WO1996009063A1

WO1996009063A1 - Use of muramyl peptide compounds

Info

Publication number: WO1996009063A1
Application number: PCT/GB1995/002237
Authority: WO
Inventors: Roger Aston; Iouri Maitchouk; Tatyana Mikhailovna Andronova
Original assignee: Peptech(Uk) Limited
Priority date: 1994-09-21
Filing date: 1995-09-20
Publication date: 1996-03-28
Also published as: AU3527495A; KR970706015A; GB9419011D0; JPH10506387A; IL115391A0; BG101325A; ZA957997B; CZ86197A3; HUT77741A; EP0782454A1; CA2200570A1; SK36697A3

Abstract

Certain muramyl peptide derivatives such as GMDP find particular use in the treatment of ophthalmic conditions such as herpetic keratitis and herpetic keratouveitis. The muramyl peptide derivatives are particularly useful for reducing or preventing corneal scarring which may occur as a result of conditions such as keratitis. Treatment with muramyl peptide compounds may be used in addition to treatment with other drugs.

Description

USE OF MURAMYL PEPTIDE COMPOUNDS

The present invention relates to the treatment of ophthalmic conditions and in particular to the treatment or prevention of corneal scarring which may be associated with bacterial or viral ophthalmic infections.

The eye is a relatively complex organ and is susceptible to many types of viral and bacterial infection and also to non-infectious conditions. Examples include conjunctivitis which is an infection of the conjunctiva and may be caused by bacteria, viruses, other microorganisms such as Chlamydia or by allergy. Disorders of the cornea include keratitis which may be caused by bacteria such as staphylococcus, streptococcus , pseudomonas or enterobacteria or by viruses such as Herpes simplex. Systemic viral illness such as measles, mumps and chicken pox can also cause keratitis but it can also result simply from dryness of the eyes. Certain eye conditions particularly keratitis, other corneal conditions and damage or infections of the eyelids can result in scarring of the cornea which can impair vision and restrict the movement of the eyelids.

Many eye conditions can be treated by known antibacterial or antiviral agents but although these are effective, corneal scarring may still result. This is thought to be because of migration of lymphocytes and macrophages into the cornea. The activity of the lymphocytes and macrophages can involve secretion of cytokines which can cause the activation of fibroblasts to lay down collagen in an inappropriate manner and cause scarring of the cornea. Scarring may also be caused by other mechanisms. It has now been discovered, however, that various muramyl peptide derivatives are useful in the treatment of ophthalmic conditions and, in particular, may be of assistance in the treatment or prevention of corneal scarring.

It has long been known that non-specific stimulation of the immune system can be brought about by exposure to bacteria, or components extracted from bacterial cells. The specific components responsible for this activity were identified as sugar-containing peptides of the cell wall, and further biochemical analysis of the peptides identified them as the peptidoglycan component of the cell wall. The smallest effective synthetic molecule was found to be an N-acetylmuramyl-L-alanyl-D-isoglutamine (Merser et al , Biochem. Biophys . Res . Comm. 66 1316 (1975) ) . The ability of this compound (now frequently referred to as "prototype muramyl dipeptide" or "prototype MDP") to protect mice against bacterial infection (Klebsiella pneumonia) has been described (Chedid et al , Proc . Na tl . Acad. Sci . USA, 74 2089 (1977) ) .

Subsequently, a wide variety of analogues of prototype muramyl dipeptide were synthesised, some of which have been proposed as treatments for the restoration of immune function or the non-specific stimulation of the immune system. These analogues, and prototype MDP itself, are muramyl peptide compounds.

Various prior art documents have dealt with the use of muramyl peptides and analogues thereof as adjuvants including Azu a et al (Adv. Exp . Med. Biol . 319, 253-263

(1992)) which teaches that MDPs are effective for use as immunoadjuvants for the potentiation of antigenicity of recombinant or component vaccines.

Allison et al (Semin . Immunol . 2(5) 369-374 (1990)) teach that an adjuvant formulation consisting of a synthetic MDP analogue in a squalane-Pluronic polymer emulsion elicits cell-mediated immunity and antibodies of protective isotypes and augments responses to various antigens.

Burke, Rev. Infect . Dis . , 13, Suppl. 11, pS906-911, Nov- Dec 1991 teaches that MTP-PE was found to be particularly useful as an adjuvant with a Herpes simplex virus subunit vaccine.

Allison et al (Mol . Immunol . , 28(3) 279-284 (1991)) relates to the use of MDP analogues with antigens to elicit cell mediated immunity in influenza, hepatitis B virus, herpes simplex virus, lentivirus and tumour vaccines.

Brynestad et al , J. Virol . , 64(2), 680-685, (1990) relates to the use of herpes simplex derived peptide coupled to a fatty acid carrier exhibited enhanced immunogenicity in mice, particularly when incorporated into liposomes and accompanied by an MDP analogue as an immunomodulator.

Ishihara et al , Vaccine, 7(4), 309-313, (1989) showed that MDP-Lys restored resistance to herpes simplex virus type-1 infection in cyclophosphamide treated mice.

Ikeda et al , Antiviral Res . , 5(4), 207-215 (1985) also teaches that MDP and MDP-Lys were able to enhance non- specific resistance to viral infection.

Many other references which disclose the use of various muramyl peptide derivatives as adjuvants.

In addition there is some suggestion in the prior art that some muramyl peptide derivatives, particularly MTP- PE, may enhance the antiviral activity of some antiviral agents.

Gangemi et al , J. Infect . Dis . , 155(3), 510-517 discloses the ability of MTP-PE to improve the therapeutic agent of the broad spectrum antiviral agent ribavirin. The effect was particularly pronounced with liposome encapsulated MTP-PE.

However, there is no teaching in any of these documents that muramyl peptide compounds may be of particular use in the treatment of ophthalmic conditions and indeed there is some suggestion in the prior art that the use of muramyl dipeptide analogues may be deleterious to the eye. For example, Lawrence et al in Exp. Eye Res . , 54(4), 105-107 (1992) discuss the break down of the anatomic barriers between the anterior segment of the eye and the blood stream during inflammation elicited by MDP. Kufoy et al (Exp . Eye Res . , 50(2), 189-195 (1990)) also studied modulation of the blood aqueous barrier by MDP and Waters et al ( Infect . Immun . , 51(3), 816-825 (1986)) state that intraocular inflammation was produced in rabbits by MDP. Thus muramyl dipeptide is known to have a deleterious effect on the eye and the same may also have been expected of its derivatives. However, it has now been found that there are unexpected benefits in a method for the treatment of ophthalmic conditions, the method comprising administering to a patient suffering from an ophthalmic condition an effective amount of a muramyl peptide derivative.

5 In a first aspect of the present invention there is provided the use of a muramyl peptide compound in the preparation of an agent for the treatment of ophthalmic diseases or conditions.

10. The expression "a muramyl peptide compound" has a clear meaning to those skilled in the art. In particular, it refers to a compound containing one or more sugar residues, at least one of the sugar residues, which will often be a muramic acid residue, being substituted with

15 at least one or more (and usually two or more) amino acid residues. Muramyl peptide compounds may be peptido- glycans which are capable of enhancing the cellular antigenicity response in mammals and which are prototype muramyl dipeptide (MDP) or analogues or derivatives

20 thereof.

The ophthalmic conditions in which muramyl peptide compounds may be of use include conjunctivitis; disorders of the cornea such as keratitis; disorders of the 25 eyelids; dryness of the eyes and eye injuries.

Muramyl peptide compounds have been found to be particularly useful in the treatment of conditions such as keratitis and keratouveitis, particularly when caused 30 by a viral, bacterial or chlamydia infection. One relatively common form of keratitis is that caused by Herpes simplex and known as Herpetic stromal keratitis. herpetic keratouveitis may also be caused by the Herpes simplex virus and muramyl peptide compounds have now been shown to be particularly effective in the treatment of both of these conditions.

It is particularly surprising that muramyl peptide compounds are useful in the treatment of keratouveitis because, as discussed above, prototype muramyl peptide is known to cause uveitis.

One reason for the usefulness of muramyl peptide compounds in the treatment of conditions such as herpetic stromal keratitis is that they are effective as antiviral agents. However, an additional benefit is that they are capable of preventing or reducing the extent of the corneal scarring which often accompanies the condition. It has been found that when administered to patients suffering from herpetic stromal keratitis, muramyl peptide compounds enhanced resolution of stromal involvement and increased the rate of vision improvement.

The mechanism by which muramyl peptide compounds confer this surprising and beneficial effect is not absolutely clear. One suggestion is that it could arise from the antiviral effects of the compounds since more rapid clearance of virally infected cells could reduce the period of infection and thus the potential for scarring. An alternative theory is that the muramyl peptide compounds may down regulate the activity of inflammatory cytokines and therefore inhibit the stimulation of fibroblasts and the deposition of scar tissue. However, the effectiveness of the invention is not affected by the accuracy or otherwise of either of these theories.

Therefore, muramyl peptide derivatives will also be useful in a method of treating or preventing corneal scarring associated with ophthalmic disease, the method comprising administering to a patient suffering from ophthalmic disease an effective amount of a muramyl peptide compound.

In a second aspect of the present invention there is provided the use of a muramyl peptide compound in the preparation of an agent for the treatment or prevention of corneal scarring associated with ophthalmic disease.

The use of a muramyl peptide compound will assist in the treatment of any ophthalmic condition from which scarring is likely to result but the ophthalmic diseases mentioned above are those in which muramyl peptide compounds are particularly useful for the prevention of scarring.

Because of their ability to prevent scarring in various ophthalmic conditions, muramyl peptide compounds are particlarly effective when used in combination with a second drug which is used for the treatment of the condition but which cannot reduce or prevent scarring in the way in which a muramyl peptide compound can.

In a third aspect of the invention there is provided a product containing a muramyl dipeptide and a second drug as a combined preparation for simultaneous, separate or sequential use in the treatment of an ophthalmic disease or condition.

The second drug may be any substance which is useful in the treatment of an ophthalmic condition: such substances are well known to those skilled in the art of ophthalmic medicine and include antiviral agents, anti-bacterial agents, steroids and nonsteroidal anti-inflammatory drugs .

The conditions for which this aspect is of especial use are those mentioned above in relation to the first and second aspects of the invention.

It can therefore be seen that the present invention will also be useful in a method for the treatment of an ophthalmic condition, the method comprising administering to a patient suffering from an ophthalmic condition an effective amount of a muramyl peptide compound in combination with an effective amount of a second drug.

As stated above, the second drug may be selected from the group consisting of antiviral agents, antibacterial agents, steroids and nonsteroidal anti-inflammatory drugs.

Agents which are particularly suitable in this aspect of the invention include acyclovir, idoxuridine, chloramphenicol, chlortetracycline, fusidic acid, gentamycin, neomycin, oflaxacin, polymixin, tetracycline, tobramycin, betamethasone, clobetasone, dexamethasone, fluormethiolone, hydrocortisone, prednisolone and flurbiprofen. Of these, acyclovir or idoxuridine are preferred.

Many muramyl peptide compounds useful in this invention fall within general formula I:

wherein:

R¹ represents a hydrogen atom or a Ci-C_jj acyl group; R² represents a hydrogen atom or a C_J-C_JJ acyl group;

R³ represents a hydrogen atom or a Ci-Cg alkyl group;

R⁴ represents a Ci-Cj_! alkyl group or a C₆ or C₁₀ aryl group;

R⁵ represents a hydrogen atom; and R represents the residue of an amino acid or a linear peptide built up of from 2 to 6 amino acid residues, at least one of the residues being optionally substituted with a lipophilic group;

Preferred acyl groups for R¹ and R² are C_x-C₅ acyl groups such as acetyl; it will be appreciated that the carbon count in the acyl group does not include the carbonyl moiety. Preferred alkyl groups for R³ are C^C_* alkyl groups such as methyl and ethyl. Preferred alkyl groups for R⁴ and C^C_s alkyl groups, particularly C₁-C₄ alkyl groups, such as methyl or ethyl; phenyl is a preferred aryl group.

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-α-aminobutyryl L-histidyl L-seryl L-glutamyl

L-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.

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^C^ (preferably C^C alkyl ester, amide or C^C, 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) -sπ-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_l-C_1Q ester group, such as a C₂-C₆ ester group: a butyl ester is an example.

Examples of muramyl dipeptides within the scope of general formula I include: muroctasin, otherwise known as MDP-Lys (L18) (N²- (N- acetylmuramyl-L-alanyl-D-isoglutaminyl) -N⁶-stearoyl- L-lysine) ;

MTP-PE (N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L- alanyl-2- (1' ,2' -dipalmitoyl-sn-glycero-3' -hydroxy- phosphoryloxy)ethylamide, monosodium) ;

murabutide (N-acetylmuramyl-L-alanyl-D-glutamine-α-

N-butyl ester) ; and

t-MDP (N-acetylmuramyl-L-threonyl-D-isoglutamine) .

The preparation of muroctasin is disclosed in EP-A-

0021367 and US-A-4317771. The preparation of MTP-PE is disclosed in EP-A-0025495. The preparation of murabutide is described in Lefrancier et al , J. Med. Chem. , 25 87

(1982) . The preparation of t-MDP can be prepared by methods known in the art. Patent publications which give details of the preparations of muramyl peptide compounds generally include BE-A-0834753, BE-A-0834754, BE- A-0847103, BE-A-0849214, DE-A-2710455, DE-A-2922533, DE- A-2747379, DE-A-2912865, FR-A-2355505, FR-A-2358159, FR- A-2375249, EP-A-0004512, EP-A-0002677, JP-A-54063016, JP- A-54073729, JP-A-55019236, US-A-4082735 and US-A-4082736. (The preparation of prototype muramyl dipeptide is disclosed in DE-A-2450355 and US-A-4235771.) All the documents referred to in this specification are incorporated herein by reference.

Not all muramyl dipeptides useful in the present invention fall within general formula I. Many fall within general formula II, which represents a very much preferred group of compounds for use in the invention:

II

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

Preferred values for R are as described above in relation to general formula I. 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.

Compounds of general formula II 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 II and is N-acetyl-D-glucosaminyl- (01-4) -N-acetylmuramyl-L-alanyl- D-isoglutamine (GMDP), the structure of which is:

GMDP

This compound (Compound II in US-A-4395399) , also known as glycopin, has already undergone preclinical toxicity testing and pharmacokinetic investigations required for licensing for clinical use in the USSR (as it then was) . The acute toxicity in mice, measured by the LD₅₀ test is 7 g/kg. This figure shows the compound to be almost an order of magnitude less toxic than muroctasin which has an LD₅₀ value in mice of 625 mg/kg. While the pyrogenicity of GMDP is sufficiently low to make it suitable for use in the present invention, and not to have prevented its clinical evaluation for other purposes, it may in some circumstances be preferable to use an even less pyrogenic analogue. Such an analogue is available, and is N-acetyl-D-glucosaminyl- (Sl-4) -N- acetylmuramyl-L-alanyl-D-glutamic acid (GMDP-A) , which is Compound III in US-A-4395399, and whose structure is as follows:

GMDP-A

Other preferred compounds within the scope of general formula II include:

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

GMDP-LL N-acetyl-D-glucosaminyl- (/31-4) -N acetylmuramyl-L-alanyl- D-glutamine n-butyl ester (GMDP-OBu) which has the structure:

GMDP-OBu

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

GMDP-Lys

N"- [N-acetyl-D-glucosaminyl- (jβl-4) -N-acetylmuramyl -L- alanyl-D-isoglutaminyl] -N^€-stearoyl-L-lysine (GMDP-

Lys (St)) which has the structure:

GMDP-Lys ( St )

Other useful compounds include:

N^α- [N-Acetyl-D-glucosaminyl- (J1--4) -N-acetyl-muramyl-L- alanyl-γ-D-glutamyl] -N^e-stearoyl-L-lysine which has the structure:

GMDPA-Lys (St )

N-Acetyl-D-glucosaminyl- (/31--4) -N-acetylmuramyl-L-alanyl- D-glutamic acid dibenzyl ester which has the structure:

GMDPA(OBzl)

N-Acetyl-D-glucosaminyl- (01--4) -N- acetylmuramyl -N-methyl- L-alanyl -D-isoglutamine which as the structure:

Me -GMDP

N-Acetyl-D-glucosaminyl- (/31--4) -N- acetylmuramyl- (/31--4) - N-acetyl-D-glucosaminyl- (31--4) -N-acetylmuramyl-bis- (L- alanyl -D-isoglutamine) which has the structure:

(GMDP)

N-Acetyl-D-glucosaminyl- (_/S1--4) -N-acetylmuramyl- (/S1--4) - N-acetyl-D-glucosaminyl- (_/31--4) -N-acetylmuramyl-bis- (L- alanyl-D-glutamic acid) which has the structure:

(GMDPA) N-Acetyl-D-glucosaminyl- (jβl--4) -N- acetylmuramyl- (/31--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- (01--4) -N- acetylmuramyl- (/31--4) N-acetyl-D-glucosaminyl- (_/S1--4) -N-acetylmuramyl-bis- [L alanyl-D-isoglutaminyl-N^€- stearoyl -L-lysine] :

[GMDP-Lys (St) ]

SUBSTITUTE SHEET (RULE 26> N-Acetyl-D-glucosaminyl- (βl --4) -N-acetylmuramyl-L alanyl -D-isoglutamine 1-adamantyl ester which has the structure :

GMDP -Ad

L-Threonyl-N^e- [N-Acetyl-D-glucosaminyl- (βl-- ) -N-acetyl- muramyl -L- alanyl -γ-D-isoglutaminyl] -L-lysyl -L-prolyl-L- arginine which has the structure :

-Pro-Arg

GMDP-tuftsin E N-Acetyl-D-glucosaminyl- (/31--4) -N-acetyl-muramyl-L- alanyl- γ-D-isoglutaminyl -L-threonyl -L-lysyl -L-prolyl-L- arginine which has the structure :

GMDP-tuftsin A

N-Acetyl-D-glucosaminyl- (01--4) -N- acetylmuramyl -L-alanyl - α -D-glutamyl -L-lysyl -L-threonyl -N^e- stearoyl -L-lysyl -L- prolyl-L-arginine which has the structure:

GMDPA-tuftsin lipophilic

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

SUBSTITUTE SHEET (RULE 6Ϊ isoGln-Lys-His-Gly-NH₂

GMDPA-bursin

N-Acetyl-D-glucosaminyl- (S1--4) -N- acetylmuramyl- L-alanyl - D- isoglut aminyl -L-glutamyl -L- tryptophan which has the structure:

GMDP-thymogen I

N-Acetyl-D-glucosaminyl- (01--4) -N-acetylmuramyl-L-alanyl - D- isoglut aminyl - e - aminohexanoyl -L-glutamyl -L- tryptophan which has the structure:

GMDP-thymogen II

N°- [N-Acetyl-D-glucosaminyl- (/31--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 :

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 II 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.

The efficacy of muramyl peptide compounds in the invention has been demonstrated using oral administration. The formulation in this instance consisted of tablets containing pharmaceutically acceptable excipients, namely lactose, starch, polyvidone, magnesium stearate and talc. Muramyl peptide compounds may be formulated for sustained and/or delayed delivery if desired. Gastric coating is another option.

The precise oral dosage for administration will always be that deemed suitable by the clinician or physician. Subject to that, a daily oral dosage in the range of from 0.1 to 50 mg per day (or per unit dose) may be found acceptable, with a range of 0.5 to 50 mg being preferred. A daily dosage of 1 to 20 mg is considered optimal.

In addition, the duration of administration may be varied. The duration will of course depend to some extent on dosage level, i.e. lower dose results in longer required duration of dosage. In general terms, the duration of dosage will be in the range of 1-60 days, preferably l-30days and most preferably 1-14 days. Furthermore, administration need not necessarily be daily: Example 2 presented herein used a regimen of GMDP once per day for three days, three days without administration of GMDP, and then GMDP once per day for a further three days. Other variations, for instance every other day, can also be used.

Since the muramyl peptide compounds are intended for the treatment of ophthalmic conditions, it is also possible for them to be formulated for topical administration to the eye. Such a formulation will generally contain pharmaceutically acceptable excipients etc usually found in such formulations.

Such formulations are new and therefore in a fourth aspect of the invention there is provided a pharmaceutical composition comprising a muramyl peptide compound together with a pharmaceutically acceptable excipient or carrier; characterised in that the composition is formulated for topical administration to the eye.

When formulated for topical administration to the eye, the compositions may comprise one or more muramyl peptides in a pharmaceutically acceptable opthalmic ointment or cream. Alternatively, the formulation can be in the form of drops which will contain appropriate buffers and preservatives common in the art and known to the skilled man.

The precise dosage for topical administration will always be that deemed suitable by the clinician or physician. Subject to that, a daily dosage in the range of from 0.1 to 100 mg per day (or per unit dose) for topical compositions may be found to be acceptable, with a range of 0.5 mg to 50 mg per day (or per unit dose) being preferred. A daily dosage of 1 to 20 mg is considered to be optimal.

The invention will now be illustrated by the following non-limiting examples and the drawings in which:

FIGURE 1: is a plot showing a comparison of the effects of the muramyl peptide compound GMDP and a placebo on the resolution of the symptoms of ophthalmic herpes infection.

FIGURE 2 : is a plot showing a comparison of the effects of the muramyl peptide compound GMDP and a placebo on the epithelial healing in patients suffering from dendritic keratitis.

FIGURE 3 : is a plot showing a comparison of the effects of the muramyl peptide compound GMDP and placebo in the incidence of complete clearing of clinical symptoms in patients with herpetic stromal keratitis.

Example 1

The use of GMDP in the treatment of ophthalmic herpes,

The study was carried out on patients with herpetic ophthalmic diseases. Twenty-five patients between 16-60 years of age took part in the study. There were 16 male and 9 female patients (Table 1) .

Patients were divided into two groups. The first group displayed dendritic herpetic keratitis (DK) with stromal lesion (13 patients) and the second group, herpetic keratouveitis (KU) (12 patients) .

Infection in patients with dendritic keratitis with stromal lesions was accompanied by involvement of the eyelid and bulbar conjunctiva, the presence in the cornea of ulcerations of the epithelium and surface stromal layers in the form of a "branching", perifocal stromal infiltration. In some patients, symptoms of descemetitis and iritis were observed.

Before the beginning of the tests, the prevailing symptoms of patients with keratouveitis was stromal inflammation, accompanied by oedema, infiltration and inflammation of the eye vascular tunic. The degree of severity of the disease varied from 14 to 21 points.

The clinical scores at the beginning of the study of patients allocated to control and GMDP groups are summarised in Table 1.

The study was placebo controlled, double blind and patients received either 1 mg GMDP per day for ten days, or identical placebo tablets. All patients who took part in the study also received local and systemic therapy appropriate for the severity and clinical form of the disease.

Before entry into the study, the following procedures were carried out: collection of case history data; clinical examination; temperature; blood samples for clinical analysis (haemoglobin, complete cell count, ESR) ; sampling from the conjunctiva of the infected eye for bacteriological analysis.

SUBSTITUTESHEET(RULE26^ Examination of the eye included testing of visual acuity, eye biomicroscopy after staining with fluorescein, internal eye examination, testing of intraocular pressure by means of Maklokov's tonometer and, if not possible due to the eye condition, by means of palpation.

Immunological examination included: taking of scrapings from the conjunctiva of the infected eye; sampling tear liquid of the infected and non-infected eye; blood samples.

After performing a complete course of clinical and immunological examination (the 1st and 2nd day of admission to the clinic) , the patient was entered into the trial and administration of drug began.

In the course of treatment and observation, daily monitoring of the patient's therapeutic status and temperature was performed, and eye biomicroscopy after fluorescein-staining was performed. Every 3 days, visual acuity was tested and tonometry was performed. Haematological and immunological examinations were repeated 3-4 days after the end of administration of the drug under test. Bacteriological analysis of the content of the conjunctival sac and other additional examinations were carried out according to the indications.

Estimation of the severity of eye disease.

In order to estimate disease severity, clinical symptoms were scored according to a three-point system: severe - 3, medium - 2, light - 1. Each symptom of the disease was estimated in points and the total intensity of inflammation was determined. Clinical scores were noted daily for each patient in addition to visual acuity scores.

Safety and tolerance of the drug.

This was evaluated on the basis of clinical examination which included subjective observations of the patient, control of allergic side reactions, objective data on the condition of internal organs, temperature, haematology results.

Estimation of clinical efficacy was performed according to the following criteria:

• degree of epitheliopathy and time to complete corneal epithelialisation

• disappearance of symptoms of iritis

• resolution of stromal infiltration

• complications in the course of inflammatory process and their character

• change in visual acuity.

Results.

(i) Safety and tolerance

No subjective complaints and side reactions attributable to the drug were observed. A generally healthy status was maintained, and no pyrexia or significant changes in clinical blood tests were observed. (ii) Efficacy and activity

The clinical outcome in treated and placebo patients in the two subgroups is compared in Table 2. For most parameters, a trend towards more rapid healing was observed in the GMDP treated group, particularly when DK and KU results were pooled. However, these results were not statistically significant. For a number of parameters, less favourable resolution was observed in the GMDP treated group of KU patients. These results appeared to be influenced to a large extent by one single individual who had a particularly slow recovery. Far greater increase in visual acuity was observed in the treated group than in the placebo group. This applied both to patients with DK and those with KU.

The progress of clinical parameters over time is shown in Figs 1 and 2. A consistent trend towards more rapid healing in the GMDP treated group is again observed.

Conclusion.

i) Clinical tests revealed a distinct advantage of GMDP in the treatment of patients with dendritic herpetic keratitis.

ii) The results of immunological control did not reveal any consistent trend that could have a bearing on the effectiveness of the drugs under study. However, the disappearance of lymphocyte reactivity to HSV is more clearly traced in the group of patients who were administered GMDP. According to the test of revealing HSV antigen in the conjunctiva of the infected eye, certain advantages are also noted in the patients who were administered GMDP.

iii) The following factors were noted: excellent tolerance of GMDP and placebo; the absence of side allergic reactions as well as the absence of changes in the general therapeutic status; temperature and clinical blood indices.

Table 1 - Main characteristics of control and GMDP treated groups.

Group

Control GMDP

Total No. 13 12

Males 9 7 Females 4 5

Age; mean (SE) 42.8 (2.9) 44.3 (3.7)

Clinical subgroup:

Dendritic keratitis 7 6 Keratouveitis 6 6

Time since initial HSV infection:

≥20 yrs 5 3 <20 yrs 4 5 1st occurrence 4 4

Duration of previous remission:

Mean (SE) (months) 27.7 (16.7) 68.4 (29.5) Range 1 mo-13 yr 2 mo-20 yr

Duration of infectious episode before entering study:

Mean (SE) (days) 29.5 (5.2) 23.6 (5.4)

Total composite score at entry to study:

Mean (SE) 16.1 (0.9) 15.1 (0.8)

Visual acuity at entry to study:

Mean (SE) 0.27 (0.07) 0.27 (0.06) Table 2 Summary of results,

Group

End point Placebo GMDP

Time to zero overall score (days l )

All 15.3 (1.6) 14.4 (2.6)

DK 13.3 (2.5) 9.8 (1.2)

KU 17.7 (1.4) 19.0 (4.4)*

Time to 50% healing (days)

All 7.2 (0.6) 6.4 (0.7)

DK 5.6 (0.6) 4.8 (1.0)

KU 9.0 (0.5) 8.0 (0.6)

Time to zero epitheliopathy (days) All 14.0 (1.5) 13.5 (2.7) DK 11.6 (2.3) 8.3 (1.7) KU 16.8 (1.3) 18.7 (4.4) *

Time to 50% epitheliopathy (days )

All 7.3 6.8

DK 5.4 5.5

KU 9.5 8.0

Time to complete epithelia.^" Lisation (days) DK 9.4 (2.9) 5.5 (1-4)

Change in visual acuity (score) All 0.22 (0.02) 0.34 (0.07) DK 0.20 (0.03) 0.28 (0.08) KU 0.25 (0.03) 0.40 (0.11)

Resolution of infiltrate (days) All 15.2 (1.6) 14.0 (2.5) DK 13.3 (2.5) 9.8 (1.2) KU 17.3 (1.4) 18.2 (4.4)*

Disappearance of iritis (days) All 8.7 (0.7) 11.2 (3.5)* DK 7.8 (1.4) 4.3 (0.3) KU 9.3 (0.8) 14.7 (4.8) * Example 2

The use of GMDP in the treatment of herpetic stromal keratitis

A second clinical trial was performed in a way broadly similar to that described in example 1. A total of 60 patients with herpetic stromal keratitis were enrolled in the study, and randomised (2 groups of 30) to treatment with GMDP tablets or identical placebo.

Dosage was 20mg GMDP per day, and administration was according to the following schedule: GMDP or placebo once per day for three days, three days rest (no GMDP/placebo) , GMDP or placebo once per day for three days.

In addition, patients received concomitant treatment (antiviral acyclovir, antibiotics etc.) appropriate to normal practice.

Evaluation of treatment was by a scoring system which examined the following parameters: stromal involvement (superficial, deep, corneal oedema, descemetitis) , corneal ulceration (punctate, area) , interior eye involvement (precipitates, hypopion, iritis) , hyperaemia (conjunctival and pericorneal) .

Results showed that GMDP treatment enhanced resolution of most clinical symptoms and accelerated improvement of visual acuity. For example, the mean time to 50% resolution of stromal involvement was 7.3 days in the placebo group and 5.8 days in the GMDP group. Mean time to complete resolution was 14.9 days and 10.6 days respectively. The time course of achieving complete clearing is plotted in Figure 3. Analysis of the incidence of clearing at successive time points (chi- squared test) showed that between days 8 and 14 inclusive, significantly (p<0.05) more patients receiving GMDP showed clearing than compared to the placebo group. Visual acuity in the GMDP treated group reached an average of 0.60 by day 10, whereas the placebo group was only 0.49.

Claims

1. The use of a muramyl peptide compound in the preparation of an agent for the treatment of ophthalmic diseases or conditions.

2. The use of a muramyl peptide compound in the preparation of an agent for the treatment or prevention of corneal scarring associated with an ophthalmic disease or condition.

3. A product containing a muramyl dipeptide and a second drug as a combined preparation for simultaneous, separate or sequential use in the treatment of an ophthalmic disease or condition.

4. A product as claimed in claim 3, wherein the second drug is an antiviral agent, anti-bacterial agent, steroid or nonsteroidal anti-inflammatory drug.

5. A product as claimed in claim 4 wherein the second drug is acyclovir, idoxuridine, chloramphenicol, chlortetracycline, fusidic acid, gentamycin, neomycin, oflaxacin, polymixin, tetracycline, tobramycin, betamethasone , clobetasone, dexamethasone , fluormethiolone, hydrocortisone, prednisolone or flurbiprofen.

6. A product as claimed in claim 5 wherein the second drug is acyclovir or idoxuridine.

7. The use as claimed in claim 1 or claim 2 or a product as claimed in any one of claims 3 to 6, wherein the ophthalmic disease or condition is conjunctivitis, a disorder of the cornea such as keratitis, a disorder of the eyelids, dryness of the eyes or an eye injury.

8. A use or product as claimed in claim 7, wherein the condition is herpetic stromal keratitis.

9. A use or product as claimed in any one of claims 1 to 8, wherein the muramyl peptide compound is of general formula I:

wherein:

R¹ represents a hydrogen atom or a C₁-C₂₂ acyl group;

R² represents a hydrogen atom or a C_x-C₂₂ acyl group;

R³ represents a hydrogen atom or a C_ι- alkyl group;

R⁴ represents a C₁-C₂₁ alkyl group or a C₆ or C₁₀ aryl group,-

R⁵ represents a hydrogen atom; and

R represents the residue of an amino acid or a linear peptide built up of from 2 to 6 amino acid residues, at least one of the residues being optionally substituted with a lipophilic group.

10. A use or product as claimed in claim 9, wherein the compound of general formula I has any or all or any compatible combination of the following substituents:

each of R¹ and R² independently represents a C_j-Cg acyl group such as acetyl;

R³ represents a C₁-C₄ alkyl group such as methyl or ethyl;

R⁴ represents a

alkyl group, particularly a C -

C₄ alkyl group, such as methyl or ethyl, or a phenyl group;

R represents a mono-, di- or tri-peptide.

11. A use or product as claimed in any one of claims 1 to 10, wherein the muramyl peptide compound is:

muroctasin, otherwise known as MDP-Lys (L18) (N²- (N- acetylmuramyl-L-alanyl-D-isoglutaminyl) -N⁶-stearoyl- L-lysine) ;

MDP-OBu(N-acetylmuramyl-L-alanyl-D-isoglutamine-n- butyl ester) ;

murabutide (N-acetylmuramyl-L-alanyl-D-glutamine -a- N-butyl ester) ; or t-MDP ⁽N-acetylmuramyl-L-threonyl-D-isoglutamine) .

12. A use or product as claimed in any one of claims 1 to 8, wherein the muramyl peptide compound conforms to general formula II:

II

wherein :

n is 1 or 2.

13. A use or product as claimed in claim 12, wherein n is 1.

14. A use or product as claimed in claim 12 or 13, wherein the proximal amino acid residue is a residue of an L-amino acid.

15. A use or product as claimed in claim 14, wherein the proximal amino acid residue (or the only amino acid residue, if there is only one) is a residue of L-alanine.

16. A use or product as claimed in any one of claims 12 to 15, wherein the second amino acid residue from the proximal end of the peptide, if present, is of the D- configuration.

17. A use or product as claimed in claim 16, wherein the said second amino acid residue is of D-glutamic or D- aspartic acid or a mono-, di- or mixed C_j-C^ (preferably C_x-C₆) alkyl ester, amide or C^C, alkyl amide thereof.

18. A use or product as claimed in claim 15, 16 or 17, wherein the said second amino acid residue is D- isoglutaminyl or D-glutamyl.

19. A use or product as claimed in any one of claims 12 to 18, wherein the third amino acid residue from the proximal end of the peptide, if present, is in the L- configuration.

20. A use or product as claimed in claim 19, wherein the third amino acid residue is L-alanyl or L-lysyl.

21. A use or product as claimed in any one of claims 12 to 20, wherein the amino acid residue or linear peptide is optionally substituted with at least one lipophilic group.

22. A use or product as claimed in claim 12, wherein the compound is N-acetyl-glucosaminyl-N-acetyl-muramyl-L- alanyl-D-isoglutamine (GMDP) .

23. A use or product as claimed in claim 12, wherein the compound is :

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

N-acetyl-D-glucosaminyl- (01-4) -N-acetylmuramyl -L- alanyl-D-glutamine n-butyl ester (GMDP-OBu) ;

N- [Nα-Acetyl-D-glucosaminyl- (_/βl-4) -N-acetylmuramyl - L-alanyl-D-isoglutaminyl] -N^e-stearoyl-L-lysine (GMDP- Lys(St));

N"- [N-Acetyl-D-glucosaminyl- (01--4) -N-acetyl -muramyl - L-alanyl -γ-D-glutamyl] -N'-stearoyl-L-lysine (GMDPA- Lys(St) ) ;

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

N-Acetyl-D-glucosaminyl- (/81--4) -N-acetylmuramyl -N- methyl-L-alanyl-D-isoglutamine (Me-GMDP) ;

N-Acetyl-D-glucosaminyl- (/Si --4) -N-acetylmuramyl- (01- -4) -N-acetyl-D-glucosaminyl- (01- -4) -N-acetylmuramyl- bis- (L-alanyl-D-isoglutamine) ( (GMDP)₂) ;

N-Acetyl-D-glucosaminyl- (01--4) -N-acetylmuramyl- (_/Sl- -4) -N-acetyl-D-glucosaminyl- (_/S1--4) -N-acetylmuramyl- bis- (L-alanyl-D-glutamic acid) ( (GMDPA) ₂);

N-Acetyl-D-glucosaminyl- (_/81--4) -N-acetylmuramyl- (01-

-4) -N-acetyl-D-glucosaminyl- (01--4) -N-acetylmuramyl- bis- (L-alanyl-D-isoglutaminyl-L-lysine) ( (GMDP ys)₂) ; N-acetyl-D-glucosaminyl- (01--4) -N-acetylmuramyl- (01- -4) -N-acetyl-D-glucosaminyl- (01--4) -N-acetylmuramyl- bis- [L-alanyl -D-isoglutaminyl -N^e- stearoyl -L-lysine] ( [GMDP-Lys (St) ]_a) ;

N-Acetyl-D-glucosaminyl- (01--4) -N-acetylmuramyl-L- alanyl -D-isoglutamine 1-adamantyl ester (GMDP-Ad) ;

L-Threonyl-N^e- [N-Acetyl-D-glucosaminyl- (01--4) -N- acetyl-muramyl-L-alanyl-γ-D-isoglutaminyl] -L-lysyl -

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

N-Acetyl-D-glucosaminyl- (01--4) -N-acetyl -muramyl -L- alanyl -γ-D-isoglutaminyl -L-threonyl -L-lysyl -L- prolyl-L-arginine (GMDP-tuftsin A) ;

N-Acetyl-D-glucosaminyl- (01--4) -N- acetylmuramyl -L- alanyl -α-D-glutamyl -L-lysyl -L-threonyl -N^e -stearoyl -L- lysyl-L-prolyl -L-arginine (GMDPA- uft sin lipophilic) ;

N^€- [N-Acetyl-D-glucosaminyl- (01--4) -N- acetyl -muramyl - L-alanyl -γ-D-isoglutaminyl] - L-lysyl -L-his t idyl- L- glycine amide (GMDPA-bursin) ;

N-Acetyl-D-glucosaminyl- (01--4) -N-acetylmuramyl-L- alanyl -D -isoglut aminyl -L- glut amyl-L- tryptophan (GMDP-thymogen I) ;

N-Acetyl-D-glucosaminyl- (01--4) -N-acetylmuramyl-L- alanyl- D-isoglutaminyl -e-aminohexanoyl -L-glutamyl -L- tryptophan (GMDP-thymogen II) ; N"- [N-Acetyl-D-glucosaminyl- (01- -4 ) -N-acetyl -muramyl - L-alanyl -D- isoglutaminyl] -N^e - 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) .

24. A method for the treatment of ophthalmic conditions, the method comprising administering to a patient suffering from an ophthalmic condition an effective amount of a muramyl peptide derivative.

25. A method of treating or preventing corneal scarring associated with ophthalmic disease, the method comprising administering to ^"a patient suffering from ophthalmic disease an effective amount of a muramyl peptide compound.

26. A method for the treatment of an ophthalmic condition, the method comprising administering to a patient suffering from an ophthalmic condition an effective amount of a muramyl peptide compound in combination with an effective amount of a second drug.

27. A method as claimed in any one of claims 24 to 26 wherein the muamyl peptide compound administered is GMDP.

28. A method as claimed in claim 27 wherein 0.1 to 50mg of GMDP is administered per day.

29. A method as claimed in claim 28 wherein 0.5 to 50mg of GMDP is administered per day.

30. A method as claimed in claim 29 wherein 1 to 20mg of GMDP is administered per day.

31. A method as claimed in any one of claims 24 to 30 wherein the duration of administration is from 1 to 60 days.

32. A method as claimed in claim 31 wherein the duration of administration is from 1 to 30 days.

33. A method as claimed in claim 32 wherein the duration of administration is from 1 to 14 days.

34. A method as claimed in any one of claims 24 to 30 wherein the compound is administered is administered as follows:

(i) three days administration;

(ii) three days without aministration;

(iii) a final three days administration.