U.S. patent application number 11/515156 was filed with the patent office on 2007-03-22 for treatment of wounds.
This patent application is currently assigned to The Secretary of State for Defence. Invention is credited to David Idris Pritchard.
Application Number | 20070066538 11/515156 |
Document ID | / |
Family ID | 10863184 |
Filed Date | 2007-03-22 |
United States Patent
Application |
20070066538 |
Kind Code |
A1 |
Pritchard; David Idris |
March 22, 2007 |
Treatment of wounds
Abstract
An isolated protein, for use in treatment of wounds, is
characterized in that it is secreted by the organism Lucilia
sericata and it exhibits proteolytic activity against FITC-casein
at a pH of 8.0 to 8.5. The protein exhibits proteolytic activity
against Tosyl-Gly-Pro-Arg-AMC but not against Suc-Ala-Ala-Phe-AMC,
and its proteolytic activity against FITC-casein and
Tosyl-Gly-Pro-Arg-AMC is inhibited by the serine proteinase
inhibitors PMSF and AMPSF. The protein is also bound by immobilized
aminobenzamidine.
Inventors: |
Pritchard; David Idris;
(Nottingham, GB) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
The Secretary of State for
Defence
Salisbury
GB
|
Family ID: |
10863184 |
Appl. No.: |
11/515156 |
Filed: |
August 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10111252 |
Aug 23, 2002 |
7144721 |
|
|
PCT/GB00/04034 |
Oct 20, 2000 |
|
|
|
11515156 |
Aug 31, 2006 |
|
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|
Current U.S.
Class: |
424/93.4 ;
424/94.64; 435/212; 514/21.2; 514/21.8; 514/9.4; 530/329 |
Current CPC
Class: |
A61L 15/32 20130101;
A61K 38/00 20130101; C07K 14/43577 20130101 |
Class at
Publication: |
514/017 ;
424/094.64; 530/329; 435/212 |
International
Class: |
A61K 38/08 20060101
A61K038/08; C07K 7/06 20060101 C07K007/06; A61K 38/48 20060101
A61K038/48; C12N 9/48 20060101 C12N009/48 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 22, 1999 |
GB |
GB 9925005.2 |
Claims
1. (canceled)
2. The method of claim 6, wherein the isolated protein has a
molecular weight of approximately 25 kDa.
3. (canceled)
4. The method of claim 6, wherein the isolated protein is secreted
by an insect in its larval stage.
5. (canceled)
6. A method for treating a wound to promote healing thereof in a
human or non-human mammal, which comprises applying to the wound a
therapeutically effective amount of: (a) an isolated protein
characterized in that: i) it is secreted by the organism Lucilia
sericata; ii) it exhibits optimum proteolytic activity against
FITC-casein at a pH of 8.0 to 8.5; iii) it exhibits proteolytic
activity against Tosyl-Gly-Pro-Arg-AMC but not against
Suc-Ala-Ala-Phe-AMC; iv) its proteolytic activity against
FITC-casein and Tosyl-Gly-Pro-Arg-AMC is inhibited by the serine
proteinase inhibitors PMSF and APMSF; and v) it is bound by
immobilized aminobenzamidine; or (b) one or more peptides selected
from the group consisting of Ser-Phe-Leu-Leu-Arg-Asn (SEQ ID NO:
1); Ser-Leu-Ile-Gly-Lys-Val (SEQ ID NO: 2); Thr-Phe-Arg-Gly-Ala-Pro
(SEQ ID NO: 3); Gly-Tyr-Pro-Gly-Gln-Val ((SEQ ID NO: 4); a peptide
having as an N-terminal sequence Ser-Phe-Leu-Leu-Arg-Asn (SEQ ID
NO: 1), Ser-Leu-Ile-Gly-Lys-Val (SEQ ID NO: 2),
Thr-Phe-Arg-Gly-Ala-Pro (SEQ ID NO: 3), or Gly-Tyr-Pro-Gly-Gln-Val
(SEQ ID NO: 4); and analogues thereof which are protected against
aminopeptidase activity.
7. The method of claim 6 wherein the isolated protein or peptide is
applied as a sterile composition comprising a therapeutically
effective amount of the isolated protein or peptide and a sterile
carrier therefore.
8. (canceled)
9. (canceled)
10. An isolated peptide selected from the group consisting of
Ser-Phe-Leu-Leu-Arg-Asn (SEQ ID NO: 1); Ser-Leu-Ile-Gly-Lys-Val
(SEQ ID NO: 2); Thr-Phe-Arg-Gly-Ala-Pro (SEQ ID NO: 3);
Gly-Tyr-Pro-Gly-Gln-Val (SEQ ID NO: 4); a peptide having as an
N-terminal sequence Ser-Phe-Leu-Leu-Arg-Asn (SEQ ID NO: 2),
Ser-Leu-Ile-Gly-Lys-Val (SEQ ID NO: 2), Thr-Phe-Arg-Gly-Ala-Pro
(SEQ ID NO:3), or Gly-Tyr-Pro-Gly-Gln-Val (SEQ ID NO: 4); and
analogues thereof which are protected against aminopeptidase
activity
11. (canceled)
12. (canceled)
13. A dressing for a wound, which comprises a sterile support and
isolated protein characterised in that: i) it is secreted by the
organism Lucilia sericata; ii) it exhibits optimum proteolytic
activity against FITC-casein at a pH of 8.0 to 8.5; iii) it
exhibits proteolytic activity against Tosyl-Gly-Pro-Arg-AMC but not
against Suc-Ala-Ala-Phe-AMC; iv) its proteolytic activity against
FITC-casein and Tosyl-Gly-Pro-Arg-AMC is inhibited by the serine
proteinase inhibitors PMSF and APMSF; and v) it is bound by
immobilised aminobenzamidine.
14. A dressing for a wound, which comprises a sterile support and
at least one peptide according to claim 10.
15. A protein which is protected against aminopeptidase activity,
wherein the protein is characterized in that: i) it is secreted by
the organism Lucilia sericata; ii) it exhibits optimum proteolytic
activity against FITC-casein at a pH of 8.0 to 8.5; iii) it
exhibits proteolytic activity against Tosyl-Gly-Pro-Arg-AMC but not
against Suc-Ala-Ala-Phe-AMC; iv) its proteolytic activity against
FITC-casein and Tosyl-Gly-Pro-Arg-AMC is inhibited by the serine
proteinase inhibitors PMSF and APMSF; and v) it is bound by
immobilized aminobenzamidine.
16. A protein which is protected against aminopeptidase activity,
wherein the protein comprises at least one peptide according to
claim 10.
17. The protein of claim 15 wherein protection against
aminopeptidase activity is achieved by amidation at the COOH
substitution in the protein using a non-coded anomalous amino
acid.
18. The protein of claim 15 wherein protection against
aminopeptidase activity is achieved by replacing a CO--NH bond by
an isostere.
19. The protein of claim 15 wherein protection against
aminopeptidase activity is achieved by amidation at the COOH
substitution in the protein using a non-coded anomalous amino acid
and by replacing a CO--NH bond by an isostere.
20. A composition for the treatment of wounds comprising a
therapeutically effective amount of a protein according to claim 6
and a sterile carrier therefore.
21. The composition of claim 20 further comprising a material
capable of delivering the protein to a wound in a slow release or
controlled release manner.
22. The composition of claim 21 wherein the material is
poly(lactide-co-glycolide) or particles of PLGA.
23. The composition of claim 20 wherein the protein is protected
against aminopeptidase activity.
24. The dressing of claim 13 wherein the protein is protected
against aminopeptidase activity.
25. The dressing of claim 13 further comprising slow-release
colloidal particles.
26. The dressing of claim 13 further comprising sponges.
27. The dressing of claim 13 which is over layered by a
conventional dressing.
Description
[0001] This application is a division of application Ser. No.
10/111,252, filed on Aug. 23, 2002, issuing, which is a U.S.
national phase application, pursuant to 35 U.S.C. .sctn.371, of PCT
international application Ser. No. PCT/GB00/04034, filed on Oct.
20, 2000, designating the United States and published in English on
May 3, 2001 as publication WO 01/31033 A2, which claims priority to
application Ser. No. GB 9925005.2, filed on Oct. 22, 1999. The
entire contents of the aforementioned patent applications are
incorporated herein by this reference.
TECHNICAL FIELD
[0002] The present invention relates to the treatment of wounds.
More particularly, it relates to substances which promote the
healing of wounds, to compositions and to dressings which
incorporate such substances and to a method of treating wounds
using such substances.
BACKGROUND TO THE INVENTION
[0003] Efficient wound healing is a complex physiological process
which involves many mechanisms including cell migration, growth
factor secretion, angiogenesis, tissue remodelling and the
intrinsic proteinase/antiproteinase balance of the wound
contributing in concert and in an apparently staged manner to
accelerate controlled tissue regeneration.
[0004] Wound care products are essential in modern medical
practice, especially for the treatment of patients with chronic
wounds or burns. Many different substances have previously been
proposed as having activities which contribute to the healing of
wounds. These previously proposed substances include streptokinase,
collagenase and streptodornase (all obtained from bacterial
sources), bromelain (from pineapples), plasmin and trypsin
(obtained from cattle) and krill enzymes (obtained from crustacea).
Clinical trial data indicate that such substances are only
partially effective in promoting the healing of wounds.
[0005] The larvae (maggots) of the green bottle fly, Lucilia
sericata, are known to have significant wound healing attributes as
live organisms. Debridement treatment using the larvae of Lucilia
sericata, has become a widely accepted clinical practice. However,
little has been reported in the literature about the way in which
these larvae go about their task of cleaning wounds to an extent
that conventionally untreatable wounds heal.
[0006] Although efficacious, live larvae are unpleasant to many
patients and the use of live larvae on wounds and the introduction
of their crude secretions into wounds, which inevitably occurs when
the larvae are used, are unacceptable to many patients and to many
medical practitioners. The use of live organisms also increases the
risk of infection or allergic reactions in the patient.
[0007] It is an object of the invention to overcome at least some
of the above problems.
SUMMARY OF THE INVENTION
[0008] We have now purified an enzyme which occurs naturally in the
crude secretions of the larvae of Lucilia sericata, and which
exhibits trysin-like serine proteinase activity and wound-healing
promotion activity. The use of the purified enzyme in the treatment
of a wound avoids the need to bring live organisms and/or their
crude secretions into contact with the wound.
[0009] The present invention provides an isolated protein
characterised in that: [0010] i) it is secreted by the organism
Lucilia sericata; [0011] ii) it exhibits optimum proteolytic
activity against FITC-casein at a pH of 8.0 to 8.5; [0012] iii) it
exhibits proteolytic activity against Tosyl-Gly-Pro-Arg-AMC but not
against Suc-Ala-Ala-Phe-AMC; [0013] iv) its proteolytic activity
against FITC-casein and Tosyl-Gly-Pro-Arg-AMC is inhibited by the
serine proteinase inhibitors PMSF and APMSF; and [0014] v) it is
bound by immobilised aminobenzamidine.
[0015] The invention also provides an isolated protein,
characterised in that it is a functional homologue of the protein
of the invention. Typically the protein will be a naturally
produced protein secreted by an insect in its larval stage.
Suitable insect are envisaged as being those that are used in their
larval form in the treatment of wounds. It is also envisaged that
clones of either the Lucilia protein, or functional homologues
thereof, will form part of the invention and may be used in wound
treatment. In this regard, the protein of the invention may be
expressed in bulk in an active recombinant form in an
insect/baculovirus expression system.
[0016] In a further aspect, the invention relates to a method for
treating a wound to promote healing thereof in a human or non-human
mammal which comprises applying to the wound a therapeutically
effective amount of a sterile composition comprising a protein
according to the invention as active ingredient. The invention also
relates to the use of a protein of the invention in the preparation
or manufacture of a medicament for the treatment of wounds.
[0017] In a further aspect, the invention relates to an isolated
peptide selected from the group consisting of;
Ser-Phe-Leu-Leu-Arg-Asn (SEQ ID NO: 1); Ser-Leu-Ile-Gly-Lys-Val
(SEQ ID NO: 2); Thr-Phe-Arg-Gly-Ala-Pro (SEQ ID NO: 3);
Gly-Tyr-Pro-Gly-Gln-Val (SEQ ID NO: 4), and a peptide having an
N-terminal sequence selected from: Ser-Phe-Leu-Leu-Arg-Asn (SEQ ID
NO: 1); Ser-Leu-Ile-Gly-Lys-Val (SEQ ID NO: 2);
Thr-Phe-Arg-Gly-Ala-Pro (SEQ ID NO: 3); or Gly-Tyr-Pro-Gly-Gln-Val
(SEQ ID NO: 4), or a protected analogue thereof which is protected
against aminopeptidase activity, and to the use of one or more of
such peptides in a method for treating a wound to promote healing
thereof in a human or non-human mammal which comprises applying to
the wound one or more of said peptides. Furthermore, the invention
relates to the use of such a peptide in the preparation or
manufacture of a medicament for the treatment of wounds.
[0018] In one embodiment of the invention, there is provided a
dressing for a wound, which comprises a sterile support carrying a
therapeutically effective amount of a protein and/or at least one
peptide according to the invention.
Where the words "comprises" and "comprising" are used herein, it is
intended that these may have the meanings "includes" and
"including", respectively, to the extent that other procedures or
other materials are not excluded.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is an elution profile of Lucilia sericata
excretory/secretory (ES) having been passed through an affinity
column.
[0020] FIG. 2 shows the residual activity of the column fractions
identified in FIG. 1.
[0021] FIG. 3 shows a picture of a gel after electrophoresis of the
column fractions identified in FIG. 1.
[0022] FIG. 4 shows FITC-Casein hydrolysis by Lucilia sericata ES
in the presence of APMSF/PMSF at pH8.
[0023] FIG. 5 demonstrates (a) Tosyl-Gly-Pro-Arg-AMC and (b)
Suc-Ala-Ala-Phe-AMC hydrolysis by Lucilia sericata in the presence
of APMSF and PMSF.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The protein of the present invention exists, in nature, in
the excretory/secretory (ES) secretions of the larvae of Lucilia
sericata.
[0025] The larval ES secretions demonstrate a classical pH optimum
of 8.0-8.5 when hydrolysing the fluorescent protein substrate
fluorescein isothiocyanate-casein (FITC-casein). By pre-incubating
the larval ES secretions, prior to monitoring the hydrolysis of
FITC-casein, with the irreversible low molecular weight inhibitors
4-(amidinophenyl) methane sulphonyl fluoride (APMSF; an inhibitor
for all trypsin-like serine proteases but not chymotrypsin-like
serine proteinases) or with phenyl methanesulphonyl fluoride (PMSF;
an inhibitor for all serine proteinases) it is shown that larval ES
secretions have two types of serine proteinase activity; a
trypsin-like activity and a chymotrypsin-like activity. The dual
activity is confirmed by monitoring the hydrolysis of the
fluorescent peptide substrates Tosyl-Gly-Pro-Arg-AMC (selective for
trypsin-like proteinases) and Suc-Ala-Ala-Pro-Phe-AMC (SEQ ID NO:
5) (selective for chymotrypsin-like proteinases), in which "AMC"
represents 7-amino-4-methyl coumarin and "Suc" represents
succinyl.
[0026] In addition to the predominant serine proteinase activity
detected in the ES secretions of Lucilia sericata other less
predominant activity is present. The presence of an aspartyl and
metalloproteinase activity has been detected though no cysteinyl
activity is shown. The aspartyl activity, shown by monitoring
FITC-casein hydrolysis, is pronounced at pH 5.0 and is successfully
inhibited by the class specific inhibitor pepstatin A. The
metalloproteinase activity present is demonstrated by the ability
of the ES secretions to hydrolyse a leucine aminopeptide, revealing
the presence of an exopeptidase. Exopeptidases recognise free
--NH.sub.2 aminoacids in peptides. Leucine aminopeptide hydrolysis
by Lucilia sericata ES is only inhibited by the Zn.sup.2+ chelator
1,10-phenanthroline, a classic metalloproteinsase inhibitor. This
inhibition reflects the presence of an exopeptidase with a
metalloproteinase enzymic nature.
[0027] The ES secretions have an .alpha.-amylase activity
calculated to be about 0.88 units/litre. Additionally, phosphatase
activity (hydrolysis of orthophosphoric monoester bond) is present
in the larval ES secretions although this activity is approximately
50 times lower when compared to the proteinases. Lipase activity
(hydrolysis of ester bonds found in fatty acid esters) is also
identified. This lipase activity is not detected when the ES
secretions are pre-incubated with the inhibitor PMSF, indicating
that this hydrolysis is due to the serine proteinase in the
secretions.
[0028] It can be concluded from our investigations that the
predominant class of activity in the larval ES secretions is serine
proteinase activity and that there are two types of serine
proteinase activity present; one derived from a chymotryptic enzyme
and one derived from a tryptic enzyme.
[0029] The protein of the present invention, which relates to the
tryptic enzyme, may be obtained in substantially pure form from the
crude ES secretions by a chromatographic procedure. The ES
secretions are collected from the larvae of Lucilia sericata and
are subjected to affinity chromatography using immobilised
aminobenzamidine. Aminobenzamidine is a reversible inhibitor of
trypsin-like serine proteinases. After collection of the
"flow-through" material from the chromatographic procedure, i.e.,
the material which is not bound by the immobilised reagent, the
enzyme which has been bound by the immobilised reagent may be
eluted by the addition of free aminobenzamidine and collected
separately.
[0030] Inhibitor studies carried out on the unbound
("flow-through") fraction and the eluted fraction using the
inhibitors APMSF and PMSF show that the unbound material has
activity characteristic of chymotryptic enzymes whereas the eluted
fraction is a tryptic serine proteinase.
[0031] We believe that tryptic serine proteinase, isolated from the
crude larval ES secretions as described above, has the ability to
initiate and promote healing events in wounds. We have found that
larval ES secretions are capable of degrading the extra cellular
matrix (ECM)/wound components, fibronectin, laminin and collagens
I, III, IV and V. These macromolecules are found in the slough of
chronic wounds and also make up the "fibrin cuffs" that are
predominant in chronic ulcers. The degradation of laminin and
fibronectin by larval secretions is inhibited by PMSF, but not
significantly by APMSF or by the metalloproteinase inhibitor
1,10-phenanthroline.
[0032] In addition, the degradation of the different forms of
collagen by the predominant serine proteinase activity indicates
the presence of a collagenase. This suggests that the serine
proteinase activity within larval ES secretions has the potential
to degrade key wound components, a requirement for the debridement
stage of healing, and is involved in preventing "fibrin cuff"
formation, stimulating angiogenesis, leucocyte invasion and
fibroplast proliferation. In contrast to the ECM components
degraded, no degradation of fibrin itself, elastin and hyaluronic
acid by ES secretions has been detected, suggesting the absence of
plasmin or elastase-like proteinase or hyaluronidase.
[0033] The trypsin-like enzyme isolated from crude larval ES
secretions, we believe, has the ability to activate healing
processes via protease-activated receptors (PARs). PAR1, which is
known to be cleaved by thrombin, trypsin and factor Xa, is present
on human endothelial cells, fibroblasts, keratinocytes, platelets,
monocytes and smooth muscle. PAR1 agonists promote endothelium
dependent vasodilation, leading to extravasation of plasma proteins
and leucocytes, to cell mitogenesis, and to the healing of inflamed
and damaged tissue. The PARs have attached ligands which are
cleaved proteolytically by the trypsin-like enzyme of the invention
to release peptides which then act to trigger other biochemical
processes involved in promoting wound healing. The arginine residue
at the cleavage site of the tethered ligand of PAR1 is susceptible
to the action of trypsin-like proteinases. Further details of the
tethered ligands and the elucidation of their sequence is given in
Am. J. Physiol. 274 (1998), c1428-c1452.
[0034] As mentioned above, PARs have tethered ligands which when
cleaved by the trypsin-like serine proteinase release one or more
peptides into the wound area.
[0035] Normally tethered ligands of PARs which are believed, by us,
to be responsible for the activation of wound healing when released
include the peptides 1 to 4 below TABLE-US-00001 1.
Ser-Phe-Leu-Leu-Arg-Asn; (SEQ ID NO: 1) 2. Ser-Leu-Ile-Gly-Lys-Val;
(SEQ ID NO: 2) 3. Thr-Phe-Arg-Gly-Ala-Pro; (SEQ ID NO: 3) and 4.
Gly-Tyr-Pro-Gly-Gln-Val. (SEQ ID NO: 4)
[0036] These four peptides are agonists of PARs 1 to 4,
respectively.
[0037] We believe that wound healing is promoted by the application
to the wound area of one or more of these peptides, or a protected
analogue thereof which is protected against aminopeptidase
activity, without the need to apply the tryptic serine proteinase
to the wound.
[0038] The peptides of the invention, as described above, can be
prepared synthetically and purified according to the usual routes
of peptide synthesis and purification known in the art. The peptide
may be protected against aminopeptidase activity to enhance
activity and/or to prolong the period within which the peptide
remains active in the wound area. Protection against aminopeptidase
activity may, for example, be achieved by the amidation at COOH
substitution in the peptide using a non-coded anomalous amino acid
and/or CO--NH amide bond replacement by an isostere.
[0039] The peptides (and/or protein) of the invention may be
applied to a wound to induce a profile of growth factors conducive
to healing. For instance, one or more peptides, either in a pure
form or in a sterile carrier, can be sprinkled over the wound area
or incorporated into a carrier to be applied to the wound. For
instance, the peptide (and/or protein) can be incorporated or
encapsulated into a suitable material capable of delivering the
peptide to a wound in a slow release or controlled release manner.
An example of such a suitable material is
poly(lactide-co-glycolide) or PLGA particles which may be
formulated to release peptides in a controlled release manner.
Alternatively, one or more peptides (and/or protein) may be
incorporated into a dressing to be applied over the wound. Examples
of such dressings include staged or layered dressings incorporating
slow-release hydrocolloid particles containing the wound healing
material or sponges containing the wound healing material
optionally overlayered by conventional dressings. Hydrocolloid
dressings of the type currently in use, for example those available
under the trademark "Granuflex", may be modified to release the
peptides to the wound.
EXPERIMENTAL METHODS
1. Isolation and Assay of the Trypsin-Like Serine Proteinase of the
Invention
[0040] The trypsin-like serine proteinase was purified by affinity
chromatography of Lucilia sericata ES on aminobenzamidine agarose.
The column matrix (1 ml) was equilibrated with 20 ml of 0.025M
Tris-HCl buffer pH 8.0 containing 0.5M NaCl. The crude ES (0.5 ml,
70 .mu.g/ml protein) was diluted with an equal volume of buffer
before application to the column. Fractions (0.5 ml) were collected
throughout the chromatography. After washing with 6.5 times column
volume of buffer to remove unbound protein, the free
aminobenzamidine ligand (2 ml 400 .mu.M) was used to elicit the
elution of bound material. Absorbance readings of the fractions at
280 nm was used to establish the positions of the unbound
(flow-through) and bound peaks which were then collected for assay.
The elution profile is shown in FIG. 1
[0041] Aminobenzamidine agarose binds trypsin-like serine
proteinases. Following application of larval enzyme secretions to
the column, unbound material passed directly through and was
collected as "flow-through" (peak I). The addition of free
aminobenzamidine to the column buffer elicited elution of the bound
proteinase (peak II). The unbound (flow-through) material contained
proteinase activity unaffacted by APMSF (possibly including a
chrymotrypsin-like enzyme), whereas the activity in the
aminobenzamidine elution peak was substantially abolished (80%) by
APMSF, indicating purification of a trypsin-like serine proteinase
activity. The residual activities of the column fractions are shown
in FIG. 2.
[0042] Column fractions were examined by electrophoresis in
non-reducing SDS sample buffer (0.5M Tris-HCl pH 6.8 containing 4%
SDS, 20% glycerol and 0.02% bromophenol blue) on 12% SDS
polyacrylamide gels containing 0.1% human haemoglobin. SDS was
removed by washing in 2.5% Triton X-100 (1 h) and distilled water
(15 min.). Proteolysis of the haemoglobin substrate in the gel by
incubation at 37.degree. C. in 0.1M Tris-HCl buffer pH 8.0
overnight produced clear bands revealed by protein staining in
Coomassie Brilliant blue corresponding to the positions of
proteinase enzymes (FIG. 3). The start and flow through fractions
each showed several proteinase activities however the
aminobenzamidine eluted a single band. Thus the trypsin-like enzyme
previously identified in the aminobenzamidine-eluted fraction (FIG.
2) was shown to have molecular weight .about.25 KDa (FIG. 3).
2. Investigation of Proteolytic Behaviour of the Larval Enzyme (ES)
with FITC-casein
[0043] The activity of Lucilia sericata ES in FITC-casein
hydrolysis at pH8 was investigated using different presentations of
ES (0.25 .mu.g) as follows:
[0044] A. ES+H.sub.2O
[0045] B. ES+ethanol
[0046] C. ES pre-incubated with 0.2 mM PMSF
[0047] D. ES pre-incubated with 0.6 mM PMSF
[0048] E. ES pre-incubated with 1 mM PMSF
[0049] F. ES pre-incubated with 0.04 mM APMSF
[0050] G. ES pre-incubated with 0.12 mM APMSF
[0051] H. ES pre-incubated with 0.2 mM APMSF
[0052] The proteolytic activity of Lucilia sericata ES was
inhibited following pre-incubation with the irreversible serine
proteinase inhibitor PMSF. It was totally inhibited in the case
where the ES had been pre-incubated with 1 mM PMSF. PMSF is
dissolved in ethanol and the effect of the solvent on the activity
of the ES was negligible. In contrast, approximately 50% of
residual serine proteinase activity from ES was detected in the
cases where the ES had been pre-incubated with the irreversible
"trysin-like" specific inhibitor APMSF. Residual activity in the
presence of APMSF indicates the presence of a chymotrypsin-like
enzyme. The activity (%) values obtained were as follows:
[0053] A. 100%
[0054] B. 85.5%
[0055] C. 13.8%
[0056] D. 18%
[0057] E. 0%
[0058] F. 43.5%
[0059] G. 47%
[0060] H. 54%
[0061] These results are shown graphically in FIG. 4.
3. Investigation of the Proteolytic Activity of the Larval Enzyme
(ES) Against Specific Substrates
[0062] The activity of Lucilia sericata ES (0.25 .mu.g) against
Tosyl-Gly-Pro-Arg-AMC (a) and against Suc-Ala-Ala-Phe-AMC (b) in
the presence of APMSF and PMSF was investigated using different
presentations of ES as follows:
[0063] (a) [0064] A. ES [0065] B. ES pre-incubated with 0.025 mM
APMSF [0066] C. ES pre-incubated with 0.05 mM APMSF [0067] D. ES
pre-incubated with 1 mM PMSF
[0068] (b) [0069] E. ES [0070] F. ES pre-incubated with 0.2 mM
APMSF [0071] G. ES pre-incubated with 1 mM PMSF
[0072] The residual activity (%) values obtained were as
follows:
[0073] (a) [0074] A. 100% [0075] B. 14.3% [0076] C. 3.6% [0077] D.
0%
[0078] (b) [0079] E. 100% [0080] F. 86.8% [0081] G. 1.3%
[0082] The results are shown graphically in FIG. 5.
[0083] The results for (a) reveal the "trypsin-like" serine
proteinase activity present in Lucilia sericata ES. The hydrolysis
of Tosyl-Glyc-Pro-Arg-AMC (selective for the serine proteinases
thrombin and plasmin) was inhibited by 1 mM PMSF and 0.05 mM APMSF.
However, the hydrolysis of the chymotryptic substrate
Suc-Ala-Ala-Phe-AMC by Lucilia sericata ES was only inhibited by
PMSF (1 mM) and not by excess APMSF (which does not inhibit
chymotrypsin). The results provide further evidence of the presence
in ES of two different sub-classes of serine proteinase.
[0084] As mentioned above, the invention also relates to functional
homologues of the Lucilia protein of the invention which may be
obtained from other insects having larval phases and which can
potentially be used in the treatment of wounds. For example, given
the teaching of the present application, it would be within the
ambit of the skilled addressee, using the experimental methods
disclosed herein, to assay larval secretions from other non-Lucilia
organisms for a functional homologue of the Lucilia protein of the
invention. Furthermore, proteinases according to the invention
produced by Lucilia which promote wound healing may be cloned and
expressed in bulk in an active recombinant form in an
insect/baculovirus expression system. Genes corresponding to the
proteinases will be identified by PCR or immunological screening
from appropriate cDNA libraries and manipulated for expression in
this system. As proteinases from diverse sources can prove
difficult to express in prokaryotic expression systems, it is
advantageous to use an insect expression system for the expression
of the insect gene corresponding to the proteinase of the
invention.
[0085] The invention is not limited to the embodiment hereinbefore
described which may be varied without departing from the invention.
Sequence CWU 1
1
5 1 6 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 1 Ser Phe Leu Leu Arg Asn 1 5 2 6 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 2 Ser
Leu Ile Gly Lys Val 1 5 3 6 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 3 Thr Phe Arg Gly Ala Pro 1 5
4 6 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 4 Gly Tyr Pro Gly Gln Val 1 5 5 4 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide
MOD_RES (1) Suc-Ala 5 Ala Ala Pro Phe 1
* * * * *