KR101736870B1 - A Complex comprising interferon conjugates and a method of producing the same - Google Patents

Abstract

The present invention relates to a complex comprising an interferon conjugate and a method for producing the same, and the complex of the present invention overcomes the drawbacks of the conventional PEG-interferon alpha 2b conjugate and has high antiviral activity and sensitivity to low hydrolysis.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a complex comprising an interferon conjugate and a method for producing the same,

Interferon conjugates and methods for their preparation.

Since polypeptides such as interferon alpha are generally poorly stable and easily denatured and degraded by proteolytic enzymes in the blood, they are easily removed through the kidney or liver. Therefore, the plasma concentration of a protein drug containing a polypeptide as a pharmacological component Protein drugs need to be given to patients more often to maintain their potency. However, in the case of protein drugs that are administered to patients in the form of injections, frequent injections to maintain blood levels of the active polypeptide cause pain to the patient. In order to solve these problems, efforts have been made to maximize the drug efficacy by increasing the stability of the drug in the blood by conjugating the protein with polyethylene glycol (PEG) and maintaining the drug concentration in the blood for a long time. Such a sustained-release preparation of a protein drug should not only increase the stability of the protein drug but also maintain the titer of the drug itself sufficiently high and cause no immune response to the patient.

Since the early 1990s, studies have been focused on increasing the half-life to reduce the frequency of interferon administration. They have shown that the use of PEG, which can react with amine groups present in interferons, can increase the half-life in the blood of interferon alfa. The use of amine-reactive PEG can react with 10 to 11 (depending on the subtype) lysine in interferon alpha, and can also bind N-terminal amino acids (Monkarsh et al., Anal Biochem. 247 ) 434-440). Moreover, these homologues may be mono-, di-, or polyunsaturated with interferon. Multiple-PEG may be combined. Patent No. 5,382,657 of Hoffmann-La Roche discloses that the larger the molecular weight of the bound PEG is, the longer the in vivo circulating half-life, and the more the PEG is bound to the interferon than the more than two PEGs, Since the amount of PEG bound to a protein decreases as the size increases, the amount released from the kidney decreases and the distribution in the body such as increased liver degradation changes (Yamaoka et al., J Pharm Sci. 83 (1994) 390 -399). Thus, there is a need for a method capable of extending half-life while maintaining the activity of interferon.

Disclosure of Invention Technical Problem [8] The present invention provides a conjugate comprising an interferon and a positional isomer conjugate bonded with an alkoxy-terminated polyalkylene oxide, a process for preparing the same, and a pharmaceutical composition containing the same.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

In one embodiment of the invention, a conjugate comprising a positional isomeric conjugate conjugated with an interferon and an alkoxy-terminated polyalkylene oxide, wherein the conjugate comprises less than 14% by weight of the total conjugate comprises an alkoxy- A polyalkylene oxide-bound complex is provided. In this embodiment, the conjugate comprises at least 60% by weight of the total conjugate, wherein the lysine of interferon alpha and the alkoxy-terminated polyalkylene oxide are conjugated to the tyrosine residue, wherein the conjugate is selected from the group consisting of lysine of interferon alpha and tyrosine wherein the polyalkylene glycol is selected from the group consisting of monomethoxy-polyethylene glycol-succinimidyl carbonate (mPEG-SC < RTI ID = 0.0 > ), Wherein the alkoxy-terminated polyalkylene oxide has a molecular weight of 0.5 to 40 Kda, wherein the interferon provides an interferon alpine complex and the interferon alfa provides a complex of interferon alpha 2b and, wherein the histidine residue provides a His ³⁴ of composite material, wherein the position The present invention provides a conjugate comprising at least three regioisomers, wherein the regiojunction conjugate provides a complex comprising at least three regioisomers, said regioisomeric conjugate comprising at least five regioisomers, said regioisomeric conjugate comprising at least 15 positional isomers And the regioisomer provides a complex selected from the group consisting of His ³⁴ , Tyr ¹²⁹ , Lys ¹³¹ , Lys ¹²¹ , and Lys ⁸³ .

In one embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of interferon-sensitized diseases comprising the complex. In this embodiment, the interferon-sensitive disease is a cancer or viral disease, wherein the viral disease is selected from hepatitis A, hepatitis B, hepatitis C, non-A / non hepatitis B, The present invention relates to a method for the treatment of human viral infections such as avian virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpesvirus type 6 (HHV-6), papilloma virus, poxvirus, picornavirus, adenovirus, Wherein the cancer is any one selected from the group consisting of sexually transmitted viruses 1 and 2 (HTLV-1 / -2), human rotavirus, rabbit, retrovirus, encephalitis and respiratory virus infection, Cell leukemia, Kaposi sarcoma, chronic myelogenous leukemia, multiple myeloma, basal cell carcinoma, malignant melanoma, ovarian cancer and skin T cell lymphoma It provides a composition studies.

In one embodiment of the present invention, there is provided a process for producing a polyalkylene oxide, comprising: subjecting an interferon and an alkoxy-terminated polyalkylene oxide to a coupling reaction in a molar ratio of 1: 1 to 1: 5; And stirring at 2 DEG C to 10 DEG C, to prepare a complex comprising an interferon and a positional isomeric conjugate bonded with an alkoxy-terminated polyalkylene oxide. In this embodiment, there is provided a method for preparing a complex comprising interposon and alkoxy-terminated polyalkylene oxide conjugated positional isomeric agitation for 1 hour to 3 hours of agitation, after which the reaction is adjusted to pH 4.4 Wherein the method further comprises the step of performing a size exclusion chromatography followed by an additional step of performing size exclusion chromatography. The method of claim 1, And a positional isomeric conjugate bonded with an alkoxy-terminated polyalkylene oxide, wherein the interferon and alkoxy-terminated polyalkylene oxide further comprise a step of performing purification by performing cation exchange chromatography Oxide conjugated positional isomeric conjugate ≪ RTI ID = 0.0 > a < / RTI >

Interferon has become known in 1957 when Isaacs and Lindenmann found that there is a virus-inhibiting factor when the chicken is infected with the influenza A virus (Isaacs, K. and Lindenmann, J., Proc. R. Soc. Lond., B147 (1957), 258-267). Human interferon is a kind of cytokine that inhibits the in vivo immune response or viral proliferation and is divided into interferon alpha, interferon beta and interferon gamma depending on the type of cells produced (Kirchner, H., et al., Tex 41 (1981), 89-93: Stanton, GJ, et al., Tex. Rep. Biol. Med., 41 (1981), 84-88). Interferon is widely known to have synergistic effects on antiviral, anticancer, NK (natural killer) cell and bone marrow cell suppression (Klimpel, et al., J. Immunol., 129 (1982) , 76-78); Fleischmann, WR, et al., J. Natl. Cancer Inst., 65 (1980), 863-966; Weigent., Et al., Infec. Immun., 40 (1980), 35-38). In addition, numerous studies have revealed that interferon acts as a regulator of the expression, structure, and function of intracellular genes, as well as antiviral functions. In addition to direct antiproliferative effects, It is known to be effective against various cancers. In recent years, the therapeutic range of drugs for chronic hepatitis B and hepatitis C has been expanding as a major drug. Interferon alpha is produced when white blood cells are stimulated by B cell mitogens, viruses or cancer cells. To date, more than 20 interferon genes have been known to be composed of 165 or 166 amino acids. In addition, interferon alpha contains some lysine in regions that are critical for binding to the receptor, and binding of PEG to one or more major lysine sites results in a decrease in antiviral activity, (Monkarsh el al., Anal Biochem. 247 (1997) 434-440). ≪ / RTI > A conjugate in which a molecule of 40 kDa high molecular weight PEG is bound to a lysine residue of interferon alpha 2a has higher antiproliferative activity of human tumor cells than interferon and other PEG interferon conjugates but antiviral activity is higher than that of original antiviral Which is a 7% level of activity. In addition, when a molecule of a low molecular weight PEG is bound only to the histidine of interferon alpha 2b, it has the disadvantage that stability is low due to the highest sensitivity to hydrolysis while maintaining the original antiviral activity of interferon alpha 2b.

In one embodiment of the invention, the interferon alpha 2b comprises recombinant interferon alpha 2b isolated from mammals and produced in prokaryotic or eukaryotic cells by recombinant DNA technology. The polyalkylene oxide may also be a reactive functional group, such as succinimidyl carbonate, N-succinimide, N-phthalimide, N-glutarimide, which facilitates conjugation of one of the hydroxyl end groups of the polymer. And most preferably monomethoxy-polyethylene glycol-succinimidyl carbonate (mPEG-SC), with a molecular weight of from 500 daltons to 45,000 daltons, preferably 12,000 daltons. The histidine residue is a His ^34, PEG 60 wt%, or 70% or more are bonded to the lysine and tyrosine residues His ³⁴ moiety, is contacted under conditions sufficient to bond to 14 wt.%, Or 10 wt% or more , Wherein the conditions comprise reacting the molecules of interferon alpha 2b at a pH between 8.0 and 8.5 with a molar excess of mPEG-SC at a low temperature.

In one embodiment of the invention, the molar excess of mPEG-SC in the preparation method is 1 to 3 times, the low temperature is 2 ° C to 8 ° C, preferably 3 ° C to 5 ° C, the interferon alpha 2b is preferably from 1 mg / ml to 2 mg / ml. In such a mixture, the PEG-interferon alpha 2b conjugate is defined as a regioisomer along with the amino acid residue to which the PEG molecule is conjugated. One molecule of PEG contains up to about 13 amino acid residues (Lys ³¹ , Lys ⁴⁹ , Lys ⁸³ , Lys ¹²¹ , Lys ¹³¹ , Lys ¹³³ , Lys ¹³⁴ , Lys ¹⁶⁴ , Tyr ¹²⁹ , His ⁷ , His ³⁴ , Cys &^lt; / RTI > ¹ , Ser ¹⁶³ ). This composition contains 14% by weight or less of positional isomers in which PEG is bonded to the histidine residue to enhance the antiviral activity of the mono-PEG-interferon alpha 2b conjugate contained therein, and positional isomers in which tyrosine and PEG are bonded to the lysine residue Is distinguished from the products of the prior art in that it comprises at least 60 wt.

In one embodiment of the invention, methods for preparing PEG-interferon alpha 2b conjugates and complexes or compositions made by such methods are included. The PEG-interferon alpha 2b conjugate is prepared by reacting a solution containing interferon alpha 2b with a sufficient amount of mPEG-SC under conditions suitable for covalently linking PEG to the Lys residue and His residues of interferon alpha 2b. This condition is carried out within a range of reaction conditions (pH, temperature, interferon alpha 2b concentration, molar ratio of interferon alpha 2b and PEG) that are sufficient for covalent attachment of a portion of PEG to the Lys and His residues of interferon alpha 2b . The conditions for performing the covalent binding include performing the conjugation reaction using a relatively small molar amount of mPEG-SC of about 2-fold to 3-fold relative to interferon alpha 2b. This condition also includes performing the reaction at a pH of 8.0 to 8.5. This condition also includes performing the reaction at a temperature of 2 ° C to 8 ° C, preferably 3 ° C to 5 ° C.

In one embodiment of the present invention, a complex is a compound or conjugate that exists in a variety of similar structures, including, but not limited to, the same structural compound, but isomeric, or a compound of the same molecular weight, ≪ / RTI >

In one embodiment of the invention, a method of treating various medical conditions in a mammal, preferably a human, is provided. The method comprises administering to a mammal in need of such treatment an effective amount of a composition comprising an IFN alpha conjugate prepared as described herein. This conjugate is useful, inter alia, for treating conditions that respond positively or positively to interferon-based therapeutic therapies, as is known in the medical arts for interferon-sensitive diseases or these terms. The condition treatable according to the invention is generally a disease susceptible to treatment with interferon alpha. For example, susceptible diseases include those that respond positively or positively to interferon alpha-based therapies as these terms are well known in the medical arts. For the purposes of the present invention, diseases that can be treated using interferon alpha therapy include those disorders in which treatment with interferon alpha has some efficacy, but side effects outweigh the benefits of such treatment do. For example, side effects associated with alpha therapy have substantially eliminated the treatment of Epstein Barr virus using interferon alpha. The practice of the present invention substantially reduces or eliminates side effects compared to conventional interferon alpha therapy.

In one embodiment of the invention, interferon-responsive disease is a disease that can be treated with interferon, including, but not limited to, cell proliferative diseases, particularly cancer (e.g., hair cell leukemia, Kaposi sarcoma, chronic myelogenous leukemia, multiple myeloma , Basal cell carcinoma and malignant melanoma, ovarian cancer and skin T cell lymphoma) and viral infection. At this time, treatment with interferon can be used to treat diseases that may benefit by inhibiting the replication of interferon-sensitive viruses. Viral infections that can be treated according to the invention include hepatitis A, hepatitis B, hepatitis C, other non-A / non hepatitis B, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV) (HTLV-1 / -), human papillomavirus type 1 and type 2 (human papillomavirus type 1, human papillomavirus type 1, papilloma virus, picornavirus, adenovirus, 2), retroviruses including human rotavirus, rabbits, and human immunodeficiency virus (HIV), encephalitis, and respiratory virus infections.

In one embodiment of the present invention, administration means introducing the composition of the present invention to a patient by any suitable method, and the administration route of the composition of the present invention is administered through any conventional route as long as it can reach the target tissue . But are not limited to, oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, intrarectal administration, intraperitoneal administration, intraperitoneal administration, Do not. The method of treatment of the present invention may comprise administering the pharmaceutical composition in a pharmaceutically effective amount. In the present invention, the effective amount may be determined depending on the kind of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of the formulation and the age, body weight, general health condition, sex and diet, And the fraction of the composition, the duration of the treatment, the drug being co-administered, and the like. In the case of an adult, 0.01 ng / kg-10 mg / kg in the case of siRNA, 0.01 ng in the case of the antisense oligonucleotide against mRNA of the gene when the gene expression inhibitor or the protein inhibitor is administered once or several times a day / kg-10 mg / kg, 0.1 ng / kg-10 mg / kg for the compound and 0.1 ng / kg-10 mg / kg for the monoclonal antibody against the protein.

The composition contains the active ingredient in an amount of 0.1 to 50% by weight based on the total weight. The compositions of the present invention may further comprise suitable carriers, excipients or diluents according to conventional methods. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method have. In detail, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant and the like which are usually used. The solid preparation for oral administration includes tablets, pills, powders, granules, capsules and the like. The solid preparation is prepared by mixing at least one excipient such as starch, calcium carbonate (calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin, and the like, but the present invention is not limited thereto.

Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The complex of the present invention overcomes the disadvantages of conventional PEG-interferon alpha 2b conjugates and has high antiviral activity and sensitivity to low hydrolysis.

1 is a chromatogram for the purity (content of di-PEG, mono-PEG and native IFN) analyzed by Size Exclusion HPLC on a complex of an embodiment of the present invention.
FIG. 2 is a chromatogram showing the content of mono-PEG analyzed by size exclusion chromatography of a PEG-interferon alpha 2b conjugate complex in which a complex of an embodiment of the present invention is separated and purified by a cation exchange chromatography method.
3 is a result of analyzing the complex of one embodiment of the present invention by a mass spectrometer (MALDI-MS). As a result, the interferon mass is about 19 kDa and the peginterferon mass is about 31 kDa, so that the PEG mass is 12 kDa.
Figure 4 is a chromatogram showing the positional isomers by analyzing the complexes of one embodiment of the present invention by cation exchange chromatography (Ion Exchange HPLC).
FIG. 5 is a chromatogram comparing the results for the regioisomer of FIG. 4 with the prior art (Wang et al., Advanced Drug Delivery Reviews 54 (2002), 547-570).
Figure 6 is a graph comparing the content of conventional interferon alpha 2b (US Pat No. 5,951, 974) liberated to compare the stability of the formulation of an embodiment of the present invention with an HPLC chromatogram comparing one embodiment of the present invention to be.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Preparation of PEG-interferon alpha 2b conjugates

To about 5 mg of interferon alpha 2b, 50 mM sodium phosphate pH 8.0 buffer solution was added to give a final concentration of 2 mg / ml. Approximately 9 mg of activated monomethoxy-polyethylene glycol-succinimidyl carbonate (mPEG-SC) having a molecular weight of about 12 KDa was added to this solution to react 3 moles of mPEG-SC per 1 mole of interferon alpha 2b. The PEG-interferon alpha 2b conjugate mixture solution was reacted with stirring at 4 ° C for 1.5 hours, and immediately the reaction mixture was brought to pH 4.4 to stop the reaction.

Isolation and purification of PEG-interferon alpha 2b conjugates

The PEG-interferon alpha 2b conjugate mixture was diluted 10-fold with 10 mM sodium acetate pH 4.4 buffer, followed by separation and purification of the PEG-interferon alpha 2b conjugate by cation exchange chromatography. The reaction mixture was analyzed by size exclusion HPLC (see Figure 1).

In addition, the PEG-interferon alpha 2b conjugate mixture was separated and purified by cation exchange chromatography as shown in Table 1 below.

step Buffer solution Volume (CV) Flow rate (CV / min) Column equilibration 10 mM sodium acetate, pH 4.4 5 One Sample loading Reaction mixture - 0.5 Column cleaning 10 mM sodium acetate, pH 4.4 5 One Dissolution 1 140 mM sodium chloride, 10 mM sodium acetate, pH 4.4 7 One Dissolution 2 2000 mM sodium chloride, 10 mM sodium acetate, pH 4.4 7 One Dissolution 3 220 mM sodium chloride, 10 mM sodium acetate, pH 4.4 5 One Dissolution 4 1000 mM sodium chloride, 10 mM sodium acetate, pH 4.4 5 One

Columns were filled with strongly acidic cation exchange resin (SP Sepharose Fast Flow; GE Healthcare) and 10 mM acetic acid sodium buffer (pH 4.4) corresponding to 10 times the column volume (CV) was passed through at 1 CV / min flow rate to equilibrate the column . After equilibration, the reaction mixture was loaded into the column by adjusting the flow rate to 0.5 CV / min. After loading, the flow rate was adjusted to 1 CV / min, and 10 mM sodium acetate buffer solution (pH 4.4) was poured by 5 times the column volume. The PEG-interferon alpha 2b conjugate was eluted with increasing the salt concentration by flowing 10 mM sodium acetate buffer (pH 4.4) containing sodium chloride 140 mM, 200 mM, 220 mM, and 1,000 mM at a flow rate of 1 CV / min , And each fraction was collected and identified by size exclusion chromatography (see FIG. 2).

The PEG-interferon alpha 2b conjugate increased molecular weight by the molecular weight of PEG by conjugating PEG to interferon alpha 2b. Thus, the molecular weight of the purified PEG-interferon alpha 2b conjugate was determined using a mass spectrometer (MALDI-MS) (see FIG. 3).

Analysis of distribution of positional isomers by PEG junction location

The positional isomer composition ratio of the PEG-interferon alpha 2b conjugate mixture purified in Example 1 according to the PEG junction position was measured using ion exchange chromatography (Ion Exchange HPLC). The column used for the cation exchange chromatography was TSKgel SP-5PW (Tosoh) and the mobile phase used a linear concentration gradient with 10 mM sodium phosphate buffer (pH 5.8) and 80 mM sodium phosphate buffer (pH 5.8). After setting the flow rate to 1 ml / min and injecting 100 μl of the sample solution of about 1 mg / ml, the eluted protein was detected at a wavelength of 214 nm. The mobile phase used a linear gradient as shown in Table 2 below.

Time (minutes) Mobile A (%) Mobile phase B (%) 0 100 0 40 0 100 100 0 100

The results of the cation exchange chromatography confirmed that the PEG-interferon alpha 2b conjugate mixture contained about 15 different peaks (see FIG. 4).

These results were compared with the conventional results (see FIG. 5, cited references (Wang et al, Advanced Drug Delivery Reviews 54 (2002) 547-570). The results indicate that the PEG-interferon alpha 2b conjugate of Example 1, Wherein at least about 70% by weight of the conjugate comprises a covalent bond at about 13 or fewer different sites on the alpha 2b molecule, wherein the PEG is bound to the? -Amino group of lysine (K) and tyrosine (Y) % Of the amino acid residues bind to the histidine (H) amino group.

Locate the PEG junction

The positional isomers of the PEG-interferon alpha 2b conjugate were separated by the method of Example 1, and individual peaks were collected. Each of the separated PEG-interferon alpha 2b conjugates was digested with proteolytic enzymes (trypsin, V8-protease, chymodripsin or subtilicin) and the PEG-conjugated fragments were separated by size exclusion chromatography and the N-terminal amino acid Sequence analysis was performed to determine the location of the PEG junction (see Grace et al., J. Biol. Chem. 280 (2005), 6327-6336).

The PEG junction positions of the peaks shown in FIG. 4 were confirmed as shown in Table 3 below.

peak Weight ratio (%) location peak Weight ratio (%) location One 3.91 Dipeg 9 13.06 K ¹²¹ 2 0.11 K ³¹ 10 28.45 K ⁸³ 3 8.31 K ¹²¹ / K ¹³⁴ 11 2.32 H ⁷ 4 1.31 K ¹³³ 12 1.77 K ⁴⁹ 5 1.66 Unknown 13 2.40 S ¹⁶³ , K ¹¹² 6 6.83 H ³⁴ 14 0.45 C ¹ 7 20.74 Y ¹²⁹ 15 2.66 N / A * 8 6.01 K ¹³¹ / K ¹⁶⁴ - - -

* No bonding occurs

These results indicate that PEG is mostly conjugated at one of the lysine amino groups and that the PEG junction at histidine is 14 wt% or less.

Formulation of PEG-interferon alpha 2b conjugates

150 mg of the PEG-interferon alpha 2b conjugate prepared in Example 1, 6.07 mg of sodium-acetate, 0.09 mg of acetic acid, 8.0 mg of NaCl and 0.05 mg of polysorbate 80 0.5 ml, and adjusted to pH 6.0 with NaOH. The following experiment was performed with the above formulation.

Antiviral activity of PEG-interferon alpha 2b conjugate

(CPE) using A549 human lung cancer cells in which the in vitro antiviral activity of the purified PEG-interferon alpha 2b conjugate of Example 1 was saturated with a virus-like stomatitis virus (ATCC NO. VR-129B) Respectively. Antiviral activity is shown in Table 4 as% of starting interferon alpha 2b with corresponding residual activity.

sample Inactive (IU /) Residual activity (%) Control group (interferon alpha 2b only) 2.21 x 10 ⁸ 100 PEG-interferon alpha 2b conjugate
(Example 1) 0.71 x 10 ⁸ 32

Antiproliferative activity of PEG-interferon alpha 2b conjugate

The in vitro antiproliferative activity of the purified PEG-interferon alpha 2b conjugate of Example 1 above was validated in human Daudi (Burkitt's lymphoma) (Borden et al., Cancer Res. 42 (1982) 4948-4953). The antiproliferative activity (IC ₅₀ ) of the PEG-interferon alpha 2b conjugate is shown in Table 5 below.

sample Antiproliferative IC ₅₀ (ng /) Increased activity Control group (interferon alpha 2b only) 0.52 One PEG-interferon alpha 2b conjugate
(Example 1) 0.02 26

As a result, it was found that the antiproliferative activity of the PEG-interferon alpha 2b conjugate of Example 2 was increased as compared with the antiproliferative activity of interferon alpha 2b.

Pharmacokinetics

The blood concentration of the purified PEG-interferon alpha 2b conjugate of Example 1 over time was examined. Sprague-Dawely rats with average weights ranging from 220 g to 240 g were isolated and fed with only 50 g of feed per day for one week. Interferon alpha 2b and the formulated PEG-interferon alpha 2b conjugate were intravenously injected intraperitoneally to the rat at 1, ug / kg, followed by 0, 5, 15, 30, 1, 3, Blood was collected after 24 hours. Serum samples were appropriately diluted and the antiviral activity at each time point was measured as in Table 4 above. At this time, the titer of the analysis condition was calculated by the statistical method using the parallel line test, and the result of the test was applied four times in succession. The plasma concentration trends of the interferon alpha 2b and the formulated PEG-interferon alpha 2b conjugates are shown in Table 6.

sample Concentration (ng /, n = 6) time 0 0.083 0.25 0.5 One 3 5 12 24 Control group
(Interferon alpha 2b only) 0.25 30.50 3.25 0.65 0.25 - - - - PEG-interferon alpha 2b conjugate
(Example 1) 0.27 125 65 26.75 15.50 4.05 1.05 0.48 0.28

Stability experiment

To compare the stability of the formulated PEG-interferon alpha 2b conjugate with the conventional PEG-interferon alpha 2b, the content of the liberated interferon alpha 2b (PEG-interferon alpha 2b) from the PEG- Lt; / RTI > The content of interferon alpha 2b liberated from PEG-interferon alpha 2b is shown in Table 7 as an area ratio (%) of peaks measured by size exclusion HPLC and HPLC chromatogram is shown in FIG. As a result, it was found that the stability of the formulated PEG-interferon alpha 2b conjugate was increased about 1.5 times as compared with the conventional interferon alpha 2b.

sample The formulated PEG-interferon alpha 2b conjugate
(Example 1) Conventional
PEG-interferon alpha 2b) Liberated interferon alpha 2b
(depegylated IFN alpha 2b) 5.15% 7.65%

Claims (21)

In a composition comprising a positional isomeric conjugate linked to an interferon and an alkoxy-terminated polyalkylene oxide,
The binding is carried out at 2-8 [deg.] C, and at a pH of 8.0-8.5,
Wherein the composition comprises 14% by weight or less of a conjugate comprising an alkoxy-terminated polyalkylene oxide bonded to a histidine residue of interferon among all the positional isomeric conjugates, thereby improving the stability, and a pharmaceutical composition for the treatment of interferon-sensitized cancer diseases or viral diseases.
The method according to claim 1,
Wherein said composition is a conjugate wherein at least 60% by weight of the total regioisomeric conjugate is conjugated to an alkoxy-terminated polyalkylene oxide at the lysine and tyrosine residues of interferon alpha.
3. The method of claim 2,
Wherein said regiomeric conjugate is a conjugate covalently bonded to an epsilon-amino group of lysine and tyrosine of interferon alpha.
The method according to claim 1,
Wherein said alkoxy-terminated polyalkylene oxide is polyethylene glycol.
5. The method of claim 4,
Wherein the polyethylene glycol is monomethoxy-polyethylene glycol-succinimidyl carbonate (mPEG-SC).
The method according to claim 1,
Wherein the alkoxy-terminated polyalkylene oxide has a molecular weight of 0.5 to 40 KDa.
The method according to claim 1,
Wherein the interferon is interferon alfa.
3. The method of claim 2,
Wherein said interferon alpha is interferon alpha 2b.
The method according to claim 1,
Wherein the histidine residue is His ³⁴ .
. The method according to claim 1,
Wherein said regiomeric conjugate comprises three or more positional isomers.
11. The method of claim 10,
Wherein said regiomeric conjugate comprises at least 5 positional isomers.
11. The method of claim 10,
Wherein said regiomeric conjugate comprises no more than 15 positional isomers.
12. The method of claim 11,
The regioisomer is selected from the group consisting of His ³⁴ , Tyr ¹²⁹ , Lys ¹³¹ , Lys ¹²¹ , and Lys ⁸³ .
delete delete The method according to claim 1,
The viral diseases are selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, non-A / non hepatitis B, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, (HHV-6), papilloma, poxvirus, picornavirus, adenovirus, linovirus, human T-leprosy virus type 1 and 2 (HTLV-1 / -2), human rotavirus , Rabbits, retroviruses, encephalitis, and respiratory virus infections.
The method according to claim 1,
Wherein said cancer is any one selected from the group consisting of hair cell leukemia, Kaposi sarcoma, chronic myelogenous leukemia, multiple myeloma, basal cell carcinoma, malignant melanoma, ovarian cancer and skin T cell lymphoma.
Subjecting interferon and alkoxy-terminated polyalkylene oxide to a coupling reaction at a molar ratio of 1: 1 to 1: 5 at a pH of from 8.0 to 8.5; And
Lt; 0 > C to 8 < 0 > C,
Wherein the total isomeric conjugate prepared after the stirring comprises interposon and alkoxy-terminated polyalkylene oxide conjugated positional isomer conjugate wherein the amount of the conjugate in which the alkoxy-terminated polyalkylene oxide is bonded to the histidine residue of interferon is 14% Or a pharmaceutically acceptable salt thereof, for the manufacture of a pharmaceutical composition for the treatment of interferon-sensitive cancer or viral diseases.
19. The method of claim 18,
Wherein the stirring is performed for 1 to 3 hours. ≪ Desc / Clms Page number 20 >
20. The method of claim 19,
Further comprising the step of adjusting the pH of the reaction mixture to 4 to 5 after the stirring to terminate the reaction. ≪ RTI ID = 0.0 > 18. < / RTI >
21. The method of claim 20,
A method for preparing a pharmaceutical composition comprising a positional isomer-linked conjugate of interferon and alkoxy-terminated polyalkylene oxide, further comprising the step of performing purification by performing cation exchange chromatography after the stirring

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