EP2155176A2

EP2155176A2 - Substrate promoting angiogenesis

Info

Publication number: EP2155176A2
Application number: EP08758535A
Authority: EP
Inventors: Michael Ahlers; Burkhard Schlosshauer; Lars Dreesmann
Original assignee: Gelita AG
Current assignee: Gelita AG
Priority date: 2007-05-16
Filing date: 2008-05-15
Publication date: 2010-02-24
Also published as: BRPI0811861A2; WO2008138612A3; IL201831A0; MX2009012324A; WO2008138612A2; DE102007024239A1; US20100056452A1; AU2008250580A1

Abstract

The aim of the invention is to design a substrate that promotes angiogenesis and is easy and inexpensive to produce. Said aim is achieved by a substrate comprising a non-porous molded element which is made of a resorbable, gelatin-containing material that is insoluble in physical conditions.

Description

Angiogenesis promoting substrate

The present invention relates to an angiogenesis-promoting substrate.

In living mammals, endothelial cells that line existing blood vessels form new capillaries wherever they are needed. The endothelial cells have the remarkable ability to adapt their number and arrangement to local requirements. Tissues are dependent on the blood supply that occurs through the blood vessel system. The vascular system in turn depends on the endothelial cells. The endothelial cells create an adaptive life assurance system that ramifies into almost all parts of the body.

While the largest blood vessels, the arteries and veins, have a thick, strong wall of connective tissue and sometimes smooth muscle and are lined inside only with a very thin, simple layer of endothelial cells, one finds in the finest branches of the vascular system, the capillaries, Walls that consist only of endothelial cells and a so-called basal lamina. Endothelial cells thus clothe the entire system of blood vessels, ranging from the heart down to the smallest capillary, and they control the passage of factors and cells into and out of the bloodstream.

Cells in tissues release oxygen deficient angiogenesis factors that stimulate the growth of new capillaries. Local (mechanical) irritation and infection also causes the proliferation of new capillaries, most of which retract and disappear as soon as the inflammation subsides. The newly developing blood vessels always arise first as capillaries, which sprout on existing small vessels. This process is called angiogenesis.

The spouting of the capillaries continues until the respective shoot encounters another capillary and can connect to it, so that blood can circulate in it (cf., for example, B. Alberts et al., Molecular Biology of the Cell, VCH Weinheim, 3rd edition 1995 , Pages 1360 - 1364).

Angiogenesis-stimulating factors are well known and include e.g. the factors HGF, FGF, VEGF and others more.

It has been proposed in the literature (cf., for example, EP 1 415 663 A1 and EP 1 555 030 A1) to apply such angiogenesis-stimulating factors in a matrix having a depot effect, the gelatin hydrogel comprising gelatin having an average molecular weight of from 100,000 to 100,000 as the depot matrix 200,000 daltons (Da) was recommended.

For different types of collagen their suitability as supporting structure in the formation of new vessels is described, as well as their anti-angiogenetic effects. As examples of this literature, see S.M. Sweeney et al., Journal of Biological Chemistry Vol. 278, no. 33, pages 30516 to 30524 (2003) and R. Xu et al. in Biochemical and Biophysical Research Communications 289, pages 264 to 268 (2001).

The object of the present invention is to provide an angiogenesis-promoting substrate which can be produced simply and inexpensively.

This object is achieved by an angiogenesis-promoting substrate which comprises a non-porous shaped body which is formed from a physiologically-insoluble, absorbable, gelatin-containing material. Gelatin-based materials have long been used for medical applications because of their good biocompatibility, for example, as a matrix material for the release of pharmaceutical agents or as a carrier material for colonization with cells. In contrast to collagen, gelatine can be produced in reproducible quality and high purity. Furthermore, it is essentially completely absorbable in the body.

As part of the present invention, it has now surprisingly been found that the gelatin-containing material as such exhibits an angiogenesis-promoting effect, i. stimulates the formation of new blood vessels in its immediate vicinity, without any further angiogenesis-promoting factors, such as e.g. the signal molecules VEGF, FGF or HGV mentioned above are needed.

It is particularly noteworthy that the angiogenesis-promoting effect of the present invention is observed in a non-porous shaped body formed of the gelatin-containing material. In previous studies by the inventors, an angiogenesis-promoting effect on porous shaped articles of gelatin-containing material had first been found, with angiogenesis taking place primarily within the shaped articles, i. ingrowth of blood vessels into the pores, cavities or spaces of the molding has been observed. The pro-angiogenic effect was therefore attributed primarily to the porous structure of the molded article (see German Patent Application No. 10 2005 054 937). Examples of such structures are sponges, fabrics or nonwovens.

In contrast, it has now been shown that according to the present invention, a non-porous shaped body as angiogenesis promoting substrate can be used, wherein the blood vessel formation is not in the molding, but in its spatial environment. Without wishing to be bound by this theory, it is believed that this effect is caused by a release of soluble components of the gelatin and therefore is largely independent of the structure of the shaped body.

Non-porous shaped articles made of a gelatin-containing material are generally easier to produce than those having a porous structure. On the other hand, the use of a shaped body of an insoluble material, which is absorbed or degraded only after a certain time, compared to the use of soluble or dissolved gelatin has the advantage that the targeted angiogenesis at a certain place, namely the environment inserted molding, can be stimulated.

Preferably, the gelatin-containing material is a gelatin-based material and consists predominantly of gelatin. This means that the gelatin makes the largest contribution to any other components of the material used.

More preferably, a gelatin-based material is used which consists essentially completely of gelatin.

Particularly suitable gelatin types are pork rind gelatin, which is preferably of high molecular weight and has a bloom value of about 160 to about 320 g.

To a much lesser extent, an angiogenesis-stimulating effect is also observed with low molecular weight, water-soluble gelatin having an average molecular weight of less than 6 kDa, but such an effect is comparatively nonspecific when compared to other also less stimulatory agents.

The gelatin used therefore preferably has an average molecular weight of more than about 6 kDa. In order to ensure optimum biocompatibility of the substrate according to the invention in medical application, a gelatin with a particularly low content of endotoxins is preferably used as starting material. Endotoxins are metabolic products or fragments of microorganisms found in the animal raw material. The endotoxin content of gelatin is expressed in international units per gram (IU / g) and determined according to the LAL test described in the fourth edition of the European Pharmacopoeia (Ph. Eur. 4).

In order to keep the content of endotoxins as low as possible, it is advantageous to kill the microorganisms as early as possible in the course of gelatin production. In addition, appropriate hygiene standards should be observed during the manufacturing process.

Thus, the endotoxin content of gelatin can be drastically reduced by certain measures in the manufacturing process. These measures include, first and foremost, the use of fresh raw materials (e.g., pork rind) to avoid storage times, the thorough cleaning of the entire production line just prior to the start of gelatine production, and, if necessary, the replacement of ion exchangers and filtration systems at the production line.

The gelatin used in the present invention preferably has an endotoxin content of about 1,200 I.U./g or less, more preferably about 200 I.U./g or less. Optimally, the endotoxin content is about 50 I.U./g or less, each determined according to the LAL test. In comparison, some commercially available gelatins have endotoxin levels of more than 20,000 I.U./g.

As already mentioned above, according to the invention, the non-porous shaped body of the angiogenesis-promoting substrate is formed from a material that is insoluble under physiological conditions, so that it has a certain period of time maintains its structural integrity and angiogenesis can be localized to the desired target area. However, since gelatin is rapidly dissolved under physiological conditions, the gelatin-containing material is preferably crosslinked.

According to another embodiment of the present invention, rapid dissolution can be counteracted by using the gelatin together with other slower dissolving components (examples of such resorbable biopolymers are chitosan and hyaluronic acid). Such components may be used for the purpose of temporarily immobilizing the gelatin moieties.

If the crosslinking is selected to stabilize the material, in particular the gelatin portion of the gelatin-containing material may be crosslinked, it being possible to resort to chemical crosslinking as well as enzymatic crosslinking.

Preferred chemical crosslinking agents are aldehydes, dialdehydes, isocyanates, carbodiimides and alkyl dihalides. Particularly preferred is formaldehyde, which simultaneously causes a sterilization of the molding.

The enzymatic crosslinking agent used is preferably the enzyme transglutaminase, which brings about linkage of the glutamine and lysine side chains of proteins, in particular also of gelatin.

The stability to resorption under the aforementioned physiological conditions to which the material is exposed when used can be reconstituted in vitro under standard physiological conditions. Here, a PBS buffer (pH 7.2) at 37 ⁰ C is used and under these conditions, the substrates can be tested for their time-dependent stability behavior and compare. Preferably, the gelatin-containing material has a predetermined degree of crosslinking. By specifying the degree of crosslinking, in particular the absorption stability of the shaped body can be adjusted, ie the time during which it receives its structural integrity under physiological conditions. Thus, for example, non-porous moldings can be used as angiogenesis-promoting substrates, which are stable depending on the degree of crosslinking of the gelatin-containing material, for example, one, three, six or twelve weeks under physiological standard conditions, depending on the period over which an angiogenic effect of attending physician is desired.

Surprisingly, it has also been shown that the higher the degree of crosslinking of the gelatin-containing material, the higher the degree of crosslinking of the gelatin-containing material, especially in the case of chemical crosslinking of the gelatin. This opens up further possibilities to selectively stimulate angiogenesis quantitatively.

The non-porous shaped body is preferably stabilized in its structure by means of a two-stage cross-linking, wherein in a first stage the gelatin-containing material is subjected in solution to a first cross-linking reaction, and then a molded article produced from this material is further cross-linked in a second cross-linking step.

While the crosslinking takes place in solution in the first crosslinking stage, crosslinking in the gas phase is particularly suitable for the second crosslinking stage, for example using formaldehyde.

The production of moldings from a gelatin-containing material by means of a two-stage crosslinking process is described in detail in the published patent application DE 10 2004 024 635 A1.

The two-stage cross-linking has the particular advantage that overall a higher degree of cross-linking can be achieved, which then also moreover can be realized substantially uniform over the entire cross section of the molded body. This has the consequence that the degradation properties of the shaped body during absorption are homogeneous, so that it retains substantially its structural integrity for the intended time dependent on the degree of crosslinking and then completely absorbed in a relatively short time with loss of structural integrity.

Due to the predetermined degree of crosslinking and the homogeneous degradation behavior described above, it is thus possible to use the angiogenesis-promoting effect of the substrate according to the invention very selectively both in terms of time and space.

For many applications, the degree of cross-linking should be such that, during 7 days, about 20% by weight or less of the gelatin-containing material degrades under the standard physiological conditions mentioned above.

The non-porous shaped body can be realized in very different forms, about which has not yet been spoken.

According to a preferred embodiment of the invention, the shaped body is a sheet material. Sheet materials can be used in a variety of ways as medical substrates in or on the body.

Particularly preferably, the shaped body is a film. Such films can be readily prepared by casting a solution of a gelatin-containing material, which method can be combined with the two-step crosslinking process described above.

Slides made of a gelatin-containing material are easy to handle and can be cut by the doctor in each case to the required size. In order to increase the flexibility of the film, the gelatin may be contained. tende material additionally contain one or more plasticizers. Preferred plasticizers are selected from glycerol, oligoglycerols, oligoglycols, sorbitol and mannitol.

The film preferably has a thickness in the range from about 20 to about 500 μm, more preferably from about 50 to about 100 μm.

According to a further embodiment of the invention, the non-porous shaped body is in the form of particles. The particles may be, for example, beads, granules or powders of a gelatin-containing material.

Preferred particles have an average diameter of about 0.1 mm to about 5 mm.

According to an advantageous embodiment of the invention, the non-porous shaped body comprises one or more non-gelatin-based pharmaceutical active substances. These may be, for example, anti-inflammatory or antibiotic agents.

In one embodiment of the invention, the non-porous shaped body is populated with cells. In this case, the substrate according to the invention can be used for cell transplantations in which angiogenesis in the area of the implanted cells is desired.

The present invention also relates to the use of a non-porous shaped article formed from a physiologically-insoluble, resorbable, gelatin-containing material for the preparation of an angiogenesis-promoting substrate intended for use in or on the human or animal body. Advantages and preferred embodiment of this use are apparent in particular from the above description of the angiogenesis-promoting substrate according to the invention. In a preferred use, the substrate is used as a wound dressing or cover. By applying the substrate to injuries or burns, especially to the skin, the angiogenic effect can contribute to faster wound healing.

According to a further preferred embodiment, the angiogenesis-promoting substrate is intended for implantation in the body. In this case, the substrate can be used intracorporally at the most diverse points of the body, wherever targeted promotion of angiogenesis is necessary or desirable.

Preferred areas of application of the angiogenesis-promoting substrate according to the invention are e.g. Transplants, the treatment of diabetes or infarction.

In carrying out therapeutic methods using the angiogenesis-promoting substrate according to the invention, a non-porous shaped body is provided in the respectively required shape and size or tailored accordingly by the attending physician, and subsequently introduced into or onto the corresponding area of the human or animal body to become.

These and other advantages of the invention will be explained below with reference to the drawings and the examples in detail. They show in detail:

FIG. 1: Photograph of blood vessel formation without an angiogenesis-promoting substrate;

FIGS. 2a to 2c are photographic representations of blood vessel formation in various angiogenesis-promoting substrates according to the invention; FIG. 3: Photograph of blood vessel formation after resorption of the angiogenesis-promoting substrate.

Production of films from a gelatin-containing material

As examples of non-porous molded articles, gelatin films having three different degrees of crosslinking (films A, B and C) were prepared by a two-step crosslinking process.

For each of the three batches 25 g of pork rind gelatin (300 g Bloom), 9 g of an 85% strength by weight glycerol solution and 66 g of distilled water were mixed and the gelatin was dissolved at a temperature of 60 ^° C. After degassing the solutions by means of ultrasound, an aqueous formaldehyde solution (2.0% strength by weight, room temperature) was added to carry out the first crosslinking step, namely 3.75 g of this solution in batch A and in each case 6.25 g of the solution approaches B and C.

The mixtures were homogenized and knife-coated at about 60 ⁰ C in a thickness of approximately 250 microns onto a polyethylene backing.

After drying at 30 ^° C. and a relative humidity of 30% for about one day, the films were stripped off from the PE base and after-dried for about 12 hours under the same conditions. The dried films (thickness about 50 microns) were exposed to the equilibrium vapor pressure of a 17 wt .-% aqueous formaldehyde solution at room temperature to perform the second crosslinking step in a desiccator. In the case of films A and B, the exposure time of Formaidehyddampfs 2 h, in the case of the film C 17 h.

Of the moldings produced in this way, the film A has a total of the lowest and the film C in total the highest degree of crosslinking, the film B is in between. This is reflected in the different degradation behavior of the films, wherein the absorption times of the described Foils under physiological conditions in animal experiments (see below) between about 14 days (slide A) and about 21 days (slide C) are.

Due to the use of glycerol as plasticizer, the films show sufficient flexibility, in particular in the hydrated state, to ensure good handling in the medical application, without fear of breakage or tearing of the films.

Evidence of the angiogenesis-promoting effect in animal experiments

The efficacy of the gelatin films A, B and C as angiogenesis promoting substrates in vivo was investigated in animal experiments. Ten weeks old mice of the strain Balb / C from Charles River (Sulzfeld) with a body weight of 20 g were used as test animals.

The substrates used were 5 × 5 mm ² pieces of the gelatin films described above. The mice were each implanted two pieces of film of a certain degree of crosslinking subcutaneously in the neck area. For this purpose, the animals were anesthetized and shaved the coat in the neck area. With a pair of tweezers, a piece of the neck skin was lifted and an incision of about 1 cm length was made. About this incision, a subcutaneous pocket was created with blunt scissors, in each of which two of the pieces of foil were inserted with tweezers. The wound closure took place by means of two single button booklets.

After 12 days, the animals were killed and the angiogenic effect of the implanted substrates was evaluated optically.

FIG. 1 shows, as a negative control, the corresponding region of the subcutaneous tissue of a mouse in which no implantation of the angiogenesis-promoting substrate was carried out. There is very little blood vessel penetration seen as normal for the subcutaneous skin tissue of the mouse. Figures 2a to 2c show photographs of the subcutaneous skin tissue in the region of the implanted pieces of foil A, B and C, respectively, after the corresponding mice were killed 12 days after implantation. The position of the pieces of film is indicated by black squares (reference numbers A, B and C, respectively, for the corresponding film) because the films themselves are poorly visible in the photograph. The films were partly dyed with Coomassie Brilliant Blue, as is visible in FIG. 2a.

All three figures show markedly increased blood vessel formation in the vicinity of the implanted pieces of foil. Both the number and the size of the blood vessels are in each case significantly higher than in the negative control in FIG. 1. This result demonstrates that angiogenesis can be achieved by non-porous shaped bodies which are formed from a material which is insoluble under physiological conditions and resorbable, containing gelatin can be locally stimulated.

In order to investigate the time frame of the angiogenesis-promoting effect, two pieces of foil B (medium degree of crosslinking) were implanted in another mouse (as described above). This mouse was killed after 21 days and the subcutaneous tissue in the area of the implants was again optically evaluated.

The result is shown in FIG. 3. The relatively thin gelatin films B are already largely absorbed after 21 days and have lost their structural integrity. At the same time, the photographic representation shows that the newly formed blood vessels, which were observed in the corresponding films after 12 days (see Figure 2b), have regressed again.

This result shows that the angiogenic effect of the non-porous shaped body is limited in time. With the progressive resorption of the angiogenesis-promoting substrate, the blood vessels also form back. However, the choice of the degree of crosslinking speed and thus also the time frame of angiogenesis.

Overall, these experiments confirm that, with the aid of the substrate according to the invention, angiogenesis in the human or animal body can be specifically stimulated both spatially and temporally.

Claims

claims

An angiogenesis-promoting substrate comprising a non-porous shaped body formed from a physiologically-insoluble, resorbable, gelatin-containing material.

2. Substrate according to claim 1, wherein the gelatin-containing material consists to a predominant proportion of gelatin.

A substrate according to claim 2, wherein the gelatin-containing material consists essentially completely of gelatin.

4. A substrate according to any one of the preceding claims, wherein the gelatin has a bloom value in the range of about 160 to about 320 g.

A substrate according to any one of the preceding claims, wherein the gelatin has a molecular weight greater than about 6 kDa.

6. Substrate according to one of the preceding claims, wherein the gelatin has a determined according to the LAL test endotoxin content of about 1,200 I.E./g or less, in particular of about 200 I.E./g or less.

7. Substrate according to one of the preceding claims, wherein the gelatin-containing material is crosslinked.

The substrate of claim 7, wherein the gelatin is crosslinked.

A substrate according to claim 7 or 8, wherein the gelatin-containing material is crosslinked using formaldehyde.

10. A substrate according to any one of claims 7 to 9, wherein the gelatin-containing material has a predetermined degree of crosslinking.

11. Substrate according to one of the preceding claims, wherein the non-porous shaped body is a sheet material.

The substrate of claim 11, wherein the sheet material is a film.

13. A substrate according to claim 12, wherein the film has a thickness in the range of about 20 to about 500 microns, preferably from about 50 to about 100 microns.

14. A substrate according to any one of claims 1 to 10, wherein the non-porous shaped body is in the form of particles.

15. The substrate of claim 14, wherein the particles have an average diameter of about 0.1 mm to about 5 mm.

16. A substrate according to any one of the preceding claims, wherein the non-porous shaped body comprises one or more non-gelatin-based pharmaceutical agents.

17. Substrate according to one of the preceding claims, wherein the non-porous shaped body is populated with cells.

18. Use of a non-porous shaped body, which is formed from a physiologically insoluble, resorbable, gelatin-containing material for the production of an angiogenesis-promoting substrate, which is intended for use in or on the human or animal body.

19. Use according to claim 18, wherein the substrate is used as a wound dressing or cover.

20. Use according to claim 18, wherein the substrate is intended for implantation into the body.

Use according to any one of claims 18 to 20, wherein the substrate is used in transplantations.

Use according to any one of claims 18 to 20, wherein the substrate is for the treatment of infarcts.

Use according to any one of claims 18 to 20, wherein the substrate is for the treatment of diabetes.