KR101191536B1 - Stabilized interferon liquid formulations - Google Patents

Abstract

Stabilized HSA-free pharmaceutical compositions are described, wherein the composition comprises interferon (IFN), and the formulation is a solution comprising buffer, amino acid and antioxidant. Preferably the interferon is human recombinant IFN-beta.

Interferon, multiple sclerosis, HSA

Description

Stabilized interferon liquid formulations

The present invention relates to HSA-free pharmaceutical compositions comprising interferon, in particular formulations of interferon-beta comprising buffers, amino acids and antioxidants.

Interferon is a cytokine, a soluble protein that plays an important role in the immune system by delivering messages between cells, helping to destroy infectious microorganisms and healing wounds that have occurred. Interferon is naturally secreted by infected cells and was first discovered in 1957. Their name derives from the fact that they "interfere" to virus replication and production.

Interferon exhibits antiviral and antiproliferative activity. On the basis of biochemical and immunological properties, naturally occurring human interferons are classified into three major categories: interferon-alpha (leukocyte: leukocyte), interferon-beta (fibroblast) and interferon-gamma (immune: immune). . Alpha-interferon is currently used in the US and other countries for hairy cell leukemia, venereal warts, Kaposi's Sarcoma) (a cancer that commonly plagues patients suffering from AIDS), chronic non-A -Approved for the treatment of chronic non-A (non-B hepatitis).

In addition, interferons (IFNs) are glycoproteins produced by the body in response to viral infections. They inhibit the growth of the virus in protected cells. IFNs composed of low molecular weight proteins are very nonspecific in their action. In other words, IFN induced by one virus is effective against a wide range of other viruses. But they are species-specific. That is, IFN induced by one species stimulates antiviral activity only in cells of the same or highly related species. IFNs were the first population of cytokines used for their potential anti-tumor and antiviral activity.

The major IFNs are represented by IFN-α, IFN-β and IFN-γ. Such major types of IFNs were initially classified according to their cellular origin (white blood cells, fibroblasts or T cells). However, it became clear that different kinds could be produced in one cell. Therefore, leukocyte IFN is currently IFN-α, fibroblast IFN is IFN-β and T cell IFN is IFN-γ. There is a fourth type of IFN, which is produced in the "Namalwa" cell line (from Burkitt's lymphoma) and produces a mixture of leukocytes and fibroblast IFN. see.

The international unit or interferon unit (U or IU, international unit) for interferon has been reported as a measure of IFN activity defined in the amount necessary for 50% protection of cells against viral damage. Assays that can be used to measure bioactivity are assays for inhibition of cytopathic effects as described (Rubinstein, et al. 1981; Familetti, P. C. et al., 1981). In the antiviral assay for interferon, about 1 unit / ml of interferon is the amount necessary to give a cytopathic effect of 50%. The unit is determined in terms of the international reference standard for Hu-IFN-beta provided by the National Institutes of Health (Pestka S. 1986) (Pestka S. 1986).

All classes of IFNs contain several distinct types. IFN-β and IFN-γ are each a single gene product.

 Proteins classified as IFN-α are the most diverse population and include about 15 types. At least 23 members, 15 of which are active, transcribed, and a cluster of IFN-α genes on chromosome 9. Mature IFN-α is not glycosylated.

 IFN-α and IFN-β are both the same length (165 or 166 amino acids) with similar biological activity. IFN-γ is 146 amino acids long and resembles less closely to the α and β classes. Only IFN- [gamma] activates macrophage or induces maturation of killer T cells. These new types of therapies are sometimes called biologic response modifiers (BRMs). Because they influence cognition through immunomodulation, they affect the response of organisms to tumors.

Human fibroblast interferon (IFN-β) has antiviral activity and can also stimulate natural killer cells against neoplastic cells. It is a polypeptide of about 20,000 Da that is induced by a virus and is double-stranded RNA. By recombinant DNA technology (Derynk et al. 1980), the amino acid sequence of the protein was derived from the nucleotide sequence of the cloned fibroblast interferon gene. It is 166 amino acids long.

Shepard et al. (1981) described several clones with mutations at base 842 (Cys-> Tyr at position 141) that eliminated antiviral activity and deletions of nucleotides 1119-1121.

Mark et al. (1984) substituted an artificial mutation by replacing base 469 (T) with (A) and replacing the amino acid at position 17 with Cys-> Ser. The resulting IFN-β was reported to be as active as 'natural IFN-β' and reported to be stable during long term storage (-70 ° C).

Levy profiles ^® (Rebif ^®) (Serono- recombinant human Interferon -β) is multiple sclerosis (multiple sclerosis: MS) interferon (IFN) generated by the recent development in interferon therapy for treatment from a mammalian cell line - a beta -1a. The recommended International Non-proprietary Name (INN) is "Interferon-beta-1a".

As with all protein-based pharmaceuticals, the major obstacle to overcome in using IFN-beta as a therapeutic agent is the loss of pharmaceutical utility, i.e. loss due to instability in pharmaceutical formulations.

Physical instability threatening polypeptide activity and efficacy in pharmaceutical formulations is the denaturation and formation of soluble and insoluble aggregates, while chemical instability is hydrolysis, imide formation, oxidation, racemization , And deamidation. Some of these changes are known to cause loss or reduction in the pharmaceutical activity of important proteins. In other cases, the exact effect of these changes is unknown, but the low quality product obtained is still considered to be pharmaceutically unacceptable due to the potential for undesirable side effects.

Stabilization of polypeptides in pharmaceutical compositions remains an area where trial and error play an important role (reviewed by Wang (1999) Int. J. Pharm. 185: 129-188; Wang and Hanson (1988) J. Parenteral Sci. Tech. 42: S3-S26). Excipients added to polypeptide pharmaceutical formulations to increase stability include buffers, sugars, surfactants, amino acids, polyethylene glycols, and polymers, but the stabilizing effects of these chemical additives vary with the type of protein.

In US 2002/0172661, IFN beta formulations are described as characterized by a low ionic strength and a pH of about 3.0-5.0.

Currently, IFN-beta formulations use HSA as a solubility enhancer of IFN-beta. However, the use of HSA has some disadvantages. HSA is a product of human blood and must be cultured from the human body. Although steps are taken to reduce risk, the use of human blood products such as HSA results in the potential introduction of human viruses such as HIV and HCV.

As a result, there is a need for additional IFN-beta pharmaceutical compositions that can enhance their pharmacological utility, including physiologically acceptable stabilizers for improving the solubility of these proteins and stabilizing the protein against aggregate formation.

The present invention relates to a stabilized pharmaceutical composition comprising interferon (IFN) and a method of making the same. These compositions are prepared without human serum albumin (HSA). Such compositions are referred to hereinafter as "HSA-free". IFN HSA-free pharmaceutical compositions comprise interferon (IFN) or isoforms, muteins, fused proteins, functional derivatives, active fractions or salts thereof, wherein the composition comprises buffers, amino acids and It is a solution containing antioxidants.

According to one embodiment of the invention, the composition also comprises a bacteriostatic agent.

As used herein, "interferon" or "IFN" is intended to include molecules defined in the literature and includes, for example, any kind of IFNs mentioned in "Background of the Invention" above. In particular, IFN-α, IFN-β and IFN-γ are included in the above definitions. IFN-β suitable in the present invention are commercially available as, and for example, Levy program ^® (Serono), AVONEX ^® can be used:: (Schering Betaferon) (Avonex Biogen) or bete Peron ^®. The use of human origin interferon is also preferred according to the invention. As used herein, the term interferon is intended to include isoforms, muteins, fusion proteins, functional derivatives, active fractions or salts thereof.

As used herein, the term “interferon-beta (IFN-beta or IFN-β)” is a salt thereof, as well as a fibroblast interferon of human origin, obtained by isolation from biological fluids or by DNA recombination techniques from eukaryotic or prokaryotic host cells. It is meant to include functional derivatives, variants, analogs and active fractions. Preferred IFN-beta is intended to mean interferon beta-1a.

As used herein, the term "mutein" refers to the substitution of one or more amino acid residues of a natural IFN with another amino acid residue or deletion, or the addition of one or more amino acid residues to the natural sequence of an IFN, resulting in greater activity of the resulting product compared to wild type IFN. Means analogs of unchanged IFNs. These muteins are prepared by known synthesis and / or site-directed mutagenesis techniques, or by other known suitable techniques. Preferred muteins include, for example, those described by Shepard et al. (1981) or Mark et al. (1984).

Such muteins have an amino acid sequence capable of sufficiently replicating a sequence of IFNs to have substantially similar or better activity against IFNs. The biological function of interferon is well known to those skilled in the art and biological standards have been established and available from the National Institute for Biological Standards and Control (http://immunology.org/links/NIBSC).

Biological assays for determining INF activity have been described. IFN assays have been described, for example, by Rubinstein et al. (1981). Therefore, this can determine whether a given mutein has a similar or better activity substantially to that of IFN by the general experimental method.

The muteins of IFNs, or nucleic acids encoding them, which can be used in accordance with the present invention are based on the techniques and information presented herein without undue experimentation, and are a finite set as substitution peptides or polynucleotides that one skilled in the art can routinely obtain. (finite set) includes a substantially corresponding sequence.

A preferred change of the mutein according to the invention is what is known as a "conservative substitution". Conservative amino acid substitutions of the polypeptides or proteins of the invention include synonymous amino acids in a population that have sufficiently similar physicochemical properties that substitutions between members of the population preserve the biological function of the molecule. The insertion and deletion of amino acids is without altering their function, in particular if the insertion or deletion consists of only a few amino acids, for example 30 or less, preferably 10 or less, and the removal of amino acids (for example cysteine residues) If there is no replacement, it is clear that it can be made within the above-defined sequence. Proteins and muteins produced by such deletions and / or insertions are within the scope of the present invention.

Preferably, said amino acid populations are defined in Table 1. More preferably the amino acid population is defined in Table 2; And the most preferred synonymous amino acid populations are defined in Table 3.

Preferred Motion Amino Acid Group amino acid        Motion amino acids Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, His Leu        Ile, Phe, Tyr, Met, Val, Leu Pro        Gly, Ala, Thr, Pro Thr Pro, Ser, Ala, Gly, His, Gln, Thr Ala        Gly, Thr, Pro, Ala Val        Met, Tyr, Phe, Ile, Leu, Val Gly        Ala, Thr, Pro, Ser, Gly Ile        Met, Tyr, Phe, Val, Leu, Ile Phe        Trp, Met, Tyr, Ile, Val, Leu, Phe Tyr        Trp, Met, Phe, Ile, Val, Leu, Tyr Cys        Ser, Thr, Cys His        Glu, Lys, Gln, Thr, Arg, His Gln        Glu, Lys, Asn, His, Thr, Arg, Gln Asn        Gln, Asp, Ser, Asn Lys        Glu, Gln, His, Arg, Lys Asp        Glu, Asn, Asp Glu        Asp, Lys, Asn, Gln, His, Arg, Glu Met        Phe, Ile, Val, Leu, Met Trp        Trp

More preferred group of amino acids  amino acid        Motion amino acids Ser        Ser Arg        His, Lys, Arg Leu        Leu, Ile, Phe, Met Pro        Ala, Pro Thr Thr Ala Pro, Ala Val        Val, Met, Ile Gly        Gly Ile        Ile, Met, Phe, Val, Leu Phe        Met, Tyr, Ile, Leu, Phe Tyr Phe, Tyr Cys        Cys, Ser His        His, Gln, Arg Gln        Glu, Gln, His Asn        Asp, Asn Lys        Lys, Arg Asp        Asp, Asn Glu        Glu, Gln Met        Met, Phe, Ile, Val, Leu Trp Trp

Most preferred group of amino acids amino acid Motion amino acids Ser Ser Arg Arg Leu Leu, Ile, Met Pro Pro Thr Thr Ala Ala Val Val Gly Gly Ile Ile, Met, Leu Phe Phe Tyr Tyr Cys Cys, Ser His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Met Met, Ile, Leu Trp Met

Examples of products of amino acid substituents in proteins that can be used to obtain muteins of IFNs for use in the present invention include US Pat. Nos. 4,959,314, 4,588,585 and 4,737,462 to Mark et al .; No. 5,116,943 to Koths et al: et al., 4,965,195 to Namen et al; 4,879,111 to Chong et al; And Lee et al. 5,017,691; And the lysine substituted proteins set forth in US Pat. No. 4,904,584 to Shaw et al. Specific muteins of INF-beta have been described, for example, by Mark (1984) and the like.

The term "fused protein" refers to a polypeptide comprising IFN, or a mutein thereof, fused to another protein, eg, another protein having an extended residence time in the blood. IFNs can therefore be fused to other proteins, polypeptide classes such as immunoglobulins or fragments thereof.

As used herein, “functional derivatives” include derivatives of IFN, their muteins and fused proteins, which can be prepared from functional groups which occur in the known art as N- or C-terminal groups or as side chains on residues, Pharmaceutically acceptable, ie included in the present invention, as long as they do not destroy the activity of proteins substantially similar to IFN activity and impart toxic properties to the composition comprising the same. These derivatives include, for example, polyethylene glycol side-chains, which mask antigenic sites and prolong the retention of IFN in the blood. Other derivatives of the free amino groups of amino acid residues formed with amides, acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) of carboxyl groups which react with aliphatic esters of carboxyl groups, ammonia or primary or secondary amines O-acyl derivatives of free hydroxyl groups (eg of seryl or threonyl residues) formed with N-acyl derivatives or acyl moieties.

As an "active fraction", or mutein, and fused protein of IFNs, the invention relates to protein molecules alone or related molecules or residues linked thereto, such as sugar or phosphate residues, or proteins thereof or Aggregates of sugar residues, fragments of all polypeptide chains or fragments of precursors, which must satisfy the condition that the fractions do not have a significant decrease in activity compared to the corresponding IFN.

The term "salt" refers to salts of carboxyl groups and acid addition salts of amino groups of the above-described proteins or analogs thereof. Salts of carboxyl groups can be formed by known techniques and include inorganic salts, for example inorganic salts such as sodium, calcium, ammonium, iron or zinc salts, and amines such as triethanolamine, arginine or lysine, piperidine And salts of organic bases formed from procaine and the like. Acid addition salts include, for example, salts of mineral acids such as hydrochloric acid or sulfuric acid and organic acid salts such as, for example, acetic acid or oxalic acid. Of course, such salts must possess the biological activity of the protein (IFN) associated with the present invention, ie the ability to bind to the corresponding receptor and initiate receptor signaling.

According to the invention, it is more preferred to use the compounds of the invention and recombinant human IFN-beta.

Certain types of interferon variants have been described recently. The so-called "consensus interferon" is a non-naturally occurring variant of IFN (US Pat. No. 6,013,253). According to a preferred embodiment of the invention, the compounds of the invention are used with a common interferon.

As used herein, human interferon consensus (IFN-con) is a non-naturally occurring polypeptide, representing IFN-alpha representing most of the naturally-ocurring human leukocyte interferon subtype sequence. Predominantly comprising amino acid residues common to a subset of, including at least one naturally occurring amino acid at at least one or more positions that do not have amino acids common to all subtypes, and otherwise at least one naturally occurring By non-naturally occurring polypeptides comprising amino acid residues that are not present at such positions in the subtype. IFN-cons include, but are not limited to, the amino acid sequences represented by IFN-con1, IFN-con2 and IFN-con3 as disclosed in US Pat. Nos. 4,695,623, 4,897,471 and 5,541,293. DNA sequences encoding IFN-cons can be prepared as described in the patents mentioned above or by other standard methods.

In another embodiment, the fusion protein comprises Ig fusion. The fusion can be done directly or via short linker peptides that are as short as 1 to 3 amino acids in length or for example 13 amino acid residues in length. The linker is a tripeptide of sequence EFM (Glu-Phe-Met), for example Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln introduced between an IFN sequence and an immunoglobulin sequence. It may be a 13-amino acid linker sequence comprising -Phe-Met. The resulting fusion protein may have improved properties such as prolongation of residence time (half life), increased specific activity, increased expression rate, or ease of purification of the fusion protein in the blood.

In another preferred embodiment, the IFN is fused to the constant region of the Ig molecule. Preferably, they are fused to heavy chain sites such as, for example, the CH2 and CH3 domains of human IgG1. Other isoforms of Ig molecules are also suitable for the production of fusion proteins according to the invention, such as isoforms IgG _{2 ,} IgG ₃ or IgG ₄ , or other Ig classes such as for example IgM or IgA. Fusion proteins can be monomeric or multimeric, hetero- or homomultimeric.

In another preferred embodiment, the functional derivative comprises at least one moiety attached to one or more functional groups, which occurs as one or more side chains at the amino acid residue. Preferably, the moiety is a polyethylene (PEG) moiety. PEGylation can be performed by known techniques, for example as disclosed in WO99 / 55377.

The dose administered to a patient as single or multiple doses will depend on several factors, including factors such as pharmacokinetic properties, route of administration, patient's condition and characteristics (gender, age, Weight, health status, height), symptoms, concurrent treatment, frequency of treatment, and desired effects.

The dosage of IFN-beta in the treatment of relapsing-remitting MS according to the invention depends on the type of IFN-beta used.

According to the invention, IFN a trademark beta seron ^® (Betaseron) kill commercially in E.Coli using recombinant IFN- beta 1b produced in possible, the dosage of (approximately) 250 ~ 300㎍ or 8MIU ~ 9.6MIU 2 It may be administered to the subcutaneous tissue every day.

According to the invention, IFN a trade name as AVONEX ^® can be commercially available, China greater long-tailed hamster ovary cells (Chinese Hamster Ovary Cell: CHO cell) in the case of the recombinant IFN- beta 1a generated by the patient (approximately) 30 Administration by intramuscular injection once a week at a dose of ˜33 μg or 6 MIU to 6.6 MIU is preferred.

According to the present invention, if IFN is recombinant IFN-beta 1a produced in Chinese silk rat ovary cells (CHO cells), commercially available under the trade name Lviv, per patient

It is desirable to administer subcutaneous tissue three times a week (TIW) at a dose of 22-44 μg or 6 MIU-12 MIU per dose.

Administration of the active ingredient according to the invention may be by intravenous, intramuscular or subcutaneous route. The preferred route of administration of IFN is the subcutaneous route. A more preferred route of administration is intramuscular administration, which may be administered once a week, for example.

IFN can also be administered daily or every other day or less frequently. Preferably, the IFN may be administered once, twice or three times a week.

The term "stability" refers to the physical, chemical, and conformalal stability (including the maintenance of biological efficacy) of the interferon formulations of the invention. Instability of protein preparations is caused by chemical degradation or agglomeration of protein molecules, thereby forming higher order polymers, deglycosylation, modification of glycosylation, oxidation or other structural modifications to form interferons included in the present invention. At least one biological activity of the polypeptide is reduced.

A “stable” solution or formulation is one in which the degree of degradation, denaturation, aggregation, loss of biological activity of the proteins contained therein is controlled to an acceptable level and does not unacceptably increase over time. Preferably the agent is one that maintains at least about 60%, preferably at least about 70%, more preferably at least about 80% of labeled interferon activity over 12-24 months. The stabilized HSA-free IFN compositions of the invention are preferably at least 6 months, 12 months, 18 months, more preferably about 20 months, more preferably at least about 22 months, most when stored at 2-8 ° C. Preferably it has a shelf life of about 24 months.

Methods for monitoring the stability of the HSA-free IFN pharmaceutical compositions of the present invention can be utilized from the prior art, including the methods described in the Examples disclosed herein. Therefore, IFN aggregate formation during storage of the liquid pharmaceutical composition of the present invention can be readily determined by measuring the change in soluble IFN in solution over time. The content of soluble polypeptide in solution can be quantified by many analytical assays tailored to the detection of IFN. Such assays include, for example, reverse phase (RP) -HPLC and UV adsorption spectroscopy, as described in the Examples below.

In liquid formulations, soluble and insoluble aggregates during storage are determined using analytical ultracentrifugation, for example, as described in the examples above, so that the soluble polypeptide portions present as soluble aggregates and the non-aggregate portions, biologically active Distinguish between parts of the molecular state. Velocity ultracentrifuation can also detect quantitatively and qualitatively non-covalent and covalent oligomers. Similarly, the new size exclusion (SE) -HPLC method (described in the Examples) can detect covalent and non-covalent oligomers, hereinafter referred to as "NEW SEC", both quantitatively and qualitatively.

The expression “use multidose” includes the use of a single vial, ampoul or cartridge of an interferon formulation for one or more injections, eg 2, 3, 4, 5, 6 or more injections. The injection is for a period of at least about 12 hours, 24 hours, 48 hours, preferably about 12 days. The injection is divided hourly, for example every 6, 12, 24, 48 or 72 hours.

The term "amino acid" means an amino acid or a combination of amino acids, wherein a given amino acid is present in its free base form or in its salt form. When amino acid combinations are used, all amino acids may exist in free base form, all amino acids may exist in form of their salts, while others may exist in free base form while others may exist in their salt form. Preferred amino acids for use in the methods or formulations of the invention carry a charged side chain, referred to herein as "charged side chain amino acids," including arginine, lysine, aspartic acid, glutamic acid, and the like. More preferably the amino acid is lysine. Stereoisomers of a particular amino acid (ie, L, D or DL isomer), or combinations of these stereoisomers, may be used in the methods or formulations of the invention as long as the particular amino acid is in its free base form or in its salt form. Can be. Preferably, L-stereoisomer is used. Analogs of these preferred amino acids can also be used in the formulations of the invention. The term "amino acid analogue" refers to a derivative of a naturally occurring amino acid. Suitable arginine analogs include, for example, aminoguanidine and N-monoethyl L-arginine. As with preferred amino acids, amino acid analogs are used in the present formulations in their free base form or in their salt form. Amino acids are also considered as stabilizers here.

The amino acids used in the formulations of the invention protect the therapeutically active polypeptide against a variety of stresses to increase or / or maintain the stability of the interferon-beta formulation. The term "stress" is not limited thereto, but heat, freezing, pH, light, vibration, oxidation, dehydration, surface, shear, cooling / thawing, pressure, heavy metals, phenolic compounds, denaturants, etc. It includes. The term stress includes any factor that modulates (ie, decreases, maintains or increases) the stability of an interferon-beta containing formulation. Increased and / or maintained stability due to amino acid addition occurs in a concentration-related manner. In other words, when an agent containing interferon-beta shows no aggregates or oligomer formation normally in the absence of amino acids, increasing the concentration of amino acids increases and / or maintains the stability of the agent containing the interferon-beta of the present invention. The content of specific amino acids used in the formulations of the present invention to reduce the formation of oligomers or aggregates to increase monomeric protein stability can be readily determined without undue experimentation using methods generally known to those skilled in the art.

The term "buffer" or "physiologically acceptable buffer" refers to a solution of a compound which is known to be stable for pharmaceutical or veterinary use in the formulation and has the effect of adjusting or maintaining the pH of the formulation to fall within the pH range desired for the formulation. it means. Suitable buffers for adjusting pH at moderately acidic pH to moderately basic pH include, but are not limited to, compounds such as phosphate, acetate, citrate, arginine, TRIS, and histidine. "TRIS" means 2-amino-2-hydroxymethyl-1,3-propanediol and pharmaceutically acceptable salts thereof. Preferred buffers are acetate buffers with saline or salts thereof.

"Isotonicity agents" are physiologically tolerant compounds that impart moderate tonicity to the formulation and prevent the net migration of water through the cell membrane in contact with the formulation. Compounds such as glycerin are commonly used for that purpose at known concentrations. Other suitable isotonic agents include, but are not limited to, mannitol, amino acids or proteins (eg glycine or albumin), salts (eg sodium chloride), and sugars (eg dextrose, mannitol, sucrose and lactose). ).

The term "antioxidant" means a compound that prevents oxygen or oxygen-derived free radicals from interacting with other materials. Antioxidants are one of many excipients that are commonly added to pharmaceutical systems to increase physical and chemical stability. Antioxidants are added to reduce or suspend the oxidative processes that occur with certain drugs or excipients when exposed to oxygen or in the presence of free radicals. These processes can sometimes be catalyzed due to light, temperature, hydrogen concentration, the presence of metal traces or the presence of peroxides. Sulfite, bissulphite, thiourea, methionine, salts of ethylenediaminetetraacetic acid (EDTA), butylated hydroxytoluene (BHT), and butylated hydroxy Anisole (Butylated hydroxy anisole: BHA) is often used as an antioxidant in drugs. Sodium EDTA has been found to increase the activity of antioxidants by chelating metal ions that would otherwise catalyze the oxidation reaction. Most preferred antioxidant is methionine. Antioxidants are also meant here as stabilizers.

Methionine may be present in its free base form or in the form of a salt thereof. Stereoisomers of methionine (ie, L, D, or DL isomers) can be used in the methods or formulations of the present invention as long as methionine is in free base form or in the form of a salt thereof. Preferably, the L-stereoisomer is used. Methionine analogs can be used in the formulations of the invention. The term "methionine analogue" refers to a derivative of methionine which occurs naturally. The methionine analogs can also be used in the formulations in their free base form or in the form of their salts.

Increased and / or maintained stability with the addition of antioxidants (eg methionine) occurs in a concentration-related manner. That is, when an agent containing interferon-beta normally exhibits oxidation or aggregate / oligomer formation when no antioxidant is normally present, an increase in the concentration of the antioxidant increases the stability of the agent containing the interferon-beta of the present invention and And / or maintained. The content of antioxidants (eg methionine) used in the formulations of the present invention to reduce oxidation or oligomer / aggregate formation can be readily determined without undue experimentation using methods generally known to those skilled in the art.

The term "bacteriostatic agent" means a compound or composition added to a formulation to act as an antimicrobial agent. As bactericides, for example, phenol, m -cresol, p -cresol, o -cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethium (benzethonium) chloride, sodium dehydroacetate and thimerosal. Preferably the bacteriostatic agent is benzyl alcohol.

The term "surfactant" means a soluble compound that reduces the surface tension of a liquid or reduces the interfacial tension between two liquids, or between a liquid and a solid, wherein the surface tension is a force acting on the surface of the liquid. And tend to minimize surface area. Surfactants include the delivery of low molecular weight population drugs and polypeptides to alter the absorbency of the drug or delivery of the drug to the tissue of interest, and have sometimes been used in pharmaceutical formulations. Well known surfactants include polysorbate (polyoxyethylene derivative: Tween) as well as Pluronic.

According to a preferred embodiment of the present invention, Pluronic F77 ^® , Pluronic F87, Pluronic F88 and Pluronic F68, particularly preferably Pluronic ^® F68 (BASF, Pluronic F68 is Ploxamer 188 Formulating interferon with a surfactant selected from also known as, a stable formulation has been obtained, which is absorbed on the surface of vials and / or delivery devices (eg, syringes, pumps, catheters, etc.) Component loss was minimized. Surfactant selected from Pluronic F77 ^® , Pluronic F87, Pluronic F88 and Pluronic F68, particularly preferably Pluronic ^® F68 (BASF, Pluronic F68 is also known as Ploxamer 188) By formulating rho interferon, a stable formulation was obtained, which is more resistant to oxidation and the formation of protein aggregates.

The pluronic surfactant is a block copolymer of ethylene oxide (EO) and propylene oxide (PO). The propylene oxide block (PO) is sandwiched between two ethylene oxide (EO) blocks.

Pluronic surfactants are synthesized by a two step process:

1. Hydrophobicity of the desired molecular weight was prepared by controlling the addition of propylene oxide to the two hydroxyl groups of propylene glycol; And'

2. Ethylene oxide was added and sandwiched between the hydrophilic groups.

Nick ^® Pluronic F77, polyoxyethylene molecular weight of the ethylene (hydrophilic) is 70% percent, hydrophobic (polyoxypropylene) is approximately of 2,306Da.

In Pluronic F87, the percentage of polyoxyethylene (hydrophilic) is 70% and the molecular weight of hydrophobic (polyoxypropylene) is about 2,644 Da.

Pluronic F88, polyoxyethylene (hydrophilic) percent is 80%, hydrophobic (polyoxypropylene) molecular weight is about 2,644 Da.

Pluronic F68, percent of polyoxyethylene (hydrophilic) is 80%, and the molecular weight of hydrophobic (polyoxypropylene) is about 1,967 Da.

Typical properties of Pluronic F77 are shown below:

Average molecular weight: 6600;

Melt / pour point: 48 ^o C;

Physical form @ 20 ^o C: solid;

Viscosity (Brookfield) cps: 480 [liquid at 25 ^o C, paste at 60 ^o C and solid at 77 ^o C];

Surface tension, dynes / cm @ 25 ^o C;

0.1% Conc. 47.0

0.01% Conc. 49.3

0.001% Conc .: 52.8

Interfacial tension, dynes / cm @ 25 ^o C vs. Nujol;

0.1% Conc. : 17.7

0.01% Conc. : 20.8

0.01% Conc. : 25.5

Drives Wetting, Seconds 25 ^o C

1.0% Conc .:> 360

0.1% Conc .:> 360

Bubble Height

Ross Miles, 0.1%, mm @ 50 ^o C: 100

Losmile, 0.1%, mm @ 26 ^o C: 47

Dynamic, 0.1%, mm @ 400 ml / min:> 600

Cloud point in aqueous solution, ⁰ C

1% Conc .:> 100

10% Conc .:> 100

HLB (hydrophile-lipophile balance): 25

Typical properties of Pluronic F87 are as follows:

Average molecular weight: 7700;

Melt / pour point: 49 ^o C;

Physical form @ 20 ^o C: solid;

Viscosity (Brookfield) cps: 700 [liquid at 25 ^o C, paste at 60 ^o C and solid at 77 ^o C];

Surface tension, dynes / cm @ 25 ^o C;

0.1% Conc. : 44.0

0.01% Conc. 47.0

0.001% Conc .: 50.2

Interfacial tension, dynes / cm @ 25 ^o C vs Nujol;

0.1% Conc. : 17.4

0.01% Conc. : 20.3

0.01% Conc. : 23.3

Drain Wetting, Seconds 25 ^o C

1.0% Conc .:> 360

0.1% Conc .:> 360

Bubble height

Lost miles, 0.1%, mm @ 50 ^o C: 80

Lost mile, 0.1%, mm @ 26 ^o C: 37

Dynamic, 0.1%, mm @ 400 ml / min:> 600

Cloud point in aqueous solution, ⁰ C

1% Conc .:> 100

10% Conc .:> 100

HLB (hydrophile-lipophile balance): 24

Typical properties of Pluronic F88 are as follows:

Average molecular weight: 11400;

Melt / pour point: 54 ^o C;

Physical form @ 20 ^o C: Solid:

Viscosity (Brookfield) cps: 2300 [liquid at 25 ^o C, paste at 60 ^o C and solid at 77 ^o C];

Surface tension, dynes / cm @ 25 ^o C;

0.1% Conc. 48.5

0.01% Conc. 52.6

0.001% Conc .: 55.7

Interfacial tension, dynes / cm @ 25 ^o C vs Nujol;

0.1% Conc. : 20.5

0.01% Conc. : 23.3

0.01% Conc. : 27.0

Drain Wetting, Seconds 25 ^o C

1.0% Conc .:> 360

0.1% Conc .:> 360

Bubble height

Loss miles, 0.1%, mm @ 50 ^o C: 80

Los Mile, 0.1%, mm @ 26 ^o C: 37

Dynamic, 0.1%, mm @ 400 ml / min:> 600

Cloud point in aqueous solution, ⁰ C

1% Conc .:> 100

10% Conc .:> 100

HLB (hydrophile-lipophile balance): 28

Typical properties of Pluronic F68 are as follows:

Average molecular weight: 8400;

Melt / pour point: 52 ^o C;

Physical form @ 20 ^o C: solid;

Viscosity (Brookfield) cps: 1000 [liquid at 25 ^o C, paste at 60 ^o C and solid at 77 ^o C];

Surface tension, dynes / cm @ 25 ^o C;

0.1% Conc. 50.3

0.01% Conc. 51.2

0.001% Conc .: 53.6

Interfacial tension, dynes / cm @ 25 ^o C vs Nujol;

0.1% Conc. : 19.8

0.01% Conc. : 24.0

0.01% Conc. : 26.0

Drain Wetting, Seconds 25 ^o C

1.0% Conc .:> 360

0.1% Conc .:> 360

Bubble height

Lost mile, 0.1%, mm @ 50 ^o C: 35

Lost mile, 0.1%, mm @ 26 ^o C: 40

Dynamic, 0.1%, mm @ 400 ml / min:> 600

Cloud point in aqueous solution, ⁰ C

1% Conc .:> 100

10% Conc .:> 100

HLB (hydrophile-lipophile balance): 29

Other polymers with properties similar to those indicated above can also be used in the formulations of the present invention. Preferred surfactants are Pluronic F68 and are surfactants with similar properties.

Pluronic, in particular Pluronic F68, has a concentration sufficient to maintain interferon stability for the desired shelf life (e.g. 12 to 24 months), and or protein loss due to adsorption on the surface, such as vials, ampoules or cartridges or syringes. It is preferred to be present at a concentration sufficient to prevent this.

Preferred concentrations of pluronic, in particular Pluronic F68, in liquid formulations are from about 0.01 mg / ml to 10 mg / ml, more preferably from 0.05 mg / ml to 5 mg / ml, even more preferably from 0.1 to 2 mg / ml, Most preferably about 0.5 mg / ml.

The preferred concentration of IFN-beta 1a in the formulation is 10 μg / ml-800 μg / ml, more preferably 20 μg / ml-500 μg / ml, even more preferably 30 μg / ml-300 μg / ml, most Preferably 22, 44, 88, or 264 μg / ml.

The formulations of the present invention have a pH between about 3.4 and 5.5, preferably about 4.7 pH. Preferred buffers are acetates with sodium or potassium ions being the preferred counterion. Acetate saline buffers are well known in the art. The concentration of buffer in the total solution can vary between about 5 mM, 9.5 mM, 10 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, and 500 mM. Preferably the concentration of the buffer is about 10 mM. A particularly preferred buffer is 10 mM buffer with acetate ions at pH 4.7 ± 0.2.

Preferably, in the compositions of the present invention, the antioxidant, for example methionine, is present at a concentration of about 0.01 to about 5.0 mg / ml, more preferably 0.05 to 0.3 mg / ml, most preferably about 0.12 mg / ml. do.

Preferably, in the composition of the present invention, the amino acid, for example lysine, is present at a concentration of about 1 to 100 mg / ml, more preferably 10 to about 50 mg / ml, most preferably about 27.3 mg / ml.

The present invention includes liquid formulations. Preferred solvent is water for injection.

Liquid formulations may be monodose or multidose. Liquid interferon formulation for multi-dose of the present invention is phenol, m -cresol, p -cresol, o -cresol, chlorocresol, benzyl alcohol, alkyl paraben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benz It is preferred to include bacteriostatic agents such as tonium chloride, sodium dehydroacetate and thimerosal. Especially preferred are phenol, benzyl alcohol and m -cresol, more preferably benzyl alcohol. The bacteriostatic agent is used in an amount capable of giving an effective concentration to maintain a sterile preparation (suitable for injection) over a period of about 12 or 24 hours to about 12 days, preferably about 6 to 12 days, between the multi-dose syringes. do. The bacteriostatic agent is preferably present at a concentration of about 0.1% to about 2.0% (mass of mass / solvent of bactericide), more preferably 0.2% to 1.0%. In the case of benzyl alcohol, a concentration of 0.2 to 0.3% is preferred.

However, the use of preservatives, such as benzyl alcohol, is not limited to multidose formulations and may also be used in monodose formulations. One embodiment of the invention consists of a single dose formulation comprising benzyl alcohol.

In another embodiment of the formulation according to the invention, at room temperature or at 28 ° C., the interferon beta is at least about 96% or at least about 98% in its monomer form (up to 4% aggregates, preferably up to 2%) Present and measured by sedimentation velocity analysis.

Preferred formulations consist of interferon-beta 1a, benzyl alcohol, lysine, methionine, Pluronic F-68 and water soluble acetate buffer, preferably adjusting the pH to 3.7-4.7.

The range of interferon in the formulations of the present invention includes those in which the concentrations that appear upon reconstitution range from about 1.0 μg / ml to about 50 mg / ml, but are capable of working at lower or higher concentrations, as well as using delivery carriers. For example, the solution formulation will differ from transdermal patch, pulmonary, transmucosal, or osmotic or micropump methods. The interferon concentration is about 5.0 μg / ml to about 2 mg / ml, more preferably 10 μg / ml to about 1 mg / ml, most preferably about 30 μg / ml to 100 μg / ml.

The formulations of the present invention preferably have an interferon activity of at least about 60%, more preferably at least about 70%, most preferably at least about 80% during the packaging time over a period of 24 months.

In another preferred embodiment, the present invention provides a method for preparing a liquid pharmaceutical composition as described above.

In another preferred embodiment, the present invention provides a method for preparing a packaged pharmaceutical composition comprising the step of adding a solution comprising the active ingredient and excipients as described above.

In another preferred embodiment, the present invention provides a vial comprising a pharmaceutical composition as described above, and a written material stating that such a solution can be maintained for 24 hours or more after the first use. It provides a product for human pharmaceutical use comprising. Preferably the written data indicates that the solution can be maintained for about 12 days.

After the first use of the multidose formulation, it can be maintained and used for at least about 24 hours, preferably at least about 4, 5 or 6 days, more preferably 12 days. After the first use, the formulation is preferably stored at room temperature or lower (ie about 25 ° C. or lower), more preferably at about 10 ° C., more preferably at 2-8 ° C. and most preferably at about 4-6 ° C.

The formulation of the present invention can be prepared by a process comprising the step of adding an interferon after adding a calculated amount of excipient to the buffer solution.

The resulting solution is then placed in a vial, ampoule or cartridge. Many variations in this process will be apparent to those skilled in the art. For example, the order of addition of the ingredients, whether additional additives are to be used, the temperature and pH at which the formulation is prepared are all factors to optimize the concentration and means of administration used.

In the case of multidose preparations, the bacteriostatic agent may be added to a solution containing the active ingredient (interferon) or optionally stored in a separate vial or cartridge and then mixed in a solution containing the active ingredient in use.

The formulations of the present invention can be administered using known devices. Examples comprising these single vial systems are auto-injector or pen for delivering a solution, such as Levy jet ^® (Rebiject) - comprises a syringe.

Products claimed in the present invention include packaging materials. The packaging material provides conditions under which the product can be used in addition to the information required by the management agency. The packaging material of the present invention provides the patient with instructions for use, if necessary, to prepare the final solution and use such solution for two vials, wet / dry, for a period of at least 24 hours. For single vial, solution products, the label indicates that such solution can be used for a period of 24 hours or more. The presently claimed products are useful for the use of human pharmaceutical products.

Stable preserved formulations can be supplied to patients in clear solutions. The solution may be single use or may be reused multiple times and may satisfy a single or multiple cycles of patient care to provide a more convenient treatment method than is currently available.

Interferons in the stable or preserved formulations or solutions described herein are selected according to the invention, including SC or IM injections; It can be administered to the patient in a variety of ways via transdermal, respiratory, mucosal, implant, osmotic pumps, cartridges, micropumps, oral or other means as would be expected by one skilled in the art.

The term "vial" means a wide range of reservoirs suitable for holding interferon in solid or liquid form in a controlled sterile state. Examples of vials used herein include ampoules, cartridges, blister packages or other reservoirs suitable for delivering interferon to a patient via injection, pump (including osmotic pressure), catheter, transdermal patch, respiratory or mucosal spray It includes. Vials suitable for parenteral, pulmonary, transmucosal or transdermal administration products are well known in the art.

The term "treatment" within the scope of the present invention means any effect that has a beneficial effect on the progression of the disease, including alleviation, reduction, reduction or attenuation of pathological development after the onset of the disease.

Pharmaceutical compositions of the present invention comprising IFN or isoforms, muteins, fusion proteins, functional derivatives, active fractions or salts can be used to diagnose, prevent and treat clinical indications in response to a therapy with the polypeptide. Useful for (local or systematic). Such clinical signs include, for example, diseases or disorders of the central nervous system (CNS), brain, and / or spinal cord, including multiple sclerosis; Autoimmune diseases including rheumatoid arthritis, psoriasis, Crohn's disease; And cancers, including breast, prostate, bladder, kidney and colon cancer.

All references cited herein, including journals or abstracts, are incorporated herein by reference in their entirety for publications of published or unpublished US or foreign patents, and registered US or foreign patents or other references, all data, tables All contents shown in the drawings, and the cited references are incorporated into the present invention. In addition, the entire contents of the references cited in the references cited herein are also incorporated herein by reference in their entirety.

With reference to known methods, no prior method steps, known method steps or conventional methods are known to be disclosed, taught or suggested in the related art.

The previous description of specific embodiments has so far clarified the general nature of the invention that others may, without undue experimentation, without deviating from the general concept of the invention, using ordinary knowledge of the relevant industry (including the contents of the references cited herein). It can be easily modified or applied to various applications as in the embodiment. Therefore, such applications and modifications are within the equivalent scope of the disclosed embodiments, based on the teachings and instructions presented herein. The phraseology or terminology herein is for the purpose of description and such usage and phraseology may be interpreted by those skilled in the art in accordance with the teachings and guidance presented herein with the knowledge of those skilled in the art.

Example  1-analytical method

Size Exclusion (SE) -HPLC, hereinafter referred to as "new SE-HPLC" or "NEW SEC", and recombinant human interferon-beta 1a (rh 1IFN-beta 1a or r-) using rate ultracentrifugation (AUC) The degree of aggregate and oligober of hβIFN-1a) was measured. The SE-HPLC and AUC methods shown below can quantitatively and qualitatively detect covalent and non-covalent oligomers.

a. SE- HPLC -Purity test

Detection of total aggregate content was carried out with a TSK G2000SWXL column (TosoHaas) or BioSuite (Waters); Eluent was performed in 50 mM sodium acetate buffer, 50 mM NaCl pH 3.8 at 0.5 mL / min in isocratic mode; The wavelength was set to 215 nm. The run time was 30 minutes.

A sample of 88 mcg / ml was analyzed with 100 μl injection.

b. Precipitation Rate Method AUC

1. Method Description

Samples were loaded intracellularly with 2-channel charcoal-epon centerpieces with 12 mm optical pathlength. The centerpiece and sapphire window were cleaned with detergent and soaked in water to have the cleanest surface possible. The corresponding placebo was loaded into a control channel (the tool functions as a dual line spectrophotometer): a dual-beam spectrophotometer. These drug-injected cells were placed in an AN-50-Ti fractionation rotor, placed in a Beckman Optima XL-1 analytical centrifuge and set to 20 ° C. The rotor is set at 3000 rpm and the sample is scanned (absorption peak at 280 nm) to confirm proper cell loading. The rotor was set at a final speed of 50000 rpm. 50 scans were recorded for each sample at the rotor speed.

The data were analyzed using the c (s) method developed at NIH by Peter Schenck and his analysis program SEDFIT (version 8.7; Schuck, P. (2000) .Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling Biophys . J. 78, 1606-1619).

In this study, many new data were applied directly to derive the distribution of sedimentation coefficients, while modeling the effect of diffusion on the data in order to increase the resolution. The method is based on the assumption that all species have the same overall hydrodynamic shape (a form defined by the coefficient of friction coefficient relative to that for a sphere, f / fo). By assigning a diffusion coefficient to each value of the sedimentation coefficient. The f / fo values are varied to find the best overall fit of the data for each sample. The distribution was calculated using 0.51 maximum-entropy smoothing.

2. Analysis Parameters

Rotor Type 8-Hole Rotor

Rotor speed 50k rpm

-Center Piece Activated Carbon Epon

Channel length 12mm

-20 ℃ during AUC operation

Detection wavelength 280nm

Sample volume 432 mcl

-Control volume 442mcl

3. Device and Software

Analytical ultracentrifuge model XL-1 (Beckman Coulter)

SEDFIT version 8.70b software (Peter Schuck-National Institutes of Health)

Origin Version 6.03 Software (Beckman Culter)

Proteome Lab XL-AXL-I Version 5.0 Software (Beckman Culter)

c. RP- HPLC - QUANT - HPLC On by IFN -β-1a quantification

The reversed phase method described below allows for quantification of IFN-β-1a in a sample.

Quantification of the protein is carried out with a C4, Wide-Pore Butyl 5 μm, 4.6 × 250 mm column (Baker: Baker); The wavelength is set to 214 nm and the eluent is performed at 1 mL / min using the next mobile phase and gradient.

A = water / Trifluoroacetic acid  0.1% -B = Acetonitrile Of Trifluoroacetic acid  0.1% -C = Acetonitrile

Samples were analyzed while injecting 50 μl of the sample itself (88 mcg / mL sample).

Time (minutes) % Eluent A % Eluent B % Eluent C 0 70 30 0 5.0 70 30 0 6.0 58 42 0 15.0 57 43 0 30.0 46 54 0 35.0 45 55 0 40.0 40 60 0 40.1 20 80 0 45.0 20 80 0 45.1 0 0 100 50.0 0 0 100 50.1 70 30 0 65.0 70 30 0

Operating time = 65 minutes

Quantification of the sample is performed against a reference curve in the range of 0.0125 mg / ml-0.2 mg / ml prepared by the control reference material.

d. Inverse phase  (RP)- HPLC - DEG Purity by / OX

The reversed-phase method described below enables the detection of IFN-β-1a oxidized forms, eluting differently from intact molecules.

Quantification of the oxidized form is carried out on a Supelcosil LC-304 column (Supelco) temperature controlled to C4, 40 ° C .; The wavelength is set to 208 nm and the elution is carried out at 1 mL / min using the next mobile phase and gradient.

A = water 60% / acetonitrile 40% / heptafluorobutyric acid 0.14% -B = water 20% / acetonitrile 80% / heptafluorobutyric acid 0.14% -C = water 20% / acetonitrile 80% / trifluoro Acetic acid 0.1%

gradient:

0' 70% A 30% B 0% C 5 ' 70% A 30% B 0% C 58 ' 62% A 38% B 0% C  Curve 6 63 ' 0% A 100% B 0% C  Curve 1 68 ' 0% A 0% B 100% C  Curve 1 69 ' 70% A 30% B 0% C  Curve 6

Operating time: 96 minutes (70 minutes +26 minutes equilibrium)

Samples were analyzed by injecting 200 μl (88 mcg / mL sample) as is.

e. Cytopathic effect  Inhibitory Biological Assay ( Cytopathic  effect inhibition bioassay- CPE )

Biological efficacy ( biopotency ) (Antiviral activity)

Antiviral activity of IFN-β-1a was measured by cytopathic effect (CPE) inhibition biological assay.

The biological activity was determined by antiviral assay based on IFN-β inducible protection of cells (WISH cell-human amniotic tissue) against cytopathic effect of virus.

The principle of biological analysis of interferon is based on the fact that numerous viruses, such as vesicular stomatitis virus (VSV), can cause cell death and can be visualized by vital staining.

The cytopathic effect can be used to quantify cell protection by interferon.

The assay is performed by direct measurement of cell death, which is assessed by the content of the dye tetrazolium salt MTT (dimethylthiotetrazolium) collected by living cells. The method utilizes a three-point parallel line assay and automatic spectrophotometric determination of the percentage of protected cells for statistical evaluation of the appropriate amount.

step:

The assay was performed on microtiter plates.

a. 50 μl of cell culture medium (MEM / 5% FBS) is added to each well.

b. 100 μl of IFN-β-1a sample or standard solution (60-100 IU hIFN-β / ml) was added to the wells and three 1: 1.5 step dilutions were performed for all rows of the plate.

c. 50 μl suspension of WISH cells (0.78-0.82 × 10 ⁶ cells / ml) is added to each well and the plates are incubated at 37 ° C. for 18-20 hours in a 5% CO ₂ humidified incubator.

d. VSV suspension was added to each well filled with MEM / 2.5% FBS, except cell control wells.

e. The plates are incubated at 37 ° C. for 24 hours in a 5% CO ₂ humidity incubator.

f. (1) at least 80% of cell damage occurs in the VSV control row and

   (2) Inverted microscope confirmed that the percent average value of protection in the presence of IFN-β standard solution was 84% in undiluted standard solution, 45% for 1: 1.5 dilution and 27% for 1: 3 dilution. after,

The cultures are stained with the specific dye MTT.

g. Color density is determined by automatic spectrophotometric reading at 592 nm.

h. To quantify IFN-β-1a activity, OD readings are analyzed by computer program (Colombo Software).

Example  2 HSA Stabilization of Free Beta-1a Formulations

The following experiments show the protective effect of a formulation comprising an antioxidant (ie methionine) and an amino acid (ie lysine) upon storage of the rh IFN-beta 1a formulation at various temperatures (2-8 ° C, 25 ° C and 40 ° C). It was performed to confirm. The following assays and methods were used on-going (see Example 1):

Biological activity (in vitro biological analysis)

Assay (RP-HPLC method)

Oxidation products (Deg-ox method)

Dimers / aggregates (NEW SEC-HPLC and AUC, both methods can detect covalent and non-covalent oligomers)

Quantitative RP-HPLC Method (Quant-HPLC)

-pH (potentiometric method)

The results are shown in Tables 6-8.

Procedure

a. The formulations were prepared using lumps stored / loaded at 2-8 ° C. The concentration of r-h IFN-beta 1a in the mass is about 0.5 mg / mL.

b. The formulation, comprising lysine (as shown in point 2), is prepared by diluting the drug substance in the carrier to obtain the final composition (r-h IFN-beta 1a concentration 88 mg / mL).

c. The compounded solution is aseptically filtered through a 0.2 micronylon membrane and collected into a sterile container, and the syringe is filtered to 0.5 mL using a filtration device.

d. The samples were stored in constant temperature cabinets of 2-8 ° C, 25 ° C and 40 ° C and tested according to the stability plan using various methods / tests for a period of about 2 months (samples stored at 40 ° C). It was.

2. Compositions of Formulations

a. 0.088 mg / mL r-h interferon-beta 1a

b. 27.3 mg / mL L-lysine monohydrochloride (Code 1.05701, Merck)

c. 0.12 mg / mL L-methionine (1.05707, Merck)

d. 0.5 mg / mL Pluronic F-68 (Lutrol F 68 DAC, USP / NF, Basf), 5163315

e. 10 mM Sodium Acetate pH 4.7

3. Results:

Storage at 2-8 ℃ 2-8 ℃ T = 0 4w 6w 8w Quant-HPLC (mcg / syr) 39.0 38.2 39.8 39.2 Deg-ox HPLC (% oxidized) 1.3 1.5 1.5 1.5 AUC (% Monomer) 98.7 - 98.8 - New-SEC (% Monomer) 100.0 100.0 100.0 100.0 PH 4.7 4.7 - 4.7 Biological Assay (MIU / mL) 24.1 24.6 - - Osmolarity (OSM / kg) 0.303 - - -

Storage at 25 ℃ 25 ℃ T = 0 2w 4w 6w 8w Quant-HPLC (mcg / syr) 39.0 42.8 38.0 39.4 37.8 Deg-ox HPLC (% oxidized) 1.3 1.5 1.4 2.3 2.3 AUC (% Monomer) 98.7 99.0 - 98.2 - New-SEC (% Monomer) 100.0 100.0 100.0 100.0 100.0 PH 4.7 4.7 4.7 - 4.7 Biological Assay (MIU / mL) 24.1 - 24.0 - -

 Storage at 40 ℃ 40 ℃ T = 0 1w 3w Quant-HPLC (mcg / syr) 39.0 37.8 34.9 Deg-ox HPLC (% oxidized) 1.3 1.9 3.4 AUC (% Monomer) 98.7 99.6 - New-SEC (% Monomer) 100.0 100.0 100.0 PH 4.7 4.7 4.6

4. Conclusion

Hypotonic studies have shown that formulations or compositions comprising lysine, methionine and plurogine F-68 remain very stable up to 8 weeks, even at 25 ° C. as well as 2-8 ° C. They remain stable at 40 ° C. for up to 3 weeks in the form of monomer percent levels (monomer percentages are always at least 98%). Two other methods confirmed these results (AUC and NEW-SE-HPLC). Therefore, the preparations of the present invention preferably comprise an amino acid and an antioxidant together. Preferably, surfactant is further added to the composition.

Preferably the amino acid is lysine and the antioxidant is methionine.

Preferably, the surface active agent is Tween (for example Tween 20: tween 20), is selected from Pluronic ^® F77, Pluronic F87, F88 and fluorine to a nick Pluronic F68. More preferably the surfactant is Pluronic F68.

Preferably the formulation is set to pH 3.5-5.5. More preferably the formulation is set at 4.7 pH.

Preferably, lysine is present at a concentration of about 1 mg / ml to about 100 mg / ml. More preferably, lysine is present at a concentration of about 27.3 mg / ml. Preferably the surfactant is present at a concentration of about 0.01 mg / ml-10 mg / ml. More preferably, the surfactant is present at a concentration of 0.5 mg / ml. Preferably the antioxidant is present at a concentration of about 0.01 to about 5.0 mg / ml. More preferably, the antioxidant is present at a concentration of about 0.12 mg / ml. Preferably the interferon is present at a concentration of about 10 μg / ml to about 800 μg / ml. Preferably the interferon is present at a concentration of about 88 μg / ml.

References

1. Derynk R. et. Al., Nature 1980; 285, 542-547

2. "A Convenient and Rapid Cytopathic Effect Inhibition Assay for interferon" [Familletti, P.C., Rubinstein, S., and Pestka S. 1981, in Methods in Enzymology, Vol. 78 (S. Pestka ed.), Academic Press, New York, 387-394;

3. Mark D.F. et al., Proc. Natl. Acad. Sci. U.S.A., 81 (18) 5662-5666 (1984)

4.Pestka S. (1986) "Interferon Standards and General Abbreviations, in Methods in Enzymology (S. Pestka, ed.), Academic Press, New York 119, 14-23.

5. Rubinstein, S., Familletti, P.C., and Pestka S. Convenient Assay for Interferons, J. Virol 1981; 37, 755-758

6. Shepard H. M. et al., Nature 1981; 294, 563-565

The present invention relates to HSA-free pharmaceutical compositions comprising interferon, in particular formulations of interferon-beta comprising buffers, amino acids and antioxidants.

Claims (31)

A stabilized HSA-free liquid pharmaceutical composition for the treatment of multiple sclerosis, wherein the composition is a solution comprising interferon (IFN) -beta 1a, benzyl alcohol, lysine, methionine, Pluronic F-68 and water soluble acetate buffer Stabilized HSA-free liquid pharmaceutical composition, characterized in that. delete delete delete The stabilized HSA-free liquid medicament of claim 1, wherein the buffer is present in an amount sufficient to maintain the pH of the composition within a positive or negative 0.5 of a specific pH, wherein the specific pH is between 3.5 and 5.5. Pharmaceutical composition. 6. The stabilized HSA-free liquid pharmaceutical composition according to claim 5, wherein the pH is 4.7 ± 0.2. The stabilized HSA-free liquid pharmaceutical composition according to claim 1, wherein the buffer is present at a concentration of 5 mM to 500 mM. 8. The stabilized HSA-free liquid pharmaceutical composition according to claim 7, wherein the buffer is present at a concentration of 10 mM. delete delete delete delete The stabilized HSA-free liquid pharmaceutical composition according to claim 1, wherein the lysine is present at a concentration of 1 mg / ml to 100 mg / ml. The stabilized HSA-free liquid pharmaceutical composition according to claim 13, wherein the lysine is present at a concentration of 27.3 mg / ml. delete delete The stabilized HSA-free liquid pharmaceutical composition according to claim 1, wherein the Pluronic F-68 is present at a concentration of 0.01 mg / ml to 10 mg / ml. 18. The stabilized HSA-free liquid pharmaceutical composition according to claim 17, wherein the Pluronic F-68 is present at a concentration of 0.5 mg / ml. delete The stabilized HSA-free liquid pharmaceutical composition according to claim 1, wherein the methionine is present at a concentration of 0.01-5.0 mg / ml. The stabilized HSA-free liquid pharmaceutical composition according to claim 20, wherein the methionine is present at a concentration of 0.12 mg / ml. The stabilized HSA-free liquid pharmaceutical composition according to claim 1, wherein the interferon is present at a concentration of 10-800 μg / ml. 23. The stabilized HSA-free liquid pharmaceutical composition according to claim 22, wherein the interferon is present at a concentration of 22, 44 or 88 μg / ml. The stabilized HSA-free liquid pharmaceutical composition according to claim 23, wherein the interferon is present at a concentration of 88 μg / ml. delete delete delete The stabilized HSA-free liquid pharmaceutical composition according to claim 1, wherein the interferon is at least 96% or 98% by mass as monomer. Storage before use comprising the liquid pharmaceutical composition according to claim 1 sealed in a sterile condition. 30. The container of claim 29, wherein the container is a prefilled syringe, vial or autoinjector. IFN composition according to claim 1 for the preparation of a medicament for the treatment of multiple sclerosis.