WO1993013791A1

WO1993013791A1 - Use of muramyl peptides for enhancing animal production

Info

Publication number: WO1993013791A1
Application number: PCT/GB1993/000123
Authority: WO
Inventors: Roger Aston
Original assignee: Peptech (Uk) Limited
Priority date: 1992-01-20
Filing date: 1993-01-20
Publication date: 1993-07-22
Also published as: AU3361093A

Abstract

A production enhancement method comprises administering to animals muramyl peptide compounds, other than prototype muramyl dipeptide. Examples of useful compounds include muroctasin, MTP-PE, murabutide, t-MDP, GMDP and GMDPA. The compounds enhance animal production and are without the disadvantages associated with conventional production enhancers.

Description

USE OF MURAMYL PEPTIDES FOR ENHANCING ANIMAL PRODUCION. This invention relates to production enhancers which lead to improved food utilisation and increased growth of animals to which they are administered and, in addition, to increased yield of products obtained from those animals.

In modern farming, where maximum efficiency is necessary to ensure profitability, it is common practice to administer production enhancers to livestock. Such production enhancers have the benefit of improving the food utilisation of the animals to which they are administered and it has been demonstrated that such animals, when compared with untreated animals, show increased body weight, and, where appropriate, increased wool production, milk production and skin growth.

Conventionally, three types of compound have been used as production enhancers; these are antibacterials, including antibiotics, hormones such as growth hormones and steroids, and ionophores, which increase permeability of the gut to nutrients. However, although these groups of compounds have been shown to be effective production enhancers, they all have serious disadvantages which have led to increasing demands for a ban on their use.

The continuous administration of antibiotics to farm animals has led to the appearance of antibiotic-resistant strains of bacteria. This poses a considerable health risk, not only to the treated animals but also to humans who may become infected with such bacteria. The serious medical repercussions which may result from the administration of hormones to livestock have been well documented. In addition, traces of antibiotics and hormones may appear in meat or milk obtained from treated animals and may pose a health risk to humans consuming such products.

Finally, there is also the problem that both antibiotics and hormones must be administered to animals in relatively high doses in order for them to be effective as production enhancers and this, of course, leads to difficulty in the administration of the product to the animal.

There is therefore a need for a production enhancer which does not have these associated disadvantages and the present invention relates to particular muramyl peptide compounds, including muramyl dipeptide derivatives (MDPs) , which may fulfil this role.

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. Natl. 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.

In DE-A-3733899, it was proposed that any immuno- stimulating agent could be used as a growth promotant and among the immunostimulating agents listed for this use is prototype muramyl peptide. However, the work of Langhans et al.. (1991), Am. J. Phvsiol.. 261, R659-R664 has subsequently shown that prototype MDP has no effect on growth. Moreover, prototype MDP has high pyrogenicity and therefore it is unsuitable for administration to animals. However, it has now, surprisingly, been discovered, in spite of the powerful disincentive to further work in the area of MDPs that this represents, that whilst prototype MDP has been reported as having no effect on animal growth, other muramyl peptide compounds are extremely effective as production enhancers.

In a first aspect of the invention there is provided a method of enhancing animal production, characterised in that it comprises administering to the said animal a muramyl peptide compound other than prototype MDP.

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 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 being prototype MDP or analogues or derivatives thereof. Many muramyl peptide compounds, as the term is used in this specification, fall within general formula I:

wherein:

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

R² represents a hydrogen atom or a

acyl group; R³ represents a hydrogen atom or a C_x-C₆ alkyl group;

R* 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;

other than prototype muramyl dipeptide.

"Production enhancing" is a term well understood by those skilled in the art and generally refers to the effect of agents which enhance any attribute which would generally be thought to be desirable in an animal. This would include both characteristics of size or growth and of the products produced by an animal. Examples of characteristics which may be enhanced by production enhancing agents are the rate and extent of growth. Production enhancement may also include selective growth where, for example, muscle tissue may grow more quickly than brain tissue. Agents which encourage selective growth are generally termed "partitioning agents". Another type of production enhancement involves the increase of food efficiency of an animal so that it achieves the same weight gain as an untreated animal whilst being fed on a smaller amount or lower quality of food. Production enhancers may also reduce mortality in treated animals. A high mortality rate is a particular problem in poultry farming. Production enhancement also includes methods of improving the quality or increasing the amounts of products obtained from animal. Examples of this include increased milk yield from lactating animals, and increased quantity or quality of the skin, wool or fur in the appropriate animals. The term "production enhancing" may include several or only one of the effects just mentioned, or indeed similar effects.

There is evidence that one area of production promotion in which muramyl peptide compounds may be particularly useful is in the treatment of shipping fever. This is a condition in which animals suffer a significant loss in weight over a relatively short period of time during transportation. It occurs especially frequently in animals which are transported in cramped conditions and seems likely to be a stress-induced condition. Muramyl peptides may also be useful in the prevention of other types of stress-induced weight loss. Since muramyl peptide compounds are immunostimulants, the method of this invention has the added advantage that the compounds combat infection by stimulating the immune system but, unlike antibiotics, the continuous administration of MDPs to animals would not be expected to result in the emergence of antibiotic-resistant strains of bacteria. The use of these production enhancers may therefore enable the dosage of antibiotics in the diet of animals to be reduced. In addition, unlike conventional production enhancers, muramyl peptide compounds leave no unmetabolised or deleterious residues in the^' tissues or milk produced by an animal and therefore the consumption of such residues by humans will not occur.

Another unexpected advantage of the compounds of the invention is that they increase growth over a prolonged period of time following a single dose. The effect is not usually seen in production enhancers. The amount of growth is generally dependent on the dose.

An added advantage is that the doses of muramyl peptide compounds required to promote animal growth are minute in comparison with required doses of growth promoting agents and this facilitates the delivery of muramyl peptide- based agents to animals.

Preferred acyl groups for R¹ and R² are Ci-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 0-^-0,, alkyl groups such as methyl and ethyl. Preferred alkyl groups for R⁴ and Ci-Cg alkyl groups, particularly C_j-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 C₁-C₂₂ (preferably C^-Cg) alkyl ester, amide or Ci-C₄ alkyl amide thereof. (The expression "mixed" is illustrated when one carboxyl group is amidated and the other esterified. D-isoglutamine and D- glutamate are preferred.

A third amino acid residue from the proximal end of the chain, if there is one, is preferably of the L- configuration, as indicated above in relation to the proximal amino acid residue. L-alanyl and L-lycyl 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)-sn-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 C¹-C¹⁰ 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-glucosaminyl-N-acetyl-muramyl-L-alanyl-D- isogluta ine (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 g/kg. Pyrogenicity is a problem which has caused difficulties for the exploitation of certain muramyl dipeptides, particularly prototype MDP. Pyrogenicity may be attenuated by appropriate formulations (see, for example, the liposomal formulations of US-A-4522811 and US-A- 4684625) , but in general it will be preferable to choose, for use in the present invention, a muramyl dipeptide which has low intrinsic pyrogenicity. While the pyrogenicity of GMDP is sufficiently low not to have prevented its clinical evaluation, it may in some circumstances be preferable to use a substantially non- pyrogenic analogue. Such an analogue is available, and is N-acetyl-glucosaminyl-N-acetyl-muramyl-L-alanyl-D- glutamic acid (GMDP-A) , which is Compound III in US-A- 4395399, and whose structure is as follows:

GMDP-A

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 Ivsodecticus and its subsequent chemical linkage to a dipeptide synthesised for example by conventional peptide chemistry. A further advantage of the use of the compounds of formula II in the growth promoting method is that they are water soluble which facilitates their administration to the animals to be treated.

The production enhancers used in the method of the invention are suitable for administration to mammals (including humans) , birds and fish, although their principal use is expected to be in the treatment of animals kept as livestock, particularly cattle, sheep, pigs and poultry.

Compositions containing one or more muramyl peptide compounds, particularly compounds of formula I or II, may be administered by any conventional route but the oral, nasal and parenteral routes are especially favourable. Preferred routes of parenteral administration are the intramuscular, subcutaneous and transdermal routes.

For oral administration, and in particular for use with poultry, the compounds may be formulated as liquid concentrates or soluble powders which can be added to the drinking water. However, in the case of mammals such as cattle, sheep and pigs, it may be more appropriate to incorporate the production promoter into a feed. Such dietary additives may be formulated as pellets or powders for example lyophilised powders and, advantageously, also include excipients such as bulking agents which have food value. A further method of oral delivery is to incorporate the production promoter into an oral implant. Such implants have been found to be particularly useful for larger animals such as cattle, especially in areas where the food supply to the animal cannot be precisely monitored. The oral implant is formulated for slow delivery, which may be either pulsed or continuous delivery and is swallowed by the animal so that it lodges in an appropriate portion of the gastrointestinal tract.

For nasal administration, which is often used for poultry, the production enhancers may be formulated as aerosols.

For parenteral administration, any one of a variety of formulations can be used. These include (usually sterile) aqueous solutions for injection, microcapsules, liposomes and biodegradable implants which may be formed from silicone, cholesterol or similar materials.

The production enhancers of the invention can also be included as an additional component in vaccines which are administered to animals for other purposes.

The dosage level of the production enhancers varies depending on the particular animal and the amount of growth required but, for oral administration, a suitable dosage rate may be from 1 to 100,000 μg/kg/day and preferably from 10 to 500 μg/kg/day. For parenteral administration, the dose will generally be smaller than for oral administration, being in the range of 0.1 to 500 μg/kg/day and preferably from 1 to 200 μg/kg/day. Sustained release preparations will, of course contain larger amounts of the MDPs than other formulations.

In a second aspect of the invention, there is provided the use of a muramyl peptide compound other than prototype MDP in the preparation of an agent for the treatment of conditions associated with lack of growth. The invention also provides an animal feed composition containing a muramyl peptide compound other than prototype MDP and the use of such a compound in a production promoting composition. The invention will now be further illustrated by the following examples.

EXAMPLE 1

Enhancement of Growth of Normal Piglets Treated With GMDP

Piglets aged 59 days were treated with doses of GMDP ranging from 15 to 150 μg/kg/day for 10 days by intramuscular injection or by oral administration (in the drinking water) . Treated animals were observed to undergo^' no change in temperature, serum albumins and globulins (α, β and γ) or in blood glucose levels. Similarly, no change in enzyme levels (aspartate aminotransferase, alanine aminotransferase or alkaline phosphatase) was observed. Measurement of blood count

(total leukocytes, eosinophils and neutrophils) revealed no changes. The effect of GMDP on the growth of the piglets is shown in Table 1.

TABLE 1 Growth promotion in piglets with GMDP Treatment

From Table 1, it can be seen that treatment of piglets with GMDP results in a dose-dependent enhancement in somatic growth. The effect is observed after both intramuscular injection and oral administration.

EXAMPLE 2

Piglets aged 90 days were divided into 3 groups with 15 animals in each group. The first group was treated with 50 μg/kg/day GMDP by intramuscular injection for 5 days, the second group was treated with 50 μg/kg/day GMDP orally for 5 days and the third group, the control group, was untreated. In each case, the weight of the piglets was determined before treatment and 20 days after the final GMDP treatment and the results are shown in Table 2.

TABLE 2

Enhancement of Growth in Piglets by GMDP

CONTROL INTRAMUSCULAR PER ORAL

Initial Weight (kg) 26.4 ± 0.89 26.3 ± 0.82 27.1 ± 1.91 Final Weight (Kg) 93.7 ± 2.96 101.3 ± 0.89 99.9 ± 0.47 Increase (Kg) 67.3 ± 2.56 75.0 ± 6.21 72.8 ± 4.59 Mean Daily Gain (g) 491.2 ± 18.7 547.4 ± 45.4 531.4 ± 33.54 Weight Gain (%) 100 111.4 108.2

It can be seen from Table 2, that the piglets treated with GMDP increased in weight considerably more than the untreated piglets. Surprisingly, the weight gain in the piglets treated by intramuscular injection was greater than the weight gain in the piglets treated by oral administration.

EXAMPLE 3

Enhancement of Growth in Chicks treated with GMDP

Four groups of chicks were each divided into three batches of 200 chicks: batch 1, a control batch; batch 2, treated orally with GMDP; and batch 3, treated with GMDP by intramuscular injection. The weight of the first group of chicks was determined when they were eight days old and a single dose of GMDP (5 μg/kg) was administered to the chicks in batches 2 and 3 of that group. The weight of the chicks was determined again when they were 19 days old. For the second group of chicks, their weight was determined at the age of 20 days and, again, a single dose of GMDP (5 μg/kg) was administered. The weights of this second group were again determined at day 30. For the third and fourth groups, a similar procedure was carried out with, for the third group, initial weighing and dosing with GMDP taking place at 31 days and final weighing taking place at 45 days and, for the fourth group initial weighing and dosing taking place at 46 days and final weighing taking place at 63 days. The results of this experiment are shown in Table 3 which gives the increases in weight of each batch of chicks in grams. TABLE 3

Enhancement of Growth in Chicks treated with GMDP (weight gain/g)

AGE OF CHICKS (DAYS)

8-19 20-30 31-45 46-63

CONTROL 16.1 ± 0.16 26.5 ± 0.22 29.3 ± 1.28 32.7 ± 1.4

PER ORAL 17.7 ± 0.18 28.1 ± 0.27 34.1 ± 1.32 38.7 ± 1.6 (%) (109.9) (106.6) (116.3) (118.3)

INTRAMUSCULAR 18.5 ± 0.11 29.0 ± 0.22 36.0 ± 1.41 40.2 ± 1.6 (%) (114.9) (109.4) (122.8) (122.8)

From Table 3, it can be seen that the chicks treated with GMDP increased in weight considerably more than the untreated chicks and, somewhat surprisingly, as with pigs, the intramuscular injection is shown to be more effective than the oral dose.

From the results given above it can be seen that muramyl peptide compounds are a most effective growth promoting agent even when given in the small doses used here.

Claims

1. A method of enhancing animal production, characterised in that it comprises administering to an animal a muramyl peptide compound other than prototype MDP.

2. A method as claimed in claim 1, wherein the muramyl peptide compound is of general formula I:

wherein:

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

R² represents a hydrogen atom or a C₁-C₂₂ acyl group; R³ represents a hydrogen atom or a C_!-C₆ alkyl group;

R⁴ represents a C₁-C_2ι alkyl group or a C₆ or C_xo 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;

other than prototype muramyl dipeptide.

3. A method as claimed in claim 2, 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 -C_S acyl group such as acetyl;

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

R* represents a Ci-Cg alkyl group, particularly a Cv C₄ alkyl group, such as methyl or ethyl, or a phenyl group; R represents a mono-, di- or tri-peptide.

4. A method as claimed in claim 1, 2 or 3, wherein the muramyl peptide compound is:

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) ; or

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

5. A method as claimed in claim 1, wherein the muramyl peptide compound conforms to general formula II:

II

wherein:

n is 1 or 2.

6. A method as claimed in claim 5, wherein n is 1.

7. A method as claimed in claim 5 or 6, wherein the proximal amino acid residue is a residue of an L-amino acid.

8. A method as claimed in claim 7, wherein the proximal amino acid residue (or the only amino acid residue, if there is only one) is a residue of L-alanine.

9. A method as claimed in any one of claims 5 to 8, wherein the second amino acid residue from the proximal end of the peptide, if present, is of the D- configuration.

10. A method as claimed in claim 9, wherein the said second amino acid residue is of D-glutamic or D-aspartic acid or a mono-, di- or mixed Cx-C^ (preferably Cj-Cβ) alkyl ester, amide or Ci-C* alkyl amide thereof.

11. A method as claimed in claim 8, 9 or 10, wherein the said second amino acid residue is D-isoglutaminyl or D- glutamyl.

12. A method as claimed in any one of claims 5 to 11, wherein the third amino acid residue from the proximal end of the peptide, if present, is in the L- configuration.

13. A method as claimed in claim 12, wherein the third amino acid residue is L-alanyl or L-lysyl.

14. A method as claimed in any one of claims 5 to 13, wherein the amino acid residue or linear peptide is optionally substituted with at least one lipophilic group.

15. A method as claimed in claim 5, wherein the compound is N-acetyl-glucosaminyl-N-acetyl-muramyl-L-alanyl-D- isoglutamine (GMDP) .

16. A method as claimed in claim 5, wherein the compound is N-acetyl-glucosaminyl-N-acetyl-muramyl-L-alanyl-D- glutamic acid (GMDP-A) .

17. A method as claimed in any one of claims 1 to 16, wherein the animal is a mammal (including a human mammal) , a bird or a fish.

18. A method as claimed in any one of claims 1 to 17, wherein the compound is administered orally.

19. A method as claimed in claim 18 wherein the dose of muramyl peptide compound is from 1 to 100,000 μg/kg/day.

20. A method as claimed in any one of claims 5 to 17, wherein the compound is administered parenterally.

21. A method as claimed in claim 20 wherein the dose of the compound is from 0.1 to 500 μg/kg/day.

22. A method as claimed in any one of claims 1 to 21, wherein the compound is administered concurrently with a vaccine.

23. The use of muramyl peptide compound other than prototype MDP in the preparation of an agent for the treatment of conditions associated with lack of growth.

24. An animal feed composition containing a muramyl peptide compound other than prototype MDP.

25. The use of a muramyl peptide compound other than prototype MDP as a growth promotant in a composition for administration to animals.