CA1250235A

CA1250235A - Drug delivery collagen synthetic vascular graft

Info

Publication number: CA1250235A
Application number: CA000473090A
Authority: CA
Inventors: Harmon Hoffman, Jr.; Kemal Schankereli; Milos Chvapil
Original assignee: Meadox Medicals Inc
Current assignee: Boston Scientific Scimed Inc
Priority date: 1984-01-30
Filing date: 1985-01-29
Publication date: 1989-02-21
Also published as: IL74180A; SE8500422D0; DE3503126C2; AU575617B2; GB2187192A; FR2558720B1; FR2558720A1; JPH0636817B2; CH670380A5; AU3819685A; NL193263C; SE464009B; SE8500422L; NL8500239A; BE901611A; NL193263B; JPS6192672A; GB2153235B; IL74180A0; GB2187192B

Abstract

A collagen-impregnated vascular graft including drug materials complexed with the collagen to be released slowly from the graft following implant. The graft is a porous synthetic vascular graft substrate having collagen fixed to the graft substrate and cross-linked in situ to render the porous substrate blood-tight. The drug materials complexed with the collagen fibrils may include antithrombic agents, antibacterial, antimicrobial agents, antifungal agents and the like.

Description

BACKGROUND OF T~E INVENTION

This invention relates to a synthetic vascular graft, and more particularly to a drug delivexy blood-tight collagen-impregnated synthetic vascular graft which does not need to be pre-clotted and which acts as a reservoir for sustained release of a drug material after implant.
The replacement of segments of human blood vessels with synthetic vascular grafts is well accepted in the art. Synthetic ~ascular grafts have taken a wide variety of conf;gurations and are formed of a wide variety of materials. Among the accepted and successful vascular graft implants are those which are formed from a biologically compatible material which retains an open lumen to permit blood to flow through-the synthetic graft after implant. The grafts may be made from biologically compatible fibers, such as Dacron~ and TeflonTM, may be knitted or woven and may be of a mono-filiment yarn, multi-filiment yarn or staple yarn.
An important factor in the selection of a particular graft substrate is the porosity of the fabric wall of which the graft is formed. Porosity is significant because it controls the tendency to hemorrhage during and after implantation and controls the ingrowth of tissue into the wall of the gxaft. It is desirable that the vascular graft substrate be sufficiently blood-tight to prevent the loss of blood during implant, yet the structure must be sufficiently porous to permit ingrowth of fibroblast and smooth muscle cells in order to attach the graft to the host tissue.
Synthetic vascular yrafts of the type described in United States Patents No. 3,805~301 and No. 4,047,252, assigned to the assignee of the subject application, are elongated flexible tubular bodies formed of a yarn such as Dacron . In the earlier patent, the graft is a warp knitted tube and in the latter issued patent it is a double-velour synthetic graft marketed under the trademark Microvel. These types of grafts have sufficiently porous struc-tures to permit ingrowth of host tissue.

The general procedure for implantation includes the step of pre-clotti.ng, wherein the graft is immersed in the blood of the patient and allowed to stand for a period of time sufficient for clotting to ensue. After pre-clotting, hemorrhaging does not occur when the graft is implanted and growth oE tissue is not impeded.
Graft infection is a most serious complication and occurs in an average of two percent of prosthetic graft placements. It is associated with a high risk of limb loss and patient mortality is as high as 75~ depending on the location of the graft. While infection usually becomes evident soon after surgery, the time may be extended which leads to more seri.ous consequences.
An absorbable collagen reinforced graft is proposed in United States PatentNo. 3,272,204 wherein the collagen i.s obtained from the deep flexor tendon of cattle. Another reinforced vascular prosthesis is descri.bed inUnited S-tates PatentNo. 3,479,670 which includes an open mesh cylindri.cal tube wrapped by an outer helical wrapping of fused polypropylene mono-filiment filled with collagen fibrils which are claimed to render the prosthesis impermeable to bacteria and fluids. The collagen fibri.ls utilized are the same as described in Patent No. 3,272,204.
The synthetic vascular grafts suggested by the prior art are claimed to be sui.table for many applications~ However~ it is desirable to provide a flexible vascular graft having zero poros-ity, one which is receptive to i.ngrowth of host tissue and serves as a reservoir for drug materials to be released slowly from the surface of the graft following implant.

SUMMARY OF THE INVENTION

A collagen-impregnated synthetic vascular graft composite which provides a reservoir for the slow release of a drug material after implant is provided. The collagen graft includes a tubular flexible porous substrate having on the inner and outer surfaces and extending through the porous structure of the substrate cross-linked collagen fibrils complexed with an effective amountof a drug ~2~

and admixed with a plasticizer. The drug material includes antibacterial agents, antithrombic agents and antiviral agents to ensure against graft infection by providing for sustained release of the drug portion of the complex after implantation.
The porous graf~ substrate may be a tubular vascular graft formed of a Dacron~ material and may be woven or knit. The collagen source i5 an aqueous fibril dispersion which may be of high purity bovine skin collagen including a plasticizer. The collagen is applied to the graft substrate by massage to cover the entire inner surface and penetrate into the substrate to insure intimate mixing of the collagen fibril complex into the porous structure of the substrate and extend over the outer surface thereof. The collagen is cross-linked preferably by exposure to formaldehyde vapor. The collagen graft provides a flexible graft with good hand.
The invention accordingly comprises the article possessing the features, properties and the relation of elements and the several steps and the relation of one or more of such steps with respect to each of the others, which are exemplified in the following detailed disclosure, and the scope of the invention will be indicated in the claims.

BRIEF DESCRIPTION OF THE DRA~ING

For a fuller understanding of the invention~ reference is had to the followiny description taken in connection with the accompanying drawing, in which:
FI~. 1 is a partial cross-sectional view of a collagen-treated synthe~ic vascular graft in accordance with the invention;
FIG. 2 is a partial cross-sectional view of a branched tubular graft of the type illustrated in Fig. l;
FIG. 3 is a graph illustrating sustained release of tetracycline from a collagen slurry in rabbits;
* Trade Mark 3~i FI&. ~ is a graph illus~rating sustained ~elease of tetracycline at differen~ collagen gal concentrations; and FIG. ~ is a graph illusteating sustained release of 4 tetracycline in a collagen gel at diffeLent con~entrations and dosage.

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DESCRIPTION OF T~E PREFERRED EMBODIMENTS

A synthetic vascular graft 10 constructed and arranged in accordance with the invention is shown in Fig. 1. Graft 10 includes a tubular substrate portion 12 which is formed of a biologically compatible filamentary synthetic material, preferably a polyethyl-ene terephthalate r such as Dacron~. Substrate 12 is a porous Dacron~ warp knit fabric having an inner and outer velour surace of the type described in U.S. Patent 4,047,252. While tubular portion 12 is formed of Dacron~, any biocompatible filimentary material may be used for the substrate provided it may be fabricated into a porous structure which will permit tissue ingrowth and maintain an open lumen for flow of blood.
The inner surface of tubular portion 12 is treated with collagen as shown at 16. Collagen layer 16 is formed from a series of at least three applications of collagen fibrils. Fig. 2 shows a bifurcated collagen-treated graft 20. Graft 20 includes a main tubular portion 22 and two branches 24. Main tubular portion 22 and bifurcated portions 24 are formed from a Dacron~ knit substrate 26.
The inner surface of substrate 26 is treated with collagen 28 also foxmed by at least three applications of collagen fibrils.
Porous vascular graft substrates suitable for use in ac-cordance with the invention, preferably are produced from Dacron~
multi-iliment yarns by knitting or weaving processes ~hich are commonly used in manufacture of these products. Generally, the porosity of the Dacron~ substrate ranges from about 2,000 to 3,000 ml/min-cm2 (purified water at 120mm Hg). The cross-linked collagen is applied to the inner surface o~ the graft by filling a tubular substrate with a slurry of collagen fibrils and plasticizer and massaging manually, removing the excess and permitting the deposited dispersion to dry. After the final application, the collagen is cross-linked by exposure to formaldehyde vapor, air dried and then vacuum dried to remove excess moisture and excess formaldehyde. The treated grafts in accordance with the invention have essentially 2ero porosity.

The following examples are set forth to illustrate the method of preparing purified collagen from bovine ski.n and treated grafts in accordance with the invention. The examples are set forth for purposes of illustration and not .intended ;.n a limiting sense.
Example 1 Fresh calf skins were mechanically stripped from young calves, fetuses or stillborns and washed in a rotating vessel with cold running water until the water was observed to be free from surface dirt, blood and/or tissues. The subcutis was mechanically cleaned to remove contaminating tissues, such as fat and blood vessels. Subsequently, the skins were cut in the longitudinal direction into strips about 12 cm wide and were placed in a wood or plastic vessel as commonly used in the leather industry.
The skins were dehaired by using a flusher solution of 1 CalOH)2 for 25 hours. Alternatively, the skin may be dehaired by mechanical means or by a combi.nati.on of chemical and mechanical means. Following the dehai.ri.ng, the skins were cut into small size pieces about 1" x 1" and were washed in cold water.
Following washing, 120 Kg of the bovine skin was placed in a vessel having 260 L water, 2 L NaOH (50%) and 0.4 ~ H2O2 (35%)-The components were mixed slowly for 12 to 15hours at 4Cand washed with an excess of tap water for 30 minutes to provide partially purified skins. The partially purified skins were treated in a solution of 260L water, 1.2L NaOH (50%~ and 1.4 Kg CaO for 5 minutes with slow mixing. This treatment was continued twice daily for 25 days. Following this treatment, the solution was decanted and di.scarded and the skins were washed with an excess of tap water for 90 minutes under constant stirring.
The ski.ns were acidifi.ed by treatment wi.th 14 kg HC1(35%) and 70 L water while subjecting the skins to vigorous stirring. The acid was allowed to penetrate the skins for about 6 hours. Follow-ing acidification, the skins were washed in an excess of tap water for about 4 hours or until a pH of 5~0 was reached. The pH of the skins was readjusted to 3.3-3.4 using acetic acid with a 0~5~
preservative. The puri.fied skin was then passed through a meat grinder and extruded under pressure through a series of fi.lter ~.æ3~

sieves of constantly decreasing mesh size. The final product was a white homogeneous smooth paste of pure bovine skin-derived co lagen.
In order to impartadequate pliability to the graEts in the dry state, plasticizers are added to the collagen slurry before application. Suitable plasticizers include glycerine, sorbitol or other biologically acceptable plasticizers. In a collagen slurry containing between about 0.5 to 5.0 percent collagen by weight, the plasticizer is present in an amount between about 4 and 12 weight percent.
~ mong the most important properties obtained when treat ing a synthetic vascular graft with collagen fibrils in accordanc~
with the invention is reduction of porosity of the porous substrate to about zero. The porosity of twenty randomly selected untreated Microvel~ DacronQ synthetic vascular grafts have a mean porosity to purified wa-ter of 1796 ml/min-cm2 at 120 mm Hg with a standard deviation of 1300 After several collagen applications, the poros-ity is reduced to zero. The following example illustrates the method of treating the graft substrate.
Example 2 A 50 cc syringe is filled with an aqueous slurry of 2%
purified bovine skin collayen prepared in accordance with Example 1. The collagen slurry includes 8~ glycerol, 17% ethanol and the remainder water and a viscosity of 30,000 cps. The syringe is placed into one end of a 2~eadox Medical Microvel~ Dacron~ graft 8 mm in diameter by approximately 12 cm in length. The slurry is injected into the lumen of the Microvel~ graft and it is massaged manually in order to cover the entire inner surface area with the collagen slurry. Any excess collagen slurry is removed through one o~ the open ends. The graft is permitted to dry f~r about 1/2 hour at room temperature. The treating and drying steps were repeated three more times.
Following the fourth application, the collagen was cross-linked by exposure to formaldehyde vapor for 5 minutes. The cross-linked graft was then air dried for 15 minutes and then vacuum dried for 24 hours to remove moisture and any excess formaldehyde.

5~;~3~

Example 3 The blood-tightness of a collagen~treated vascular graft prepared in accordance with ~xample 2 was tested as follows. A
Microvel~ graft 8 mm x 12 cm was attached to a blood reservoir at a pressure of 120 mm Hg due to the height of the reservoir. Heprin stabilized blood was passed through the graft and blood collected through the grafts was determined and expressed in ml per min-cm2.
The porosity over 5 runs was determined to be 0.04, 0.0, 0.0, 0.04 and 0.03. This represents a mean porosity of 0.022 ml/min-cm2 which was considered zero, as the value is within the experimental error of the study.
In order to compare this result with the blood loss for untreated Microvel grafts, the e~periment was repeated using an untreated graft. The mean porosity was 36 ml/min-cm2.
The antimicrobial activity of a collagen treated fabric graft prepared in accordance with the invention is demonstrated as follows.
Example ~
The porosity of a collagen treated fabric graft is reduced to less than about 1 percent of an untreated graft after three coatings. A standard water porosity test used to measure water porosity of a graft is as follows. A column of water equivalent to 120 mm Hg pressure is allowed to flow through a one-half cm2 orifice having a sample o~graft over the orifice for one minute. The amount of water collected was measured. The milliliters of water collected per minute per cm squared area was calculated. Several readings are taken for each sample. The porosity is reported as follows-porosity = ml/min/cm2 The water porosity of a Microvel~ graft fabric was about1,900 ml/min/cm2. The porosity after treatment was as follows:
~umber of CoatingsPorosity o 1,900

2 146

3 14 ~%~

4 5 2.~

In each case the collagen applied was a bovine skin derived-plastici~ed slurry prepared in accordance with the compo-sition described in Example 2. Based on these results, it is preferable to provide collagen of at least three or four layers of fibrils, and most preferably four or five layers with dryingbetween each application and cross-linking to fi~ the collagen to the sub-strate.
In accordance with the invention, each layer of the collagen and at least the last two layers applied to a porous substrate are chemically modified to incorporate a drug or an antithrombic agent, such as heprin, in order to prevent infection and to inhibit clotting along the inner surface of the prosthesis.
As noted, the collagen may be comple~ed with a variety of drugs, such as antibacterial agents, antimicrobial agents or antifungal agents in order to prevent graft infection. Typical antibacterial agents which may be utilized include oxacillin, gentamicin, tetra-cycline, cephalosporin and the like which may be complexed with the collagen fibrils prior to application to the graft substrate.
In addition to reduced porosity, collagen treated vas-cular grafts in accordance with the invention exhibit reduced thrombogenicity compared to untreated grafts.
Example 5 A homogeneous slurry of bovine skin derived collagen pre-pared in accordance with Example 1 was prepared containing 1% bovine skin derived collagen, 8% glycerol, 17~ ethanol with the remainder water. Ceclor~, a cephalosporin antibiotic of Eli Lilly and Company which inhibits the growth of Staphylococcus aureus and Escherichia coli, was blended into the sluxry at a concentration of 20 mg per ml. The collagen slurry including the Ceclor~ was massaged onto a double velour Dacron~ fabric on both sides with 1/2 hour drying periods between treatments. The treatment resulted in the addition of 3.1 mg collagen per cm~.

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As a control, Dacron~ double velou.r fabric was also impregnated with the same collagen slurry omitting the Ceclor~
antibiotic. This control had a coating of 4.1 mg of collagen per cm2 ~
Both pieces of treated fabric were immersed for 1 minute in 4% formaldehyde, 10% glycerol solution, vacuum desiccated for 64 hours and sterilized using gamma radi.ation.
The antimicrobial activity of the collagen treated Dacron~ vascular graft fabric, i.mpregnated with Ceclor~, was determined in an agar diffusion assay. Fabric swatches of 1 cm2 were placed on i.nnoculated agar surfaces resulting in growth inhibition zones whi.ch indicated that the anti.biotic was active against SO aureus (34 mm zone of inhibition) and E. coli (29 mm zone of inhibition). The untreated control collagen-vascular graft fabric did not exhibi.t any anitimicrobi.al effect. The results are tabulated in the following Tables I and II.
TABLE I
FABRIC TREATED WITH COLLAGEN AND ANTIBIOTIC

=
_ aureus 36mm 31mm 35mm 34mm E. coli 33mm 28mm 27mm 29mm TABI,E II
FABRIC TREATED WITH COLL~GEN

_ aureus 0 0 0 0 E coli 0 0 Example 6 A collagen slurry prepared in accordance with Example 1 containing 13.2% collagen protein (determi.ned by i.ts hydroxypro-line content) was mixed in a 1:3 ratio with water (W) to form a 3.3 -10- ` ~2~:35 weight percent homogeneous collagen gel (G). The pH of the collagen gel was adjusted to 3.8 and 20mg of tetracycline (TC) was added per millimeter of gel. Immediately before injection into two rabbits, the collagen gel-tetracycline complex was mixed with glutaralde-hyde (0.3 ml of 3% glutaraldehyde per ml of the gel) and injected through 18 gage needles into the subcutis. Two rabbits as controls were injected with a similar dose of tetracycline and water, 20 mg TC/ml water/kg body weight.
In order to study the rate of tetracycline released from the injected site, blood was collected at various time intervals from the rabbit's ear vein. The content of TC in the blood was measured according to the procedure of Wilson, et al. (Clin. Chem.
~cta., 36; 260, 1972). The results of the TC analysis in the blood of the total of four rabbits collected within 2 hours to 7 days post-injection are set forth in Fig. 3.
Fig. 3 sho~s that after injection of TC in water the drug reaches its maximum in the serum within two hours as shown by Curve Ao At 11 hours the TC is no longer detectable. When tetracycline was administered in a collagen gel cross-linked with glutaraldehyde (lOG ~ 30W), the level of serum TC remained stable for about 6 days as shown by Curve B. Thus, administration of TC in collagen gel prolonged the efEective release of the drug 25 times compared with injection in an aqueous medium onlyO
_ample 7 The test described in Example 5 was repeated using colla-gen gel at two different concentrations for the final injection.
Additionally, the tetracycline content was 30 mg oxyte-tracycline (OTC)/ml gel/kg body weight at a dose of 1 ml/kg of body weight, or 50~ more tetracycline per dose than Example 5. The results illustrated in Fig. 4 show that the concentration of collagen in the gel affects the rate of OTC release from the collagen matrix. The denser the collayen gel, the slower is the release of the drug. In this Example, the kinetics of the OTC
release was studied for a total of 124 hours after injection of the tested complex in the subcutis of a total of six rabbits.

In Fig~ 4 Curve A shows that the OTC in water reaches its maximum in the serum shortly ater injection and is not detectable after 18 or 20 hours. Curve B shows OTC serum concentration for OTC
complexed with a collagen matrix at a weight ratio of gel complex to water of 1:20 and Curve C at 3:20. Release of the OTC is more rapid for the less concentrated gel of Curve B.
Example 8 Collagen gel contai.ning 3% collagen, measured as a dry substance, was mixed with tetracycline to form two concentrations, containing (A) 50mg TC/ml and (B) lOOmg TC/ml gel. After mixing with 0.3 ml of 3% glutaraldehyde (Gl) per ml gel (G), complex A was injected at a dosage of 2 ml/kg body wei.ght and complex B was injected at a dosage o 1 ml/kg. Plasma level concentrations of l'C
in mg/ml are shown in Curves A and B of Fig. 5. Ccmplex A was also injected at a dosage of 1 ml/kg and is shown by Curve C in Fig. 5.
The actual plasma levels of tetracycline during the period up to 5 days post-injection are shown in ~ig. 5.
The data of Fig. 5 shcw that both the actual concentration of tetracycline as well as the surface geometry of the implant affects the level o magnitude of drug release from the gel and the level of tetracycline in the plasma.
It will thus be seen that the objects set forth above, among those made apparent from the preceding descripti.on, are efficiently attained and, si.nce certain changes may be made in the article and in carrying out the above process set forth without departing from the spirit and scope of the invention, it isi.ntended that all matter contained in the above descript.ion and shown in the accompanying drawings shall be interpreted as illustrati.ve and not in a limiting sense.
It is also to be understood that the following claims are i.ntended to cover all of the generic and speci.fic features of the invention herein described and all statements of the scope of the invention which, as a matter of language, might be sai.d to fall therebetween.
Particularly it is to be understood that in said claims, ingredients or compounds recited in the singular are intended to include compatible mixtures of such ingredients wherever the sense permits.

Claims

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

1. A synthetic vascular graft comprising:
a tubular flexible porous graft substrate of a synthetic fiber having a porosity of less than about 3,000 m1/min.-cm2 (purified water at 120 mm Hg);
the graft substrate having on the inner surface and extending through the porous substrate to the outer surface cross-linked collagen fibrils complexed with an effective amount of a drug and admixed with a plasticizer for rendering the graft blood-tight and flexible and providing for sustained release of the drug portion of the complex after implantation.

2. The vascular graft of claim 1, wherein the drug portion of the complex is a pharmaceutical agent selected from the group consisting of antimicrobial agents, antibacterial agents, antifungal agents, antithrombogenic agents, cell-proliferation promoting agents and mixtures thereof.

3. The vascular graft of claim 1, wherein the collagen fibril-drug complex is applied in at least three applications formed by applying aqueous slurries of collagen fibrils which have been dried between applications and cross-linked after application.

4. The vascular graft of claim 1, wherein the porous substrate is polyethylene terephthalate.

5. The vascular graft of claim 4, wherein the porous substrate is knitted.

6. The vascular graft of claim 4, wherein the porous substrate is woven.

7. The vascular graft of claim 4, wherein the inner and outer surface of the substrate have a velour surface.

8. The vascular graft of claim 1, wherein the collagen fibrils-drug complex is cross-linked by exposure to formaldehyde vapor.

9. The vascular graft of claim 1, wherein the plasticizer is a biologically compatible polyhydric material.

10. The vascular graft of claim 9, wherein the plasticizer is sorbitol.

11. The vascular graft of claim 9, wherein the plasticizer is glycerine.

12. A process for preparing a drug delivery collagen-containing synthetic vascular graft comerising:
providing a porous tubular flexible synthetic graft substrate;
placing an aqueous slurry of a complex of collagen fibrils complexed with a drug and admixed with a plasticizer onto at least the inner surface of the substrate massaging the slurry into the substrate to insure intimate mixing of the collagen fibril complex into the porous structure of the substrate:
drying the collagen;
cross-linking the collagen; and vacuum drying said graft.

13. The process of claim 12, wherein the steps of placing an aqueous slurry of collagen fibrils onto the substrate, massaging and drying is repeated at least three times.

14. A synthetic vascular graft comprising:
a tubular flexible porous polyethylene terephthalate graft substrate having an initial porosity of less than 3,000 m1/min.-cm2 (purified water at 120 mm Hg):
the inner surface thereof having a coating of at least five layers of cross-linked collagen fibrils complexed with a drug and admixed with a plasticizer which extends into the porous structure of the substrate and covers the outer surface:
each layer formed from an aqueous slurry containing between about 1.5 to 4.0 weight percent collagen fibrils and between about 6 and 10 weight percent plasticizer.

15. A slurry for forming a drug delivery blood-tight synthetic vascular graft comprising about 0.5 to 5.0 percent collagen fibrils complexed with an least an effective amount of a drug material, 4.0 to 12.0 percent plasticizer and the balance water.

16. The process of claim 12, wherein the cross-linking is effected with formaldehyde vapor.

17. The process of claim 12, wherein an aqueous slurry of water-insoluble collagen fibrils is applied to the lumen and massaged and dried prior to application of the collagen complexed with a drug.