WO1990000401A2

WO1990000401A2 - Neutral protease inhibitors for promoting innervation of muscle fibers

Info

Publication number: WO1990000401A2
Application number: PCT/GB1989/000878
Authority: WO
Inventors: Gerda Vrbova; Theresa Jane Fisher
Original assignee: University College London
Priority date: 1988-07-05
Filing date: 1989-07-04
Publication date: 1990-01-25
Also published as: GB8815962D0; WO1990000401A3

Abstract

A method is provided for promoting synapse formation and innervation (and particularly reinnervation) of muscle fibers which comprises administering an inhibitor of calcium activated neutral protease (CANP), for example Leupeptin or E64, to provide an effective concentration of said inhibitor to the at least partially denervated area of a muscle. Also provided are compositions for use in the above method.

Description

PHARMACEUTICAL PREPARATION AND METHOD FOR PROMOTING INNERVATION

OF MUSCLE FIBERS This invention relates to a novel therapeutic method for promoting innervation of muscle fibers, particularly reinnervation of muscle fibers following nerve trauma due, for example, to injury or disease. The invention further relates to pharmaceutical preparations (including implantable devices) for use in carrying out the aforementioned method.

It is known that during early development, muscle fibers are innervated de novo by a mechanism which involves the growth of numerous axons and which initially results in polyneuronal innervation of the muscle fibers. Subsequently all but a single nerve connection to each muscle fibre are selectively withdrawn by a process, known as synaptic remodelling, which requires the presence of certain specific enzymes, including CANP (calcium activated neutral proteases) .

Calcium activated neutral protease (CANP) is a calcium dependent thiol protease found in a wide variety of tie-sues including muscle and nerve tissue. It appears to be involved in regulating the turnover and degradation of muscle myofibrillar proteins and neuronal cytoskeletal elements. In particular, the role of this enzyme in the developmental elimination of polyneuronal innervation and in the maintenance of the adult neuromuscular junction has been studied.

During the early stages of postnatal development, mammalian muscle fibers are innervated by more than one axon (known as polyneuronal innervation) . However as development proceeds there is a transition to mononeuronal innervation and it is suggested that CANP enzymes are involved in the elimination of the excess innervation. In the adult, the remaining single mature nerve ending continues to change its terminal branching pattern by gradually becoming more complex, a process termed "synapse remodelling". Calcium activated neutral proteases have also been considered to be involved in this reorganisation of mature mammalian neuromuscular junctions.

In studying the role of CANPs in the above processes, several exogenous inhibitors of this enzyme have been utilized, for example leupeptin and E64. These inhibitors have been found to slow down the elimination of polyneuronal innervation during development and to result in nerve endings .becoming more complex than usual during synapse remodelling in the adult.

The use of specific inhibitors of thiol proteases (a class of proteases which include the CANPs referred to above) , have previously been proposed for use in the treatment of Duchenne Muscular Dystrophy. This proposed therapeutic use was suggested on the basis that degradation of myofibrils in muscle may be attributable to elevated activities of thiol proteases, including CANPs. However this treatment has not been found to be especially successful.

Once the initial innervation and synaptic modelling processes are essentially complete (i.e. following normal development in early childhood continuing to adult life) , the capacity of nerve fibers to re-innervate muscles and form synapses following trauma or diseases is limited and slow. Consequently a therapeutic method of promoting reinnervation of muscle fibers and synapse formation would be desirable. inhibitors, e.g. inhibitors of the proteases involved in synaptic modelling are also able to accelerate recovery after nerve trauma. Moreover, in conditions where the nerve supply to a muscle is partly damaged through injury or disease, CANP inhibitors have been found to promote the replacement of lost contacts by the remaining healthy motor nerves.

Thus according to the present invention there is provided the use of an inhibitor of calcium activated neutral protease (CANP) in the manufacture of a pharmaceutical composition or device for use in promoting synapse formation and innervation of muscle fibers, particularly synapse formation and reinnervation of muscle fibres following nerve trauma.

There is further provided a method of promoting synapse formation and innervation of muscle fibers, particularly synapse formation and reinnervation of muscle fibers following nerve trauma which comprises administering an inhibitor of calcium activated neutral protease (CANP) so as to provide an effective concentration of said inhibitor to the at least partially dennervated area of a muscle.

The inhibitor of calcium activated neutral protease (CANP) used in the present invention is preferably an oligopeptide comprising at least one amino acid residue. As used herein the term "oligopeptide" refers to a molecule containing a residue of an amino acid HN_ααC00H linked via at least one peptide bond (-C0.NH-) to another amino acid or to an amine. The or each amino acid is preferably an amino acid of normal metabolism. For the purposes of treatment it is desirable that the inhibitor is of low-toxicity, is non-immunogenic and is able to penetrate cell membranes and enter nerve terminals. A class of peptide inhibitors which have been found to be particularly effective comprises peptides which contain one or more branched chain amino acid residues and optionally one or more residues of basic amino acids or amines. The branched chain amino acid residues are preferably selected from valine, leucine and isoleucine.

Preferably the N-terminus of said oligopeptide is blocked, for example by an acyl group derived from a C. _,- carboxylic acid. Examples of suitable acyl groups include acyl groups derived from acetyl, propionyl, cis- or trans-oxiranedicarboxylic acid or a C₁_i, alkyl half-ester of cis- or trans-oxiranedicarboxylic acid. In a preferred class of CANP inhibitors the oligopeptide comprises at least one residue derived from an aliphatic polyamine containing 1 to 6 carbon atoms, preferably an aliphatic polyamine residue having the structure

-NH(CR,R ) R_

1 2 m 3 wherein R. and R_ may be the same or different and represent hydrogen, C, _j. alkyl or, C. alkoxy and wherein individual groups (CR_R_) may be the same or different, R represents H, NH₂, OH, C00H, C00R' , CHO, COROR", CH_0H, CH DR' , C_^ alkyl, guanidyl or amino-(C. alkyl), wherein each of R' and R" independently represents C, n alkyl and m is an integer from 1 to 4. re e w invention are represented by the formula

R

wherein R₁ , R_ and R, and m are as defined above,

R^ is an acyl group derived from acetyl, propionyl, cis- or trans-oxiranedicarboxylic acid or a

C. alkyl half-ester of cis- or trans-oxirane dicarboxylic acid and

R_ is prop-2-yl, 2-methyl-proD-l-yl or 1-methvl-prop-l-yl. 0

The group -NH-(CR R_) R_ preferably has one of the following structures ϋ*

NH-

CH„

NH-(CH₂)₂-NH-CH^'

CH. and

•NH

NH-CH-(CH ) -NH-C;

'NH,

One example of an inhibitor represented by formula I is the compound known as "E64", an epoxy compound which was originally isolated from Aspergillus japonicus. The structure of E64 is shown below.

Another example of an inhibitor represented by formula I is the compound known as E64c, a derivative of E6 wherein the agmatine residue is replaced by an N-isopropyl (ethylene diamine) residue. The structure of E64c is shown below.

For a discussion of the CANP inhibitor E6 and its analogues (which may also be used in the method of the invention) see "In Vitro and In Vivo Inhibition of Cysteine Proteinases by EST, a New Analogue of E64", Tamai, M. et al. , J. Pharmacobio-Dyn. , 672-677 (1986) and "Inhibitions of E64 Derivatives of Rat Liver Cathepsin B and Cathepsin L In Vitro and In Vivo", Hashida, S. et al. , J. Biochem 88, I8O5-I8H (I98O). ur er c ass o c may e use are e so-called "leupeptins", a class of oligopeptides which have been isolated f om various species of Actinomycetes. One compound in the class is itself known as leupeptin and has the structure N-acetyl-L-leucyl-Lleucyl-L-arginal, i.e.

CH, CO

Other related leupeptins are described in the literature, see e.g. "Isolation and Characterisation of Leupeptins Produced by Actinomycetes", Kondo, S. et al. , Chem. Pharm. Bull. 17(9), 1896-1901 (1969) and "Structures and Syntheses of Leupeptins Pr-LL and Ac-LL", Kawamurak et al. , Chem. Pharm. Bull. 17(9). 1902-1909

(1969).

In the present invention the pharmaceutical preparation ma3^r be in the form of an injectable solution comprising the inhibitor together with a suitable excipient, for example sterile, pyrogen free water or isotonic saline.

Preferably the quantity of CANP inhibitor administered should be sufficient to provided a concentration in the

-~ -z> extracellular fluid of 10 to 5 x 10 ^J g/ml of CANP inhibitor. Most preferably the quantity administered should provide a concentration of 10 to 10 ^ g/ml.

Alternatively, the pharmaceutical preparation may be in the form of an implantable device from which the inhibitor is released. The device preferably comprises a polymer matrix which acts as a non-toxic carrier which releases the inhibitor over a period of time. More specifically the implantable device may comprise a silicone rubber strip containing the inhibitor and can, for example, be formed by mixing the inhibitor in powdered form with a rubber solution and allowing the mixture to set. The^" amount of inhibitor used and the size of the implant will depend on the extent of nerve trauma.

To administer the inhibitor to denervated muscle fibres, the device may be implanted onto the surface of the muscle adjacent the denervated region.

Although the precise mode of action of the inhibitors used in accordance with the invention has not been determined, it is believed that the inhibitor penetrates cell membranes and enters nerve terminals and thus inhibits calcium activated neutral protease at these locations. As a result this inhibition the reinnervation of muscle fibres and synapse formation is promoted.

The following examples involve experiments carried out in rats and illustrate the method of the invention.

In the following experiments the implant strips were prepared by mixing predetermined amounts of the test compound (in powder form) with a calculated quantity of rubber solution (Dow Corning 3 ^0) and allowing the mixture to set overnight. When dry, small strips (about 3 nun x 1 mm x 0.5 mm, weighing 1-1.5 mg) were cut from the resulting flexible strips of rubber. EXAMPLE 1

Effect of administering leupeptin after partial denervation of the soleus muscle in 4-6 day old rats

A group of rats (weight 11-15 g) aged 4-6 days (an age which is prior to the elimination of polyneuronal innervation) were partially denervated by removal of the L v.r.^"0ne day later a second operation was performed to implant a 1 mg silicon rubber strip containing the test compound alongside the soleus muscle on the operated side of the rat adjacent the traumatised nerve. The test compounds in each 1 mg strip were as follows:

Leupeptin 62.5 μg.

Leucine (42 μg) and arginine (20.5 μg) as a mixture having the same molar proportions of these amino acids as present in leupeptin

The effects of the above treatments on the soleus muscle were observed 2-4 months later in the following tests.

a) Maximum Tetanic Tension

The maximum tetanic tension was measured for the operated muscles and expressed as a percentage of the contralateral muscle tension.

The soleus muscles of both legs were dissected free, and the distal tendon attached to a strain gauge, with a silk thread. Isometric contractions were elicited by stimulating the soleus motor nerve via bipolar silver electrodes. A pulse duration of 50us was used, and the voltage was increased to produce maximum contractions. Tetanic tensions (20-100H ; 0.6s) were then measured. The output of the strain gauge was amplified and displayed on an oscilloscope. Leupeptin 89# ± 5-7% (n=l6)

Leu/Arg mixture 70/- ± 6.3% (n=12)

This illustrates an increased recovery of the operated muscle tension for the leupeptin treated group compared to the control group. The difference is significant using a "t" test (0.05 > P >0.02).

b) Mean Motor Unit Size

The mean motor unit size (a parameter indicative of degree of recovery from nerve trauma) was estimated from the oscilloscope traces of twitch tension recordings from the soleus muscle. The number of motor units in each operated and contralateral muscle was determined. This was achieved by gradually increasing the stimulus intensity to the motor nerve, and recording stepwise increments in twitch tension, due to the successive recruitment of individual motor units, by stimulating axons with different thresholds. This was continued until a plateau was reached when all the motor axons were recruited and the tension could not be increased further. The average motor unit size was then found by dividing the number of motor units found in the operated muscle into the maximum tetanic tension (g) for that muscle. This value can then be expressed as a percentage of the mean size of a motor unit from the contralateral muscle, obtained by the same method.

The leupeptin treated operated muscles showed a 3"fold increase of mean motor unit size compared to their contralateral muscles, whereas the control group showed only a 2-fold increase. The difference is significant using a "t" test (0.02 > p >0.01) . c) Pattern of Innervation

Sections of the operated and contralateral soleus muscles stained using the combined Ag/Acetylcholine esterase method showed that the total amount of axonal sprouting in the leupeptin treated and control groups was very low.

EXAMPLE 2

Effect of leupeptin after partial denervation of the soleus muscle in 17-19 day old rats

A group of rats (weigh 27~39 g) aged 17-19 days (an age which is after the elimination of polyneural innervation) were partially denervated and one day later the test (leupeptin) and control (leucine/arginine) strips were implanted as described in Example 1.

The effects of the treatments on the soleus muscle were observed 2 weeks later, and in another case 2 months later, to observe the longer term effect of leupeptin in rats of this age group.

a) Maximum Tetanic Tension

Expressed as a percentage of the contralateral muscle tension the results were as follows:

2 Weeks

Leupeptin 89.5% ± 8.9 (n=ll)

Leu/Arg 61.6% ± 8.9 (n=7)

This difference is significant using "t" test (0.05 > p >0.02)

2 Months

Leupeptin 89..' Thus, as in the younger group of rats in Example 1, leupeptin increased the recovery of the operated muscle tension compared to the control group. The maximum tentanic tension at 2 months for the leupeptin operated muscle was very similar to the results obtained at 2 weeks which suggests that the improvement, caused by treatment with leupeptin is permanent.

b) Mean Motor Unit Size

As in Example 1, the leupeptin treated operated muscles showed a 3-fold increase of mean motor unit size compared to their contralateral muscles which was greater than that shown by the control partially denervated soleus muscles.

c) Pattern of Innervation

Stained sections of the operated and contralateral soleus muscles showed that the total amount of sprouting in the operated muscles following leupeptin treatment is 32.5% ± 1.9 (n=5) compared to 21% in the Leu/Arg treated muscles.

Thus it may be concluded that the increase in muscle tension in the leupeptin treated rats operated at 17-19 days is achieved by an enlargement of the territory occupied by the motorneurones due to an increase in sprouting of their motor axons. EXAMPLE 3

Effect of Leupeptin On Unoperated Soleus Muscle

By way of comparison, a long silicon rubber strip containing leupeptin (62.5 μg) was implanted onto the surface of a non- denervated soleus muscle of d y old rats.

Leupeptin was found to have no significant effect on maximum tetanic tension, mean motor unit size or the pattern of innervation.

Claims

1. The use of an inhibitor of calcium activated neutral protease (CANP) in the manufacture of a pharmaceutical composition or device for use in promoting synapse formation and innervation of muscle fibers.

2. The use claimed in Claim 1 in the manufacture of a pharmaceutical composition or device for use in promoting synapse formation and reinnervation of muscle fibers following nerve trauma synapse formation and reinnervation of muscle fibres following nerve trauma.

3. The use claimed in Claim 1 or Claim 2 wherein the inhibitor of CANP is an oligopeptide comprising at least one amino acid residue.

4. The use claimed in Claim 3 wherein said at least one amino acid residue is a branched chain amino acid selected from valine, leucine and isoleucine.

5. The use claimed in Claim 3 or Claim 4 wherein the N-terminus of said oligopeptide is blocked.

6. The use claimed in Claim wherein the N-terminus is blocked by an acyl group derived from a C. r carboxylic acid.

7- The use claimed in Claim 6 wherein the N-terminus is blocked by an acyl group derived from acetyl, propionyl, cis- or trans- oxirane dicarboxylic acid or a C. _ alkyl half-ester of cis- or trans-oxirane dicarboxylic acid.

8. The use claimed in any of Claims 3 to 7 wherein the oligopeptide comprises at least one residue derived from an aliphatic polyamine containing 1 to 6 carbon atoms. 9, The use claimed in Claim 8 wherein the aliphatic polyamine residue has the structure

-NHΪCR^)^ wherein R₁ and R_ may be the same or different and represent hydrogen, C. _j, alkyl or C ^ alkoxy and wherein individual groups (CR_R_) may be the same or different, R represents H, NH_, OH, C00H, C00R' , CHO, COROR", CH-OH, CH-OR', C. _j, alkyl, guanidyl or amino-(C,_, alkyl), wherein each of R' and R" independently represents C, _ , alkyl and m is an integer from 1 to 4.

10. The use as claimed in any preceding claim wherein the inhibitor is represented by the formula

R - CO- NH-CIΪ-C0- NH-(CR,R R_n 1 2 m 3

R. n

wherein R. , R_ and R_ and m are as defined in Claim 8.

R^ is an acyl group derived from acetyl, propionyl, cis- or trans-oxirane dicarboxylic acid or a C, i, alkyl half-ester of cis- or trans-oxirane dicarboxylic acid, and

R_^ is prop-2-yl, 2-methyl-prop-l-yl or 1-methyl-prop-l-yl. 11. The use as claimed in Claim 8 or Claim 9 wherein the group

-NH- CR. R-,) R_ has one of the following structures 1 m 3

NH-

and

12. The use claimed in Claim 1 or Claim 2 wherein the inhibitor is E64.

13- The use claimed in Claim 1 or Claim 2 wherein the inhibitor is E64c.

14. The use claimed in Claim 1 or Claim 2 wher iii the inhibitor is leupeptin.

15. The use claimed in any of Claims 1 to 14 wherein the inhibitor is in the form of an injectable solution which comprises the inhibitor together with a suitable excipient.

16. The use claimed in any of Claims 1 to 14 wherein the inhibitor is in the form of an implantable device.

17. The use claimed in Claim 16 wherein said implantable device comprises the inhibitor dispersed in a polymer matrix.

18. The use claimed in Claim 17 wherein said polymer matrix is silicone rubber. 19. A method of promoting synapse formation and innervation of muscle fibres which comprises administering an inhibitor of calcium activated neutral protease (CANP) to provide an effective concentration of said inhibitor to the at least partially denervated area of a muscle.

20. A method of promoting synapse formation and reinnervation of muscle fibres following nerve trauma which comprises administering an inhibitor of calcium activated neutral protease (CANP) to provide an effective concentration of said inhibitor to the at least partially denervated area of a muscle.

21. A method according to Claim 19 wherein the inhibitor of CANP is as defined in any of Claims 3 to 14.

22. A method according to any of Claims 19 to 21 wherein the inhibitor is in the form of an injectable solution which comprises the inhibitor together with a suitable excipient.

23. A method according to any of Claims 18 to 20 wherein the inhibitor is in the form of an implantable device.

24. - A method according to Claim 23 wherein said implantable device comprises the inhibitor dispersed in a polymer matrix.

2 . A method according to Claim 24 wherein said polymer matrix is silicon rubber.