Lyophilized immunocytokine-containing preparation

FIELD: chemical and pharmaceutical industry.

SUBSTANCE: invention relates to lyophilized pharmaceutical immunocytokine composition including immunocytokine and containing as cytokine component interleukin-2 (IL-2), sugar or aminosugar, amino acid and surfactant, wherein said composition contains: immunocytokines from 0.1 to 25 mg/ml; sugar or aminosugar 1-200 mg/ml; amino acid 1-200 mMol/l; and surfactant 0.001-1 mass %.

EFFECT: composition for parantheral administering with prolonged storage time even at increased temperatures.

13 cl, 8 ex, 4 tbl, 2 dwg

 

The present invention relates to stable liofilizirovannom pharmaceutical preparation containing immunocytokine, and to the preparation of a lyophilized pharmaceutical product.

Immunocytokine are conjugates consisting of antibodies and cytokines, in which each carboxyl ends of the two heavy chains of immunoglobulins antibodies associated with the N-terminal parts of the cytokine.

Antibodies are glycoproteins that possess protective action, which are found in the blood, lymph and secretions of the body as a result of immunization with antigens and immediately subjected to the reaction of antigen-antibody. Antibodies belong to the immunoglobulin (Ig) and can be divided into 5 classes: IgA, IgD, IgE, IgG and IgM, some of them, in turn, can be subdivided into additional subclasses (isotypes), such as IgGI, IgG2, IgG3, IgG4, IgA, and IgA2. Immunocytokine include all IgG antibodies. They encompass monoclonal antibodies, polyclonal antibodies and polyspecific antibodies, such as, for example, bespecifically antibodies.

Cytokines are polypeptides that are secreted endocrine or paracrine, that is, are secreted by cells into the blood or into the surrounding tissue and after binding with specific receptors have functional actions (in most of the cases on the division and growth, and also, for example, the movement of other cells. In some cases, the cytokine-producing cells themselves subjected to such effect (i.e. an autocrine). Cytokines, in particular, regulate the complex interaction of cells of the immune system.

Examples of cytokines are lymphokines, Monokini and ordinary polypetide hormones. Cytokines include growth hormones such as human growth hormone, N-nationally the human growth hormone and the growth hormone bovine parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prolactin, glycoprotein hormones such as follicle stimulating hormone (FSH), tireotropin (TSH) and lutropin (LH), growth factor, hepatocyte, fibroblast growth factor, prolactin, placental lactogenic, mouse gonadotropin-associated peptide, inhibin, activin, growth factor vascular endothelial (VEGF), integrin, thrombopoietin (SRW), growth factors, nerve cells, such as NGFβ, platelet growth factor, transforming growth factors (TGF)such as TGFα and TGFβ, erythropoietin (EPO), interferons such as IFNα, IFNβ and IFNγ, hematopoietic growth factors, such as M-CSF, GM-CSF and G-CSF, interleukins (IL)such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, and factors tumor necrosis (TNF)such as TNFαor TNFβ.

Like the aforementioned antibodies and cytokines immune the cytokines are peptide active ingredients, and, therefore, can not be absorbed enterline. Therefore, for therapeutic applications, as a rule, they must be administered parenterally in the form of a solution.

One of the problems with the formulations of medicines on the basis of the solutions containing the peptide active ingredients, is their tendency to aggregation and the formation of protein multimers. However, this problem mainly depends on the physicochemical properties of the concrete used active ingredients. Whereas proteins with hydrophilic properties, have a relatively low tendency to form aggregates in aqueous solutions, proteins having hydrophobic properties, have a high propensity for aggregation.

Antibodies consist of 2 antiparallel folded structures that are similar to "sandwich" with respect to each other (conservative domains). Hydrophobic and hydrophilic amino acids are folded structures with hydrophobic side chains of the two folded structures, which are in each case oriented to each other and, thus, are directed inside the "sandwich"structure and hydrophilic amino acids in each case directed outward (J.Klein, Immunologie [Immunology], Verlag Chemie, Weinheim, 1991). Hydrophilic amino acids, externally directed, participate in the solubilization of antibodies in the same solution and, therefore, prevents the interaction between different antibodies. Therefore, antibodies have low hydrophobicity and propensity for aggregation.

Due to the above described properties is relatively easy to obtain stable solutions of antibodies. One example of a commercially available product is Rituxan®, aqueous preparation containing monoclonal antibody rituximab, inorganic buffer and Polysorbate. Removing water freeze-drying allows to obtain a stable aqueous solutions of antibodies that are already relatively stable per se, and this stability is further enhanced. Before the introduction of the received lyophilizate restore in aqueous solutions by adding water. An example of a product of this type is Remicade®that in addition to the monoclonal antibody infliximab, inorganic buffer and Polysorbate additionally contains sugar as a means of protecting from freezing, or strukturoobrazovatelja.

In the application WO 98/22136 A2 describes a freeze-dried preparation containing the antibody, sugar or amino sugar, an amino acid and a surfactant. Despite the fact that the drug is claimed to antibodies in General, only the drugs, which include monoclonal antibodies directed against hepatitis b virus (AK HBV), and in each case, the drug containing the second antibody to L-selectin (anti-L-selectin) and antibody to a growth factor receptor L nerve cells (anti-L-NGFR), disclosed as working examples.

Cytokines do not contain conservative domains, which can cause a good solubility in water, as is the case for antibodies. So they have a high propensity for aggregation in aqueous solution. This is particularly true for cytokines that contain a bundle of four α-helices as a common structural features (so-called 4 α-spiral beam cytokines) and have a pronounced hydrophobicity due to such a structural feature. Hydrophobic interactions that accompany hydrophobicity, are often, in turn, cause/mechanism of aggregation (Hora-MS and Chen-B, (1999), Bio-pharm. Ind. Perspect., 217-248). Cytokines that contain a bundle of four α-helices as a common structural features, are many interleukins, in particular IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11 and IL-12, interferons, in particular IFNβ and IFNγ, hematopoietic growth factors, such as M-CSF, GM-CSF, G-CSF, erythropoietin (EPO) and the stem cell factor (SCF). Cytokines, which are discussed in detail in the literature described as hydrophobic, are, for example, IL-2 (Robb-RJ and others (1983) PNAS 80:5990-4; US 5580856), IL-4 (Sharma-S and others (1987) 235:1489-92), IL-5 (Takatsu-K and others (1985) L 134:382-9), G-CSF (US 510465).

This strong tendency of cytokines, in particular cytokines with a bunch of 4 α-spirals, requires special conditions is to stabilize them. For example, the product Proleukin®, lyophilized, containing the active ingredient interleukin-2, contains as adjuvants sugar, inorganic buffer and anionic detergent (sodium lauryl sulphate). However, anionic detergents, particularly if the drug is intended for parenteral administration, are extremely questionable from a Toxicological point of view.

Further by example, which indicates the special difficulties in stabilizing drugs, containing cytokines are products on the market that contain interferon β (Avonex®, Betaferon®, Rebif®). All these products are stabilized by albumin, which can also be attributed to extremely critical from a Toxicological point of view, in particular, in connection with unwanted immune reactions.

Due to the above differences between cytokines and antibodies immunocytokine, each of which consists of one antibody and two cytokines, are also quite different physico-chemical properties of antibodies. In particular, immunocytokine containing cytokine, which has four α-spiral beam, have a strong tendency to form agglomerates in aqueous solutions, due to their pronounced hydrophobicity, and difficult to stabilize.

The object on which the present invention is to obtain a stabilized preparation of immunocytokines. The drug should not contain any toxicologically unacceptable adjuvants, it should be stable for a long period of time in conditions of high stress, such as increased temperature and atmospheric humidity, and should be restored with the help of aqueous solvent to obtain a solution, ready for the introduction, having a high content of the active component.

Suddenly it is possible to obtain the product, which meets these requirements, by freeze-drying aqueous buffer solution, which in addition to immunocytokine contains sugar or amino sugar, an amino acid and a surfactant. The present invention thus relates to a stable liofilizirovannom the drug, which contains immunocytokine, sugar or amino sugar, an amino acid and a surfactant.

Preferably the product contains immunocytokine that as a cytokine component contains cytokines selected from the group comprising cytokines, which have as a common structural features of a bunch of 4 α-helices, in particular interleukin, preferably IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11 and/or IL-12, interferon, preferably IFNJ3 and/or IFNy, and/or hematopoietic growth factor, preferably M-CSF, GM-CSF, G-CSF, EPO or SCF. Bole is preferably the composition contains immunocytokine, containing interleukin-2 (IL-2).

The drug in accordance with the invention is a physiologically well-tolerated, can be easily prepared, accurately dosed and is stable under study in respect of the decay products and components during long-term storage and even after repeated processes of freezing and thawing. It is stable when stored for a period of at least three months to two years when stored at refrigerator temperature (2-8° (C) and at room temperature (23-27°C, 60%relative atmospheric humidity (ov)). It has been unexpectedly discovered that the drug in accordance with the invention is also stable when stored for a specified period of time at elevated temperatures and higher atmospheric humidity, for example at a temperature of 40°C and 75%relative humidity.

Dried product can be recovered in a simple way to get ready for the introduction of a solution that does not contain particles by adding an aqueous solvent, for example water, for the purpose of injection or isotonic aqueous solution. The recovered solution is stable over a period of approximately 5 days, but, in particular, more preferably within 24 hours.

The recovery of the drug in compliance and with the invention using an aqueous solvent mainly allows to obtain a drug containing immunocytokine solutions which have a pH value in the range of 5 to 8, preferably those which have a pH value of from 5.6 to 7.4, particularly preferably a pH value of 6-7, and the osmolarity of 250 to 350 mosmol/kg Restored the drug may therefore is injected directly intravenously, intraarterially, as well as subcutaneously without significant pain. In addition, the drug can also be added to the solutions for infusion, such as, for example, a glucose solution, isotonic saline solution or ringer's solution, which can also include additional active ingredients, thus allowing a relatively large amount of the active component.

In accordance with a preferred embodiment of the invention lyophilized pharmaceutical preparation essentially consists of immunocytokine, sugar or amino sugar, amino acids, buffers and surfactants.

The preparation according to the invention allows to obtain the drug solutions immunocytokine, which corresponds to its concentration clinical needs. Preferred are solutions immunocytokine that have a concentration immunocytokine from about 0.1 to 25 mg/ml, more preferably from 1 to 10 mg/ml, most preferably from 1 to 5 mg/ml

Sugar, which is used in the preparation is in accordance with the invention, can be a mono-, di-, or trisaccharide. These sugars may be used alone or in mixture with polyalcohols, xylytol (e.g., mannitol). Examples of monosaccharides which may be mentioned are glucose, mannose, galactose, fructose and sorbose, examples of disaccharides that may be mentioned include sucrose, lactose, maltose or trehalose as an example of trisaccharide that can be mentioned is raffinose. Preferred are sucrose, lactose, maltose or trehalose, especially preferred are sucrose and maltose.

It is also possible presence of amino sugars, i.e. monosaccharides that contain primary, secondary or tertiary amino group or the acylated amino group (-NH-CO-R) instead of the hydroxyl group. For the purposes of the present invention, more preferred are glucosamine, N-methylglucamine, galactosamine and Narimanova acid.

Sugar/amino sugar present in the product in accordance with the invention in such quantity that he was present in the solution obtained after recovery using the volume of solvent in a concentration from approximately 1 to 200 mg/ml Sugar is preferably present in the recovered solution in a concentration of from 15 to 30 mg/ml

Suitable amino acids that are used ACC is accordance with the invention, represent a basic, acidic or neutral amino acids such as arginine, histidine, ornithine, lysine, glycine, among others. It is preferable to use amino acids in the form of their inorganic salts (mainly in the form of salts of hydrochloric acid, that is in the form of hydrochloride amino acids). In the case when using free amino acids, the desired pH value is adjusted by adding a physiologically acceptable portable buffer substances, such as, for example, organic or inorganic acid, such as citric acid and phosphoric acid, sulfuric acid, acetic acid, formic acid, or their salts. Preferred are the citrates and phosphates, which get particularly stable lyophilizate.

Preferred amino acids are arginine, lysine and ornithine. In addition, it also is possible to use acidic amino acids, such as glutamic acid and aspartic acid, or neutral amino acids, such as isoleucine, leucine and alanine, or aromatic amino acids, such as phenylalanine, tyrosine or tryptophan. The amino acid content in the product in accordance with the invention is from 1 to 200 mmol/l, preferably from 40 to 100 mmol/l, particularly preferably 40-80 mmol/l (based the each case on the recovered solution).

Surfactants that can be used are all surface-active substances, which are normally used in pharmaceutical preparations, preferably nonionic surfactants, in particular Polysorbate and polymers of polyoxyethylene-polyoxypropylene. More preferred are esters of polyoxyethylenesorbitan and fatty acids, in particular monolaurate polyoxyethylene(20)sorbitol and monooleate polyoxyethylene(20)sorbitol. In accordance with the invention, the preparation comprises from 0.001 to 1 wt.%, preferably 0.005 to 0.5 wt.% and particularly preferably from 0.01 to 0.15 wt.% (based in each case on the recovered solution).

If the product in accordance with the invention includes buffers, they can, in principle, be any physiologically tolerated substances that are acceptable for establishing the desired pH values. The amount of buffer substance is chosen so that after recovery of lyophilized, for example, using water for injection, the resulting aqueous solution has a concentration of the buffer from 5 to 50 mmol/l, preferably from 10 to 20 mmol/L. the Preferred buffers are citrate buffers or phosphate buffers. Acceptable phosphate buffers are solutions of salts of phosphorous to the slots of mono - and/or disodium and potassium, such as disodium hydrogen phosphate or potassium dihydrophosphate, as well as mixtures of salts of sodium and potassium, such as, for example, a mixture of disodium hydrogen phosphate and potassium dihydrophosphate.

If the recovered solution is not isotonic due to the osmotic properties immunocytokine and as a result used adjuvants for stabilization, isotonic agent, preferably physiologically tolerated salt, such as, for example, sodium chloride or potassium chloride, or a physiologically tolerable polyol or sugar, such as, for example, glucose, glycerol or mannitol may also be present in an amount necessary to establish isotonicity.

In addition, lyophilizate in accordance with the invention may include additional physiologically tolerated adjuvants, such as, for example, antioxidants, such as ascorbic acid or glutathione, preservatives, such as phenol, m-cresol, methyl - or propylparaben, chlorobutanol, thiomersal or benzalkonium chloride, or more stabilizers, builders, solvents, such as glycols (PEG)such as PEG 3000, 3350, 4000 or 6000, or cyclodextrins, such as hydroxypropyl-β-cyclodextrin, sulphobutylether-β-cyclodextrin or γ-cyclodextrin, or dextrans.

The drug in accordance with the invention can be PR is prepared by obtaining water preparation containing immunocytokine as an active ingredient, and a sugar or amino sugar, an amino acid and a surfactant, and, if it is desirable, optional pharmaceutical adjuvants, followed by lyophilization of the solution.

Water preparation can be prepared by adding the indicated adjuvants to the solution containing immunocytokine. To this end, certain uterine volumes of solutions containing these additional adjuvants in certain concentrations, mainly added to a solution having a certain concentration immunocytokine, as obtained from its preparation, and the mixture, if it is desired, diluted to the pre-calculated concentrations of water. Alternative adjuvants can also be added as solids to the original solution containing immunocytokine. If immunocytokine is in the form of solids, for example in the form of a lyophilisate, the product in accordance with the invention can be prepared first by dissolving the appropriate immunocytokine in water or in an aqueous solution containing one or more additional adjuvants, followed by the addition in each case of the amounts of mother solutions that contain additional adjuvants, additional adjuvants in solid form and/the water. Immunocytokine can mainly be dissolved directly in a solution containing any additional adjuvants. One or more adjuvants, which are present in the preparation in accordance with the invention, are mainly to be added during or at the end of the cooking process of the preparation of the private immunocytokine. This is preferably carried out at the final stage of cleaning, which is carried out after its receipt by dissolving immunocytokine directly in an aqueous solution containing one, several or all of these additional adjuvants, or re-tebufelone by suitable methods, such as filtration tangential flow. In order to get the drug, the corresponding(s) additional(s) component(s) (s) need(s)is added in a smaller amount in each case and/or not added at all. Especially preferable for the respective component to be dissolved directly in an aqueous solution, which contains all additional adjuvants, at the final stage of cleaning, which is done after cooking, directly receiving the solution, which is subjected to lyophilization.

The pH value of the solution containing the appropriate immunocytokine and adjuvants, set at the level of 5 to 8, sterile filtration is the situation and dried by freezing.

The obtained dried product can be recovered by adding water to the solvent receiving water preparation, which can be entered directly, in particular, parenteral. The present invention also relates to aqueous pharmaceutical preparation of immunocytokines, which is obtained by restoring the freeze-dried in accordance with the invention, the aqueous solvent.

The recovered aqueous pharmaceutical preparation preferably has a pH in the range of 5-8, preferably pH of 5.6-7.4 and most preferably pH 6,0-7,0.

The examples explain the invention without limiting it.

Example 1 (batch 8020)

Lyophilized from an aqueous solution containing:

0.7 mg/ml EMD 273066 (huKS-IL2)

5 mmol/l citric acid

100 mmol/l arginine HCl

1.5 wt.% sucrose

0.01 wt.% monooleate polyoxyethylene(20)sorbitan (tween 80).

The preparation was carried out by mixing certain amounts of aqueous solutions containing the appropriate adjuvants in certain concentrations. Used the following solutions:

A solution (solution of the active ingredient)containing:

5 mg/ml EMD 273066

5 mmol/l citric acid

100 mmol/l arginine HCl

0.01 wt.% monooleate polyoxyethylene(20)sorbitan (tween 80)

NaOH q.s. to pH 7.0.

Solution (a solution adjuvant):

1,744 mA is.% sucrose

5 mmol/l citric acid

100 mmol/l arginine HCl

0.01 wt.% monooleate polyoxyethylene(20)sorbitan (tween 80)

NaOH q.s. to pH 7.0.

To prepare the drug in accordance with the invention 100 ml of solution a and 614 ml of the solution was combined with each other.

The prepared solution was sterilized by filtration before packing. Vials 6 ml each were filled with 4 ml of solution. Then the vials previously have sealed using plugs and liofilizirovanny. After freeze-drying vials hermetically sealed.

Example 2 (party 8021)

Lyophilized from an aqueous solution containing:

0.7 mg/ml EMD 273066 (huKS-IL2)

5 mmol/l citric acid

100 mmol/l arginine HCl

1.5 wt.% maltose

0.01 wt.% monooleate polyoxyethylene(20)sorbitan (tween 80).

The preparation was carried out by mixing certain amounts of aqueous solutions containing the appropriate adjuvants in certain concentrations. Used the following solutions:

A solution (solution of the active ingredient)containing:

5 mg/ml EMD 273066

5 mmol/l citric acid

100 mmol/l arginine HCl

0.01 wt.% monooleate polyoxyethylene(20)sorbitan (tween 80)

NaOH q.s. to pH 7.0.

Solution (a solution adjuvant):

1,744 wt.% maltose

5 mmol/l citric acid

100 mmol/l arginine HCl

0.01 wt.% monooleate of polyaxial the flax(20)sorbitan (tween 80)

NaOH q.s. to pH 7.0.

To prepare the drug in accordance with the invention 100 ml of solution a and 614 ml of the solution was combined with each other.

The prepared solution was sterilized by filtration before packing. Vials 6 ml each were filled with 4 ml of solution. Then the vials previously have sealed using plugs and liofilizirovanny. After freeze-drying vials hermetically sealed.

Example 3 (party 8431)

Lyophilized from an aqueous solution containing:

1 mg/ml EMD 273066 (huKS-IL2)

5 mmol/l citric acid

100 mmol/l arginine HCl

1.5 wt.% sucrose

0.01 wt.% monooleate polyoxyethylene(20)sorbitan (tween 80).

The preparation was carried out by mixing certain amounts of aqueous solutions containing the appropriate adjuvants in certain concentrations. Used the following solutions:

A solution (solution of the active ingredient)containing:

of 1.45 mg/ml EMD 273066

1.5 wt.% sucrose

5 mmol/l citric acid

NaOH q.s. to pH 7.0.

Solution (a solution adjuvant):

1.5 wt.% sucrose

5 mmol/l citric acid

287 mmol/l arginine HCl

0,0283 wt.% monooleate polyoxyethylene(20)sorbitan (Tween 80)

NaOH q.s. to pH 7.0.

To prepare the drug in accordance with the invention 46,9 ml of solution a and 25.1 ml of the solution was combined with each other.

Prigot is undertaken the solution was sterilized by filtration before packing. Vials 6 ml each were filled with 2 ml of solution. Then the vials previously have sealed using plugs and liofilizirovanny. After freeze-drying vials hermetically sealed.

Example 4 (party 8591)

Lyophilized from an aqueous solution containing:

4 mg/ml EMD 273066 (huKS-IL2)

12.5 mmol/l citric acid

80 mmol/l arginine HCl

1.8 wt.% sucrose

0,008 wt.% monooleate polyoxyethylene(20)sorbitan (tween 80).

The preparation was carried out by mixing certain amounts of aqueous solutions containing the appropriate adjuvants in certain concentrations. Used the following solutions:

A solution (solution of the active ingredient)containing:

5 mg/ml EMD 273066

5 mmol/l citric acid

100 mmol/l arginine HCl

0.01 wt.% monooleate polyoxyethylene(20)sorbitan (tween 80)

NaOH q.s. to a pH of 6.0.

Solution (a solution adjuvant):

to 8.7 wt.% sucrose

41 mmol/l citric acid

NaOH q.s. to a pH of 6.0.

To prepare the drug in accordance with the invention, 4 ml of solution a and 15.5 ml of the solution was combined with each other.

The prepared solution was sterilized by filtration before packing. Vials of 2 ml each were filled with 1 ml of solution. Then the vials previously have sealed using plugs and liofilizirovanny. After freeze-drying LAF the ons hermetically sealed.

Example 5 (comparative product 1 with mannitol instead of sucrose, party 8008)

Lyophilized from an aqueous solution containing:

0.7 mg/ml EMD 273066 (huKS-IL2)

5 mmol/l citric acid

100 mmol/l arginine HCl

4 wt.% mannitol

0.01 wt.% monooleate polyoxyethylene(20)sorbitan (tween 80)

to pH 7.0 using NaOH.

The drug was received directly freeze-drying of a solution of the active component having the above composition.

The resulting solution was filtered using a sterile filter before packing. Vials 6 ml was filled with 4 ml of solution. Then the vials previously have sealed using plugs and liofilizirovanny. After freeze-drying vials hermetically sealed.

Example 6 (comparative example 2, part 8434, corresponds to the composition of the party 8431 without adding arginine)

Lyophilized from an aqueous solution containing:

1 mg/ml EMD 273066 (huKS-IL2)

5 mmol/l citric acid

1.5 wt.% sucrose

0.01 wt.% monooleate polyoxyethylene(20)sorbitan (tween 80).

The preparation was carried out by mixing certain amounts of aqueous solutions containing the appropriate adjuvants in certain concentrations. Used the following solutions:

A solution (solution of the active ingredient)containing:

of 1.45 mg/ml EMD 273066

1.5 wt.% sucrose

5 mmol/l is imoney acid

NaOH q.s. to pH 7.0.

Solution (a solution adjuvant):

1.5 wt.% sucrose

5 mmol/l citric acid

0,0283 wt.% monooleate polyoxyethylene(20)sorbitan (tween 80)

NaOH q.s. to pH 7.0.

To prepare the drug in accordance with the invention, 46,9 ml of solution a and 25.1 ml of the solution was combined with each other.

The prepared solution was sterilized by filtration before packing. Vials 6 ml each were filled with 2 ml of solution. Then the vials previously have sealed using plugs and liofilizirovanny. After freeze-drying vials hermetically sealed.

Example 7 (comparative preparation 3, party 8430, corresponds to the composition of the party 8431 without the addition of tween 80)

Lyophilized from an aqueous solution containing:

1 mg/ml EMD 273066 (huKS-IL2)

5 mmol/l citric acid

100 mmol/l arginine HCl

1.5 wt.% sucrose.

The preparation was carried out by mixing certain amounts of aqueous solutions containing the appropriate adjuvants in certain concentrations. Used the following solutions:

A solution (solution of the active ingredient)containing:

of 1.45 mg/ml EMD 273066

1.5 wt.% sucrose

5 mmol/l citric acid

NaOH q.s. to pH 7.0.

Solution (a solution adjuvant):

1.5 wt.% sucrose

5 mmol/l citric acid

287 mmol/l arginine HCl

NaOH q.s. to pH 7.0.

<> To prepare the drug in accordance with the invention 46,9 ml of solution a and 25.1 ml of the solution was combined with each other.

The prepared solution was sterilized by filtration before packing. Vials 6 ml each were filled with 2 ml of solution. Then the vials previously have sealed using plugs and liofilizirovanny. After freeze-drying vials hermetically sealed.

Example 8 (comparative preparation 4, party 8429, corresponds to the composition of the party 8431 without the addition of tween 80 and without the addition of arginine)

Lyophilized from an aqueous solution containing:

1 mg/ml EMD 273066 (huKS-IL2)

5 mmol/l citric acid

1.5 wt.% sucrose.

The preparation was carried out by mixing certain amounts of aqueous solutions containing the appropriate adjuvants in certain concentrations. Used the following solutions:

A solution (solution of the active ingredient)containing:

of 1.45 mg/ml EMD 273066

1.5 wt.% sucrose

5 mmol/l citric acid

NaOH q.s. to pH 7.0.

Solution (a solution adjuvant):

1.5 wt.% sucrose

5 mmol/l citric acid

NaOH q.s. to pH 7.0.

To prepare the drug in accordance with the invention 46,9 ml of solution a and 25.1 ml of the solution was combined with each other.

The prepared solution was sterilized by filtration before packing. The bottles along the con 6 ml each were filled with 2 ml of solution. Then the vials previously have sealed using plugs and liofilizirovanny. After freeze-drying vials hermetically sealed.

Stability studies of drugs

The stability of the preparations according to the invention was investigated in the stability testing. For this cooked lyophilizate some time kept at different temperatures and investigated using the appropriate analytical methods. Climatic conditions - temperature 40°C and a relative atmospheric humidity (O.V.) 75% were selected as the voltage conditions for the rapid achievement of differences in stability for different drugs. Possible instability of drugs immunocytokines manifested mainly in the formation of aggregates and the formation of degradation products. The decay products and soluble aggregates preferably determined by displacement chromatography based on size (HPLC-SEC), whereas visual observation and turbidity measurement used to determine the visible units. Similarly, the ELISA test used for evaluation of medicines, served to verify the integrity and ability to bind with the receptor. Together with UV photometry at a wavelength of 280 nm it is additionally used to determine the content of substances.

Analytical methods:/p>

Appearance

Cooked preparations were visually examined to determine the presence of luminescent particles and manifestations of possible turbidity.

Protein concentration: A280 nm

The absorbance of the obtained protein solution at a wavelength of 280 nm was used to determine the concentration of the prepared drugs. The extinction coefficient of 1.41 for the active component huKS-IL2 (EMD 273066) was determined by quantitative amino acid analysis. To determine the protein solutions were diluted three times as long as the absorbance of the studied solutions was not in the range between 0.1 and 1.0 (which corresponds to a protein concentration of 0.5 mg/ml) units of absorbance. The absorbance of the studied solutions containing the active ingredient, measured relative to the corresponding comparative solution that does not contain the active component.

Purity: pressure chromatography based on size, SEC-HPLC

Displacement chromatography based on size (SEC) is an analytical method which can be used to determine the purity and the ratio of monomer/aggregate cooked preparations. Components of the investigated solutions were divided on the basis of the size of their molecules using HPLC column with specific pore sizes. Relatively large molecule was loirevalley together with the displacement amount is, while relatively small molecules pass through the pores of the stationary phase is different depending on the size and therefore held more or less firmly. Relatively small molecules, such as decay products huKS-IL2, therefore, had a greater retention time than huKS-IL2 monomers, and in particular, than aggregates of huKS-IL2 who first loirevalley with the column.

The concentration and integrity of the molecules of the active ingredient: KSA-ELISA

In this analytical method (ELISA, enzyme-linked immunosorbent assay), microtiter tablets were coated with a specific antigen for huKS-IL2 (ERSAM or KSA antigen). Molecules huKS-IL2 in the test solution was determined by binding to the antigen component parts-antibodies and, thus, binding to the microtiter plate. After adding biotinylated anti-IL2 antisera, anti-IL2 antibodies interacted with IL2 parts associated molecules huKS-IL2. Excess anti-IL2 molecules were removed by washing microtiter plate. Added conjugate streptavidin-peroxidase was associated with Biotin and oxidized leiko-form tetramethylbenzidine dye (TMB)is added at a later stage, causing staining in blue. The oxidation reaction was stopped after a certain period of time by adding phospho what Noah acid. There is staining solution in a yellow color, which can be quantitatively measured at a wavelength of 450 nm. The protein concentration in the test solutions thus proportional to the absorbance determined at this wavelength.

The results:

The results presented in tables 1 and 2 (party 8020 and 8021) clearly confirm the quality and stability of the prepared drugs. Climatic conditions - temperature 40°C and a relative atmospheric humidity (O.V.) 75% were selected as the voltage conditions for the rapid achievement of differences in stability for different drugs. Unexpectedly the above preparations were stable even at elevated storage temperatures and high relative atmospheric humidity (40°C/75% O.V.) during the period more than 6 months.

The corresponding comparative preparation 1 (table 3, part 8008, example 5), in which the sugar alcohols mannitol was used as an amendment instead of disaccharides (sucrose or maltose), after 4 weeks of storage in appropriate conditions voltage formed aggregates. After storage for 26 weeks at room temperature (25°C, 60% O.V.) also occurred visible units in the respective climatic conditions. Thus, the comparative product 1 may be stored only when cooling in contrast to p is eparate in accordance with the invention.

The product from example 4 (party 8591) is an additional example of the excellent stability of the drug in accordance with the invention and also suggests that the drug may also be used at higher protein concentrations (see table 4). This drug is kept at a temperature of 40°With (75% O.V.) for 14 weeks.

In further experiments investigated whether all adjuvants in the preparations according to the invention is indeed necessary for stabilization. Experimental batch 8431 contained all the components of the drug in accordance with the invention, whereas in the comparative drugs were no separate components:

- the drug in accordance with example 3 (party 8431): contains all the components;

- comparative preparation 2 (example 6, the party 8434): does not contain arginine;

- comparative preparation 3 (example 7, the party 8430): does not contain tween 80;

- comparative preparation 4 (example 8, the party 8429): does not contain tween 80 and arginine.

The results, shown in figure 1, no doubt confirm that adding amino acids essential for the stability of the drug in accordance with the invention. According to this figure while adding tween 80 does not seem necessary, it is not quite true. Tween 80 is added initially, and for cleaning of the protein p and the preparation of solutions of active ingredients to prevent, in particular, visible aggregates. Applied concentration of tween 80 is above the critical micellar concentration (KMC for tween 80 (0.001%). Experimental attempts were made to remove as arginine and twin by the so-called filtering method in a tangential flow. When this buffer exchange was carried out by diafiltration through the membrane 50 kDa. However, micelles of tween 80 in some cases exceed the limit of displacement of the membrane and, thus, cannot be completely removed.

The addition of tween 80 to the drug in accordance with the invention is essential to the re-dissolution of liofilizatow and for the stability of the solution obtained after recovery, which is confirmed by further research. Based on the solution of the active component EMD 273066 (huKS-IL2)containing tween 80, adjuvant tween 80 was separated by affinity chromatography using a protein a column A. tween 80 was added in higher amounts to the obtained solution, not containing tween 80. The resultant preparations were subjected to stress during the period of 21 days at a temperature of 25°in vials of 2 ml using a laboratory shaker. Drugs, subjected to tension, daily analyzed visually and photometrically to determine them in the concentration of the emission of the protein. The results of this study shown in figure 2. For drugs without the addition of tween 80, a slight turbidity after one day, which markedly enhanced by visual observation. After 21 days it is also observed a significant decrease in the protein content, as shown in the figure.

Table 1

The stability of the drug 8020 in accordance with example 1
Example 1
Storage time (weeks)Determination of proteinHPLC-SECKSA-ELISAThe appearance of the freeze-dried after the dissolution of 4.0 ml bidest. waterTurbidity is determined by photometry A
[week][mg/ml][%][mg/ml]dissolved
The original solution0,79896,830,870-0,0036
Lyophilized after recip.0,84996,680,857transparent p-p, slightly opalescent0,0033
FREEZING

Store at -20°
4-6,72 -Transparent colourless solution0,0019
8-96,89---
13-97,02-Transparent colourless solution-
260,80897,380,685Transparent colourless solution0,0021
REFRIGERATOR

Store at 2-8°
4-96,70-Transparent colourless solution0,0015
8-97,01---
13-97,01-Transparent colourless solution-
260,80697,250,735Transparent colourless solution0,0011
Store at 25°C/60% ov
4-96,66-Transparent colourless solution0,0011
8-97,060,757--
13 -96,93-Transparent colourless solution-
260,80997,23determined as 0.720Transparent colourless solution0,0017
Storage at 40°C/75% ov
4-RUB 96.62-transparent colourless solution0,0013
8-97,000,827--
13-96,99-transparent colourless solution-
260,81697,190,775Transparent colourless solution0,0007

Table 2

The stability of the drug 8021 in accordance with example 2
Example 2
Storage time (weeks)Determination of proteinHPLC-SECKSA-ELISAThe appearance of the freeze-dried after the dissolution of 4.0 ml bidest. WaterTurbidity is determined by photometry A
[week][mg/ml][] [mg/ml]Dissolved
The original solution0,81096,870,865-0,0042
Lyophilized after recip.0,86896,700,817A clear solution0,0034
FREEZING

Store at -20°
4-97,12-Transparent colourless solution0,0013
8-97,19---
13-97,11-Transparent colourless solution-
26-----
REFRIGERATOR

Store at 2-8°
4-97,27-Transparent colourless solution0,0018
8-97,07---
13-97,07-Transparent colourless solution-
26 ----
Store at 25°C/60% ov
4-97,27-Transparent colourless solution0,0015
8-97,140,753--
13-96,96-transparent colourless solution-
26-----
Storage at 40°C/75% ov
4-97,17-transparent colourless solution0,0013
8-97,210,820--
13-96,91-transparent colourless solution-
26-

-
Table 3

The comparative stability of the drug 1 (party 8008 in accordance with example 5)
Party: 00808
Storage time (weeks)Determination of proteinHPLC-SECKSA-ELISAThe appearance of the freeze-dried after the dissolution of 4.0 ml bidest. WaterTurbidity is determined by photometry A
[week][mg/ml][%][mg/ml]Dissolved
Lyophilized after recip.0,76394,790,667a clear solution0,0033
FREEZING

Store at -20°
4-----
8-----
21-96,63-transparent colourless solution0,0035
260,76696,16-transparent colourless solution0,0040
Refrigerator

Store at 2-8°
4---transparent colourless solution-
8----
21-96,42-transparent colourless solution0,0037
260,80695,74-transparent colourless solution0,0043
Store at 25°C/60% ov
4---transparent colourless solution-
8--transparent colourless solution-
21-91,34-Slightly hopeless. transparent p-p0,0064
26-81,33-Slightly turbid whitish solution0,0209
Storage at 40°C/75% ov
4---A cloudy solution-
8---A cloudy solution-
13---A cloudy solution-
26- --A cloudy solution-

Table 4

The stability of the preparation in accordance with example 4 (party 8591)
Example 4
Storage time (weeks)Determination of proteinHPLC-SECKSA-ELISAThe appearance of the freeze-dried after the dissolution of 4.0 ml bidest. waterTurbidity is determined by photometry A
[week][mg/ml][%][mg/ml]dissolved
Lyophilized after recip.0,84996,14-transparent colourless solution-
Storage at 40°C/75% ov
14-94,424.13transparent colourless solution-

1. Lyophilized pharmaceutical composition of immunocytokines, including immunocytokine containing as a cytokine component interleukin-2 (IL-2), a sugar or amino sugar, an amino acid and a surfactant, and it can be recovered in the form of an aqueous FA the pharmaceutical composition, containing from 0.1 to 25 mg/ml immunocytokine, from 1 to 200 mg/ml sugar/amino sugar, 1-200 mmol/l amino acids and 0.001-1 wt.% surface-active substances.

2. Lyophilized pharmaceutical composition according to claim 1, characterized in that it essentially consists of immunocytokine, sugar or amino sugar, amino acids, buffers and surfactants.

3. Lyophilized pharmaceutical composition according to claim 1 and/or 2, characterized in that the sugar is a mono-, di - or trisaccharide, preferably sucrose, lactose, maltrose or trehalose

4. Lyophilized pharmaceutical composition according to claim 1 and/or 2, characterized in that the amino sugar is a glucosamine, N-methylglucamine, galactosamine or Narimanovo acid.

5. Lyophilized pharmaceutical composition according to claim 1 and/or 2, characterized in that the amino acid is a basic, acidic or neutral amino acid, preferably arginine, lysine or ornithine.

6. Lyophilized pharmaceutical composition according to claim 1 and/or 2, characterized in that the surfactant is a nonionic surfactant.

7. Lyophilized pharmaceutical composition according to claim 6, characterized in that the surfactant is a Polysorbate or a polymer of polyaxial the Lena-polyoxypropylene.

8. Lyophilized pharmaceutical composition according to claim 7, characterized in that the surfactant is an ester of polyoxyethylenesorbitan and fatty acid monooleate polyoxyethylene(20)sorbitol or monolaurate polyoxyethylene(20)sorbitol.

9. Lyophilized pharmaceutical composition according to claim 1 and/or 2, characterized in that it further comprises an isotonic agent in the amount necessary to establish isotonicity.

10. Aqueous pharmaceutical composition of immunocytokines, resulting from the recovery of freeze-dried for one or more of claims 1 to 9 with an aqueous solvent.

11. Aqueous pharmaceutical composition of claim 10, wherein the solution has a pH value of 5-8, preferably about 5.6 to 7.4.

12. Aqueous pharmaceutical composition according to claim 11, characterized in that the solution has a pH value of 6-7.

13. A method of obtaining a lyophilized pharmaceutical composition according to one or more of claims 1 to 9, characterized in that the prepared aqueous composition comprising immunocytokine, sugar or amino sugar, an amino acid, a surfactant and, if it is desirable, optional pharmaceutical adjuvants, and then the solution is subjected to lyophilization.



 

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