CA2140834A1 - Method for removing the prions in collagens and collagens thereby obtained - Google Patents
Method for removing the prions in collagens and collagens thereby obtainedInfo
- Publication number
- CA2140834A1 CA2140834A1 CA 2140834 CA2140834A CA2140834A1 CA 2140834 A1 CA2140834 A1 CA 2140834A1 CA 2140834 CA2140834 CA 2140834 CA 2140834 A CA2140834 A CA 2140834A CA 2140834 A1 CA2140834 A1 CA 2140834A1
- Authority
- CA
- Canada
- Prior art keywords
- collagen
- collagens
- uta
- sodium hydroxide
- creutzfeldt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2310/00—Prostheses classified in A61F2/28 or A61F2/30 - A61F2/44 being constructed from or coated with a particular material
- A61F2310/00005—The prosthesis being constructed from a particular material
- A61F2310/00365—Proteins; Polypeptides; Degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to a method for the preparatlon of collagens, in whlch collagenous tissues are extracted and the collagen is solubilized, the collagen being subjected to an alkali treatment, characterlzed in that, for the purpose of removal of the UTA, it comprises the steps consisting of:
- removing the tissue or cell debris present in the collagen solution obtained;
- subjecting the collagen in solution to an alkali treatment;
- isolatilng the collagen free from risks of transmission of UTA.
- removing the tissue or cell debris present in the collagen solution obtained;
- subjecting the collagen in solution to an alkali treatment;
- isolatilng the collagen free from risks of transmission of UTA.
Description
~ 21~0834 h~l~O~ FOR REMOVING THE PRIONS IN COLLAGENS
AND COT.T~G~NS THEREBY OBTAINED
The invention relates to removal of the riskæ of contamination of biological extraction products with unconventional transmissible agents (UTA), also known as "prions". It relates more especially to a method which ensures removal of the prions for the preparation of collagens intended, in particular, for the manufacture of biomaterials.
~ The risks of cont~m;n~tion of biological extrac-tion products with unconventional transmissible agents (UTA) currently form the subject of detailed studies.
These risks have been confirmed in several ~n;m~l species and in man.
As regards ~n;~-l species, scrapie of sheep has existed on farms for several hundred years. Since 1986, in the United Ringdom, an epidemic of bovine spongiform encephalopathy (BSE) has affected cattle, and more than 70,000 cases have been reported to date. The hypothesis adopted today to explain this catastrophic epidemic of BSE appears to be the use of meat meals of cont~m; n~ ted sheep, originating from British knackeries, in the feed of young calves. This hypothesis seems to indicate the pos~ibility of transfer from one species to another, even orally, which obviously has repercussions, at least in people's minds and attracting media coverage, as regards the risks of transmission to man.
In man, a comparable and fatal degenerative disease has been known for some decades. It is Creutzfeldt-Jakob disease (CJD), the prevalence of which in the world is approximately 0.6 cases per million inhabitants. CJD has been stable at this level ever since it was identified and subject to census; there appears to be no relationship to the cases of scrapie of sheep or BSE epidemic. However, the very long incubation of these diseases, which can extend to se~eral decades, does not facilitate correlation studies or isolated observations, and strengthens doubts and the importance of preventive - - 2 - .214083~
measures.
Cases of accidental transmission of CJD to man have been reported. ~uru disease, a form of ~ Lology ~n to CJD and restricted to certain man-eating tribes of New Guinea, disappeared once the rites of removal and con-sumption of the brains of deceased ancestors were aban-doned, thanks to the discoveries and action of Dr Gad-jusek. Cases of transmission of CJD have been reported in neurosurgery in patients who have been in contact with poorly sterilized, cont~m;n~ted instruments (BERNOUILLI
et al., 1977; FONCIN et al., 1980; WIL~ et al., 1982), or who have received corneal or dura mater grafts removed from cadavers (DUFFY et al., 1974; PRICHARD et al., 1992;
MASULO et al., 1989; MIYASHITA et al., 1991; NISBET et al., 1989; POC~T~RT et al., 1992). Transmissions to neurosurgeons or to their collaborators have also been described (SCHOENE et al., 1981; MTrT~R, 1988; SITWELL et al., 1988; GORMAN et al., 1992).
Moreover, the use of hormones extracted from human pituitaries and associated nervous tissues ha~ led to several tens of cases of contamination of children treated for dwarfism with growth hormone (POWELL-JACRSON
et al., 1985; ~OCX et al., 1985; GIBBS et al., 1985;
TINTNER et al., 1986; CROXSON et al., 1988; MARZEWSRI et al., 1988; NEW et al., 1988; MACARIO et al., 1991;
FRADgIN et al., 1991; BUCHANAN et al., 1991; BROWN et al., 1992; BILLETTE DE VTTT~MT~UR et al., 1992), and of two women treated for sterility with gonadotrophins (COCHIUS et al., 1990).
To date, only nervous tissue is recognized unani-mously by experts as the major, if not exclusive, source of UTA. Many other human or ~n;m~l tissues are used in the biological industry and, for the time being, no non-nervous tissue has been the source of a documented and confirmed transmission of ~TA. Doubts exist about the risks of transmission of UTA from organs rich in lymphoid cells, and a classification of tissues in terms of these risks has been proposed by the W~O: WHO 1991.
Using particular ~nim~l models, various 214083~
publications report the presence of transmissible prions in the placenta of infected ewes (PATTISON et al., 1972 and 1974), as well as in the placenta, plasma and lymphocytes of a patient suffering from CJD (TAMAI et al., 1992), and in the leukocyte concentrates or whole blood of patients suffering from CJD (MANUELIDIS et al., 1985) or of persons in good health (MANUELIDIS et al., 1993). These various studies and results have not been confirmed by other authors (BROWN et al., 1984), and the criticisms voiced regarding the experimental conditions, suspected of laboratory contamination, demand con-firmation before any definitive conclusions can be drawn.
Regardless of whether or not these risks are real, and in the absence of prior diagnosis of these UTA, the most reliable safety factor for the future lies in the quality of the purification and/or inactivation methods used in the preparation of biological extraction products. Thus, it becomes necessary, under pressure from the regulatory bodies and bearing in mind the safety stAn~Ards introduced, to be able to guarantee the effi-cacy of these methods as regards their capacity to remove UTA. In addition, the safety requirements for a product obviously depend on the risks associated with this product, but also on the benefits provided to the patients. Thus, in all cases where the benefit to the patient is not a major feature, or in cases where equally effective alternative products exist, biological extrac-tion products will have to provide ~-~;mll~ guarantees.
The collagens, of human or An;~l origin, which are used nowadays in surgery in many biomaterials, are among these biological products for which it is sought to ensure removal of the prions. The properties of collagens enable them to be used as hemostatic agents, tissue repair guides, filling products, adhesives, corneal lenses and tissues reconstituted by crossl;nk;ng methods.
The value of collagens is their excellent biocompat-ibility, which enables them to exert the desired function and then to disappear by absorption in a few days, a few weeks or a few months, dep~n~;ng upon their mode of CrO881; nk; ng.
It is generally accepted that ~n;m~l collagens lack the risks of tran~mission of UTA, in particular for the following reasons:
- the hide or t~n~on~ of young calves which are used for the preparation of bovine collagen (mainly type I) have never been considered to be carriers of UTA, even when they come from sick ~n;~ls (WHO 1991);
- the ~n;~-ls which are the sources of these tissues come from controlled farms unaffected by BSE, and are subject to strict health controls.
In addition, the ~n;~-l tissues used are some-times subjected to prior alkali treatments intended to remove the hairs from the hide and some soluble im-purities under these conditions, especially keratinous and elastic proteinaceous substances (French Patent No. 1,568,829, Nov. 1967). The authors point out that the collagenous substances are relatively intact after separation.
For some preparations, young ox hide is sub-jected, before t~nn;ng~ to dipping in an aqueous solution comprising 0.3 to 1.0 N sodium hydroxide with 10 - 25%
(w/w) of sodium sulfate and a 0.05 - 0.3 M concentration of an amino compound at a temperature of 15 - 25C; the action time varies from a few hours to several days (Japanese Patent No. 140,582 of 1976, NIPPI In-corporated). Under these conditions, the authors assert that products intended for medical applications may be prepared from the collagen obtained, 80 as to display a very weak antigenic power, by prolonging the alkali treatment and promoting the decomposition of telopep-tides.
Similarly, US Patent No. 4, 511,653 describes the preparation of human collagens by treatment of placental tissues with 0.5 M sodium hydroxide for 48 hours at a temperature below 10C. One of the advantages put forward by the authors is the removal of ~iruses such as that of hepatitis B under these alkaline conditions.
French Patent Application No. 92/00,739 describes 21~0834 a method of preparation of collagens by alkali treatment of ~n;~-l hides with sodium hydroxide (or potassium hydroxide) at a concentration of 1 N for 0.5 to 1.5 hours at a temperature not exceeding 30 to 32C, before ex-traction of the collagen. The authors seem surprised thatthere i8 no modification of the helical structure of the collagen or of its molecular structure. They also assert that they obtain collagen fibers which display a hemo-static power 1.5 to 2.5 times as great as that of collagen fibers obt~;ne~ by a method differing only in the absence of the Al k~l; treatment step.
It is generally accepted that alkali treatments are effective for inactivating UTA. Treatment with 1 N
sodium hydroxide for 1 hour at 20C is nowadays acknow-ledged to be one of the few possible approaches fordecontaminating biological products. This treatment is, moreover, recomm~n~ed by the WHO (WHO 1991) whenever it is possible.
However, the efficacy of this sodium hydroxide treatment depends on the experimental conditions and on the UTA strains (extracted from brains of infected An;m~lg) uged in the ~n;m-l models.
In effect, P. BROWN et al. (1984) describe a reduction in infectivity o 5.5 log1O LD50 of a CJD strain after treatment with 0.1 N or 1 N sodium hydroxide for 1 hour. With this CJD strain, no residual infectivity is detectable.
P. BROWN et al. (1986) also describe reductions in infectivity of more than 5 loglO LD50 for a CJD strain, and more than 6.8 log1O LD50 for a sheep scrapie strain, after treatment with 1 N sodium hydroxide for 1 hour. No residual infectivity is detectable in either case. A
residual infectivity is observed in the case of treatment with 0.1 N sodium hydroxide.
While DI MARTINO et al. (1992) describe a reduc-tion in infectivity of 6 log1O LD50 for a scrapie strain after treatment with 1 N sodium hydroxide for 1 hour at room temperature, with, however, a detectable residual infectivity in the sodium hydroxide-treated contAm;n~ted sample injected undiluted.
In view of this state of the art, the Applicant wanted to test the relative efficacy of treatment with 1 N NaOH for 1 hour at a temperature in the region of 20C, especially for collagens of placental origin.
This treatment was applied to a ground prepara-tion of placental tissues cont~m;n~ted with a ground preparation of mouse brain infected with sheep scrapie strain NIH C 506/M3 at the sixth passage. The experi-mental ~n;~-l was the C57B16 mouse. After treatment of the tissues with 5 volumes of 1.2 N NaOH for one hour, the collagens were precipitated by ~;ng HCl to a pH in the region of 3 at +8C. The precipitate obtained was collected by centrifugation, and then subjected to several washes at room temperature with an 80:20 v/v acetone/water mixture and lastly w~h;ng with pure acetone to obtain, after drying under l~m;n~ flow, a collagenous powder. This collagenous powder was digested with collagenase 80 as to obtain a fluid solution, and injected in its entirety in 20 ~1 portions into mice intracerebrally in the right hippocampus.
Tests were carried out in parallel on con-t~m;n~ted tissues not subjected to the alkali treatment, serving as a positive control, or other tests carried out using uncont~ ; n~ ted placental tissues, serving as a negative control.
It was possible in this way to conclude that 1 N
sodium hydroxide treatment applied for 1 hour at a temperature in the region of 20C does not enable the scrapie strain used to be inactivated completely. From an initial infectious titer of 108 LDs0/g of ground brain preparation, only an inactivation of approximately 4 logl0 LD50 was observed.
Hence, contrary to the previous, more optimistic, published results, it is not obvious that collagen preparations completely freed from the risks of residual presence of UTA can be obtained from tissues inten-tionally cont~m;n~ted beforehand with UTA.
These results unquestionably leave very _ 7 _ 21 4 083~
considerable doubt hanging over the possibility of validating the methods of purification of collagens, even when an alkali treatment step i8 present.
The object of the present invention is to provide a method for the preparation of collagens, of ~n;m~l or human origin, which ensures a complete and reliable removal of UTA.
The object of the invention is also to carry out a complete and reliable inactivation of UTA while pre-serving the structure and properties of the solublecollagen molecules.
Another object of the invention is, lastly, to define the optimal conditions of the alkali treatment applied to collagens in solution.
To thiæ end, the subject of the invention is a method for the preparation of collagens, according to which collagenous tissues are extracted and the collagen is solubilized, the collagen being subjected to an alkali treatment, ~ld~ e~ in that, for the ~l~e of r3~n~1 of the UTA, it ~ the steps ~ sisting in :
- extracting collagenous tissues;
- solubilizing the collagen;
- removing the tissue or cell debris present in the collagen solution obtained; 5 - subjecting the collagen in solution to an alkali treatment;
- isolating the collagen free from risks of trans-mission of UTA.
The subject of the invention is also the composi-tions based on collagen or its derivati~es which areobtained by the abovementioned method, free from risks of transmission of UTA, as well as the biomaterials produced from the collagens obtained.
The inventors discovered that, surprisingly, all risk of residual cont~m; n~ tion with UTA could be elimi-nated during the preparation of collagens, whether of ~n; ~-1 or h~ n origin, on the one hand by removing the tissue or cell debris present with the collagen prior to its alkali treatment, and on the other hand by carrying 214083~
out said alkali treatment under specific conditions.
Thus, according to the invention, the problem of the efficacy and reliability of the UTA-inactivation treatment is solved, in particular, by carrying out, after extraction of collagenous tissues according to st~n~rd methods, removal of the tissue or cell debris by filtration through a membrane of porosity less than or equal to 1.2 ~, or by any suitable means for removal of the debris, for example centrifugation, and by then carrying out the alkali treatment of the collagen in ~olution.
This filtration step makes it obligatory, how-ever, to use only collagens which have been solubilized beforehand, either by enzymatic digestion of the covalent bonds link;ng the collagen ch~; n~ to one another, or by alkaline cleavage of these same bonds.
It is essential to note here that the methods of alkali treatment of collagens known hitherto were always applied to solid tissues, since their object was, at best, to solubilize these collagens partially and to remove some impurities.
In addition, the conditions of sodium hydroxide treatment enabling the UTA to be inactivated and, con-comitantly, the properties of collagen solutions to be preserved were not yet known.
An important objective of the invention is hence to ensure the effective inactivation of UTA while pre-serving as far as possible the structure and properties of collagen.
Thus, according to the invention, only the alkali treatment applied to a previously solubilized and fil-tered collagen solution enables all trace of UTA to be removed when there has been intentional cont~m;n~tion of the initial collagenous tissue.
The alkali treatment according to the invention consists in ~;ng to a collagen solution sodium hydroxide whose concentration is between 0.1 N and 2 N, and in allowing the action to proceed for approximately 1 hour with stirring at a temperature of the order of g According to the invention, the treatment condi-tions are advantageously defined in accordance with the collagen type.
5For type I or III collagens in solution and previously filtered in the dilute state through membranes of porosity less than or equal to 1.2 microns, the alkali treatment conditions are advantageously a time of 60 to 70 minutes, a temperature of 20C +3C and a sodium hydroxide concentration of between 0.1 N and 2 N, and p-eferably 1 N. If the sodium hydroxide concentration is increased above 2 N, or if the contact time is increased beyond 2 to 3 hours, the isoelectric point and the electrophoretic migration of the collagen molecules become distinctly modified. The modified properties of the collagens then make some applications more difficult.
For type IV collagen in solution and previously filtered in the dilute state through m~mhranes of poro-sity less than or equal to 1.2 microns, the alkali treatment conditions are advantageouæly a time of 60 to 70 minutes, a temperature of 20C +3C and a sodium hydroxide concentration in the region of 0.1 N. If the alkali treatment time i8 increased or the sodium hydroxide concentration is increased above 0.1 N, the viscosity of the collagen IV solutions decreases greatly and no longer enables gels which are of sufficient viscosity in some applications to be obtained. For applications independent of viscosity, treatment with 1 N
sodium hydroxide is possible and provides a further formal guarantee of the removal of UTA.
The inactivated collagen is then precipitated and isolated in the desired form, such as, for example, powder or gel.
Collagen is thereby obtained according to a preparation method which can be officially validated by the health regulatory bodies.
The collagen thereby obtained no longer presents a risk of transmission of UTA, e~en in cases of acci-dental cont~m; n~ tion of the collagenous tissues. Its , 2140834 helical and molecular structure is prefierved. Its properties such as isoelectric point or alternatively viscosity can also be preserved.
The collagen according to the in~ention may be used for the preparation of compositions of biomaterials for medical or surgical use, such as injectable products, hemostatic products, biological glues for bonding tissues to one another or to an implanted biomaterial, filling products, cicatrizing products, and the like.
A better underst~n~;ng of the invention will be gained on reading the examples given below by way of illustration and without implied limitation.
EXAMPLES
I - PREPARATION OF INTE~MEDIATE COLLAGENS
Exampl e 1 .
Human type I, III or IV collagens are extracted according to the methods described in French Patent Application No. 85/1~,004, by digestion of placental tissu~ with pepsin, then separation and purification of the three collagen types by salt precipitations at acid and neutral pH values.
Example 2.
~ovine collagen i8 prepared from dermis or tendons of young calves, by the method described in French Patent Application No. 81/22,691, according to which the collagen is solubilized by the action of sodium hydroxide.
Calf hides originating from freshly slaughtered ~n~m~l S are washed with water by stirring for 1 hour in a vat. The hairs and the subcutaneous tissue are separ-ated from the dermis using a rotating-strip splitting machine. The recovered dermis is chopped and ground. The ground preparation is washed in three successive baths of pH 7.8 phosphate buffer. Between each bath, the ground 35 preparation is separated from the solution by continuous centrifugation at between 1,000 and 4,000 rpm. The residue i8 then rinsed in two successive baths of softened water, and the liguid is separated from the ground preparation by centrifugation. Those first washes 214083~
serve to remove non-collagenous substances. The tissue is then placed in a vat cont~;n;ng 1 N sodium hydroxide solution at a temperature in the region of +4C for a period of 1 to 10 days. The medium is then acidified with hydrochloric acid to a pH below 3, adding sodium chloride so as to achieve a concentration of 100 g/l. The precipi-tated collagen is dialyzed against softened water.
This product which has undergone a prolonged alkali attack is not preferred when the electrical properties of the collagen molecule have to be as close as possible to the initial state, and taking into account the heterogeneity of the molecules obtained (some being monomers, others polymers, others insoluble aggregates, modified to a greater or lesser extent in accordance with the conditions of greater or lesser intensity of action of sodium hydroxide on the tissue).
Exampl e 3 .
Bo~ine collagen is prepared from dermis of young calves, washed as abo~e and subjected to pepsin digestion in 0.05 M citric acid buffer, pH 2.4. The dose of pepsin is approximately 40 g per kg of collagenous tissue. The digestion time is approximately 60 hours at 17C +2C.
As in the previous examples, the solubilized collagen is then purified by salt precipitation at acid pH and at neutral pH. Separations of precipitate are accomplished by continuous centrifugation.
II - INACTIVATION OF COLLAGENS
Example 4.
The precipitates of h~ n or bo~ine type I or III
collagen obtained according to ~Y~mple 1, 2 or 3 are solubilized in 0.05 M citric acid at a concentration of 1 to 2 g/l. After dissolution for at lea~t 8 hours, the solution obtained is centrifuged to remove insoluble aggregates, and then filtered through membranes having a porosity of up to approximately 1.2 microns (a porosity of 0.45 micron is preferable whenever bacteriological sterility is sought).
The filtered acid solution i8 treated with sodium chloride at a concentration of 41 g/l (the sodium chloride content should be increased to 100 g/l for collagens pretreated for a long time with sodium hydrox-ide). After the mixture has stood overnight at a tem-perature of approximately 10C, the collagen precipitate i8 recovered by continuous centrifugation. The precipi-tate is redissolved with 0.01 N hydrochloric acid at a concentration of approximately 8 g/l. The clear solution obtA;ne~ is neutralized to pH 7 by adding 2 N sodium hydroxide.
1 volume of 2 N sodium hydroxide is added to 1 volume of the above collagenous solution, and the mixture is kept stirring for 60 to 70 minutes at 20C +3C, giving a final sodium hydroxide concentration of 1 N.
The mixture is then diluted 5 times with demineralized water and immediately neutralized to pH 7.5 with 1 M citric acid. The collagen is precipitated by adjustment to 100 g/l of NaCl at 20C and at neutral pH, or by adjustment to 41 g/l of NaCl at pH 2.8 and at 10C.
The collagen precipitate is washed with acetone to obtain a powder enabling various known biomaterials to be prepared according to st~n~rd methods.
Exampl e 5 .
For type IV collagen, the procedure is as in Example 4, except that the final sodium hydroxide concen-tration is reduced to a value of 0.1 N.
III - TEST OF EFFICACY OF THE METHOD ACCORDING TO THE
lN v~l-lON
To test the efficacy of the method, collagens may be prepared from dermis or from placentas previously mixed with 1/lOth the weight of brain of mouse infected with scrapie strain NIH C 506/M3 at the sixth passage and assaying at approximately 108 LD50/g of ground brain preparation.
The collagen acetone powders obt~;ne~ according to the above examples are digested with collagenase 80 as to obtain a fluid solution which is nontoxic to the brain, and injected in their entirety in 20 ~1 portions into mice intracerebrally, in the right hippocampus.
The absence of symptoms of acute degenerative - 2l40834 encephalopathy after an interval of 18 months will be established, whereas the control mice have all died.
211l~8~
BIBLIOGRAPHY
- C. BERNOULLI et al., (1977) THE LANCET, i, 478-479, "Danger of accidental person-to-person transmission of Creutzfeldt-Jakob disease by surgery", - T. BILLETTE DE VILLEMEURE et al., (1992) REV. NEUROL., 148, 5, 328-334, "Maladie de Creutzfeldt-Jakob chez quatre enfants traites par l'hormone de croissance"
[Creutzfeldt-Jakob disease in four children treated with growth hormone], - P. BROWN et al., (1984) ANN. NEUROL., 16, 295, ~!Creutzfeldt-Jakob disease of long duration.
Clinicopathological characteristics. Transmissibility and differential diagnosis", - P. BROWN et al., (1984) THE NEW ENGLAND JOURNAL OF
MEDICINE, 310, 11, 727, "Sodium hydroxide decont~min~tion of Creutzfeldt-Jakob disease virus", - P. BROWN et al., (1986) THE ~OuKNAh OF INFECTIOUS
DISEASE, 153, 6, 1145-1148, "Newer data on the in-activation of Scrapie or Creutzfeldt-Jakob disease virus in brain tissue", - P. BROWN et al., (1992) THE LANCET, 340, 24-27, "Friendly fire in medicine : hormones, homografts, and Creutzfeldt-Jakob disease", - C.R. BU~N~N et al., (1991) BR. MED. J., 302, 824-828, "Mortality, neoplasia, and Creutzfeldt-Jakob disease in patients treated with human pituitary growth hormone in the United Ringdom", - J.I. COCHIUS et al., (1990) AUST. N. Z. J. MED., 20, 592-593, "Creutzfeldt-Jakob disease in a recipient of human pituitary-derived gonadotropin?"
- M. CROXSON et al., (1988) NEUROLOGY, 38, 1128-1130, "A
new case of Creutzfeldt-Jakob disease associated with hllm~n growth hormone therapy in New Zealand", - A. DI MARTINO et al., (1992) ARC~IVE OF VIROLOGY 124, 111-121, "Purification of non i~fectious ganglioside preparations from Scrapie-infected brain tissues", - P. DUFFY et al., (1974) N. ENGL. J. MED., 290, 692-693, "Possible person-to-person transmission of Creutzfeldt-Jakob disease", 21gO834 - J.F. FONCIN et al., (1980) REV. NEUROL., 136, 280, "Transmission iatrogane interhumaine possible de Creutzfeldt-Jakob avec atteinte des grains du cervelet"
~Possible interhuman iatrogenic transmission of Creutzfeldt-Jakob disease affecting the granules of the cerebellum], - J.E. FRADRIN et al., (1991) JAMA, 265, 880-884, "Creutzfeldt-Jakob disease in pituitary growth hormone recipients in United States", - D.J. GIBBS et al., (1985) N. ENGL. J. MED., 313, 734-7~38, "Clinical and pathological features and laboratory confirmation of Creutzfeldt-Jakob disease in a recipient of pituitary-derived human growth hormone?", - D.G. GORMAN et al., (1992) NEUROLOGY, 42, 463, "Creutzfeldt-Jakob disease in a pathologist", - T.~. ROCH et al., (1985) N. ENGL. J. MED., 313, 731-733, "Creutzfeldt-Jakob disease in a young adult with idiotypic hypopituitarism. Possible relation to the administration of cadaveric human growth hormone", - M.E. MACARIO et al., (1991) BR. MED. J., 302, 1149, "Pituitary growth hormone and Creutzfeldt-Jakob disease", - E.E. MANUELIDIS et al., (1985) THE LANCET, October 19, 896-897, "Transmission to ~ni~-l S of Creutzfeldt-Jakob disease from human blood", - E.E. MANUELIDIS et al., (1993) PRAC. NATL. ACAD. SCI., 90, 896-897, "A transmissible Creutzfeldt-Jakob disease-like agent is prevalent in the hllm~n population", - C. MASULLO et al., (1989) J. NEUROSURG. 71, 954-955, "Transmission of Creutzfeldt-Jakob disease by dural cadaveric graft", - D.J. MARZEWSKI et al., (1988~ NEUROLOGY, 38, 1131-1133, "Creutzfeldt-Jakob disease following pituitary-derived human growth hormone therapy : a ~ew American case", - D.C. ~TT-T-~, (1988) N. ENGL. J. MED., 318, 853-857, "Creutzfeldt-Jakob disease in histopathologist technicians", - R. MIYASHITA et al., (1991) NE~ROLOGY, 41, 940-941, "Creutzfeldt-Jakob disease in a patient with a cadaveric dural graft", - M.I. NEW et al., (1988) NEUROLOGY, 38, 1133-1134, "Preclinical Creutzfeldt-Jakob disease discovered at autopsy in a human growth hormone recipient", - T.J. NISBET et al., (1989) JAMA, 261, 11-18, "Creutzfeldt-Jakob disease in a second patient who received a cadaveric dura mater graft", - I.H. PATTISON et al., (1972) THE VET~TN~RY RECORD, 90, 465-468, "Spread of Scrapie to sheep and goats by oral dosing with foetal membranes from Scrapie-affected sheep", '9 I.H. PATTISON et al., (1974) BR. VET. J., 130, Ixv, "Further observations on the production of scrapie in sheep by oral dosing with foetal m~hranes from scrapie-affected sheep", - M. POC~T~T et al., (1992) THE LANCET, 340, 614-615, "Creutzfeldt-Jakob disease after non commercial dura mater graft", - J. POWELL-JACRSON et al., (1985) THE LANCET, ii, 244-246, "Creutzfeldt-Jakob disease after administration of human growth hormone", - J. PRICHARD et al., (1992) JAMA, Feb. 27, 1036, "Dementia in dura mater graft patient", - C. SCHOENE et al., (1981) ARCH. NEUROL., 38, 473-477, "Transmissible spongiform encephalopathy (Creutzfeldt-Jakob disease). Atypical clinical and pathological findings", - L. SITWELL et al., (1988) N. ENGL. J. MED., 318, 854 "Creutzfeldt-Jakob disease in histopathology technicians", - Y. TAMAI et al., (1992) THE NEW ENGLAND ~O~KNAL OF
MEDICINE, 327, 9, 649, "Demonstration of the trans-missible agent in tissue from a pregnant woman with Creutzfeldt-Jakob disease", - R. llNLN~K et al., (1986) NE~ROLOGY, 36, 932-936, "Neuropathologic verification of Creutzfeldt-Jakob disease in the exhumed American recipient of human pituitary growth hormone; epidemiologic and pathogenic implications?", - R.G. WILL et al., (1982) J. NEUROL, NEUROSURG.
PSYCEIAT., 45, 235-238, "Evidence for case-to-case transmi~sion of Creutzfeldt-Jakob disease", - WEO/CDS/VPH/92.104, 12-14 November 1991 "Report of a WHO consultation on public health issues related to ~n;m~l and human spongiform encephalopathies", Geneva.
AND COT.T~G~NS THEREBY OBTAINED
The invention relates to removal of the riskæ of contamination of biological extraction products with unconventional transmissible agents (UTA), also known as "prions". It relates more especially to a method which ensures removal of the prions for the preparation of collagens intended, in particular, for the manufacture of biomaterials.
~ The risks of cont~m;n~tion of biological extrac-tion products with unconventional transmissible agents (UTA) currently form the subject of detailed studies.
These risks have been confirmed in several ~n;m~l species and in man.
As regards ~n;~-l species, scrapie of sheep has existed on farms for several hundred years. Since 1986, in the United Ringdom, an epidemic of bovine spongiform encephalopathy (BSE) has affected cattle, and more than 70,000 cases have been reported to date. The hypothesis adopted today to explain this catastrophic epidemic of BSE appears to be the use of meat meals of cont~m; n~ ted sheep, originating from British knackeries, in the feed of young calves. This hypothesis seems to indicate the pos~ibility of transfer from one species to another, even orally, which obviously has repercussions, at least in people's minds and attracting media coverage, as regards the risks of transmission to man.
In man, a comparable and fatal degenerative disease has been known for some decades. It is Creutzfeldt-Jakob disease (CJD), the prevalence of which in the world is approximately 0.6 cases per million inhabitants. CJD has been stable at this level ever since it was identified and subject to census; there appears to be no relationship to the cases of scrapie of sheep or BSE epidemic. However, the very long incubation of these diseases, which can extend to se~eral decades, does not facilitate correlation studies or isolated observations, and strengthens doubts and the importance of preventive - - 2 - .214083~
measures.
Cases of accidental transmission of CJD to man have been reported. ~uru disease, a form of ~ Lology ~n to CJD and restricted to certain man-eating tribes of New Guinea, disappeared once the rites of removal and con-sumption of the brains of deceased ancestors were aban-doned, thanks to the discoveries and action of Dr Gad-jusek. Cases of transmission of CJD have been reported in neurosurgery in patients who have been in contact with poorly sterilized, cont~m;n~ted instruments (BERNOUILLI
et al., 1977; FONCIN et al., 1980; WIL~ et al., 1982), or who have received corneal or dura mater grafts removed from cadavers (DUFFY et al., 1974; PRICHARD et al., 1992;
MASULO et al., 1989; MIYASHITA et al., 1991; NISBET et al., 1989; POC~T~RT et al., 1992). Transmissions to neurosurgeons or to their collaborators have also been described (SCHOENE et al., 1981; MTrT~R, 1988; SITWELL et al., 1988; GORMAN et al., 1992).
Moreover, the use of hormones extracted from human pituitaries and associated nervous tissues ha~ led to several tens of cases of contamination of children treated for dwarfism with growth hormone (POWELL-JACRSON
et al., 1985; ~OCX et al., 1985; GIBBS et al., 1985;
TINTNER et al., 1986; CROXSON et al., 1988; MARZEWSRI et al., 1988; NEW et al., 1988; MACARIO et al., 1991;
FRADgIN et al., 1991; BUCHANAN et al., 1991; BROWN et al., 1992; BILLETTE DE VTTT~MT~UR et al., 1992), and of two women treated for sterility with gonadotrophins (COCHIUS et al., 1990).
To date, only nervous tissue is recognized unani-mously by experts as the major, if not exclusive, source of UTA. Many other human or ~n;m~l tissues are used in the biological industry and, for the time being, no non-nervous tissue has been the source of a documented and confirmed transmission of ~TA. Doubts exist about the risks of transmission of UTA from organs rich in lymphoid cells, and a classification of tissues in terms of these risks has been proposed by the W~O: WHO 1991.
Using particular ~nim~l models, various 214083~
publications report the presence of transmissible prions in the placenta of infected ewes (PATTISON et al., 1972 and 1974), as well as in the placenta, plasma and lymphocytes of a patient suffering from CJD (TAMAI et al., 1992), and in the leukocyte concentrates or whole blood of patients suffering from CJD (MANUELIDIS et al., 1985) or of persons in good health (MANUELIDIS et al., 1993). These various studies and results have not been confirmed by other authors (BROWN et al., 1984), and the criticisms voiced regarding the experimental conditions, suspected of laboratory contamination, demand con-firmation before any definitive conclusions can be drawn.
Regardless of whether or not these risks are real, and in the absence of prior diagnosis of these UTA, the most reliable safety factor for the future lies in the quality of the purification and/or inactivation methods used in the preparation of biological extraction products. Thus, it becomes necessary, under pressure from the regulatory bodies and bearing in mind the safety stAn~Ards introduced, to be able to guarantee the effi-cacy of these methods as regards their capacity to remove UTA. In addition, the safety requirements for a product obviously depend on the risks associated with this product, but also on the benefits provided to the patients. Thus, in all cases where the benefit to the patient is not a major feature, or in cases where equally effective alternative products exist, biological extrac-tion products will have to provide ~-~;mll~ guarantees.
The collagens, of human or An;~l origin, which are used nowadays in surgery in many biomaterials, are among these biological products for which it is sought to ensure removal of the prions. The properties of collagens enable them to be used as hemostatic agents, tissue repair guides, filling products, adhesives, corneal lenses and tissues reconstituted by crossl;nk;ng methods.
The value of collagens is their excellent biocompat-ibility, which enables them to exert the desired function and then to disappear by absorption in a few days, a few weeks or a few months, dep~n~;ng upon their mode of CrO881; nk; ng.
It is generally accepted that ~n;m~l collagens lack the risks of tran~mission of UTA, in particular for the following reasons:
- the hide or t~n~on~ of young calves which are used for the preparation of bovine collagen (mainly type I) have never been considered to be carriers of UTA, even when they come from sick ~n;~ls (WHO 1991);
- the ~n;~-ls which are the sources of these tissues come from controlled farms unaffected by BSE, and are subject to strict health controls.
In addition, the ~n;~-l tissues used are some-times subjected to prior alkali treatments intended to remove the hairs from the hide and some soluble im-purities under these conditions, especially keratinous and elastic proteinaceous substances (French Patent No. 1,568,829, Nov. 1967). The authors point out that the collagenous substances are relatively intact after separation.
For some preparations, young ox hide is sub-jected, before t~nn;ng~ to dipping in an aqueous solution comprising 0.3 to 1.0 N sodium hydroxide with 10 - 25%
(w/w) of sodium sulfate and a 0.05 - 0.3 M concentration of an amino compound at a temperature of 15 - 25C; the action time varies from a few hours to several days (Japanese Patent No. 140,582 of 1976, NIPPI In-corporated). Under these conditions, the authors assert that products intended for medical applications may be prepared from the collagen obtained, 80 as to display a very weak antigenic power, by prolonging the alkali treatment and promoting the decomposition of telopep-tides.
Similarly, US Patent No. 4, 511,653 describes the preparation of human collagens by treatment of placental tissues with 0.5 M sodium hydroxide for 48 hours at a temperature below 10C. One of the advantages put forward by the authors is the removal of ~iruses such as that of hepatitis B under these alkaline conditions.
French Patent Application No. 92/00,739 describes 21~0834 a method of preparation of collagens by alkali treatment of ~n;~-l hides with sodium hydroxide (or potassium hydroxide) at a concentration of 1 N for 0.5 to 1.5 hours at a temperature not exceeding 30 to 32C, before ex-traction of the collagen. The authors seem surprised thatthere i8 no modification of the helical structure of the collagen or of its molecular structure. They also assert that they obtain collagen fibers which display a hemo-static power 1.5 to 2.5 times as great as that of collagen fibers obt~;ne~ by a method differing only in the absence of the Al k~l; treatment step.
It is generally accepted that alkali treatments are effective for inactivating UTA. Treatment with 1 N
sodium hydroxide for 1 hour at 20C is nowadays acknow-ledged to be one of the few possible approaches fordecontaminating biological products. This treatment is, moreover, recomm~n~ed by the WHO (WHO 1991) whenever it is possible.
However, the efficacy of this sodium hydroxide treatment depends on the experimental conditions and on the UTA strains (extracted from brains of infected An;m~lg) uged in the ~n;m-l models.
In effect, P. BROWN et al. (1984) describe a reduction in infectivity o 5.5 log1O LD50 of a CJD strain after treatment with 0.1 N or 1 N sodium hydroxide for 1 hour. With this CJD strain, no residual infectivity is detectable.
P. BROWN et al. (1986) also describe reductions in infectivity of more than 5 loglO LD50 for a CJD strain, and more than 6.8 log1O LD50 for a sheep scrapie strain, after treatment with 1 N sodium hydroxide for 1 hour. No residual infectivity is detectable in either case. A
residual infectivity is observed in the case of treatment with 0.1 N sodium hydroxide.
While DI MARTINO et al. (1992) describe a reduc-tion in infectivity of 6 log1O LD50 for a scrapie strain after treatment with 1 N sodium hydroxide for 1 hour at room temperature, with, however, a detectable residual infectivity in the sodium hydroxide-treated contAm;n~ted sample injected undiluted.
In view of this state of the art, the Applicant wanted to test the relative efficacy of treatment with 1 N NaOH for 1 hour at a temperature in the region of 20C, especially for collagens of placental origin.
This treatment was applied to a ground prepara-tion of placental tissues cont~m;n~ted with a ground preparation of mouse brain infected with sheep scrapie strain NIH C 506/M3 at the sixth passage. The experi-mental ~n;~-l was the C57B16 mouse. After treatment of the tissues with 5 volumes of 1.2 N NaOH for one hour, the collagens were precipitated by ~;ng HCl to a pH in the region of 3 at +8C. The precipitate obtained was collected by centrifugation, and then subjected to several washes at room temperature with an 80:20 v/v acetone/water mixture and lastly w~h;ng with pure acetone to obtain, after drying under l~m;n~ flow, a collagenous powder. This collagenous powder was digested with collagenase 80 as to obtain a fluid solution, and injected in its entirety in 20 ~1 portions into mice intracerebrally in the right hippocampus.
Tests were carried out in parallel on con-t~m;n~ted tissues not subjected to the alkali treatment, serving as a positive control, or other tests carried out using uncont~ ; n~ ted placental tissues, serving as a negative control.
It was possible in this way to conclude that 1 N
sodium hydroxide treatment applied for 1 hour at a temperature in the region of 20C does not enable the scrapie strain used to be inactivated completely. From an initial infectious titer of 108 LDs0/g of ground brain preparation, only an inactivation of approximately 4 logl0 LD50 was observed.
Hence, contrary to the previous, more optimistic, published results, it is not obvious that collagen preparations completely freed from the risks of residual presence of UTA can be obtained from tissues inten-tionally cont~m;n~ted beforehand with UTA.
These results unquestionably leave very _ 7 _ 21 4 083~
considerable doubt hanging over the possibility of validating the methods of purification of collagens, even when an alkali treatment step i8 present.
The object of the present invention is to provide a method for the preparation of collagens, of ~n;m~l or human origin, which ensures a complete and reliable removal of UTA.
The object of the invention is also to carry out a complete and reliable inactivation of UTA while pre-serving the structure and properties of the solublecollagen molecules.
Another object of the invention is, lastly, to define the optimal conditions of the alkali treatment applied to collagens in solution.
To thiæ end, the subject of the invention is a method for the preparation of collagens, according to which collagenous tissues are extracted and the collagen is solubilized, the collagen being subjected to an alkali treatment, ~ld~ e~ in that, for the ~l~e of r3~n~1 of the UTA, it ~ the steps ~ sisting in :
- extracting collagenous tissues;
- solubilizing the collagen;
- removing the tissue or cell debris present in the collagen solution obtained; 5 - subjecting the collagen in solution to an alkali treatment;
- isolating the collagen free from risks of trans-mission of UTA.
The subject of the invention is also the composi-tions based on collagen or its derivati~es which areobtained by the abovementioned method, free from risks of transmission of UTA, as well as the biomaterials produced from the collagens obtained.
The inventors discovered that, surprisingly, all risk of residual cont~m; n~ tion with UTA could be elimi-nated during the preparation of collagens, whether of ~n; ~-1 or h~ n origin, on the one hand by removing the tissue or cell debris present with the collagen prior to its alkali treatment, and on the other hand by carrying 214083~
out said alkali treatment under specific conditions.
Thus, according to the invention, the problem of the efficacy and reliability of the UTA-inactivation treatment is solved, in particular, by carrying out, after extraction of collagenous tissues according to st~n~rd methods, removal of the tissue or cell debris by filtration through a membrane of porosity less than or equal to 1.2 ~, or by any suitable means for removal of the debris, for example centrifugation, and by then carrying out the alkali treatment of the collagen in ~olution.
This filtration step makes it obligatory, how-ever, to use only collagens which have been solubilized beforehand, either by enzymatic digestion of the covalent bonds link;ng the collagen ch~; n~ to one another, or by alkaline cleavage of these same bonds.
It is essential to note here that the methods of alkali treatment of collagens known hitherto were always applied to solid tissues, since their object was, at best, to solubilize these collagens partially and to remove some impurities.
In addition, the conditions of sodium hydroxide treatment enabling the UTA to be inactivated and, con-comitantly, the properties of collagen solutions to be preserved were not yet known.
An important objective of the invention is hence to ensure the effective inactivation of UTA while pre-serving as far as possible the structure and properties of collagen.
Thus, according to the invention, only the alkali treatment applied to a previously solubilized and fil-tered collagen solution enables all trace of UTA to be removed when there has been intentional cont~m;n~tion of the initial collagenous tissue.
The alkali treatment according to the invention consists in ~;ng to a collagen solution sodium hydroxide whose concentration is between 0.1 N and 2 N, and in allowing the action to proceed for approximately 1 hour with stirring at a temperature of the order of g According to the invention, the treatment condi-tions are advantageously defined in accordance with the collagen type.
5For type I or III collagens in solution and previously filtered in the dilute state through membranes of porosity less than or equal to 1.2 microns, the alkali treatment conditions are advantageously a time of 60 to 70 minutes, a temperature of 20C +3C and a sodium hydroxide concentration of between 0.1 N and 2 N, and p-eferably 1 N. If the sodium hydroxide concentration is increased above 2 N, or if the contact time is increased beyond 2 to 3 hours, the isoelectric point and the electrophoretic migration of the collagen molecules become distinctly modified. The modified properties of the collagens then make some applications more difficult.
For type IV collagen in solution and previously filtered in the dilute state through m~mhranes of poro-sity less than or equal to 1.2 microns, the alkali treatment conditions are advantageouæly a time of 60 to 70 minutes, a temperature of 20C +3C and a sodium hydroxide concentration in the region of 0.1 N. If the alkali treatment time i8 increased or the sodium hydroxide concentration is increased above 0.1 N, the viscosity of the collagen IV solutions decreases greatly and no longer enables gels which are of sufficient viscosity in some applications to be obtained. For applications independent of viscosity, treatment with 1 N
sodium hydroxide is possible and provides a further formal guarantee of the removal of UTA.
The inactivated collagen is then precipitated and isolated in the desired form, such as, for example, powder or gel.
Collagen is thereby obtained according to a preparation method which can be officially validated by the health regulatory bodies.
The collagen thereby obtained no longer presents a risk of transmission of UTA, e~en in cases of acci-dental cont~m; n~ tion of the collagenous tissues. Its , 2140834 helical and molecular structure is prefierved. Its properties such as isoelectric point or alternatively viscosity can also be preserved.
The collagen according to the in~ention may be used for the preparation of compositions of biomaterials for medical or surgical use, such as injectable products, hemostatic products, biological glues for bonding tissues to one another or to an implanted biomaterial, filling products, cicatrizing products, and the like.
A better underst~n~;ng of the invention will be gained on reading the examples given below by way of illustration and without implied limitation.
EXAMPLES
I - PREPARATION OF INTE~MEDIATE COLLAGENS
Exampl e 1 .
Human type I, III or IV collagens are extracted according to the methods described in French Patent Application No. 85/1~,004, by digestion of placental tissu~ with pepsin, then separation and purification of the three collagen types by salt precipitations at acid and neutral pH values.
Example 2.
~ovine collagen i8 prepared from dermis or tendons of young calves, by the method described in French Patent Application No. 81/22,691, according to which the collagen is solubilized by the action of sodium hydroxide.
Calf hides originating from freshly slaughtered ~n~m~l S are washed with water by stirring for 1 hour in a vat. The hairs and the subcutaneous tissue are separ-ated from the dermis using a rotating-strip splitting machine. The recovered dermis is chopped and ground. The ground preparation is washed in three successive baths of pH 7.8 phosphate buffer. Between each bath, the ground 35 preparation is separated from the solution by continuous centrifugation at between 1,000 and 4,000 rpm. The residue i8 then rinsed in two successive baths of softened water, and the liguid is separated from the ground preparation by centrifugation. Those first washes 214083~
serve to remove non-collagenous substances. The tissue is then placed in a vat cont~;n;ng 1 N sodium hydroxide solution at a temperature in the region of +4C for a period of 1 to 10 days. The medium is then acidified with hydrochloric acid to a pH below 3, adding sodium chloride so as to achieve a concentration of 100 g/l. The precipi-tated collagen is dialyzed against softened water.
This product which has undergone a prolonged alkali attack is not preferred when the electrical properties of the collagen molecule have to be as close as possible to the initial state, and taking into account the heterogeneity of the molecules obtained (some being monomers, others polymers, others insoluble aggregates, modified to a greater or lesser extent in accordance with the conditions of greater or lesser intensity of action of sodium hydroxide on the tissue).
Exampl e 3 .
Bo~ine collagen is prepared from dermis of young calves, washed as abo~e and subjected to pepsin digestion in 0.05 M citric acid buffer, pH 2.4. The dose of pepsin is approximately 40 g per kg of collagenous tissue. The digestion time is approximately 60 hours at 17C +2C.
As in the previous examples, the solubilized collagen is then purified by salt precipitation at acid pH and at neutral pH. Separations of precipitate are accomplished by continuous centrifugation.
II - INACTIVATION OF COLLAGENS
Example 4.
The precipitates of h~ n or bo~ine type I or III
collagen obtained according to ~Y~mple 1, 2 or 3 are solubilized in 0.05 M citric acid at a concentration of 1 to 2 g/l. After dissolution for at lea~t 8 hours, the solution obtained is centrifuged to remove insoluble aggregates, and then filtered through membranes having a porosity of up to approximately 1.2 microns (a porosity of 0.45 micron is preferable whenever bacteriological sterility is sought).
The filtered acid solution i8 treated with sodium chloride at a concentration of 41 g/l (the sodium chloride content should be increased to 100 g/l for collagens pretreated for a long time with sodium hydrox-ide). After the mixture has stood overnight at a tem-perature of approximately 10C, the collagen precipitate i8 recovered by continuous centrifugation. The precipi-tate is redissolved with 0.01 N hydrochloric acid at a concentration of approximately 8 g/l. The clear solution obtA;ne~ is neutralized to pH 7 by adding 2 N sodium hydroxide.
1 volume of 2 N sodium hydroxide is added to 1 volume of the above collagenous solution, and the mixture is kept stirring for 60 to 70 minutes at 20C +3C, giving a final sodium hydroxide concentration of 1 N.
The mixture is then diluted 5 times with demineralized water and immediately neutralized to pH 7.5 with 1 M citric acid. The collagen is precipitated by adjustment to 100 g/l of NaCl at 20C and at neutral pH, or by adjustment to 41 g/l of NaCl at pH 2.8 and at 10C.
The collagen precipitate is washed with acetone to obtain a powder enabling various known biomaterials to be prepared according to st~n~rd methods.
Exampl e 5 .
For type IV collagen, the procedure is as in Example 4, except that the final sodium hydroxide concen-tration is reduced to a value of 0.1 N.
III - TEST OF EFFICACY OF THE METHOD ACCORDING TO THE
lN v~l-lON
To test the efficacy of the method, collagens may be prepared from dermis or from placentas previously mixed with 1/lOth the weight of brain of mouse infected with scrapie strain NIH C 506/M3 at the sixth passage and assaying at approximately 108 LD50/g of ground brain preparation.
The collagen acetone powders obt~;ne~ according to the above examples are digested with collagenase 80 as to obtain a fluid solution which is nontoxic to the brain, and injected in their entirety in 20 ~1 portions into mice intracerebrally, in the right hippocampus.
The absence of symptoms of acute degenerative - 2l40834 encephalopathy after an interval of 18 months will be established, whereas the control mice have all died.
211l~8~
BIBLIOGRAPHY
- C. BERNOULLI et al., (1977) THE LANCET, i, 478-479, "Danger of accidental person-to-person transmission of Creutzfeldt-Jakob disease by surgery", - T. BILLETTE DE VILLEMEURE et al., (1992) REV. NEUROL., 148, 5, 328-334, "Maladie de Creutzfeldt-Jakob chez quatre enfants traites par l'hormone de croissance"
[Creutzfeldt-Jakob disease in four children treated with growth hormone], - P. BROWN et al., (1984) ANN. NEUROL., 16, 295, ~!Creutzfeldt-Jakob disease of long duration.
Clinicopathological characteristics. Transmissibility and differential diagnosis", - P. BROWN et al., (1984) THE NEW ENGLAND JOURNAL OF
MEDICINE, 310, 11, 727, "Sodium hydroxide decont~min~tion of Creutzfeldt-Jakob disease virus", - P. BROWN et al., (1986) THE ~OuKNAh OF INFECTIOUS
DISEASE, 153, 6, 1145-1148, "Newer data on the in-activation of Scrapie or Creutzfeldt-Jakob disease virus in brain tissue", - P. BROWN et al., (1992) THE LANCET, 340, 24-27, "Friendly fire in medicine : hormones, homografts, and Creutzfeldt-Jakob disease", - C.R. BU~N~N et al., (1991) BR. MED. J., 302, 824-828, "Mortality, neoplasia, and Creutzfeldt-Jakob disease in patients treated with human pituitary growth hormone in the United Ringdom", - J.I. COCHIUS et al., (1990) AUST. N. Z. J. MED., 20, 592-593, "Creutzfeldt-Jakob disease in a recipient of human pituitary-derived gonadotropin?"
- M. CROXSON et al., (1988) NEUROLOGY, 38, 1128-1130, "A
new case of Creutzfeldt-Jakob disease associated with hllm~n growth hormone therapy in New Zealand", - A. DI MARTINO et al., (1992) ARC~IVE OF VIROLOGY 124, 111-121, "Purification of non i~fectious ganglioside preparations from Scrapie-infected brain tissues", - P. DUFFY et al., (1974) N. ENGL. J. MED., 290, 692-693, "Possible person-to-person transmission of Creutzfeldt-Jakob disease", 21gO834 - J.F. FONCIN et al., (1980) REV. NEUROL., 136, 280, "Transmission iatrogane interhumaine possible de Creutzfeldt-Jakob avec atteinte des grains du cervelet"
~Possible interhuman iatrogenic transmission of Creutzfeldt-Jakob disease affecting the granules of the cerebellum], - J.E. FRADRIN et al., (1991) JAMA, 265, 880-884, "Creutzfeldt-Jakob disease in pituitary growth hormone recipients in United States", - D.J. GIBBS et al., (1985) N. ENGL. J. MED., 313, 734-7~38, "Clinical and pathological features and laboratory confirmation of Creutzfeldt-Jakob disease in a recipient of pituitary-derived human growth hormone?", - D.G. GORMAN et al., (1992) NEUROLOGY, 42, 463, "Creutzfeldt-Jakob disease in a pathologist", - T.~. ROCH et al., (1985) N. ENGL. J. MED., 313, 731-733, "Creutzfeldt-Jakob disease in a young adult with idiotypic hypopituitarism. Possible relation to the administration of cadaveric human growth hormone", - M.E. MACARIO et al., (1991) BR. MED. J., 302, 1149, "Pituitary growth hormone and Creutzfeldt-Jakob disease", - E.E. MANUELIDIS et al., (1985) THE LANCET, October 19, 896-897, "Transmission to ~ni~-l S of Creutzfeldt-Jakob disease from human blood", - E.E. MANUELIDIS et al., (1993) PRAC. NATL. ACAD. SCI., 90, 896-897, "A transmissible Creutzfeldt-Jakob disease-like agent is prevalent in the hllm~n population", - C. MASULLO et al., (1989) J. NEUROSURG. 71, 954-955, "Transmission of Creutzfeldt-Jakob disease by dural cadaveric graft", - D.J. MARZEWSKI et al., (1988~ NEUROLOGY, 38, 1131-1133, "Creutzfeldt-Jakob disease following pituitary-derived human growth hormone therapy : a ~ew American case", - D.C. ~TT-T-~, (1988) N. ENGL. J. MED., 318, 853-857, "Creutzfeldt-Jakob disease in histopathologist technicians", - R. MIYASHITA et al., (1991) NE~ROLOGY, 41, 940-941, "Creutzfeldt-Jakob disease in a patient with a cadaveric dural graft", - M.I. NEW et al., (1988) NEUROLOGY, 38, 1133-1134, "Preclinical Creutzfeldt-Jakob disease discovered at autopsy in a human growth hormone recipient", - T.J. NISBET et al., (1989) JAMA, 261, 11-18, "Creutzfeldt-Jakob disease in a second patient who received a cadaveric dura mater graft", - I.H. PATTISON et al., (1972) THE VET~TN~RY RECORD, 90, 465-468, "Spread of Scrapie to sheep and goats by oral dosing with foetal membranes from Scrapie-affected sheep", '9 I.H. PATTISON et al., (1974) BR. VET. J., 130, Ixv, "Further observations on the production of scrapie in sheep by oral dosing with foetal m~hranes from scrapie-affected sheep", - M. POC~T~T et al., (1992) THE LANCET, 340, 614-615, "Creutzfeldt-Jakob disease after non commercial dura mater graft", - J. POWELL-JACRSON et al., (1985) THE LANCET, ii, 244-246, "Creutzfeldt-Jakob disease after administration of human growth hormone", - J. PRICHARD et al., (1992) JAMA, Feb. 27, 1036, "Dementia in dura mater graft patient", - C. SCHOENE et al., (1981) ARCH. NEUROL., 38, 473-477, "Transmissible spongiform encephalopathy (Creutzfeldt-Jakob disease). Atypical clinical and pathological findings", - L. SITWELL et al., (1988) N. ENGL. J. MED., 318, 854 "Creutzfeldt-Jakob disease in histopathology technicians", - Y. TAMAI et al., (1992) THE NEW ENGLAND ~O~KNAL OF
MEDICINE, 327, 9, 649, "Demonstration of the trans-missible agent in tissue from a pregnant woman with Creutzfeldt-Jakob disease", - R. llNLN~K et al., (1986) NE~ROLOGY, 36, 932-936, "Neuropathologic verification of Creutzfeldt-Jakob disease in the exhumed American recipient of human pituitary growth hormone; epidemiologic and pathogenic implications?", - R.G. WILL et al., (1982) J. NEUROL, NEUROSURG.
PSYCEIAT., 45, 235-238, "Evidence for case-to-case transmi~sion of Creutzfeldt-Jakob disease", - WEO/CDS/VPH/92.104, 12-14 November 1991 "Report of a WHO consultation on public health issues related to ~n;m~l and human spongiform encephalopathies", Geneva.
Claims (11)
1. A method for the preparation of collagens, in which collagenous tissues are extracted and the collagen is solubilized, the collagen being subjected to an alkali treatment, characterized in that, for the purpose of removal of the UTA, it comprises the steps consisting in :
- removing the tissue or cell debris present in the collagen solution obtained;
- subjecting the collagen in solution to an alkali treatment;
- isolating the collagen free from risks of trans-mission of UTA.
- removing the tissue or cell debris present in the collagen solution obtained;
- subjecting the collagen in solution to an alkali treatment;
- isolating the collagen free from risks of trans-mission of UTA.
2. The method as claimed in claim 1, characterized in that the collagenous tissues are of animal or human origin.
3. The method as claimed in either of claims 1 and 2, characterized in that the collagen is solubilized by enzymatic digestion.
4. The method as claimed in either of claims 1 and 2, characterized in that the collagen is solubilized by enzymatic alkali treatment of the collagenous tissues.
5. The method as claimed in any one of the preceding claims, characterized in that, in order to remove the tissue or cell debris, the collagen solution is filtered through a membrane of porosity less than or equal to 1.2 microns.
6. The method as claimed in claim 5, characterized in that the filtration is carried out through a membrane of porosity approximately 0.45 micron.
7. The method as claimed in any one of the preceding claims, characterized in that the collagen in solution freed from the tissue or cell debris is subjected to the action of sodium hydroxide at a concentration of between 0.1 N and 2 N, for approximately 1 hour at a temperature of the order of 20°C.
8. The method as claimed in claim 7, characterized in that, for type I or III collagen, sodium hydroxide is preferably used at a concentration of approximately 1 N.
9. The method as claimed in claim 7, characterized in that, for type IV collagen, sodium hydroxide is used at a concen-tration of approximately 0.1 N.
10. Collagen in powder or gel form, obtained by the method as claimed in any one of claims 1 to 9.
11. Biomaterial produced from collagen obtained by the method as claimed in any one of claims 1 to 9.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9400716 | 1994-01-24 | ||
| FR9400716A FR2715405B1 (en) | 1994-01-24 | 1994-01-24 | Process for the elimination of prions in collagens and collagens thus obtained. |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2140834A1 true CA2140834A1 (en) | 1995-07-25 |
Family
ID=9459320
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2140834 Abandoned CA2140834A1 (en) | 1994-01-24 | 1995-01-23 | Method for removing the prions in collagens and collagens thereby obtained |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0667352A1 (en) |
| JP (1) | JPH0841425A (en) |
| CA (1) | CA2140834A1 (en) |
| FR (1) | FR2715405B1 (en) |
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| US8039591B2 (en) | 2008-04-23 | 2011-10-18 | Codman & Shurtleff, Inc. | Flowable collagen material for dural closure |
| US8795710B2 (en) | 2004-02-09 | 2014-08-05 | Codman & Shurtleff, Inc. | Collagen device and method of preparing the same |
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| FR2867388B1 (en) * | 2004-03-12 | 2006-06-23 | Agronomique Inst Nat Rech | COMPOSITIONS AND METHODS FOR DESTRUCTION OF INFECTIOUS PRION PROTEINS. |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2516927B1 (en) * | 1981-11-26 | 1986-05-23 | Merieux Fond | PROCESS FOR THE INDUSTRIAL PREPARATION OF COLLAGENIC MATERIALS FROM HUMAN PLACENTARY TISSUES, HUMAN COLLAGENIC MATERIALS OBTAINED, THEIR APPLICATION AS BIOMATERIALS |
| FR2517315B1 (en) * | 1981-11-30 | 1985-12-20 | Tech Cuir Centre | PROCESS FOR THE PREPARATION OF NEW FORMS OF COLLAGEN, NATIVE OR DERETICULATED, WITH PRESERVED HELICOIDAL STRUCTURE, ASSOCIATED WITH MUCOPOLYSACCHARIDES AND THEIR APPLICATIONS IN PARTICULAR IN THE COSMETOLOGICAL, PHARMACEUTICAL, ANALYTICAL AND OTHER FIELDS |
| CA1259914A (en) * | 1984-07-06 | 1989-09-26 | Donald G. Wallace | Methods of bone repair using collagen |
| US4642117A (en) * | 1985-03-22 | 1987-02-10 | Collagen Corporation | Mechanically sheared collagen implant material and method |
| JPH0662679B2 (en) * | 1985-06-21 | 1994-08-17 | 新田ゼラチン株式会社 | Tissue-friendly collagen and its manufacturing method |
| FR2586703B1 (en) * | 1985-09-02 | 1989-12-01 | Merieux Inst | PROCESS FOR EXTRACTING PLACENTAL COLLAGENS, COLLAGENS OBTAINED IN PARTICULAR IN THE FORM OF GELS OR SOLUTIONS AND THEIR APPLICATIONS |
| US4969912A (en) * | 1988-02-18 | 1990-11-13 | Kelman Charles D | Human collagen processing and autoimplant use |
-
1994
- 1994-01-24 FR FR9400716A patent/FR2715405B1/en not_active Expired - Lifetime
-
1995
- 1995-01-20 EP EP95400117A patent/EP0667352A1/en not_active Withdrawn
- 1995-01-23 CA CA 2140834 patent/CA2140834A1/en not_active Abandoned
- 1995-01-24 JP JP2745695A patent/JPH0841425A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5756678A (en) * | 1995-05-01 | 1998-05-26 | Cohesion Technologies, Inc. | Prion inactivation in connective tissue materials |
| US8795710B2 (en) | 2004-02-09 | 2014-08-05 | Codman & Shurtleff, Inc. | Collagen device and method of preparing the same |
| US8039591B2 (en) | 2008-04-23 | 2011-10-18 | Codman & Shurtleff, Inc. | Flowable collagen material for dural closure |
Also Published As
| Publication number | Publication date |
|---|---|
| FR2715405A1 (en) | 1995-07-28 |
| FR2715405B1 (en) | 1996-04-05 |
| JPH0841425A (en) | 1996-02-13 |
| EP0667352A1 (en) | 1995-08-16 |
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