CZ86197A3 - Use of muramyl peptide compounds - Google Patents

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

Technical field

The invention relates to the treatment of ocular diseases, and in particular to the treatment or prevention of corneal scarring, which may be associated with bacterial or viral eye infections.

BACKGROUND OF THE INVENTION

The eye is a relatively complex organ and is sensitive to many types of viral and bacterial infections as well as to non-infectious conditions. For example, conjunctivitis, which is a conjunctival infection, may be caused by bacteria, viruses, other microorganisms such as Chlamydia, or allergies. Corneal disorders include keratitis, which may be caused by bacteria such as staphylococci, streptococci, pseudomonads or enterobacteria or viruses such as Herpes simplex. Systemic viral diseases such as measles, mumps and chickenpox can also cause keratitis, but it can also be caused simply by dry eye. Certain ocular conditions, particularly keratitis, other conditions of the cornea and eyelid disorders or infections, may result in corneal scarring, which may impair vision and reduce eyelid movement.

Many ophthalmic conditions can be treated with known antibacterial and antiviral drugs, but although these drugs are effective, corneal scar may still occur. This is believed to be due to the migration of lymphocytes and macrophages into the cornea. Lymphocyte and macrophage activity may include secretion of cytokines, which may cause fibroblast activation, which disproportionately affects collagen and causes corneal scarring. Scarring may also be caused by another mechanism.

However, it has now been found that various muramyl peptide derivatives are useful in the treatment of ophthalmic conditions and, in particular, may be involved in the treatment or prevention of corneal scarring.

It has long been known that non-specific stimulation of the immune system can be achieved by exposing the system to the presence of bacteria or components derived from bacterial cell walls. The particular compounds responsible for this activity were identified as cell wall peptides containing sugars, and further biochemical analysis of the peptides identified them as the peptidoglycan component of the cell wall. The smallest active synthetic molecule has been found to be N-acetylmuramyl-Lalanyl-D-isoglutamine (Merser et al., Biochem. Biophys. Res. Comm. 66, 1316 (1975)). The ability of this compound (now often referred to as muramyl dipeptide or MDP prototype) to protect mice from bacterial infection (Klebsiella pneumonia) has been described (Chedid et al., Proc. Natl. Acad.

Sci. USA, 74, 2089 (1977)).

Subsequently, a wide variety of analogues of the prototype muramyldipeptide were synthesized, some of which were designed to enhance immune functions or to stimulate the immune system non-specifically. These analogues and the MDP prototype itself are muramyl peptide compounds.

Many authors have discussed the use of muramyl peptides and their analogs as adjuvants, for example Azuma et al. (Adv. Exp. Med. Biol. 319: 253-263 (1992)) which discloses that MDP compounds are suitable for use as an immunoadjuvant to enhance the antigenicity of recombinant or component vaccines.

Aliison et al. (Semin. Immunol. 2 (5), 369-374 (1990)) discloses that adjuvant mixtures composed of synthetic MDP analogues in emulsion with squalene-Pluronic polymer induce cell-mediated immunity and protective isotype antibodies and enhance response to various antigens.

Burke (Rev. Infect. Dis., 13, Suppl. 11, 906-911, November-December 1991) report that MTP-PE is particularly useful as an adjuvant with a Herpes simplex virus subunit vaccine.

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

Brynestad et al., J. Virol., 64 (2), 680-685, (1990) report that the use of a Herpes simplex-derived peptide in conjunction with a fatty acid carrier exhibits increased 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) report that MDP-Lys restores resistance to type 1 Herpes simplex virus infection in cyclophosphamide treated mice.

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

A number of other reports describe the use of various muramyl peptide derivatives as adjuvants.

In addition, there is an incentive in the 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 properties of the broad-spectrum antiviral drug ribavirin. The effect was particularly pronounced with liposome-encapsulated MTP-PE.

However, none of these documents discloses that muramyl peptide compounds may be particularly useful in the treatment of ophthalmic conditions, and in fact it is believed in the art that the use of muramyldipeptide analogs may be detrimental to the eyes. For example, Lawrence et al. in Exp. Eye Res., 54 (4), 105-107 (1992) discusses the removal of anatomical barriers between the ventral segment of the eye and the bloodstream during MDP-induced inflammation. Kufoy et al. (Exp. Eye Res., 50 (2), 189-195 (1990)) also studied MDP-induced blood water barrier modulation and Waters et al. (Infect. Immun., 51 (3), 816 825 (1986)) reported that intraocular inflammation was induced in rabbits by MDP. This muramyl peptide is known to have a deleterious effect on the eye, and the same effect can be expected with its derivatives. However, it has now been found that a method of treating ophthalmic conditions which involves administering an effective amount of a muramyl peptide derivative to a patient suffering from an ophthalmic condition has an unexpectedly positive effect.

One embodiment of the invention is the use of a muramyl peptide compound for the manufacture of a medicament for the treatment of ophthalmic diseases and conditions.

The term muramyl peptide compound has a clear meaning to one skilled in the art. In particular, it is a compound comprising one or more sugar residues, at least one sugar residue, which will often be a muramic acid residue that is substituted with at least one or more (and usually two or more) amino acid residues. Muramyl peptide compounds may be peptidoglycans which are capable of enhancing the cellular antigenic response in mammals and which are prototypes of muramyldipeptide (MDP) or analogs or derivatives thereof.

Ophthalmic conditions in which muramyl peptide compounds may be employed include conjunctivitis; corneal disorders such as keratitis; eyelid disorders; eye drying and eye injury.

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

It is particularly surprising that muramyl peptide compounds are suitable for the treatment of keratouveitis because, as mentioned above, the prototype muramyl peptide is known to act in 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, another advantage is that they are able to prevent or reduce the extent of corneal scarring that often accompanies this condition. It has been found that when administered to patients suffering from herpetic stromal keratitis, muramyl peptide compounds accelerate the disappearance of stromal damage and increase the rate of improvement in vision.

The mechanism by which muramyl peptide compounds exert these surprising and beneficial effects is not entirely clear. According to one theory, this effect is due to the antiviral effects of these compounds, since faster eradication of virally infected cells can reduce the period of infection and thus the possibility of scarring. According to another theory, muramyl peptide compounds can regulate the activity of inflammatory cytokines and thus inhibit fibroblast stimulation and scar tissue deposition. However, the effectiveness of the present invention is not affected by the accuracy or other aspect of these theories.

Muramyl peptide derivatives are therefore also suitable for a method of treating or preventing corneal scarring associated with ophthalmic diseases, the method comprising administering an effective amount of a muramyl peptide compound to a patient suffering from ophthalmic disease.

As a second aspect of the invention, there is provided the use of a muramyl peptide compound for the preparation of a medicament for treating or preventing corneal scarring associated with ophthalmic diseases.

The use of the muramyl peptide compound cooperates in the treatment of any ophthalmic condition likely to result in scarring, but the above ophthalmic diseases are those in which the muramyl peptide compounds are particularly useful for preventing scarring.

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

A third object of the invention is to provide a product comprising muramyldipeptide and a second drug substance as a combined preparation for simultaneous, separate or sequential use in the treatment of an ophthalmic disease or condition.

The second drug substance may be any substance that is suitable for the treatment of the ophthalmic condition, such substances are well known to the person skilled in the art of ophthalmology, and include antiviral agents, antibacterial agents, steroids and non-steroidal anti-inflammatory agents.

Conditions for which this aspect of the invention is particularly useful are those listed above in connection with the first and second aspects of the invention.

Thus, it is understood that the present invention will also be useful in a method of treating ophthalmic conditions, the method comprising administering an effective amount of a muramyl peptide compound in combination with an effective amount of a second therapeutic agent to a patient suffering from an ophthalmic condition.

As mentioned above, the second therapeutic agent may be selected from the group consisting of antiviral agents, antibacterial agents, steroids, and non-steroidal anti-inflammatory agents.

Particularly suitable agents for this embodiment of the invention include acyclovir, idoxuridine, chloramphenicol, chlortetracycline, fusidic acid, gentamycin, neomycin, oflaxacin, polymixin, tetracycline, tobramycin, betamethasone, clobetasone, dexethethethone, fluretisethethone, floroxisethethone, floromisethethone, fluoromethisone, fluoromethisone, fluoroportethone, fluoromethisone, fluoromethone. Of these, acyclovir or idoxuridine are preferred.

Many muramyl peptide compounds useful in the present invention fall within Formula I:

where:

R ¹ represents a hydrogen atom or an acyl group containing 1 to 22 carbon atoms;

R ² is hydrogen or acyl containing 1 to 22 carbon atoms;

R ³ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms;

R ⁴ represents an alkyl group having 1 to 21 carbon atoms or an aryl group having 6 or 10 carbon atoms;

R ⁵ represents a hydrogen atom; and

R represents an amino acid residue or a linear peptide consisting of 2 to 6 amino acid residues, wherein at least one of the residues is optionally substituted with a lipophilic group;

Preferred acyl groups for R ¹ and R ² are acyl groups containing 1 to 5 carbon atoms such as acetyl; wherein it is desirable that the number of carbons in the acyl group does not include a carbonyl moiety. Preferred alkyl groups for R ³ are C 1 -C 4 alkyl groups such as methyl and ethyl. Preferred alkyl groups for R are alkyl groups having 1 to 6 carbon atoms, preferably alkyl groups having 1 to 4 carbon atoms, such as methyl or ethyl; a preferred aryl group is phenyl.

Preferably R is mono-, di- or tripeptide. The proximal peptide residue (or a single peptide residue, if any) is preferably an L-amino acid. Examples include:

L-alanyl

L-valyl

L-leucyl

L-isoleucyl

L-α-aminobutyryl

L-seryl

L-threonyl

L-methionyl

L-cysteinyl

L-phenylalanyl

L-tyrosyl

L-tryptophanyl

L-lysyl

L-ornithyl

L-arginyl

L-histidyl

L-glutamyl

L-glutaminyl

L-aspartyl

L-asparaginyl

L-prolyl

L-hydroxyprolyl

L-alanyl and L-threonyl are preferred.

The additional amino acid from the proximal end of the peptide preferably has a D-configuration. It is preferably acidic and may be D-glutamic or D-aspartic acid or mono-, or mixed alkyl esters thereof having 1 to 22 carbon atoms (preferably 1 to 6 carbon atoms), amides or alkyl amides having 1 to 4 carbon atoms. (The term mixed is used when one carboxyl group is amidated and the other esterified.) D-isoglutamine and Dglutamate are preferably used.

The third amino acid residue from the proximal end of the chain, if present, is preferably in the L configuration as mentioned 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 moiety. The lipophilic group may be an acyl group containing 10 to 22 carbon atoms such as stearoyl or a di-acyl-sn-glycero-3'hydroxyphosphoryloxy group containing from 10 to 22 carbon atoms in acyl, for example each of the C 10 -C 22 acyl groups may be a palmitoyl group.

The lipophilic group may alternatively (or additionally, since more than one substitution may be present) an ester group having 1 to 10 carbon atoms, such as an ester group having 2 to 6 carbon atoms: for example, a butyl ester group.

Examples of muramyl dipeptides within formula I include:

muroctasin, otherwise known as MDP-Lys (L18) (N ² - (Nacetylmuramyl-L-alanyl-D-isoglutaminyl) -N ⁶ -stearoyl-L-lysine);

MTP-PE (N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanyl-2 (1 ', 2'-dipalmitoyl-sn-glycero-3' hydroxyphosphoryloxy) 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 described in the patent

EP-A-0 021 367 and US-A-4 317 771. The preparation of MTP-PE is described in EP-A-0 025 495. The preparation of murabutide is described in Lefrancier et al., J. Med. Chem., 25, 87 (1982). Preparation of t-MDP may be accomplished by methods known in the art. Examples of patent applications which give details of the preparation of muramyl peptide compounds are, in principle, cited

BE-A-0 834 753, BE-A-0 834 754, BE-A-0 847 103, BE-A-0 849 214, DE-A-2 710 455, DE-A-2 922 533, DE-A-2 747 379, DE-A-2 912 865, FR-A-2 355 505, FR-A-2 358 159, FR-A-2 375 249, EP-A-0 004 512, EP-A-0 002 677, JP-A-5 063 í 016 , JP-A- 54,073,729

JP-A-55 019 236, US-A-4 082 735 and US-A-4 082 736. (Preparation of a prototype muramyldipeptide is described in DE-A-2 450 355 and US-A-4 235 771). All documents referred to in this specification are hereby incorporated by reference.

Not all muramyldipeptides useful in the present invention fall within the general formula (I).

H

HOCH ₂

NHAc

OH

II where:

R represents an amino acid residue or a linear peptide of 2 to 6 amino acid residues, wherein at least one of the residues is optionally substituted with a lipophilic group;

and n is 1 or 2.

Preferred R values are given above in relation to formula I. It is particularly preferred that the peptide R corresponds to a peptide in the prototype MDP (L-Ala-D-isoGln). Alternatively, in another preferred embodiment, R may be L-Ala-D-Glu.

The preferred value of n is 1.

Compounds of formula (II) are disclosed in U.S. Pat. No. 4,395,399 and the preferred embodiments herein are preferred embodiments of the invention. In addition, in the present invention, the R group may be lipophilically substituted as described above.

One of the most preferred compounds for use in the present invention is of formula II and is N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) having the structure:

BOY:

NHAc

GMDP

This compound (compound II in US-A-4,395,399), also known as glycopine, has already undergone preclinical toxicity testing and pharmacokinetic observations required for authorization for clinical use in the USSR (former). The acute toxicity in mice, as measured by the LD ₅₀ assay, is 7 g / kg. This figure shows that the compound is almost of the order of magnitude less toxic than muroctasin, which has an LD ₅₀ value in mice of 625 mg / kg.

While the pyrogenicity of GMDP is low enough to be suitable for use in the present invention and does not prevent its clinical use for other purposes, even less pyrogenic analogs may be preferred in certain circumstances. Such an analog is available and is N-acetyl-D-glucosaminyl- (β1-4) -N-acetylmuramyl-L-alanyl-D-glutamic acid (GMDP-A), which corresponds to compound III of US-A-4,395 399 and having the following structure:

hoch ₂

GMDP-A

Other preferred compounds of formula II include

N-acetyl-D-glucosaminyl- (β1-4) -N acetylmuramyl-L-alanyl-Lisoglutamine (GMDP-LL) having the structure:

NHAc

GMDP-LL N-acetyl-D-glucosaminyl- (β-4) -N acetylmuramyl n-butyl ester

L-alanyl-D-glutamine (GMDP-OBu) having the structure:

BOY;

NHAc

GMDP-OBu

N-acetyl-D-glucosaminyl- (β-4) -N acetylmuramyl-L-alanyl-Disoglutaminyl-L-lysine (GMDP-Lys) having the structure:

GMDP-Lys

N - [N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-L-alanyl-D-isoglutaminyl] -N ^s -stearoyl-L-lysine (GMDP-Lys (St)) having the structure:

GMDP-Lys (Wed)

Other suitable compounds are:

^And N - [N-Acetyl-D-glucosaminyl- (βΐ-4) -N-acetyl-muramyl-L-alanyl-YD-glutamyl] -N @ ^{-stearoyl-L-S-lysine} (GMDPA-Lys (St)) which has structure:

BOY, BOY ₂

NHAc

CHCO-L-Ala-D-Glu-L-Lys-COC ₇ H _{3 5} 'Na ⁺

AND

CH,

HIM

OH

NHAc

GMDPA-Lys (St) dibenzyl ester N-acetyl-D-glucosaminyl- (βΐ-4) -Nacetylmuramyl-L-alanyl-D-glutamic acid (GMDPA (OBzl) ₂ ) / having the structure:

HOCH ₂

NHAc

GMDPA (OBzl) ₂

N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-N-methyl-L alanyl-D-isoglutamine (Me-GMDP) having the structure:

BOY;

NHAc

Me-GMDP

N-acetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl- (βΐ-4) -Nacetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl-bis- (L-alanyl D-isoglutamine) ((GMDP) ₂ ), which has the structure:

HOCH,, 0

BOY,

NHAc

BOY, BOY ₂

NHAc

CHCO-L-Ala-D-isoGln

AND

CH,

NHAc

CHCO-L-Ala-D-isoGln

AND

CH ₃

HIM

OH

NHAc (GMDP) ₂

N-acetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl- (βΐ -acetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl-bis- (LD-glutamic acid) ((GMDPA) ₂ ), which has the structure:

4) -Nalanyl boy ₂ /

hoch ₂

° \ 0H

NHAc

CHCO-L-Ala-D-Glu

I ch ₃ (GMDPA) ₂

N-acetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl- (βΐ-4) -Nacetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl-bis- (L-alanyl D-isoglutaminyl- L-lysine) ((GMDP Lys) ₂ ) having the structure:

KOCH ₂ / - ° \ boy ₂

NHAo

NHAc

CHCO-L-Ala-D-isoGln-L-Lys

AND

_CH3 (GMDP Lys) ₂

N-acetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl- (βΐ-4) -Nacetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl-bis- [L-alanylD-isoglutaminyl-N ^with -stearoyl-L-lysine] ([GMDP-Lys (St)] ₂ ), having the structure:

BOY,

BOY,

BOY,

HIM

OH

NHAc

BOY,

NHAc \ OH

NHAc

CHCO-L-Ala-D-isoGln-L-Lys-COC ₁₇ H ₃₅ 'Na ⁺

CH ₃

NHAc

CHCO-L-Ala-D-isoGln-L-Lys-COC ₁₇ H ₃₅ 'Na ⁺

_CH3 [GMDP-Lys (St)] ₂

N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP-Ad) 1-adamantylester having the structure:

hoch ₂

NHAc

GMDP-Ad

L-Threonyl-N ^S - [N-acetyl-D-glucosaminyl- (βΐ-4) -Nacetylmuramyl-L-alanyl-γ-D-isoglutaiainyl] -L-lysyl-L-prolyl-Larginine (GMDP-tuftsin Ε), which has the structure:

hoch ₂

Λ

NHAc boy ₂

CHCO-L-Ala-D-isoGln-Lys (Thr) -Pro-Arg

CH ₃

HIM

OH

NHAc

GMDP-Tuftsin E

N-acetyl-D-glucosaminyl- (βΐ-4) -N-acetyl-muramyl-L-alanyl-γD-isogiutaminyl-L-threonyl-L-lysyl-L-prolyl-L-arginine (GMDPtuftsin A) having structure:

GMDP-Tuftsin A

N-acetyl-D-glucosaminyl- (βΐ - 4) -N-acetyl-L-alanyl-D-glutamyl-L-lysyl-L-threonyl-N ^S -stearoyl-L-lysyl-L-prolyl-L-arginine (GMDPA lipořilní -tuftsin), which has the structure:

BOY,

BOY,

HIM

OH

NHAc \ NHAc

CHCO-L-Ala-D-Glu-Thr-Lys (COC ₁₇ H ₃₅ ) -Pro-Arg

CH, lipophilic GMDPA-tuftsin

N- [N-acetyl-D-glucosaminyl- (β-4) -N-acetyl-muramyl-Lalanyl-γ-D-isoglutaminyl] -L-lysyl-L-histidyl-L-glycinamide (GMDPA-bursin) having the structure :

HOCH ₂ /

HOCH, \ NHAc

CHCO-L-Ala-D-isoGln-Lys-His-Gly-NH 2

CH ₃

HIM

OH

NHAc

GMDPA-Bursin

N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-L-alanyl-Disoglutaminyl-L-glutamyl-L-tryptophan (GMDP-thymogen I) having the structure:

₂ ° _X OH ₂

NHAc

CHCO-L-Ala-D-isoGln-Glu-Trp

CH ₃

HIM

OH

NHAc

GMDP-thymogen I

N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-L-alanyl-Disoglutaminyl-s-aminohexanoyl-L-glutamyl-L-tryptophan (GMDPthymogen II) having the structure:

GMDP-thymogen II

^And N - [N-Acetyl-D-glucosaminyl- (βΐ - 4) -N-acetyl-muramyl-L-alanyl-L-alanyl-D-isoglutaminyl] -N @ -stearoyl ^with L-lysyl-Lglutamyl-L-tryptophan ( GMDP-thymogen III), which has the structure of boy ₂ '

HOCH ₂ y NHAc

CHCO-L-Ala-D-isoGln-Lys (COCl ₇ H ₃₅ ) -Glu-Trp

CH ₃

HIM

OH

NHAc

GMDP-thymogen III

N-acetylmuramyl-L-threonyl-D-isoglutamine (THR-MDP) having the structure:

HOCH ₂

CHCO-L-Thr-D-isoGln

AND

CH ₃

N-acetylmuramyl-L-alanyl-D-glutamine thr-MDP n-butyl ester (Murabutid) having the structure:

CHCO-L-Ala-D-Gln-OC ₄ H ₉

I ch ₃

Murabutid

The following abbreviations are used in the above structures:

Bzl - benzyl;

Me-methyl;

Ahx - ε-aminohexanoyl.

The most preferred compound is GMDP followed by GMDP-A and murabutide.

Glucosaminyl muramyl dipeptides within the scope of Formula II can be prepared relatively inexpensively and in sufficient quantity as described in U.S. Pat. No. 4,395,399. The preparation process is based on the extraction and purification of the disaccharide component from Micrococcus lysodecticus and its subsequent chemical treatment. binding to a dipeptide synthesized, for example, by a method conventional in the art of peptide synthesis. The disaccharide can also be equally well synthesized using the standard sugar chemistry method.

The efficacy of the muramyl peptide compounds of the invention was demonstrated using oral administration. The composition of the tablets of the formulation in this case comprises a pharmaceutically acceptable excipient, in particular lactose, starch, polyvidone, magnesium stearate and talc. Muramyl peptide compounds may be tailored to sustained or delayed release requirements. Another option is gastric coating.

The exact oral dosage for administration will always depend on the clinician or attending physician. Accordingly, an acceptable daily oral dose in the range of 0.1 to 50 mg per day (or per dosage unit) may be found, with a preferred dose being 0.5 to 50 mg. An amount of 1 to 20 mg is considered to be the optimal daily dose.

In addition, the duration of administration may vary. Of course, the duration of administration will depend to some extent on the dosage level, i.e., a lower dosage results in a longer required administration period. Generally, the period of administration will be in the range of 1 to 60 days, preferably 1 to 30 days, and particularly preferably 1 to 14 days. In addition, administration may not necessarily be daily: Example 2 herein uses the GMDP administration system three days once daily, three days without GMDP administration, and then for another three days GMDP once daily administration. Other variations, such as administration every other day, may also be used.

Since muramyl peptide compounds are intended for the treatment of ophthalmic conditions, it is also possible to formulate them for topical administration to the eye. Such a formulation will generally comprise a pharmaceutically acceptable excipient and other substances customary in such compositions.

Such formulations are novel, and therefore, in four aspects of the present invention, pharmaceutical compositions comprising muramyl peptide compounds together with a pharmaceutically acceptable excipient or carrier are described; characterized in that the composition is formulated for topical administration to the eye.

When formulated for topical administration to the eye, the composition may comprise one or more muramyl peptides in a pharmaceutically acceptable ophthalmic ointment or cream. Alternatively, the composition may be in the form of drops containing suitable buffers and preservatives known in the art and known to those skilled in the art.

The exact dosage for topical administration will always depend on the clinician or attending physician. Accordingly, an acceptable daily dose for topical compositions ranging from 0.1 to 100 mg per day (or per dosage unit) may be found, with a preferred dose being 0.5 to 50 mg per day (or per dosage unit). . An amount of 1 to 20 mg is considered to be the optimal daily dose.

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

Figure 1: is a graph showing a comparison of the effects of muramyl peptide GMDP and placebo on the resolution of symptoms of ocular herpes infection.

Figure 2: is a graph showing a comparison of the effects of muramyl peptide GMDP and placebo on healing of the epithelium of patients suffering from dendritic keratitis.

Figure 3: is a graph showing a comparison of the effects of muramyl peptide GMDP and placebo in the event of complete resolution of clinical symptoms in patients with herpes stromal keratitis.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Use of GMDP in the treatment of ophthalmic herpes infection

The study was conducted in patients with herpetic ophthalmic diseases. Twenty-five patients aged 16 to 60 years participated in the study. There were 16 men and 9 women in the group (Table 1).

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

Infection in patients with dendritic keratitis with stromal lesions was accompanied by eyelid and conjunctival involvement, corneal epithelial ulcers and branched stromal layers, perifocal stromal infiltration. Symptoms of descemetitis and iritis have been observed in some patients.

Before the start of the tests, the prevalent symptoms in keratouveitis patients were stromal inflammation accompanied by edema, infiltration and inflammation of the ocular vascular envelope. The degree of disease severity ranged from 14 to 21 points.

Clinical values at baseline in the control and GMDP treated patients are summarized in Table 1.

Placebo was used as a control in the study, with double-blind tests and patients receiving either 1 mg GMDP per day or identical placebo tablets for 10 days. All patients who participated in the study also received topical and systemic therapy adapted to the severity and clinical form of the disease.

The following procedures were performed prior to the start of the study: collection of case data; clinical tests; temperature detection; blood sampling for clinical analysis (hemoglobin, total cell count, ESR); taking samples from the conjunctiva of the infected eye for bacteriological analysis.

Eye examinations included visual acuity testing, fluorescein stained eye biomicroscopy, internal eye examination, intraocular pressure determination using a Maklocon tonometer and, if this was not possible due to eye condition, palpation.

Immunological examinations included: collecting swabs from the conjunctiva of the infected eye; collecting tear samples from the infected and uninfected eye and blood samples.

After completing a complete set of clinical and immunological examinations (on the first and second days after admission to the clinic), the patient was initiated and medication started.

During treatment and observation, the health status of patients, their temperature and ocular biomicroscopy after fluorescein staining were examined daily. Every three days, visual acuity was tested and tonometry was performed. Haematological and immunological examinations were repeated 3 to 4 days after the administration of test substances. Bacteriological analysis of the conjunctival sac contents and other additional examinations were performed as indicated.

Estimation of the severity of eye disease

To estimate the severity of ocular disease, clinical symptoms were assessed by a three-point system: severe - 3, moderate - 2, mild -1. Each of the symptoms of the disease was expressed by a point value and the overall intensity of inflammation was determined.

Clinical trials were performed daily for each patient in addition to visual acuity assessments.

Safety and tolerance of substances

The safety and tolerability of the substances was assessed on the basis of clinical trials, which included subjective observation of the patient, control of allergic side reactions, objective data on the condition of internal organs, temperature, haematological results.

Clinical efficacy was determined according to the following criteria:

- the degree of epithelopathy and the time required for total corneal epithelization

- disappearance of symptoms of iritis

- disappearance of stromal infiltrations

- complications during the inflammatory process and their nature

- changes in visual acuity

Results (i) Safety and Tolerance

No subjective problems and side effects attributable to these agents were observed. Generally good health was maintained and no feverish conditions or substantial changes in clinical blood tests were observed.

(ii) Efficacy and activity

Clinical results of patients in the placebo and placebo groups are compared in Table 2. In most parameters, a trend towards faster healing was observed in the GMDP-treated group, especially when the results were combined in the DK and KU. However, these results were not statistically significant. For many parameters, worse results were obtained in the group of patients suffering from KU treated with GMDP. These results appear to be largely influenced by one case that showed a particularly slow improvement in health. There was a much greater improvement in visual acuity in the treatment group compared to the placebo group. This was true of both DK patients and KU patients.

Figures 1 and 2 show the development of clinical parameters over time. Again, there is a consistent trend towards faster healing in the GMDP-treated group.

Conclusion

i) Clinical trials have shown clear benefits of GMDP in the treatment of patients with dendritic herpes keratitis.

(ii) The results of the immunological control did not show any steady trend that could be related to the efficacy of the test substance. However, the disappearance of lymphocyte reactivity to HSV is more evident in the group of patients receiving GMDP. According to the test for the detection of HSV antigen in the conjunctiva of the infected eye, some benefits were also observed in patients receiving GMDP.

(iii) The following were noted: excellent tolerance to GMDP and placebo; the absence of allergic side reactions as well as the absence of changes in general health; temperature and clinical blood values.

Table 1: Main features of the control and GMDP treated groups

Group Control GMDP Total amount 13 12 men 9 7 women 4 5 Age; average (standard) deviation) 42.8 (2.9) 44.3 (3.7) Clinical subgroup Dendritic keratitis 7 6 Keratouvetis 6 6 Time since first HSV infection > 20 years 5 3 <20 years 4 5 first case 4 4 Time of previous remission average (standard deviation) (months) 27.7 (16.7) 68.4 (29.5) range 1 month to 2 months to 13 years 20 years Duration of infectious condition before the test: average (standard deviation) (days) 29.5 (5.2) 23.6 (5.4) Total score on the beginning of the test average (standard deviation) 16.1 (0.9) 15.1 (0.8) Visual acuity at the beginning test average (standard deviation) 0.27 (0, 07) 0.27 (0.06)

Table 2: Overview of results

Group Time Placebo GMDP Time to Total Score 0 (days) total 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 reach 50% healing (days) total 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 reach zero epithelopathies (days) total 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 reach 50% epithelopathy (days) total 7.3 6, 8 DK 5.4 5, 5 KU 9.5 O 00 Time to complete Financial epithelization (days) DK 9.4 (2.9) 5.5 (1.4) Changes in visual acuity (score) total 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) Dissolution of infiltrate (days) total 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) total 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

Use of GMDP in the treatment of herpetic stromal keratitis

A second clinical trial was performed in a manner substantially similar to that described in Example 1. A total of 60 patients suffering from herpetic stromal keratitis were tested. These patients were randomly divided into two groups (2 groups of 30) for the administration of GMDP tablets and identical placebo tablets.

The dose was 20 mg GMDP daily, with the administration system as follows: GMDP or placebo once daily for 3 days, 3 days delay (no GMDP / placebo), GMDP or placebo once daily for 3 days.

In addition, patients received concomitant treatment (antiviral acyclovir, antibiotics, and so on) according to routine practice.

Treatment evaluation was performed by scoring, which monitored the following parameters: stromal affection (superficial, deep, corneal edema, descemetitis), corneal ulcers (point, area), affection of the inside of the eye (precipitates, hypopion, iritis), hyperaemia (conjunctival and pericorneal) .

The results show that GMDP treatment improved the disappearance of most clinical symptoms and accelerated improvement in visual acuity. For example, the mean time to 50% disappearance of stromal involvement was 7.3 days in the placebo group and 5.8 days in the GMDP group. The average time to complete cure was 14.9 days for the first group and 10.6 days for the second group. The time course of complete resolution of symptoms is shown in Figure 3. Analysis of the extent of symptom resolution at subsequent time points (chisquared test) shows that between 8 and 14 days inclusive, significantly (p <0.05) more patients receiving GMDP show symptom resolution as compared to with a group of patients receiving placebo. Visual acuity in the GMDP-treated group averaged 0.60 over 10 days, while in the placebo-treated group only 0.49.

Claims (34)

PATENT CLAIMS ..... ~ L · Use of a muramyl peptide compound for the preparation of a composition for the treatment of ophthalmic diseases or conditions. Use of a muramyl peptide compound for the preparation of a composition for treating or preventing corneal scarring associated with an ophthalmic disease or condition. 3. A product comprising muramyldipeptide and a second drug substance as a combined preparation for simultaneous, separate or sequential use in the treatment of an ophthalmic disease or condition. The product of claim 3, wherein the second therapeutic agent is an antiviral agent, an antibacterial agent, a steroidal or non-steroidal anti-inflammatory drug. Product according to claim 4, characterized in that the second drug substance is acyclovir, idoxuridine, chloramphenicol, chlortetracycline, fusidic acid, gentamycin, neomycin, oflaxacin, polymixin, tetracycline, tobramycin, betamethasone, clobetasone, dexethethonone, hydrocethethone, fluorotethone, fluorisethonone, hydrocethethone, fluorotetone, fluorotetone, hydrocetone, or flurbiprofen. Product according to claim 5, characterized in that the second drug substance is acyclovir or idoxuridine. The use according to claim 1 or claim 2 or the product according to any one of claims 3 to 6, wherein the ophthalmic disease or condition is conjunctivitis, corneal disease such as keratitis, eyelid disease, eye dryness or eye damage. The use or product of claim 7, wherein the condition is herpetic stromal keratitis. Use or product according to any one of claims 1 to 8, wherein the muramyl peptide compound is a compound of formula I: where: R ¹ represents a hydrogen atom or an acyl group containing 1 to 22 carbon atoms; R ² is hydrogen or acyl containing 1 to 22 carbon atoms; R ³ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ⁴ represents an alkyl group having 1 to 21 carbon atoms or an aryl group having 6 or 10 carbon atoms; R represents a hydrogen atom; and R represents an amino acid residue or a linear peptide consisting of 2 to 6 amino acid residues, wherein at least one of the residues is optionally substituted with a lipophilic group. The use or product of claim 9, wherein the compound of formula I comprises some or all or any compatible combination of the following substituents: each of R ¹ and R ² represents acyl groups containing 1 to 5 carbon atoms such as acetyl; R ³ represents an alkyl group having 1 to 4 carbon atoms such as methyl and ethyl; R ⁴ represents an alkyl group having 1 to 6 carbon atoms, preferably an alkyl group having 1 to 4 carbon atoms, such as methyl or ethyl, or a phenyl group; R is a mono-, di- or tripeptide. Use or product according to any one of claims 1 to 10, wherein the muramyl peptide compound is: muroctasin, otherwise known as MDP-Lys (L18) (N ² - (Nacetylmuramyl-L-alanyl-D-isoglutaminyl) -N ⁶ -stearoyl-L-lysine); MTP-PE (N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanyl-2 (1 ', 2'-dipalmitoyl-sn-glycero-3'-hydroxyphosphoryloxy) monosodium ethylamide); MDP-OBu (N-acetylmuramyl-L-alanyl-D-isoglutamine-n-butyl ester) murabutide (N-acetylmuramyl-L-alanyl-D-glutamine-α-N-butyl ester); or t-MDP (N-acetylmuramyl-L-threonyl-D-isoglutamine). Use or product according to any one of claims 1 to 8, wherein the muramyl peptide compound corresponds to the general formula II: H HOCH ₂ NHAc OH where: II R represents an amino acid residue or a linear peptide of 2 to 6 amino acid residues, wherein at least one of the residues is optionally substituted with a lipophilic group and n is 1 or 2. The use or product of claim 12, wherein n is 1 The use or product of claim 12 or 13, wherein the proximal amino acid residue is an amino acid residue in the L-configuration. The use or product of claim 14, wherein the proximal amino acid residue or the single amino acid residue, if only one is present, is an L-alanine residue. The use or product of any one of claims 12 to 15, wherein the second amino acid residue from the proximal end of the peptide, if present, is in the D-configuration. Use or product according to claim 16, wherein said second amino acid residue is D-glutamic acid or Dasparic acid or a mono-, di- or mixed alkyl ester thereof containing 1 to 22 carbon atoms, preferably 1 to 6 carbon atoms, an amide or an alkyl amide containing 1 to 6 carbon atoms. The use or product of claim 15, 16 or 17, wherein said second amino acid residue is D-isoglutaminyl or D-glutamyl. The use or product of 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. The use or product of claim 19, wherein the third amino acid residue is L-alnanyl or L-lysyl. The use or product of any one of claims 12 to 20, wherein the amino acid residue or linear peptide is optionally substituted with at least one lipophilic moiety. The use or product of claim 12, wherein the muramyl peptide compound is N-acetyl-D-glucosaminyl-Nacetylmuramyl-L-alanyl-D-isoglutamine (GMDP). The use or product of claim 12, wherein the compound is: N-acetyl-D-glucosaminyl-N-acetylmuramyl-L-alanyl-D-glutamic acid (GMDP-A); N-acetyl-D-glucosaminyl- (β1-4) -N-acetylmuramyl-L-alanyl-D-glutamine n-butyl ester (GMDP-OBu); ^And N - [N-Acetyl-D-glucosaminyl- (βΐ-4) -N-acetyl-L-alanyl-D-isoglutaminyl] -N ^with -stearoyl-L-lysine (GMDP-Lys (St)); ^And N - [N-Acetyl-D-glucosaminyl- (βΐ-4) -N-acetyl-muramyl-L-alanyl-YD-glutamyl] -N ^with -stearoyl-L-lysine (GMDPA-Lys (St)); N-acetyl-D-glucosaminyl- (β-4) -Nacetylmuramyl-L-alanyl-D-glutamic acid dibenzyl ester (GMDPA (OBzl) ₂ ); N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-N-methyl-Lalanyl-D-isoglutamine (Me-GMDP); N-acetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl- (βΐ-4) -Nacetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl-bis- (L-alanylD-isoglutamine) ( (GMDP) ₂ ); N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl- (β-4) -Nacetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-bis- (L-alanyl-D-glutamic acid) ((GMDPA) ₂ ); N-acetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl- (βΐ-4) -Nacetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl-bis- (L-alanylD-isoglutaminyl-L -lysine) ((GMDP Lys) ₂ ); N-acetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl- (βΐ-4) -Nacetyl-D-glucosaminyl- (βΐ-4) -N-acetylmuramyl-bis- [L-alanylD-isoglutaminyl-N ^S- stearoyl-L-lysine] ([GMDP-Lys (St)] ₂ ); N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-L-alanyl-D-isoglutamine 1-adamantyl ester (GMDP-Ad); L-threonyl-N ^e - [N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-L-alanyl-γ-D-isoglutaminyl] -L-lysyl-L-prolyl-Larginine (GMDP-tufftsin E); N-acetyl-D-glucosaminyl- (β-4) -N-acetyl-muramyl-L-alanyl-γD-isoglutaminyl-L-threonyl-L-lysyl-L-prolyl-L-arginine (GMDPtuftsin A); N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-L-alanyl-α-D glutamyl-L-lysyl-L-threonyl-N ^with -stearoyl-L-lysyl-L-prolyl-Larginine ( lipophilic GMDPA-tuftsin); ^With N - [N-Acetyl-D-glucosaminyl- (βΐ-4) -N-acetyl-muramyl-L-alanyl-YD-isoglutaminyl] -L-lysyl-L-histidyl-L-glycine amide (GMDPA-Bursin); N-acetyl-D-glucosaminyl- (β-4) -N-acetylmuramyl-L-alanyl-Disoglutaminyl-L-glutamyl-L-tryptophan (GMDP-thymogen I); N-acetyl-D-glucosaminyl- (β1-4) -N-acetylmuramyl-L-alanyl-Disoglutamines Ι-ε-aminohexanoyl-L-glutamyl-L-tryptophan (GMDPthymogen II); N - [N-acetyl-D-glucosaminyl- (β-4) -N-acetyl-muramyl-Lg alanyl-L-alanyl-D-isoglutaminyl] -N-stearoyl-L-lysyl-Lglutamyl-L-tryptophan (GMDP) -thymogen III); N-acetylmuramyl-L-threonyl-D-isoglutamine (Thr-MDP); or N-acetylmuramyl-L-alanyl-D-glutamine n-butyl ester (Murabutid). 24. A method of treating ophthalmic conditions 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 an ophthalmic disease, comprising administering an effective amount of a muramyl peptide compound to a patient suffering from ophthalmic disease. 26. A method of treating an ophthalmic condition 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 therapeutic agent. The method of any one of claims 24 to 26, wherein the muramyl peptide compound administered is GMDP. 28. The method of claim 27, wherein 0.1 to 50 mg of GMDP is administered daily. 29. The method of claim 28 wherein 0.5 to 50 mg of GMDP is administered daily. The method of claim 29, wherein 1 to 20 mg of GMDP is administered daily. Method according to any one of claims 24 to 30, characterized in that the period of administration is 1 to 60 days The method of claim 31, wherein the administration period is from 1 to 30 days. The method of claim 32, wherein the administration period is 1 to 14 days. The method of any one of claims 24 to 30, wherein the compound is administered as follows: i) three days of administration ii) three days without administration iii) final three days of administration.