EP1439844A1

EP1439844A1 - Pharmaceutical preparation comprising a growth factor and/or a cytokine and an antiseptic biguanide

Info

Publication number: EP1439844A1
Application number: EP02783001A
Authority: EP
Inventors: Wolfram Eichner; Frank Jethon
Original assignee: Fresenius Kabi Deutschland GmbH
Current assignee: Fresenius Kabi Deutschland GmbH
Priority date: 2001-10-26
Filing date: 2002-10-24
Publication date: 2004-07-28
Also published as: PL369229A1; NO20042099L; WO2003037356A1; DE10152956A1

Abstract

The invention relates to a pharmaceutical preparation comprising a growth factor and/or a cytokine and an antiseptic biguanide.

Description

A pharmaceutical composition comprising a growth factor and / or a cytokine and an antiseptic biguanide

The present invention relates to a pharmaceutical composition comprising a growth factor and / or a cytokine and an antiseptic biguanide. The present invention further relates to the use of a corresponding composition for treating wounds.

The treatment of wounds and wound healing disorders, for example after severe surgical interventions, as well as for acute or chronic wound infections is an acute problem in everyday clinical practice. In recent years, this problem has been exacerbated by the emergence of infectious microorganisms resistant to a variety of antibiotics.

Various local antiseptics and antibiotics are used in the prior art to treat corresponding wounds. However, the fundamental problem any antiseptic wound treatment is ^as' ss chemical substances with good bactericidal action also act cytotoxic generally and the For this reason, wound healing is impaired. The rinsing of wounds with antiseptic solutions, for example with chlorine-containing solutions, organic silver compounds, alcoholic mixtures with quaternary ammonium compounds or iodophores, was initially unsuccessful in everyday clinical practice due to the side effects (Hernandez-Richter, HJ, Struck, H.: wound healing, Georg Thieme Verlag, Stuttgart, 1970).

A larger study finally found that a poiy (hexamethylene) biguanide (hereinafter referred to as PHMB) is particularly suitable for local antiseptic wound treatment (Willenegger, H. Unfallchirurgie, 1994, 20 (2): 94-110; and GB 1,202,495 ). The combination of low cytotoxic effects and good antiseptic effects has led to PHMB becoming established for the local treatment of infected wounds and also for prophylactic treatment during or after an operation (see Roth and Baltzer, Z. Unfall - Chir. Versicherungsmed. 1990, 83 (4): 224-6; and Wagner, M., Orthopädie, 1995, 24 (4): 319-325).

For this pharmaceutical use, PHMB has been mixed with various other active ingredients, as well as carriers and auxiliaries. From DE 3 537 627 it is known, for example, that a combination of PHMB with polyethylene glycol can be used as a disinfectant and local antiseptic. In contrast, WO97 / 00076 relates to the production of pharmaceutical compositions for the topical treatment of microbial infections, the pharmaceutical compositions containing a PHMB salt, for example the hydrochloride salt. In contrast, EP 450 117 uses a lactate-free Ringer solution which contains 0.1-0.2% of a concentrate from a 20% aqueous PHMB hydrochloride solution.

WO00 / 33829 discloses that PHMB can also be used to heal wounds that are free from infections, since the active substance has a granulation-promoting activity and thus itself has a healing effect. PHMB is also used in other areas as a disinfectant, for example for cleaning and disinfecting rooms and equipment in the food industry, for stabilizing beverages, and for fighting algae and bacterial growth in swimming pools (Wallhäusers, KH, practice of sterilization, disinfection , Preservation; germ identification - industrial hygiene; Thieme 1995).

Polyhexanide acts on bacterial cells by triggering changes in permeability of the cell wall and charge shifts in the cell membrane, inhibiting membrane-constant metabolic processes and causing the coagulation of cell contents (Davies, et al. J. appl. Bact. (1969) 32, 233- 243; Broxton, Woodcock et al. Microbios. (1983) 40. 187-193; Broxton, Woodcock et al. Microbios. (1984) 41. 15-22; Broxton, Woodcock et al. J. appl. Bact.

(1983) 54, 345-353; Broxton, Woodcock et al. J. appl. Bact.

(1984) 57. 115-125; Mitchinson et al. Biochem. -Biophys. Acta. (1983) 31-36; Ikeda et al. , Biochem. -Biophys. Acta. (1983) 380-386; Ikeda et al. , Biochem. -Biophys. Acta. (1984) 57-66; Ikeda et al., Bull. Chem. Soc. Jpn. (1985) 58. 705-709; Gilbert et al. J. appl. Bact. (1990) 69 (4). 585-592; Gilbert et al. J. appl. Bact. (1990) 69 (4). 593-598).

These effects of polyhexanide can only be observed in a weakened form in eukaryotic cells which have a different cell membrane and in particular primarily neutral lipid structures in the membrane.

In the prior art there are of course a variety of alternative treatment options for acute and chronic wounds. Non-infected, chronic wounds are treated with growth factors, for example.

Growth factors are polypeptides that interact with certain cell receptors and can stimulate cell division.

The wound healing process in particular is controlled by growth factors, which have mitogenic properties, for example. and stimulate cellular poliferation, have angiogenic properties and stimulate the growth of new blood vessels, and control the production and degradation of the extracellular matrix.

The growth factor PDGF ("platelet derived growth factor") obtained from platelets is present as an essential growth factor in serum, but not in plasma (Antoniades et al., Processings of the National Academy of Sciences of the United States of America, 1975, vol. 72: 2635-2639; and Ross and Vogel, Cell, 1978, vol. 14: 203-210).

PDGF is mainly secreted by thrombocytes, but also by other cell types (Pierce et al., Journal of Cellular Biochemistry, 1991, Vol. 45: 319-326) and acts as a mitogen on the cells of the connective tissue and on most mesenchy cells - paint origin (Pierce and Mustoe, Annual Review of Medicin, 1995, Vol. 46: 467-481).

Three different isoforms of PDGF have been isolated, each homo- or heterodimeric combinations of two different peptide chains, designated A and B (Meyer-Ingold and Eichner, Cell Biology International, 1995, Vol. 19, 389-398).

It could be shown that the addition of PDGF improves wound closure (see Greenhalgh, D.G., The Journal of Trauma, Injury, Infection and Critical Care, 1996, Vol. 41: 159- 167). In the meantime, a gel containing recombinant PDGF-BB in a concentration of 0.01% has been approved by the US FDA and other regulatory authorities for the treatment of diabetic foot ulcer.

Recombinant PDGF-BB is highly active in vitro, but it has been shown that in vivo activity, that is to say an effect which promotes wound healing, is difficult to achieve. This is primarily due to the complex situation in the wound in which for example, proteases are present in a relatively high concentration, which attack and inhibit PDGF. In the prior art, a protease inhibitor has therefore been applied together with the growth factor in order to prevent proteolytic degradation of the growth factor (Wlaschek et al., Br. J. Deratol., 137 (1997), 646; Kiyohara et al ., Biol. Pharm. Bull., 16 (1993), 73-76; Kiyohara et al., J. Pharmacobiodyn, 14 (1993), 47-52).

Gels which contain PDGF as the active ingredient and which are suitable for use as a wound dressing are described, for example, in WO 97/12601. The gels consist of a pharmaceutically acceptable cellulose polymer and, in addition to the PDGF, can contain a preservative and, as a stabilizer, a charged group. Combinations of different growth factors or combinations of growth factors with other active substances have also been proposed in the prior art for wound treatment. For example, WO 92/16181 discloses a pharmaceutical composition which contains PDGF and dexamethasone, and their use for tissue regeneration.

However, growth factors are not used to treat infected wounds, since the growth factor as a protein can itself be a source of nutrients for the microorganisms.

In relation to this prior art, the object of the present invention is to provide pharmaceutical compositions which enable improved wound healing, in particular improved healing of infected wounds.

This task has now been solved by pharmaceutical compositions comprising a growth factor and / or a cytokine and an antiseptic biguanide. In the context of the present invention it has surprisingly been possible to show that growth factors and cytokines are stable and active over a long period in the presence of antiseptic concentrations of the biguanide. The present invention thus provides an advantageous combination of active ingredients and their use for the treatment of acute and chronic wounds.

A variety of growth factors and cytokines are known in the art. The pharmaceutical composition according to the invention can contain one or more of the known growth factors and / or one or more of the known cytokines. The corresponding selection can easily be made by the person skilled in the art depending on the planned medical use. The use of PDGF-AA, PDGF-BB, PDGF-AB, factor XIII, CSF, TGF, TGF-ß, EGF, TNF, somatomedin and / or a lymphokine is preferred. A combination of different growth factors, different isoforms of a growth factor, different cytokines or mixtures thereof can of course also be used. The use of the growth factor PDGF-BB is particularly preferred.

Biguanides are compounds that are derived from biguanide (C ₂ H ₇ N ₅ ), especially its polymers. Antiseptic biguanides are compounds that have an antimicrobial effect, that is to say act as bacteriostatics or preferably as bactericides. The compounds preferably have a broad activity against many bacteria and can be characterized by an antimicrobial activity against E. coli of at least 0.5 μg / ml, preferably at least 12 or at least 25 μg / ml (minimum microbicidal concentration or MMK; measured in the suspension test ). However, the MMK can reach maximum values of up to 1000 or 2000μg / ml.

According to a preferred embodiment, the pharmaceutical composition according to the invention comprises a poly (imino [imidocarbonyl] iminopolymethylene), the use of poly (hexame- thylene) biguanide (PHMB), which is also known as polyhexanide, is particularly preferred as an antiseptic biguanide. The compounds can be present as racemates or pure isoforms.

The concentration of the growth factor and the biguanide in the pharmaceutical composition according to the invention can be selected by the person skilled in the art by simple experiments depending on the intended use, in particular depending on the type of formulation of the composition (cream, gel, etc.). According to the invention, it is preferred that the growth factor is present in a concentration of 1.0 μg / g to 700 μg / g of pharmaceutical composition, a concentration of 50 to 90 μg / g being preferred.

The biguanide can be present, for example, in a concentration of 0.0001 to 0.1% by weight, based on the weight of the pharmaceutical composition, a concentration of 0.0005 to 0.006% by weight being preferred. The biguanide can have any molecular weight distribution, an average molecular weight distribution up to 15,000, in particular in the range from 1,000 to 8,000, being preferred. An average molecular weight of 2,800 is particularly preferred.

The type of pharmaceutical composition according to the invention depends on the intended medical use. The composition can be formulated as a cream, oil, oil in water or water in oil emulsion, plaster, wound dressing or gel. The formulation as a gel, in particular as a hydroxyethyl cellulose gel, is particularly preferred. The preparation of a gel according to the invention is shown in Example 4.

The pharmaceutical composition according to the invention can be used for the treatment of chronic or acute wounds, soft tissue wounds, in particular for the treatment of infected wounds or for the treatment of the consequences of a surgical intervention. The compositions according to the invention are particularly suitable for chronic, infected and difficult to heal wounds, such as diabetic ulcer, especially on the foot.

The present invention further relates to processes for producing a pharmaceutical composition, in which a growth factor and an antiseptic biguanide are mixed with pharmaceutically acceptable carriers and / or auxiliaries.

Brief description of the figures:

Fig. 1: PHMB cytotoxicity on Balbc-3T3 cells;

Fig. 2: PHMB cytotoxicity on HUVE cells;

3: PDGF on an SDS-PAGE gel after incubation with different concentrations of Lavasept;

Fig. 4: Mitogenic activity of the PDGF after incubation with different concentrations of Lavasept.

Example 1 Cytotoxic Effect of Lavasept

The cytotoxic effect of Lavasept (Lavasept concentrate No.4927031, batch IBX 00301.2001, Fresenius) was tested on Balbc-3T3 fibroblasts (mouse cell line) and examined. For this, cells were seeded in a 96-well microtiter plate and contacted with different concentrations of Lavasept (0.001%, 0.002%, 0.005%, 0.01%, 0.05%, 0.1% and 0.2%; these and all The following percentages relate to% by volume; all dilutions were made in BSA-containing PBS buffer, "phosphate buffered saline", pH 7.4) and the cytotoxicity was examined by microscopic and macroscopic observation of the cells and the color of the medium. It was found that at a concentration of 0.1% and 0.2% Lavasept no or hardly any cells can be seen; all cells are lysed. In 0.05% Lavasept, sporadic cell extensions are identified, while the cytotoxic effect decreases with a lower concentration. However, a low cytotoxicity was found at a concentration of 0.005% Lavasept and an incubation of 6 days.

These observations are confirmed by the evaluation of the color of the medium, which show that cytotoxicity starts at 0.004% Lavasept.

Example 2 Cytotoxic Effect of Lavasept

Lavasept cytotoxicity on Balbc-3T3 mouse cells and human vascular endothelial cells ("human umbelical vein endothelial cells", HUVE cells) was also determined using the sensitive MTT test. The MTT test is based on the reduction of the dye 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (thiazolyl blue), which leads to a change in color (yellow to violet). The reduction occurs in living cells through active mitochondrial dehydrogenases; A corresponding conversion of the dye is not possible in dead cells. A photometric measurement allows the percentage of living cells to be determined (cf. Shi et al., J. Immunol. Methods, 164 (1993), 149-154).

Balbc-3T3 fibroblasts are excellently stimulated with PDGF and are known in the art as a robust cell type. In contrast, human endothelial cells are much more sensitive and demanding to cultivate and pass through.

The results are shown in Tables 1 to 3 and in FIGS. 1 and 2. Table 1 MTT test using Bal c-3T3 cells

Table 2 MTT test using Balbc-3T3 cells

Table 3 MTT test using HUVEC cells

In summary, it can be stated that the microscopic evaluation of 3T3 cells after Lavasept treatment shows clearly cytotoxic effects in the form of cell detachment or a loss of the fine structure even at a concentration between 0.005 and 0.01%. Higher concentrations lead to cell death, cell lysis and denaturation of the proteins. The MTT test showed that Lavasept concentrations in the range of 0.002 to 0.005% already have a cytotoxic effect.

Lavasept, however, is tolerated by Balbc-3T3 cells up to a concentration of approximately 0.01% and only leads to an inhibition of cell growth of approximately 40 to 70%.

Human primary endothelial cells are more sensitive to the addition of Lavasept. Concentrations up to 0.001% can be tolerated and ^"lead to an inhibition of cell growth of 22%. Increasing the Lavasept concentration to 0.005% but is no longer tolerated. Example 3 Stability of the human PDGF-AB in the presence of Lavasept

In the present experiment, the stability of recombinant, human PDGF-AB in the presence of Lavasept was examined. For this, the protein was incubated with different Lavasept concentrations. The integrity of the protein was then determined using SDS gel electrophoresis and the activity was determined using the established mitogen assay.

The PDGF-AB was obtained from Strathmann (code PDGF-AB-10) and was reconstituted in PBS according to the manufacturer. In addition, an excess of BSA was added. The BSA is intended to protect the growth factor as a carrier protein at low protein concentrations. The protein concentration of the reconstituted PDGF was 100 μg / ml. For the analysis, PDGF was diluted with Lavasept and adjusted to a concentration of 50 μg / ml (500 ng / μl). The final concentration of the Lavasept was 0.0%, 0.005%, 0.025%, 0.05% and 0.25%. The concentration of the growth factor was a constant 50 μg / ml.

The growth factor / Lavasept solution was stored at -20 ° C for at least one and at most eight weeks.

For the analysis of the stability of the protein, all fractions were separated on a polyacrylamide gel and the proteins were visualized using the silver stain. As FIG. 3 shows, PDGF-AB is very stable under the chosen conditions. Incubation of the PDGF with 0.005% up to 0.05% Lavasept does not lead to a degradation of the protein. At a final concentration of 0.25% Lavasept, no degradation products can be seen, but the protein band is somewhat weaker, which may be due to a partial breakdown. Overall, however, it can be clearly seen that the selected concentration range has no significant influence on the stability of the protein. This result is also confirmed by the mitogenicity test. FIG. 4 shows the uptake of 3 H-tymidine by Balbc-3T3 cells in the presence of a combination of PDGF-AB and different concentrations of the Lavasept. To perform this test, the growth factor was pre-incubated with Lavasept in the range of 0.01% to 0.5% and diluted to a final concentration before the start of the test, which is not cytotoxic, but at which the growth factor is detectable (10-25 ng / ml).

As FIG. 4 shows, the mixture of PDGF with Lavasept has no negative influence on the activity of the growth factor.

Example 4 Preparation of a wound dressing gel containing Lavasept and PDGF

The parts:

No. of raw materials quantity

1 glycerin 100% (anhydrous),

DAB quality 85.00 g

2 Lavasept concentrate (Fresenius) 2.00-5.00 g

3 Hydoxyethylcellulose, Ph.Eur. 32.50 g 4 PDGF-BB 0.05-0.1 g

5 Ringer's solution ad 1000.00 g

are mixed together in the following procedure:

Ringer's solution is placed in the batch container; the glycerin is stirred in. Lavasept concentrate is stirred in and the mixture is ^" heated to 40-70 ° C. to avoid lump formation when sprinkling in the hydroxyethyl cellulose. Powdery hydroxyethyl cellulose and PDGF are slowly added with stirring. The mixture is homogenized until the gel is clear.

The gel is filled into suitable tubes, syringes etc., which are provided with a corresponding label, whereby only completely clear gel is used!

Claims

claims

L. A pharmaceutical composition comprising a growth factor and / or a cytokine and an antiseptic biguanide.

i. Pharmaceutical composition according to claim 1, characterized in that the growth factor is PDGF-AA, PDGF-BB, PDGF-AB, factor XIII, CSF, TGF, TGF-ß, EGF, TNF, somatomedin and / or a lymphokine.

!. Pharmaceutical composition according to claim 1 or 2, characterized in that the growth factor is present in a concentration of 1.0 μg / g to 700 μg / g, preferably in a concentration of 50 μg / g to 90 μg / g pharmaceutical composition.

:. Pharmaceutical composition according to one of claims 1 to 3, characterized in that the antiseptic biguanide is poly (hexamethylene) biguanide (PHMB).

Pharmaceutical composition according to one of claims 1 to 4, characterized in that the antiseptic biguanide is present in a concentration of 0.0001 to 0.1% by weight, based on the weight of the pharmaceutical composition.

Pharmaceutical composition according to one of claims 1 to 5, characterized in that the biguanide has an average molecular weight distribution in the range from 1,000 to 8,000.

A pharmaceutical composition ^' ^"that the 6, characterized ^in' pharmaceutical composition is a cream, oil, oil in water or water in oil emulsion, patches, wound dressing or gel formulated according to any one of claims 1 to.

8. Pharmaceutical composition according to one of claims 1 to 7, characterized in that the gel is a hydroxyethyl cellulose gel.

9. Use of a pharmaceutical composition according to any one of claims 1 to 8 for the treatment of chronic or acute wounds.

10. Use according to claim 9, characterized in that the wound is the result of a surgical intervention.

11. Use according to claim 9 or 10, characterized in that the wound is infected.

12. Use according to any one of claims 9 to 11, characterized in that the wound is a chronic, infected wound.

13. Use according to one of claims 9 to 12, characterized in that the wound is a soft tissue wound.

14. Use according to one of claims 9 to 13, characterized in that the wound is a diabetic ulcer, in particular a diabetic ulcer on the foot.

15. A method for producing a pharmaceutical composition, characterized in that a growth factor and an antiseptic biguanide are mixed with pharmaceutically acceptable carriers and / or auxiliaries.