Conjugates of interferon

 

(57) Abstract:

The invention relates to medicine, in particular to the conjugates of interferon formula I

< / BR>
where R and R', independently of one another, represent lower alkyl; X represents NH or O; n and n' are integers, the sum of which ranges from 600 to 1500; and the average molecular weight parts of polyethylene glycol in the conjugate is from 26000 to 66000 Yes, and to a method for producing a conjugate of PEG--IFN possessing antiproliferative activity, as well as to a method of treatment or prevention immunomodulatory diseases. Conjugate PEG--IFN has an extended half-life in blood flow and residence time in plasma, reduced immunogenicity, reduced clearance and increased antiproliferative activity, which is accompanied by reduced antiviral activity. 5 S. and 9 C.p. f-crystals, 6 ill., table 4.

Interferon, in particular 2a-interferon is a pharmaceutically active protein, which has antiviral and antiproliferative activity. For example, the interferon is used to treat leukemic reticulata and Kaposi's sarcoma, and it also shows activity against HBV. To improve stability is to be conjugated to the polymer polyethylene glycol (PEG).

Biological availability of therapies with the use of proteins is often limited by their short half-lives in plasma, which prevents the achievement of their maximum therapeutic action. In recent years, it has been confirmed that PEG-conjugated to biological molecules possess useful therapeutic properties (Inada and others, J. Bioact. and Compatible Polymers 5, 343 (1990); Delgado and others, Critical Reviews in Therapeutic Drug Carrier Systems 9, 249 (1992); Katre, Advanced Drug Delivery Systems 10, 91 (1993). They have the best physical stability and resistance to heat, protected (insensitive) from enzymatic degradation have increased solubility, longer half-life in blood flow in vivo, reduced clearance and increased efficiency. Published evidence that branched PEG-conjugates have a higher pH value and temperature resistance and greater stability towards proteolytic degradation compared to linear PEG-conjugates (Monfardini and others, Bioconjugate Chem. 6, 62 (1995)). Other properties Paglierani proteins are reduced immunogenicity and antigenicity, as well as reduced toxicity. Another effect of Paglierani specific proteins can be reduced activity in vitro, followed by Uwe the Satake-Ishikawa and others, Cell Structure and Function 17, 157-160 (1992), IL-2 (interleukin-2) (Katre and others , the OEWG. Natl. Acad. Sci. USA 84, 1487 (1987)), TNF- (tumor necrosis factor) (Tsutsumi and others, Jpn. J. Cancer Res. 85, 9 (1994)), IL-6 (interleukin-6) (Inoue and others, J. Lab. Clin. Med. 124, 529 (1994)) and CD4-IgG (antigen T-cell immunoglobulin G) (Chamow and others, Bioconj. Chem. 5, 133 (1994)), and others.

It was found that in the case of interferon Pegylation reduces antiviral activity in vitro, but increases antiproliferative activity against tumor cells of human rights. However, the new PEG-conjugated interferon of the present invention have unexpected properties, which consists in the fact that the antiproliferative activity of PEG-interferon significantly higher than not only that of the interferon, but other PEG-conjugates of interferon. Although the antiproliferative activity of the conjugate was significantly increased compared with the activity of other PEG-conjugates of interferon, however, in this case, there is a similar reduction in antiviral activity. In addition, PEG-conjugate-interferon according to the present invention has no immunogenicity, resulting in fact in his introduction antibodies are formed. In contrast, other PEG-conjugates of interferon in certain Steg-derivatives-interferon (-IFN). The conjugate of the present invention, as described below, includes PEG, which has a branched structure. Branched PEG has the advantage that it has the ability to join two molecules of linear PEG at a single point, thus doubling the mass of the attached PEG and without increasing the number of points Paglierani.

Compared to the unmodified-IFN (i.e.- IFN without an attached PEG) conjugate has an increased half-life in blood flow and residence time in plasma, reduced immunogenicity, reduced clearance and increased antiproliferative activity, which is accompanied by reduced antiviral activity in vitro. The conjugate of the present invention in comparison with other conjugates PEG--IFN has a significantly higher antiproliferative activity unrelated proportional dependence with increased or decreased other characteristics, and in fact has no immunogenicity.

Physiologically active conjugate PEG--IFN according to the present invention have the formula:

< / BR>
The conjugate of the present invention has the same applications as-IFN, such as antiproliferative funds. In Cheney, such as diseases related to the tumor, for example, leukemia retikulez, chronic myelogenous leukemia (CML) and Kaposi's sarcoma, and infectious diseases using the same method, which is used to treat these diseases-interferon (in particular 2a-IFN). However, the conjugate of the present invention has improved properties, including better stability, higher solubility, increased half-life in the bloodstream and increasing the residence time in the plasma. In addition, these conjugates possess antiproliferative activity superior to that of a-IFN. It was also confirmed that the conjugate exhibits an unexpected division of antiviral and antiproliferative action. This property provides an additional advantage in case you want to strengthen a specific activity of the conjugate and at the same time to reduce or eliminate unwanted activity. For example, if the unwanted side effects associated with antiviral activity, the elimination of this activity will be to eliminate this side effect, while maintaining the antiproliferative activity. Thus, the present invention also includes pharmaceutical compositions based on compounds of the tion, used to treat or prevent diseases include conjugate of interferon General formula I and a therapeutically inert non-toxic and therapeutically acceptable carrier. Designed for use in the pharmaceutical compositions can be prepared and measured in accordance with accepted medical practice, taking into account subject to the treatment of the disease, the condition of the individual patient, the site of delivery of the conjugate protein, the method of administration and other factors known to specialists in this field of technology.

The conjugate of the present invention is a physiologically active conjugate PEG--IFN those having the formula:

< / BR>
where R and R', independently of one another, represent lower alkyl; X represents NH or O (X denotes at least one of the functional groups in the molecule is IFN selected from NH2or HE); n and n' are integers, the sum of which ranges from 600 to 1500; and the average molecular weight parts of polyethylene glycol in the conjugate is from about 26000 Yes to approximately 66000 Yes. The conjugate of formula I has a branched structure in which two fragments of PEG attached to the protein via a simple link.

The numbers n and n' you is this activity may be the same higher or be a part of the activity of unmodified-IFN. The numbers n and n' (with n and n' may be the same or different) represent the number of units of ethylene glycol in PEG. One link PEG, co2CH2has a molecular weight of approximately 44 Yes. Molecular weight of the conjugate (except for molecular weight-IFN) depends on the numbers n and n'. The sum of n and n' for a conjugate of formula I is from 600 to 1500, which results in obtaining a conjugate having a total average molecular weight of the links of the PEG from approximately 26000 up to 66000 and preferably from approximately 35,000 to 45,000 Da, in particular from approximately 39000 to 40,000 Da, and especially preferred molecular weight of 40,000 Da. The preferred amount of n and n' ranges from approximately 800 to 1,200, and the average amount is from about 850 to 1000, and the preferred amount is approximately 910. Each of the numbers n and n' independently may be equal to 420 or 520, or both can be equal to 420 or 520, or both can be equal to 455. The preferred ratio of n to n' can be from about 0.5 to 1.5, with the most preferred ratio is from about 0.8 to about 1.2. The concept of "when the acceptable region this value, according to standard analytical methods.

Also preferred is a conjugate of formula I, in which-IFN is a 2a-IFN, conjugate, in which R and R' represent methyl, conjugate, in which X represents NH, and conjugate, in which n and n' each or both together equal or 420 or 520. Such a conjugate having the above characteristics, is especially preferred.

R and R' can denote any of the lower alkyl represents an alkyl group having from 1 to 6 carbon atoms, such as methyl, ethyl, isopropyl, etc. This group also includes extensive alkali. The preferred alkyl is methyl. In two groups the PEG formula I R and R' may be the same or different.

Under-IFN (interferon) and its varieties 2a-IFN understand natural or recombinant protein, preferably human, derived from any conventional source, such as the tissue by chemical synthesis of the protein from cell culture using native and recombinant cells. Under the scope of the present invention is subject to any protein having the activity-IFN including mutiny or modified in some other way proteins. M is. 21, 1 (1983)). Preferred-IFN, as indicated above, is 2a-IFN produced by known methods (Pestka, Sci. Am. 249, 36 (1983); European patent 43980)).

Physiologically active conjugate of formula I have activity-IFN, which is defined as any partial or enhanced well-known activity-IFN determined in various ways known in the art. In particular, the conjugates of the present invention have activity-IFN, namely, as shown antiproliferative activity against tumor cells and antiviral activity against cells infected with the virus. These activities are known activities, svojstvennymi-IFN. This activity of the conjugate can be determined by methods well known in the art, for example, the methods described below (see , also Rubinstein and others, J. Virol. 37, 755 (1981); Borden and others, Cane. Res. 42, 4948 (1982)). One of the objects of the present invention is a conjugate of formula I, with a higher antiproliferative activity and less antiviral activity compared to the unmodified-IFN.

The conjugate of formula I are obtained by covalent binding-IFN with PEG, which can be activated by substitution of hydroc the de N-hydroxysuccinimide ether (in particular monomethoxy-PEG) of formula II. The reagent can be obtained by conventional methods (Monfardini and others , see above). Binding occurs through an amide or ether linkage. In the preferred conjugate binding occurs through an amide bond (X represents NH). One of the objects of the present invention is a method of strengthening the antiproliferative activity of interferon while reducing antiviral activity-IFN by linking-IFN, as described above, with a reagent of formula II with obtaining conjugate PEG-IFN.

X indicates the place of attachment of on-IFN, through which the PEG-reagent of formula II is covalently associated with a-IFN. The reagents are attached to the primary amino groups (HN = NH2), for example, lysine or N-ends-IFN. The reagents can also be attached to the hydroxyl (HN = HE), for example, serine (see diagram 1 at the end of the description).

The reagent of formula II (PEG-NHS), which in General two circuit monomethoxy-PEG (m-PEG) linked with lysine, each with - and-amino groups via urethane (urethane) links and which contains the carboxyl group of lysine activated to Succinimidyl ether may be obtained by conventional methods in accordance with known methods (Monfardini and others, see above) applicable to the reagent, in which R obozrenie, pieces Alabama). The preferred average molecular weight of the obtained PEG is approximately 20000 Yes that provides total weight of PEG in PEG-NHS approximately 40000 Yes (polymers with other molecular weights can be obtained by traditional methods by changing the value of n in the source materials for the reagent of formula II, representing alcohol PEG).

The reagent of formula II can be anywhereman with-IFN traditional ways. In particular, the reagent of formula II is first subjected to interaction with one or more primary amino groups (for example, with N-terminal groups or side chains of lysine) -IFN (for example, 2a-IFN) for the formation of amide bond between a-IFN and polymer-based PEG. The reaction Paglierani can also occur between PEG-NHS and free (if present) with hydroxyl groups (e.g., serine) -IFN with the formation of ester bonds. The reaction mechanism described above. Reaction conditions are common for experts in the field of technology and in more detail below. The PEG-reagent together with a-IFN in mean conditions at low temperature and under conditions suitable for nucleophilic substitution, resulting conjugate is estreno traditional methods. Can be used with PEG of the present invention with any of the selected molecular masses. Reaction conditions can be chosen in such a way as to obtain the conjugate according to the invention with one attached reagent. The conjugate of formula I, to which is attached one reagent of formula II, separated from unmodified-IFN and conjugates that have more than one attached molecules of the reagent, the traditional ways.

To separate conjugates on the basis of differences in charges may apply cleaning methods, such as cation exchange chromatography, which allows effective sharing of the conjugates on the basis of their different molecular masses. The content of fractions obtained by cation-exchange chromatography, can be determined by molecular weight using accepted methods, for example, using mass spectroscopy, electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS page-ordinator) or other known techniques used for molecular separation of enantiomers on molecular weight. Then accordingly determine the fraction, which contains a conjugate of formula I, purified from unmodified-IFN and conjugates, and the support one molecule of lysine

molecule of the reagent, so that the number of molecules of lysine during the hydrolysis shows the number of PEG groups attached to the protein, and thus can be confirmed by the number of molecules of the reagent attached to the conjugate.

Below Sobranie illustrated in the examples which do not limit its scope. In these examples 2a used-IFN. Other species-IFN can also be conjugated to PEG by the methods described in the examples.

Description of drawings

Fig. 1: Antitumor activity PEG-2a-IFN, studied in deprived fur mice that were subcutaneously implanted renal cells lines A. All animals for 33 days before the start of the experiment (day 33) was subcutaneously injected implant containing 2x106renal human cell lines A. Processing using PEG-2a-IFN was started on day 0 of the experiment. The given amounts (30, 60, 120 or 300 µg) PEG-2a-IFN was administered subcutaneously in the opposite relative to the tumor side, 1 time per week during the 4-week period.

Fig. 2: Antitumor activity 2a-IFN, studied in deprived fur mice that were subcutaneously implanted renal cells lines A. All animals for 33 days before the start of the experiment (day 33) is started on day 0 of the experiment. The given amounts (10, 20, 40 or 100 µg) 2a-IFN was administered subcutaneously in the opposite relative to the tumor side, 3 times a week during the 4-week period.

Fig. 3: Antitumor activity PEG-2a-IFN, studied in deprived fur mice that were subcutaneously implanted renal cells lines ACHN. All animals 25 days before the start of the experiment (day 25) were injected subcutaneously implant containing 2x106renal human cell line ACHN. Processing using PEG-2a-IFN was started on day 0 of the experiment. The given amounts (30, 60, 120 or 300 µg) PEG-2a-IFN was administered subcutaneously in the opposite relative to the tumor side, 1 time per week during the 5-week period.

Fig. 4: Antitumor activity 2a-IFN, studied in deprived fur mice that were subcutaneously implanted renal cells lines ACHN. All animals 25 days before the start of the experiment (day 25) were injected subcutaneously implant containing 2x106renal human cell line ACHN. Treatment with 2a-IFN was started on day 0 of the experiment. The given amounts (10, 20, 40 or 100 µg) 2a-IFN was administered subcutaneously in the opposite relative to the tumor side, 3 times a week during the 5-week period.

Fig. 5: Protivoopujolevy man line G402. All animals for 45 days before the beginning of the experiment (day 45) were injected subcutaneously implant containing 2x106renal human cell lines G402. Processing using PEG-2a-IFN was started on day 0 of the experiment. The given amounts (30, 60, 120 or 300 µg) PEG-2a-IFN was administered subcutaneously in the opposite relative to the tumor side, 1 time per week during the 5-week period.

Fig. 6: Antitumor activity 2a-IFN, studied in deprived fur mice that were subcutaneously implanted renal cells lines G402. All animals for 45 days before the beginning of the experiment (day 45) were injected subcutaneously implant containing 2x106renal human cell lines G402. Treatment with 2a-IFN was started on day 0 of the experiment. The given amounts (10, 20, 40 or 100 µg) 2a-IFN was administered subcutaneously in the opposite relative to the tumor side, 3 times a week during the 5-week period.

Example 1

Obtaining a conjugate of the formula I

Materials

2a-Interferon was obtained by known methods (Pestka, see above). Polietilenglikolya (PEG) reagent of formula II was purchased from the firm's Shearwater Polymers, Inc. (Huntsville, PCs Alabama). Resin FractogelEMD CM 650(S) with a particle size of 25-40 μm, supplied by the firm EAT Separations (Gibbstown, ioWhittaker (Walkersville, MD). Sets of gels for electrophoresis in polyacrylamide gel in the presence of dodecyl(lauryl) sulfate sodium (PAG-ordinator) and devices for electrophoresis were purchased from company NOVEX (San Diego, PCs California). The concentrated dye Fast Stain for staining protein PEG-conjugates during electrophoresis in SDS page-ordinator was purchased from the firm Zoion Research, Inc. (Newton, MA). Kit for determination of endotoxin in the test using a lysate of amoebocytes (LAL-test) was purchased from company Associates of Cape Cod, Inc. (Woods Hole, MA). All other used chemicals were of the highest available quality. Rats with cannulae, implanted in the jugular vein, and line mouse BDF-1 were purchased from the firm Charles River Laboratories (Wilmington, MA).

The experimental technique

A. Small-scale obtaining a conjugate of the formula I

208 mg (5.2 mmol) of the reagent of formula II (average molecular weight of 40,000 Da) was added to 50 mg (2.6 μmol) -IFN in 10 ml of 100 mm borate buffer, pH 8.0. The final molar ratio of protein:reagent was 1: 2. The reaction mixture was stirred at 4oC for 2 hours. The reaction was stopped by bringing the pH to 4.5 using glacial acetic acid.

The reaction mixture was 50-fold diluted with water, filtered through the spine of flow 20 ml/min The column was pre-balanced with 10 mm ammonium acetate, pH 4.5. Emerging from the column, the product was analyzed using UV absorbance at 280 nm. Then the column washed with equilibrating buffer until until UV absorbance returned to baseline level. The conjugates of PEG-IFN with more than one attached to the reagent of formula II (oligomers of PEG-IFN) were suirable with 40 mm ammonium acetate, pH 4.5, and the conjugate of formula I has suirable with 0.12 M NaCl in 40 mm ammonium acetate buffer. Remaining on the column unmodified IFN was suirable with 0.5 M NaCl in the same buffer. The column was regenerated by washing with 1.0 M NaCl, followed by rinsing equilibrating buffer. Combined fractions conjugates of formula I concentrated using a vacuum filter for mixed cell type Amicon, equipped with a YM10 membrane type, to the concentration of approximately 1 mg/ml

Applied for treatment of cation exchange resin Fractogel 650 CM(S) was effective in adsorbing PEG and unmodified IFN. The intensity of adsorption depended on the degree of Paglierani. The conjugates were associated less strongly compared to the unmodified IFN. Oligomers of PEG-IFN was suirable with 40 mm ammonium acetate, while preparations contained <5 in this case (endotoxin units)/mg of endotoxin. The resulting preparation contained >99% of the conjugate of formula I and was deprived of unmodified IFN.

B. large-Scale obtaining a conjugate of the formula I

6240 mg (156 mcmole) reagent of formula II (average molecular weight of 40,000 Da) was dissolved in 4oWith 63 ml of 1 mm model HC1 was added to 125 ml of a solution containing 1000 mg (52 μmol) of interferon in 50 mm borate buffer, pH 9.0 in. The final ratio of protein/reagent was 1:3, and the final concentration of protein in the reaction mixture was 5.3 mg/ml of the Reaction mixture was stirred at 4oC for 2 hours. The reaction was stopped by bringing the pH to 4.5 using glacial acetic acid.

The reaction mixture was 10-fold diluted with water and applied on a column Packed with 600 ml of resin Fractogel EMD CM 650 (M) and pre-equilibrated to 20 mm sodium acetate buffer, pH 4.5, at a linear velocity of 1.3 cm/min. and the Column washed with equilibrating buffer and then with 10 mm NaCl to remove excess reagent and by-products of the reaction and oligomers of PEG-IFN. The conjugate of formula I has suirable with an equilibrium buffer containing 200 mm NaCl. Unmodified interferon, yet preserved in the column were removed by washing 0.75 M NaCl in equilibrating buffer. about the preparation of a medicinal product was filtered by dialysis in 20 mm sodium acetate buffer, pH 5.0, containing 150 mm NaCl. The overall yield of the conjugate of formula I was 40-45%.

Purified PEG-IFN from the drug in krupnomasshtabno method of obtaining consists of more than 99% of the conjugate of formula I. the Average molecular weight of the conjugate of formula I of this example is 62000 Yes, including the molecular weight of 2a-IFN, which is equal to 19241 Yes, and the average molecular weight of the reagent, which is between 40000 and 45000 Yes, comprising approximately 43000 Yes.

Example 2

Characterization of the conjugate of formula I

Determination of protein

The concentration of protein was determined using the value AS280equal to 1, for a solution with a concentration of 2a-IFN 1 mg/ml

Analysis using SDS page-ordinator

The conjugate was analyzed by electrophoresis in polyacrylamide gel (8-16%) in the presence of dodecyl(lauryl) sulfate sodium reduction conditions in accordance with the methods of Laemmli (Nature 227, 680 (1970)). PAG-LTO-gels containing PEG-conjugates, were stained for determination of protein using the dye Fast Stain (firm Zoion Research, Inc.), in accordance with the manufacturer's instructions.

Determination of the levels of endotoxin

The levels of endotoxin was determined using the LAL test in sootvetstyovujushchie activity in vitro conjugate of the formula I

Antiviral activity in bovine kidney cells

Antiviral activity in vitro 2a and IFN conjugate of formula I, obtained as described in example 1.And the method was determined using the biological analysis of cell culture, using bovine kidney cell line Madin-Darby (MDBK), infected with vesicular stomatitis virus (Rubinstein and others , see above). Indicators of antiviral activities listed in table 1 along with the corresponding residual activity in% in relation to the original IFN.

Antiproliferative activity in vitro on tumor cells of a person

Antiproliferative activity in vitro was determined in a human cell line Daudi (Burkitt's lymphoma), as described by Borden and other Human cell lines Daudi supported in a stationary suspension cultures in RPMI medium 1540, supplemented with 10% fetal bovine serum and 2 mm glutamine (Grand Island Biologicals, Grand island, PCs, new York. Cells were subjected to screening and found that they are deprived of Mycoplasma. Cells (2x104) were added to the wells tablets for micrometrology (Costar, MA) with 100 ál of medium. Various concentrations of IFN and the conjugate of formula I, obtained as described in example 1.And technique, was added to the wells of 100 MK is Ali3H-thymidine (New England Nuclear, Boston, MA) at a dose of 0.25 µci/well. Cells were collected on glass filters and measured the radioactivity using a liquid scintillation counter. The results were expressed as % inhibition, calculated by the formula:

% inhibition = [(A-b)/a]X100,

where And denotes the number of pulses per minute in the control culture (cells incubated in a clean environment);

In denotes the number of pulses per minute in the experimental culture.

Experiments were performed in four replicates, and the standard deviation in all cases was less than 20% from the average value. The experiments were repeated at least twice, obtaining comparable results.

Antiproliferative activity (IC50) And IFN conjugate of formula I are given in table 2. The data show that there is a 28-fold increase in antiproliferative activity of the conjugate of formula I in comparison with that of IFN.

Example 4

Pharmacokinetics

Female rats Sprague Dawley with an average body weight 240-260 g, which is surgically implanted cannula in the jugular vein, were placed individually, providing free access to food and water and were kept at 1. the next day after the introduction of a cannula of 0.15-0.2 ml SPR rats were injected with 2x106units-IFN in 0.2-0.4 ml SPR, and then were injected with 0.15 to 0.2 ml SPR to ensure receipt of the entire amount of the drug in the body of the animal. Thus, each animal received a dose 8x106units-IFN/kg of body weight.

After 5, 15 and 30 minutes, and 1, 3, 5, 12 and 24 hours after injection and IFN conjugate of formula I selected blood samples. After discarding the first portion of the blood volume of 0.15-0.2 ml via the jugular cannula every time selected aliquot of the blood volume of 0.5 ml using a new syringe. At room temperature, the samples were placed in test tubes to separate the serum. Once all samples at a given moment of time were collected, the tubes were centrifuged at 14000xg for 10 minutes in an Eppendorf centrifuge with cooling. The separated serum was transferred into microcentrifuge tubes 1.5 ml and frozen at -80oTo perform biological analysis. Serum samples appropriately diluted and for each time point was determined antiviral activity, as described previously. From the plot of activity with time was determined concatena its location in the plasma.

The final period t1/2was determined by the method of linear regression in the logarithmic scale.

Example 5

Immunogenicity

Normal mice BDF-1 (ten animals per group) were injected with intraperitoneal once a day five times a week various interferon preparations containing 300000 units of antiviral activity. Some mice were also injected with the compound form 2a-IFN, which is more immunogenic than the Monomeric form. Blood samples were taken on the 19th day after the last injection and analyzed the serum for the presence of neutralizing antibodies.

As can be seen from table 4, the mice that were injected with 2a-IFN, developed neutralizing antibodies, and this reaction was strongly increased in mice that were injected with the compound form of interferon. In most animals, which were injected with the conjugate of the present invention, was not detected antibodies.

Example 6

Antitumor activity in vivo

Antitumor activity in vivo conjugate of formula I (PEG-2a-IFN) and unmodified 2a-IFN was assessed by determining their ability to reduce the size of the various human tumor cells, subcutaneously implanted in mice. The results show is over, was subcutaneously injected into the back of the left side of the implant containing 2x106renal human cell lines A (Fig. 1 and 2), renal human cell lines ACNH (Fig. 3 and 4) or renal human cell lines G402 (Fig. 5 and 6). Within 3-6 weeks, tumors were allowed to be as follows. The criterion of size, appropriate for the study ranged from 0.05 to 0.5 cm3. The mice were injected total daily dose PEG-2a-IFN or unmodified 2a-IFN, components 30, 60, 120 or 300 µg. If PEG-2a-IFN mice were treated once a week (on Monday), using 30, 60, 120 or 300 µg PEG-2a-IFN for processing. In the case of unmodified 2a-IFN mice were treated three times per week (Monday, Wednesday and Friday), using 10, 20, 40 or 100 µg 2a-IFN for processing. The processing time was 4-5 weeks depending on the intensity of growth (aggressive) tumors. The tumor volumes were measured every Monday to treatments.

Results: PEG-2a-IFN resulted in a marked decrease in tumor size induced cells A, compared to the unmodified 2a-IFN for all studied levels of weekly doses for 7 day, 14 day, 21 day and 28 day after the start of treatment (Fig. 1 and 2). Treatment was continued for 4 h GAWSIS processing PEG-2a-IFN, found no residual tumor. In mice exposed to processing unmodified 2a-interferon, tumor weight, caused by cells A amounted to 1.28 g, 0,62 g and 1.60 g, respectively, at each of the three mice. The weight of tumors caused by cell A, totalled 2.32 g, 2.37 g and of 1.94 g of each of the three mice in the control. 80 the day after the end of the four-week period of treatment the tumors was determined by palpation of the seven mice. Palpation in all seven mice did not reveal the presence of neoplastic tissue.

PEG-2a-IFN resulted in a significant decrease in tumor size induced cells ACNH, compared to the unmodified 2a-IFN at weekly doses of 60, 120 and 300 μg per 14 day 21 day 28 day 35 day (Fig. 3 and 4).

PEG-2a-IFN resulted in a significant decrease in tumor size induced cells G402, compared to the unmodified 2a-IFN at weekly doses of 60 and 120 mcg per 14 day 21 day 28 day 35 day (Fig. 5 and 6).

1. Physiologically active conjugate PEG-IFN--, having formula

< / BR>
where R and R' independently of one another represent lower alkyl;

X represents NH or O;

n and n' are integers, the sum of which ranges from 600 to 1500,

and the average molecular weight of the links of the popular weight parts of polyethylene glycol is from 35000 to 45000 Yes.

3. Conjugate under item 2, in which the molecular weight parts of polyethylene glycol is 40000 Yes.

4. Conjugate under item 1, in which R and R' represent methyl.

5. Conjugate under item 1, in which X represents NH.

6. Conjugate under item 1, in which IFN is a 2a-IFN.

7. Conjugate under item 1, in which the average sum of n and n' ranges from 850 to 1000.

8. Conjugate under item 1, in which R and R' denotes methyl, X denotes NH, -IFN is a-2a-IFN and one or both n and n' is equal to 420.

9. Conjugate under item 1, in which R and R' denotes methyl, X denotes NH, -IFN is a-2a-IFN and one or both n and n' is equal to 520.

10. Conjugate under item 1, having antiproliferative activity, surpassing that of y-IFN and reduced antiviral activity compared with IFN.

11. A method of obtaining a conjugate PEG--IFN possessing antiproliferative activity exceeding that of y-IFN and reduced antiviral activity compared with IFN, namely, that covalently bind to the reagent of formula II

< / BR>
c-IFN getting conjugate PEG--IFN.

12. Pharmaceutical composition having anti-proliferative and anti-virus activity, containing conjugate PEG--IFN according to any one of paragraphs. 1-10 is an immune-based disorders, such as diseases related to tumors and infectious diseases, containing conjugate PEG--IFN according to any one of paragraphs. 1-10 and a therapeutically inert carrier.

14. Method for the treatment or prevention of immune-based diseases, including the introduction of conjugate PEG--IFN according to any one of paragraphs. 1-10.

 

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