Drug – bio-affecting and body treating compositions – Enkephalin or endorphin or derivatives
Reexamination Certificate
1999-11-26
2003-07-22
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Enkephalin or endorphin or derivatives
C514S002600, C514S012200, C530S302000
Reexamination Certificate
active
06596778
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national stage application of International Application PCT/GB98/00616, filed Feb. 26, 1998, which international application was published on Sep. 3, 1998, as International Publication WO 98/38215 in the English language. The International Application claims priority of Great Britain Patent Application 9704174.3 filed Feb. 28, 1997.
BACKGROUND AND SUMMARY OF THE INVENTION
This invention relates to an agent for alleviating the effects of muscular dystrophy and other muscle-wasting diseases (eg. motor neurone disease) in which there is leakage of enzymes such as creatine phosphokinase (CPK) from the muscle into the blood (hereinafter simply called “a muscle-wasting disease”).
The present invention resides in the use of an agent which is an active &bgr;-endorphin fragment or an analogue of such a fragment, in the manufacture of a medicament for alleviating the effects of muscle-wasting disease.
The present invention also resides in the use of an agent which is an active &bgr;-endorphin fragment or an analogue of such a fragment, in the manufacture of a medicament for inhibiting or reducing leakage of an enzyme (preferably CPK) from a living muscle.
The present invention also resides in the use of an agent which is an active &bgr;-endorphin fragment or an analogue of such a fragment, in the manufacture of a medicament for improving the contractile response of muscle.
&bgr;-Endorphin is a 31 amino acid peptide which is processed in vivo by cleavage of basic amino acid residues to &bgr;-endorphin
1-7
, and it has been assumed that cleavage of adjacent basic residues results in the production of the C-terminal dipeptide, &bgr;-endorphin
30-31
.
The effect of these active &bgr;-endorphin fragments is particularly surprising because it has previously been observed (see Smith, M. E. and Hughes, S., “The Effect of &bgr;-Endorphin and &agr;-Melanotropin on Muscle Wasting in Mice”, Journal of the Neurological Sciences (1995),129 (suppl.), p127) that intravenous injection of &bgr;-endorphin alone produced little effect in preventing CPK leakage into the blood.
Suitable active &bgr;-endorphin fragments for use in the present invention are fragments at the C-terminal end of the &bgr;-endorphin molecule, such as &bgr;-endorphin (30-31; glycylglutamic acid), (29-31) and (28-31). In &bgr;-endorphin, the amino acids 28, 29, 30 and 31 are lysine, lysine, glycine and glutamic acid (human) or glutamine (most other mammals), respectively.
As far as the analogues of the above active &bgr;-endorphin fragments are concerned, stabilised analogues thereof are preferred wherein one or more of the following substitutions may be made:
Amino Acid
Substituting moiety
28 (Lys)
Orn, MeLys, des-NH
2
, Nle or D-Lys
29 (Lys)
Orn, D-Lys, MeLys or Nle
30 (Gly)
Sar, AzGly, Ala, D-Ala, D-Ser or Pro
31 (Glu)
Gln.
Additionally, the hydrogen terminating the N-terminal end (preferably the &bgr;-endorphin 28-amino acid-N-terminal) of the &bgr;-endorphin fragment may be substituted by Ac, &bgr;-Ala, HOOC(CH
2
)
2
CO—, Tyr, benzylcarbonyl (C
6
H
5
CH
2
CO—), malonyl or R (wherein Ac is an acyl group, for example acetyl, and R is a fatty acyl group.
Of such analogues, those where amino acid 30 (Gly) is replaced by Sar, those where amino acid 29 (Lys) is replaced by D-Lys, and those where the N-terminal (28) end of the fragment is Ac-Lys, are preferred. Particularly preferred is the stabilised analogue in which all three substitutions have been made, i.e. Ac-Lys-D-Lys-Sar-Glu, most preferably CH
3
CO-Lys-D-Lys-Sar-Glu.
Furthermore, the above C-terminal peptides (i.e. the above-mentioned &bgr;-endorphin fragments are small molecules compared to &bgr;-endorphin and analogues of these fragments can be made which are stable to proteolytic digestion and therefore have a relatively long half-life in the blood enabling their actions to be long lasting. Their resistance to proteolytic digestion may also make them effective via oral administration. In addition, the C-terminal peptides do not contain the opioid amino acid sequence and therefore are not likely to have the side effects of &bgr;-endorphin which are due to its opioid actions.
Thus, it is considered that the active &bgr;-endorphin fragments and analogues thereof may be administered intravenously, subcutaneously or intramuscularly, although stabilised analogues, such as Ac-Lys-D-Lys-Sar-Glu, may possibly be administered orally.
In one series of experiments, the C-terminal peptide, glycylglutamine, was injected intravenously into the blood of dystrophic mice 4 to 5 times a week in an amount of approximately 1.5 microgram/g body weight each time over a seven week period. Control mice were injected with the same volume of saline at the same times. There were six mice in each group.
The following measurements were made:
1. Change in body weight (the dystrophic mice would usually not gain significant weight over seven weeks and might even lose weight-depending on their age).
2. Weight of the extensor digitorum longus (EDL) muscle.
3. Level of CPK in the blood (a diagnostic test for muscular dystrophy is to measure the level of CPK in the blood. In muscle diseases, the blood levels of enzymes such as CPK are elevated and in muscular dystrophy the levels can be very high).
4. CPK enzyme levels in the muscle.
The following results were obtained:
1. The mice treated with glycylglutamine showed a statistically significant increase in body weight (of approximately 12%), whilst the saline controls showed a slight decrease in body weight (0.04% which is non-significant).
2. There were slight increases in EDL muscle weight (approximately 5%) which were not statistically significant.
3. The CPK level in the blood was reduced by 25%, indicating an improvement in the health of the muscle (i.e. less damage).
4. The enzyme concentration in the EDL muscle was increased by 55% (when calculated on a wet weight basis).
The above findings, particularly in result 4 above, indicate that chronic injections of this peptide could be therapeutic in muscular dystrophy. In Duchenne muscular dystrophy—the most common muscular dystrophy in humans—it appears to be the fast muscle fibres which are most involved in the disease process. The muscle examined (the EDL) is a fast muscle which contains a high proportion of fast fibres.
The effects of different &bgr;-endorphin-derived peptides on the contractile responses in mouse diaphragm muscle have been further examined in order to see which regions of the parent molecule might be important for its effect.
REFERENCES:
Khan et al., Effect of beta-endorphin 1-27 and beta-endorphin 30-31 on the contractile responses of mouse diaphragm in vitro, Journal of Physiology, vol. 487, p. 163P, 1995.*
Khan et al., Action of beta-endorphin peptides on the contractions of mouse diaphragm muscle, Peptides, vol. 18, No. 1, pp. 8787-8792, 1997.*
Smith et al., Effect of beta-endorphin and alpha-melanotropin on muscle wasting in mice, Journal of Neurological Sciences, vol. 129, Suppl., pp. 127-130, 1995.*
Protein Information Resource (PIR) accession No. I51630, 1997.*
Patent Abstracts of Japan vol. 017, No. 067 (C-1025), Feb. 10, 1993 & JP 04 273896 A (Taisho Pharmaceut Co Ltd.), Sep. 30, 1992.
Effect of beta-endorphin 1-27 and beta-endorphin 30-31 on the contractile responses of mouse diaphragm in vitro: J. Physiol., vol. 487, 1995, p. 163P XO002069247, Kahn et al.
:Action of beta-endorphin peptides on the contractions of mouse diaphragm muscle Peptides, vol. 18, No. 1, 1997. pp. 87-92, XP002069248, Khan et al.
“Effect of beta-endorphin and alpha-melanotropin on muscle wasting in mice”, J. Neurological Sciences. vol. 129, No. Suppl. 1995. pp. 127-130, XP002069249.
Andrus Sceales Starke & Sawall LLP
Carlson Karen Cochrane
The University of Birmingham
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