WO1981000516A1

WO1981000516A1 - Gel containing fibrine and antibiotic for treating infected bones and preparation process thereof

Info

Publication number: WO1981000516A1
Application number: PCT/EP1980/000083
Authority: WO
Inventors: A Braun
Original assignee: Merck Patent Gmbh; A Braun
Priority date: 1979-08-31
Filing date: 1980-08-27
Publication date: 1981-03-05
Also published as: EP0037814A1; JPS56501129A

Abstract

A gel based on fibrine and antibiotic for the treatment of an infected bone contains fibrinogene, a solution containing thrombin and enriched with calcium ions as well as an antibiotic of the tobramycin, gentiamycin group and/or one of their physiologically acceptable salts. The gel may be prepared by mixing the fibrinogene in the form of a lyophilisate or cryoprecipitate with an antibiotic in sterile conditions and then adding a thrombin solution enriched with calcium ions.

Description

Fibrin antibiotic gel for the treatment of infected bone and process for its preparation

The invention relates to a fibrin antibiotic gel for the treatment of infected, in particular staphylococcal, infected bone and a method for its production.

It is known to add antibiotics to polymethyl methacrylate ("PMMA"; bone cement) for the local therapy of bone infections. The mixture of gentamycin with PMMA in the form of gentamycin-PMMA ball chains is particularly suitable for the local antibacterial chemotherapy of bone and soft tissue infections.

Since the implanted balls represent foreign bodies, they must be removed again. This can be done, for example, by letting the last ball protrude through the skin closure and then pulling out the whole chain after ten to fourteen days. Overall, however, the removal of the balls is a stressful and therefore undesirable intervention for the patient. Attempts have already been made to eliminate the disadvantages of using PMMA ball chains and to show the possibility of an easier treatment route.

So it is e.g. known (cf. Bosch et al., Archive for Orthopedic and Trauma Surgery, Volume 90 (1977), pages 63-75), in connection with bone grafts for filling bone defects, to use a fibrin adhesive system, the fibrin only being used at the place of choice, is formed directly in the bone cavity by adding thrombin to a fibrinogen solution. Commercially available combination preparations of neomycin in powder form were also added if necessary.

It has now been found that, surprisingly, two certain antibiotics, namely tobramycin and gentamicin and their physiologically acceptable salts, ^"form with Fibri an F ibrin antibiotic gel that is exceptionally well suited for the treatment of infected bone and extremely effective. It is not necessary to first make this fibrin antibiotic gel in the body (directly in the bone cavity); the ingredients are rather appropriately mixed extracorporeally. It is possible to control the coagulation process of the fibrin by varying the concentration of the thrombin.

The invention accordingly relates to a fibrin antibiotic gel for the treatment of infected bone with a content of fibrinogen, a thrombin-containing solution enriched with calcium ions and an antibiotic from the group of the aminoglycosides, characterized in that it is an antibiotic tobramycin, gentamycin and / or one of their physiologically acceptable salts. The sulfates are particularly suitable as physiologically acceptable salts. All components of the gel are expediently used in the form of customary commercial preparations.

For example, human fibrinogen in the form of a cryoprecipitate containing about 90 mg of thrombinable protein or as a lyophilisate (e.g. obtained from human blood from pooled donor plasma).

The thrombin-containing solution is expediently prepared by dissolving thrombin (e.g. in the form of a powder) in an aqueous calcium chloride solution. The concentration of calcium chloride is preferably about 30 to 50, in particular 40, mmol / l. The concentration of thrombin is preferably between about 10 and about 500 NIH units / ml.

The antibiotic is expediently used in an amount based on body weight, whereby the maximum daily dose should be observed. The concentration of the antibiotic in the gel according to the invention is preferably between about 1 and about 10, in particular between 2 and 5 percent by weight.

The gel can be prepared by mixing fibrinogen with an antibiotic (for example, tobramycin sulfate or gentamycin sulfate, each about 5 mg / kg body weight) under sterile conditions and then adding the calcium ion-enriched thrombin-containing solution to polymerize the fibrinogen to fibrin. The gel is preferably produced extracorporeally, ie before it is actually used in the bone cavity. The clotting time depends on the thrombin concentration. The plastic deformability of the resulting clot can be maintained for a period of 1/2 to 1 minute if a thrombin concentration of about 150 NIH units per ml is used.

The flow properties of the gel are determined by lower thrombin concentration (10-15 NIH units / ml). Maintained much longer (up to about 3 minutes). Fibrin coagulation is slowed down and the tensile strength of the polymer is increased.

The fibrin antibiotic gel with primary cancellous bone plastic not only controls the infection, but also improves the osteogenic potency of the biological implant.

It is of course possible to also bones at risk of infection, e.g. after open fractures, to treat the infection with the ir'ibrin antibiotic gel. The antibiotic achieves a high local active ingredient level.

example 1

I. Test animals

60 rabbits of the breed "White New Zealanders" were chosen as experimental animals. Of these, 22 were female and 38 were male. The average body weight was 4130 g. I. Experimental planning

Thirty test animals were subjected to treatment with the fibrin antibiotic gel after randomization (group A). The remaining 30 experimental animals served as a control group and were treated with fibrin only. (Group B). In both groups, the bone was contaminated with Staphylococcus aureus. The animals were sacrificed on the 7th postoperative day and the findings were examined macroscopically, bacteriologically and histomorphologically.

III. Production of the fibrin antibiotic gel

1. Fibrin glue

Fibrin glue (commercial preparation) is produced from human donor plasma by cold precipitation. For storage, the finished product is deep-frozen and stored at -18 ° C or colder. 1 ml of the solution contains an average of 90 mg of thrombinable protein. The total protein content of the solution is about 10%. In addition to the precipitable fibrinogen, 1 ml of fibrin glue contains 5 - 12% cold-insoluble globulins, a maximum of 5% albumin and about 10 units of factor XIII, as well as traces of plasminogen and coagulation factors of the intrinsic system. The cryopreserved human fibrinogen is thawed at room temperature about 20-30 minutes before its intended use. The viscosity of the preparation decreases with increasing temperature. Storage at room temperature for 4 hours requires activation of the coagulation factors on the one hand (coagulation) and the plasminogen on the other. The thawed, sol-like human fibrinogen is available in a pre-filled syringe. 0.25 ml per test animal are placed on a petri dish for further processing. 2. Tobramycin solution

15 mg of tobramycin sulfate dry matter per test animal are dissolved in 0.1 ml of distilled water and mixed well with 0.25 ml of fibrin glue.

3. Calcium-enriched, thrombin-containing solution

3000 NIH units of thrombin dry substance (commercial preparation) are dissolved in 20 ml of an aqueous calcium chloride solution which contains 35 mmol / 1 CaCl ₂ .

IV. Operative technique

As premedication for anesthesia, 0.2 mg atropine sulfate and 0.5 ml / kg xylazine were injected intramuscularly. Sufficient anesthesia could be achieved with 50 mg / kg ketamm for about 45 minutes for the surgical intervention on the bone.

After sterile covering of the shaved knee joint, the distal femoral metaphysis was exposed from the side. The corticalis was perforated to a size of 4 x 8 mm with a drill and milling machine and spongy bones were milled out, so that the defect could be filled with approx. 0.5 ml. The marrow was tamped with bone wax for hemostasis. A 4 x 4 x 1 mm collagen fleece was inoculated with 0.05 ml staphylococcal suspension and the bone cavity was contaminated with it. This corresponds to a germ density of 0.5 - 2 x 10 ⁷ colonies. After randomization, the bone defect was closed in group A with the fibrin-antibiotic gel and in group B with fibrin. With good plastic deformability during the polymerization, the distal femoral metaphysis was included in group A. Fill in 0.45 ml fibrin antibiotic gel and compress the fibrin clot over the corticalis window manually for 2 minutes. In group B (control group), 0.1 ml of tobramycin-containing solution was replaced by 0.1 ml of distilled water, so that 0.25 ml of fibrinogen was coagulated by 0.2 ml of thrombin-containing solution.

Bacteriological methodology

As a test germ, a coagulase-positive, tobramycin-sensitive (MIC <0.125 mcg / ml) Staphylococcus aureus isolated from a wound swab from the Orthopedic University Clinic in Heidelberg was used Cultivated 37 ° C, washed off with sterile physiological saline and the bacterial wash-off by turbidity comparison (tube no. 1 of the barium sulfate turbidity series after

McFarland) to a germ density of approx. 3 x 10 ⁸ germs / ml. The exact number of bacteria (colony-forming units) of the suspension was determined using the plate casting method at the time of application to the animal. A nutrient agar of the following composition was used as the bacterial counting storage (each gram per 1000 ml water):

Meat extract 3, Witte Peptone 5, sodium chloride 5, agar 12; pH 7.2.

For evaluation, smears from the medullary canal and extra-articular tissue were taken with sterile cotton swabs and each in 0.2% glucose broth and on agar plates (10% mutton blood addition). After an incubation time of 18-24 hours at 37 C, the growth media were checked for growth and incubated for a further 24 hours if there was no turbidity or colony formation. The material was described as sterile if no growth had been shown up to this point. The grown staphylococci were identified by the microscopic preparation, shape and color of the colonies, hemolysis like and detection of the bound coagulase (clumping factor).

VI. Histological technique

The pieces of bone taken from the section of the test animals were fixed in 4% neutral, buffered formalin and then carefully decalcified. The decalcifying solution consisted of 200 g of ethylenediaminetetraacetic acid tetrasodium salt, made up to 1000 ml with water and adjusted to pH 7.0-7.2 with citric acid. After decalcification, the samples were embedded in paraffin in the usual way. 3 - 5 u thick layers were stained with hematoxylin-eosin, van Gieson, Di PAS, Giemsa, Ladewig and according to the Weigert's method of presentation of fibrin.

Results

The test animals were sacrificed on the 7th postoperative day and the findings were evaluated macroscopically, bacteriologically and histomorphologically. Of the animals in control group B, 15 died of sepsis between the 2nd and 7th day. The decrease in body weight in this group averaged 530 g. In the rabbits (group A) treated with fibrin tobramycin, no animal died from the consequences of an infection. The body weight had only decreased by 136 g on average. I. Macroscopic finding

In group A, the macroscopic findings of bone and soft tissue in 22 test animals showed no signs of purulent inflammation in situ. The finding was questionable in 6 animals; phlegmonous inflammation had to be assumed only in two cases. The bone showed a firm consistency when sawing. In most cases, fibrin residues of the composite were still easy to detect, but infiltrated with hemorrhage. All animals in control group B showed the classic symptoms of purulent inflammation. In some cases, pus spontaneously emptied from the wound that had become dehiscent. In all cases, a pronounced purulent, sometimes abscessing, sometimes phlegmonous inflammation was found in the periarticular soft tissue of the knee joint. The bone was spontaneously fractured in 7 animals at the level of the corticalis window. Soft, atrophic bone tissue was evident when opening the medullary cavity with the oscillating saw. In the distal metaphysis of the femur, an inflammatory destructive marrow was found. Fibrin residues of the composite were still partially detectable and also infiltrated hemorrhagically. II. Bacteriological finding

In all animals, a swab was taken intramedullarily from the surgical area both in the periarticular soft tissue and in the distal femur for bacteriological examination. Of the 30 rabbits in group A, the smears were sterile in 27 animals and infected in 3 animals. there were found once subcutaneously, once intraosseously and once subcutaneously and intraosseously staphylococci. Of the 30 animals in the control group (group B), all smears were staphylococcal infected. The results of the bacteriological examination can be summarized in a four-field table. The evaluation according to the modified x ² test of the Geigy tables p. 192 (1973) showed x ² = 59.64. The significance barrier is x = 6.635 for the defined significance probability (P = 0.01). The test group and control group thus differ with regard to the treatment procedure with an error probability of at most 1%. The local use of Tobra mycin in the fibrin antibiotic gel can be regarded as exceptionally effective according to this statistical statement. III. Histological finding

The histological examination revealed non-specific inflammation in all animals in the operated area. According to the choice of the pathogen, it was a purulent inflammation, which, however, occurred in different degrees of severity. In group A animals, on average, inflammation was far less severe. An extensive fibrin residue was typically detected in this group. A granulation tissue had already developed around it, which was also permeated with granulocytes and some macrophages. In some cases, newly formed bone trabeculae made of braided bone could be detected. As a rule, the infected area was enclosed by this granulation tissue, the distal epiphyseal joint undamaged, so that one could speak of a localized osteomyelitic process. The extent of cellular infiltration changed, neighboring bone and soft tissue structures were only included in the inflammatory process in individual cases. In these cases, the pathogen detection was still positive. In contrast, less fibrin was usually detected in group B treated with fibrin only. There was a severe purulent, often necrotizing osteomyelitis, which in numerous cases destroyed the distal epiphyseal plate, spread to the epiphysis and involved the surrounding soft tissues. The process also proceeded proximally. Both the compact and the remaining cancellous bone showed extensive necrosis in the sense of sequestration. Healing processes in the sense of the onset of new bone formation could not be demonstrated in any case, whereas in the case of test animals treated locally with antibiotics, the inflammation was usually limited to the portal of entry and in some cases new bone formation was observed after the short test period, leading to those treated only with fibrin Animals the virulent pathogen to a phlegmonous-necrotizing osteomyelitis that spreads many times uninhibited. The histological picture suggests that the antibiotic from the matrix material passes into the environment and obviously has an effect on the pathogen.

Example 2

The procedure is analogous to Example 1, but the tobramycin sulfate is replaced by 15 mg of gentamycin sulfate and comparable results are obtained. Example 3

Application set

The application set contains the following individual components:

(a) Calcium chloride solution 40 mmol / 1 2 ml

(b) Thrombin, lyophilized 300 NIH units

(c) Tobramycin sulfate 350 mg

Dissolve (c) in 2 ml of distilled water, mix this solution with 4 ml of thawed approx. 9% fibrinogen cryoprecipitate and combine the mixture with a freshly prepared solution of (b) in (a) shortly before use.

Example 4

Application set

The application set contains the following individual components:

(a) Calcium chloride solution 40 mmol / 1 4 ml

(b) Thrombin, lyophilized 600 NIH units

(c) Gentamycin sulfate 600 mg

(d) Fibrinogen, lyophilized 800 mg

Dissolve (c) in 4 ml of distilled water, mix this solution with a solution of (d) in 8 ml of distilled water and add a freshly prepared solution of (b) in. (A) shortly before use.

Claims

Claims:

1. fibrin-antibiotic gel for the treatment of infected bone containing fibrinogen, a thrombin-containing solution enriched with calcium ions and an antibiotic from the group of the aminoglycoside, characterized in that it is an antibiotic tobramycin, gentamycin and / or one contains their physiologically acceptable salts.

2. A process for the preparation of the gel according to claim 1, characterized in that a human fibrinogen in the form of a cryoprecipitate or a lyophilizate is mixed with the antibiotic under sterile conditions and then a thrombin-containing solution enriched with calcium ions is added.