MXPA97004012A - Conjugados de interfe - Google Patents

Abstract

Physiologically active PEG-IFNalfa conjugates, with the following formula (

Description

INTERFERON CONJUGATES DESCRIPTION OF THE INVENTION Interferon, in particular interferon a2a, is a pharmaceutically active protein having an antiviral and antiproliferative activity. For example, interferon is used to treat capillary cell leukemia and Kaposi's sarcoma and is active against hepatitis. In order to increase their stability and solubility and reduce immunogenicity, pharmaceutically active proteins such as interferon can be conjugated with the polyethylene glycol (PEG) polymer. The bioavailability of protein therapeutics is often limited due to the short period of half-life, thus preventing them from developing their clinical potency to the fullest. In recent years it has been shown that biomolecules conjugated with PEG possess useful clinical properties (Inada et al., J. Bioact. And Compatible Polymers 5, 343 (1990); Delgado et al., Critical Revie s in Therapeutic Drug Carrier Systems. 9, 249 (1992), - Katre, Advanced Drug Delivery Systems 10, 91 (1993)). Among these, they have better physical and thermal stability, protection against susceptibility to enzymatic degradation, greater solubility, longer period of circulating half-life in vivo, less clearance and greater potency. It has been reported that branched PEG conjugates have a higher pH and thermal stability and greater stability to proteolytic digestion than linear PEG conjugates (Monfardini et al., Bioconjugate Chem. 6, 62 (1995)). Other properties of PEG proteins are reduced immunogenicity and antigenicity, as well as reduced toxicity. Another REF: 24605 effect of PEGuylation of certain proteins may be a reduced in vitro activity accompanied by enhanced in vivo activity. This has been observed in G-CSF (Satake-Ishikawa et al., Cell Structure and Function 17, 157-160 (1992)), IL-2 (Katre et al., Proc. Nati. Acad. Sci. USA 84, 1487 (1987)), TNF-a (Tsutsumi et al., Japan J. Cancer Res. 85, 9 (1994)), IL-6 (Inoue et al., J. Lab. Clin. Med. 124, 529 ( 1994)) and CD4-IgG (Chamo et al., Bioconj. Chem. 5, 133 (1994)), among others. It has now been observed that in the case of interferon, PEGuylation reduces in vitro the antiviral activity but increases the antiproliferative activity of human tumor cells. However, the novel PEG interferon conjugate of this invention has surprising properties since the antiproliferative activity of PEG interferon is much greater not only than interferon but other conjugates of PEG interferon. Although the anti-proliferative activity of the conjugate is greatly increased over other PEG-terferon-oi conjugates, the reduction in antiviral activity is still similar. In addition, the PEG interferon-a conjugate of this invention is non-immunogenic, virtually induces the formation of antibodies. On the contrary, other PEG interferon-a conjugates induce limited antibody formation. Accordingly, the invention constitutes a new class of PEG derivatives of interferon (IFNa). The conjugate of the present invention has a branched PEG structure, as can be seen below. The branched PEG has the advantage that it allows the union of 2 linear molecules of PEG at an individual site, thus doubling the mass of PEG bound without the need for multiple pegylation sites. Compared with unmodified IFNa (ie, IFNa without a bound PEG), the conjugate has a longer period of circulating half-life and a longer residence time in plasma, a lower immunogenicity, a lower clearance and a higher antiproliferative activity, simultaneously with less antiviral activity in vi tro. In comparison with other PEG-IFNa conjugates, the conjugate of this invention has a much higher antiproliferative activity, disproportionate to the increase or reduction that occurs with its remaining characteristics, and virtually no immunogenicity. The physiologically active PEG-IFNa conjugate species of the present invention have the formula: The conjugate of the present invention has the same uses as IFNa, for example, in antiproliferative applications. In particular, the PEG interferon-a conjugates of the present invention are useful for treating immunomodulatory disorders such as neoplastic diseases, for example, capillary cell leukemia, CML, Kaposi's sarcoma, and infectious diseases, in the same way Different IFNa (especially IFNa2a) are used to treat these diseases. However, the conjugate of the present invention has increased certain properties including superior stability, increased solubility, a circulating half-life and enhanced plasma residence times. In addition, these conjugates have an antiproliferative activity that is superior to that of IFNa. Also as it was said, the conjugate presents a surprising dissociation of antiviral and antiproliferative effects. This property is additionally useful for enhancing a desired activity of a conjugate, while decreasing or eliminating an undesired activity. For example, if an unwanted side effect is associated with an antiviral activity, the elimination of this activity would eliminate the side effect, while maintaining the antiproliferative activity. Therefore, the present invention also comprises pharmaceutical compositions made from compounds of formula I or their salts and the methods for producing them. The pharmaceutical compositions of the present invention employed for the control or prevention of diseases comprise an interferon conjugate of general formula I and a therapeutically inert, non-toxic and therapeutically acceptable loading material. The pharmaceutical compositions "Which are used can be formulated and dosed in a manner consistent with good medical practice taking into account the disorder to be treated, the condition of the individual patient, the place of delivery of the protein conjugate, the method of administration and other known factors. the specialists . The claimed conjugate is the physiologically active PEG-IFNa conjugate, of formula: wherein R and R 'are independently lower alkyl; X is NH or 0 (X is at least one of the functional groups of the IFNa molecule selected from NH2 or OH); n and n 'are integers whose sum is 600 to 1500; and the average molecular weight of the polyethylene glycol units in said conjugate is about 26,000 daltons to about 66,000 daltons. The conjugate of formula I has a ched structure in which two PEG units are bound to the protein by a single bond. The numbers n and n 'are selected so that the resulting conjugate of formula I has a physiological activity of IFNα, which activity can represent the same as, rather than, or a part of, the corresponding activity of the unmodified IFNα, nyn' ( n and n 'can be the same number or different numbers) represent the number of ethylene glycol units in the PEG. An individual PEG unit of OCH-CH- has a molecular weight of about 44 daltons. The molecular weight of the conjugate (excluding the molecular weight of IFNa) depends on the numbers n and n '. The sum of nyn 'for the conjugate of formula I is 600 to 1500, whereby a conjugate having a total average molecular weight of PEG units is obtained from about 26,000 to 66,000, preferably from about 35,000 to 45,000 daltons, and more especially from about 39,000 to 45,000 daltons, with 40,000 daltons being the most preferred. A preferred sum of nyn 'is approximately 800 to 1200, with an average sum of approximately 850 to 1000, with a preferred sum being approximately 910. nyn' can individually be 420 or 520 or both can be 420 or 520, or both they can be 455. The preferred ratio between n and n is about 0.5 to 1.5, with an especially preferred ratio of from about 0.8 to about 1.2. A molecular weight of "approximately" a certain number means that it oscillates within a reasonable range of this number, determined by conventional analysis techniques. Also preferred is a conjugate of formula I wherein IFNa is IFNa2a, a conjugate wherein R and R 'are methyl, a conjugate wherein X is NH, and a conjugate wherein n and n are individually or both, or 420 or 520. Especially preferred is that conjugate having all the characteristics mentioned above. R and R 'may be any lower alkyl, understood as such an alkyl group having from one to six carbon atoms as p. ex. methyl, ethyl, isopropyl, etc. Branched alkyls are included. A preferred alkyl is methyl. As regards the two PEG groups of formula I, R and R 'may be the same or different. By IFNa (interferon a) and its IFNa2a species is meant the natural or recombinant protein, preferably human, which is obtained from any conventional source, such as tissues, protein synthesis, cell culture with natural or recombinant cells. Any protein that has IFNa activity, such as muteins or other modified protein classes, is included. The obtaining and separation of IFNα from natural or recombinant sources is well known (Pestka, Arch. Biochem. Biophys. 221, I (1983)). A preferred IFNa is IFNa2a, which, as indicated above, is obtained by known methods (Pestka, Sci. Am. 249, 36 (1983); European Patent No. 43 980)). The physiologically active conjugate of formula I has IFNa activity, by which is meant any fraction or multiple of any known IFNa activity, determined by various assays known in the art. In particular, the conjugates of this invention have IFNa activity as demonstrated by antiproliferative activity against tumor cells and antiviral activity against cells infected with a virus. These are known activities of the IFNa. Said activity in a conjugate can be determined by assays well known in the art, for example the assays described below (see also Rubinstein et al., J. Virol., 37, 755 (1981); Borden et al., Canc. Res. 42, 4948 (1982)). Part of this invention is a conjugate of formula I which has a higher antiproliferative activity and a lower antiviral activity than unmodified IFNa. The conjugate of formula I is obtained by the covalent attachment of IFNa to PEG which has been activated by replacing the hydroxyl of PEG with a linking group, forming a reactant which is an ester of N-hydroxy succinimide derived from PEG (in particular monomethoxy PEG) of formula II. The reagent can be obtained by conventional methods (Monfardini et al., See above). The binding takes place by means of an amide or ester bond. In a preferred conjugate, the binding takes place via an amide bond (X is NH). Part of this invention is a method for increasing the antiproliferative activity of IFNα while reducing the antiviral activity of IFNα, by binding IFNα as described above to a reagent of formula II to give a PEG-IFN conjugate. X represents the binding site on IFNa by which the PEG reagent of formula II is covalently bound to IFNa. The reactants are bound to primary amino groups (XH = NH.sub.25 on for example the N-terminal or the N-terminal of the IFNof.) The reactants can also bind to a hydroxyl (XH = OH) on for example serine. or ROCH2CH2 (OCH2CH2) n - O- C- NH The reagent of formula II (PEG2-NHS), in which a total of 2 mono-methoxy PEG chains (m-PEG) are attached to the lysine, each to the groups o? and amino by carbamate (urethane) linkages and having the carboxyl group of activated lysine as succinimidyl ester, can be obtained by conventional methods, according to known procedures (Monfardini et al., see above) applicable to a reagent with R as lower alkyl, and a desired n. The reagent can be purchased from Shearwater Polymers, Inc. (Huntsville, Alabama). The average of the MW (molecular weights) preferred of the PEG obtained is approximately 20,000 daltons, which provides a total mass of PEG of approximately 40,000 daltons in PEG2-NHS (other MW can be obtained by varying n for the materials starting from the PEG- alcohol for the reagent of formula II, by conventional methods). The reagent of formula II can be conjugated to iFNa by conventional methods. Specifically, the reagent of formula II reacts primarily with one or more primary amino groups (e.g., N-terminal and lysine side chains) of iFNa (e.g. IDNa2a) to form an amide linkage between IFNa and the basic structure of the polymer of the PEG. The pegylation reaction can also take place between the PEG2-NHS and the free hydroxyl groups (if any) (for example serine) of IFNa? to form an ester bond. The reaction mechanism is shown above. The reaction conditions are conventional for an expert and are described in detail below. The PEG reagent is combined with IFNa under slightly basic conditions at a low temperature under conditions suitable for a nucleophilic substitution which will produce the conjugate of formula I.

This is also shown in the above reaction mechanism.

The binding of the reactants to IFNa can be carried out by conventional methods. PEGs of any type can be used Selected PM, of this invention. The reaction conditions can be selected to achieve the claimed conjugate with a bound reagent. The conjugate of formula I, which has a single reagent of formula II attached, is separated from the unmodified IFNα and conjugated by having more than one reagent molecule bound by conventional methods. Purification methods such as cation exchange chromatography can be employed to separate the conjugates by charge difference, which effectively separates the conjugates at their various molecular weights. The content of the fractions obtained by cation exchange chromatography can be identified by molecular weight using conventional methods, for example, mass spectroscopy, SDS-PAGE, or other known methods for the separation of molecular entities by molecular weight. Next, a fraction containing the conjugate of formula I purified free of unmodified IFNar and of conjugates with more than one bound reagent is identified accordingly. In addition, the reagents of formula II release a lysine per reagent during acid hydrolysis, so that the number of lysines in the hydrolysis indicates the number of PEGs bound to the protein, whereby the number of reagent molecules attached can be determined to the conjugate. The following examples are described for the purpose of illustrating the invention and do not limit it in any way. IFNa2a is used in these examples. Other species of IFNof can also be conjugated to the PEG, by the methods described in the examples. DESCRIPTION OF THE DRAWINGS Figure 1: Anti-tumor activity of PEG2-IFNalfa2a in immunodepressed mice implanted subcutaneously with human renal A498 cells. All animals received a subcutaneous implant of 2 x 10s human renal A498 cells on day 33 of the study. On day 0 of the study, treatment with PEG-IFNalfa2a was initiated. The indicated amount (30, 60, 120 or 300 μg) of PEG2-IFNalfa2a was administered subcutaneously under the side opposite the tumor, once a week for a period of four weeks. Figure 2: Anti-tumor activity of IFNalpha2a in the immunocompromised mouse implanted subcutaneously with human renal A498 cells. All animals received a subcutaneous implant of 2 x 10 * human renal A498 cells on day 33 of the study. On day 0 of the study, treatment with IFNalfa2a was initiated. The indicated amount (10, 20, 40 or 100 μg) of IFNalpha2a was administered subcutaneously below the side opposite the tumor, 3 times per week for a period of four weeks. Figure 3: Anti-tumor activity of PEG2-IFNalpha2a in immunodepressed mice implanted subcutaneously with human renal ACHN cells. All animals received a subcutaneous implant of 2 x 10 * human renal ACHN cells on day 25 of the study. On day 0 of the study, treatment with PEG2-IFNalfa2a was initiated. The indicated amount (30, 60, 120 or 300 μg) of PEG2-IFNalfa2a was administered subcutaneously under the opposite flank to the tumor, 1 time per week for a period of five weeks. Figure 4: Anti-tumor activity of IFNalpha2a in immunodepressed mice implanted subcutaneously with human renal ACHN cells. All animals received a subcutaneous implant of 2 x 10 * human renal ACHN cells on day 25 of the study. On day 0 of the study, treatment with IFNalfa2a was started. The indicated amount (10, 20, 40 or 100 μg) of IFNalpha2a was administered subcutaneously below the side opposite the tumor, 3 times per week for a period of five weeks. Figure 5: Anti-tumor activity of PEG2-IFNalfa2a in immunodepressed mice implanted subcutaneously with human kidney G402 cells. All animals received a subcutaneous implant of 2 x 10 * human G402 renal cells on day 45 of the study. On day 0 of the study, treatment with PEG2-IFNalfa2a was initiated. The indicated amount (30, 60, 120 or 300 μg) of PEG2-IFNalfa2a was administered subcutaneously below the side opposite the tumor, 1 time per week for a period of five weeks. Figure 6: Anti-tumor activity of IFNalpha2a in immunodepressed mice implanted subcutaneously with human renal G402 cells. All animals received a subcutaneous implant of 2 x 10 * human G402 renal cells on day 45 of the study. On day 0 of the study, treatment with IFNalfa2a was started. The indicated amount (10, 20, 40 or 100 μg) of IFNalpha2a was administered subcutaneously below the side opposite the tumor, 3 times per week for a period of five weeks. Example 1 Preparation of the conjugate of formula I Materials Interferon2a was prepared by known methods (Pestka, see above). The polyethylene glycol (PEG) reagent of formula II was purchased from Shearwater Polymers, Inc.

(Huntsville, Ala). Fractogel® EMD CM 650 (S) resin, with particles of size 25-40 μm, was supplied by EM Separations (Gibbstown, MA). Concentrated buffered saline phosphate (10X) (PBS), pH 7.3, was purchased from BioWhittaker (Walkersville, MD). The prefabricated gels for electrophoresis of dodecyl (lauryl) sodium sulfate / poly-acrylamide gel (SDS-PAGE) and the electrophoresis units were purchased from NOVEX (San Diego, CA). Concentrated Fast Stain ("fast concentrated dye") for protein staining of PEG conjugates on SDS-PAGE was purchased from Zoion Research, Inc. (Newton, MA). The LAL endotoxin assay kit was purchased from Associates of Cape Cod, Inc. (oods Hole, MA). The rest of the reagents used were of the highest quality available in the market. Cannulated rats in the jugular and BDF-1 mice were supplied by Charles River Laboratories (Wilmington, MA). Experimental procedures A. Small scale preparation of the conjugate of formula I. Two hundred and eight milligrams (5.2 μmoles) of the reagent of formula II (average MW of 40,000 daltons) were added, to 50 mg (2.6 μmoles) of IFNa in 10 ml of 100 mM borate, pH 8.0. The final protein / reagent molar ratio was 1: 2. The reaction mixture was stirred at 4 ° C for 2 hours. The reaction was stopped by adjusting the pH to 4.5 with glacial acetic acid. The reaction mixture was diluted to 50 times its volume with water, filtered through a 0.2 μ filter and applied to an Amicon column packed with 100 ml (3.2 x 13 cm) of Fractogel EMD CM 650 (S), with a flow velocity of 20 mi / minute. The column was previously equilibrated with 10 Mm ammonium acetate, pH 4.5. The effluent from the column was monitored by UV absorbance at 280 nm. The column was then washed with the equilibration buffer until the UV absorbance returned to the baseline. The PEG-IFN conjugates with more than one reagent of formula II bound (PEG-IFN oligomers) were eluted with 40 Mm ammonium acetate, pH 4.5 and the conjugate of formula I was eluted with 0.12 M NaCl in buffer. 40 M ammonium acetate. The unmodified IFN that remained in the column was eluted with 0.5 M NaCl in the same buffer. The column was regenerated by washing with 1.0 M NaCl followed by a washing with equilibration buffer. The pooled fractions of the conjugate of formula I were concentrated in a stirred Amicon cell concentrator equipped with a YM10 membrane at approximately a concentration of 1 mg / ml. The cation exchange resin, the Fractogel CM 650 (S) used for the purification, effectively adsorbed the PEG and the unmodified IFN. The adsorption strength depended on the degree of pegylation. The conjugate bound less strongly than the unmodified IFN. The PEG-IFN oligomers were eluted with 40 Mm ammonium acetate, while the conjugate of formula I eluted with 0.12 M NaCl. The unmodified IFN eluted with 0.5 M NaCl. All the preparations contained < 5 EU / mg of endotoxins. The resulting preparation contained > 99% conjugate of formula I and was free of unmodified IFN.

B. Large scale preparation of the conjugate of formula I. Six thousand two hundred forty milligrams (156 μmoles) of the reagent of formula II (average molecular weight of 40,000 daltons) was dissolved in 63 ml of 1 mM HCl at 4 ° C and added rapidly to 125 ml of a solution containing 1000 mg (52 μmol) of interferon in 50 mM borate buffer, pH 9.0. The final protein / reagent ratio was 1: 3 and the final protein concentration of the reaction mixture was 5.3 mg / ml. The reaction mixture was stirred for 2 hours at 4 ° C. The reaction was stopped by adjusting the pH to 4.5 with glacial acetic acid. The reaction mixture was diluted to 10 times its volume with water and applied on a column packed with 600 ml of Fractogel EMD CM 650 (M) previously equilibrated with 20 mM sodium acetate, pH 4.5 at a linear velocity of 1 , 3 cm / min. The column was washed with the equilibration buffer followed by 10 mM NaCl to remove excess reagent, the secondary products of the reaction and the PEG-IFN oligomers. The conjugate of formula I was eluted with equilibration buffer containing 200 mM NaCl. The unmodified interferon still adsorbed on the column was removed by washing with 0.75 M NaCl in the equilibration buffer. The conjugate of formula I, which was eluted at 0.3-0.5 mg / ml, was subsequently concentrated and diafiltered in the final formulation buffer, 20 M sodium acetate, pH 5.0, containing 150 mM NaCl. The total yield of the conjugate of formula I was 40-45%. Purified PEG-IFN from the large-scale preparation consists of >99% conjugate of formula I. The average molecular weight of the conjugate of formula I of this example is 62,000 daltons, including the molecular weight of IFNa2a which is 19,241 daltons, and the average molecular weight of the reagent which is between 40,000 and 45,000 daltons, around 43,000 daltons. E-Example 2 Characterization of the conjugate of formula I Determination of protein Protein concentrations were determined using an A-β0 value of 1.0 for a 1 mg / ml solution of a2a IFNa. SDS-PAGE Analysis The conjugate was analyzed by gel electrophoresis (8-16%) of sodium dodecyl (lauryl) sulfate / polyacrylamide, under reducing conditions, according to the methods of Laemmli (Nature 227, 680 (1970)). SDS.-PAGE analysis containing PEG conjugates was performed in search of proteins using Fast Stain ("fast staining") (Zoion Research, Inc.), according to the manufacturer's instructions. Determination of endotoxin levels Endotoxin levels were determined using the LAL method according to the manufacturer's instructions. All preparations contained < 5 EU / mg of endotoxins. EXAMPLE 3 Bioactivities in vi tro of the conjugate of formula I Antiviral activity in bovine renal cells The antiviral activity was determined in vi tro of IFNa2a and the conjugate of formula I as prepared in example lA, in a cell culture bioassay using cells Madin-Darby bovine kidney (MDBK) activated with vesicular stomatitis virus (Rubinstein et al., see above). The antiviral activities are summarized in Table 1, together with their corresponding residual activities, as a percentage of the starting IFN. Table 1 Antiviral activities Antiproliferative activity in vi tro in human tumor cells. Antiproliferative activities in vi tro were tested on human Daudi cells (Burkitt's lymphoma), as described by Borden et al. Human Daudi cells were maintained in stationary suspension cultures in RPMI 1540 medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Grand Island Biologicals, Grand Island, NY). The cells were screened and determined to be free of mycoplasmas. Cells (2 x 104) were added to wells of microtiter plates (Costar, MA) in 100 μl of medium. Various concentrations of IFN and the conjugate of formula I prepared according to example I.A. were added to the wells in a volume of 100 μl. Plates were incubated at 37 ° C in 5% CO. for 72 hours. The cells were pulsed with 0.25 μCi / well of 3 H-thymidine (New England Nuclear, Boston, MA), sixteen hours before collection of the cells. The cells were collected on glass filters and counted in a liquid scintillation counter. The results were expressed as% inhibition, calculated by the formula:% inhibition = [(A - B) / A] x 100, where A = cpm in the control culture (cells incubated only with the medium) B = cpm in the cultures of the experiment The test was carried out on samples in quadruplicate and the standard deviation was less than 20% of the average of all the cases. Experiments were performed at least twice with comparable results. The antiproliferative activities (ICS0) of IFN and the conjugate are listed in Table 2. The data indicate that there is a 28-fold increase in antiproliferative activity for the conjugate of formula I, with respect to that of IFN. Table 2 Antiproliferative activities in vi tro in human Daudi cell lines (Burkitt's lymphoma) IC50 (ng / ml) Activity Antiproliferative Sample Increase IFNa2a 0.56 lx Conjugate of formula I 0.02 28x Example 4 Pharmacokinetics Female Sprague Dawley rats, surgically implanted with jugular cannulas, with an average body weight of 240-260 g, were individually housed, with free access to food and water and kept in a light cycle - dark of 12 hours. Within 4-6 hours after arrival the jugular cannulas were washed with PBS. The next day, after washing with 0.15-0.2 ml of PBS, 2x10 * units of IFNa were injected in 0.2-0.4 ml of PBS, followed by an injection of 0.15-0.2. My PBS to ensure that all the drug had been washed inside the animal. Thus, each animal received a dosage of 8x10 * units of IFNa / kg of body weight. Blood samples were taken at 5, 15 and 30 minutes, as well as after 1, 3, 5, 12 and 24 hours after the injection of IFN and, the conjugate of formula I. At all the times indicated, after discarding the first 0.15-0.2 ml of blood, an aliquot of 0.5 ml of blood was extracted using a new syringe through the jugular cannula. Samples were poured separately into serum tubes at room temperature. Once all the samples were collected at the indicated times, the tubes were centrifuged at 14,000 x g in an Eppendorf refrigerated centrifuge for 10 minutes. The serum was transferred separately to 1.5 microfuge tubes and frozen at -80 ° C, until they were used for the bioassay. The serum samples were appropriately diluted and the antiviral activity was determined at each of the indicated times, as described. From the graph of time versus activity, the terminal half-lives of the conjugate of formula I and of the iFNa were determined and recorded in table 3, which also includes residence times in plasma. Table 3 Terminal half-lives (t12) and mean residence time in plasma Time of residence Sample 1/2 (hours) in plasma (hours) IFNa2a 2.1 1.0 Conjugate of formula I 15.0 20.0 t1 / 2 terminal estimated by logarithmic linear regression. EXAMPLE 5 Immunogenicity ^ Normal BDF-1 mice (ten per group) were injected intraperitoneally once per day five times per week with various preparations of interferon containing 300,000 units of antiviral activity. Some mice were also injected with the added form of IFNa2a which is more immunogenic than the monomeric form. Blood samples were taken after 19 days after the last injection, and the serum was evaluated to detect the existence of neutralizing antibodies. As seen in Table 4, mice injected with IFNar2a produced neutralizing antibodies, and this response was greatly increased in mice injected with interferon aggregates. No antibodies were detected in the majority of animals injected with the conjugate of this invention. Table 4 Immunogenicity Antibody (UNI / ml) * Median Treatment Margin IFN? Í2a 2,400 217 - 8,533 Aggregates of IFNa2a 42,667 8,000 - 768,000 Conjugate of Formula 1 0-0.133 * Interferon neutralizing units / ml Example 6: In vivo anti-tumor activity The in vivo anti-tumor activity of a conjugate of formula I (PEG-IFNalfa2a) and of IFNalpha2a was evaluated by determining its ability to reduce the size of different human tumor cells implanted subcutaneously in mice. The results are indicated in figures 1-6. Procedure: Athymic immunosuppressed mice (Harian) received a subcutaneous implant under the left rear side of 2 x 10 * human kidney cells A498 (FIGS. 1 and 2), human kidney cells ACHN (FIGS. and 4), or human G402 kidney cells (figures 5 and 6). They were allowed to elapse from 3 to 6 weeks for the tumors to settle, as indicated. The size criterion for acceptance in the study was 0.05 to 0.50 cubic centimeters. Mice were treated with weekly total doses of PEG2-IFNalfa2a or IFNalfa2a unmodified, 30, 60, 120 or 300 μg. In the case of PEG2-IFNalfa2a mice were treated once a week (Monday) with 30, 60, 120 or 300 μg of PEG2-IFNalfa2a per treatment. In the case of IFNalfa2a unmodified mice were treated three times a week (Monday, Wednesday, Friday) with 10, 20, 40 or 100 g of IFNalfa2a per treatment. The duration of the treatment was 4 to 5 weeks depending on the aggressiveness of the tumor. Tumor volumes were measured every Monday before treatments. Results: The PEG2-IFNalfa2a showed a marked redueción of the size of the tumor A498 compared with the IFNalfa2a without modifying in all the levels of weekly dosage tested, to the 7 days, 14 days, 21 days and 28 days after beginning the treatment (figures 1 and 2) . The treatment continued for four weeks. Seven days after the treatment was discontinued, three mice were sacrificed from each group. In the three mice treated with PEG2-IFNalfa2a no residual tumor was observed. In mice treated with IFN-alpha2a without modifying the tumor weight A498, it was 1.28 grams, 0.62 grams and 1.60 grams respectively in each of the three mice. The tumor weight A498 was 2.32 grams, 2.37 grams, and 1.94 grams in each of the three control mice. At 80 days after the end of the four-week treatment period, the presence or absence of tumors in seven mice was determined by palpation. The seven mice were found to be free of tumor tissue by palpation. PEG2-IFNalfa2a showed a significant reduction in the size of the ACHN tumor compared to the unmodified IFNalfa2a, for the weekly dosage levels of 60, 120, and 300 μg, at 14 days, 21 days, 28 days, and 35 days (Figures 3 and 4). PEG2-IFNalfa2a showed a significant reduction in the size of the G402 tumor compared to the unmodified IFNalfa2a, for the weekly dosage levels of 60 and 120 μg, at 14 days, 21 days, 28 days and 35 days (Figs. and 6). ***** It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (17)

1. CLAIMS l. A physiologically active PEG-IFNa conjugate of formula: characterized in that R and R 'are independently from each other, lower alkyl; X is NH or O; n and n 'are integers whose sum is 600 to 1500; and the average molecular weight of the polyethylene glycol units in said conjugate is about

   26. 000 daltons to approximately 66,000 daltons.
2. 2. A conjugate of claim 1, characterized in that the molecular weight of the polyethylene glycol units is from about 35,000 to about 45,000 daltons.
3. 3. A conjugate of claim 2, characterized in that the molecular weight of the polyethylene glycol units is approximately 40,000 daltons.
4. 4. A conjugate of claim 1, characterized in that R and R 'are methyl.
5. 5. A conjugate of claim 1, characterized in that X is NH
6. 6. A conjugate of claim 1, characterized in that IFNa is IFNa2a.
7. 7. A conjugate of claim 1, characterized in that the average sum of n and n 'is 850 to 1000.
8. 8. A conjugate of claim 1, characterized in that R and R' are methyl; X is NH; IFNa is IFNa2a; and one or both n and n 'is 420.
9. 9. A conjugate of claim 1, characterized in that R and R' are methyl; X is NH; IFNa is IFNa2a; and one or both n and n 'is 520.
10. 10. A conjugate of claim 1, characterized in that it has a higher antiproliferative activity than IFNa and less antiviral activity than IFNa.
11. 11. A method for obtaining the PEG-IFNa conjugate with a higher antiproliferative activity and a lower antiviral activity compared to IFN * c, characterized in that the method consists in the covalent attachment of a reagent of formula II to iFNa to obtain said conjugate PEG-IFNa.
12. 12 - Pharmaceutical compositions, characterized in that they contain the PEG-IFNot conjugate as claimed in any one of claims 1-10 and a therapeutically inert filler.
13. 13. Pharmaceutical compositions for the treatment or prophylaxis of immunomodulatory disorders such as neoplastic diseases or infectious diseases, characterized in that they contain a PEG-IFNa conjugate as claimed in any one of claims 1-10 and a therapeutically inert filler.
14. 14. The use of a PEG-IFNa conjugate according to any one of claims 1-10, for the preparation of medicaments for use in the treatment or prophylaxis of diseases.
15. 15. A PEG-IFNa conjugate as claimed in claims 1-10, provided that it is prepared according to the method claimed in claim 11.
16. 16. A PEG-IFNc conjugate. as claimed in claims 1-10, for the preparation of medicaments for use in the treatment or prophylaxis of diseases.
17. 17. The products, pharmaceutical compositions, processes and methods, as have been substantially described thus far.