CA1147726A

CA1147726A - Process for preparing macromolecular biologically active collagen

Info

Publication number: CA1147726A
Application number: CA000359671A
Authority: CA
Inventors: Gheorghe Cioca
Original assignee: Seton Co
Current assignee: Seton Co
Priority date: 1979-09-12
Filing date: 1980-09-05
Publication date: 1983-06-07
Also published as: FR2464962B1; ES494963A0; JPS5695195A; GB2116978B; GB2058084B; CH647947A5; IT8049654A0; GB2116978A; GB2058084A; FR2464962A1; US4279812A; JPS642120B2; GB8302017D0; DE3034273A1; IT1128187B; DE3034273C2; ES8106909A1

Abstract

ABSTRACT
Macromolecular reconstituted collagen is prepared by treat-ing natural insoluble collagen with an aqueous solution comprised of an alkali sulfate salt and an alkali metal hydroxide for at least 48 hours to saponify fats suspended within the natural insoluble collagen. The fat free collagen is then treated with an aqueous solution comprised of an alkalie metal sulfate for at least four hours to stabilize the interfibular bonds between individual polypeptide chains. The collagen is then dis-solved in an aqueous acid solution and frozen at a rate of -20°C/hour.
The frozen collagen is vacuum dried at 10-3 to 10-5 torr for at least 16 hours to produce a biologically active collagen article. Various bio-logically active materials may be added to the aqueous acid solution prior to freezing. The collagen product may then be implanted into an animal or the like and the medication slowly released. The article can remain within the biological system and it will slowly dissolve due to enzymatic digestion and through other biological processes.

Description

7;~6 BACKGROUND OF THE INVENTION
This invention relates to collagen and more particularly to macromolecular reconstituted collagen fiber.
~ 'Natural insoluble collagen" as used herein means and referff to collagen which cannot be dissolved in an aqueous alkaline or in any inorganic ~alt solution without chemical modification and includes hides, ffplit~, and other mammillian or reptilian coverings. More particularly, "natural insoluble collagen" means and refers to the corium which is the intermediate layer of a bovine hide between the grain and the flesh 10 ~ides.
Collagen constitutes the connective tissue and iA the major type of fibrous protein in higher vertebrae. Collagen in its natural state exiffts in a triple chain helix along with a confftant periodicity be-tween aligned triple chains. The triple helical configuration of colla-gen i~ sometimes referred to as a fibril and the fibrils align with an 1, 0 axial periodicity of about 640 A.
Although there are ~everal types of collagen, the major type i8 referred to aff "type I" which is the major collagen of skin, bones and tendonff. The type I collagen has a chain composition of ~0~1 (I)2~
20 2]. Thed~l (I) andd~2 chains are homologous.
In young animals there is little intermolecular and interfib-rilar crosslinking which provides for some degree of solubility of the collagen. However, during the aging process both intermolecular and interfibrilar crosslinking occurs, thus making the collagen insoluble, The use of collagen in substantially pure form has been pro-posed for many uses including for burn dressings as is disclosed in U.
S. Patents Noq. 3, 939, 831 and 3, 514, 518, and similar medical appli-cations as is disclosed in U. S. Patents Nos. 3, 157, 524 and 3, 628, 974, 11477;~6 along with its use as an additive to food.
While it has been known that collagen can be purified by the depolymerization of natural insoluble collagen along with subsequent reconstitution, the yields have been somewhat low and the resultant product is not necessarily biologically active.
United States Patent No. 3,637,642 is exemplary of a process for dl8solving insoluble collagen and regenerating the fiber.
Further, collagen and related materials have found utility in the food, cosmetic and pharmaceutical fields.
Further, methods have been proposed for solubilizing and reconstituting collagen with the use of enzymes to sever intra- and inter-fibular bonds such as is disclosed in United States Patent No. 3,034,852.
Further, processes have been proposed for converting collagen fibrous masses to sheet-like material such as in United States Patents No. 2,934,447 and 2,934,446.
According to United States Patents Nos. 3,939,831 and 3,742,955 medicinal dressings can be prepared from collagen having dispersed therein antibiotics and the like to aid in healing of skin which has been burned.
In accordance with the present invention a process for dissolving and regenerating collagen flber is provided which removes substantially all impurities from the collagen source and provides a substantially pure collagen product which is biologically active and substantially non-antigenic.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with a broad aspect of the present invention, there is provided a process of preparing macromolecular reconstituted collagen fiber comprising: (a) treating natural insoluble collagen fiber with an aqueous solution comprised of an alkali sulfate salt and an alkali metal hydroxide for at least 48 hours to saponify fats suspended within the natural insoluble collagen fiber to produce fat free collagen fiber, the collagen fibers being uniformly swelled; (b) treating the fat free collagen ~-- .
~ - 3 -~1~7~26 fiber with an aqueous solution comprised of an alkali metal sulfate for at least four hours to stabilize interfibular bonds; (c) dissolving the collagen fiber in an aqueous solution and; (d) freezing said solution to effect a temperature reduction rate of -18C to -24C/hour; (e) vacuum drying said solution at 10 3 to 10 5 torr for at least 12 hours. Step (d) is preferablY carried out at a rate of -20C/hour, down to a temperature of 60 C.
Thus, the present invention provides a regenerated collagen product having an average molecular weight of 383,000 to 460,000 with 4% to 6%
of the polypeptide chains having a molecular weight of 30,000 to 60,000 and 8% to 12% of the polypeptide chains having a molecular weight of 1,000,000 to 1,500,000 whenever produced by the process described above, or by an obvious chemical equivalent thereof.
Various biologically active materials may be added to the aqueous acid solution prior to freezing. The collagen product may then be implanted into an animal or the like and the medication slowly released. The article can remain within the biological system and it will slowly dissolve due to enzymatic digestion and through other biological processes.

In accordance with the present invention the alkali sulfates u8eful in the practice of the invention are the alkali metal sulfates such as so~ium sulfate, potassium sulfate, and the alkali earch metal sulfates such as calcium sulfate, magnesium sulfate and the like. The most preferred alkali sulfate is sodium sulfate. The alkali metal hydroxides useful in the practice of the invention are sodium and potassium hydroxide, and more preferably, sodium hydroxide. Alkaline earth metal sulfates such as calcium hydroxide and magnesium hydroxide may be substituted in part for the alkali metal hydroxides; however, sufficient potassium and/or sodium hydroxide must be provided.
The ~queou~ s~lution of the alkali sulf~te and the alkali metal B _ 4 _ 1~477Z6 hydroxide i8 composed of 1 molar to 2.5 molar of alkali metal hydroxide, 0.5 molar to 1 molar of alkali sulfate and 0.1 molar to 0.5 molar of other salt constituents; and more preferably, 2.0 molar to - 4a -7;~6

2. S molar alkali metal hydroxide, 0. 9 molar to l. 0 molar alkali sul-fate and 0. l molar to 0. 2 molar of other salt constituents. The alkali metal hydroxide and alkali sulfate should be at an initial pH of about 12 to 13, The other salt constituents may include alkali metal chloride such as sodium chloride and potassium chloride, alkaline earth metal chlorides such as magnesiùm chloride, calcium chloride and the like.
Care should be taken to properly proportion the alkali sulfate to the al-kali metal hydroxide in order to provide for complete saponification of 10 fats suspended within the natural insoluble collagen while retaining the native characteristics of the collagen and controlling the swelling of the collagen fiberY. If too much sodium hydroxide is used the collagen will be denatured and intermolecular bonds will be severed. If insuffi-cient sodium hydroxide is used the collagen product will have retained impurities such as fats and other hydrolyzable materials which are un-desirable.
In treatlng the natural insoluble collagen with the aqueous solution of the alkali sulfate salt and the alkali metal hydroxide, the natural insoluble collagen should be cut into pieces which are sufficient-20 ly small so that the aqueous solution may penetrate and rcact therein.The natural collagen pieces should be about ten cubic centimeters or less, and more preferably five cubic centimeters or less. Further, the treatment should take place at ambient temperature for at least 48 hours in order to completely saponify all of the fats suspended within the natural insoluble collagen and to provide a uniform degree of swell-ing of the collagen fiber. Care should be taken that the initial treat-ment with the alkali sulfate salt and the alkali metal hydroxide is not too prolonged or the polypeptide chains will be attacked and the colla-11~'7726 gen will be denatured. For example, when the natural insoluble colla-gen is cut into pieces of five cubic centimeters the initial treatment should take no more than 96 hours elsewise the collag~n fiber will de-grade into lower molecular weight components and be denatured. After thi~ initial treatment the natural insoluble collagen become~ very soft and tran~parent.
After the first treating solution is removed, the collagen i6 treated with a solution of an alkali metal sulfate or alkali earth metal sulfate or a combination thereof at a substantially neutral pH. The con-10 centration of sulfate ~hould be about 0. 5 to 1. 0 molar. Other salts suchas sodium chloride, potassium chloride, magnesium chloride and the like may be added to thi~ salt treatment ~o long as a sufficient quantity of alkali metal sulfate, preferably sodium sulfate, is used to stabilize the interfibular bonds of the collagen. This treatment with the alkali metal salt fihould be for at least four hours.
Preferably the collagen is then neutralized with an aqueous acid ~olution having a pH between 3 and 4. The acids used to form the aqueou~ acid solutlon are typically boric acid, tartaric acid, acetic acld or the like. The washing takes place for about six hours to re-20 move re~idual salt~ and base constituents. The pH of the collagen sub-~equent to neutralization is about ~even.
The collagen is then washed with tap water. To remove re-sidual 6alts within the collagen, it is washed with distilled water sever-al times. Preferably, each washing cycle takes about 4 hours. After each 4 hour cycle the water is decanted and fresh distilled water i9 added. Normally 4 to 7 cycles are required to remove the residual s alts .
The collagen i9 then dissolved in an aqueous acid solution '726 preferably a solution of 1% to 1. 5% by -weight collagen is prepared.
The acids useful for dissolving the collagen fiber are the weak organic acids such as acetic, citric, lactic, ascorbic and tartaric acids. Pre-ferably the pH is adjusted to below four in order to obtain good solubility.
In the ca~e of ascorbic acid a 1% ~olution i~ ~ufficient and in the case of acetlc or tartaric acid a 0. 5% solution is sufficient. The pH of the aque-ou~ ~olution should be about 3 to 4.
The collagen solution i9 then frozen to reduce its temperature at a rate of _18C to -24C/hour until it is at a temperature of -60Cto 10 -70C.
The freezing with a temperature reduction of about -18C to -24C/hour is required ~o that the ice crystals formed are extremely small and do not ~ub6tantially ~ever the collagen chains to provide re-duced molecular weight to the final collagen product.
To obtain the de~ired rate of freezing the collagen solution i~ placed in a freezer at -60C to -70C.
The frozen Eolution is then placed in a freeze dryer with an inltial temperature of -60C to -70C and vacuum ~ublimated at 10-3 to 10-5 torr. The freeze drying proce~ requires about 12 to 24 hours 20 with a final temperature of 30C.
In addition, although there is a prevention of extreme de-struction of collagen chain~ by freezing, minor amounts of cryogenic destruction i9 required in order to form reactive and as~ociative sites which provide crosslinking yielding enhanced mechanical and enzymatic stability to the final porduct and for combination with other additives to be retained in the collagen.
The collagen prepared in accordance with the invention is one which has a weight average molecular weight of about 450, 000 and ~1~77'~6 axial periodicity between triple helix fibrils of about 750 A to 850 A
and more preferably about 800 A as opposed to about 640 A for tropo-collagen. Surprisingly, the collagen prepared in accordance with the invention retains substantially the same biological activity as natural collagen which has been purified by long and tedious processes.
The product prepared in accordance with the invention is a spongy tenaceous mass.
Further, the average molecular weight of collagen prepared in accordance with the invention is 383, 000 to 460, 000 as compared with 300, 000 for tropocollagen. The collagen prepared in accordance with the invention has ~ to 6% of the polypeptide chains having a molec-ular weight of 30, 000 to 60, 000 and 8 to 12% 1, 000, 000 to 1, 500, 000.
In another aspect of the invention biologically active materials may be added to the collagen solution or dispersion prior to freezing.
These materials may be drugs or the like. Because the collagen ar-ticles produced in accordance with the invention proximate natural or reconstituted collagen, the release of the biologically active material from the collagen approximates the absorption of the material by a bio-logical system which has the collagen implanted or in contact therewith.
It is believed that the severance of polypeptide chain~ during freezing causes the formation of radicals which associate with particu-lar medicaments and can thus be released in vivo.
Further, after a collagen article having a medicament there-in is implanted and the medicament is spent, the collagen article is slowly dissolved and/or decomposed by enzymatic digestion or other biological process thus eliminating the need for removal of the implant.
One particular use of the process of preparing collagen ar-ticles useful in the practice of the invention is the blocking of estrus in ~7'7~6 - animals. An appropriate hormone such as chlormadinone acetate, di-methisterone, ethisterone, hydroxyprogesterone, hydroxyprogesterone caproate, medroxyprogesterone, norethindrone, norethynodrel, pro-gesterone, 3 - ethylenedioxy, 17 - acetoxy, 6 - methyl - pregn- 5 -ene - 20 - one, and the like, is added to the collagen solution or dis-persion prior to freezing. The hormone is added at an effective amount to block estrus preferably at a level of 1 part/40, 000 parts of collagen solution to l part/50, 000 parts of collagen solution.
The collagen article is inserted into the uterus of the animal.
10 After 14 to 18 days the hormone is spent from the collagen article. Two to seven days following the withdrawal of hormone application an en-hanced estrus occurs and the animal may then be serviced. Further, the animal may also be artifically inseminated at this time.
In a further embodiment antibiotics such as penicillin, te-tracycline, oxytetracycline, chlorotetracycline, chloramphenicol, sul-fonamides and the like may be added along with the hormone to prevent infection during insemination and subsequent pregnancy.
2 to 14 days after insertion of the collagen article, the en-zymatic proce~ses in the uterus begins to biochemically degrade the 20 collagen. Glutaraldehyde may be added to the collagen to increase the time required for biochemical degradation.
In another application for using collagen articles prepared in accordance with the invention, a spermicide (such a~ nonylphenoxy polyoxyethylene ethanol) i9 added to the collagen solution in the same manner as a hormone is added. The collagen article may be inserted into the vagina and the spermicide kills the sperm injected into the va-gina thus preventing conception. The enzymes present in the vagina slowly dissolve the collagen thus removal of the insert is unnecessary.
_9_ ~7'7~6 Further, as previously described glutaraldehyde or other crosslinking agents may be added to the collagen to slow the dissolution rate of the collagen in vivo.
A further embodiment of the present invention involves the addition of an antibiotic, a bacteriostat or a bacteriocide to the collagen di~perYion of ~olution prior to freezing. After freeze drying the colla-gen article may be u~ed for healing of burns or the like.
In general, variou~ medicaments can be added to the collagen dispersion and the collagen article implanted to effect biological, phy-10 siological or psychological effects in the biological system in which iti~ implanted.
The invention will be more fully de~cribed with reference to the specific example~ herein set forth.
EXAMP~E I
One kilogram of corium derived from raw cow hides i~ cut into piece~ of 5 cm3. The 5 cm3 piece~ are treated in a vat with a ~olution having the following compo~ition:
In~redient Amount Water 3000 ml Calcium hydroxide 150 g Potas ~ium hydroxide 50 g Sodium hydroxide 100 g Sodium ~ulfate 144 g Sodium chloride 100 g Pota~sium chloride 200 g Calcium sulfate 100 g The natural insoluble collagen is treated in the above ~olution with agitation for 48 houræ. Observation and testing after treatment showed 11~7~7Z6 saponification of all fats and a uniform degree of swelling of the colla-gen. The corium became very soft, transparent and porous.
The treating vat was drained of the solution and a second solution was charged to the vat having the following composition:
Ingredient Amount Water 6000 ml Sodium sulfate 144 g Sodium chloride 100 g Pota~sium chloride 100 g Calcium sulfate 100 g The corium was treated with the ~odium ~ulfate solution for at lea~t four hours to stabilize the interfibular collagen bonds, The ~econd solution wa~ drained from the vat by screening and the vat was recharged with a solution of 3000 ml of water and 90 g of boric acid to wa~h and acidify the twice-treated corium. This treat-ment took six hour~ until the collagen had a neutral pH. The collagen was then ~eparated from the liquid and treated with ten liters of tap water for four hours. The tap water was drained and the collagen wa~
again treated with 15 liters of distilled water for at least 4 hour~ to 20 wa~h any residual ~alts therefrom; the water was decanted and 15 li-ter~ of di~tilled water was added. This procedure was repeated until six washing cycles were accomplished. The collagen was dissol~ed in ten liters of 1% ascorbic acid and stirred until homogeneous. The collagen solution was poured into form~ 1 cm high and 2. 5 cm in dia-meter. The forms were frozen at a temperature of -60C to -70C to effect a temperature reduction rate of the collagen of -20C/hour to a temperature reduction rate of the collagen of -20C/hour to a temper-ature of -60C. The collagen was ~en freeze dryed with an initial -11_ temperature of -60C and a final temperature of 30C after 16 hours.
The collagen prepared in accordance with Example I had an average molecular weight of 450, 000. The axial period between colla-gen fibrils was about 800 A.
The collagen also prepared in accordance with Example I
wa~ readily dige6ted by collagenase and upon testing showed no anti-genic activity.
The collagen prepared in accordance with Example I had good mechanical propertie~ including ela~ticity, compressability and 10 ~oftness.
EXAMPLE II
Example I was repeated except that 0. 025 gram~ of med-roxyproge~terone acetate wa~ added to 1000 ml of the collagen solution prior to pouring into the forms.
The collagen pellets having ~he hormone uniformly disper~ed therethrough were implanted into the uteri of cow~ to induce e~trus therein. Becau~e of Ihe complexation of the collagen with the hormone the hormone i~ relea6ed over a period of 2 to 14 day~. After this per-iod the cow~ are artificially in~eminated. The collagen pellet i~ allow-20 ed to remain in the uteru~ and ~lowly dissolve~ due to enzymatic di-ge~tion thereof.
EXAMP LE III
Example II was repeated except that 0. 25 gram~ of chloro-mycetin was added to the collagen di~persion prior to freezing.
The chloromycetin was effective in preventing infection with-in the cattle during insemination and ~ubsequent pregnancy.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process of preparing macromolecular reconstituted collagen fiber comprising:
(a) treating natural insoluble collagen fiber with an aqueous solution comprised of an alkali sulfate salt and an alkali metal hydroxide for at least 48 hours to saponify fats suspended within the natural insoluble collagen fiber to produce fat free collagen fiber, the collagen fibers being uniformly swelled;
(b) treating the fat free collagen fiber with an aqueous solution comprised of an alkali metal sulfate for at least four hours to stabilize interfibular bonds;
(c) dissolving the collagen fiber in an aqueous solution and;
(d) freezing said solution to effect a temperature reduction rate of -18°C to -24 C/hour;
(e) vacuum drying said solution at 10-3 to 10-5 torr for at least 12 hours.

2. The process according to claim 1 including treating the fat free collagen with distilled water to remove salts therefrom prior to dissolving said collagen.

3. The process of claim 2 including treating with distilled water for at least 4 hours, decanting the water, and retreating with distilled water at least 4 times for 4 hours for each treatment.

4. The process according to claim 1 wherein said alkali sulfate is sodium sulfate.

5. The process of claim 4 wherein said sodium sulfate is present at a level of 0. 5 molar to 1 molar.

6. The process of claim 1 wherein said alkali metal hy-droxide is sodium hydroxide.

7. The process of claim 6 wherein said sodium hydroxide is present at a level of 1 molar to 2. 5 molar.

8. The process of claim 1 wherein said alkali sulfate is present at a level of 0. 5 molar to 1, 0 molar when stabilizing said in-terfibular bonds,

9. The process of claim 1 wherein said collagen is neu-tralized subsequent to stabilizing the interfibular bonds by washing with an aqueous acid solution.

10. The process of claim 1 wherein said aqueous solution for dissolving said collagen is an aqueous acid solution.

11. The process of claim 10 wherein the acid in said aque-ous acid solution is ascorbic acid.

12. The process of claim 11 wherein said aqueous acid solution has a pH of 3 to 4.

13. The process of claim 1 wherein said natural insoluble collagen is a bovine corium.

14. The process of claim 1 including adding a crosslinking agent to said collagen fiber.

15. The process of claim 1 including adding a minor amount of a biologically active substance to said collagen solution prior to freezing.

16. The process of claim 14 including adding a minor amount of a biologically active substance to said collagen solution prior to freezing.

17. A process of claim 15 wherein said biologically active substance is a hormone.

18. The process of claim 16 wherein said biologically active substance is a hormone.

19. The process of claim 15 wherein said biologically active substance is an antibiotic.

20. The process of claim 16 wherein said biologically active substance is an antibiotic.

21. The process of claim 15 wherein said biologically active substance is a spermicide.

22. The process of claim 16 wherein said biologically active substance is spermicide.

23. The process of claim 14 wherein said crosslinking agent is glutaraldehyde.

24. A regenerated collagen product having an average molecular weight of 383,000 to 460,000 with 4% to 6% of the polypeptide chains having a molecular weight of 30,000 to 60,000 and 8% to 12% of the polypeptide chains having a molecular weight of 1,000,000 to 1,500,000 whenever produced by the process of claim 1, or by an obvious chemical equivalent thereof.

25. The product of claim 24 wherein said product has enhanced cross-linking when produced by the process of claim 14 or by an obvious chemical equivalent thereof.

26. The product of claim 24 which includes a minor amount of a biologically active substance when produced by the process of claim 15 or by an obvious chemical equivalent thereof.

27. The product of claim 24 wherein said product has enhanced cross-linking and includes a minor amount of a biologically active substance when produced by the process of claim 16 or by an obvious chemical equivalent thereof.

28. The product of claim 24 which includes a minor amount of a hormone when produced by the process of claim 17 or by an obvious chemical equivalent thereof.

29. The product of claim 24 wherein said product has enhanced cross-linking and includes a minor amount of a hormone when produced by the process of claim 18 or by an obvious chemical equivalent thereof.

30. The product of claim 24 which includes a minor amount of an anti-biotic when produced by the process of claim 19 or by an obvious chemical equivalent thereof.

31. The product of claim 24 wherein said product has enhanced cross-linking and includes a minor amount of an antibiotic when produced by the process of claim 20 or by an obvious chemical equivalent thereof.

32. The product of claim 24 which includes a minor amount of a spermicide when produced by the process of claim 21 or by an obvious chemical equivalent thereof.

33. The product of claim 24 wherein said product has enchanced cross-linking and includes a minor amount of a spermicide when produced by the process of claim 22 or by an obvious chemical equivalent thereof.