SK89998A3 - Immunologically active mistletoe extract preparations - Google Patents

Abstract

The invention relates to pharmaceutical preparations from mistletoe extracts with an immunologically active level of mistletoe lectin which obtain a high level of transit time stability due to stabilising additives. The stable preparations are adjusted to a level of mistletoe lectin which is the lowest possible to avoid side effects but has an optimal effect.

Description

IMMUNOLOGICAL EFFECTIVE PREPARATIONS FROM IMELA EXTRACT

Technical field

The invention relates to medicaments for the parenteral administration of mistletoe lectin and further comprises mistletoe extract pharmaceutical compositions which are characterized by having as little immunologically active mistletoe lectin content as possible to prevent side effects and having long-term stability in use due to stabilizing ingredients.

BACKGROUND OF THE INVENTION

Mistletoe (V / scum album) has long been known as a medicinal plant. Mistletoe extracts are used in preparations for the treatment of rheumatic diseases and arthrosis. Aqueous mistletoe preparations are used as an injectable solution in the palliative therapy of malignant tumors. Cancer-attenuating properties of mistletoe have been studied and described many times. Mistletoe lectins have been identified as the active ingredient. [Hajto, T., Hostanska, K., Gabius, W - J., Modulators Potency of the β - Galactoside - Specific Lectin from Miesteltoe Extract (Iscador) on the Host Defense System in Vivo in Rabbits and Patients. Cancer Res. 49 (1989), 4803-4808, Beuth, J., Ko, H. L., Tunggal, L., Geisel, J., Pulverer, G. Vergleichende Untersuchungen zur immunaktiven Wirkung von Galaktosid - Spezifischem Mistelektin: Arzneim. Forsxh, 43 (1993), 166-169,].

Mistletoe lectins possess both cytotoxic and immune-modulating properties, so that, at appropriate dosages, they are able to positively affect various parameters of the immune system. They belong to the class of glycoproteins, and consist of lighter ¹ component (A chain) with those ribosome inactivating (RIP) and the heavy tail (chain B), characterized by a specific activity of binding sugar. The components are linked together by disulfide bridges (Luther, P., Becker, H., Die Mistel, Berlin: Springer-Veriag 1987, 58-119,).

The high toxicity of mistletoe extracts and the constituents contained therein requires precise dosing for therapeutic use to ensure the tolerability of mistletoe preparations and to prevent the induction of immune suppressing effects due to overdose. So far, 1 ng of lactine per kg body weight has been reported as the optimal dose

30995 / H masses [Hajto, T., Hostanska, K., Gabius, W-J., Modulators Potency of the β Galactoside-specific Lectin from Miesteltoe Extract (Iscador) on the Host Defense System in Vivo in Rabbits and Patients. Cancer Res. 49 (1989), 4803-4808, Hajto, T, Hostanska K, Erfender, Madaus AG, Anmelder. Lektinkonzentrate aus Mistelextrakten und entsprechende, stabilisierte Mistellektinpräparate. Verfahren zu ihrer Herstellung sowie diese enthaltende Arzneimittel und deren Verwendung zur Erhôhung der natiiriichen Immunresistenz und / oder in der Tumor-Therapie. EP 0 602 686 A2. Offenlegung 22.06.1984, Beuth, J., Ko, H. L., Tunggal, L., Geisel, J., Pulverer, G. Vergleichende Untersuchungen zur Immunaktiven Wirkung von Galaktosid - Spezifischem Mistellektin: Arzneim. Forsxh, 43 (1993), 166-169].

Based on the current state of scientific knowledge, parenteral medicaments containing aqueous mistletoe preparations are not either adapted to a certain mistletoe lectin content or contain mistletoe lectin from 50 mg / ml to 350 mg / ml (EP 0 602 686 A2), or even more, an example of iscador Q 5 mg spezial with 375 ng / ml.

The essential components of mistletoe extracts, mistletoe lectins, are proteins. As such, they are only short-term stable in aqueous solutions which are the basis for use as parenteral drugs. Dissolved proteins are exposed in various ways to chemical or physical changes [Franks, F. Conformational Stability of Proteins. In Franks F. Editor of Protein Biotechnology. Totowa, New Jersey, Humana Press 1993: 395-43, Franks F. Storage Stabilization of Proteins. In Franks F. Editor: Protein Biotechnology. Totowa, New Jersey, Humana Press 1993: 468-532, Wang, Y-CJ., Hanson MA. Parenteral Formulations by proteins and Peptides. Stability and Stabilizers. J. Parenter. Sci. Techn. 1998suppl, 42: S3-S26.] It is therefore essential that proteins that are used as drug active substances in aqueous solutions be stabilized.

EP 0 602 686 A2 discloses the possibility of stabilizing aqueous solutions having a mistletoe lectin content. While these stabilizers have been able to ensure limited solution stability with respect to adsorption on surfaces, it has only been possible for a certain period of time that is too short to meet the requirements of a safe drug, such as set out in the drug testing guidelines [Allgemeine Verwaltungsvorschrift zur Anwendung der Arzeineimittelprufrichtlinien vom 14. Dezember 1989. Bundesanz. 1989, 41, 243a]. Thus, stability has not been achieved for a sufficient time at either 25 ° C storage temperatures or cooling (4-8 ° C) (Fig. 1). Thus, the medicaments produced according to EP 0 602 686 A2 meet only the general requirements for the safety of the medicaments for a short period, where reproducible applications of the active substance are required during the shelf life of the medicament.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION It is an object of the present invention to provide a pharmaceutical composition for positively influencing immunological parameters in the treatment of tumors or infectious diseases, having an optimally effective and safely administrable content of mistletoe extracts, wherein the active ingredients contained are reproducible in the drug and stable.

This problem has been solved by providing a pharmaceutical composition comprising aqueous leach from mistletoe twigs and having a long shelf life due to stabilizing ingredients in an amount of <0.1 mg / ml.

The mistletoe extract preparation according to the invention comprises a solution containing 5 to 50 mg / ml, preferably 20-30 ng / ml, of an immunologically active galactoside-binding mistletoe lectin in an appropriate amount and has, by the addition of stabilizers, in concentration ranges from 0.001 to 0, 1 mg / ml, preferably 0.01 mg / ml, the desired stability with respect to storage surface stability.

The mistletoe lectin preparations may, in another embodiment, also be provided as a lyophilisate.

Surprisingly, according to the invention, it has been found that in the region of considerably lower doses of from 15 to 375 pg / kg body weight, the optimum efficacy is manifested, especially after repeated administration over a longer period. These low dosages substantially reduce the usual side effects.

The medicaments according to the invention are also characterized by a dosage unit of from 15 to 375 pg / kg body weight with a suitable content of biologically active mistletoe lectin.

As a dosage unit, an analytically controlled content of galactoside specific mistletoe lectin for administration from 25 to 200 pg / kg body weight, in particular 75 pg / kg body weight, is preferred.

.V.995 / H

Thanks to the compositions according to the invention, it is for the first time possible to reproduce parenteral compositions which allow safe application throughout the shelf life, also in the form of disposable syringes, where otherwise the adsorption of protein on lipophilic surfaces of plastics is very problematic. These are used to improve immune parameters in both humans and mammals, for tumor therapy, as well as for viral diseases.

Determination of optimally effective content of mistletoe lectin:

For clinical trials, an aqueous solution for injection from non-wooded mistletoe twigs with leaves containing 30 ng / ml of mistletoe lectin binding galactoside was determined to be mistletoe lectin I. The duration of use in cancer patients was 4 weeks for two treatments week. Surprisingly, it was found that even at a dose of 75 pg / kg body weight, the strongest positive changes in the immune parameters in serum cancer patients could be observed. These findings are novel because it has to be assumed in the prior art that up to a significantly higher dosage of 1 ng / kg body weight induces positive changes in immune parameters. Comparative tests have shown that both larger and lower dosages (15 pg / kg and 375 pg / kg, respectively) induce minor changes in the immune parameters of CD 3 + HLA - DR + and LGL cell numbers - (Large granular lymphocytes) (cf. Figures 2 and 3).

Stabilization of mistletoe lectin:

Alkanols such as polyols and carbohydrates, hydrophilic polymers such as polyvinylpyrrolidone, polyvinyl alcohol, non-ionic surfactants such as polyalkylene glycols (poloxamers), fatty acid esters with polyoxyethylene (poloxyethylene) (polyoxyethylene) are used as stabilizers of aqueous mistletoe lectin preparations. , such as albumin, especially human serum albumin.

Stabilizing additives for mistletoe lectin additions are known from EP 0 602 686 A2, where they are added to the solution in a concentration range of from 0.1 mg / ml to 10 mg / ml, preferably 5 mg / l. EP 0 602 686 A2 was fully taken into account when analyzing the solutions according to the invention.

It has now surprisingly been found that concentrations of said stabilizers from 0.01 mg / ml and below behave equally in stabilizing effect. It could

5J995 / H shown, for example, on polyvidone (PVP) and albumin stabilizers known in the literature. These stabilizers increase protein stability with respect to surface binding to lipophilic surfaces (Figure 4). In addition, a comparable quality for storage stability was also achieved when lower concentrations of excipients (e.g. polyvidone) were used (Figure 5).

Furthermore, it has surprisingly been found that nonionic surfactants, such as polyalkylene glycols (block polymers of ethylene oxide and petroleum oxide, e.g. Pluronic) and polyoxyethylene fatty acid esters (e.g. Tween 20), also act as stabilizers in the same concentration ranges.

Mistletoe extract preparations are used for sustained therapy. Reducing the content of excipients for stabilization substantially contributes to the safety of the drug because, for example, polymers such as polyvidone and polyalkylene glycols based on ethylene oxide and propylene oxide are slowly eliminated after parenteral administration in renal impairment. In addition, there is a risk that these substances accumulate. This danger is substantially reduced by reducing the concentration to an exemplary 0.01 mg / ml.

Chelating agents such as nitriloalkane carboxylic acids and derivatives thereof are used in the compositions of the invention to increase the shelf life of the injectable solutions. Surprisingly, the use of these compounds, in particular in the form of alkali metal salts, in addition to the stabilizers mentioned above, has resulted in a drastically prolonged shelf-life of mist-extract preparations. The influence of chelating agents which have been used in parenteral dosage forms mainly for metal complexation (Wang, Hanseri) on mistletoe extract preparations is unknown in the prior art.

Suitable compounds are NTA (nitriloacetic acid and its salts), EDTA (edetic acid and its salts), CDTA (1,2-diaminocyclohexane - N, N tetraacetic acid and its salts), N - (2-hydroxyethyl) - ethyldiamine - N, N, N triacetic acid and its salts and TTHA (triethylenetetramine hexaacetic acid and its salts). One or more such compounds may be used.

In a preferred embodiment, edetic acid is used as the nitriloalkanecarboxylic acid derivative, in particular as the disodium salt. preferably

3.-Λ95 / Η disodium edetate is used in the concentration range from 0.001 mg / ml to 0.1 mg / ml, in particular 0.01 mg / ml.

The superiority of the combination of different stabilizing agents over a single compound is evident in terms of storage stability of mistletoe formulations according to the decrease in mistletoe lectin content, for example for polyvidone or polyvidone and disodium edetate. (Figure 7).

The addition of stabilizers such as polyvidone and disodium edetate does not demonstrably affect the activity of mistletoe extract formulations. Thus, mistletoe preparations having the same mistletoe lectin content, both with and without the addition of PVP and disodium edetate, have the same activity in the MOLT-4 cytotoxicity assay and in the release of cytokines from human blood cells. The ingredients themselves show no activity in this assay. This ensures that the ingredients do not affect the pharmacological action of mistletoe extract formulations.

The following examples of formulations of stable formulations illustrate the invention without limiting it.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Solution for injection (5 L)

Water extract (1: 1,1-1,5) from non-wooded mistletoe twigs with leaves corresponding to 30 ng / ml active mistletoe lectin 0,2 - 0,7 g

Sodium monohydrogen phosphate. 12 H2O 20,13g

Sodium dihydrogen phosphate. 2H2O. 1.88 g

Sodium chloride 37,50g

Polyvidone K17PF 0.05g

Disodium edetate 0,05g

Water 4965 g

Example 2: Solution for injection (6 L)

Water extract (1: 1,1-1,5) from non-wooded mistletoe twigs with leaves corresponding to 30 ng / ml active mistletoe lectin 0,2 - 0,7 g

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Sodium monohydrogen phosphate. 12 H2O 20,13g

Sodium dihydrogen phosphate. 2H ₂ O 1.88 g

Sodium chloride 37,50g

Pluronic F 68 0.05g

Disodium edetate 0,05g

Water 4965g

Example 3: Solution for injection (51)

Water extract (1: 1,1-1,5) from non-wooded mistletoe twigs with leaves corresponding to 30 ng / ml active mistletoe lectin 0,2 - 0,7 g

Sodium monohydrogen phosphate. 12 H2O 20,13g

Sodium dihydrogen phosphate. 2H ₂ O 1.88g

Sodium chloride 37,50g

Tween 20 0.05g

Disodium edetate 0,05g

Water 4965 g

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Storage stability of an aqueous leachate aqueous preparation (1: 1, 11.5) from non-wooded mistletoe twigs, adjusted to 65 ng / ml containing 0.1 mg Polyvidone K17PF / ml, determination of mistletoe lectin by the modified ELLA method ( Vang., PiiLarsen K., Bog - Hansen TC, and New Quantitative and Highly Specific Assay for Lectinbinding Activity, In Bog - Hansen TC, van Druesche E. editors, Lactins Biology - Biochemistry - Clinical Biochemicstry Vol 5 Berlin Wde Gruyter, 1986637-644).

Figure 2 Mean LGL cell counts during four-week subcutaneous PS76A2 administration:

day: before application day: end of study

Figure 3 Mean course of activated lymphocytes (CD3 + HLA - DR +) during four weeks of subcutaneous PS76A2 administration

0: before application starts

1: 24 hours after application start

39995 / H

2: 48 hours after application start

3: 28 days after application start

Figure 4 Loss of mistletoe lectin content as a percentage of injections containing aqueous preparation of aqueous extract (1: 1,1-1,5) of woody mistletoe twigs with leaves, adjusted to 65 ng / ml, depending on residence time in disposable syringes ( PE / PP) with rubber piston.

Figure 5 Storage stability of mistletoe formulation in 20 mM phosphate buffer pH 7.4 as measured by mistletoe lectin content in percent.

Figure 6 Storage stability of mistletoe formulation in 20 mM phosphate buffer pH 7.4 measured by percentage of mistletoe lectin, storage at 48 ° C.

Figure 7 Storage stability of injectable solutions containing aqueous preparation of aqueous leach (1: 1.1-1.5) from non-wooded mistletoe twigs with leaves in 20 mM phosphate buffer, storage at 4-8 ° C.

Claims (10)

A medicament for parenteral administration of zimeis extracts as an aqueous extract of mistletoe twigs, characterized in that it contains as a solution from 5 to 50 ng / ml, preferably 20 to 30 ng / ml, of an immunologically active galactoside-binding mistletoe lectin. Medicinal composition according to claim 1, characterized in that it is in the form of an aqueous extract of an immunologically active lectin from galactoside-binding mistletoe containing stabilizers in an amount of <0.01 mg / ml. Medicament composition according to claim 2, characterized in that it has a stabilizing surfactant content of about 0.01 mg / ml. Medicament according to claims 2 and 3, characterized in that it contains alkanols, in particular polyols and carbohydrates, hydrophilic polymers, in particular polyvinylpyrrolidone, polyvinyl alcohol, non-ionic surfactants, in particular polyalkylene glycols and polyoxyethylene fatty acid esters and / or proteins, in particular albumin, in particular, human serum albumin, as stabilizers. Medicament preparation according to one of the preceding claims, characterized in that it has an additional content of chelating compounds in an amount of from 0.1 to 0.001 mg / ml, in particular 0.01 mg / ml. Medicinal product according to claim 6, characterized in that it has an alkali metal salt of nitriloalkanecarboxylic acids as chelating compounds. Pharmaceutical preparation according to claim 6, characterized in that it contains edetic acid, preferably as the disodium salt, as the nitriloalkanecarboxylic acid derivative. Medicament according to any one of the preceding claims, characterized in that it is present in a lyophilized form. Medicinal product according to any one of the preceding claims, in unit dosage form, characterized in that it has a biologically active content. 3,995 / H galactoid-binding mistletoe lectin for administration of 15 to 375 pg / kg body weight. Medicinal product according to claim 8 in the form of a unit dose, characterized in that it has a lectin content for administration of 25 to 200 pg / kg, in particular about 75 pg / kg body weight.