WO1994005702A1

WO1994005702A1 - Extraction of collagen

Info

Publication number: WO1994005702A1
Application number: PCT/EP1993/002247
Authority: WO
Inventors: Gianfranco Fedeli; Luigi Cislaghi; Guido Vandoni
Original assignee: Edmond Pharma Srl
Priority date: 1992-09-03
Filing date: 1993-08-20
Publication date: 1994-03-17
Also published as: EP0610481A1

Abstract

Herein described is a new form of extraction collagen having the following characteristics: optical rotation, measured on 1 % autoclaved, in 0.5 M acetic acid, corresponding to a [α]D25°=-145° ± 5, toluidine blue metachromasia = negative, it reconstitutes by neutralization even from a solution in 0.1N HCl left at 37 °C during 60', it hydrolyses completely within 40 minutes by the action of pepsine, it is stable under the proteolytic action of pancreatin during over 24 hours. This form of collagen is obtained preceding its extraction from tissues by a treatment with sodium hydrate, and following extraction itself with a precipitation of collagen at pH 4.9 - 5.5 and a treatment with carbon dioxide. The thus obtained new form of collagen, among all known ones, is the closest to negative collagen and the most suitable for pharmaceutical and cosmetic use.

Description

Extraction of collagen.

The present invention Rovers a new form of collagen which, compared to known forms, is closer to native collagen.

Collagen is a scleroprotein widely diffuse in animal world, such as to constitute one third of the total proteins of mammals' organism.

Being the basic material of all supporting and tegumental structures of vertebrates, its chemical structure, very complex, has been the subject of intensive chemico-physical studies, with further X-ray and electronic microscope investigation. Such studies contributed in detecting the chemical structures forming collagen frame responsible for the extraorci nary mechanical properties of tissues formedd by collagen.

Through the above studies it was possible to ascertain that histologic native collagen has a structure based on three polypeptide spiralwinding chains, each formed by about 1000 aminoacids, called tropocollagen. To this structure other protein, polysaccharide or glycopeptide molecules, especially glycosaminoglycanes, elastin and reticuline are bound. The main studies on collagen structure and on its properties are reported by :

A. Rich - Bioph. Science 1959

J. Gross - Scient Americ 1961

M. Chvapil - Biology of Collagen, Academic Press 1980.

Several "collagens" for pharmaceutical use are on the market, and companies specialized in the manufacture of laboratory reagents put "collagen" at disposal of researchers.

However, the procedures leading to the isolation of "collagen" are based on extraction techniques forcedly drastic, and anyhow denaturating, which imply the demolition of the otherwise unattackable histological structure. The thus obtained "collagen" is therefore totally different from native collagen of which, incidentally, at least eleven structurally different types are known, but still keeps some of its characteristics.

The characteristics of extractive collagen clearly depend on the extraction technique, since such technique on one hand must break bonds which are resistant in themselves, and on the other, avoid the demolition of collagen or its transformation into jelly or glue.

Collagen extraction procedures are described in US. patent 2,979,438 and in U.S. patent 2,973,302; the latter ciescribes also its purification. According to the techniques currently in use, collagenous tissue is subjected to a swelling, with dispersion in acid medium of collagen contained in said tissue, suitably treated.

In all procedures the final product is an aqueous dispersion, slightly acid - normally through acetic acid - from which the various collagen preparations are then obtained.

These procedures have several inconveniences in common:

- swelling of the material and extraction are treatment that take a long time; it is therefore difficult to control degradation;

- the final product is obtained with relatively low yields (e.g. C. Menicagli on Il Farmaco 1987, vol. 42, page 325, reports yields of 7-10% of the treated material);

- said final product always contains a certain amount of acid, which rises risks of a possible degradation of collagen and, besides giving the product an unpleasant smell, does not allow its application directly on wouncis.

To increase the extraction speed, and to obtain higher yields, preliminary enzymatic treatments have been applied, in fact facilitating extraction (U.S. P 2,9733)2 and 2,979,428 mentioneci above).

However, even with these methods it is impossible to avoid inconveniences, since extraction time lasts anyhow some ten hours, and it is known, besides, that enzymatic treatment alters the delicate internal equilibrium of the molecule, with modifications and degradations that alter its frame (C. Menicagli, Il Farmaco 12, 325-333, 1987).

Finally, antigenicity risk, which may berive from an insufficient elimination of macromolecules tightly binding collagen to tissues, has to be taken into consideration as well.

It has now been found that, subjecting the tissue to a β-elimination reaction (J. Kiss, Adv. In Carbohydrate Chemistry and Biochemistry 29, 229, Acad. Press 1974) before extraction, said extraction becomes much faster and more selective.

Surprisingly, it has also been found that the thus obtained collagen has characteristics which, on the whole, are not encountered in any of the collagens described in literature or commercially available. Finally, it has been found that a suspension of the product, obtained by suitably precipitating collagen, subjected to a treatment with carbon dioxide to saturation, yields an odourless aqueous gel, stable, and consistent with the conditions of a damaged cutis.

Therefore, according to one of its aspects, the present invention plays a form of extractive collagen characterized in that :

(A) it has a specific rotation, measured on the autoclaved at 121º, at a 1% concentration in 05 M acetic acid, equal to [α]_D ^25º = -145º ±5

(B) it gives a negative metachromatic reaction at toluidine blue

(C) it reconstitutes precipitating by neutralization even from a 0.1 N hydrochloric sacid solution kept at 37ºC during 1 hour

(D) it is regularly hydrolyzed by pepsine in less than 40 minutes

(E) it is resistent to the proteolytic action of pancreatin during over 24 hours;

The specific rotation is measured according to conventional techniques. For the determination of [α]_D ^25º of the products described in the following exemples, a 1% solution of collagen in 0.5 M acetic acid, autoclaved at 121ºC during 30' and filtered, has been used; the filtrate has been examined at a Polax-D Atago polarimeter in 1 dm optical tube; the measured degrees of the rotation angle are related to the dry basis.

The metachromatic reaction is determined on the product proteolyzed with Akalase at 60º, pH 8, during 4 hours, filtered and dilute with distilled water at a concentration of 4 mg per ml; 5 ml of this solution are reacted with 5 ml of a toluidine blue solution in

0.0024 N hydrochloric acid; the metachromatic reaction is positive when the colour turns from intense blue to violet or carmine-purple.

A positive reaction indicates the presence of mupolysaccharide residues in the collagen structure. The collagen of the present invention is negative at the metachromatic reaction.

The reconstitution of a solution in hydrochloric acid indicates that collagen is not degradated, and has maintained its tertiary structure.

The test is performed by dissolving 0.25% collagen in 0.1 N hydrochloric acid, allowing this solution to rest for one hour at 37ºC, and subsequently neutralizing this solution to pH 7 with about 2.6 ml of 6 N sodium hydrate. The collagen according to the present invention precipitates completely in a filamentous form.

Pepsine hydrolysis is performed by leaving at 37ºC during 40' a

0.25% solution of collagen in 0.1 N hydrochloric acid, containing 1.25 u.FIP per ml of pepsine.

The presence of undissolved residues stands as an indication that extractive collagen still contains foreign proteins resistent to pepsine, such as elastase and reticulin.

The collagen of the present invention is completely digested by pepsine, without leaving undissolved residues.

Treatment with panαreatine is performed by leaving 250 mg of collagen in 100 ml of 0.015 M phosphate buffer pH 8 containing 250 mg of 6 NF pancreatine during 24 hours at 37ºC. If digested by pancxeatin under the above conditions, said collagen has lost its scleroprotein characteristics, and particularly its tertiary structure. Collagen according to the present invention resists pancreatic proteolysis at least 24 hours.

Although in literature extractive collagen is mentioned as "native collagen" or "purified non-degradated collagen", none of the known products has the five above reported characteristics, which stand as an indication that the new form of purified extractive collagen of the present invention, though being devoid of the "secondary" structures supporting its frame in tissues, is the closest one to collagen present in tissues, since it keeps the mechanical properties of the latter. More particularly, in the new form of collagen of the present invention, glycosaminoglycanes, glycopeptides, elastine and reticuline are absent. However, surprisingly, it displays those properties one often tries to recover by treatment of extractive collagen with tanning agents that, by creating artificial cross-links, yield the so-called "reconstituted collagen".

The new form of collagen of the present invention has also other characteristics that make it closer to the collagen present in tissues. These characteristics demonstrate that the tertiary structure of collagen of the present invention is entire, despite the absence of the bonds formed by glycosaminoglycanes and by glycoproteins, reticulines and elastines.

Furthermore, the new form of collagen of the present invention shows the following characteristics:

(F) at a 1% concentration in 05 M acetic acid it yields a complete and homogeneous gel in less than 60 minutes (re-gelification capacity);

(G) at a 0.1% concentration in 0.1 M acetic acid and centrifugation at 3000 rpm during 30 minutes, over 90% of dissolved collagen concentrates in the heavy liquid phase, the volume of which is less than 20% the centrifuged volume (resistence to dilution) (H) up to a temperature of 80ºC and during a time up to 30' it does not turn to jelly (gelatinic degradation point);

(I) at the ultraviolet the solution autoclaved at 121ºC during 30' in

0.1 N sodium hydrate, shows a spectrum with the following characteristics:

λ max at 292 nm ± 1 with E^1% _lcm = 1.5 ± 0.1

λ min at 275 nm + 2

E₂₇₅/E₂₉₂ = 0.85 ±0.05

The regelification capacity (F) is practically determined by putting 100 mg of anhydrous collagen in 10 ml of 05 M acetic acid. Under these conditions the collagen of the present invention yields, within 60 minutes, a homogeneous gel, very viscous, barely opalescent and translucid. This property indicates the presence of a structure consistent with the native one, since it is the same occurring at the extraction of collagen from tissues; many collagen-based commercial preparations have lost such capability to regelify, and reconstituted collagen shows it but at a very reduced rate (M. Chvapil, Biology of Collagen, Academic Press pages 312-324, 1980).

The principle of resistence to dilution is based on the fact that entire structure collagen, when diluted (to 0.1%) in solution induces a coordination field versus the solvent water molecules in opposition to the diffusion of solute; at centrifugation the dilute solutions separate two liquid phases the mass ratios of which demonstrated correlation with the entireness status of the collagenous structure.

Resistence to dilution is determined by dissolving 1% collagen in gel form in 0.5 M acetic acid; 10 g of this gel are diluted with 90 g of 0.056 M acetic acid, to obtain a 0.1 M final acetic concentration, then homogenized; the thus obtained homogeneous solution is centrifuged during 30 minuted at 3000 rpm in refrigerated centrifuge.

The new form of collagen of the present invention, diluted at 0.1% in 0.1 M acetic medium, after centrifugation separates two phases, the bottom one of which, heavier, does not take more than 20% of the total volume and contains over 90% of the total dissolved collagen. The gelatinic degradation point, indicating the thermal degradation to irreversible jelly, consists of the determination of temperature and time necessary for a 1% collagen gel to transform into jelly which, unlike collagen, cannot any longer precipitate by neutralization as collagen does; this test too, possible only if the product satisfies test (F), can give indications on the structural state of dispersed collagen, since time and temperature vary inversely to degradation. The new form of collagen of the present invention shows a gelatinic degradation point higher than 80ºC during 30 minutes.

The U.V. absorption spectrum is carried out on 1% collagen in 0.5 M acetic acid subjected to steam autoclaving at 121ºC during 30 minutes and diluted after filtration, if needed, with 4 volumes of 0.25 M sodium hydrate, to obtain a final concentration of 0.2% of the material in 0.1 N sodium hydrate.

The U.V. spectra performed under these conditions have confirmed that the various forms of collagen contained in commercial preparations are different among them, in that the spectra have different absorption features.

The ratio among such differences of UV absorption and the structural state of collagen contained in the various commercial preparations has not been clarified. Under the above specified conditions, the new form of collagen of the present invention has, at

UV spectrum, the profile reported under item (I) above.

Figure no. 1 shows, by comparison, the spectrographic trace of collagen according to the present invention and that of three different commercial collagen-based preparations.

As specified under item (F), the new form of collagen of the present invention may be easily gelified at a 1% concentration in 0.5 M acetic acid.

It has however been ascertained that, although the new form of collagen of the present invention is more stable than known commercial preparations, the presence of acetic acid gives the product, in the gel form like known preparations, an unpleasant smell and an acidity (pH co mprised between 25 and 3) that make it unacceptable for cosmetic use and for a direct therapeutical use. Thus, according to another of its aspects, the present invention concerns an aqueous collagen-based gel, characterized in that said aqueous gel is formed from carbon dioxide. By preference, said gel is saturated through carbon dioxide.

The new collagen-based aqueous gel is homogeneous and preferably contains the new form of collagen of the present invention, but may even contain any other industrial collagen, provided it is suitable for pharmaceutical and cosmetic use.

The aqueous suspension is obtained by precipitating collagen from its solutions at a pH preferably comprised between 4.9 and 5.6, and the suspension at this pH is the one being treated and advantageously saturated with carbon dioxide.

Under these conditions of pH the collagen mass keeps the maximum degree of hydration, allowing gelification with excess of carbon dioxide.

The new collagenous aqueous gel of the present invention displays interesting properties that make it of easy use in the pharmaceutical or cosmetic industry and that enable it, in the medical area, to treat cases in which acid-based gels, with a very low pH, are not adequate. More particularly, the new collagen-based aqueous gel, saturated with carbon dioxide, affords:

- a good stability of the extracted collagenous structure, due to a lower acidity of the gel;

- the availability, for use on a wounded or scalded skin, since its pH of 4.9 - 5.6 corresponds to the mean physiological one of human skin, whilst common jelly preparations of collagen the pH of which is normally comprised between 2.5 - 3, are irritating and poorly indicated for repair processes;

- an atoxic bacteriostasis due to carbon dioxide;

- an easy reconstitution to sponges or tablets by freeze-drying, since the criodrying is facilitated by the absence of acids, particularly of acetic acid, the stripping of which required high temperatures, often results incomplete and may lead to denaturation.

According to another of its aspects, the present invention concerns a procedure to prepare extractive collagen, characterized in that

(a) it is dealing with a collagenous tissue with a solution of an alkaline hydroxide;

(b) the mass is neutralized to a pH from 55 to 9 and the residue is separated;

(c) collagen is extracted from the residue in acid medium, after purification - if needed - with proteolytic enzymes;

(d) the thus obtained mass is centrifuged and the collagen present in the supernatant liquid at a pH 4.9 - 5.5 is precipitated;

(e) the thus obtained collagenous mass is subjected to:

(e₁) single or consecutive washings with an anhydrous alcohol, with ketone or an ether, and to drying to obtain an anhydrous collagen

(e₂) alternatively, after washing with water, to a tratment of an aqueous suspension of it with carbon dioxide, to obtain collagen as aqueous gel.

As starting material any collagenous tissue is used, though Achilles tendon is the one providing the highest yield.

In step (a) the collagenous tissue, previously deaned from extraneous tissues and preferably grinded or minced, is treated with an alkaline hydroxide, preferably with a 1 N solution of sodium hydroxide.

Normally, after 45-90 minutes at room temperature the reaction is complete.

In step (b), the reaction mass is simply neutralized to a pH from 55 to 9, using, for instance, hydrochloric or acetic acid. This neutralization may not be indispensable, but is useful since, especially in case of extraction, in step (c), with acids, it avoids the use of great quantities of acid, particularly of acetic acid.

In step (c), the extraction takes place according to the traditional techniques, particularly by treatment with acids, after purification with enzymes.

However, according to the process of the present invention, the above treatments are being carried out under conditions different to those of known processes. In fact, as for extraction in acid medium, the time of such extraction is short, in that, after 4 - 8 hours, it is completed and collagen can be separated. As regards the enzyme pre-treatment, it could be performed by an enzyme as pancreatin, papain or alkalase without collagen undergoing degradation.

The extraction in acid environment is preferably carried out by acetic acid.

At the end of extraction a viscous gel is obtained and it is diluted with water for collagen separation.

Enzymatic treatment is carried out starting from the mass, such as obtained at the end of step (b), having a pH compatible with the enzyme. So, if pancreatin is used, it is advantageous for the starting mass to have a pH comprised between 8.5 - 9, preferably 85 - 8.7; if papain is used, the preferred pH is 55 - 6; if alkalase is used, pH of the mass coming from step (b) is preferably 7.9 - 8.1. In case of enzyme pre-treatment, reaction time is 5 - 8 hours at a temperature of 40 - 50º. However, considering the characteristics of the material obtained at the end of step (b), the treatment with pancreatin could be protracted from 5 to 40 - 50 hours without the collagen degrading. In practice, it is preferred to operate at about 42ºC for 5 - 50 hours employing pancreatin, at about 45ºC for 5 - 6 hours if papain is used and at about 45ºC for 6 hours employing alkalase.

Also in case of enzymatic treatment, at the end of step (c) a viscous gel is obtained and it is preferably diluted with water before the next step.

In step (d), the mass containing collagen is centrifuged and the separated liquid is treated with a base up to a pH 4.9 - 5.6. By preference, ammonium hydroxide, indicatively 1 N, is used as base. In such a way the collagen precipitates and in step (e) it can be isolated at the anhydrous state or in aqueous gel form, directly utilized in pharmaceutical technique or for cosmetic preparations. In step (e₁), collagen is isolated at an anhydrous state appropriately washing the precipitate mass with an alcohol, e.g. anhydrous methanol or ethanol, with a ketone, e.g. acetone, or an ether as diethyl ether. By drying, collagen is obtained as a white and cottony solid.

Alternatively, in step (e₂), the obtained mass at the end of step (d), after washing with water if needed, is diluted with water, and carbon dioxide is bubbled in the thus obtained suspension until saturation to obtain a homogeneous and translucent gel, directly utilizable for the preparation of pharmaceutical or cosmetic compositions.

Anhydrous collagen obtained at the end of step (e₁) can be easily re-gelified, as described in point (F) above.

The procedure of the present invention provides an extremely pure collagen, having characteristics closer to the collagen present in the tissues. In particular, it has all the above mentioned characteristics (A) and (E), and, by preference, the characteristics (A) and (I).

Compared to the well known procedures used for collagen extraction, the procedure of the present invention shows advantages which, on an industrial scale, do realize not only the obtaining of a new collagen form, but also a reaction time saving and higher yields. More particularly, the preliminary treatment with 1 N sodium hydrate hugely facilitates the next collagen extraction operations, making swifter the extraction phase and allowing yields which, starting, for example, from equine, ovine or bovine Achilles tendon, are equal to 25-30% of the origin tissue, even higher than those given by the procedures facilitated by a prior enzymatic treatment

Such a surprising result is due to the alkaline division of the collagen bond with the polysaccharidic chain which supports this protein in the tissue. This division, known as "β alkaline elimination" has released the proteic part of interest. This makes the collagen more available for extraction, which is completed in a few hours and not in days as happens with the traditional technique. Such a decrease of acid extraction times reduces to a minimum the risk to damage the collagen structure.

Considering the preventive enzymatic purification, the procedure of the present invention better shelters the collagen from possible enzymatic damages because salinity resulting from neutralization of sodium carbonate better thickens the collagen, making it nonsensitive to a possible enzymatic attack. These conditions allowed to employ advantageously also an energetic enzymatic purification as the digestion protracted 48 hours with entire 6 NF pancreatin, that is wholly containing its enzymes complex, to eliminate totally the related histic antigens. In such conditions more effective enzymatic systems can be employed, as papain and alkalase, which are not described in literature for collagen preparation probably because too energetic.

This surprising result confirms that the use of β-alkaline elimination gives a great flexibility to the procedure of the present invention since it allows, on one hand to decrease the acid extraction times, and on the other hand, in case of preventive enzymatic purification. either to make harmless a prolonged exposure of the so treated biological material to pancreatin action, or to utilize other proteolytic enzymes.

Flexibility and reliability of the present invention procedure are confimed by a further characteristic of the new collagen form so obtained: tied up hydroxyproline rate is not lower than 12% and varies from 12 to 14%, preferably from 12.5% to 14%, normally from

12.7% to 13.3%.

The so obtained new collagen form is particularly indicated for the preparation of pharmaceutical or cosmetic compositions containing it.

In particular, the new collagen form of the present invention is suitable for the preparation of new formulations , for local or rectal use, useful for the treatments of intestinal wounds or inflammation.

The new collagen form of the present invention can be employed for spray preparations.

Moreover, it was surprisingly found that the new collagen form according to the present invention can be administered by enema without provoking undesired side-effects.

Finally, the new collagen form of the present invention can be utilized for lyophilized sponges, in case with disinfectants as chlorhexidine or one of its salts or with antibiotics as aminoglycosides, for example gentamycin, neomycin or amikacin, macrolydes, for example erythromycine, or with clindamycin.

For a spray collagen-based preparation, the thus obtained aqueous solution is introduced, according to the usual industrial techniques, in the suitable bottles with the appropriate propellent gas, for example a chlorofluorocarbides, propane, hydrogen butane, air or carbon dioxide mixture.

Collagen aqueous solution for spray form may contain other active ingredients, for example anti-inflammatories, anti-histaminics, anti-allergies, disinfectants or antiseptics, as dorhexidine or one of its salt as dihydrochloride, diacetate or digluconate, or trichlocarban.

Preferably, the collagen aqueous solution contains chlorhexidine digluconate at a concentration starting from 0.01 to 0.3%, advantageously from 0.05% to 0.2%, preferably from 0.07% to 0.15%. The collagen aqueous solution can be employed as enemas, in bottels or tubes containing from 30 to 200 ml of 0,1-5% collagen solution, preferably from 0.2 to 2% or from 0.8 to 15%, or in singledose preparation containing from 5 to 20 ml of a solution at the above-mentioned concentrations.

Such solutions are administared by rectal route to patients suffering from rhagades, proctitis. Collagen aqueous solution for rectal use may contain other active ingredients.

Preferably, it contains an anti-inflammatory derived from 5-aminosalicylic acid.

According to a preferential aspect of the present invention, the collagen aqueous solution at the above-mentioned concentrations contains 5-aminosalicylic acid (mesalazine) at 0.5 - 8%, more particularly from 1 to 7%, preferably from 2 to 4%, usually in a quantity of 2 or 4 g per enema. Such solution is particularly indicated as enemas for patients suffering from inflammatory or self-immune origin diseases, as ulcerative colitis and Chron disease.

The following examples show the invention without however limiting it The indicated percentages both of the above description and the following examples are given as weight (g) volume {100 ml). Example 1

40 g grinded and washed bovine Achilles tendons have been suspended for 60' with 150 ml 1 N sodium hydrate, at room temperature. Expired the time, the mass has been neutralized at pH 8.6 with 73 ml 2 N hydrochloric acid, gradually added under accurate stirring. To the mass, put in 500 ml flask, 0.8 g 6 NF pancreatin have been added suspended in 40 ml 1 N-Sodium chloride solution. After the addition of some thymol crystals as bacteriostatic, the flask containing the suspension has been put in thermostatic bath at 42ºC. The mass has been left in these conditions for 8 hours keeping pH between 8 - 85 with 1 N ammonia addition. After this time further 0.8 g 6 NF pancreatin have been added, operating as for the first addition.

Pancreatic digestion has been protracted for other 15 hours.

Expired the time, the non-digested mass has been collected on buchn filtering under vacuum the digested liquid.

The residue has been washed twice with 400 ml distilled water each time. Thereafter, the washed residue has been suspended again in 400 ml distilled water containing 4 ml 34% hydrogen peroxide; regulated at pH 9, the suspension has been kept in these conditions for 60'.

Thereafter the liquid has been eliminated by filtration and the residue has been washed four more times by filtration employing 400 ml distilled water each time.

The so washed residue has been suspended by shaker in 05 N acetic acid obtaining in 3 hours a very dense practically homogeneous gel. This gel has been diluted with 4 liters distilled water and centrifuged to eliminate traces of undissolved matter.

The dear surnatant has been precipitated with ammonia at pH 53. After separation and washing, the precipitate has been regelified with carbon dioxide bubbling gas through a porous septum for 5 hours.

At the end 1018 g translucid homogeneous gel have been obtained containing a 1.1% dry collagen residue, for a dry collagen yield, compared to worked tendons, equal to about 28%.

The obtained collagen showed the following characteristics:

(A) [α]^25º _D = - 148º±3º

(B) toluidine blue metachromasia = negative

(C) reconstitution from 0.1 N HCl = total

(D)pepsinic hydrolysis = in 40 minutes

(E) resistance to pancreatin > 24 hours

(F) regelification in 0.5 M acetic acid = complete in 60 minutes.

(G) dilution resistance: heavy phase volume = 15%, recovered collagen = 92% (H) gelatinic degradation point = 80º × 60 minutes

(I) UV spectrum:

1%

E = 1.46, E₂₇₆/E₂₉₂ = 0.82

1 cm

- bound hydroxyproline = 13.2%

Example 2

Operating as in the example 1, raw papain was used instead of pancreatin.

In this case the digestion has been carried out at pH 55 - 6, at 45ºC for only 5 hours.

At the end after the washings, the acetic extraction (occurred in 3 hours), the centrifugation, the ammonia precipitation at pH 4.9 and the regelification with carbon dioxide, 1050 g of translucent homogeneous gel have been obtained with a 1.15% dry residue, for a yield on the tissue equal to about 30.19%.

The obtained collagen showed the following characteristics:

(A) [α]^25º _D = - 146º±3º

(B) toluidine blue metachromasia = negative

(C) reconstittion from 0.1 N HCl = total

(D)pepsinic hydrolysis = in 30 minutes

(E) resistance to pancreatin > 24 hours

(F) regelification in 0.5 M acetic acid = complete in 60 minutes.

(G) dilution resistance: heavy phase volume = 19%, recovered collagen = 92%

(H) gelatinic degradation point = 80º × 60 minutes (I) UV spectrum:

1%

E = 157, E₂₇₆/E₂₉₂ = 0.89

1 cm

- bound hydroxyproline = 13.3%

Example 3

Operating as in the example 1, NOVO alkalase was used, as enzyme for the purification from contaminating proteic material.

Digestion conditions in this case have been the following: pH 8, temperature at 45ºCC time 6 hours.

The non-diluted residue in the acetic extraction phase, divided by centrifugation, resulted nearly negligible.

At the end of this test 972 g of translucid homogeneous gel have been obtained with a 1.11% dry residue for a yield compared to the worked tissue equal to 27%.

The obtained collagen showed the following characteristics:

(A) [α]^25º _D = - 144º±3º

(B) toluidine blue metachromasia = negative

(C) reconstitution from 0.1 N HCl = total

(D)pepsinic hydrolysis = in 35 minutes

(E) pancreatin resistance > 24 hours

(F) regelification in 05 M acetic acid = completed in 60 minutes.

(G) dilution resistance: heavy phase volume = 17%, recovered collagen = 93%

(H) gelatinic degradation point = 80º × 60 minutes

(I) UV spectrum: 1%

E = 1.46, E₂₇₆/E₂₉₂ = 0.88

1 cm

- bound hydroxyproline = 13.2%

Example 4

Operating as in the example 1, until precipitation with ammonia at pH 53 of the centrifugation surnatant. At this point, the precipitated mass has been washed several times with anhydrous methanol and acetone, and once with diethyl ether, letting the cottony white solid residue dry at the air.

At the end, 12.6 g of cottony white mass have been obtained having a

13.6% of loss on drying, thus obtaining an anhydrous collages yield of 27.2% of the worked starting material.

The obtained collagen showed the following characteristics:

(A) [α]^25º _D = - 143º±3º

(B) toluidine blue metachromasia = negative

(C) reconstitution from 0.1 N HCl = total

(D) pepsinic hydrolysis = in 40 minutes

(E) resistance to pancreatin > 24 hours

(F) regelification in 0.5 M acetic acid = complete in 60 minutes.

(G) dilution resistance: heavy phase volume = 16%, recovered collagen = 91%

(H)gelatinic degradation point = 80º × 60 minutes

(I) UV spectrum:

1%

E = 1.58, E_276/E₂₉₂ = 0.86

1 cm

- bound hydroxyproline = 12.7% Example 5

Operating as in example 3, completing the test as in example 4, the aim was to obtain a filamentous soft mass as cotton flocks. In this case the yield was 26.6%, compared to the weight of initially worked tendons. The so obtained collagen showed the following characteristics:

(A) [α]25º_D = - 145º±3º

(B) toluidine blue metachromasia = negative

(C) reconstitution from 0.1 N HCl = total

(D)pepsinic hydrolysis = in 40 minutes

(E) resistance to pancreatin > 24 hours

(F) regelification in 0.5 M acetic acid = complete in 60 minutes.

(G) dilution resistance: heavy phase volume = 18%, recovered collagen = 90.8%

(H) gelatinic degradation point = 80º × 60 minutes

(I) UV spectrum:

1%

E = 1.58, E₂₇₆/E₂₉₂ = 0.88

1 cm

- bound hydroxyproline = 13.1%

Example 6

According to the above-mentioned A - I parameters, collagen preparations, obtained according to the present invention, and various anhydrous collagen preparations existing on the market or obtained according to some known methods, are compared in this example. The comparison has been extended also to the bound hydroxyproline content. The examined products have been the following:

1) anhydrous collagen obtained from example 4;

2) anhydrous collagen obtained from example 5;

3) the collagen obtained, with previous purification by means of enzymatic digestion, according to the procedure described in example 1 of USP 2, 973, 302, subjected to anhydrification as indicated in examples 4 and 5 of the present invention;

4) the collagen obtained according to the description published by C.

Manicagli on Il Farmaco ed. PRAΗCA 42, no. 12, pages 325 - 333 (1987) and subjected to anhydrification as indicated in examples 4 and 5 of the present invention;

5) the lyophylized collagen FLUKA code no. 27662, analysis no.

2786640/1 292;

6) the dried collagen π SIGMA type code no. 8886, lot 108C8015 (obtained according to R.E. Neuman, Arch, of Biochem 24, 1949);

7) The dried collagen V SIGMA type code no. 4387, lot 69F0269 (obtained according to J. Einbinder and M. Schubert, J. Biol. Chem., 188, 335, 1951);

8) the lyophilyzed pharmaceutical collagen CONDRESS (Gentili, Italia) lot no. 137 (November 91);

9) the lyophilyzed pharmaceutical collagen HEMOSTOP (Herix, Uruguai) lot no.89123788.

The operating conditions for single parameters have been: (A) Optical rotation (Opt rot [α]^25º _D)

500 mg test anhydrous collagen have been dispersed and diluted to

50 ml with 0.5 M acetic acid trying, when possible, to obtain the gel.

Anyhow the obtained dispersions have been autoclaved with steam at 1 psi (121º C) for 30 minutes.

After cooling, the solutions, suitably filtered, have been subjected to the measurement of α rotation angle of polarized light in the PolaxD

Atago polarimeter employing a 1 dm optical tube.

Optical rotation has been expressed as specific rotation (on dry) according to the following formula:

(B) Toluidine blue metachromasia (t.b.metachromasia)

250 mg test anhydrous collagen have been digested for four hours at

60ºC with 40 mg Alkalase Novo dispersed in 25 ml of 0.15 M phosphate buffer pH 8.

At the end the proteolysis, heating the solution at 90º for 15 minutes, has been interrupted.

After cooling the digested solution has been filtered. 10 ml filtrate have been diluted to 25 ml with 15 ml of distilled water containing

0.2 ml of 0.01 N hydrochloric acid.

5 ml of this solution, corresponding to 20 mg initially weighed sample, have been mixed with 5 ml of a solution containing 0.05 mg/ml of toluidine blue in 0.0025 N hydrochloric acid. The reaction has been judged "very positive" when the colour resulted sharply carmine.

(C) Reconstitution from 0.1N hydrochloric acid

250 mg test anhydrous collagen have been dispersed in 100 ml of

0.1N hydrochloric acid. The dispersion has been kept in thermostated bath at 37ºC for one hour; liquids which after this treatment were not dear, had to be darified by centrifugation, before proceeding in the test.

After cooling and possible clarification, the solution has been neutralized at pH 7 adding dropwise about 2.6 ml of 6 N sodium hydrate.

The obtained precipitate has been separated by centrifugation and washed five times by centrifugation with 50 ml each time of a 1:1 water and methanol mixture.

The washed precipitate has been subsequently dried. The recovery of the reconstituted collagen has been considered complete and total if its weight did not result lower than 225 mg, arbitrarily judging as possible a 10% standard loss for mechanical reasons.

(D) Pepsinic hydrolysis

250 mg test anhydrous collagen have been dispersed in 100 ml of 0.1 N hydrochloric acid containing 125 μ FTP pepsine. The dispersion has been put in thermoregulated bath at 37ºC. After 20 minutes incubation, every 5 minutes, 5 ml aliquotes of the supernatant were neutralized with 0.5 ml 1 N sodium hydrate added dropwise. The proteolysis was judged complete when precipitate by neutralization of dear supernatant was no longer detected.

(E) Resistance to pancreatin

250 mg test anhydrous collagen have been put in 100 ml 0.015 M phosphate buffer at pH 8 containing 250 mg 6 NF pancreatin . The whole has been put in a thermoregulated bath at 37ºC, occasionally stirring, for 24 hours. The pancreatin-proof sample after this time must result undiluted and practically unaltered.

A quantitative verification has been performed determining α-amino nitrogen according to Sorensen(USP XXII Ed., page 1185: V α-amino-nitrogen), after the first hour of incubation on a supernatant aliquot and expressing the hydrolysis degree as per cent of α-amino nitrogen on the total nitrogen.

(F) Regelification in 0.5 M acetic add (regel. in 0.5 M acetic acid)

To 100 mg test anhydrous collagen put in a 100 ml flask, 10 ml of 0.1

M acetic add have been added. The whole has been vigorously stirred until homogeneous and solid gel formation occurred.

(G) Dilution resistance

The gel formed in the test (F) has been diluted with 90 ml of 0.056 M acetic acid to obtain a final concentration of 0.1% collagen in 0.1 M acetic acid. After homogenization the dilution has been kept at rest for one hour to verify the stability of the obtained solution.

Subsequently this solution has been centrifuged at 3000 rpm for 30 minutes in a refrigerated centrifuge. Thereafter the supernatant has been accurately divided and the volume of the heavy liquid phase has been measured, from which collagen was recovered by precipitation with two volumes of methanol containing 1% ammonia. At the end, the precipitate was anhydrified with methanol, dried and weighed.

(H) Gelatinic degradation point

The test has been carried out only if the sample provided a solid gel, also if notcomplete, in the test (F).

10 g aliquotes of collagen sample, gelified in 0.5 M acetic acid, have been put in bath at 80ºC for 15 - 30 - 45 - 60 - 75 - 90 minutes. At the end of each period the individual tests have been neutralized with

2.4 ml 2 N hydrate sodium, added dropwise. The gelatinic degradation was considered complete in such tests which did not show precipitation at the neutralization.

(I) UV spectrum

The UV spectrum has been determined on solutions coming from test (A) (optical rotation) diluted 15 with four volumes of 0.25 M sodium hydrate to obtain a 0.2% final concentration, from collagen autoclaved in 0.1 N sodium hydrate.

Extinction values at the maximum wavelength, have been expressed as E_{1 cm} and, when possible, also the E_min/E_max ratio has been calculated when minimum extinction beyond maximum towards UV extreme was observed.

- Bound hydroxyproline

This determination has been carried out by colorimetric way with p-dimethylaminobenzaldehyde on sample hydrolized with hydrochloric acid and oxidized according to Mc Farlane and Guest (Canad. J. Research 17, 139, 1939) and Waldschmidt-Leitz

Ztschr.f.physiol.Chem. 219, 273, 1933).

In Table 1 the results obtained on different tested samples are reported.

As you can note only the two rapresentative samples of the present invention fully satisfy the above mentioned parameters entirety.

Example 7

A 100 g weighed quantity of collagen obtained as described in the

Example 1 has diluted for pharmaceutical preparations, taking the volume to 101. After possible filtration, the solution is divided mechanically in 1000 tubes per clisma which are subsequently welded.

Example 8

At 10 kg collagen obtained as described in Example 1, water under stirring is added until complete solution. After possible filtration to eliminate undissolved residues, 20 kg 5-amminosalicylic acid are added and taken to volume at 10001.

The whole is divided in vials containing 100 ml of 1% collagen aqueous suspension and 2% 5-amminosalicylic acid.

In the same way, from 30 kg 5-amminosalicylic acid, 10,000 bottles of

100 ml aqueous suspension at 1% collagen and 3% 5-amminosalicylic acid are prepared.

Example 9

Collagen-based clisma and 5-amminosalicylic acid having the following composition:

collagen

5-amminosalicylic acid g 0.250

polyethylenglycol g 4,000

benzoic acid g 2,000

ascorbic acid g 0,350

water as for to ml 100 At 25 collagen obtained as described in Example 1, water is added until complete solution, then other 150 1 water and the calculated quantity of other additives are added, under stirring. At the end, 40 kg 5-amminosalicylic acid are added and taken to volume at 1000 1. The so obtained mixture is divided in 10,000 vials per clisma ready to use, at 0.25% collagen and 4% 5-amminosalicylic acid.

Example 10

At 10 kg collagen obtained as described in example 1, water is added, as described in the example 8, taking to volume at 1000 1. At the solution so obtained 1 kg digluconate chlorhexidine is added and the whole is divided in spray cylinders with butane propellent gas, containing a 1% collagen solution and 0.1% chlorhexidine digluconate .

Claims

C LA IMS

1. Procedure for the preparation of extractive collagen, characterized by:

(a) it is a collagineous tissue with an alkaline hydroxy solution

(b) neutralize the mass to obtain a pH comprised between 5.5 - 9 and divide the residue;

(c) extract the collagen from the residue in acid environment, before possible purification with proteolytic enzymes;

(d) centrifuge the so obtained mass and precipitate the collagen present in the surnatant liquid at a pH comprised between 4.9 - 5.6;

(e) subject the so obtained collagineous mass:

(e₁) or at single or at subsequent washings with anhydrous alcohol, with ketone or ether and at drying to obtain an anhydrous collagen;

(e₂) or, after washing with water, at a treatment of its aqueous suspension with carbon dioxide to obtain collagen as aqueous gel.

2. Procedure according to the claim 1, diaracterized by the fact that, in the step (a), as alkaline hydrate is employed 1 N sodium.

3. Procedure according to the claims 1 or 2, characterized by the fact that reaction with alkaline hydroxide is carried out at room temperature for 45 - 90 minutes.

4. Procedure according to one of the claims from 1 to 3 characterized by the fact that, in the step (b), die reaction mass is neutralized at pH 6 - 7.

5. Procedure according to one of the claim from 1 to 3 characterized by the fact that, in the step (b), the reaction mass is neutralized at pH 85 - 9.

6. Procedure according to one of the claims from 1 to 3 characterized by the fact that, in the step (b), the reaction mass is neutralized at pH 55 - 6.

7. Procedure according to one of the claims from 1 to 3 characterized by the fad that, in the step (b), the reaction mass is neutralized at pH 7.9 - 8.1.

8. Procedure according to one of the claims from 1 to 3 and 5, chiaracterized by the fact that the step (c) is carried out with acetic acid at room temperature for a time from 4 to 8 hours.

9. Procedure according to the claims 1 - 3, 5 and 8, characterized by the fact that in step (c) a pre-treatment with pancreatin is carried out.

10. Procedure according to the claim 9 diaracterized by the fact that pre-treatment is carried out at about 42ºC for a time from 5 to 50 hours.

11. Procedure according to one of the claims 1 - 3, 6 - 8, characterized by the fart that in step (c) a pre-treatment with papaine is carried out.

12. Procedure according to the claim 11, characterized by the fact that the so-called pre-treatment is carried out at about 45ºC for 5 - 6 hours.

13. Procedure according to one of the claims 1 - 3, 7 - 8, diaracterized by the fact that, in step (c),a pre-treatment with alkalase is carried out .

14. Procedure according to the claim 13, diaracterized by the fact that the so-called pre-treatment is characterized at about 45 *C for 6 hours.

15. Procedure according to one of the claims from 1 to 14, characterized by the fact that, in step (d) the collagen precipitation is provoked by a base addition.

16. Procedure according to the claim 15, characterized by the fact that, as base, ammonium hydroxide is employed.

17. Procedure according to one of the claims from 1 to 16, diaracterized by the fact that in step (e₁) following washings with methanol or ethanol, acetone and ethilic ether are employed.

18. Procedure according to one of the claims from 1 to 16, diaracterized by the fact that in step (e₂) the mass obtained at the end of the step (d), in case before washing in water, is diluted with water and in the thus obtained suspension carbon dioxide is made bubbled preferably until saturation.

19. Extractive collagen form characterized by the fact that

(A) to have an optical rotation, measured on the autodaved at 121º for 30 minutes and at 1% concentration in 0.5 M acetic acid, equal to [a]^25º _D = - 145º ± 5;

(B) to give negative toluidine blue metachromasia reaction; (C) to reconstitute by precipitating by neutralization also by a 0.1 N chloridric acid kept at 37ºC for one hour;

(D) to be regularly hydrolized by pepsine in less than 40 minutes;

(E) to be resistant to the proteoiitic action of pancreatin further to 24 hours.

20. Collagen form according to the claim 19, diaracterized by the fact that:

(F) at 1% concentration in 05 M acetic add it provides a homogeneous and complete gel in less than 60 minutes (regdation capacity);

(G) at 0.1% concentration in 0.1 M acetic add and centrifugation at 3000 turns for 30 minutes, more than 90% of the undissolved collagen is concentrated and recovered in the heavy liquid phase in which the volume is less than 20% of the centrifuged volume (dilution resistance);

(H) till a temperature of 80º and for a period till 30' it does not change in gelatin (gelatin degradation point);

(I) at the ultraviolet, the solution autoclaved at 121ºC for 30 minutes and diluted in 0.1 N sodium hydrate, shows a spectrum with the following characteristics:

λ max at nm 292 ± 1 with

λ min at nm 275 ± 2

E₂₇₅/E₂₉₂ = 0-85 ± 0.05.

21. Collagen form according to the claim 19 or 20, obtainable in anhydrous form as from collagineous tissue according to the claims from 1 to 17.

22. Aqueous based collagen gel, characterized to be constituted by a collagen aqueous suspension treated with carbon dioxide.

23. Aqueous based collagen gel according to the claim 22, saturated with carbon dioxide.

24. Aqueous based collagen gel, saturated by carbon dioxide.

25. Aqueous based collagen gel according to one of the claims from 22 to 24, obtainable precipitating the collagen at pH 4.9 - 5.6 by its solution and treating the so obtained suspension with carbon dioxide.

26. Aqueous gel according to one of the claims from 22 to 25 and collagen based according to the claims from 19 to 20.

27. Aqueous gel according to the claim 26, obtainable as from collagineous tissue according to the claims from 1 70 16 and 18.

28. A pharmaceutical or cosmetic composition containing, as active ingredient, an extractive collagen according to claims 18 to 21.

29. A pharmaceutical or cosmetic composition containing an aqueous extractive collagen gel according to claims 22 to 27.

30. A pharmaceutical or cosmetic composition containing, as active ingredient, an extractive collagen having the following characteristics:

(A) it has an optical rotation, measured on the autoclaved at 121º for 30 minutes and at 1% concentration in 05 M acetic acid, equal to [α^]25º _D = - 145 ±5;

(B) it gives negative toluidine blue metachromasia reaction;

(O to reconstitute by precipitating by neutralization also by a 0.1

N chloridric add kept at 37ºC for one hour;

(D) it is regularly hydrolized by pepsine in less than 40 minutes;

(E) to be resistant to the proteolytic action of pancreatin further to 24 hours in admiture with a pharmaceutical or cosmetic excipient or vehicl .

31. A composition according to claim 30 in which the extractive collagen has the following characteristics:

(F) at 1% concentration in 0.5 M acetic acid it provides a homogeneous and complete gel in less than 60 minutes (regelation capacity);

(G) at 0.1% concentration in 0.1 M acetic acid and centrifugation at 3000 turns for 30 minutes, more than 90% of the undissolved collagen is concentrated and recovered in the heavy liquid phase in which the volume is less than 20% of the centrifuged volume

(dilution resistance);

(H) till a temperature of 80º and a period till 30' it does not change in gelatin (gelatinic degradation point);

λ max at nm 292 + 1 with

λ min at nm 275 ± 2

E₂₇₅/E₂₉₂ = 0.85 ± 0.05.

32. A composition according to claim 30 or 31 in which the collagen contained therein is obtainable in anhydrous form starting collagenic tissue by:

(a) division by β-elimination by means of a treatment with an alkaline hydroxide solution;

(b) neutralization of the mass till pH 5.5 - 7.8 and residue separation;

(c) collagen extraction in acid environment, also before purification with proteolytic enzymes;

(d) centrifugation of the so obtained mass and collagen precipitation present in the surnatant liquid at a pH 4.9 - 5.6; (e₁) washing with anhydrous alcohol, with acetone and ether and exsiccation at the air.

33. A pharmaceutical or cosmetic composition containing, as active ingredient, an aqueous gel of collagen consisting of a collagen aqueous suspension treated with carbon dioxide.

34. A composition according to claim 33 in which said collagen aqueous suspension is saturated with carbon dioxide.

35. A composition according to claim 30 or 31 in which the collagen contained therein is obtainable in form of an aqueous gel by:

(a) division by β-elimination by means of treatment with an alkaline hydroxide solution;

(b) neutralization of the mass till pH comprised between 55 - 8.7 and residue separation;

(d) centrifugation of the so obtained mass and precipitation of collagen present in the surnatant liquid at pH comprised between 4.9 - 5.6 and (e₂) washing with water and treatment of an aqueous suspension of the collagen thus obtained with carbon dioxide.

36. A composition according to claim 30 or 31 in form of enema containing 0.1 to 5% or 0.2 to 2% or 0.8 to 1.5% of collagen in water for pharmaceutical preparation.

37. A composition according to claim 36, also containing 05 - 8% or 1 - 7% or 2 - 4% of 5-aminosalicylic acid.

38. A composition according to claim 37 in which each enema contains 2 or 4 g of 5-aminosalicylic acid.

39. A composition according to claim 30 or 31 in form of spray containing 0.1 to 5% or 0.2 to 2% or 0.8 to 1.5% of collagen in water for pharmaceutical preparations and a pharmaceutically acceptable propellent.

40. A composition according to claim 30 also containing 0.01 to 03% or 0.05 to 0.2% or 0.07 to 0.15% of chlorhexidine or one of its salts.

41. A composition according to claim 40 in which said chlorhexidine salt is the digluconate.