl—N—methyl—L—alanyl—D—isoglutamine which has the structure:30CA 02265890 1999-03-04W0 98l09989 PCTIGB97I02422l 5HO CH2OOHOO NHAC5 HOCH2./ O CHCO—MeAlo—D—isoGln“OOH éH3NHAc1 0 Me —GMDP15202530N—Acetyl—D—glucosaminyl—(31——4)—N-acetylmuramyl—(Bl——4)—N—acetyl—D—g1ucosaminyl—(Bl——4)—N—acetylmuramyl—bis-(L-alanyl—D—isoglutamine) which has the structure:HOCH2,{-—-OOHOO NHACHO CH2 0CHCO—L—No—D—boGmOH IHC30 NHACHOCH2OO/OH O $HCO'L‘NO“D-EOGMHOCH3NHAc(GMDP) 2N—Acetyl—D—glucosaminy1—(Bl——4)—N-acety1muramyl—(Bl——4)-N—acetyl—D—glucosaminy1—(Bl——4)—N-acetylmuramyl—bis-(L-alanyl—D-glutamic acid) which has the structure:CA 02265890 1999-03-04wo 98/09989 PCTIGB97/024221 6HOCH20‘OH0HOCH2 O NHAC5 OH O $HCO-L—No—D—GmCH3NHACHOCH2 Ooo10 O NHACHOCH2/iH O $HCO—L—No—D—GwHOCH3NHAC(GMDPA)21 52O253035N-Acetyl-D—glucosaminyl—(Bl——4)—N-acetylmuramyl—(Bl——4)—N—acetyl-D—glucosaminyl—(Bl——4)~N-acetylmuramyl-bis—(L-alanyl—D—isoglutaminyl—L-lysine) which.has the structure: HOCH20.OHONHACHOCH2OCHCO—L—No~D—homn—L—LysOH 5.3NHAC}-E0$HCO-L—No—D—EoQn—L-Q6CH3(GMDP Lys)2N~acetyl—D—glucosaminyl—(Bl——4)-N—acetylmuramyl-(Bl--4)-N—acetyl—D—glucosaminyl—(Bl——4)—N-acetylmuramy1—bis-[Lalanyl~D—isoglutaminyl—N‘—stearoyl—L—lysine]:1015202530WO 98/09989CA 02265890 1999-03-04 PCT/GB97/024221 7HOCH2OOHoHOCH2 0 NHACOH O tHco—L—No—D—momn—L—uscoc17H3§No*ca}NHACCHCO—L—No-D—$omn—L—LysCOC17H35_No+[GMDP-Lys(St)]2N-Acetyl-D-glucosaminyl'(Bl¢—4)—N—acetylmuramyl—Lalanyl-D—isoglutamine l—adamantyl ester which. has thestructure:HOCH2O‘OHOO NHACHOCH2+——OOH $HCO-L~Alo—D-—isoG|n—OAd~H0‘ CH3NHACGMDP -AdL—Threonyl-N‘-[N-Acetyl—D—glucosaminy1—(Bl-—4)—N~acetyl—muramyl-L-a1any1—y—D—isoglutaminyl]—L—lysyl—L—prolyl—L—arginine which has the structure:CA 02265890 1999-03-04WO 98/09989 PCT/GB97/0242218HOCH20OHOO N HAG5 HO C H 2O/ CHCO—L—Na-D—bomn—msUhfl—Pm—A@“OOH $H5NHAC10 GMDP-tuftsin E15202530N—Acetyl—D-glucosaminy1-(Bl——4)—N—acety1—muramyl—L-alanyl—y-D-isoglutaminyl~L—threonyl—L—lysyl~L—prolyl-L-arginine which has the structure:HOCHZOO ‘OHNHAHOCH2 0 °k——OCHCO—L—No-D—$oCm—Thw%ys~W©-AmHOOH ICH3NHACGMDP—tuftsin AN—Acetyl-D-glucosaminyl-(B1——4)—N—acetylmuramyl—L—alanyl-a—D—glutamyl—L-lysyl—L—threonyl—N‘—stearoyl—L—lysyl—L-prolyl—L-arginine which has the structure:CA 02265890 1999-03-04WO 98/09989 PCT/GB97/02422l9HOCH2OCH0O N HAC5 H 0 C H 2O CHCO—L—No—D—Gm—ThP¢yflCOC17H35%4%o-Am“OOH cH3N HA C10 GMDPA—tuftsin lipophilic15202530N‘-[N—Acety1—D-glucosaminyl—(Bl——4)—N—acetyl—muramyl-L-alanyl—y—D—isoglutaminyl]~L—lysyl-L—histidyl—L—glycineamide which has the structure:HOCH20‘OHOO NHACHOCH2OCHCO—L—No—D—EoGn—ws—HS—Ow—NH2H60“ asNHACGMDPA—bursinN—Acetyl—D—glucosaminyl—(Bl——4)—N—acetylmuramyl—L-alanyl-D—isoglutaminyl—L-glutamyl—L—tryptophan which has thestructure:CA 02265890 1999-03-04WO 98/09989 PCT/GB97l0242220HO CH20OHOO NHAC5 HO CH2+——OOH Cl3HCO—L--Alo-D—isoG|n-G|u—TrpHOCH3NHAC10 GMDP—thymogen I15202530N-Acetyl-D-glucosaminyl—(Bl--4)—N—acety1muramyl—L—alanyl—D—isoglutaminyl—e~aminoheXanoyl—L—glutamyl—L—tryptophanwhich has the structure:HOCH20‘OHOHOCH2 O NHAC*-OCHCO—L-No-D—BoGn—Nw—Gm—Tm“OOH cu-13NHACGMDP—thymogen IIN“-[N—Acetyl—D-glucosaminyl—(B1--4)—N—acety1—muramyl—L—a1anyl—D—isoglutaminyl]—N‘—stearoyl—L—1ysyl—L—g1utamyl-L-tryptophan which has the structure:HO CH2 0O OHNHAcHOCH2 0X—‘O .OH CIIHCO—L-A|o—D——IsoG|n—Lys(COC1 7H35) —G|u —-TrpWO 98/099891O1520253035CA 02265890 1999-03-04PCT/GB97/0242221GMDP—thymogen IIIN-ecetylmuramyl-L—threonyl—D-isoglutamine which has thestructure:HOCHZO‘OHOOH NHAC?HCO—L‘ThF—D-SOONCH3 'Thr—MDPN-aoetylmuramyl—L—alanyl—D—glutamine n—butyl ester whichhas the structure:HOCH20OHOOH NHAc$HCO—L—No—D~Gm—OC4HgCH3MurabutideIn the above structures, the following abbreviations areused:Bzl — benzyl;Me — methyl;Ahx — e-aminohexanoyl.The most preferred compound is GMDP followed by GMDP—A,and murabutide.WO 98/099891015202530CA 02265890 1999-03-04PCTIGB97/0242222Glucosaminyl—muramyl dipeptides within the scope ofgeneral formula I can be prepared relatively cheaply andin reasonably large quantities by the process disclosedin US-A—4395399. The preparation disclosed is based onthe extraction and purification of the disaccharidecomponent from the bacterium Micrococcus lysodecticus andits subsequent chemicallinkage to a dipeptidesynthesised for example by conventional peptidechemistry. However, the disaccharide may equally well bechemically synthesised using standard sugar chemistry.Preferably, the agent will comprise l—5mg of the muramylpeptide compound and most preferably 5mg.The agent can in fact comprise one or more muramylpeptide compounds. In addition, the agent could alsocomprise one or more other active ingredients capable ofalleviating immunosuppression.The invention is exemplified herein using GMDP formulatedinto an elementary tablet for oral administration. Forexample, the composition of a tablet suitable for oraladministration could be as follows:Muramyl peptide (eg GMDP) 5mg;lactose 145 mg;maize starch 84 mg;polyvidone 5.5 mg;magnesium stearate 2.5 mg;talc 7.6 mg;It has been determined that such tablets dissolve rapidlyand release 90% of the muramyl peptide compound within 15minutes. One skilled in the art will appreciate thatWO 98/099891015202530CA 02265890 1999-03-04PCT/GB97/0242223tablets with different compositions but which immediatelyrelease their contents could be formulated, and that suchtablets would. also find use in the practice of theinvention.Similarly, the skilled man will appreciate that the agentcomprising the muramyl peptide compound could be in theform of a syrup or solution which would also be suitablefor oral administration.It is known that when delivered to the stomach (as wouldbe the case with a quick dispersing tablets), GMDP has arelatively low bioavailability, (measured at 10% in therat and the dog). It is possible that GMDP could beformulated into more sophisticated oral deliverycompositions that gave improved bioavailability. If thiswere done, the absolute dose of GMDP necessary to have ananti-myelosuppressive effect would be different. Thus,for example, if GMDP bioavailability were increased by,for example, twofold, the preferred dose of GMDP would bereduced by half.In another aspect, the present invention provides amethod for the alleviation of myelosuppression in asubject which comprises administering to the subject 0.5-50mg of a muramyl peptide compound. Preferably, themethod comprises administering 1-5, and most preferablySmg of a muramyl peptide compound as defined herein tothe subject. In a preferred embodiment the muramylpeptide compound is GMDP.The invention will now be described with reference to thefollowing examples which should not be construed as inl01520253035WO 98/09989CA 02265890 1999-03-04PCT/GB97/0242224any way limiting the invention.The examples refer to the figures wherein:FIGURE 1: shows the results of administration ofGMDP in mice which have been renderedimmunosuppressed.by treatment with cyclophosphamide;andFIGURE 2: shows the effect on neutrophil count ofvarying doses of GMDP on mice which had been treatedwith cyclophosphamide.Example 1Mice were rendered immunosuppressed by treatment with30mg/kg).Following that, mice were challenged with Candida (4.8 Xcyclophosphamide (3 successive days at10‘ cfu/mouse) and then three successive daily treatmentswith GMDP or G—CSF. Mortality was scored, and neutrophilcounts.Neutrophil counts (mean)Day 0 Day 3 Day 6 Day 9Vehicle control 2289 1912 5303 9303CYA alone 322 5984 llll2 8552CYA + GMDP 611 5756 7844 9282CYA + G—CSF 530 3596 12736 6517The CYA treatment effectively induced severe neutropeniain the mice (day 0 levels compared to vehicle control).GMDP did not increase neutrophils numbers above thespontaneous increase in cyclophosphamide control.MortalityMortality of mice as a result of infection is given inFig 1. Vehicle control showed high survival. CYA101520253035WO 98/09989CA 02265890 1999-03-04PCT/GB97/0242225control showed lowsurvival because they areimmunosuppressed CYA + G—CSF showed enhanced survival.Surprisingly, CYA + GMDP showed higher survival control,despite the fact that no increase in neutrophils was seenabove CYA control. Conclusion This result was mostsurprising. As expected, G—CSF showed an excellentenhancement of neutrophils, which was associated with acertain measure of protection from Candida—inducedlethality. GMDP did not show such a dramaticneutrophil peak at day" 6. NEVERTHELESS, GMDP wasalmost as effective as G—CSF at protecting from Candidalethality.Example 2Five groups of 35 mice each received a dose of the cancerchemotherapeutic agent cyclophosphamide which was knownto be sufficient to induce severe leukopenia (reductionin numbers of white cells). The groups then receiveddifferent treatments. These were:Group 1 — no treatment (controls)Group 2 — injections of GMDP at a dose of 0.025 mg/kgGroup 3 — injections of GMDP at a dose of 2.5 mg/kgGroup 4mg/kgGroup 5 — oral GMDP (in water supply) at a dose of 2.5mg/kgAt regular intervals, 5 mice from each group were— oral GMDP (in water supply) at a dose of 0.025sacrificed, and blood taken. A complete blood count wasperformed to enumerate the number and proportions of thecell types present. The results are shown in Figure 2 Itwas found that GMDP administered both by injection andorally were capable of boosting the number of white cellsin the blood. Surprisingly the 0.025 mg/kg oral dose wasas effective as the injected dose.Example 3‘WO 98/099891015202530CA 02265890 1999-03-04PCTIGB97/0242226Healthy ‘volunteers were given. sterile, endotoXin—freeinjections of GMDP dissolved in saline. Blood sampleswere taken at intervals and temperature was monitored.Pat No _\ Dose(g GMDP) Leukocytes* Temperature**V01 2 125 0.2 0.5V02 125 2.4 0.2VO3 250 -0.1 0.6VO4 250 5.0 1.5* Change in number of cells x 19/L** Increase from baseline in degrees CExample 4Healthy volunteers were given tablets of GMDP with aglass of water as part of a Phase I safety trial. Inaddition to monitoring general status, bloodbiochemistry, temperature was taken at regular intervalsover 24 hours.No significant incidence of fever was observed at any ofthe doses tested, up to 50 mg.Dose of GMDP Number of subjects Incidence of fever(mg)0 32 None10-20 25 None25-32 10 None40 6 None50 6 NoneExample 5Thirteen patients with incurable breast cancer wererandomised to receive placebo or GMDP (2 mg/day) for 6days immediately following a treatment course with cancerWO 98/099891015202530CA 02265890 1999-03-04PCT/GB97/0242227chemotherapy (mitomycin, methotrexate, mitoxanthrone).It was foundthat GMDP was able to reduce the duration and severity ofthe depression of white blood cellsBlood counts were performed periodically.induced bychemotherapy.Example 6A total of 150 patients were treated with placebo, or oneof three doses of GMDP (5, 25 or 50mg). Counts of bloodcell types were made before treatment, or 24 hours afterreceiving the single oral dose. The following changeswere seen in neutrophils, monocytes, lymphocytes andeosinophilsChange in 24 h following dosing (cells X 109/litre)Dose Neutro Mono. Eosino Baso Thrombo(mg)0 -0.2 0 0.02 -0.01 -35 .8 0.02 0 -0.06 -325 .0 0.14 -0.01 -0.02 -2450 .8 0.27 -0.01 -0.12 -10Example 7In a clinical trial 112 patients scheduled to undergoresection of the large bowel were randomised to receiveGMDP or placebo each day for 10 days leading up to theday of surgery.Their primary tumours, the chemotherapy they hadundergone, and the effect of surgery combined to renderthem generally immunosuppressed, and a number succumbedto infection (Table)Complication GMDP PlaceboWound sepsis 7 13 NSSignificanceCA 02265890 1999-03-04W0 98/09989 PCT/GB97/0242228Generalised sepsis 12 18 NSPneumonia 13 27 <0.011015202530Although. the trend was for GMDP to protect from allinfections, the oily significant result was for pulmonaryinfection. This represent a surprising efficacy in thissub—group.Example 8Groups of mice received various combinations of treatment(200mg/kg) to(lOmg/kg,according to the treatment schedule shown in the tablewith cyclophosphamide inducemyelosuppression, and GMDP subcutaneous)below. All mice were challenged with 7 x 105 organisms ofE. coli on day 5 and sacrificed on day 6, at which timethe number of E. coli organisms present in the liver wasdetermined.It was found that after treatment with cyclophosphamidealone (Group 1), many more organisms were present in theliver than in normal animals that were challenged but didnot have cyclophosphamide treatment (Group 3). GMDP wasable to eliminate this effect and protect against themassive increase in bacteria (Group 2).At first it was hypothesised that this protection was dueto the ability of GMDP to enhance neutrophil numbers.However, when a parallel series of animals was examined,it was found that on the day of bacterial challenge (day5) restoration. of neutrophils in the GMDP group wasnegligible, and by no means could explain the protectionagainst E. coli.CA 02265890 1999-03-04WO 98/09989 PCT/GB97/0242229Clearly, it could not have been predicted that GMDP wouldbe of value in the protection of the myelosuppressedanimals from infectious challenge on day 5.5 pgy o 1 2 3 4 5 6TreatmentGroup 1 CYA — — — — - —Group 2 CYA — — GMDP GMDP GDMP GMDPGroup 3 - — — — — — —10 Results - Bacterial count in the liver (log E. coli)Group 1 ND ND ND ND ND ND 6.35Group 2 ND ND ND ND ND ND 3.46Group 3 ND ND ND ND ND ND 3.47Results — Neutrophils (cell number x 1,000/pl)15 Group la 3.2 ND ND ND 0.1 0.2 2.5Group 2a 3.2 ND ND ND 0.1 0.4 4.2Group 3a . . . . . . . . . . . ..(baseline) . . . . . . . . . . . ..Abbreviations:CYA — cyclophosphamide20 ND — Not done