JPS58170796A - Injectable bridged collagen, preparation and transplantation substance of collagen - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to compositions and methods for treating body tissue. FIELD OF THE INVENTION This invention relates to collagen implants for soft tissue augmentation in mammals, particularly those that are injectable.

Collagen is used as a pharmaceutical carrier, surgical prostheses (sutures and dressings), and implants. (Chv.
apil, et al, Intl Rev of
ConneC-tive Ti5sue Res (1
973) 6:1) In many cases, collagen is
In order to improve mechanical properties, reduce immunogenicity and improve absorption resistance, it is cross-linked by chemicals, radiation or other means.This known cross-linked collagen has solid properties. There is.

Implants made from solid cross-linked collagen were surgically implanted. (i.e., implanted by incision.) 01iver et al.
edics & Re-1ated Re5earch
(1976) 115:291-302.

Br J Exp Path (1980) 61:5
44-549. and Conn Ti5su Res (
1981) 9:59-62, describe implants obtained by treating the skin with trypsin and then crosslinking with aldehydes. The resulting skeletal collagen implants are reported to maintain their size long after implantation. The major problem with such body implants is that they must be surgically implanted. Other problems are that the injectable implant is not easily deformed and that residual glutaraldehyde can cause the implant to lose its flexibility due to continued cross-linking. be.

5chechter et al. Br J Plas Sur
(1975) 28: 198-202 discloses skin crosslinked with glutaraldehyde which is soaked in L-alanine after crosslinking. This article postulates that exposing the skin to L-alanine blocks residual reactive groups of aldehydes, thus preventing the release of toxic molecules generated by such reactive groups.

US Pat. No. 3,949,073 describes the use of collagen atelopeptide solutions as injectable implants for soft tissue augmentation. According to the description in this publication, collagen is reconstituted before transplantation, and once transplanted, it becomes a fibrous tissue. Furthermore, this publication suggests adding insoluble collagen microfibrils to control the contraction of the fibrous bodies formed at the augmentation site. A commercially available product of the material described in the above publication is ZYDERM (trade name) Collagen 42, which consists of a reconstituted aqueous atelopeptide sodium chloride solution containing a small amount of local anesthetic. Although this commercially available product is highly effective, it may shrink when implanted, primarily due to its liquid component being absorbed into body tissue. Therefore, if volume constancy, sometimes referred to as "permanence," is important, replacement implant material must be added by injection (not necessarily once).

A primary object of the present invention is to provide collagen that is useful for skin augmentation and that (1) is homogeneous, i.e., the collagen has only a single physical shape; (2) is injectable; 3)
Z Y D E RM - To provide a cross-linked collagen implant material that has superior permanence and resistance to physical deformation compared to collagen implants.

One aspect of the present invention is a novel chemically crosslinked atelopeptide collagen that is characterized as a viscous fluid at 22°C and substantially free of residual crosslinkers. This collagen generally has a temperature of about 700 ~
It has a viscosity of about 3000 cp and contains less than about 20 ppm residual crosslinker.

Another aspect of the invention is a collagen graft material for use in augmenting soft tissues, characterized in that it consists of a dispersion of chemically cross-linked atelopeptide collagen as described above.

Another aspect of the invention is to neutralize the acidic aqueous solution at a reduced temperature and hypotonic ionic strength (2).
Reconstituting atelopeptide collagen from its solution and producing cross-linked collagen, which is a viscous fluid at 22°C: At sufficient concentration and conditions, the reconstituted atelopeptide collagen shares with collagen. The above-mentioned method is characterized by crosslinking in an aqueous medium with a binding crosslinking agent, quenching the crosslinking reaction with a quenching agent that reacts with the crosslinking agent, and separating the viscous crosslinked atelopeptide collagen from the reaction mixture. A method for preparing chemically crosslinked atelopeptide collagen.

Cross-linked collagen for use in the present invention can be derived from collagen harvested from many mammalian sources. The donor need not be genetically similar to the recipient into which the collagen material will ultimately be implanted.

Cow or pig dermis is usually used because it is readily available. The first step in making cross-linked collagen is to prepare an atelopeptide collagen solution from the dermis. The animal's skin is soaked in a mild acid;
The skin is then softened by a soufflage to remove hair, epidermis, and fat.

The depilated skin is then soaked again in a moderate acid and pulverized by physical methods such as grinding, cutting, or milling.This pulverization leaves the skin in a state ready for solubilization.

When the ground tissue is dispersed in an acidic aqueous medium and digested with a proteolytic enzyme other than Kola N genease, it can be solubilized without being denatured.

At low temperatures, dilute acid solutions are usually used to avoid denaturation. Mineral acids such as HCl and carboxylic acids such as acetic acid, malonic acid, and lactic acid have a pH range of about 1.5 to 5, about 5 to 25
Can be used in a temperature range of ℃. 1 to 5 at 20°C and approximately pH 2
Preferably, the ground tissue is dispersed in HCl to a concentration of 11/13. After dispersion of the tissue, enzymes are added and the mixture is incubated to allow the enzymes to digest the telopeptide portion of the collagen and other solubilized components of the tissue. The enzyme used is one that digests the telopeptide portion without denaturing the collagen helix. Examples of such enzymes are trypsin, pepsin, chymotrypsin and papain. Pepsin is preferred because it is relatively easy to deactivate and separate from solubilized collagen. Normally, the enzyme concentration is about 0.1 to 42 collagen.
-10% by weight (2 is sufficient.Culture time may generally be selected from about 2 days to 2 weeks.

The progress of solubilization can be checked by determining the viscosity of the solution. The point at which the viscosity reaches a nearly constant value is the end point of solubilization, at which point the enzyme is deactivated (denatured) and separated.

Inactivation of the enzyme can be accomplished by adding an alkaline substance such as sodium hydroxide to bring the pH of the solution to at least about 7. After the enzyme is inactivated (denatured), the solution is processed to separate the denatured enzyme and the tissue portion that was digested during solubilization. Various dialysis methods, sedimentation methods, and filtration methods can be applied to this separation. In a preferred embodiment, the acid is first added to p.
After lowering the H, the solution is clarified by diatomaceous earth precipitation.

Marry the precipitate and concentrate the lachrymal fluid. The concentrate is then fractionated by ion-exchange chromatography and further concentrated to a substantially pure atelopeptide collagen solution that can be used to make cross-linked collagen.

The next step in making cross-linked collagen is to reconstitute the atelopeptide collagen from solution. Reconstitution is preferably carried out by neutralizing the solution at a low temperature, preferably about 10°C to 25°C. Preferably, the ionic strength of the neutralizing solution is low compared to physiological conditions.

Ionic strengths of about 0.03 to about 0.1, preferably about 0.06 are commonly used. Neutralization involves raising the pH of the solution by adding a suitable base or buffer, such as Na2HPO4 or NaOH, to a level where the collagen solution reassociates with fibrils. 111 fibers are formed under these conditions in the pH range of about 469 to about 10.0. Preferably, the final pH is in the range of about 5-8. If the pH is less than about 7 within this range, thin (and soft) fibrils are likely to form;
is 7 or more, coarser fibrils are likely to be formed. Soft fibrils. Such organization makes soft fibril dispersions easier to inject. The time required for the fibril formation step typically ranges from about V2 to about 18 hours.

The reconstituted ateloproptide fibrillar collagen gel suspension is crosslinked with a crosslinking agent that forms covalent bonds with the collagen. Typically, crosslinkers are polyfunctional, although difunctionality is more common. The crosslinking conditions provide an implant that can be formulated as an injectable fluid and has superior permanence to implants made from comparable formulations of non-crosslinked fibrillar atelopeptide collagen. The conditions are such that they produce viscous, covalently cross-linked collagen. When crosslinking reaches this extent, the crosslinking reaction is quenched by the addition of a quenching agent. The quenching agent forms a non-hazardous water-soluble adduct with the crosslinking agent. The concentration of the crosslinking agent and the duration of the crosslinking reaction are important process conditions in obtaining a degree of crosslinking that provides a viscous fluid product that is injectable as such. Rapid cooling is important to prevent further crosslinking and changes in product viscosity. Inactivation of the reactive groups of the crosslinking agent also removes such reactive groups from the product, rendering the product less immunogenic compared to non-quenched crosslinked collagen. Aldehydes are suitable crosslinking agents. Examples of aldehydes used to crosslink collagen include formaldehyde, glutaraldehyde, acetaldehyde, glyoxalpyruvate aldehyde, and dialdehyde starch. Glutaraldehyde is particularly preferred. Compounds having functional groups that react with functional groups (eg, aldehyde groups) of the crosslinking agent to form water-soluble adducts can be used to quench the crosslinking reaction. Quenching agents with free amino groups such as amino acids are preferred. Glycine is particularly preferred. The concentration of glutaraldehyde in the reaction mixture generally ranges from about 0.001 to about 0.000.
05% by weight. The duration of the crosslinking reaction is generally from half an hour to about one week. The reaction is usually carried out at about 0°C to about 35°C. The quenching agent is added in small amounts (at least in stoichiometric proportion) to the crosslinking agent. It is preferable to add the quenching agent in excess.

A particularly preferred crosslinking and quenching step procedure is as follows: about 0.01% by weight glutaraldehyde at about 22°C.
for about 16 hours and quenched with excess glycine to about 0.2M at about 22° C. for 1 to 2 hours.

After quenching is complete, the crosslinked atelopeptide collagen product is washed with a buffered aqueous solution to remove aldehyde-quenchant adduct, unreacted aldehyde, aldehyde polymer, and unreacted quenchant. A sodium phosphate-sodium chloride buffer solution with a pH of 6.9 to 7.4 is preferred. The washed product has a suitable protein concentration, typically about 20 to
Concentrate by filtration or centrifugation to a size of 50 mm, preferably about 25 to 40 mm. The protein concentration can be adjusted to the above range by adding a buffer or further concentrating, depending on the case. The washed product is about 20
Contains less than ppm free aldehydes and has a steady flow viscosity (
An oscillating disk viscometer (oscil), which measures kinematic viscosity, rather than a steady flow viscosity
(lating disk viscosit neter)
(Nametre Co, 7.006PBD type)
(=Measured accordingly. About 700 to about 3 at 22°C
It has a viscosity in the range of 000 cp.

The final formulation of the quenched aqueous suspension of cross-linked collagen involves adjusting the ionic strength of the suspension to isophoric (i.e., about 0.15 to about 0.2) and localization during injection. and adding a local anesthetic such as ridecaine to a concentration of about 0.3% by weight to reduce pain. The suspension is then filled into a syringe fitted with a 27 gauge needle or larger. The term "injectable" as used herein means that the formulation can be dispensed from the syringe with normal manual pressure.

Process of preparing a novel injectable cross-linked collagen (
Preferably, the above steps in step 2 are performed under sterile conditions using sterile materials.

The collagen graft material of the present invention described above can be injected subcutaneously or subcutaneously for the purpose of augmenting soft tissue, treating or correcting congenital abnormalities, acquired defects, and cosmetic defects.

Examples of these include facial hypoplasia, acidogenesis of the cheeks and zygomatic bones, -individual breast acidogenesis, pectus excavatum, hypoplasia or agenesis of the pectoral muscles (Boland's anomaly), and cleft palate or submucosal cleft palate. Treatment (as a retropharyngeal graft) (= associated congenital anomalies such as velopharyngeal dysfunction, depression scars, (e.g. DLE)
Discoid lupus erythematosus ζ2) mucosal atrophy, enucleated eye enophthalmia (sophthalmia), facial blackout, submucosal atrophy, itchy linear intensity (scleroderma), saddle nose syndrome, Rompergue Acquired defects (after trauma, surgery, infection) such as disease or unilateral vocal cord paralysis, as well as glabellar frown lines, deep nasolabial folds, perioral geometric wrinkles, sunken cheeks, and acid formation in the breasts. Cosmetic defects such as symptoms.

The following examples illustrate the advantages of viscous crosslinked collagen, implants made from the collagen, methods for using the implants, and implants made from the implants. The examples are not limited.

Preparation of Bovine Aterobapted Collage/Solution Bovine skin was softened and hair removed by HcJ treatment. The depilated skin was then ground and dispersed in Hel at pH 2 in an amount of 8-11 g/l. Pepsin, 0.1% by weight based on total protein, was added to this dispersion and the mixture was stirred at 15-20°C for about 100% by weight.
Cultured for ~300 hours. NaOH was then added to raise the pH of the culture medium to approximately 7, thus stopping the digestion. The inactivated (denatured) enzyme was removed from the reaction mixture by precipitation at low pH. The solution was then purified and concentrated by filtration and chromatography to obtain a solution of bovine atelopeptide collagen/3119 in dilute hydrochloric acid (PH1-4).
/d was created. This solution is hereinafter referred to as CIS.

Preparation of fibrillar collagen 0.02 M Na2HP in CIS at 18°C to 20°C
Add O4 and adjust its pH to 7.4-70.2 or 5.8-6
．． 5 to reconstitute fibrillar collagen from CIS. Fibers were allowed to form for 1-2 hours.

Preparation of cross-linked viscous collagen A, neutral reconstituted fibrous collagen suspension 1605 w/w when using reconstituted collagen at pH 7.4 ± 0.2
1.0% glutaraldehyde aqueous solution at pH 3 1.6
Added 2d. The glutaraldehyde solution was gradually added (20%), and finally (20%).

After 16 hours of reaction time, the reaction was quenched by adding 3M glycino to 0.2M. The quenching time was 1 hour. Next, the cross-linked collagen/
At 7,4, buffer approximately 100 d, 0.02 M N with centrifugation at 17000.9 for 5-7 min between each wash.
a1ilPO4,0,13M (Washed 3 times with DNA
m@tve Co,, 7.006 FBDIjl,
measured at a shear rate of approximately 5000 mm''1) and was found to be approximately 700 cp at 22°C. After the final wash and centrifugation, the collagen has a protein concentration of approximately 2
8-38 Gin-a: Resuspended in buffer until solid. This dispersion was filled into a syringe equipped with a φ27 gauge needle. This collagen 11 preparation will be referred to as preparation C hereinafter.

Preparation of injectable crosslinked collagen fluids was performed as described above using various crosslinking reaction times and glutaraldehyde concentrations. These preparations are shown in the table below.

Preparation agent glutar Reaction time name Aldehyde (hours) A O,01 and'! IC0
, 051 G O, 0516 B, When using collagen reconstituted at pH & 8 to 6.5: Gradually add a 1% aqueous glutaraldehyde solution at pH 3 to the reconstituted fibrous collagen dispersion while stirring;
The final glutaraldehyde concentration was 0.005%~o,
It was set to oio%. The crosslinking was carried out for 16 hours while stirring. Next, 2M glycine was added to a final concentration of 0.2
The mixture was added with stirring until the concentration reached M~0.3M, and the mixture was rapidly cooled. The quenching lasted 2-3 hours with stirring. Next, 16,000 g of rapidly cooled crosslinked collagen was added to
Centrifuged for 0 min and collected, 0.02 M Na,
HPO4゜0, 13 M Nacl, pH 7.4 1
Resuspend in 0-40 volumes. 16,000 yen of this suspension
0. ! Centrifugation for 5-20 min at i' gives the final
A viscous, crosslinked collagen product was obtained. The protein concentration after the final centrifugation was 45±5 m9/d.

In vivo test of collagen preparation 5pra9u, 45-50 days after birth, weight 120±20g
e-Dawley female rats were used as transplant recipients.

Groups of three rats were implanted with Preparations A, C, E, and G and ZYDERM Collagen implants as a control agent. Each rat was implanted at the following two locations: the cross-linked collagen preparation was implanted in the right dorsal cranial region, and the control agent was implanted in the left dorsal cranial region. Approximately 1 cc of the preparation was injected subcutaneously per site.

All preparations were removed from the body 15 days after implantation.

The donor tissue was carefully dissected from the explant and its wet weight was recorded. next,
Weight recovery rate (persistence) was calculated from the implanted weight.

The weighed specimens were then embedded, sectioned, and stained for histological examination. The dyes are hemoxylin, eosin, and Gomori trichrome.
e) was used.

Test Results A summary of the histological examination of Preparations A, C, E and G is given below.

Formulation A: This preparation was more cross-linked and had a much more uniform lace-like appearance compared to the implants of the preparation that was not quenched with Glyci/. For preparations that are more cross-linked and not quenched with glycine, the fissures generally form large, closely packed areas with intervening eyes. Fissures also occurred between the implants of Preparation A, but were smaller and fewer in number. In the implant material of Preparation A, the intervening small fissures were well infiltrated with fibroblasts, even within the eye. The fissures looked like new collagen was being synthesized within the eye. Few round cells were observed. Vascularization was moderate in the peripheral half of the A preparation implant and was not confined to the new collagen synthesis zone. No evidence of encapsulation was seen. There were no epithelioid cells and multinucleations.

Formulation C: This formulation was more lacy and porous than Formulation A. This preparation showed excellent dispersive cell infiltration with areas of new collagen synthesis. These properties, along with the generally small number of round cells, are the reasons why Conditioner C is considered the best of the four preparations from a histological point of view.

Formulation E: This formulation was generally less lacy than the one crosslinked with 0.01% glutaraldehyde. Although fibroblasts were distributed diffusely, their numbers were small and angiogenesis was generally limited to the periphery of the implant. Areas of new collagen synthesis may also be affected by other preparations;
Nirubete shuban (2 was not observed).

Formulation G: This formulation exhibited the most uniform lacy porous appearance of all formulations crosslinked with 0.05% glutaraldehyde. Although the migration of cells into this preparation was good, an increase in the number of multinucleated focal areas and round cells was also evident. This was not observed with the preparation cross-linked with 0.001% glutaraldehyde.

The following table reports the persistence (percent wet weight recovery of carefully dissected explants relative to the wet weight of the implants) of the preparation and Koitrol agents.

114H agent Persistence A 771g (68K gold 511B8282% 0 64*61 control (average) 30~40g! Applicant Collagen Corboley Vi1 Daibajin Patent attorney Ka Toasa Michi Continuation of page 1 Priority claim 01982 May 6, ΦUnited States (US)■3
75665 0 Inventor Donald Grayson Wallace 38 Menlo Park Hedge Road, California 94025, USA Procedural amendment (voluntary) April 6, 1985 Commissioner of the Patent Office Kazuo Wakasugi 1 Participant indication 1988 Patent application No. 038117 (filed on March 8, 1982) 2 Title of the invention Injectable cross-linked collagen, its preparation method, and implant material using the collagen 3 Relationship with the case of the person making the amendment Patent applicant 8 Contents of the amendment Clear copy of statement (no change in contents) z7m

Claims (1)

[Claims] (]) (al is a viscous fluid at 22°C,
(b) A chemically crosslinked atelopeptide collagen characterized in that it is substantially free of residual crosslinkers. (2) Collagen has a temperature of about 700 to about 3 at 22℃
Collagen according to claim 1, characterized in that it has a viscosity of 0.000 cp. (3) The amount of residual crosslinking agent in collagen is approximately 20p.
The collagen according to claim 1 or 2, characterized in that the collagen is less than pm. (4) The collagen according to claim 1, 2, or 3, wherein the crosslinking agent is glutaraldehyde. (5) a chemically crosslinked azolopeptide collagen that is a viscous fluid at 22°C and is substantially free of residual crosslinkers;
Preparation method: (a) reconstituting azolopeptide collagen from an acidic aqueous solution by neutralizing the acidic aqueous solution at low temperature and low tonic ionic strength; (b) producing cross-linked collagen, which is a viscous fluid at 22°C. (c) crosslinking the reconstituted atelopeptide collagen in an aqueous medium with a crosslinking agent that forms a covalent bond with the collagen at concentrations and conditions sufficient to and (d) separating the viscous crosslinked atelopeptide collagen from the reaction mixture. (6) the cross-linked atelopeptide collagen has a viscosity of about 700 to about 3000 cp at 22°C, and 20 pp
6. The method according to claim 5, characterized in that the reactive crosslinking agent contains m or less. (7) The temperature in step (a) is about 0°C to about 25°C, and the ionic strength of step (at) is about 0.03 to about 0.
．． 1, and step (final pH of al is about 49 ~
10.0, the crosslinking agent is glutaraldehyde, and the concentration of glutaraldehyde in the crosslinking reaction mixture is about 0.
．． 7. A method as claimed in claim 6, characterized in that the amount is about 0.001% by weight to about 0.05% by weight. (8) Collagen is collagen from cow dermis,
The temperature of step (a) is about 0°C to about 25°C, the ionic strength of step (a) is about 0.03 to about 0.1, and the final pH of step (a) is about 5°C. to about 8, the crosslinking agent is glutaraldehyde, and the concentration of glutaraldehyde in the crosslinking reaction mixture is about 0. O(1
1% by weight to about 0.05% by weight, the crosslinking reaction is carried out for about one hour to about one week, the quenching agent is glinone, and the separation is performed by washing the viscous crosslinked atelopeptide collagen and preparatory to other reactions. the cross-linked atelopeptide collagen has a viscosity of about 700 to about 3000 cp at 22°C and about 20 ppp.
6. The method according to claim 5, characterized in that it contains m or less reactive aldehydes. (9) (a) It is a viscous fluid at 22°C;
(b) A collagen implant material for use in mammalian soft tissue augmentation, characterized in that it consists of an aqueous suspension of chemically crosslinked atelopeptide collagen substantially free of residual crosslinking agents. αQ Collage/ is about 700 to about 300 at 22℃
Implant material according to claim 9, characterized in that it has a viscosity of 0 cp. aυ The amount of residual crosslinking agent in collagen is approximately 20 ppm
The implant material according to claim 9, characterized in that: 02. The implant material according to claim 9, 10 or 11, wherein the crosslinking agent is glutaraldehyde.