WO1997035608A1

WO1997035608A1 - Diabetes treatment

Info

Publication number: WO1997035608A1
Application number: PCT/GB1997/000795
Authority: WO
Inventors: Margaret Elizabeth Smith
Original assignee: The University Of Birmingham
Priority date: 1996-03-22
Filing date: 1997-03-21
Publication date: 1997-10-02
Also published as: EP0895479A1; AU2037697A; US6184209B1; GB9606076D0; AU720956B2; JP2000510821A; CA2249454A1

Abstract

Diabetes mellitus type I or II is treated by use of a peptide including an amino acid sequence corresponding to that of an active C-terminal fragment of β-endorphin or an active analogue of a C-terminal fragment of β-endorphin.

Description

DIABETES TREATMENT

This invention relates to diabetes treatment and is more particularly concerned with diabetes mellitus. The present invention is considered to be suitable for the treatment of either type I (insulin-dependent) or type II (non-insulin-dependent) diabetes mellitus.

It has been observed by R. L. Reid et al ("β-endorphin stimulates the secretion of Insulin and Glucagon in Diabetes Mellitus", Metabolism, Vol 33, No 3 (March), 1984, pages 197-199) that administration of human β- endorphin (2.5 mg by intravenous bolus injection) to three subjects with non-insulin dependent diabetes mellitus (type I!) induced prompt and simultaneous increments in the plasma concentrations of insulin and glucagon lasting up to 90 minutes and that, in contrast to the hyperglycemic response previously observed in normal subjects following administration of β-endorphin, these diabetics showed a progressive decline in plasma glucose throughout a three hour study period. However, this progressive decline in plasma glucose was substantially unaffected by the administration of β-endorphin which took place one hour after the start oi the study period.

It has been observed by the inventor that, in normal mice, there are only a small number of β-endorphin receptors on the muscle fibres, whereas in obese (ob/ob) diabetic mice, there is a dramatically higher density of β-eπdorphin receptors on the muscles. The obese (ob/ob) mice inherit diabetes mellitus which resembles type II (non-insulin-dependent) diabetes mellitus seen in humans. In both the human and the mouse with this condition, the muscles are insulin-resistant. The present invention is based on the surprising discovery that blood glucose levels can be reduced by administration of β-endorphin fragments which do not include the opioid (N-terminal) region of β-endorphin, and that such fragments act by enhancing the uptake of glucose into the muscle by a non-insulin dependent route .

The present invention resides in the use of a peptide including an amino acid sequence corresponding to that of an active C-terminal fragment of β-endorphin or an active analogue of a C-terminal fragment of β- endorphin, in the manufacture of a medicament for the treatment of diabetes mellitus (type I or II).

The present invention also resides in the use of a peptide including an amino acid sequence corresponding to that of an active C-terminal fragment of β-endorphin or an active analogue of a C-terminal fragment of β-endorphin, in the manufacture of a medicament for increasing uptake of blood glucose into muscle.

The term "active" refers to the activity for the uses intended in the present invention.

The peptide preferably has β-endorphin 31 (Glu or Gin) as the C-terminal amino acid residue. More preferably, the peptide includes or consists of at least β-endorphin 30-31 (Gly-Glu or Gly-GIn), even more preferably β-endorphin 29-31 (Lys-Gly-Glu or Lys-Gly-GIn), and most preferably β- endorphin 28-31 (Lys-Lys-Gly-Glu or Lys-Lys-Gly-GIn). However, it is within the scope of the present invention for the peptide to include further amino acid residues in its N-terminal region. For example, the peptide may be β-endorphin 22-31 , 23-31 , 24-31 , 25-31 , 26-31 or 27-31.

The peptide preferably has no more than 10 amino acid residues, but more preferably has less, eg up to nine, eight, seven, six or five amino acid residues; or even more preferably, less than six amino acid residues, Thus, most preferably, the peptide is a di-, tri-, tetra- or penta-peptide.

The use of active analogues of the above-mentioned peptides is also within the scope of the present invention. In particular, 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) Gin

(wherein 28 to 31 relate to the numbering for the corresponding amino acids in the β-endorphin amino acid sequence).

One of hydrogen atoms at the N-terminal end of the peptide (preferably the 28-N-terminal end of β-endorphin 28-31) may be substituted by β- Ala, HOOC(CH₂)₂CO-, Tyr, benzylcarbonyl, malonyl, acetyl, fatty acid acyl or other acyl group. Hereinafter, these acyl groups will be referred to as Ac.

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 CH₃CO-Lys-D-Lys-Sar-Glu or CH₃CO-Lys-D-Lys-Sar-Gln.

Furthermore, the above peptides are small molecules compared to insulin (or β-endorphin). The stabilised analogues include those 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 peptides usable in the present invention do not contain the opioid amino acid sequence and therefore are not likely to have those side effects of β-endorphin which are due to its opioid actions.

β-endorphin 28-31 is a per se known compound and is sometimes referred to as melanotrophin-potentiating factor (MPF). It is a putative neurotrophic agent. Stabilised analogues of MPF, such as Ac-Lys-D-Lys- Sar-Glu, are also known, see for example J.S. Morley et al in "MPF analogue with high stability to proteolysis", Neuropeptides 2:109-1 14, 1981 and D. M. Ensor et al, Brain Research, 610 (1993), pages 166-168. The latter report MPF and Ac-Lys-D-Lys-Sar-Glu as causing significant reductions on the turning response of dopamine-depleted rats to D- amphetamine. MPF and certain analogues thereof are also reported as promoting urodele limb regeneration (see J. S. Morley et al, Life Sciences, Vol. 45, pages 1341-1347).

The peptides used in the present invention can be administered intravenously, subcutaneously or intramuscularly, although stabilised analogues, such as Ac-Lys-D-Lys-Sar-Glu, may possibly be administered orally.

The peptides used in the present invention can be synthesised in a per se known manner, for example by use of the solid-phase method of R. Bruce Merrifield where amino acids are added stepwise to a growing peptide chain linked to an insoluble resin matrix, using (i) dicyclohexylcarbodiimide to activate the carboxyl groups to be subjected to peptidisation at the appropriate stages, and (ii) a t-butyloxycarbonyl group and trifluoroacetic acid respectively to block and deblock the amino groups to be protected at the appropriate stages during synthesis.

In the case of the N-substituted peptides noted above, N-substitution may also be effected in a manner known per se by a simple peptidisation reaction with appropriate blocking, if necessary, depending upon the nature of the substituent group.

The invention further resides in a method of treating a patient with diabetes, comprising the step of administering to such patient an effective amount of a peptide as defined above.

The invention also resides in a method of increasing uptake of blood glucose into muscle in a patient, comprising the step of administering to such patient an effective amount of a peptide as defined above.

The peptides may be administered in amounts in the range of 0.1 to 100 nmoles of peptide per kg body weight, more preferably 1 to 10 nmoles/kg body weight. The present invention is based on sets of experiments whose results are reported in the following Examples.

Example 1

Isolated soleus, EDL (extensor digitorum longus) or diaphragm muscles of normal male and female mice were incubated for 30 min at 37°C in oxygenated Krebs buffer containing 2-deoxy-D-[1-³H]glucose. 2-Deoxy- D-glucose is a derivative of D-glucose which is taken up into muscle by carrier-mediated transport and which accumulates in muscle as 2-deoxy- D-glucose-6-phosphate without further metabolism. The tritiated derivative can therefore be used to determine the uptake into muscle of D-glucose (which itself is metabolised). A COOH-terminal (C-terminal) fragment of β-endorphin and an analogue of a β-endorphin fragment were added to the Krebs buffer. These were the C-terminal dipeptide (glycyl-L-glutamine or glycyl-L-glutamic acid) and CH₃CO-Lys-D-Lys-Sar- Glu, a stable analogue of the C-terminal tetrapeptide of β-endorphin. Pyruvate (2 mM) and 2% bovine serum albumen were also present in the bathing medium surrounding the muscles. The extracellular space in the muscles was determined by including L-[1-¹ C] glucose, which is not transported into muscle via the D-glucose membrane carrier.

At the end of the incubation period, the tissue samples were digested in 1 M NaOH at 90°C and the radioactivity counted by liquid scintillation counting.

There was an increase in the uptake of 2-deoxy-D-glucose into the muscles in the presence of either of these peptides. a) Isolated soleus muscles - male mice

CH₃CO-Lys-D-Lys-Sar-Glu was effective at concentrations of 10^"" M, 10^"'° M and 10^"9 M. At 10^"9 M, CH₃CO-Lys-D-Lys-Sar-Glu produced a 9-fold (800%) increase in the uptake of 2-deoxy-D-glucose into the muscles. In comparison, insulin at 10^"9 M gave only a 6-fold (500%) increase.

b) Isolated soleus muscles - female mice

CH₃CO-Lys-D-Lys-Sar-Glu was effective at concentrations of 10^"8 M and 10^"7 M. At 10^"8 M, CH₃CO-Lys-D-Lys-Sar-Glu produced an approximately 2-fold (100%) increase in the uptake of 2-deoxy-D-glucose into the muscles. Insulin at 10^"β M (optimum concentration) gave a similar approximately 2-fold (100%) increase.

c) EDL muscles - male mice

CH₃CO-Lys-D-Lys-Sar-Glu at a concentrations of 10^"9 M produced a 7- fold (600%) increase in the uptake of 2-deoxy-D-glucose into the muscles. In comparison, insulin at 10^'9 M gave only a 2.5-fold (150%) increase.

d) EDL muscles - female mice

CH₃CO-Lys-D-Lys-Sar-Glu was effective at concentrations of 10^"6 M, 10'⁷ M and 10^"* M. At 10^"7 M, CH₃CO-Lys-D-Lys-Sar-Glu produced a 7-fold (600%) increase in the uptake of 2-deoxy-D-glucose into the muscles. In comparison, insulin at 10^"7 M gave only a 3.5-fold (250%) increase.

e) Diaphragm muscles - female mice

CH₃CO-Lys-D-Lys-Sar-Glu was effective at concentrations of 10^"7 M, 10^"8 M and 10^"9 M. At 10^β M, CH₃CO-Lys-D-Lys-Sar-Glu produced a 4-fold (300%) increase in the uptake of 2-deoxy-D-glucose into the muscles, which is similar to that produced by insulin at the same concentration.

Example 2

Following the procedure outlined in Example 1 , it was shown that Gly- Gln in a concentration of 10^'7 M caused a 180% increase in 2-deoxy-D- glucose uptake in EDL muscles of normal female mice, whilst with Gly- Glu there was an 80% increase.

Example 3

Following the procedure outlined in Example 1 , it was shown that CH₃CO-Lys-D-Lys-Sar-Glu in a concentration of 10'⁷ M caused an approximately 2-fold increase in 2-deoxy-D-glucose uptake in soleus muscles of obese-diabetic (ob/ob) mice.

Example 4

Following the procedure outlined in Example 1 using neonatal normal mouse abdominal muscle strips, it was shown that CH₃CO-Lys-D-Lys-Sar- Glu caused increases in the uptake of 2-deoxy-D-glucose into the muscle strips at concentrations of 10^"* M, 10^"9 M and 10^'10 M, the increase being 40% at 10^"10 M.

Example 5

In another experiment on the muscles of obese-diabetic mice, following the procedure outlined in Example 1 , it was shown that CH₃CO-Lys-D- Lys-Sar-Glu in a concentration of 10^"7 M caused a 70% increase in 2- deoxy-D-glucose uptake in the EDL muscles of these obese-diabetic mice. Example 6

Following the procedure outlined in Example 1 using rat L6 cells (a commercially available cell line derived from rat muscle), it was shown that CH₃CO-Lys-D-Lys-Sar-Glu was effective at concentrations of 10^"12 M to 10^'8 M. At 10^'9 M, CH₃CO-Lys-D-Lys-Sar-Glu produced an approximately 40% increase in the uptake of 2-deoxy-D-glucose into the muscles.

In the presence of 10^"8 M insulin, CH₃CO-Lys-D-Lys-Sar-Glu at a concentration of 10^'9 M caused a 30% increase in glucose uptake over that produced by the insulin alone.

From the above, it will be understood that the peptide acts to increase the uptake of blood glucose into muscle independently of insulin. Thus, it is considered that the present invention is also suitable as an alternative to insulin administration for the treatment of type I diabetes mellitus.

Key

Tyr = L-tyrosine; Lys = L-lysine; D-Lys = D-lysine; Ac =acyl, preferably acetyl; Orn = L-ornithine; MeLys =■ N-α-methyl-lysine; des-NH₂ = desamino-lysine; Nle -= ε-amino group of lysine replaced by Me; AzGIy - α-azaglycine; D-Ser = D-serine; Ala « L-alanine; D-Ala = D-alanine; Pro - proline; Sar = sarcosine, otherwise known as N-methyl-glycine (N-MeGly).

Claims

1 . The use of a peptide including an amino acid sequence corresponding to that of an active C-terminal fragment of β-endorphin or an active analogue of a C-terminal fragment of β-endorphin, in the manufacture of a medicament for the treatment of diabetes mellitus.

2. The use as claimed in claim 1 , in the manufacture of a medicament for the treatment of type II diabetes mellitus.

3. The use as claimed in claim 1 , in the manufacture of a medicament for the treatment of type I diabetes mellitus.

4. The use of a peptide including an amino acid sequence corresponding to that of an active C-terminal fragment of β-endorphin or an active analogue of a C-terminal fragment of β-endorphin, in the manufacture of a medicament for increasing uptake of blood glucose into muscle.

5. The use as claimed in any preceding claim, wherein the peptide has Glu or Gin as the C-terminal amino acid.

6. The use as claimed in any preceding claim, wherein the peptide includes Gly-GIu or Gly-GIn as its C-terminal region, or consists of Gly- Glu or Gly-GIn.

7. The use as claimed in any one of claims 1 to 5, wherein the peptide includes Lys-Gly-Glu or Lys-Gly-GIn as its C-terminal region, or consists of Lys-Gly-Glu or Lys-Gly-GIn.

8. The use as claimed in any one of claims 1 to 5, wherein the peptide includes Lys-Lys-Gly-Glu or Lys-Lys-Gly-GIn as its C-terminal region, or consists of Lys-Lys-Gly-Glu or Lys-Lys-Gly-GIn.

9. The use as claimed in any one of claims 1 to 4, wherein the peptide has an amino acid sequence corresponding to that of an analogue of a C-terminal fragment of β-endorphin.

10. The use as claimed in claim 9, wherein the active analogue is a stabilised analogue, wherein one or more of the following substitutions are made:-

Amino Acid Substituting moiety

28 (Lys) Orn, MeLys, des-NH₂, Nle or D-Lys

29 (Lys) Orn, D-Lys, MeLys or Nle

30 (Gly) Sar, AzGIy, Ala, D-Ala, D-Ser or Pro

31 (Glu) Gin

11. The use as claimed in any preceding claim, wherein a hydrogen at the N-terminal end of the peptide is substituted by β-Ala, HOOC(CH₂)₂CO-, Tyr, benzylcarbonyl, malonyl, acetyl, fatty acid acyl or other acyl group.

12. The use as claimed in claim 10, wherein the peptide includes Ac-Lys-D-Lys-Sar-Glu, where Ac is an acyl group.

13. The use as claimed in claim 10, wherein the peptide is CH₃CO- Lys-D-Lys-Sar-Glu.

14. The use as claimed in any one of claims 1 to 12, wherein the peptide has not more than ten amino acid residues.

15. The use as claimed in claim 14, wherein the peptide has no more than five amino acid residues.

16. A method of treating a patient with diabetes mellitus, comprising the step of administering to such patient an effective amount of a peptide as defined in any preceding claim.

17. A method of increasing uptake of blood glucose into muscle in a patient, comprising the step of administering to such patient an effective amount of a peptide as defined in any one of claims 1 to 15.