WO1998009989A1

WO1998009989A1 - The use of muramylpeptides in the treatment of myelosuppressed or otherwise immunocompromised states

Info

Publication number: WO1998009989A1
Application number: PCT/GB1997/002422
Authority: WO
Inventors: Philip Ledger; Roger Aston
Original assignee: Peptech (Uk) Limited
Priority date: 1996-09-06
Filing date: 1997-09-08
Publication date: 1998-03-12
Also published as: GB9618673D0; CA2265890A1; AU4307397A; ZA978027B

Abstract

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

Description

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

The present invention relates to the use of Muramyl peptides in the preparation of an agent for the alleviation of myelosuppression. In particular, it relates to such use in the preparation of an agent for alleviating myelosuppression following treatment of cancer patients by radiation or chemotherapy.

The muramyl peptides are a class of glycopeptide compounds which in nature constitute part of bacterial cell walls (Ellouz et al , Biochem Biophys Res Comm, 59:1317-1325, (1974)). This term has also come to include synthetic variants of naturally occurring forms. Muramyl peptides were discovered by virtue of their ability to act as adjuvants to injected vaccines (Adam, Synthetic adjuvants, In: Modern concepts in Immunology, Vol 1, (1985)) . As studies progressed, it was also found that these molecules also possessed other immunostimulant properties, and had the ability to enhance nonspecific immunity to infectious organisms (Chedid et al , PNAS USA, 74:2089, (1977)) possibly by activating specific cell types of the immune system, causing them for example to ingest foreign particles with greater efficiency (Andronova and Ivanov, Sov Med Revi ew D . Immunol ogy 4:1-63, (1991) ) .

More recently, it has been found that under some conditions, certain muramyl dipeptides can also have anti-inflammatory or immunosuppressive properties, which is contrary to their usual immunostimulant effect

(Adeleye et al , APMIS, 102:145-52, (1994)).

Thus, the utility of this class of molecule in the treatment of myelosuppression has been very limited as evidenced by the lack of commercial products .

The smallest functional muramyl peptide, N-acetylmuramyl -L-alanyl-D-isoglutamine (MDP) and its close analogues contain one sugar residue, N-acetylmuramic acid. Another class of muramyl peptide contains a disaccharide, namely N-acetylglucosaminyl -N-acetylmuramic acid. The simplest of these compounds is N-acetylglucosaminyl-N- acetylmuramyl-L-alanyl-D-isoglutamine, abbreviated as GMDP . These forms are exemplified by the range of compounds described in US patent 4,395,399. Some studies have shown that GMDP has properties distinct from MDP. For example, GMDP is less pyrogenic (fever inducing) than MDP, and in some circumstances has more distinct anti-inflammatory properties (Adeleye et al , (1994), supra) .

The development of chemotherapeutic drugs has greatly increased the medical profession's ability to successfully treat numerous types of cancer. Similarly, irradiation of the human body is used for certain cancers. The principal of these treatments is to kill rapidly dividing cells, such as the rapidly dividing cells of a tumour. Unfortunately, certain healthy cells of the body also divide rapidly, and their damage by the cancer treatment methods results in unwanted side effects of the treatment. For example, skin and hair bulb cells, and the cells lining the gut are rapidly dividing, and disturbances in these tissues are well known in patients undergoing cancer therapy. Of particular concern is damage caused to so-called hematopoietic ("blood forming") tissues, the consequence of which is a reduction in the number of circulating cells of the immune system. While this is not harmful per se, the reduced immune function renders these patients more susceptible to life threatening infections.

The seriousness of this so called myelosuppressive side-effect is evidenced by the fact that repeated cycles of anti-cancer treatment are often reduced in intensity or delayed in time, in order for immune function to become restored to an acceptable level. Therapeutic agents have therefore been developed which can counteract the myelosuppressed state. The best known and currently most widely used of these are the so-called "colony stimulating factors", which cause precursors of the immune cells to differentiate and multiply, and therefore replenish the cells killed by the radiation or chemotherapy. These factors are proteinaceous, expensive to produce, and have to be administered by repeat injection or infusion (Gabrilove et al , New England J Med, 318:1414-22, (1988)).

The ability of MDPs and GMDP to counteract states of myelosuppression arising from the use of radiation therapy and chemotherapy has been illustrated in animal models (Azuma and Otani, Medicinal Res Revi ews , 14:401-14, (1994); Adrianova et al , Radiobiologia ,

32:566-70, (1992)) and, in the case of the hydrophobic analogue MDP-stearoyl-L-lysine, has even been used for specific therapeutic advantage in cancer patients

(Tsubura et al , Arzneim. -Forsch/Drug Res , 38:1070-74, WO89/01778) . In all these cases, the drug has been administered to patients by injection, and in doses effective for human use has been associated with the occurrence of side effects such as fever and local reactions at the site of injection (Tsubura et al , supra) .

The most effective agents for the restoration of neutrophils and protection from neutropenia are the colony stimulating factors (CSF) described above, of which granulocyte - CSF (G-CSF) is the most commonly used in the clinic at the present time. These agents specifically target precursor cells, thereby inducing formation of neutrophils, and it might be anticipated that as such they would be more effective than a muramyl peptide, including GMDP, which does not in itself have the ability to induce precursor cells, but needs to cause release of messengers which in turn induce precursor cells. The limited ability of MDPs to increase neutrophil numbers is highlighted even by marketed products such as Romurtide, which perform less well than G-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 rendered neutropenic with cyclophosphamide and subsequently treated with GMDP than was obtained with G-CSF. In fact, in this model where mice rendered neutropenic with cyclophosphamide (CYA) were challenged with an infection of Candida, GMDP did not increase neutrophil numbers above the CYA controls. However, most surprisingly, although the effect on neutrophil number was lower, when these same animals were challenged with a potentially lethal dose of the micro-organism Candida, GMDP gave a very effective protection against lethality, compared to the high mortality seen in animals receiving CYA alone. Surprisingly, it would therefore appear that neutrophil induction alone is not a good indicator of overall efficacy against "neutropenia", in which the most important feature is protection from the clinical consequences of immune suppression, not just the cell count per se . Clearly, "immunostimulation" is not a single activity, or G-CSF would have been more effective than GMDP. Therefore the knowledge that GMDP is an immunostimulator is not sufficient to predict that it would be so effective at treating chemotherapy- induced neutropenic immunosuppression. Vast numbers of i munostimulators have been identified over the years however only a very small handful have found utility in the market place. The correct balance of immune cell activation appears to be critical for the identification of drugs with clinical utility.

As has been discussed, muramyl peptides are able to increase neutrophil numbers in animals rendered immunosuppressed by chemotherapy, when delivered by injection. However, such an effect has never been observed following delivery by the oral route. In Example 2 we have shown that in a mouse model, most surprisingly, a strong effect on neutrophils is seen after oral administration of GMDP as well as after injection of GMDP. The significance of this observation needs to be considered in the light of human clinical data. Thus, in human clinical Phase I studies (example

3) , it was found that an intramuscular injection of as little as 250 micrograms of GMDP induced fever

(temperatures increased by 1.5oC) and flu like symptoms.

When temperatures were raised, white blood cell counts were elevated, but clearly the side effects would preclude the use of this route of administration as a treatment for neutropenia.

In other Phase I trials, GMDP was administered as oral tablets. Doses of up to 50 gm were given with no significant induction of fever or side effects (Example 4) . From animal studies, it is known that when administered by the oral route typically about 10% of GMDP enters the systemic circulation. However, the human oral dose cited above (50mg) , is 200 times greater than the 250 micrograms which causes fever by injection. This absence of side effects from oral administration is therefore most surprising, meaning that it has the potential to provide a useful treatment for neutropenia, particularly in situations where patients are already suffering from infections i.e. febrile neutropenia. The absence of pyrexia, despite the substantial dose of GMDP in the circulation following oral administration, is paradoxical and as yet no specific mechanistic account for it exists. It is possible however that the oral route limits exposure of endothelial cells to the drug whilst increasing availability to organs such as the liver .

Effective treatments for post -chemotherapeutic neutropenia are typically expected to induce neutrophils. We have demonstrated that this can be done with oral GMDP in Example 5, in which the intensity and period of post- chemotherapeutic neutropenia was reduced by GMDP treatment. Moreover, this occurred in the absence of significant side effects. A surprising finding related to this is disclosed in Example 6: Thus, as expected, neutrophil numbers are increased by treatment of patients with oral GMDP. However, in addition the number of monocytes is also increased. It is well known that monocytes are important in phagocytosing infectious agents, and their increase in number is therefore relevant to protection from infection. Eosinophils and basophils did not increase, indicating that this is not simply a generalised effect of GMDP on all blood cell types. Killion et al , { Oncology Research, 6:357-364, (1994) ) have shown that the lipophilic muramyl peptide analogue MTP-PE is capable of increasing monocyte numbers following cytotoxic therapy in a mouse model, but in this instance no beneficial effect on neutrophils was seen. In fact, a slight but insignificant decrease in total white blood cells occurred. Also, analogues such as MTP-PE are specifically designed to be targeted to monocytes by addition of a hydrophobic tail. This increases their effectiveness, but also increases the side effect of fever that they induce. As further illustration of this novelty, the MDP analogue MDP lysyl stearate has been shown to induce thrombocytes (US 5,037,804), whereas GMDP had no beneficial effect on these cells.

Finally, although it is known that GMDP is effective at protecting from infection in general, and this has been disclosed in Khaitov et al , (In Immunology of Infections, Ed Masihi pp205-215 Pub Dekker, (1994)), infection occurring during the i mm u n o s upp r e s s e d post-chemotherapeutic state can take several forms, and the highest occurrence is of pulmonary infection or pneumonia. To our surprise, we have found in clinical trials (Example 7) that GMDP is particularly effective at preventing such pulmonary infections. The accumulation of this evidence is that (1) GMDP shows a surprising degree of efficacy in the treatment of functional consequences of neutropenia (i.e. susceptibility to infection) compared to agents such as G-CSF which are more potent in the induction of neutrophils, (2) it is active orally for this indication at doses which would be potentially lethal when applied to other MDPs, (3) this activity occurs in the absence of significant side effects which are nevertheless seen when GMDP is injected and (4) it is particularly effective against pulmonary infections, which are a characteristic consequence of chemotherapy induced neutropenia. Without wishing to be bound by any particular theory, it appears that these surprising findings are based on the hitherto unrecognised discovery that administration by the oral route presents GMDP to different tissues of the body and with a different kinetic profile than after injection, provoking a therapeutically advantageous induction of subsets of immune cells and resulting in less effect on those tissues such as endothelial cells which could mediate harmful or otherwise unwanted side effects .

Muramyl peptides show a diverse range of properties and it is impossible to predict in which clinical circumstance a particular biological property can be exploited as a useful treatment. This fact is particularly highlighted by the observation that although MDPs were discovered over 20 years ago, to this day they have only found limited clinical utility. The specific use of GMDP as an effective method for the treatment of neutropenia therefore represents a non-obvious and novel invention over the existing state of the art .

Thus, in a first aspect, the present invention provides the use of a muramyl peptide compound of general formula

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, wherein the agent is adapted for oral administration and comprises 0.5-50mg of the muramyl peptide.

In the context of the present invention, the term "alleviation of myelosuppression" will be clear to the skilled man. In general it will mean that the agent comprising the muramyl peptide compound will be capable of restoring immune function in patients who will potentially be myelocompromised, for instance following radiation treatment or chemotherapy. Moreover, this should be associated with an increased resistance to the clinical consequence of myelosuppression, ie infection.

R preferably represents a mono-, di- or tri -peptide. The proximal peptide residue (or the only peptide residue, if there is only one) is preferably that of an L-amino acid. Examples include: L-alanyl L-tryptophanyl

L-valyl L-lysyl

L-leucyl L-ornithyl

L-isoleucyl L-arginyl

L- -aminobutyryl L-histidyl

L-seryl -glutamyl -threonyl L-glutaminyl

L-methionyl L-aspartyl

L-cysteinyl L-asparaginyl

L-phenylalanyl L-prolyl

L-tyrosyl L-hydroxyprolyl

L-alanyl is preferred, as is L-threonyl.

It is particularly preferred that the peptide R correspond 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 peptide is preferably of the D-configuration. It is preferably acidic and may be D-glutamic or D-aspartic acid or a mono-, di- or mixed C_j^C^ (preferably C^-Cg) alkyl ester, amide or C_x-C₄ alkyl amide thereof. (The expression "mixed" is illustrated when one carboxyl group is amidated and the other esterified. D-isoglutamine and D- glutamate are preferred.

A third amino acid residue from the proximal end of the chain, if there is one, is preferably of the L- configuration, as indicated above in relation to the proximal amino acid residue. L-alanyl and L-lysyl are preferred.

The amino acid residue or linear peptide is optionally substituted with at least one lipophilic group. The lipophilic group may be a C₁₀ - C₂₂ acyl group such as stearoyl or a di- (C₁₀-C₂₂ acyl) -srz-glycero-3 ' -hydroxy- phospheryloxy-group wherein for example each of the C₁₀- C₂₂ acyl groups can be a palmitoyl group. The lipophilic group may alternatively (or in addition, as more than one substitution may be present) be a C, -C₁₀ ester group, such as 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 are equally preferred in the present invention. Additionally, in this invention, the group R may be substituted lipophilically as described above.

One of the most preferred compounds for use in the present invention falls within general formula I and is N-acetyl-D-glucosaminyl- (/31-4) -N-acetylmuramyl-L-alanyl- D-isoglutamine (GMDP), the structure of which is:

- D-isoGIn

GMDP

Other preferred compounds within the scope of genera] formula I include : N-acetyl-D-glucosaminyl- (61-4) -N-acetylmuramyl-L-alanyl - D-glutamic acid (GMDP-A) which has the structure:

-D-Glu

GMDP-A

N-acetyl-D-glucosaminyl- (31-4) -N acetylmuramyl -L-alanyl L-isoglutamine (GMDP-LL) which has the structure:

GMDP-LL

N-acetyl-D-glucosaminyl- (,61-4) -N acetylmuramyl -L-alanyl - D-glutamine n-butyl ester (GMDP-OBu) which has the structure :

GMDP-OBu N-acetyl-D-glucosaminyl- (<3l-4) -N acetylmuramyl -L-alanyl - D-isoglutaminyl-L-lysine (GMDP-Lys) which has the structure :

GMDP-Lys

N^α- [N-acetyl-D-glucosaminyl- (61-4) -N-acetylmuramyl-L- alanyl-D-isoglutaminyl] -N^f-stearoyl-L-lysine (GMDP-

Lys (St) ) which has the structure:

GMDP-Lys (St!

Other useful compounds include

N°- [N-Acetyl-D-glucosaminyl- (61- -4) -N-acetyl-muramyl -L- alanyl-γ-D-glutamyl] -N^£-stearoyl-L-lysine which has the structure :

GMDPA-Lys (St)

N-Acetyl-D-glucosaminyl- (61--4) -N- acetylmuramyl -L-alanyl - D-glutamic acid dibenzyl ester which has the structure:

GMDPA(OBzl)₂

N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl-N-methyl- L-alanyl-D-isoglutamine which has the structure:

Me -GMDP

N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl- (61--4) - N-acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl-bis- (L- alanyl-D-isoglutamine) which has the structure:

(GMDP)

N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl- (61--4) -

N-acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl-bis- (L- alanyl-D-glutamic acid) which has the structure: -D-Glu

(GMDPA)

N-Acetyl-D-glucosaminyl- (61- -4) -N-acetylmuramyl- (61- -4) - N-acetyl-D-glucosaminyl- (31--4) -N-acetylmuramyl-bis- (L- alanyl-D-isoglutaminyl-L-lysine) which has the structure:

(GMDP Lys) ₂ N-acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl- (61--4) N-acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl-bis- [L alanyl-D-isoglutaminyl-N^£-stearoyl-L- lysine] :

[GMDP-Lys (St ) ] ₂

N-Acetyl-D-glucosaminyl- (61- -4) -N-acetylmuramyl-L alanyl-D-isoglutamine 1-adamantyl ester which has the structure :

GMDP-Ad

L-Threonyl-N^f- [N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl-L-alanyl -γ-D-isoglutaminyl] -L-lysyl -L-prolyl-L- arginine which has the structure:

GMDP-tuftsin E

N-Acetyl-D-glucosaminyl- (61--4) -N-acetyl -muramyl -L- alanyl-γ-D-isoglutaminyl -L-threonyl -L-lysyl -L-prolyl-L- arginine which has the structure:

-Thr-Lys-Pro-Arg

GMDP-tuftsin A

N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl -L-alanyl - -D-glutamyl -L-lysyl -L-threonyl -N^£-stearoyl -L-lysyl -L- prolyl-L-arginine which has the structure: ₁₇H_J5)-Pro-Arg

GMDPA-tuftsin lipophilic

N^e- [N-Acetyl-D-glucosaminyl- (61--4) -N-acetyl-muramyl-L- alanyl-γ-D-isoglutaminyl] -L- lysyl-L- hist idyl -L-glycine amide which has the structure :

His-Gly-NH₂

GMDPA-bursin

N-Acetyl-D-glucosaminyl- ( 1--4) -N-acetylmuramyl-L-alanyl- D-isoglutaminyl-L-glutamyl-L-tryptophan which has the structure :

GMDP-thy ogen I

N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl -L-alanyl - D-isoglutaminyl- e -aminohexanoyl-L-glutamyl-L-tryptophan which has the structure:

GMDP-thymogen II

N^α- [N-Acetyl-D-glucosaminyl- (01--4) -N-acetyl-muramyl-L- alanyl -D-isoglutaminyl] -N^£-stearoyl -L-lysyl -L-glutamyl-L- tryptophan which has the structure:

GMDP-thymogen III

N-acetylmuramyl-L-threonyl-D-isoglutamine which has the structure :

r-D-isoGlπ

Thr-MDP

N-acetylmuramyl-L-alanyl-D-glutamine n-butyl ester which has the structure:

Murabutide

In the above structures, the following abbreviations are used:

Bzl - benzyl; Me - methyl ; Ahx - e-aminohexanoyl .

The most preferred compound is GMDP followed by GMDP-A, and murabutide. Glucosaminyl-muramyl dipeptides within the scope of general formula I can be prepared relatively cheaply and in reasonably large quantities by the process disclosed in US-A-4395399. The preparation disclosed is based on the extraction and purification of the disaccharide component from the bacterium Micrococcus lysodecticus and its subsequent chemical linkage to a dipeptide synthesised for example by conventional peptide chemistry. However, the disaccharide may equally well be chemically synthesised using standard sugar chemistry.

Preferably, the agent will comprise l-5mg of the muramyl peptide compound and most preferably 5mg .

The agent can in fact comprise one or more muramyl peptide compounds. In addition, the agent could also comprise one or more other active ingredients capable of alleviating immunosuppression.

The invention is exemplified herein using GMDP formulated into an elementary tablet for oral administration. For example, the composition of a tablet suitable for oral administration 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 g;

It has been determined that such tablets dissolve rapidly and release 90% of the muramyl peptide compound within 15 minutes. One skilled in the art will appreciate that tablets with different compositions but which immediately release their contents could be formulated, and that such tablets would also find use in the practice of the invention.

Similarly, the skilled man will appreciate that the agent comprising the muramyl peptide compound could be in the form of a syrup or solution which would also be suitable for oral administration.

It is known that when delivered to the stomach (as would be the case with a quick dispersing tablets) , GMDP has a relatively low bioavailability, (measured at 10% in the rat and the dog) . It is possible that GMDP could be formulated into more sophisticated oral delivery compositions that gave improved bioavailability. If this were done, the absolute dose of GMDP necessary to have an anti -myelosuppressive effect would be different. Thus, for example, if GMDP bioavailability were increased by, for example, twofold, the preferred dose of GMDP would be reduced by half.

In another aspect, the present invention provides a method for the alleviation of myelosuppression in a subject which comprises administering to the subject 0.5- 50mg of a muramyl peptide compound. Preferably, the method comprises administering 1-5, and most preferably 5mg of a muramyl peptide compound as defined herein to the subject. In a preferred embodiment the muramyl peptide compound is GMDP.

The invention will now be described with reference to the following examples which should not be construed as in any way limiting the invention.

The examples refer to the figures wherein:

FIGURE 1: shows the results of administration of

GMDP in mice which have been rendered immunosuppressed by treatment with cyclophosphamide; and

FIGURE 2: shows the effect on neutrophil count of varying doses of GMDP on mice which had been treated with cyclophosphamide .

Example 1 Mice were rendered immunosuppressed by treatment with cyclophosphamide (3 successive days at 30mg/kg) . Following that, mice were challenged with Candida (4.8 x 10⁶ cfu/mouse) and then three successive daily treatments with GMDP or G-CSF. Mortality was scored, and neutrophil counts.

Neutrophil counts (mean)

Day 0 Day 3 Day 6 Day 9

Vehicle control 2289 1912 5303 9303

CYA alone 322 5984 11112 8552

CYA + GMDP 611 5756 7844 9282

CYA + G-CSF 530 3596 12736 6517

The CYA treatment effectively induced severe neutropenia in the mice (day 0 levels compared to vehicle control) . GMDP did not increase neutrophils numbers above the spontaneous increase in cyclophosphamide control .

Mortality Mortality of mice as a result of infection is given in Fig 1. Vehicle control showed high survival. CYA control showed low survival because they are immunosuppressed CYA + G-CSF showed enhanced survival. Surprisingly, CYA + GMDP showed higher survival control, despite the fact that no increase in neutrophils was seen above CYA control. Conclusion This result was most surprising. As expected, G-CSF showed an excellent enhancement of neutrophils, which was associated with a certain measure of protection from Candida-induced lethality. GMDP did not show such a dramatic neutrophil peak at day 6. NEVERTHELESS, GMDP was almost as effective as G-CSF at protecting from Candida lethality.

Example 2 Five groups of 35 mice each received a dose of the cancer chemotherapeutic agent cyclophosphamide which was known to be sufficient to induce severe leukopenia (reduction in numbers of white cells) . The groups then received different treatments. These were: Group 1 - no treatment (controls)

Group 2 - injections of GMDP at a dose of 0.025 mg/kg Group 3 - injections of GMDP at a dose of 2.5 mg/kg Group 4 - oral GMDP (in water supply) at a dose of 0.025 mg/kg Group 5 - oral GMDP (in water supply) at a dose of 2.5 mg/kg

At regular intervals, 5 mice from each group were sacrificed, and blood taken. A complete blood count was performed to enumerate the number and proportions of the cell types present. The results are shown in Figure 2 It was found that GMDP administered both by injection and orally were capable of boosting the number of white cells in the blood. Surprisingly the 0.025 mg/kg oral dose was as effective as the injected dose.

Example 3 Healthy volunteers were given sterile, endotoxin-free injections of GMDP dissolved in saline. Blood samples were taken at intervals and temperature was monitored.

Pat No Dose (g GMDP) Leukocytes* Temperature**

VOl 125 0.2 0.5

V02 125 2.4 0.2

V03 250 -0.1 0.6

V04 250 5.0 1.5 * Change in number of cells x 1⁹/L

** Increase from baseline in degrees C

Example 4

Healthy volunteers were given tablets of GMDP with a glass of water as part of a Phase I safety trial. In addition to monitoring general status, blood biochemistry, temperature was taken at regular intervals over 24 hours.

No significant incidence of fever was observed at any of the doses tested, up to 50 mg.

Dose of GMDP Number of subjects Incidence of fever

(mg) 0 32 None

10-20 25 None

25-32 10 None

40 6 None

50 6 None

Example 5

Thirteen patients with incurable breast cancer were randomised to receive placebo or GMDP (2 mg/day) for 6 days immediately following a treatment course with cancer chemotherapy (mitomycin, methotrexate, mitoxanthrone) . Blood counts were performed periodically. It was found that GMDP was able to reduce the duration and severity of the depression of white blood cells induced by chemotherapy.

Example 6

A total of 150 patients were treated with placebo, or one of three doses of GMDP (5, 25 or 50mg) . Counts of blood cell types were made before treatment, or 24 hours after receiving the single oral dose. The following changes were seen in neutrophils, monocytes, lymphocytes and eosinophils

Change in 24 h following dosing (cells x 10⁹/litre)

Dose Neutro Mono. Eosino Baso Thrombo

(mg)

0 -0.2 0 0.02 -0.01 -3

5 0.8 0.02 0 -0.06 -3

25 4.0 0.14 -0.01 -0.02 -24

50 5.8 0.27 -0.01 -0.12 -10

Example ≥ 7

In a clinical trial 112 patients scheduled to undergo resection of the large bowel were randomised to receive GMDP or placebo each day for 10 days leading up to the day of surgery.

Their primary tumours, the chemotherapy they had undergone, and the effect of surgery combined to render them generally immunosuppressed, and a number succumbed to infection (Table)

Complication GMDP Placebo Significance Wound sepsis 7 13 NS Generalised sepsis 12 18 NS

Pneumonia 13 27 <0.01

Although the trend was for GMDP to protect from all infections, the oily significant result' as for pulmonary infection. This represent a surprising efficacy in this sub-group .

Example 8

Groups of mice received various combinations of treatment with cyclophosphamide (200mg/kg) to induce myelosuppression, and GMDP (lOmg/kg, subcutaneous) according to the treatment schedule shown in the table below. All mice were challenged with 7 x 10⁵ organisms of E . coli on day 5 and sacrificed on day 6, at which time the number of E. coli organisms present in the liver was determined.

It was found that after treatment with cyclophosphamide alone (Group 1) , many more organisms were present in the liver than in normal animals that were challenged but did not have cyclophosphamide treatment (Group 3) . GMDP was able to eliminate this effect and protect against the massive increase in bacteria (Group 2) .

At first it was hypothesised that this protection was due to 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 (day 5) restoration of neutrophils in the GMDP group was negligible, and by no means could explain the protection against E . coli . Clearly, it could not have been predicted that GMDP would be of value in the protection of the myelosuppressed animals from infectious challenge on day 5.

Day __ 0 1 2 3 4 5 6 Treatment Group 1 CYA - - - - - -

Group 2 CYA - - GMDP GMDP GDMP GMDP Group 3 - - - - - - - Results - Bacterial count in the liver (log E. coli) Group 1 ND ND ND ND ND ND 6.35 Group 2 ND ND ND ND ND ND 3.46 Group 3 ND ND ND ND ND ND 3.47 Results - Neutrophils (cell number x 1,000/μl) Group la 3.2 ND ND ND 0.1 0.2 2.5 Group 2a 3.2 ND ND ND 0.1 0.4 4.2

Group 3a (baseline)

Abbreviations :

CYA - cyclophosphamide ND - Not done

Claims

CLAIMS :

1. The use of a muramyl peptide compound of general formula I :

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.

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 C_x-C₂₂ (preferably C-. -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 .

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- ace tyl- glucosaminyl -N-acetyl -muramyl -L-alanyl -D- glutamic acid (GMDP-A) ;

N-acetyl-D-glucosaminyl- (61-4) -N-acetylmuramyl-L- alanyl -D-glutamine n-butyl ester (GMDP-OBu) ;

N- [Noj-Acetyl-D-glucosaminyl- (61-4) -N-acetylmuramyl - L-alanyl-D-isoglutaminyl] -N'-stearoyl-L- lysine (GMDP- Lys(St) ) ;

W- [N-Acetyl-D-glucosaminyl- (βl--4) -N-acetyl-muramyl- L-alanyl-γ-D-glutamyl] -N^£-stearoyl-L-lysine (GMDPA- Lys(St) ) ;

N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl-L- alanyl-D-glutamic acid dibenzyl ester (GMDPA(OBzl) ₂) ;

N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl -N- methyl-L-alanyl-D-isoglutamine (Me-GMDP) ;

N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl- (61- -4) -N-acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl- bis- (L-alanyl-D-isoglutamine) ( (GMDP) ₂) ;

N-Acetyl-D-glucosaminyl- ( 1- -4) -N-acetylmuramyl- (61- -4) -N-acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl- bis- (L-alanyl-D-glutamic acid) ((GMDPA)₂); N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl- (61- -4) -N-acetyl-D-glucosaminyl- (βl--4) -N-acetylmuramyl- bis- (L-alanyl-D-isoglutaminyl-L-lysine) ( (GMDP Lys ) ₂ ) ; __

N-acetyl-D-glucosaminyl- (βl--4) -N-acetylmuramyl- (61- -4) -N-acetyl-D-glucosaminyl- (/31--4) -N-acetylmuramyl- bis- [L-alanyl -D- isoglutaminyl -N^€-stearoyl-L- lysine] ( [GMDP-Lys (St) ]₂) ;

N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl -L- alanyl-D-isoglutamine 1-adamantyl ester (GMDP-Ad) ;

L-Threonyl-N'- [N-Acetyl-D-glucosaminyl- (61--4) -N- acetyl-muramyl-L-alanyl-γ-D-isoglutaminyl] -L-lysyl -

L-prolyl-L-arginine (GMDP-tuftsin E) ;

N-Acetyl-D-glucosaminyl- (61- -4) -N-acetyl -muramyl -L- alanyl -γ -D- isoglutaminyl -L-threonyl -L- lysyl -L- prolyl-L-arginine (GMDP-tuftsin A) ;

N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl -L- alanyl -α-D-glutamyl -L-lysyl -L-threonyl -N^e-stearoyl- L- lysyl-L-prolyl-L- arginine (GMDPA- tuft sin lipophilic) ;

N^f- [N-Acetyl-D-glucosaminyl- (61 --4) -N-acetyl -muramyl - L- alanyl -γ -D-isoglutaminyl] -L-lysyl -L- hist idyl -L- glycine amide (GMDPA-bursin) ;

N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl-L- alanyl -D-isoglutaminyl -L-glutamyl -L- tryptophan (GMDP-thymogen I) ; N-Acetyl-D-glucosaminyl- (61--4) -N-acetylmuramyl-L- alanyl-D-isoglutaminyl- e -aminohexanoyl-L-glutamyl-L- tryptophan (GMDP-thymogen II) ;

N°- [N-Acetyl-D-glucosaminyl- (61- -4) -N-acetyl-muramyl-

L-alanyl -D-isoglutaminyl] -N' -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 l-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.

19. A method as claimed in claim 18 wherein l-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.