Peg-inf-alpha interferon conjugates, method of obtaining and pharmaceutic compositions containing them

The present invention relates to a physiologically active PEG-IFN-α conjugate, a method for its preparation, and pharmaceutical compositions containing it.

Interferon, in particular interferon-α2a, is a pharmaceutically active protein with antiviral and antiproliferative activity. For example, interferon is used to treat hairy cell leukemia and Kaposi's sarcoma and is active in hepatitis. In order to improve stability and solubility and reduce immunogenicity, pharmaceutically active proteins such as interferon can be coupled with a polyethylene glycol (PEG) polymer.

The bioavailability of protein drugs is often limited due to their short plasma half-life, which prevents the maximum clinical effect being utilized. In recent years, biological molecules conjugated to PEG have been shown to exhibit clinically useful properties (Inada et al., J. Bioact. And Compatible Polymers 5, 343 (1990); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9, 249 (1992); Katre et al., Advanced Drug Delivery Systems 10, 91 (1993)). These include better physical and thermal stability, resistance to enzymatic degradation, increased solubility, longer in vivo circulatory half-life, reduced clearance and increased potency. Branched PEG conjugates have been reported to have greater thermal and pH stability and greater resistance to proteolytic degradation compared to conjugates with linear PEG (Monfardini et al., Bioconjugate Chem. 6, 62 (1995)). Other properties of PEG protein conjugates are reduced immunogenicity and antigenicity as well as reduced toxicity. Other effects of PEGylation of certain proteins may be lower in vitro activity combined with increased in vivo activity. This phenomenon was observed

among others for G-CSF (Satake-Ishikawa et al., Cell Structure and Function 17, 157-160 (1992)), IL-2 (Katre et al., Proc. Natl. Acad. Sci. USA 84, 1487 (1987 )), TNF-α (Tsutsumi et al., Jpn. J. Cancer Res. 85, 9 (1994)), IL-6 (Inoue et al., J. Lab. Glin. Med. 124, 529 (1994) ) and CD4-IgG (Chamów et al., Bioconjugate Chem. 5, 133 (1994)).

It has now been observed that in the case of interferon, PEGylation reduces antiviral activity in vitro but increases the antiproliferative activity against human tumor cells. However, the new interferon and PEG conjugates of the present invention show unexpected properties in that their antiproliferative activity is not only significantly stronger than interferon but also other interferon conjugates with PEG. Although the antiproliferative effect of the conjugate is much higher than that of other PEG-interferon conjugates, the reduction in antiviral activity is similar. In addition, the PEG interferon-a conjugate of the invention is not immunogenic and has virtually no antibody formation. In contrast, other PEG interferon-α conjugates cause limited antibody formation.

Accordingly, the invention is a new class of PEG interferon-α (IFN-a) derivatives. The conjugate of the invention has a branched PEG structure as seen below. Branched PEG has the advantage of allowing the attachment of two linear PEG molecules in a single site, thus doubling the amount of PEG without the need for multiple PEGylation sites.

Compared to unmodified IFN-α (i.e. IFN-α without PEG attached), the conjugate has an extended circulatory half life and plasma residence time, reduced immunogenicity, reduced clearance and increased antiproliferative activity with reduced in vitro antiviral activity. Compared with other PEG-IFN-α conjugates, the conjugate of the invention exhibits significantly higher antiproliferative activity, disproportionate to other differences in its characteristics, and virtually no immunogenicity.

The physiologically active type of PEG-IFN-α conjugates according to the invention has formula 1 shown in the figure.

The conjugate of the invention has the same uses as IFN-α, for example antiproliferative applications. In particular, PEG interferon-α conjugates of the invention are useful in the treatment of immunomodulatory diseases such as cancer, for example, hairy cell leukemia, CML and Kaposi's sarcoma, and infectious diseases, in the same manner as IFN-α is used (a especially IFNa2a). However, the conjugates of the invention show improved characteristics, including better stability, greater solubility, extended circulatory half-life, and extended plasma residence time. In addition, these conjugates have more antiproliferative activity than IFN-α. As also noted, the conjugates show an unexpected separation of antiviral and antiproliferative activities. This property is further useful to increase the desired conjugate effect while reducing or eliminating the undesirable effect. For example, if an unwanted side effect is associated with antiviral activity, the elimination of this effect should eliminate this side effect while maintaining antiproliferative effects. Hence, the present invention also includes pharmaceutical compositions based on compounds of formula I or their salts, and methods for their preparation.

The pharmaceutical compositions of the present invention used to control or prevent disease include an interferon conjugate of general formula I and a therapeutically inert, non-toxic and therapeutically acceptable carrier. The pharmaceutical compositions used can be prepared and dosed in a manner consistent with good me4 practice

186 949, taking into account the disease being treated, the individual condition of the patient, the place of administration of the protein conjugate, the method of administration and other factors known to those skilled in the art.

The claimed conjugate is a physiologically active PEG-IFN-α conjugate of formula 1 in which R and R 'are independently C 1 -C 6 alkyl; X is NH or O (X is at least one functional group in the IFNa molecule selected from the group consisting of NH2 or OH); nin 'are integers whose sum is between 600 and 1500; and the average molecular weight of the polyethylene glycol units in the conjugate is from about 26,000 Da to about 66,000 Da. The conjugate of Formula 1 has a branched structure such that the two PEG molecules are attached to the protein by a single bond.

The numbers nin 'have been chosen in such a way that the resulting conjugate of formula 1 has physiological IFN-α activity, which activity is the same, greater or a fraction of the corresponding activity of unmodified IFN-α. nin '(nin' values ​​can be the same or different) means the number of ethylene glycol units in PEG. A single PEG unit - OCH2 CH2, has a molecular weight of about 44 Da. The molecular weight of the conjugate (excluding the molecular weight of IFN-a) depends on the numbers n '. The sum of nin 'for the conjugate of Formula 1 is from 600 to 1500, resulting in a conjugate with a total molecular weight of PEG units from about 26000 to 66000, preferably from about 35000 to 45000 Da, in particular from 39000 to 45000 Da, with 40,000 Da is particularly preferred. The preferred sum of n 'is from about 800 to 1200, with the average total being about 850 to 1000, and the preferred sum is 910. Both n and n' may be 420 or 520, or both may be 420 or 520 , or both can be 455. A preferred ratio of n to n 'is from about 0.5 to about 1.5, and a particularly preferred ratio is from about 0.8 to 1.2. The molecular weight of "about" a particular number means that it is within a reasonable range of that number as determined by conventional analytical methods.

Also preferred is a conjugate of formula 1 in which IFN-α is IFN-a2, a conjugate in which R and R 'are methyl, a conjugate in which X is NH, and a conjugate in which nin' separately or both are 420 or 520. Particularly preferred is a conjugate with all of the above characteristics.

R and R 'may be any C 1 -C 6 alkyl, by which is meant an alkyl group having from one to six carbon atoms, such as methyl, ethyl, isopropyl, etc. This includes branched alkyl groups. The preferred alkyl group is methyl. With respect to the two PEG groups in Formula 1, R and R 'may be the same or different.

By IFN-α (interferon a) and its type of IFN-a2a, is meant a natural or recombinant protein, preferably a human protein obtained from any conventional source such as tissues, protein synthesis, cell culture of natural or recombinant cells. Included is any protein having IFN-α activity, such as muteins or other modified proteins. The preparation and isolation of IFN-α from natural and recombinant sources is well known (Pestka, Arch. Biochem. Biophys. 221, 1 (1983)). The preferred IFN-α is IFN-a2a, which can be obtained by known methods (Pestka, Sci. Am. 249, 36 (1983); European Patent No. 43980)).

The physiologically active conjugate of Formula 1 has IFN-α activity, which means any fraction or numerous activities among known IFN-α activities, which can be confirmed by various known tests. In particular, the conjugates of the invention show IFN-α activity as demonstrated by antiproliferative activity against various tumor cells and anti-viral activity against virus infected cells. These are known IFN-α activities. Such activity in the conjugate can be determined by known tests, for example the tests described below (see also, Rubinstein et al., J. Virol. 37, 755 (1981); Borden et al., Canc. Res. 42, 4948 (1982)) . Part of the invention is a conjugate of Formula 1 having greater antiproliferative activity and lower antiviral activity than unmodified IFN-α.

The conjugate of formula 1 is produced by the IFN-α covalent bond with PEG, which has been activated by substituting the PEG hydroxyl group with a binding group to form a reagent that is an N-hydroxyl succinate ester derivative of PEG (in particular

186 949 monomethoxy-PEG) of formula 2. The reagent can be obtained by conventional methods (Monfardini et al., Supra). Binding occurs due to amide or ester binding. In a preferred conjugate, binding occurs due to an amide bond (X is NH). Part of the invention is a method of increasing IFN-α antiproliferative activity, while reducing IFN-α antiviral activity, by coupling it as described above with a reagent of Formula 2 to form a PEG-IFN conjugate.

X is the IFN-α attachment site where the PEG reagent of formula II is covalently bound to IFN-α. The reagents bind to the primary amine groups (XH = NH2) for example at lysine or at the N-terminus of IFN-α. Reagents can also bind to a hydroxyl group (XH = OH) on serine, for example.

The reagent of formula 2 (PEG2-NHS), in which two mono-methoxy PEG chains (m-PEG) are bound in total to lysine, one of which through the amino group and the other through the amino group with carbam (urethane) bonds, and having a lysine carboxyl group activated to a succinimidyl ester can be obtained by conventional methods according to known procedures (Monfardini et al., supra) suitable for use with a C 1 -C 6 alkyl reagent and the desired number n. Reagent may be obtained from Shearwater Polymers, Inc. (Huntsville, Alabama). The preferred average MW of PEG obtained is about 20,000 Da, providing a total PEG weight of about 40,000 Da in PEG2-NHS (other MW can be obtained by changing n for PEG alcohol substrates for reagent of formula 2 by conventional methods).

The reagent of formula II can be coupled with IFN-α by conventional methods. Specifically, the reagent of formula I primarily reacts with primary amine groups (e.g., N-terminal and lysine side chains) of IFN-α (e.g. IFN-α2a), forming an amide bond between IFN-α and the PEG polymer backbone. The PEGylation reaction may also occur between PEG2-NHS and free (if present) hydroxyl groups (e.g. serines) of IFN-α forming an ester bond. The reaction mechanism is shown in the diagram in the figure. The reaction conditions are conventional for the skilled person and are detailed below. The PEG reagent is combined with IFN-α under slightly basic conditions at low temperature, under conditions suitable for nucleophilic substitution that results in the formation of the conjugate of formula 1. This is also shown in the reaction scheme.

Attachment of the reagent to IFN-α can be achieved by conventional methods. PEGs with different MWs can be used according to the invention. The reaction conditions can be selected to give a conjugate with one reagent attached. The conjugate of Formula 1 with one reactant of Formula 2 attached is separated from unmodified IFN-α and conjugates with more than one reactant molecule attached by conventional methods. Purification methods such as cation exchange chromatography can be used to separate conjugates on the basis of differences in charge, which effectively separates the conjugates by molecular weight. The content of fractions obtained by cation exchange chromatography can be determined on the basis of molecular weight by using conventional methods, for example, mass spectrometry, SDS-PAGE or other known method of separating molecules based on their molecular weight. Then, the fraction that contains the conjugate of formula 1 free of unmodified IFN-α and conjugates with more than one reagent attached is identified. In addition, the reagent of formula 2 under the influence of acid hydrolysis releases one lysine per reagent molecule such that the number of lysines in the hydrolysis product indicates the number of PEG attached to the protein, so that the number of reagent molecules associated with the conjugate can be checked.

The following examples are provided to illustrate the invention, not to limit it. IFN-a2a was used in these examples. Other types of IFN-α may also be coupled to PEG, for example, by the methods given.

Description of drawings

Figure 1: Antitumor activity of PEG2-IFN-a2a in nude mice implanted subcutaneously with A498 human renal cells. All animals received

186 949 subcutaneous implant 2x10⁶ A498 human kidney cells on study day -33. On study day 0, PEG2-IFN- treatment was started (x2a. Marked amounts (30, 60, 120 and 300 pg) PEG2-IFN-a2a was administered subcutaneously on the opposite side of the tumor once a week for four weeks.

Figure 2: Antitumor activity of PEG2-IFN-a2a in nude mice implanted subcutaneously with A498 human renal cells. All animals received a subcutaneous implant of 2x106 A498 human kidney cells on study day -33. On study day 0, PEG2-IFN-a2a treatment was started. Marked amounts (10, 20, 40 and 100 pg) of PEG2IFN-a2a were administered subcutaneously on the opposite side of the tumor, 3 times a week for four weeks.

Figure 3: Antitumor activity of PEG2-IFN-a2a in nude mice implanted subcutaneously with ACHN human renal cells. All animals received a subcutaneous implant of 2x106 human renal ACHN cells on study day -25. On study day 0, PEG2-IFN-a2a treatment was started. Marked amounts (30, 60, 120 and 300 pg) of PEG2-IFN-a2a were administered subcutaneously on the opposite side of the tumor, once a week for five weeks.

Figure 4: Antitumor activity of PEG2-IFN-α2a in nude mice implanted subcutaneously with ACHN human renal cells. All animals received a subcutaneous implant of 2x106 human renal ACHN cells on study day -25. On study day 0, PEG2-IFN-a2a treatment was started. Marked amounts (10, 20, 40 and 100 pg) of PEG2IFN-tx2a were administered subcutaneously on the opposite side of the tumor, 3 times a week for five weeks.

Figure 5: Antitumor activity of PEG2-IFN-a2a in nude mice implanted subcutaneously with G402 human renal cells. All animals received a subcutaneous implant of 2x106 human G402 kidney cells on study day -45. On study day 0, PEG2-IFN-a2a treatment was started. Marked amounts (30, 60, 120 and 300 pg) of PEG2-IFN-a.2a were administered subcutaneously on the opposite side of the tumor, once a week for five weeks.

Figure 6: Antitumor activity of PEG2-E⁷N-a2a in nude mice implanted subcutaneously with G402 human renal cells. All animals received a subcutaneous implant of 2x106 human G402 kidney cells on study day -45. On study day 0, PEG2-IFN-a2a treatment was started. Marked amounts (10, 20, 40 and 100 pg) of PEG2IFN-cx2a were administered subcutaneously on the opposite side of the tumor, 3 times a week for five weeks.

Example 1. Preparation of the conjugate of formula 1

materials

Interferon-a2a was prepared by a known method (Pestka, supra). A polyethylene glycol (PEG) reagent of formula 2 was purchased from Shearwater Polymers, Inc. (Huntsville, Alabama). Fractogel® EMD CM 650 (S) resin, 25-40 pm particle size, was purchased from EM Separations (Gibbstown, MA). Concentrated (10x) phosphate buffered saline (PBS) was purchased from BioWhittaker (Walkersville, MD). Pre-prepared SDS-PAGE gels and electrophoresis devices were purchased from NOVEX (San Diego, CA). Concentrated Fast Stain for staining PEG conjugate protein on SDS-PAGE was purchased from Zoion Research, Inc. (Newton, MA). The LAL endotoxin test kit was purchased from Associates of Cape Cod, Inc. (Woods Hole, MA). Other reagents used were of the highest quality available. Rats with cannulated clavicular veins and BDF-1 mice were purchased from Charles River Laboratories (Wilmington, MA).

Experimental procedures

A. Small-scale production of the conjugate of Formula 1

208 milligrams (5.2 (tmol) of the reagent of formula 2 (average MW of 40,000 Da) was added to 50 mg (2.6 pmol) of IFN-a in 10 ml of 100 mM borate, pH 8.0. Final protein to reagent molar ratio 1: 2. The reaction mixture was stirred at 4 ° C. for 2 hours The reaction was stopped by adjusting the pH to 4.5 using glacial acetic acid.

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The reaction mixture was diluted 50-fold with water, filtered through a 0.2 µm filter and loaded onto an Amicon column filled with 100 ml (3.2x13 cm) Fractogel EMD CM 650 (S) at a flow of 20 ml / min. The column was previously equilibrated with 10 mM ammonium acetate, pH 4.5. The eluent leaving the column was tested by measuring UV absorbance at 280 nm. Then, the column was washed with equilibration buffer until UV absorbance returned to baseline. PEG-IFN conjugates with more than one reagent of formula 2 attached (PEG-IFN oligomers) were eluted with 40 mM ammonium acetate, pH 4.5 and the conjugate of formula 1 eluted with 0.12 M NaCl in 40 mM ammonium acetate buffer. The unmodified IFN remaining 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 washing with equilibration buffer. The combined fractions of the conjugate of Formula 1 were concentrated in an Amicon mixing chamber equipped with a YM10 membrane to a concentration of about 1 mg / ml.

The Fractogel CM 650 (S) cation exchange resin used for purification effectively adsorbed PEG and unmodified IFN. The strength of adsorption depended on the degree of PEGylation. Conjugates bound less strongly than unmodified IFN. PEG-IFN oligomers were eluted with 40 mM ammonium acetate, while conjugates of formula 1 were eluted with 0.12 M NaCl. Unmodified IFN was eluted with 0.5 M NaCl. All preparations contained <5EU / mg endotoxins. The resulting preparations contained> 99% conjugate of Formula 1 and were free of unmodified IFN.

B. Large scale production of the conjugate of Formula 1

6240 milligrams (156 pmol) of the reagent of formula 2 (average molecular weight of 40,000 Da) was dissolved in 63 ml of 1 mM HCl at 4 ° C and then 125 ml of a solution containing 1000 mg (52 pmol) of interferon in 50 mM borate buffer, pH 9 was quickly added. , 0. The final protein to reagent ratio was 1: 3 and the final protein concentration in 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 reaction pH to 4.5 using glacial acetic acid.

The reaction mixture was diluted 10-fold with water and added to a column filled with 600 ml Fractogel EMD CM 650 (M), previously equilibrated with 20 mM sodium acetate, pH 4.5, at a linear rate of 1.3 cm / min. The column was washed with equilibration buffer followed by 10 mM NaCl to remove excess reagent, by-products and PEG-IFN oligomers. The conjugate of Formula 1 was eluted with equilibration buffer containing 200 mM NaCl. The unmodified IFN adsorbed on the column was removed by washing with 0.75 M NaCl in equilibration buffer. The conjugate of formula 1 eluted at 0.3-0.5 mg / ml was further concentrated and diafiltered in final buffer, 20 mM sodium acetate, pH 5.0, containing 150 mM NaCl. The total yield of conjugate of formula was 40-45%.

The purified PEG-IFN from large-scale production consisted of> 99% of the conjugate of formula 1. The average molecular weight of the conjugate of formula 1 according to the example was 62,000 Da, of which the molecular weight of IFN-a2a was 19,241 Da and the average molecular weight of the reagent , from 40,000 Da to 45,000 Da, was 43,000 Da.

Example II Characterization of the conjugate of formula 1

Protein determination

Protein concentrations were determined using an A280 value of 1.0 for a 1 mg / ml IFN-α2a solution.

SDS-PAGE analysis

The conjugate was analyzed by SDS-polyacrylamide gel electrophoresis (8-16%) under reducing conditions, according to the Laemmli method (Nature 227, 680 (1970)). SDS-PAGE containing PEG conjugates was stained using Fast Stain (Zoion Research, Inc.) according to the manufacturer's instructions.

Determining the amount of endotoxin

Endotoxin concentration was determined using the LAL method according to the manufacturer's instructions. All formulations contained> 5 EU / mg endotoxin.

Example III. In vitro biological activities of the conjugate of Formula 1

Antiviral activity against bovine kidney cells

186 949

In vitro antiviral activity of IFN-α2a and the conjugate of Formula 1 as prepared in Example IA was tested in a cell culture bioassay using Madin-Darby bovine kidney cells (MDBK) exposed to foot-and-mouth disease virus (Rubinstein et al., Supra). Antiviral activities are given in Table 1, together with their respective residual activities, as a percentage of the starting IFN.

Table 1

Antiviral activities

<td>samples</td><td>Type of PEG</td><td>Total PEG (kDa)</td><td>number Rev. lys</td><td>Specific activity (U / mg)</td><td>Activity residual (%)</td>

<td>IFN-o.2a</td><td> -</td><td> -</td><td> -</td><td>2.00 x 10⁸</td><td> 100</td>

<td>Conjugate of formula 1</td><td>branched</td><td> 40</td><td> 1</td><td>1.40 x 10⁷</td><td> 7</td>

In vitro antiproliferative activity against human tumor cells

In vitro antiproliferative activities were tested on human Daudi cells (Burkitt's lymphoma) as described in Borden et al. Human Daudi cells were maintained as stationary cultures suspended in wRPMI 1640 supplemented with 10% fetal bovine serum and 2 mM glutamine (Grand Island Biologicals, Grand Island, NY). Cells were examined for mycoplasma and found to be uninfected. Cells (2x10⁴) were plated into wells of microtiter plates (Costar, MA) in 100 µl medium. Different wells of IFN and the conjugate of Formula 1 as prepared in Example IA were added to the wells in a volume of 100 μΐ. Plates were incubated at 37 ° C in 5% CO₂ for 72 hours. Cells were pulsed with 0.25 pCi / well³H-thymidine (New England Nuclear, Boston, MA), sixteen hours before cell collection. Cells were harvested on glass filters and counted in a scintillation counter. The results are given as% inhibition calculated according to the formula:

% inhibition = [(AB) / A] x 100, where;

A = cpm in control culture (cells incubated in medium alone)

B = cpm in experimental culture

Samples were tested in quadruplicate and the standard deviation was lower than 20% of the mean of all forfeits. Experiments were performed at least twice with comparable results.

The antiproliferative activities (IC50) of IFN and the conjugate are given in Table 2. The data indicate a 28-fold increase in antiproliferative activity for the conjugate of formula 1 compared to IFN.

Table 2

In vitro antiproliferative activities against the human Daudi cell line (Burkitt's lymphoma)

<td>A sample</td><td>Antiprofessional IC50 (ng / ml)</td><td>Increase in activity</td>

<td>IFN (x2a</td><td> 0,56</td><td>1x</td>

<td>Conjugate of formula 1</td><td> 0,02</td><td>28x</td>

Example IV pharmacokinetics

Female Sprague Dawley rats, with surgically implanted cannulas in the subclavian veins, with an average body weight of 240-260 g, were housed separately, with free access to food and water, on a 12-hour day-night cycle. Within 4-6 hours after arrival, the subclavian cannula was rinsed with PBS. The next day after rinsing with 0.150.2 ml PBS, 2x10 was injected⁶ IFN-α units in 0.2-0.4 ml PBS, followed by 0.15-0.2 ml PBS to make sure all drug was given to the animal. Thus, each animal received a dose of 8x1 (0 'IFN-α units per kilogram of body weight).

186 949

Blood samples were taken at 5, 15 and 30 minutes, and 1, 3, 5, 12 and 24 hours after the injection of IFN and the conjugate of formula 1. At all time points, after removal of the first 0.15-0.2 ml of blood, fresh 0.5 ml aliquot of blood was withdrawn via a subclavian cannula. Samples were separated into serum separation tubes at room temperature. After collecting samples for all time points, the tubes were centrifuged at 14000xg in a refrigerated Eppendorf centrifuge for 10 minutes. The separated serum was transferred to 1.5 ml microcentrifuge tubes and frozen at -80 ° C until use in the bioassay. Serum samples were appropriately diluted and antiviral activity tested at each time point as described. From the time versus activity graph, the final half life of the conjugate of Formula 1 and IFN-α was determined, and is given in Table 3 together with the plasma residence time.

Table 3

Final half-life (t] /₂) and average plasma residence time

<td>A sample</td><td>i.e. / 2 (hours)</td><td>Retention period in plasma (hours)</td>

<td>IFN-a2a</td><td> 2,1</td><td> 1,0</td>

<td>Conjugate of formula 1</td><td> 15,0</td><td> 20,0</td>

Final t1 / 2 was determined by linear regression.

Example V. Immunogenicity

Normal BDF-1 mice (ten per group) were injected intraperitoneally once daily for five days a week with various preparations of interferon in an amount of 300,000 units of antiviral activity. Some mice were also injected with the aggregated form of IFN-a2a, which is more immunogenic than the monomeric form. Blood samples were taken 19 days after the last injection and the sera were tested for neutralizing antibodies.

As can be seen from Table 4, IFN-α2a injected mice produced neutralizing antibodies, and this response was significantly enhanced in mice injected with interferon aggregate. No antibodies were detected in most animals injected with the conjugate of the invention.

Table 4 Immunogenicity

Antibodies (INU / ml) *

Median treatment

IFN-a2a 2400

IFN-a2a 42667 aggregates

Conjugate Formula 10

Range

217-8533

8000-768000

0-1133 * Interferon / ml neutralizing units

Example VI. In vivo anti-tumor activity

In vivo antitumor activity of the conjugate of formula 1 (PEG2-IFN-a2a) and unmodified IFN-a2a was tested by determining their ability to reduce tumors from various human tumor cells implanted subcutaneously in mice. The results are shown in Figures 1-6.

Procedure: athymic nude mice (Harlan) received a subcutaneous implant in the left flank with 2x10⁶ A498 human renal cells (Figures 1 and 2), ACHN human renal cells (Figures 3 and 4) or G402 human renal cells (Figures 5 and 6). The tumors were allowed to grow within 3-6 weeks as indicated. The size criterion for inclusion in the study was 0.05-0.5 cubic centimeters. Weekly doses of PEG2-IFN-a2a or unmodified IFN-α2a of 30, 60, 120 or 300 pg were administered to the mice. For PEG2-IFN-a2a, mice were treated once a week (Monday) with 30, 60, 120

186 949 or 300 ug per dose. In the case of unmodified IFN-a2a, mice were given three times a week (Monday, Wednesday, Friday) at 10, 20, 40 or 100 (Leagues per dose. The duration of treatment was 4-5 weeks depending on the aggressiveness of the tumor. Tumor volume measured every Monday before treatment.

Results: PEG2-IFN-α2a showed a significant reduction in A498 tumor size compared to unmodified IFN-α2a at each of the doses used at 7, 14, 21 and 28 days after the start of treatment (Figures 1 and 2). Treatment continued for four weeks. Seven days after stopping treatment, three mice per group were killed. No residual tumor was observed in three PEG2-IFN-α2a-treated mice. In mice treated with unmodified IFN-α2a, the weight of A498 tumors was 1.28, 0.62 and 1.6 grams in each of the three mice, respectively. The weight of A498 tumors in three control mice was 2.32, 2.37 and 1.94 grams, respectively. 80 days after the end of treatment, palpation occurred in 7 mice. All mice were tumor free.

PEG2-IFN-α2a caused a significant reduction in the size of ACHN tumors compared to unmodified IFN-α2a for weekly doses of 60, 120 and 300 µg on days 14, 21, 28 and 35 (Figures 3 and 4).

PEG2-IFN-α2a caused a significant reduction in the size of G402 tumors compared to unmodified IFN-α2a at 60 and 120 pg on days 14, 21, 28 and 35 (Figures 5 and 6).

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