CA2194678A1 - Use of muramyl peptide compounds - Google Patents

Abstract

Muramyl peptide compounds, particularly N-acetyl-D-glucosaminyl-(.beta.1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP), are useful in the treatment or prophylaxis of inflammatory dermatological conditions such as psoriasis and in the treatment or prophylaxis of immune-related diseases of the skin and mucous membranes.

Description

21 9~67~

USE OF MURAMYL PEPTIDE COMPOUNDS _ =

The present invention relates to the treatment of inflammatory dermatological conditions and in particular to the treatment of psoriasis.

Psoriasis is a non-infective, usually chronic ;nfl. -tory skin disease occurring in about 2~ of the population. It has a number of clinical manifestations, the most common of which is raised, red, rongh~n~
plaques covered by silvery scales. The nails are involved in about 50~ of cases, and arthritic involvement may occur. The ~t~rnAl changes in the skin are associated with histological changes in the disposition of the epidermis, and vascularisation of sub-epidermal tissues. Infiltration of lymphocytes commonly occurs.
The occurrence of psoriasis results from a combination of envi,l ~1 and genetic factors. Numerous studies have demonstrated a range of changes in functionality of the epidermis and immune system of psoriatics, but no comprehensive theory yet exists which can accounts for all the observed abnormalities.

Current therapy for psoriasis can be topical (;n~ln~ing steroids, coal tar, dithranol, vitamin ~3 derivatives) oral (including retinoids, methotrexate, cyclosporine) or phototherapy (W13 alone, or W A with psoralen).
Unfortunately, the more effective preparations (eg.
steroids, methotrexate, cyclosporine) have the potential to cause severe side effects. The less potent, palliative preparations, which tend to be topical, although being less dangerous, are frequently messy and inconvenient to use.

.

It is therefore evident that an effective, safe, oral treatment for psoriasis i9 a very desirable medication and would have extreme utility.

It has now been discovered that various muramyl peptide derivatives are useful in the treatment of psoriasis.

It has long been known that non-specific 8t; l~tinn of the immune system can be brought about by exposure to bacteria, ~or components extracted from bacterial cell walls. The specific .~ _ ~ntc responsible for this activity were identified as sugar-cnnt~;n;ng peptides of the cell wall, and further biochemical analysis of the peptide identified then as the peptidoglycan ~ ~nt of the cell wall. The smallest effective synthetic molecule was found to be an N-acetylmuramyl-~-alanyl-D-isoglutamine (Merser et al, Biochem. Piophys. Pes. Comm.
66 1316 (1975)). The ability of this ~ ~ ' lnow fre~uently referred to as ~prototype muramyl dipeptide"
or "prototype MDP") to protect mice against bacterial infection_ (~lebsiella pn~n~nni~) has been described (Chedid et al, Proc. Nat'l. Acad. Sci. USA, 74 2089 (1977)).

Subseauently, a wide variety of analogues of prototype muramyl dipeptide were synthesised, some of which have been proposed as treatments for the restnr~t;nn of immune function or the non-specific st;~nlst;nn of the immune system. These analogues, and prototype MDP itself, are muramyl peptide compounds.

Various authors have dealt with the use of muramyl peptides and analogues thereof as adjuvants, including Azuma et al (Adv. Exp. Med. i3iol. 319, 253-263 tl992)), 2 1~ ~ 6 7 8 r, ~ ~1619 .

who teach that MDPs are effective for use as 1 n~juvants for the potentiation of antiyenicity 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 ~mlllcinn elicits cell-mediated immunity and ~nt;ho~;es Of protective isotypes and ~nr,~~ntq 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 (~ol. 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.

Muramyl peptides have also been proposed for use as anti viral agents (Ikeda et al, Anti~iral ~es 5:207-15 (1985)) and in the treatment of cancers (Phillips NC and Tsao, M-S, Cancer Immunol Immunother. 33:85-90 (1991)).

In the t~srh;nr of these documents, little is disclosed cnnrr~n;ng the use of muramyl peptide ~ , ~c for diseases of the skin, and none of these have involved psoriasis or other ; nfl; tory conditions. Most skin applications have involved the potential anti-cancer benefits of this class of compound. For example, Talmadge, JE et al ~Cancer Res 46:1160-3 (1986)) found ~, that tke muramyl pepeide analcsue ~TP-?E ~as a~le to retard -he grcw~:l of pri~ary skin cancers. In anos..er study (Gree nals;. D and Gamelli, RL, Jourual of ~rauma 27:5;0-L !lga7)), the use of .~DP was ~YA~in~ n wound healing. Howel~er, in this case neither br~triA7 nor deleterious ef-ects ~ere seen. Elsewhere, the local injection of ~DP with an antigen has been shown to enhance the subsequent cutaneous ~delayed type hypersensitivit~'' response to that antigen (Tsu~imoto, M
et al, Mic-obiol l~unol. 23:933-936 (1979)).

US-A-4357322 tiCrlos~ the use of various muramyl and desmethylmuramyl dipeptides in treating ;nfl tt~n. ~0 . mention, however. is made o_ GMDP or psoriasis.
Azneimittel Forschung Drug Research, 38, 7A: 1002-lO09 (1988) provides a review of the phArmArologica properties of Muroctasin.

EP-A-0406175 discloses a compound, 3-0-[N-acetyl-muramyl -L-threonyl-D-isoglutaminyl~-1,2-di-0-palmitoyl-sn-glycerol, which is disclosed as having immune-~nhAnr~ng actlvity.

DE-A-3129759 discloses the use o~ certain muramyl peptide compounds as anti-inflammatory agents.

~O-A-93/10148 discloses the use o~ muramyl peptide cu~yuunds in the treatment of septic shock.

- In a fi5sr aspec of the present invention there is provided the use of a muramyl peptlde ~ ~ ' in the prepara~ion of an agen~ for the treatment or prophylaxis of an ; nf~ ~ory dermatoiogica~ condition.

~MENDEDSHEET

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _, _ _ _ _ 4a Many muramyl pep~ide compounds useful in this invention fall within general formula I:

R lo CH~

F-' ~OR~
\ NHCOR~
P~CHCOR

wherein:
R' represents a hydrogen atom or a C,-C~ acyl group;

~MEhDED SHEET

WO96/01645 2 ~ 9 4 6 7 8 PCT/GB9S/01619 .

R2 represents a hydrogen atom or a Cl-C2l acyl group;
R3 represents a hydrogen atom or a Cl-C6 alkyl group;
R4 represents a Cl-C2l alkyl group or a Cs or C,~ aryl group;
Rs 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;
other than prototype muramyl dipeptide and desmethylmuramyl dipeptide.

Preferred acyl groups for Rl and R2 are Cl-Cs 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 Cl-C~ alkyl groups such as methyl and ethyl. Preferred alkyl groups for R~ and Cl-C6 alkyl groups, particularly Cl-Cc 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-~-aminobutyryl L-histidyl L-seryl L-glutamyl L-threonyl L-glutaminyl WO96/016~5 2 1 ~ 4 6 78 PCT/GB9~/01619 L-methionyl L-aspartyl L-cysteinyl L-asparaginyl L-phenylalanyl L-prolyl L-tyrosyl L-hydroxyprolyl L-alanyl is preferred, as is D-threonyl.

The next amino acid from the proximal end of the peptide is preferably of the D-configuration~ It is preferably acidic a~a may be D-glutamic or D-aspartic acid or a mono-, di- or mixed C1-C22 (preferably C,-Cs) alkyl ester, amide or -C1-C~ alkyl amide thereof. ~The expression ~'mixed" is illustrated when one carboxyl group is amidated and the other esterified. D-isoglutamine and D-gl~1t~m~te ~are preferred.

A third amino acid -esidue 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 li porh; l; c group. The lipophilic group may be a C10-C22 acyl group such as stearoyl or a di-(C1o-Cz2 acyl)-sn-glycero-3~-hydroxy-phn5phPryloxy-group wherein for example each of the C10-C22 acyl groups can be a palmitoyl group. The l; rorh; l; c group may alternatively (or in addition, as more than one substitution may be present) be a C1-C10 ester group, such as a C,-C6ièster group: a butyl ester is an example.

Examples of muramyl dipeptides within the scope of general formula I include:

WO9610164S 2 1 9 4 6 7 ~ P~ 3i l6~9 muroctasin, otherwise known as MDP-Lys (L18) IN2-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N6-stearoyl-L-lysine);

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

murabutide (N-acetylmuramyl-L-alanyl-D-glutamine-~-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 i8 disclosed in EP-A-0025495. The preparation of murabutide i8 described in ~,~frAnrler et al, ~. 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 s ,,~ ds 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 ~r~ ' C 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:

WO 96/01645 2 1 9 4 6 7 8 ~ c cl6l9 ~OCH~ --~ ~ ~ OH

1~ \NHAc ~ ~¦ H3CCHCOR
H ~
~HAc n 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 residueg being opt;nn~lly substituted with a l;rnrhil;c group; and n is l or 2.

Preferred values for R are as described above in relation to general formula I. It is particularly yLefeLL~d that the peptide R correspond to the peptide in prototype MDP
~L-Ala-D-isoGln). Alternatively, in another yLefe '_'; , R may Leylese.-L h-Ala-D-Glu.
The preferred value for n is 1.

~c __ '~ of general formula II are ~; ~rl o~r~ in US-A-4395399 and the preferences set out in that c'~ are equally preferred in the present invention.
Additionally, in this invention, the group R may be substituted l irorh; l; cally as described above.

One of the most preferred I _ ~ for use in the W096/01645 2 1 9~678 F~~ .'Cl619 present invention falls within general formula II and i8 N-acetyl-D-gl ucor--; nyl - (~1-4)-N-acetylmuramyl-L-alanyl-D-;co~ cm;n~ (GMDP), the structure of which is:
HG Ch-~ ~

HOCH_ ~ ! \!lHAc ~ sl ,HC0-L-Alo-D-isoGln 11~ 'H_ ~J HAc 1 0 Gr~mP

This ~ ol1n~ II in US-A-4395399), also known as glycopin, has already nn~rcJnn~ pre~l ;n;r~l toxicity testing and phcrr-nok;n~;c investigations required for licensing for ~l;n;r~l use in the USSR (as it then was).
The acute toxicity in mice, measured by the LDso test is 7 g/kg. This figure shows the I ~_ ' to be almost an order of magnitude less toxic than muroctasin which has an LDso value in mice of 625 mg/kg.

While the pyrogenicity of GMDP is sllff;~;~ntly low to make it suitable for use in the present invention, and not to have prevented its rl ;n;~l eV~ln~tinn for other purposes, it may in some circumstances be preferable to use an even less pyrogenic AnllOcJu~ Such an ~nclo~l~ is available, and is N-acetyl-D-glll~ nyl- (~1-4)-N-acetylmuramyl-L-alinyl-D-glutamic acid ~GMDP-A), which is ~ ~ ' III in ~S-A-4395399, and whose structure is as follows:
.

WO96101645 2 ~ 9 4 6 7 8 PCT1GB95101619 HCi CH~
~' ' jri \ . IHAc H~ ,HCO- L--Al o--[)--Glu NHAc GMDE -A

Other preferred _ '- within the scope of general formula II include:

N-acetyl-D-gl~ R~minyl-(~l-4)-N acetylmuramyl-L-alanyl-L-isoglut~m;n~ (GMDP-LL) which has the structure:

,~OOH

HOClH2 o l HO~ CHCO--L--Alo-L-i~oGln NHAc GMDP - LI, N-acetyl-D-gl 7l~R~m; nyl - (~l-4)-N acetylmuramyl-L-alanyl-D-gl ut~m; n~ n-~utyl ester (GMDP-OBu) which has the ~LLU~;LULI:::

WO 96101645 2 i 9 4 6 7 8 ~ 619 HO CH~
k~OOH
\W

~ \ CHCO--L--Ala-D--Gln--OElu HO~ CH3 NHAc GMDP - OBu 1~
N-acetyl-D-glucosaminyl-(~1-4)-N acetylmuramyl-L-alanyl-D-isoglutaminyl-L-lysine (GMDP-Lys) which has the structure:

HOCH~

o~HOAc ~-- I CHCO--L--Alo--D-isoGln-L--Lys HO~ CH3 NHAc GMDP - Ly~

2~ D~-[N-acetyl-D-glucosaminyl-(~1-4)-N-acetylmuramyl-L-alanyl-D-isoglutaminyl]-N'-stearoyl-L-lysine (GMDP-Lys(St)) which has the structure:

WO 96101645 2 1 9 ~ 6 7 ~ r~ 6l9 ~OOH
~$~
HOCH O \NHAc HO~ CHCO-L-Alc-D-isoGln-L-Lys-COC1 7H35 Na+
NHAc GMDP-Lys ~St~

Other useful compounds include:

N~-[N-Acetyl-D-glucosaminyl-(~1--4)-N-acetyl-muramyl-L-alanyl-~-D-glutamyl]-N'-stearoyl-L-lysine which has the structure:

HO CH., ~OOH
\S~

HOCH2 ( ) ~NHAc Ho$~ CHCO-L--Ac--C--Gll--L-Ly~--COC~7H35 NHAc GMDPA-Lys(St) N-Acetyl-D-glncnc~;nyl-(~1--4)-N-acetylmuramyl-L-alanyl-D-glutamic acid dibenzyl ester which has the structure:

WO96/0164~i 2 1 9 4 6 7 8 PCT/GB9Y01619 .

HOCll2 ~OOH

)~HA

HO~ CHCO--L--Alc--û--Clu--(OB11)2 1~ HAc GMDPA~OBzl)z N-Acetyl-D-glurmr~m~nyl-(~1--4)-N-acetylmuramyl-N-methyl-~-alanyl-D-isoglutamine which has the structure:

~OOH

HOCH 0 \NHAc Ho$~ CHC0-MeAI~I-D-isoGln NHAc Me-~MDP
N-Acetyl-D-glucosaminyl-(~1--4)-N-acetylmuramyl-(~1--4)-N-acetyl-D-glucosaminyl-(~1--4)-N-acetylmuramyl-bis-(~-alanyl-D-isoglutamine) which has the structure:

WO96/01645 2 1 ~ 4 6 7 8 PCT/GB9S/01619 .

~OOH

~NHAc ~~¦ CHCO--L--Alo-D--isoCln ~ CH3 HOCH2 ~ NHAc HOCH ~ \
r~ I CHCO--L-Ac-D-isoGln HO~ CH3 N HAc (GMDP) 2 N-Acetyl-D-gls~.q~mi nyl- (~1- -4)-X-acetylmuramyl-(~ 4)-N-acetyl-D-glucosaminyl-(~1--4)-N-acetylmuramyl-bis-(L-alanyl-D-glutamic acid) which has the structure:

,~OOH
~5~
HOCH2 ( ) ~NHAc ,~o\l CHCO-L-~Ja-D-Glu =~ ~ ~ CH3 O NHAc HO~ ~
1~
HOCH ~ \NHAc ~0 ¦ CHCO--L--Alo--D--Glu HO~ CH3 NHAc (GMDPA), WO961016~5 2 1 9 4 6 7 8 . ~I~Dg il~l6l9 N-Acetyl-D-gl~l~ns~m;~yl-(~1--4)-N-acetylmuramyl-(~1--4)-N-acetyl-D-glucosaminyl-(~~1--4)-N-acetylmuramyl-bis-(L-alanyl-D-isoglutaminyl-L-lysine) which has the structure:

~OH

HO~ \CHCO L Alo-D-lsoGln-L-Lys HOCH2 ~ NHAc HOC~ O \NHAc HO~ CHCO-L-Alo--D-isoGln-L-LYs NHAc (GMDP Lys) 2 N-acetyl-D-glucosaminyl-(~1--4)-N-acetylmuramyl-(p~1--4)-N-acetyl-D-gln~s~mlnyl-(~1--4)-N-acetylmuramyl-bis-[L
alanyl-D-isoglutaminyl-N~-stearoyl-L-lysine~:

/~ CHCO-----Alo--D--isoGln L LycCOC17 j5 HOCH

3 0 Ho CH2 Ho~ CLICO-L-AIo--D--~soGln--L--4sCOC17H~5 Nc NLAc [GMDP-Lys(St)] 2 WO96101645 2~ 9~678 ~ 5cl6l9 N-Acetyl-D-glucosaminyl-(~1--4)-N-acetylmuramyl-L
alanyl-D-isoglutamine l-adamantyl ester which has the structure-~OOH

HO CH O \NHAc Ho$~ CHCO--L--Alo--D-isoGln--OAd N HAc GMDP-Ad L-Threonyl-N'-[N-Acetyl-D-glllrrs~m1nyl-(~ 4)-N-acetyl-muramyl-L-alanyl-~-D-isoglutaminyl]-L-lysyl-L-prolyl-L-arginine ~hich has the structure:

HO CH,7 ,~OOH
\~O~
2'i I \ ~
HOCH ~ ) \NHAc H~ CHCO-L--Alo--D-isoGln-Lys(rnr)--Pro-Arg NHAc GMDP-tuftsin E

N-Acetyl-D-glucosaminyl-(~1--4)-N-acetyl-muramyl-L-alanyl-~-D-isoglutaminyl-L-threonyl-L-lysyl-~-prolyl-L-WO96101645 21 94678 r~ J~ 1619 .

arginine which has the structure:

5 ~O~ H

HO CH O \NHAC
~ ~I CHCO-L-Ala-D--isoGln-Thr-Lys-Pro-Arq HO~ CH3 NHAc GMDP-tuftsin A

N-Acetyl-D-gl u~r C,~m; nyl- ~1- -4~-N-acetylmuramyl-L-alanyl-~-D-glutamyl-L-lysyl-L-threonyl-N'-stearoyl-L-lysyl-L-prolyl-L-arginine which has the structure:

~OOH
1'~
HOCH o \NHAC
H0~ cHco-L-Ala-D-Glu-Thr-Lys(cocl7H3s)-pro-Arr NHAC

GMDPA-tuftsin lipophilic N'-[N-Acetyl-D-glllrr~a~minyl-(~1--4)-N-acetyl-muramyl-L-alanyl-v-D-isoglutaminyl]-L-lysyl-L-histidyl-L-glycine amide which has the structure:

WO 96101645 2 1 9 ~ 6 7 8 P~ JI S3 ~I619 ~OOH

\ NHAc HOCH2 ( ) ./~~ CHCO-L-AlrJ-D-isoGln-Lys-His-Gly-NH2 Ht~ CH3 N HAc GMDPA-bursin N-Acetyl-D-gll-nns~m;nyl-(~1--4)-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-glutamyl-L-try~tophan which has the structure:

~OOH

HOCH o ~NHAc ~ ~¦ CHC0-L-AkJ-D-isoGIn-GIu-Trp HO~ CH3 NHAc GMDP-thymogen I

N-Acetyl-D-gl--rn.sr~minyl-(~1--4)-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-~-aminohexanoyl-L-glutamyl-L-tryptophan which has the structure:

WO96/016~5 219 4 67~ r .~ 1619 ,k~OOH

~ ~¦ CHC0-L-Alo-D--isoGln-Ahx--Glu--Trp H0~ CH~
NHAc GMDP-thymogen II

N~-[N-Acetyl-D-glucosaminyl-(~1--4)-N-acetyl-muramyl-L-alanyl-D-isoglutaminyl]-N'-stearoyl-L-lysyl-L-glutamyl-L-tryptophan which has the structure:

~OOH

HO CH ~ \NHAC
HO~ CHCO--L--Alo--D--icoGln--Lyc(COC1 ~H35)--Glu--rSp N HAc 2 5 GMDP-thymogen III

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

WO96101645 2 I q~78 r~ 1619 ~O OH

OH~NHAc CHCO- L--~hr-D -isoGln Thr-MDP

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

,~OOH
~5~
bH~NHAc CHCO-L--Ala-D-Gln--OC, Hg Murabutide In the above structures, the following abbreviations are used:

Bzl - benzyl;
Me - methyl;
Ahx - ~-Am;n~An~yl The most preferred compound ~s GMJP followed by GMDP-A, and murrbutide.

,ilu-m,-~minyl-muramyl dipeptides within the scope of general formula II can be prepared relatively cheaply and in reasonably large quantities by the process disclosed in ~S-A-4395399. The preparation disclosed is based on the extraction and purification of the ~;c~c-hcride -o~nPnt from the bacterium Micrococcu- 7ysodecticus and its subsequent chemical linkage to a dipeptide 8ynthGc; CQ~ for example by conventional peptide chemistry. ~owever, the disaccharide may equally well be chemically synthesised using standard sugar chemistry.

Tnfl. tory dermatological conditions treatable, or preventable, by means of the invention include all types of psoriasis, including discoid or plaque psoriasis, flexural psoriasis, scalp psoriasis, palmar/plantar psoriasis, guttate psoriasis, erythrodermic psoriasis and pustular psoriasis, psoriatic arthritis and changes in the nails resulting from psoriasis.

The effect of GMDP in psoriasis has been studied in clinical trials, which are described in more detail in Example l below. The PASI (psoriasis area and severity index) of treated individuals tended to fall during treatment, and a number of individuals even showed complete clearing of lesions. Articular symptoms of psoriatic arthritis were also improved by GMDP.
Individuals receiving placebo tablets showed minimal ~ LOV. ', some worsening or no change.

As with many treatments for psoriasis, the exact ~ -h~ni~m by which compounds use~ul in the invention WO96101645 2 t 9 4 6 7 8 PCT1GB95101619 causes iL~ Lt is not known. In fact, lt is extremely surprising that muramyl peptide compounds should have this b~n~ effect as muramyl peptides are generally considered to be pro-inflammatory ; nstimulants, whereas regression of ;n~ d lesions would generally be considered as re~uiring anti-inflammatory treatment. It has even been described in the literature that treatment with interferon, another "immunostimulator" can in fact ~c~rha~e psoriasis (Funk J; Langeland T; Schrumpf E; Hanssen LE Br Dermatolo~y 12S: 463-5 (l99l), Pauluzzi P, Kokelj, F, Perkan, V, Pozzato, ~, Moretti, M. Acta Dermatol Venereol 73:395 (1993)).

Some experimental studies have been performed which provide insight into the possible m~rh~n; ~m of action of muramyl peptide compounds, particularly GMDP, ~n the skin, and also address the potential scope cf the invention. However, the effectiveness of the invention is not a~fected by the relevance of these experiments or the accuracy of their interpretation.

Thus, as is described in detail in Example 2 below, the muramyl peptide compound GMDP was applied to the skin of mice in solution in ethanol. At control sites, ethanol alone was applied. Some groups of animals received irradiatlon with ultra violet light (UVB) or received an application of cis urocanic acid (cUCA), both treatments known to provoke changes in the cutaneous immune system.
Like UVL3 and cUCA, GMDP treatment caused a loss of ATPase staining and retraction of dendritic processes of the epidermal dendritic cells, which is generally associated with a loss of immunological function. i.e., the GMDP

WO96101645 2 ~ 9 4 6 ?~ r~ ,5,~1619 .

reduced the potential i ~activity of the skin. In this respect, it is observed that W 3 phototherapy alone is a treatment for psoriasis, thereby supporting the efficacy of muramyl peptide compounds in the invention.

In other experiments, it was determined that unlike ~3, GMDP does not cause migration of dendritic cells from the epidermis to draining lymph nodes.

From these experiments it can be ~n~ P~ that GMDP i8 having a fnn~ t~l effect on dendritic Langerhans cells, which are the primary immune component of the epidermis. Since there is evidence to support a role for S
Langerhans cells in the pathogenesis of psoriasis (Placek et al, Acta Derm Venereol (Stockh) 68:369-77 (1988)), it is possible that a down-regulation of the activity of these cells is able to reduce the severity of the disease. The experiments with the mice were performed by topical application of GMDP, but GMDP is kno~n to be orally bioavailable, and so GMDP orally administered, as in the clinical trial, could be expected to exert an effect in the skin.

3ecause of the demonstrated effect on the ~ut~nPovq immune system on the one hand and an immune system-related human skin disease (psoriasis) on the other, it can be expected that muramyl peptides will have utility in the treatment of a range of immunologically based ;nfl. tory skin diseases for which it has never previously been proposed. Moreover, as the mucous membranes lining the buccal cavity, the vagina and the uterine cervix also contain T,~ngPrh~nq cells, these too are target organs for muramyl peptide treatment.

WO96/01645 2 t 9 4 6 7 8 PCT/GB95/01619 In the second aspect of the present invention there is provided the use of muramyl peptide compound in the preparation of an agent for the treatment or prophylaxis of immun-e:-related diseases of the skin and mucous S membranes. These include, but are not restricted to, endogenous eczema (otherwise known as atopic eczema or atopic ~ermatitis), seborrhoeic eczema, pompholyx, contact dermatitis, urticaria, erythroderma, lichen planus, vitiligo and alopoecia areata.

The efficacy of muramyl peptide ~ , ~c in the invention has been demonstrated using oral administ~a~ion. The formnl~;nn in this instance consisted of tablets ~nnt~in;ng 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 tha~ deemed suitable by the clinician or physician.
Subject to that, a daily oral dosage in the range of from O.l to ~D0 mg per day (or per unit dose) may be found acceptable, with a range of 0.5 to 50 mg being preferred.
Within this preferred range, an optimal daily doeage would be within the range 2 to 3Q mg or indeed 2 to 20 mg.

In addition, the duration of ~m;n;qtnation 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, prefera'oly 1-30 days and most preferably 1-14 days.

W096101645 2 1 9 4 6 7 8 A ~ 1~ ~, ~1619 .

It has been demonstrated that muramyl peptide compounds do have an affect on the cutaneous immune system after topical administration. Thus, despite the advantages inherent in an oral dosage form, it may be ~tPrminod that for certain specific instances a topical formulation of muramyl peptide compound(s) will be preferred, when for example functioning of thé gastrointestinal tract of a patient is compromised by disease or surgery, or in the case of particularly recalcitrant skin diseases where a particularly high local concentration of muramyl peptide compound is desired.

According to a third aspect of the invention, there is provided a topical formulation of a muramyl peptide compound.

The formulation, which may be presented as an o; n ~, lotion or cream, may contain phar~cont~A7ly acceptable excipients or carriers, with due regard being taken of the ability of the formulation effectively to release the muramyl peptide ir.to the skin. The formulation may even enhance the passage of said muramyl uu,..~uu~ds by the incorporation of so called pe~ ti~n onhAn~or~, Muramyl peptide compounds may be used either singly or in combination with each other in the invention. Also, muramyl peptide compounds may be used in combination with other ~omro-ln~c~ whether formulated together or separately; for example, a muramyl peptide c ~ ' may be administered orally and another _ ~ administered topically. When ~-sed in combination, either with each other or with other compounds, administration can be simultaneous, separate or se~uential.

In a final aspect, the present invention provides a method for the prophylaxis or treatment of an Wo96tO1645 2 ~ 9 ~ ~ 7 g PCT/GB9~01619 in~1 tory dermatological condition, comprising administering to a patient a muramyl peptide compound.

Preferred features of each aspec~ of the invention are as for each other aspect, mutatis mutandis.

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

FIGURE l: is a plot showing changes in psoriasis severity and area index (PASI) during the course of treatment with GMDP or placebo.

EXA~P~E 1 The use of GMDP i~ the treatment of psoriasis.

A Open label trial A preliminary trial in the use of GMDP for psoriasis was conducted in 8 patients who received GMDP in doses of lO
or 20 mg for a period of 9 to 18 days. In general, effective reduction of the cutaneous symptoms of psoriasis was observed. One of the patients had the variant of psoriasis known as psoriatic arthritis, and this patient observed a reduction in ~oint paln and morning s~iffness of joints.

s Placebo controlled trial MET~ODS
~ineteen individuals with psoriasis (pla~ue psoriasis, pustular psoriasis or erythrodermic psoriasis) were r~n~ qP~ either to oral treatment with GMDP tablets (at WO96101645 2 1 ~ ~ 6 7 ~ PCT/GB95/0l6l9 a dose of 20 mg per day for 10 days, followed by 20 mg every other day for a further ten days, (i.e. treatment on days 1,2,3,4,5,6,7,8,9,10,12,14,16, 8 and 20) or an identical placebo tablet. The trial was "double blind", in that neither the patient nor the investigator knew which preparation (GMDP or placebo) was given to particular patients. Common practice in modern clinical trials, this double hl ;n~;ng is particularly important in psoriasis trials where a strong psychological element can cause i , UV~ t in patients who "know" they are receiving a potential treatment.

The severity and area of the psoriasis in all of the patients was measured at entry to the trial and at intervals out to about 20 days according to the internationally recognised PASI (psoriasis area and severity index) system (Frederiksson and Pettersson, Dermatologica 157:238-244 (1978)). Changes in individual patients are shown in Fig 1. In this graph, the starting condition of patients was standardised at "100~", and then at subse~uent ~min~tinnq the PASI score is shown as a percent of starting score. It is clear that the PASI of treated individuals tended to fall during treatment, and a number of individuals even showed complete clearing of lesions. Individuals receiving placebo tablets showed minimal i _ uv~ ~, some worsening or no change. At the time of last , in~t;on~
the mean score for treated patients was 6.0 and the mean score for control patients was almost twice that, 11.7.

W096/016~5 2 1 ~ 4 ~ 7 8 PCT1GB95/01619 ~PLE 2 The use of OEMDP in the treatment of psoriasis - n~d with the Eequential use of other p30riasis treatments.
~ ~
In a double blind, placebo-controlled clinical trial of GMDP, 14.=patients received active ~GMDP) treatment (30 mg/day for lO days). Of these, all except one subject showed i1~1~L~V~ 1L as measured by a decrease in their PASI score.

Following~ initial ill~L~V~ t during the lO days' treatment with GMDP, several of the patients elected to receive I~rther psoriasis therapy other than GMDP. This included W irradiation, topical salicylic acid, oral magnesium sulphate and vitamin A derivatives.
I111~L~V. o~ntin11o~ in these patients, clearly demonstrating that GMDP tro~t~ t can be safely and eifectively followed by other treatments, i~ that is considered appropriate by the administering physician.

~MPLE 3 The eifect of GMDP on ~TPase expression by ~pi~or~-dendritic cells in the mouse.

~Q~
The method used in this experiment has been described in detail by Norval et al ~Norval, M, Gilmour, J W, and Simpson T J, Photodermatol Phot~ ~7 Photomed 7:243-8 (1990) ) GMDP was dissolved in ethanol at l mg/ml and O.l mg/ml.
Twenty microlitres o~ this solution, or vehicle (ethanol) WO96/01645 219467~ P~ 6l9 .

control was painted on to the dorsal sides of the ears of mice which had previously been "stripped" with adhesive tape to remove the superficial barrier layers. Other mice were irradiated with a dose of W3 (144 mJ/cm2) known to 1 nfl nPn~e Langerhans cell (LC) ATPase expression. In another experiment of this type, the positive control was cis-urocanic acid (cUCA), a substance generated in the epidermis under the influence of UV5, and believed to be implicated in the mediation of UV3-induced changes in ~C
function.

Twenty four hours later mice were killed, epidermis was removed from the dorsal surface of the ears, and stained to reveal ATPase activity. Cells showing staining were counted and expressed as cells/mm2. The morphology of the cells was also noted.

RESULTS AND DISCUSSION
Results are summarised in Tables 2 and 3. GMDP at the two doses studied significantly reduced the number of AT~ase positive cells as compared to vehicle controls.
The positive control treatments (UV3 irradiation and c-UCA) also reduced cells, as expected.

TA3~E 2 Treat~ent No of ATPa~e -ve cells/mm2 (meanlSD) Ethanol 420 (30) GMDP 1 mg/ml 350 (20) GMDP 0.1 mg/ml 325 (20) UV3 160 (25) WO96/016~5 2 ~ ~ 4 6 7 ~ . s 16l9 Treatment No oL ATP~se +ve cells/~m2 (mean+SD) Ethanol 445 (30) GMDP 1 mg/ml 35D (30) GMDP 0.I mg/ml 280 (20) c-UCA 325 (30) MPT.Ti! 4 The effect oL GMDP on the migration oL ~ri~r~ 7 dendritic cell~ to draining lymph node~.

In this example, the ability of GMDP to provoke migration of dendritic epidermal cells wa8 ~ inr~r1, Mice were treated with ethanol, GMDP at 1 mg/ml in ethanol, or U~3 at g6 mJ/cm2. Forty eight hours later mice were killed, the auricular (ndraining~) lymph nodes excised, and dendritic cells purified and counted.

RESULTS ~Nn DIS W SSION

Results are summarised in Table 4.

Treatment No Of dendritic cells per lyrph node (~ean_SD) Ethanol 1750 ~400) GMDP 1 mg/ml 2200 (250) U~3 8000 (1800) u~n3 signlficantly r~nh~nr r~r1 the number of dendritic cells in lymph nodes. GMDP showed no significant effect.

Claims (31)

1. The use of a muramyl peptide compound other than 3-0-[N-acetylmuramyl-L-lysyl-D-isoglutaminyl]-1,2-di-0-palmitoyl-sn-glycerol, in the preparation of an agent for the treatment or prophylaxis of an inflammatory dermatological condition.

2. The use as claimed in claim 1, wherein the muramyl peptide compound is a compound of general formula I:

wherein:

R1 represents a hydrogen atom or a C1-C22 acyl group;
R2 represents a hydrogen atom or a C1-C22 acyl group;
R3 represents a hydrogen atom or a C1-C6 alkyl group;
R4 represents a C1-C31 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.

3. The use as claimed in claim 2, wherein the muramyl peptide compound is:

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-.alpha.-N-butyl ester); or t-MDP (N-acetylmuramyl-L-threonyl-D-isoglutamine) .

4. The use as claimed in claim 1, wherein the muramyl peptide compound is:

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.

5. The use as claimed in claim 1, wherein the muramyl peptide compound is N-acetyl-D-glucosaminyl-(.beta.1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP).

6. The use as claimed in claim 1, wherein the muramyl peptide compound is N-acetyl-D-glucosaminyl-(.beta.1-4)-N-acetylmuramyl-L-alanyl-D-glutamic acid (GMDP-A).

7. The use as claimed in claim 1, wherein the muramyl peptide compound is:

N-acetyl-D-glucosaminyl-(.beta.1-4)-N acetylmuramyl-L-alanyl-L-isoglutamine (GMDP-LL);

N-acetyl-D-glucosaminyl-(.beta.1-4)-N acetylmuramyl-L-alanyl-D-glutamine n-butyl ester (GMDP-OBu);

N-acetyl-D-glucosaminyl-(.beta.1-4)-N acetylmuramyl-L-alanyl-D-isoglutaminyl-L-lysine (GMDP-Lys); or N~-[N-acetyl-D-glucosaminyl-(.beta.1-4)-N-acetylmuramyl-L-alanyl-D-isoglutaminyl]-N~-stearoyl-L-lysine (GMDP-Lys(St)).

8. The use as claimed in claim 1, wherein the muramyl peptide compound is:

N~-[N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetyl-muramyl-L-alanyl-.gamma.-D-glutamyl]-N~-stearoyl-L-lysine (GMDPA-Lys(St));

N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-L-alanyl-D-glutamic acid dibenzyl ester (GMDPA(OBz1)2);

N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-N-methyl-L-alanyl-D-isoglutamine (Me-GMDP);

N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-(.beta.1--4)-N-acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-bis-(L-alanyl-D-isoglutamine)((GMDP)2);

N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-(.beta.1--4)-N-acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-bis-(L-alanyl-D-glutamic acid) ((GMDPA)2);

N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-(.beta.1--4)-N-acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-bis-(L-alanyl-D-isoglutaminyl-L-lysine) ((GMDP Lys)2);

N-acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-(.beta.1--4)-N-acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-bis-[L-alanyl-D-isoglutaminyl-N.epsilon.-stearoyl-L-lysine]
([GMDP-Lys(St)]2);

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

L-Threonyl-N.epsilon.-[N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetyl-muramyl-L-alanyl-.gamma.-D-isoglutaminyl]-L-lysyl-L-prolyl-L-arginine (GMDP-tuftsin E);

N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetyl-muramyl-L-alanyl-.gamma.-D-isoglutaminyl-L-threonyl-L-lysyl-L-prolyl-L-arginine (GMDP-tuftsin A);

N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-L-alanyl-.alpha.-D-glutamyl-L-lysyl-L-threonyl-N.epsilon.-stearoyl-L-lysyl-L-prolyl-L-arginine (GMDPA-tuftsin lipophilic);

N'-[N-Acetyl-D-glucosaminyl-(.beta.l--4)-N-acetyl-muramyl-L-alanyl-y-D-isoglutaminyl]-L-lysyl-L-histidyl-L-glycine amide (GMDPA-bursin);

N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-glutamyl-L-tryptophan (GMDP-thymogen I);

N-Acetyl-D-glucosaminyl-(.beta.1--4)-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-.epsilon.-aminohexanoyl-L-glutamyl-L-tryptophan (GMDP-thymogen II);

N~-[N-Acetyl-D-glucosaminyl-(.beta.1--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).

9. The use as claimed in any one of claims 1 to 8, wherein the inflammatory dermatological condition is psoriasis.

10. The use as claimed in claim 9, wherein the psoriasis is discoid or plaque psoriasis, flexural psoriasis, scalp psoriasis, palmar/plantar psoriasis, guttate psoriasis, erythrodermic psoriasis or pustular psoriasis, psoriatic arthritis, or involves changes in the nails resulting from psoriasis.

11. The use of muramyl peptide compound, other than 3-O-[N-acetylmuramyl-L-lysyl-D-isoglutaminyl]-1,2-di-O-palmitoyl-sn-glycerol, in the preparation of an agent for the treatment or prophylaxis of immune-related diseases of the skin and mucous membranes.

12. The use as claimed in claim 11, wherein the muramyl peptide compound is as defined in any one of claims 2 to 8.

13. The use as claimed in claim 11, wherein the immune-related disease is endogenous eczema (otherwise known as atopic eczema or atopic dermatitis), seborrheic eczema, pompholyx, contact dermatitis, urticaria, erythroderma, lichen planus, vitiligo or alopoecia areata.

14. The use as claimed in any one of claims 1 to 13 wherein the agent is for topical administration.

15. The use as claimed in claim 14 wherein the agent is an ointment, lotion or cream.

16. A method for the prophylaxis or treatment of an inflammatory dermatological condition, which comprises administering to a patient a muramyl peptide compound, other than 3-0-[N-acetylmuramyl-L-lysyl-D-isoglutaminyl]-1,2-di-O-palmitoyl-sn-glycerol.

17. A method as claimed in claim 16, wherein the muramyl peptide compound is as defined in any one of claims 2 to 8.

18. A method as claimed in claim 16 or claim 17, wherein the inflammatory dermatological condition is psoriasis.

19. A method as claimed in claim 18, wherein the psoriasis is discoid or plaque psoriasis, flexural psoriasis, scalp psoriasis, palmar/plantar psoriasis, guttate psoriasis, erythrodermic psoriasis or pustular psoriasis, psoriatic arthritis, or involves changes in the nails resulting from psoriasis.

20. A method as claimed in any one of claims 16 to 19, wherein the muramyl compound is administered at a daily dose of from 0.1 to 100 mg.

21. A method as claimed in claim 20, wherein the daily dose is from 0.5 to 50 mg.

22. A method as claimed in claim 21, wherein the daily dose is from 2 to 30 mg.

23. A method as claimed in claim 22, wherein the daily dose is from 2 to 20 mg.

24. A method as claimed in any one of claims 16 to 23, wherein the muramyl peptide compound is administered for 1-60 days.

25. A method as claimed in claim 24, wherein the muramyl peptide compound is administered for 1-30 days.

26. A method as claimed in claim 25, wherein the muramyl peptide compound is administered for 1-14 days.

27. A method as claimed in any one of claims 16 to 26, wherein the muramyl peptide compound is administered together with at least one other therapeutic agent used in the treatment or prophylaxis of an inflammatory dermatological condition.

28. A method as claimed in claim 27, wherein the two agents are admnistered simultaneously, separately or sequentially.

29. A pharmaceutical composition comprising a muramyl peptide compound, other than 3-0-[N-acetylmuramyl-L-lysyl-D-isoglutaminyl]-1,2-di-O-palmitoyl-sn-glycerol, together with at least one other therapeutic agent used in the treatment or prophylaxis of an inflammatory dermatological condition, for simultaneous, separate or sequential use in the treatment or prophylaxis of an inflammatory condition.

30. A pharmaceutical composition as claimed in claim 29, modified by any one or more of the features of claims 2 to 10.

31. A pharmaceutical formulation as claimed in claim 29 or claim 30, wherein the other therapeutic agent is salicylic acid, magnesium sulphate or a vitamin A
derivative.