Preparation with collagenolytic activity having high activity and pharmaceutical compositions containing it

The invention relates to a pharmaceutical composition containing in association with a pharmaceutical vehicle a collagenase of high specific activity and capable of being inhibited at least partly by myosine. It is useful for the treatment of pathologies manifested by an uncontrolled alteration of the collagen-rich structures in man or animal.

This application is a continuation of International Application No. 
PCT/FR84/00004 filed Jan. 5, 1984, now abandoned. 
The invention relates to a preparation with collagenolytic activity, more 
particularly to pharmaceutical compositions containing this preparation, 
in association with a pharmaceutical vehicle, particularly for use in the 
treatment of pathologies manifested by an uncontrolled alteration in the 
structures rich in collagen, in man or animal. 
Several types of micro-organisms producing collagenolytic enzyme or 
"collagenase" have already been described in the literature. It is however 
often difficult to control the active constituent contents of these 
preparations. These preparations can besides not be free of a certain 
number of toxic constituents, for example neurotoxins, which render them 
unsuited for use in compositions intended to be pharmaceutical. 
It is an object of the invention to provide a pharmaceutical composition 
permitting selective enzymatic activity with respect to pathogenic 
collagenic structures when applied to lesions manifested by uncontrolled 
alteration of collagen. It is also an object to produce compositions in 
which the relative proportions of the principal constituents of the 
collagenolytic preparation are equilibrated in predetermined proportions 
as a function of the quality of the desired therapeutic results. 
The pharmaceutical composition according to the invention comprises an 
effective dose of a collagenase of high specific activity recognising 
specifically an X-glycyl-L-prolyl peptide sequence, in which X is a 
natural amino acid residue linked to the N-terminal end of the glycyl 
residue. The peptide sequence is cut specifically by the collagenase at 
the X-glycyl linkage. Moreover, the collagenase is inhibited at least 
partly by myosin. 
A preferred collagenase for use in the pharmaceutical composition according 
to the invention can be obtained from cultures of Vibrio alginolyticus 
chemovar iophagus, of which the principal taxonomic characteristics will 
be recalled below, or from any micro-organisms derived from the latter or 
capable like the latter of producing such a collagenase. More generally 
still, collagenases which can be employed in the pharmaceutical 
composition according to the invention are those which are capable of 
reacting with selective antibodies prepared against the collagenase called 
"Achromobacter collagenase" listed under the number EC. 3.4.24.8 in 
"Enzyme Nomenclature Edition IU-IUB (1978). This collagenase is 
obtained from the strain of Vibrio alginolyticus chemovar iophagus which 
has been deposited in the National Collection of Micro-organism Cultures 
of the PASTEUR INSTITUTE (C.N.C.M.) under number I-029. 
This collagenase is distinguished from other known collagenases by its high 
specific activity. The latter can reach 2 units microkatal/mg (.mu.kat/mg) 
of protein. It has been described, for example, in the article entitled 
"Subunit structure of Achromobacter Collagenase", of V. KEIL-DLOUHA and B. 
KEIL, which appeared in Biochim. Biophys. Acta 522, 218-228 (1978). 
Reference can also be made, as regards the chemical characterization of 
this enzyme as well as of the producing micro-organism, to the articles 
entitled "Chemical characterization and study of the autodigestion of pure 
collagenase from Achromobacter iophagus" of Vera KEIL-DLOUHA, which 
appeared in Biochimica et Biophysica Acta, 429 (1976) 239-251, and "Some 
newly characterized collagenases from procaryotes and lower eucaryotes" of 
Borivoj KEIL, which appeared in Molecular & Cellular Biochemistry, Jan. 
26, 1979, volume 23, nr. 2.

The invention relates more particularly to pharmaceutical compositions 
containing doses expressed by activity of the collagenase, related to the 
total weight of the composition, at least equal: 
to 0.05 .mu.kat/g of the pharmaceutical composition for emulsions, pomades, 
powders or any compositions not truly liquid, in the sense usually given 
to this expression, and 
to 0.05 .mu.kat/ml of the pharmaceutical composition for solutions 
formulated for topical use. 
Preferred pomades according to the invention contain particularly from 0.1 
to 2, preferably from 0.5 to 1 .mu.kat of collagenase per gram of pomade. 
Preferred solutions for topical applications according to the invention 
contain from 0.1 to 2, preferably 0.5 to 1 .mu.kat of collagenase per ml 
of solution. 
Advantageous compositions according to the invention contain in addition, 
on the one hand, neutral proteases and, on the other hand, endonucleases. 
Preferably, the proportions of proteases and endonucleases with respect to 
0.350 .mu.kat of collagenase do not exceed 200 caseinolytic units for 
proteases and 500 nucleasic units for endonucleases. 
Suitable compositions for application of the invention contain the three 
types of constituents, preferably in the relative proportions 
corresponding: 
to at least 0.350 .mu.kat of collagenase, 
to 20 to 200 caseinolytic units as regards their content of neutral 
proteases and 
30 to 500 nucleasic units, as regards the endonucleases. 
In still more preferred compositions, the relative proportions with respect 
to one another of the above constituents correspond respectively to 
activities: 
of at least 0.350 .mu.kat as regards collagenase, 
of 20 to 50 caseinolytic units as regards the neutral proteases and 
30 to 60 nucleasic units, as regards endonucleases. 
The use of the compositions according to the invention in the treatment of 
lesions of the above indicated type (for example burns, ulcers, eschars, 
hard eschars or white eschars based on collagen, cheloids, such as those 
produced after surgical operations) has been found to be particularly 
beneficial. 
In its therapeutic action, the composition according to the invention 
preferentially decomposes the collagen of the conjunctive tissue. It does 
not attack the muscular mass, in particular the myofibrils of the muscles, 
its collagenolytic component being inhibited by the myosin of the muscle. 
The composition according to the invention will sometimes, below, be 
called "Achromase" particularly when it contains proteases and 
endonucleases, especially in the above-indicated proportions, in addition 
to the collagenase. 
The action of Achromase is characterized by the production, from 
macromolecular substrates of the necrosis, of fragments which have 
chemotactic action on the blood elements. It is by this chemotactic action 
that in particular the macrophages and the leucocytes are accumulated at 
the periphery of the zone of action of Achromase. This accumulation of the 
elements of the blood is favorable to the process of tissue regeneration 
(for example, in ulcers, in tissues damaged by burns). 
Another characteristic of the action of Achromase with respect to tissues 
altered to a necrotic character is that it stops at the level of the 
living tissue. The action of the Achromase stops after having decomposed 
the dead tissue at the limit of the living tissue. It does not cause, by 
the action of its proteolytic components, lesions of the living tissue. 
These effects are produced particularly due to the inhibition systems 
which exist in the living tissue, particularly to the three systems 
involving: 
(1) collagenase--inhibitors of a polypeptidic nature present in the 
undamaged tissue, 
(2) the myosin present in the muscular tissue, and 
(3) nonspecific inhibitors of collagenase proteases, in particular of 
.alpha..sub.2 -macroglobulin, present in the blood stream. 
It is in this respect important to stress that to be effective the 
pharmaceutical compositions must have a high collagenolytic activity, 
having particularly the orders of magnitudes which have been indicated 
above. It has in fact been established that, contrary to what could have 
been feared, the selectivity of action of the collagenolytic preparation 
of the invention with respect to only necrosed tissues or, more generally, 
any unhealthy tissue, was not affected even at high doses of collagenase. 
In fact the natural barriers that healthy tissue offers against the action 
of collagenase or regulators of the activity of the latter that the 
natural barriers contain, were found to be fully effective against what 
could a priori be considered as an "overdose" of the enzyme. 
The neutral proteases, which form the second active constituent of 
Achromase, complete the action of the collagenase by degrading still 
further the fragments of the initial collagenolytic molecular substrate. 
When the preparation employed in the pharmaceutical composition according 
to the invention originates from Vibrio alginolyticus chemovar iophagus, 
said proteases are devoid of undesirable constituents. 
Finally, the third constituent of Achromase, essentially formed of 
endonucleases, contributes to the destruction of the DNA releasable by the 
cells on the occasion of an infectious process, e.g., the pus of which the 
DNA released forms a larger part. 
Preferably, the proteases and the endonucleases associated with the 
collagenase come from the same micro-organisms, and are obtained 
simultaneously with the collagenase in the culture media of the 
micro-organism. 
The conditions under which the mutual proportions of these constituents can 
be adjusted are described below. 
The invention relates also to preferred collagenolytic compositions in 
which the above-said collagenase is associated with effective preparations 
of hydrolysate of collagen, particularly enzymatic hydrolysate of 
collagen. 
Essentially these hydrolysates are composed from collagen fragments, for 
example from calf skins composed of peptide molecules of which the average 
molecular weights are less than 10,000 daltons and particularly between 
6,000 and 8,000 daltons. These hydrolysates can also be characterized by 
certain of their constituent amino acids and more particularly by the 
presence of hydroxy-proline. For example, a characteristic collagen 
hydrolysate comprises the following amino acids (originating from 
collagen) in the following proportions: 
glycine: 33% 
proline: 8.4% 
hydroxyproline: 11.4% 
These collagen hydrolysates can be obtained by any suitable fragmentation 
process of the denatured or undenatured collagenase. The term 
"hydrolysate" is not limited to products obtained by chemical or enzymatic 
hydrolysis of denatured or undenatured collagen. The hydrolysates of the 
type concerned are for example obtained by atomising gelatin. 
It is to be noted that these collagen hydrolysates are of the same nature 
as the products resulting from the degradation of fibrous or necrotic 
collagen contained in wounds that the collagenolytic compositions 
according to the invention seek to hydrolyse. 
It has been observed that the addition to the collagenase of the invention 
of substantial proportions of collagen hydrolysate such as mentioned above 
is manifested by an increase in the curative activity of the compositions 
according to the invention. 
Another very important advantage of the addition of these collagen 
hydrolysates to the collagenase compositions according to the invention 
resides in the considerable increase in the stability on storage of the 
collagenolytic compositions according to the invention. This increase in 
stability is manifested particularly fully on the occasion of the 
lyophilisation of collagenase preparations according to the invention. It 
will hence be particularly interesting to associate these collagen 
hydrolysates, very soluble in the cold, with collagenase preparations in 
the terminal phase of the preparation of the latter. In particular these 
collagen hydrolysates can be added directly to collagenase solutions 
obtained from culture media of the producing micro-organism. It is 
particularly desirable to add the collagen hydrolysates to the collagenase 
solutions after carrying out additional ultra-filtration and purification 
operations of the solutions obtained, which are required to remove 
undesirable insoluble and soluble constituents contained in the culture 
media removed, but before the final freeze drying of the collagen 
preparations obtained. 
Preferred pharmaceutical compositions of the invention contain collagen 
hydrolysates and collagenase in the proportion of 1 to 25 mg, preferably 2 
to 10 mg of collagen hydrolysate per 1 mg of collagenase. It is also 
possible to express preferred ratios in the following manner. The 
preferred compositions contain from 1 to 25, particularly from 2 to 10, 
for example 5 mg of collagen hydrolysate per microtal of collagen. 
It goes without saying that these ranges of proportions apply without 
distinction to the case where the collagen is associated or to the case 
where it is not associated with the above-indicated proteases and 
endonucleases. 
The pharmaceutical composition according to the invention hence plays an 
essential role where tissue wastes which tend to accumulate in the region 
of lesions of the type concerned are destroyed, this destruction 
(detersion) necessarily preceding the start of the natural or induced 
cicatrisation, for example by graft. 
The invention relates also to a process for the production of an active 
preparation according to the invention, containing more particularly three 
types of constituents which have been envisaged above. This process 
employs more particularly the aerobic bacteria Vibrio alginolyticus 
chemovar iophagus. This process consists of cultivating this 
micro-organism under conditions known in themselves and inducing also in a 
manner known in itself the production of collagenase by the addition to 
the culture medium of a suitable inductor, more particularly collagen or 
collagen fragments having a molecular weight still sufficient to form a 
secondary structure characteristic of the helicoidal collagen chains. This 
culture is continued until after induction in the culture medium of 
production of said neutral proteases, the culture being interruptable when 
the relative proportions of collagenase and proteinase have reached 
desired values comprised within the above-indicated respective intervals, 
the active preparation then being recoverable by techniques known in 
themselves from the culture medium. In particular, it is possible, after 
removal of the cells, to precipitate proteins from the supernatant liquor 
with a sufficient concentration of ammonium sulfate which can, for 
example, reach 60% of the saturation level of this salt in solution. The 
precipitate replaced in suspension can be dialysed against distilled water 
to remove the ions extracted in the course of the precipitation, before 
being finally freeze dried. As a modification, the active principle can be 
purified by ultrafiltration. 
In the foregoing, it is by modifying the duration of the culture that it 
has been proposed to adjust the relative proportions of collagenase or of 
protease (or proteinase). It can also be envisaged to supplement the 
collagenase with proteinases and endonucleases of external origin, or on 
the contrary of withdrawing the excess of proteinase and endonuclease for 
the case where this solution could be purified, having regard to the 
increased yield of collagenase that could be expected from a prolongation 
of the time of culture. 
This latter operation can be carried out by taking advantage of the very 
different molecular weights of the collagenase and the proteinases: In 
particular, these molecular weights are higher than 30,000 for collagenase 
and less than 30,000 for proteases, so that their separation is easy, for 
example on suitable ultrafiltration membranes. In particular, the culture 
medium obtained after interruption of the culture and separation of the 
bacteria, can be the subject of three successive ultrafiltrations, the 
first being in order to remove the residual bacteria and obtain a clear 
liquid containing collagenase and proteases, the second being on an 
ultrafiltration membrane separating the proteins from the higher molecular 
weights, at 30,000, particularly the collagenases and the third being on a 
filtration membrane enabling the separation of molecular weights higher 
than 10,000, which enables the proteases to be recovered. The collagenase 
and the proteases can then be remixed in the desired proportions. 
In accordance with the additional feature already mentioned above of the 
invention it is advantageous prior to recovery of collagenases which are 
further purified by freeze drying to add collagen hydrolysates prior to 
the freeze-drying, preferably in the proportions which have been 
indicated. In other words it is those solutions supplemented with collagen 
hydrolysates, which are then subjected to freeze drying. 
The addition of these collagen hydrolysates to a collagenase in solution 
before its recovery from this solution, can be analyzed as representing an 
essential step of a process seeking to stabilize said collagenase. This 
stabilization process is hence characterized by the step which constitutes 
the lyophilisation of the solution containing the collagenase, in the 
presence of the abovesaid collagen hydrolysates, taken preferably in the 
relative proportions which have been indicated above with respect to 
collagenase. 
To assay the various constituents of the composition, recourse may be had 
to the techniques described below which employ the substrates, the 
reagents and the methods of calculation also mentioned below. 
ASSAY OF THE COLLAGENASE (Wunsch E. and Heidrich H. G. (1963) Z. fur 
Physiol. Chem. 333, 149-151) 
SUBSTRATE 
The substrate used was 
4-phenylazo-benzyloxycarbonyl-L-Pro-Leu-Gly-L-Pro-D-Arg. HCl (PZ-PLGPR), 
marketed by the Fluka Company. 
REAGENTS 
The reagents employed where the following: 
A. Buffer: 
the buffer was formulated from the following three solutions: 
solution A1: prepared from 160 mM of veronal (sodium salt of 
5.5-diethylbarbituric acid) and 143 mM of sodium acetate, 3H.sub.2 O; 
solution A2: 1N HCl; 
solution A3: 8.5% NaCl; 
the buffer was obtained by mixing 200 ml of solution A1 with 80 ml solution 
A3, and by adjusting the pH to 8.5 with the solution A2. It was up to one 
liter by adding water and CaCl.sub.2, 2H.sub.2 O was added to obtain a 
final concentration of 4 mM. 
B. Substrate solution: 
It was obtained by dissolving 10 mg of PZ-PLGPR in 100 .mu.l of methanol. 
Buffer was added to bring it to 10 ml and the substrate solution so 
obtained remained stable for one week. 
C. Collagenase sample: 
The collagenase sample was obtained by preserving 1 mg of solution in 1 ml 
of buffer; by means of samples of unknown specific activity. It was 
diluted according to the preliminary readings. The optimal dilutions must 
give colorimetric values of the optical density at 320 nm (see below) 
within the range of 0.1 to 1.0. 
D. Citric acid: 
It was formed of a 0.5% solution in water. 
E. Ethyl acetate. 
ASSAY TECHNIQUE 
It proceeded as follows: 
1. the solution B was preheated to 37.degree. C.; 
2. 0.1 ml of the collagenase solution (correctly diluted) was mixed with 
0.4 ml of preheated solution B and the solution was incubated for 15 min. 
at 37.degree. C. 
3. 1 ml of solution D was added and 
4. 5 ml of ethyl acetate were added and vigorous mixing was carried out. 
5. The upper organic layer was then removed and it was dried by adding 
anhydrous Na.sub.2 SO.sub.4 ; 
6. The optical density at 320 nm was measured with respect to that of a 
control obtained under the same conditions as the sample studied, by the 
procedure indicated under points 1 through 5, except that buffer was used 
in place of the substrate (solution B). 
METHOD OF CALCULATION 
##EQU1## 
1.0 .mu.kat=90,000 WH units 
The coefficient was deduced from a calibration curve obtained with the 
product which cuts up the peptide (PZ-Pro-Leu) (compound B marketed by 
Fluka). 
II DETERMINATION OF THE PROTEASES (Laskowski M. (1955) Methods in 
enzymology II, 32; Kunitz M. (1947) J. Gen. Physiol. 30, 291) 
SUBSTRATE 
The substrate used was casein for biochemical use (marketed by Merck, under 
number 2 244). 
REAGENTS 
The reagents used were as follows: 
solution A: 
it was composed of: 
Aa: boric acid 0.2M (12.4 g/1,000 ml); 
Ab: 0.05 borax (19.05 g/1,000 ml); To obtain solution A, 100 ml of solution 
Aa were adjusted to pH 7.6 by means of solution Ab (about 4 ml); 
solution B: 
1 g of casein was suspended in 100 ml of solution A. It was heated in a 
water bath to 100.degree. C. until completely dissolved (about 15 min). 
The opalescent solution so obtained can be stored for a week at 4.degree. 
C.; 
solution C: 
5% trichloroacetic acid in water. 
PROTEINASE SAMPLE 
Dilution of samples containing proteinase must be adjusted according to the 
preliminary assay to an equivalent of 2-10 .mu.g of trypsin/ml. 
ASSAY TECHNIQUE 
It proceeded as follows: 
1. the tubes were prepared (Vidal plastic tubes 11.times.70), in number 
corresponding to twice the number of samples, in order that to each sample 
there may correspond its own control; 
2. 0.5 ml of solution B were preincubated in each tube, for 5 min at 
37.degree. C.; 
3. to each "sample" tube, were added 0.5 ml of sample. It was incubated for 
20 min at 37.degree. C. with stirring; 1.5 ml of solution C were added; 
4. To each control tube were added 1.5 ml solution C. It was incubated 5 
min at 37.degree. C. and 5 ml of sample was added; 
5. All the tubes were left at 4.degree. C. throughout the night. 
6. It was centrifuged for 10 min at 8,000 rpm at room temperature. 
7. the optical density at 280 (OD.sub.280) of the supernatant liquid of the 
sample with respect to the corresponding control was read. 
CALCULATION 
1. Calibration curve: 
A stored solution of pure trypsin was diluted (0.1 mg/ml of HCl 10.sup.-3 
M) to obtain final concentrations of 1, 2, 4, 6, 8 .mu.g of trypsin per 
ml. Then the steps 1 to 7 mentioned in the preceding paragraph were 
carried out. 
2. One unit of activity corresponds to 1 .mu.g of trypsin; one unit of 
specific activity corresponds to 1 .mu.g of trypsin per mg of protein. 
III DETERMINATION OF DEOXYRIBONUCLEASE (Kunitz M. (1950) J. Gen. Physiol. 
33, 349-363) 
REAGENTS 
The reagents used were as follows: 
deoxyribonuclease I (marketed by BOEHRINGER) of quality II (2,000 U/mg); 
deoxyribonucleic acid (marketed by BOEHRINGER) at 3 ml/10 mg. 
BUFFER 
The buffer was formulated as follows: 100 mM of Tris. HCl; 4 mM of 
MgSO.sub.4 ; 7H.sub.2 O; 1.8 mM of CaCl.sub.2.2H.sub.2 O were adjusted to 
pH 7.5 with hydrochloric acid. 
SOLUTIONS 
Standard enzyme: 1 mg of deoxyribonuclease I was dissolved in 1 ml of 
buffer. 
Substrate: 150 .mu.l in 50 ml of buffer was diluted. 
ASSAY TECHNIQUE 
It proceeded as follows: 
1. the absorbance at 260 nm of 3 ml of substrate solution was adjusted to 
0. 
2. 100 .mu.l of standard enzyme were added and mixed; 
3. the variation in absorbance over 10 min was recorded. 
4. The ratio .DELTA.A.sub.260 was determined (variation of absorbance at 
260 nm)/min; 
5. Steps 1 to 4 are repeated with fresh substrate solution and an unknown 
enzyme sample and the value of .DELTA.A.sub.260 /min was determined. 
CALCULATION 
mg/ml=optical density at 280 nm (OD.sub.280).times.0.9 
##EQU2## 
IV DETERMINATION OF PROTEINS (Lowry H. O., Roseborough N. J. and Randall 
R. J. (1951), J. Biol. Chem. 193, 265) 
REAGENTS 
The reagents used were as follows: 
solution A: 2% Na.sub.2 CO in 0.1M NaOH; 
solution B: 2% Na, potassium tartrate; 
solution C: 1% CuSO.sub.4 ; 
solution D: 1 ml of solution B+1 ml of solution C, diluted to 100 ml with 
solution A; 
solution D: foline reagent diluted to 1/1. 
CALIBRATIONS CURVE 
1. The reference protein solution was prepared from 1 mg of bovine albumin 
serum (BSA, marketed by Sigma) in 2 ml of water. 
2. A series of reference solution dilutions were prepared from 10 to 80 
.mu.l to a final volume of 400 .mu.l in water. 
3. 2 ml of solution D were added. It was mixed and left for 10 min at the 
temperature of the laboratory; 
4. 200 .mu.l of solution E were added. It was left to incubate in darkness 
at 50.degree. C. for 10 min; 
5. The optical density at 750 nM was read with respect to the control 
(water instead of the assay solution used in step 2). In this way the 
calibration curve was constructed. 
DETERMINATION 
The unknown sample was treated at different dilutions (final volume 400 
.mu.l, step 2). 
CALCULATIONS 
The protein content of the unknown sample was expressed as equivalent in 
weight of reference bovine albumin serum (BSA). 
Other features of the invention will appear also in the course of the 
description which follows of the culture conditions of Vibrio 
alginolyticus chemovar iophagus. Reference will be made to the figures in 
which: 
FIG. 1 depicts curves representing relative concentrations of different 
enzymes produced by culturing the abovesaid bacteria, as a function of 
time; 
FIG. 2 depicts comparative curves aiming at establishing the inhibiting 
character of myosine with respect to collagenase. 
The Vibrio alginolyticus chemovar iophagus strain is voluntary aerobic and 
appears in the form of gram negative rods 2-2.5 .mu.m in length and 1-1.5 
.mu.m in width, motile by means of single polar flagellae. It forms round 
and yellow colonies 2-3 mm in diameter, after incubation at 30.degree. C. 
for one night on a thiosulfate-citrate-bile-sucrose medium (TCBS). 
It has a positive response in the following tests: cytochrome oxydase, 
Voges-Proskauer reaction, indole production, lysine decarboxylase, 
gelatinase (skin test), esterase of the wetting agent marketed under the 
name TWEEN 80, tetrathionate reductase, nitrate reduction, production of 
acid from glucose, maltose, mannitol, sucrose, trehalose, mannose and 
glycerol, citrate utilization (Simmons). It is sensitive to the action of 
aminoglycosidic antibiotics, to chloramphenicol, tetracyclines, colistine, 
rifampicine, to sulfonamides, to nalixide acid, to nitrofurantoin and to 
the vibriostatic agent 0/129 in the disk test, but it resists 
trimethoprime. 
It gives a negative response in the following tests: reaction to methyl 
red, arginine dihydrolase, urease, phenylalanine deaminase, ONPG test, 
production of H.sub.2 S (Kligler iron agar), production of gas from 
glucose, production of acid from xylose, arabinose, adonital, rhamnose, 
sorbose, sorbitol, dulcitol, lactose, inositol, salicine, raffinose, 
mellibiose, .alpha.-methylglucoside, mucate. It does not use malonate. 
The strain I-029 is very halophilic with an optimum growth which occurs in 
13% sodium chloride and an apparent tolerance to 15% sodium chloride. It 
is also fully resistant to ampicilline, carbenicilline, in part to 
cephalotine. It shows positive reactions in tests with ornithine 
decarboxylase and in cellobiose fermentation tests. 
CULTURE CONDITIONS 
The cells were cultivated under stirring and forced aeration (1.4 
atmospheres) at 30.degree. C. in a medium containing 0.1M Tris. HCl, 
0.4NaCl, 2 mM of CaCl, at pH 7, containing 2.5% casamino acids (Difco 
Labs., Detroit, Mich.). Two hours after the start of the culture, there 
was added to the medium the composition marketed under the name ASF 
(fragments of calf skin collagen peptide; average molecular weight 7000, 
ROUSSELOT S.A., France) to obtain a concentration of 1.5% by weight. For 
the controls of growth, the turbidity was measured by absorbance per cm at 
600 nm in samples extracted at one hour intervals. 
PRODUCTION OF COLLAGENASE, PROTEINASE AND NUCLEASE 
The strain produced caseinolytic proteinase extra-cellular nuclease and, as 
soon as ASF has been added, collagenase. The activity level of the 
collagenase then rises to reach 60 nkat/ml. 
The curves of FIG. 1 are representative as regards: 
curve a, cellular growth, 
curve b, collagenase activity, 
curve c, the activity of the caseinolytic proteinase, and 
curve d, the activity of the nuclease, as a function of time. 
The culture is preferably interrupted, when the activity of the collagenase 
passes through a maximum, particularly after five hours in the case shown. 
The collagenase was concentrated and recovered from this medium. It can be 
further purified as described in the book of KEIL-DLOUHA, V. 1976, 
"Chemical characterization and study of the autodigestion of pure 
collagenase from Achromobacter iophagus, Biochem. Biophys. Acta, 429, 
239-305 and of LECROISEY A., V. KEIL-DLOUHA, D. R. WOODS, D. PERRIN and B. 
KEIL, 1975, "Purification, stability and inhibition of the collagenase 
from Achromobacter iophagus, FEBS Letters 59, 167-172. 
A typical preparation of Achromase contains 0.312 mg of protein (dry 
weight) per mg of the total product. It is characterized by the following 
relative activities, per mg of dry weight of protein: 
collagenase: 0.554 .mu.kat 
proteases: 145 caseinolytic units 
endonucleases: 477 nucleasic units 
STABILIZATION OF COLLAGENOLYTIC ACTIVITY 
The introduction in a collagenase solution of collagen hydrolysate has the 
effect of stabilizing the collagenolytic activity when the preparation is 
freeze dried. The effects of this stabilization, expressed in percentages 
of preservation of enzymatic activity of a collagenase preparation, before 
and after freeze drying, have been evaluated in tests in which there were 
employed variable proportions of collagen hydrolysate with respect to a 
particular dose of collagenase. The collagen hydrolysate used was that 
marketed in France under the name ASF by Etablissement Rousselot S.A. It 
is an atomized gelatin soluble in the cold in water. 
The results obtained are indicated in the table below for the relative 
proportions of Achromase and of ASF which result from the left hand column 
of the table. 
______________________________________ 
ENZYMATIC ACTIVITY IN % 
Achromase:ASF 
Before freeze-drying 
After freeze-drying 
______________________________________ 
1:0 100 50 
1:1 100 63 
1:5 100 96.6 
1:10 100 94 
1:25 100 78 
______________________________________ 
It follows from examination of this table that: 
the freeze-drying of an Achromase solution, in the absence of ASF, resulted 
in a 50% loss of collagenolytic activity and 
the addition of ASF substantially reduced the losses of activity. 
Particularly favorable results were observed for proportions of 2 to 10 
parts by weight of ASF with respect to 1 part by weight of Achromase. For 
the proportion of 5 parts of ASF to one part of Achromase there was 
observed an almost complete preservation of the enzymatic activity of the 
initial solution. 
To preserve the enzymatic activity in the course of freeze drying and to 
confer remarkable added stability there is also added to the preparations 
of freeze dried collagenase the hydrolysates of collagen, when the freeze 
dried collagenase is stored. It has thus been observed that the loss of 
collagenolytic activity, in percent, at the temperature of 4.degree. C., 
was of 25% for a collagenase free from collagen hydrolysate after seven 
months. After the same period and at the same temperature the 
collagenolytic activity of collagenase preparations containing from 1 to 
10 parts by weight of collagen hydrolysate for one part by weight of 
collagenase was preserved to 100%. 
The same effects of collagenolytic hydrolysates on stability were observed 
at the temperature of 20.degree. C. instead of 4.degree. C. Thus, after 
seven months there was a loss of activity of 30% for the collagenase 
preparation free from collagenolytic hydrolysates and 5% only for 
preparations containing the same proportions of collagenolytic hydrolysate 
as above. 
Similar stabilizations have been observed in other types of preparations 
based on collagenase (or Achromase), particularly when the latter are in 
ready-for-use form, such as those which will be considered below. 
BIOLOGICAL PROPERTIES 
The collagenase preparations obtained show, besides their above-indicated 
collagenolytic activity, the characteristic of being inhibited by myosine, 
which plays the role of non-competitive inhibitor for the collagenase. 
This property can be exploited by techniques employing the kinetic study 
of the effect of myosin on the activity of collagenase with respect to 
collagen. 
The degradation of the collagen by the collagenase is followed by the 
determination of the hydroxyproline released after hydrolysis of the 
collagenase fragments solubilized by digestion. 
Hydroxyproline being a specific amino acid of collagen, the other proteins 
contained in the reaction mixture do not interfere during the assay. 
The mixture below was incubated in a 0.02M Tris. HCl, 0.23M NaCl, 
5.times.10.sup.-3 M CaCl.sub.2 buffer, pH 7.4 for one hour at 37.degree. 
C.: 
muscular collagen fiber 5 to 30 mg, 
collagenase at 0.1 mg/ml in CaCl 10.sup.-4 : 1 ml 
incubation buffer or 
myosine suspension at 5 mg/ml in this same buffer: 5 ml. 
After incubation, the reaction mixture was centrifuged 5 minutes at 12,000 
g, 0.5 ml of the supernatant liquor was hydrolysed overnight at 
110.degree. C. with 0.5 ml of 12N HCl. The samples were then evaporated 
under vacuum and taken up again with one volume of water so that the 
determination of the hydroxyproline was made possible. 
The result of such a test with collagenase of titer 0.098 .mu.kat/ml is 
illustrated by the curves of FIG. 2, in which the curve e is 
representative of the variations of the ratio 1/v (v representing the 
number of moles of collagen digested per hour at 37.degree. C.) as a 
function of the ratio 1/s (s representing the number of moles of collagen 
employed), placed in the presence of collagenase, in the absence of 
myosine (interpretation according to LINWEAVER BURK). 
The curve f is representative of the development of the digestion of the 
same preparations respectively in the presence of 5 mg of myosine. 
Examination of these curves shows the very significant effects of the 
inhibition of the activity of the collagenase preparations concerned with 
respect to the collagen. 
The collagenase employed according to the invention is devoid of any 
toxicity. This can be demonstrated by study of the lethal doses carried 
out in a mouse according to the method of J. T. LICHTFIELD and F. W. 
WILCOXON. 
The LD-50s measured with a preparation injected intravenously with 
collagenolytic activity according to the invention, titrating 0.17 
.mu.kat/mg of protein, were the following: 
8.58 mg/kg of mouse, which corresponds to 
1.46 .mu.kat per kg of mouse, when this lethal dose is related to the 
collagenolytic activity units of the preparation. 
The remarkable collagenolytic activity of the preparations according to the 
invention has been demonstrated in pharmacological in vivo tests indicated 
below. 
These tests consisted of the treatment, with a preparation containing the 
collagenase according to the invention, of burns previously formed on the 
skin of pigs, by the application of water at the temperature of 80.degree. 
C., at several points on the back of these animals for 15 seconds (6 
lesions on the right side and 6 lesions on the left side of the pigs). The 
dead epidermis was then removed from the scalded surfaces and the lesions 
were exposed to air for 48 hours. Each lesion had a diameter of the order 
of 3.8 cm. Tests carried out with the pomade containing collagenase were 
duplicated by tests carried out with a similar pomade, but which was 
devoid of collagenase, this pomade having been used as a control. 
The compositions of these pomades were the following: 
For the control pomade: 
______________________________________ 
polyoxyethylenic alcohol 
13 g 
vaseline oil 20 g 
water 100 g 
sodium hydroxyde to adjust the pH to 7. 
______________________________________ 
The pomade containing the collagenase contained the same excipients, and 
0.25 .mu.kat of collagenase per g of pomade. 
The pomade containing the collagenase was applied to the lesions on the 
right side of the pigs, the control pomade on those of the left side. 
The pomade applications were repeated respectively 3 days, 4 days, 6 days 
and 13 days after the formation of the burns. 
The following observations were made 6 days and 13 days after the formation 
of the burns: 
State of the burns 6 days later 
The depth of the burn and the thickness of the tissue which have survived 
were approximately the same in the burns treated by the pomade and those 
which had received the control composition. 
In the burns treated with the control pomade, the presence of eschars 
containing intact collagen fibers was noted in 6 burns. The burns treated 
with the pomade containing the collagenase according to the invention did 
not include any. 
There was observed the presence of inflammatory cells infiltrated into the 
upper part of the dermis on 6 burns treated by the pomade with 
collagenase. The same observation could only be made on 3 control burns. 
The afflux of inflammatory cells testifies to a cicatrization process more 
advanced than in the burns where these infiltrations had not taken place. 
There was only observed however few differences at the level of the repair 
of the epidermis and of the conjunctive tissue, between the treated burns 
and the control burns, except that this repair appeared a little more 
advanced in the case of the treated burns. 
State of the burns 13 days later 
It was observed in the treated animals that the burns were covered with a 
thin film of degraded tissue. New conjunctive tissue had developed over 
the whole of the surface of the burns, immediately under this film as well 
as in greater depth, in the dermis. The epidermis was developed in the 
form of a granulation tissue from the edges of the lesions, immediately 
beneath the film of degraded tissue. 
It was observed in the treated lesions that the collagen portion of the 
eschars was dissolved by the collagenase, so that the regeneration of the 
conjunctive tissue could be developed at the surface, under conditions 
similar to those which are observed in the case of wounds through cuts. 
The epidermis formed can migrate over the surface of this granulation 
tissue. 
It is observed on the contrary that the control burns are covered with deep 
eschars of compact collagen which adheres to the tissue and covers it. New 
conjunctive tissue was formed only under the new epidermis layer which 
formed only on the edges of the burns. The epidermis had migrated from the 
edges of the lesion, through the dermis, beneath the abovementioned 
eschars, thus separating the latter from the deep tissue. Hence, the 
sources of new epidermis were only observed at the edge of the lesions, 
which would require a surgical skin graft to ensure the complete 
cicatrization of these lesions. 
One of the advantages of the pomade containing collagenase resides in the 
fact that the enzymatic treatment produces automatically a correct 
equilibrium between the necrotic tissues and the living tissues, which 
equilibrium would be difficult to recreate surgically. 
The pharmaceutical preparations according to the invention can be presented 
in any form usable by external application: gels, emulsions, particularly 
such as pomades and creams, or solutions or sprayable forms (aerosols in 
powder), the collagenase being in each preparation associated with 
suitable excipients according to the particular forms. In the case of 
solutions for topical application, recourse is advantageously had to 
placing collagenase in solution in an isotonic medium, preferably a 
physiologically acceptable buffer of pH 7-8.5, and preferably sterile. 
The collagenase preparations in the form of solutions in an injectable 
sterile vehicle can be administered subcutaneously, close to the wounds to 
be treated or to cheloids. The use of these injectable solutions can also 
be envisaged for deep administrations, particularly for the destruction of 
a pathological accumulation of collagen in the tissues, like for example 
in intervertebral discs. 
The pharmaceutical compositions according to the invention are hence 
applicable to all types of lesions manifested by an uncontrolled 
alteration of the structures rich in collagen. By way of examples of 
lesions which can be treated, are burns, ulcers, eschars, hard or white 
eschars based on collagen, cheloids, such as those produced after surgical 
operations, necroses, in particular in the case of decubitus or ulcers. 
The pharmaceutical compositions according to the invention can also be 
applied preventively, particularly prior to surgical operations to form 
plastic or other grafts, or in operations of aesthetic surgery, and 
generally to any wounds of the skin. 
The collagenase solutions according to the invention can also be applied to 
the treatment of dental caries. It is known in fact that the dental pulp 
is essentially formed from compact calcified collagen, dental caries being 
manifested by fissurization or a hole in the tooth, through which calcium 
escapes. The decalcified web of collagen which persists then becomes 
porous and risks becoming the seat of bacterial infection. 
By the introduction of a concentrated solution of collagenase according to 
the invention into this web, the dissolution of the porous collagen is 
induced which can then be removed by rinsing. The action of the 
collagenase is interrupted by itself at the level of the healthy calcified 
collagen. 
The collagenase solution used, buffered to pH 7-8.5, particularly with 
borate, contains preferably at least 1 .mu.kat of collagenase per ml of 
solution. At this concentration and a fortiori for higher concentrations, 
the porous collagen is rapidly dissolved. 
After rinsing and disinfection, the treated tooth can be closed again by 
applying techniques well known to the man skilled in the art. The possible 
residual traces of collagenase retained in the tooth are progressively 
inhibited by the dominating acid pH which is restored in the midst of the 
tooth. 
As is self-evident and as results besides already from the foregoing, the 
invention is in no way limited to the cases which have been envisaged in 
the foregoing. It encompasses on the contrary all modifications, 
particularly those where the stabilizing properties of the collagen 
hydrolysates are applied with respect to any other collagenases capable of 
being employed in the formulation of pharmaceutical compositions. These 
collagenases possess at the same time, selective activities which have 
been defined in the foregoing and the desirable innocuousness at 
sufficient doses at which the selective collagenolytic activity can be 
manifested. In this respect the invention relates also to any association 
of a collagenase corresponding to the foregoing conditions, whatever the 
source, particularly cultures of micro-organisms from which it has been 
obtained, in association with collagen hydrolysates. These compositions 
associating the two types of principles, particularly in proportions 
equivalent to the preferred intervals of proportions which have been 
indicated, constitute equivalents of the associations more particularly 
claimed in some of the claims which follow. 
In the same way the claims which relate more particularly to the processes 
of stabilization of a collagenase and more particularly of Achromase, 
especially by freeze-drying their solutions in the presence of collagen 
hydrolysates extend their effects to the stabilization of any form of 
collagenase, particularly on the occasion of the lyophilization of their 
solutions, as soon as collagen hydrolysates would be employed in the same 
way, particularly within the intervals of relative proportions defined 
above.