Stable galenic freeze-dried pharmaceutical preparation of recombinant carbohydrate-binding polypeptides

This application is a national stage filing under 35 U.S.C. 371 of PCT application PCT/EP02/11093, filed Oct. 2, 2002, which claims the benefit of priority from German Application No. 101 49 030.5, filed Oct. 5, 2001, the specifications of each of which are incorporated by reference herein in their entirety. PCT Application PCT/EP02/11093 was published under PCT Article 21(2) in German.

The invention relates to a method for the production of a medicament containing a polypeptide, comprising at least one recombinant carbohydrate-binding polypeptide or a functional fragment or derivative of said carbohydrate-binding polypeptide in a form stable for storage. Said polypeptide comprises polypeptides or functional derivatives thereof fused to cytotoxically effective peptides to give fusion proteins or which are connected to a further polypeptide having cytotoxic activity. The invention further relates to the further formulation of the disclosed medicaments to give medicaments of various dosage forms.

In recent years, medicinal research has uncovered a broad spectrum of diseases that can be treated with recombinant proteins. Examples of proteins of human origin are insulin, EPO and G-CSF the dosage forms and kinds of application of which have been described in various European patents. EP 0 430 200 B1 describes the application of human proteins for subcutaneous and intramuscular administration. Medicaments with stabilised human. proteins which contain, amongst others, urea or different amino acids, are known from EP 0 306 824 B1. In this patent, EPO and G-CSF are given as examples. EP 0 607 156 B1 describes the production of conserved medicaments with human proteins for infusion or injection purposes.

In general, the term “recombinant” refers to proteins which are prepared using the recombinant DNA technique. These methods comprise cloning of the gene encoding the protein in question, inserting corresponding cDNA or genomic DNA in a suitable vector system and transforming/transfecting said vectors in suitable host organisms (bacteria or eukaryotic cells). If the cloned gene is expressed in the host organism, the corresponding protein can be recovered from the culture supernatant (if the protein expressed is secreted) or from a homogenate of the host organism (if the corresponding protein is expressed in an intracellular manner). Methods for producing recombinant proteins have been described for both animal and plant proteins. An example of the exact procedure for producing a dimeric plant protein is described in EP 0 751 221 B1. This patent describes, amongst others, the unprecedented successful cloning of the genes encoding the ML-subunits. Furthermore, in this patent, the use of said dimeric plant proteins produced recombinantly for the preparation of medicaments is described, too.

The use of mistletoe extracts (extracts of Viscum album) as a curative has been known for centuries. Active ingredients called lectins have been identified as effective components of these extracts. These lectins are proteins which recognise very specific carbohydrate structures also in lipid- or protein-bound form and which bind thereto. Mistletoe lectin which has been characterised as ribosome-inactivating class II protein is pharmacologically effective by the interplay of its two subunits. The B-chain of the mistletoe lectin which has sequence motifs with specific carbohydrate-binding properties is, in this case, responsible for the transport of the protein to the target cell. In the target cell, the A-subunit then blocks the ribosomal metabolism of the cell due to its enzymatic rRNA-N-glycosidase activity and, in this way, it triggers a programmed cell death (apoptosis) in said cell.

The pharmaceutical preparations which have so far been known from the state of the art generally contain human proteins, humanised proteins, extracts containing plant proteins or proteins isolated from plants. It is decisive for the effectiveness of preparations containing proteins that the biological activity of said proteins is maintained. For rViscumin, it is, for example, the dimeric structure and the activities that are to be attributed to the single chains and the specific pharmacological mode of action of said molecules that are to be maintained. Maintaining these biological activities strongly depends on the pH of the solution containing the proteins (cf. FIG. 1). Furthermore, storage conditions of the preparation in question influence the stability of a drug/medicament.

The mode of action of the mistletoe plant and the extracts obtained therefrom for treating diseases has been described in European patent EP 0 602 686 B1. As explained in this specification, mistletoe extracts have been used for therapeutic purposes for centuries. Since the beginning of this century, mistletoe preparations are used in cancer therapy with varying success (Bocci, 1993; Gabius et al., Gabius & Gabius, 1994; Ganguly & Das, 1994). Hajto et al. (1989, 1990) were able to show that the therapeutic effects are mediated in particular by so-called mistletoe lectins (viscumins, Viscum album agglutinins, VAA). Apart from a cytotoxic effect, nowadays, in particular an (unspecific) immunostimulation is discussed, the positive effects of which are utilised for an accompanying therapy and for aftercare of tumour patients. An increase in the life quality of such patients is possibly mediated by the release of enogenous endorphins (Heiny and Beuth, 1994).

Numerous tests in vitro (Hajto et al., 1990; Männel et al., 1991; Beuth et al., 1993a) and in vivo (Hajto, 1986; Hajto et al., 1989; Beuth et al., 1991; Beuth et al., 1992), as well as clinical studies (Beuth et al., 1992) prove the increased release of inflammatory cytokines (TNF-α, IL-1, IL-6) mediated by mistletoe lectin as well as an activation of cellular components of the immune system (TH cells, NK cells).

Today a 60 kDa mistletoe lectin protein is considered an active principle of the mistletoe extracts, wherein the mistletoe lectin can be recovered from extracts in a biochemical manner Franz et al., 1977; Gabius et al., 1992). The ML protein consists of two covalently S-S-coupled subunits, the A-chain of which is responsible for an enzymatic inactivation of ribosomes (Endo et al., 1988) and the B-chain of which is responsible for the carbohydrate binding. The biological activity is correlated with obtaining the lectin activity of the B-chain (Hajto et al., 1990).

The use of a form of the medicament or a pharmaceutical preparation with rViscumin as an active component is an interesting an advantageous alternative for a plant preparation as it is now possible to use a chemically classified substance as a medicament. It is with regard to the high toxicity of the mistletoe lectin that the use of recombinantly produced proteins makes a good tolerance possible thanks to exact dosage. In this case, a form of the medicament or a pharmaceutical preparation is of particular advantage which is storage-stable over a long period of time, i.e. several months and preferably at least one year. Storage of the form of the medicament or the pharmaceutical preparation in said storage-stable form should moreover be simple and should be possible without sophisticated technology. Furthermore, it should be possible to simply further formulate the form of the medicament or the pharmaceutical preparation to a corresponding dosage form if its storage-stable form is not the same as its dosage form. With aqueous formulations according to the state of the art, storage periods of less than 10 weeks (2.5 months) are realistic under storage conditions of 2-8° C. (fridge).

Therefore, the technical problem underlying the present invention was to provide a method for producing a medicament or pharmaceutical preparation in a stable form for long-term storage which guarantees simple use both with regard to storage and administration and, optionally, its preparation. In this case, the medicament of the invention is to comprise at least one recombinant carbohydrate-binding polypeptide or a functional fragment or derivative of said polypeptide, furthermore, optionally, containing a pharmacologically acceptable carrier.

This technical problem is solved by the embodiments characterised in the claims.

As a consequence, the present invention relates to a method for producing a medicament containing a polypeptide comprising at least one recombinant carbohydrate-binding polypeptide or functional fragment or derivative of said polypeptide in a form stable for long-time storage, moreover, optionally, containing a pharmaceutically acceptable carrier comprising the step of cooling, freezing, spray drying or lyophilising while retaining the pharmacological properties of the polypeptide in the solution, wherein the solution is characterised in that the pH value of the solution is higher than pH 6.0 and a buffer system contained in the solvent guarantees that this pH-value is maintained.

Starting from the corresponding cloned genes, recombinant polypeptides and proteins can be presented using conventional methods of molecular biology. Amongst others, these have been described in the textbook “Gentechnologie” (Old and Primrose, 1992) or in the laboratory manuals “Methods for General and Molecular Bacteriology” (Gerhardt et al., Chapter 18) or “Molecular Cloning: A Laboratory Manual” (Sambrook et al. 1993).

In accordance with the invention, a “carbohydrate-binding polypeptide” is a polypeptide which has the property that it specifically binds to certain carbohydrates. Examples of such carbohydrates are galactose, N-acetyl-galactosamine, modified galactose, neuraminic acids, low-molecular saccharides and oligosaccharides with terminal galactose and/or terminal galactosamine moieties or modified galactose moieties or terminal neuraminic acid moieties, and peptides and lipids having a corresponding carbohydrate function. “Functional fragments or derivatives of said polypeptide” of the invention are characterised in that they also have a specificity for binding to the above-mentioned carbohydrates.

The use of the polypeptides of the invention, such as e.g. rViscumin and other plant, dimeric class II polypeptides of ribosome-inactivating proteins (RIP II) for producing highly effective medicaments has been described amongst others in EP 0 751 221 B1. However, said medicaments are preferred to be administered one year after preparation at most.

Within the meaning of the invention, a medicament or pharmaceutical preparation is considered storage-stable if it can be stored over al long period of time, i.e. several months, that is more than six months, without a significant change of the specific properties of the pharmaceutical preparation and the polypeptide and, therefore, the effectiveness of said medicament or preparation being observed. In this context, a storage-stable form of the medicaments or pharmaceutical preparations according to the invention, which are stored over a period for 1, 2, 3, 4 or 5 years, is preferred. Preferably they can be stored under storage conditions that are common in the market and to be adhered to by distributors and applicants (2-8° C. and/or ambient temperature below 25° C.) without a significant change in the specific properties of the pharmaceutical preparation and the polypeptide and, therefore, the effectiveness of said medicament or preparation being observed. Thus, the invention relates to storage and transport forms of the polypeptides described herein, which are very well manageable.

The formulation of the medicament of the invention is optionally effected in combination with a “pharmacologically acceptable carrier” and/or diluent. Examples of particularly suitable pharmacologically acceptable carriers are known to those skilled in the art and comprise buffered saline solutions, water, emulsions such as e.g. oil/water emulsions, various kinds of detergents, sterile solutions, etc. Medicaments that comprise such carriers can be formulated using known conventional techniques. These medicaments can be administered to an individual in a suitable dose. Administration can be effected orally or parentally, e.g. intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, intranasally, intrabronchially or intradermally or via a catheter at a site in an artery. The kind of dosage is determined by the attending physician in accordance with the clinical factors. The person skilled in the art knows that the kind of dosage depends on various factors such as, e.g. the patient\'s height or weight, body surface, age, sex or general health, but also on the particular agent to be administered, the duration and kind of administration and on other medicaments which are possibly administered in parallel. A typical dose can, for instance, range from 0.001 to 1000 μg wherein doses below or above this exemplary range are thinkable, in particular when the aforementioned factors are taken into consideration. In general, if the composition of the invention is administered regularly, the dose should range from 10 ng to 10 mg units per day or per application interval. If the composition is to be administered intravenously, the dose should range from 1 ng and 0.1 mg units per kilogram body weight per minute.

The composition of the invention can be administered locally or systemically. Preparations for parenteral administration comprise sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, plant oils such as, e.g. olive oil, and organic ester compounds such as, e.g. ethyloleate, which are suitable for injections. Aqueous carriers include water, alcoholic-aqueous solutions, emulsions, suspensions, salt solutions and buffered media. Parenteral carriers comprise sodium chloride solutions, Ringer\'s dextrose, dextrose and sodium chloride, Ringer\'s lactate and bound oils. Intravenous carriers comprise, e.g. fluid supplements, nutrient supplements and electrolyte supplements (such as, e.g. those based on Ringer\'s dextrose). Furthermore, the composition of the invention can comprise preservatives and other additives such as, e.g. anti-microbial compounds, antioxidants, complex-forming agents and inert gasses. Furthermore, depending on the intended use, compounds such as, e.g. interleukins, growth factors, differentiation factors, interferons, chemotactic proteins or an unspecific immunomodulary agent.

The buffer substances used are suitable to maintain the adjusted pH within the ranges described during the phase of cooling, freezing, spray drying or lyophilising. The buffer substances are preferred to be selected in such a way that, with a low buffer capacity, it is not possible for the pH to change to lower values during freezing. By maintaining a high pH range during lyophilisation, the stability of the polypeptide is guaranteed. A low buffer capacity is moreover preferred for an injection solution ready for application. In Example 1, a method for checking the pH during cooling or freezing of pharmaceutical preparations is described. By means of this or similar methods, buffer substances can be determined which are suitable for the method of the invention.

In the state of the art, a plurality of medicaments are described which contain low-molecular, oligomeric compounds (including peptides) and high-molecular compounds (including polypeptides) in buffered solutions. In addition, for a plurality of such medicaments which contain corresponding compounds that are stable in a wide pH range methods for improving the storage properties have been described and are known to the skilled person. Examples thereof are methods comprising freezing, spray drying or lyophilising of medicaments. Due to said pH-independent stability, it has so far not been described that a specific control of the pH during lyophilising or spray drying was necessary. Moreover, conventional lyophilisation devices for producing medicaments und pharmaceutical preparations have not been supplied with means for controlling the pH.