Interferon conjugates

Interferon-conjugated substances

Full description

Background of the invention

The present invention relates to interferons, particularly interferon α2a, a pharmacologically active protein which has antiviral and antiproliferative activity. For example, interferon is used to treat hairy cell leukemia and Caposi's cancer, and is active against hepatitis C. To improve solubility and reduce immunogenicity, pharmacologically active proteins such as interferon may be conjugated with a PEG polymer.

The bioavailability of protein drugs is usually limited due to their short half-life in plasma, thus preventing them from reaching maximum clinical efficacy. In recent years, it has been found that molecules

:Inada et al., J. Bioact. and Compatible Polymers 5, 343 (1990); Delgado et al., Critical Reviews in Therapeutic Grug Carrier Systems 9, 249 (1992); Katre, Advanced Drug Delivery Systems 10, 91 (1993).

Among these properties we find better thermal and physical properties, protection against exposure to enzymatic degradation, increased melting point, longer half-life inside the body, ease of disposal and enhanced effectiveness. It has been reported that branched PEG conjugates showed increased pH, thermal stability, and greater stability to proteolytic digestion than linear PEG conjugates ((1995) 62,6, EP-A 0 400 472 and Monfardini et al Bioconjugate Chem). ). Other properties of PEG proteins are lack of immunogenicity and antigenicity, in addition to lack of toxicity.

Another effect of the association of certain proteins with PEG may be a decrease in their activity outside the body

By increasing its activity within the body. 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. Natl. Acad. Sci. USA 84, 1487 (1987)), TNF -α (Ttsutsumi et al., Jpn. J. Cancer Res. 85, 9 (1994)), IL-6 (Inoue et al., J. Lab. Clin. Med. 124, 529 (1994)) and CD4 -IgG (Chamow et al., Bioconj. Chem. 5, 133 (1994)),

Among others.

It has now been observed that in the case of interferon, conjugation with PEG reduces antiviral activity outside the body but increases antiproliferative activity in human tumor cells. However, the new PEG-conjugated interferon in this invention has amazing properties in that the antiproliferative activity of the PEG interferon is greater than not only the interferon but also of the other PEG-conjugated interferons. Although the antiproliferative activity of the conjugate is much greater than that of the PEG interferona-α α conjugate, so far the lack of antiproliferative activity

Equal to viruses. In addition, the interferon-α PEG conjugate of this invention is non-immunogenic, as it does not form antibodies. In contrast, other PEG-interferon-α conjugates produce limited antibodies.

General description of the invention

Accordingly, the invention is a new group of PEG derivatives of interferon-α (IFNα).

The conjugate substance of this invention has a branched PEG structure, as will be demonstrated below. Branched PEG has the advantage of allowing two linear PEG molecules to bind at one site, thus doubling the mass of bound PEG without the multiple sites of PEG.

Compared to unmodified IFNα (i.e. IFNα is not bound to PEG); The conjugated substance has an increased half-life and plasma residence time, decreased immunogenicity, ease of elimination, and increased activity. Anti-proliferative, accompanied by a lack of antiviral activity outside the body. Compared to other PEG-IFNα conjugates; The substance associated with this invention has better anti-proliferative activity, regardless of the increase or decrease that occurs in its other properties, and actually has no ability to stimulate immunity.

The types of physiologically active PEG-IFNα conjugates of this invention have the formula:

<img file="SA466B1_D0001.tif" />

The conjugate substance of this invention has the same uses as IFNα, for example, antiproliferative uses. In particular, the interferon-α PEG conjugates of the invention are useful in the treatment of immunomodulatory disorders such as neoplastic diseases, e.g., hairy cell leukemia, CML, Kaposi's carcinoma, and infectious diseases, and in the same manner IFNαs (particularly IFNα2a) are used in the treatment of these Diseases. However, the conjugate material of this invention has improved properties including higher stability, higher melting point, increased circulation half-life and plasma residence time. In addition, these conjugates have antiproliferative activity that is higher than that of IFNα. Also as mentioned the conjugated substance has amazing separation of antiviral and anti-proliferative effects. This property is additionally useful for increasing the desired activity of the conjugated substance, and for reducing or eliminating undesired activity. For example, if an unwanted side effect was accompanied by antiviral activity, removing that activity would eliminate the side effect, while the antiproliferative activity would remain. Therefore, the present invention also includes pharmaceutical compositions based on compounds of Formula I or their salts

And its production methods.

Pharmaceutical compositions of the present invention that are used to control or prevent diseases include an interferon conjugate of Formula I and a pharmaceutically inert, non-toxic and pharmaceutically acceptable carrier substance. For use, drug formulations may be formulated and dosed in a manner consistent with good clinical practice, taking into account the disorder to be treated, the condition of the individual patient, the site of delivery of the protein conjugate, the route of administration, and other factors known to the practitioner.

The protected substance is the physiologically active PEG-IFNα conjugate which has the formula:

<img file="SA466B1_D0002.tif" />

Where R and 'R are each individually the lower alkyl, From 600 to 1,500, and the average molecular weight of the polyethylene glycol units in the aforementioned conjugated material is from 26,000 daltons to 66,000 daltons. The formula I conjugate has a branched structure, in which two PEG molecules are linked to the protein by a single bond.

The numbers n and 'n are selected such that the resulting formula I conjugate has the physiological activity of IFNα, the activity of which is the same as, greater than, or a fraction of the corresponding activity of unmodified IFNα. n and 'n (n and 'n may be the same or different) represent the number of ethylene glycol units in PEG. A single PEG unit of OCH2CH2 has a molecular weight of about 44 daltons. The molecular weight of the conjugated substance (excluding the molecular weight of IFNα) depends on the number of n and 'n. The sum of n and 'n for the conjugate habit of formula I ranges from 600 to 1,500, yielding a conjugate having an average full molecular weight of PEG units of about 26,000 to 66,000, preferably 35,000 to 45,000 daltons, and especially to 45,000 daltons, especially preferably 40,000 daltons. The preferred sum of n and 'n ranges from 800 to 1200, with the average sum from 850 to 1000, and the preferred sum is about 910. Either n or 'n may be 420 or 520, or both may be 420 or 520, or both may be 455. The preferred ratio of n and 'n ranges from 0.5 to 1.5, and especially the preferred ratio ranges from 0.80 to about 1.2. A molecular weight of about a certain number means that it is in the appropriate range for this

The number as developed by traditional analytical methods.

It is also preferable for the conjugate substance to be of the form I in which IFNα is IFNα2a, the conjugate substance in which R and 'R are methyl, and usually the conjugate substance in which X is NH,

The paired substance in which n and 'n are either separately or both 420 or 520. This combined material that has all of the above characteristics is particularly preferable.

R and 'R may be lower alkyl, which means any alkyl group with one to six carbon atoms such as methyl, ethyl, isopropyl, etc. These include branched alkyl groups. The preferred alkyl is methyl. Considering that two of the PEG groups are of formula I, R and 'R may be the same or different.

IFNα (interferon a) and its type IFNα2a means natural or recombinant protein, preferably human, such as taken from any familiar source such as tissue, protein synthesis, cell culture with normal cells or recombinant cells. Any protein with IFNα activity, such as muteins or other modified proteins, is included here. Obtaining and isolating IFNα from natural or recombinant sources is well known:

((1983) 1, 221. Pestka, Arch. Biochem. Biophys)

The preferred IFNα is IFNα2a, which as mentioned above is taken up by known means:

((980 43. Emopean Patent No. (1983); 36, 249. Pestka, Sci. Am)

The conjugated substance of physiologically active Formula I has IFNα activity, meaning any portion or parts of any known IFNα activity, which has been determined by various experiments known in the art, in particular, the conjugated substance of this invention having IFNα activity which is demonstrated in antiproliferative activity against cells Tumor and antiviral activity against virus-infected cells. These are the known activities of IFNα. This activity in the coupled matter can be determined by experiments well known in the art, for example the experiments described below, see also:

4948 ,42 .Borden et al., Canc. Res (1981) 755, 37. Rubinstein et al., J. Virol)

.((1982)

Part of this invention is the conjugated substance of Formula I, which has better antiproliferative activity and less favorable antiviral activity than unmodified IFNα.

The conjugate of formula I is produced by covalent bonding of IFNα with PEG, which is activated by replacing the hydroxyl of PEG with a bonding group, to form a factor.

The reagent is N-hydroxy succinimide ester derived from PEG (particularly PEG monomethoxy) of formula II. The reagent can be obtained by ordinary methods (Monfardini et al., above). Bonding is via an amide or ester bond. In the preferred conjugate, the bonding is via an amide bond (X is NH). Part of this invention is a method for increasing the antiproliferative activity of IFNα and reducing the antiviral activity of IFNα, by attaching the IFNα described above to a formula II reagent to produce a PEG-IFN conjugate.

The Primary amino reagents (XH = NH2), for example, bind to lysine or to the N-terminus of IFNα. Reagents can bind to hydroxyls (OH = XH (hydroxyl), for example serine

<img file="SA466B1_D0003.tif" />

The reagent agent of formula II (PEG2-NHS), in which the sum of two...

The PEG mono-methoxy chains are linked to lysine, each at the a and ε amino groups, via carbamate (urethane) bonds, in which the lysine carboxyl group is activated to the succinimidyl ester. It is possible to obtain It is determined by ordinary methods, according to well-known procedures (Monfardini et al., above) adapted to a reagent in which R such as the lower alkyl n and the lower alkyl are required. The reagent can be obtained from an institution

Huntsville, Alabama) Shearwater Polymers. The preferred average molecular weight (MW) obtained for PEG is about 20,000 Daltons, and the total mass of PEG is given as about 40,000 Daltons in PEG2-NHS (other molecular weights can be obtained by different starting materials PEG alcohol for formula II reagent, by normal methods).

The formula II reagent may be conjugated to IFNα by ordinary methods, in particular, the formula II reagent initially reacts with one or more of the primary amino groups (e.g., N-terminus and lysine side chains) of normally IFNα (e.g., IFNα2a). ) to form an amide bond between IFNα and the backbone of the PEG polymer. A PEG addition reaction can occur between PEG2-NHS and the free hydroxyl groups (if present) (eg serine) of IFNα to create an ester bond. The reaction method is described above. The reaction conditions are normal for a skilled person and are described in detail below. The PEG reagent monomer combines with IFNα under simple basic conditions at low temperature under conditions suitable for nucleophilic substitution which will produce a conjugate of formula I. This is also shown in the reaction method above.

Binding of IFNα reagents may be accomplished by standard methods. PEGs having any MW chosen may be used in this invention. The reaction conditions may be chosen to give the called conjugate with the binding of a single reagent. The formula I conjugate, which has a single bound reagent of formula II, is separated from unmodified IFNα and the conjugates that have more than one bound molecular reagent by ordinary methods. Selection methods such as positively charged ion exchange chromatography may be used to separate conjugated substances with different charges, which effectively separates conjugated substances into their different molecular weights. The content of the fractions obtained by positively charged ion exchange chromatography may be determined by molecular weight using standard methods, for example, mass spectrometry, SDS-PAGE, or other known methods for separating molecules by molecular weight. A fraction is defined accordingly which contains the form I conjugate purified from unmodified IFNα and from conjugates having more than one bound reagent agent. In addition, Formula II reagents yield one lysine per reagent upon acid analysis, so that the number of lysines in the analysis indicates the number of PEGs bound to the protein, and thus the number of reagent molecules bound to the conjugate may be verified.

The following examples are provided to illustrate the invention without limiting it in any way. IFNα2a is used in these examples. Other types of IFNα may bind to PEG in similar ways.

Brief explanation of the drawings

Figure 1: Antitumor activity of PEG2-IFNapha2a in light beige mice implanted under

Its skin is made of human kidney A498 cells. All animals received a subcutaneous dose of 2 x 610 human kidney A498 cells on study day-33. On study day 0, PEG2IFNalpha2a treatment begins. The required amount (30, 60, 120 or 300 micrograms) of PEG2IFNalpha2a is given below:

The skin under the side opposite the tumor, once a week for four weeks.

Figure 2: Antitumor activity of IFNalpha2a in light beige mice implanted subcutaneously with human kidney A498 cells. All animals received a subcutaneous dose of 2 x 610 human kidney A498 cells on study day 33. On study day 0, IFNapha2a treatment begins. The required amount (10, 20, .4 or 100 micrograms) of IFNapha2a is given subcutaneously under the side opposite the tumor, 3 times a week for four weeks.

Figure 3: Antitumor activity of PEG2-IFNalpha2a in light beige mice implanted under

Skinned with human ACHN kidney cells. All animals were given a subcutaneous dose of 2 x 610 human ACHN kidney cells on study day-25. On study day 0, PEG2-IFNalpha2a treatment begins. The required quantity (30, 60, 120 or 300 micrograms) of a substance is given

PEG2-INalpha2a is administered subcutaneously under the opposite side of the tumor, once a week for five weeks.

Figure 4: Anti-tumor activity of IFNalpha2a in light beige mice with human ACHN kidney cells implanted under their skin. All animals were given a subcutaneous dose of 2 x 610 human ACHN kidney cells on study day-25. On study day zero, subject treatment begins

The required amount (10, 20, 40 or 100 micrograms) of IFNalpha2a is given subcutaneously under the side opposite the tumor, 3 times a week for five weeks.

Figure 5: Antitumor activity of PEG2-IFNalpha2a in light beige mice implanted under

Its skin is G402 human kidney cells. All animals were given a subcutaneous dose of 2 x 610 kidney cells

G402 Human beings on study day-45. On study day 0, PEG2-IFNα2a treatment begins. The required amount (30, 60, 120 or 300 micrograms) of PEG2-IFNalpa2a is given. Subcutaneously under the side opposite the tumor, once a week for five weeks.

Figure 6: Anti-tumor activity of IFNalpha2a in light beige mice with G402 human kidney cells implanted under their skin. All animals were given a subcutaneous dose of 2 x 610 human G402 kidney cells on study day-45. On study day 0, IFNalpha2a treatment begins. The required amount (10, 20, 40 or 100 micrograms) of IFNalpha2a is given subcutaneously under the side opposite the tumor, 3 times a week for five weeks.

Detailed description

Example 1

Preparation of a conjugated substance of formula I

Materials

Interferon2αa αa is prepared by well-known methods (Pestka, above). He buys

PEG (polyethylene glycol) reagent formula II from Huntsville, Ala. (Shearwater Polymer). Fractogel® EMD CM 650(S) resin, with particle sizes 25-40 µm, is produced by Gibbstown, MA EM Separations. Purchase 10 times concentrated phosphate buffered saline (PBS), pH 7.3, from MD Bio Whittaker, Walkersville. Sodium dodecyl (lauryl) sulfate/polyacrylamide Ch

Sodium dodecyl (laurel) sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) Prepared gels and electrophoresis units were from NOVEX (San Diego, CA). Concentrated Fast Stain for protein staining of PEG-conjugated materials on SDS-PAGE was purchased from Zoion Research (Woods Hole, MA). The LAL Endotoxin Calibration Kit was purchased from Woods Hole Associates of Cape Cod, MA. All other reagents used were of the highest quality available. Index finger vein needle and 1-BDF mice are from Charles River Laboratories (Wilmington, MA).

Steps of the experiment

(a) Small scale preparation of the conjugate of formula I

Mabin and eight mg (5.2 µmol) of formula II reagent (average MW of 40,000 Daltons) added to 50 mg (2.6 µmol) of IFNα in 10 milliliters of 100 mM borate, pH 8. It was the ratio

Final molecular molarity of protein reagent factor 2:1. Stir the reaction mixture at 4°C for two hours. The reaction is stopped by adjusting the pH to 4.5 with icy acetic acid.

The reaction mixture was diluted 50 times with water, filtered through a 0.2 µm filter and placed on an Amicon column filled with 100 ml (3.2 x 13 cm) Fractogel EMD CM 650(s), at a flow rate of 20 ml/min. The previously prepared column Equilibrium with 0-1 mM ammonium acetate, pH 4.5. The flowing column is measured by UV absorbance at 280 nm. The column is then washed with neutral buffer until the UV absorbance returns to the starting point. PEG-IFN conjugates that have more than one Formula II reagent bound (PEG-IFN oligomers) are separated by 40 mM ammonium acetate, pH 4.5 and the Formula I conjugate is separated by 0.12. 1 mol NaCl in 40 mol of neutral ammonium acetate solution. Unmodified IFN remaining in the column was separated by 0.5 mol NaCl in the same neutral solution. The column is regenerated by washing with 1 mol NaCl, followed by washing with neutral equilibrium solution. The aggregated fractions of the conjugated substance of Formula I are concentrated in the Amicon cell stirrer sealed by a YM10 membrane to about 1 mg/ml.

concentration.

The cation-substituted resin (Fractogel CM 650(s) used for prophylaxis effectively distinguishes unmodified PEG and IFN. The strength of adsorption depends on the degree of association with PEG. The combination of conjugates is less tight than with unmodified IFN. The PEG-IFN oligomers are separated by 0.4 Ammonium acetate, while the conjugate I is separated by 0.12 mM NaCl. Unmodified IFN is separated by 0.5 mM NaCl. All preparations contain &lt;5 EU/mg endotoxins. The resulting preparation contains &gt; 99% of the conjugate is form I and is free of unmodified IFN.

(b) Large scale preparation of the conjugate of formula I

Six thousand two hundred and forty mg (156 micromol) of formula II reagent (average molecular weight 40,000 daltons) is dissolved in 63 milliliters of 1 mM HCl at 4 C and quickly added to 125 milliliters of a solution containing 1,000 mg (52

Micromol) of interferon in 50 mmol neutral borate solution, pH 9. The final protein/reagent ratio was 3:1 and the final protein concentration of the reaction mixture was 5.3 mg/ml. Stir the agent mixture for 2 hours at 4°C. The reaction is stopped by adjusting the pH to 4.5 with glacial acetic acid.

Dilute the reaction mixture 10 times with water and add to a column filled with 0.6 ml of Fractogel EMD CM 650(M), previously equilibrated with 2 0 mM sodium acetate, pH 4.5, at a linear speed of 1.3 cm/s. The column is washed with neutral equilibrium followed by 10 mM NaCl to remove excess reagent, side products and PEG-IFN oligomers. The conjugate of formula I is washed with neutral equilibrium containing 200 mM NaCl. Unmodified interferon still adsorbed on the column was removed by being washed with 0.75 mol NaCl in neutral equilibrium buffer. The conjugated material of Formula I, which was washed at a rate of 0.3-0.5 mg/ml, was concentrated and filtered again for the final neutral solution composition.

20 mM sodium acetate, pH 5, contains

150 mM NaCl. The total yield of formula I adduct is 40-45%.

PEG-IFN purified from the bulk preparation scale consists of &gt; 99% of the conjugate is of Formula I. The average molecular weight of the conjugate of Formula I for this example is 62,000 daltons, including the molecular weight of IFNα2a, which is 19,241 daltons, and the average molecular weight of the reagent between 40,000 and 45,000 daltons, about 43,000 daltons.

Example 2

Properties of the associated substance of formula I

Protein identification

Protein concentrations are determined using an A280 value equal to 1 for a 1 mg/ml solution of a substance

IFNaα2a.

SDS-PAGE lysis

Decomposition of the conjugate by sodium doceyl (lauryl) sulfate/polyacrylamide (8-16%) gel electrophoresis, under

Reducing conditions, according to the methods of Nature 227, 680 (1970)) Laemmli. PEG-containing SDS-PAGE conjugates were stained for protein using Fast Stain (Zoion Research) according to manufacturing conditions.

Determination of endotoxin levels

Endotoxin levels were determined using the LAL method according to manufacturing conditions. All preparations contain &lt; 5 EU/mg endotoxins.

Example 3

Biological activities of the conjugate of formula I beyond the determination of antiviral activity in bovine kidney cells

The antiviral activity of ex vivo IFNα2a and the formula I conjugate as prepared in Example 1a is determined in a bioassay of a cell culture containing Madin-Darby kidney cells.

bovis (MDBK) elicited by vesicular stomatitis virus (Rubinstein et al., above). The antiviral activities are recorded in Table 1, along with their corresponding residual activities as a percentage of the IFN starting material.

<img file="SA466B1_D0004.tif" />

Antiproliferative activity in vitro in human tumor cells

Antiproliferative activities in vitro were assayed in human Daudi cells (Burkitt's lymphoma, as described in Borden et al. Human Daudi cells were maintained as fixed suspension cultures in RPMI 1540 supplemented with 10% fetal bovine blood and 2 mM l -glutamine Grand Island Biologicals, Grand Island, NY) glutamine). The cells were wiped and found to be free of mycoplasma. Cells (2 x 410) are added to the surfaces of small standard size vessels

(Costar, MA) in 100 μl of medium. Different concentrations of IFN and formula I conjugate as prepared in Example 1a are added to 100-µl vessels. The surfaces were incubated at 37°C in 5% CO2 for 72 hours. Cells were driven with 0.25 μCi/pot of thymidine-3H (NewEngland Nuclear, Boston, MA), sixteen hours before cell collection. Cells are collected on glass filters and counted in a liquid pulse counter. Results are expressed as a percentage of prevention calculated using the formula:

% Blocking = [(A/BA] x 100,

where:

cpm =A in the comparison culture (cells are incubated in the medium only) cpm =B in the experimental culture

Samples are conducted 4 times, and the standard deviation value is less than 20% of the average for all cases. Experiments are conducted at least twice and the results are compared.

The antiproliferative activities (IC50) of the IFN substance and the conjugate substance are recorded in Table 2. Information indicates that there is a 28-fold increase in the anti-proliferative activity of Formula I conjugate when compared with IFN.

<img file="SA466B1_D0005.tif" />

Example 4

Pharmacokinetics Phamracokinetics

Female Sprague Dawley rats, surgically implanted with carotid intravenous cannulae, have an average body weight of 240 to 260 g and are raised individually, allowed freedom of access to food and water and continued on a 12-hour light-dark cycle. Within 4-6 hours after arrival, the carotid intravenous cannulas are infused with PBS. The next day, after immersion with 0.15-0.2 ml PBS, inject 2 x 610 units of IFNα in 0.2-0.4 ml PBS, then inject 0.15-0.2 ml

PBS, make sure that all the drug has entered the animal. Thus, each animal is given a dose of 8 x 610 IFNα units per kg of body weight.

Blood samples are drawn after 5, 15, and 30 minutes, as well as 3, 1, 5, 12, and 24 hours after the injection of IFN and formula I conjugate. At all time points, after the first 0.15-0.2 milliliters of blood have been removed. Qasim Tam draws 0.5 milliliters of blood using a new syringe through a carotid intravenous cannula. Samples are placed in blood apheresis tubes at room temperature. When all samples are collected for all times, centrifuge the tubes at 14,000 × g in an ice-cold Eppendorf centrifuge for 10 minutes. The separated blood is transferred into 1.5 milliliter opaque tubes and iced at -80°C, until it is ready for bioassay. Blood samples were appropriately diluted and antiviral activity was determined at each time as described. From plotting time versus activity, the final half-life of the IFNα and I conjugate is determined and is reported in Table 3, which also includes residence times in the blood.

<img file="SA466B1_D0006.tif" />

Example 5 Stimulate immunity

Normal 1-BDF mice (ten per group) were injected intraperitoneally once a day five times a week with different interferon preparations having 300,000 units of antiviral activity. Some mice are also injected with a bulk form of IFNα2a, which is more immunogenic than the monomer form. Blood samples are taken 19 days after the last injection, and the blood is evaluated for the presence of neutralizing antibodies.

As shown in Table 4, mice injected with IFNα2a produce neutralizing antibodies and this response is clearly increased in mice injected with combined interferon. No antibodies were observed in the majority of animals injected with the conjugate of this invention.

Table 4

<img file="SA466B1_D0007.tif" />

Example 6 Antitumor activity inside the body The in vivo antitumor activity of the formula I conjugate (PEG2-IFNalpha2a)

The unmodified substance is evaluated by determining its ability to reduce the size of different human tumors

Implanted subcutaneously in mice. The results are shown in Figures 1-6.

Steps: Inject a light beige, dethymicized mouse subcutaneously under the back of its left side with 2 x 610 human A498 kidney cells (Figures 1 and 2), human ACHN kidney cells (Figures 3 and 4), or human G402 kidney cells (Figures 5 and 6). . Leave the tumors 3 to 6 weeks to become firm, as desired. The volume characteristic for admission into the study is 0.05 to 0.5 cm3. Mice are given weekly full doses of PEG2-IFNalpha2a or non-IFNalpha2a.

Modified 30, 60, 120 or 300 micrograms. In the case of PEG2-IFNalpha2a, mice are treated once a week (on Monday) with 30, 60, 120 or 300 micrograms of PEG2-IFNalpha2a each treatment. In the case of unmodified IFNalpha2a, mice were treated three times a week (Monday, Wednesday, Friday) with 10, 20, 40 or 100 micrograms of IFNalpha2a per treatment. The duration of treatment is 4 to 5 weeks, depending on the aggressiveness of the tumor. Tumor sizes were measured every Monday before treatment.

Results: PEG2-IFNalpha2a showed a clear decrease in A498 tumor size when...

It was compared with unadjusted IFNalpha2a for all weekly dose levels tested, at 7 days, 14 days, 21 days and 28 days after the start of treatment (Figures 1 and 2). The treatment continues for four weeks. Seven days after stopping treatment, three mice in each group are slaughtered. In the three mice treated with PEG2-IFNalpha2a no residual tumor was observed. In mice treated with

Unmodified IFNalpha2a, A498 tumor weight was 1.28 g, 0.62 g, and 1.6 g, respectively, in each of the three mice. The weight of tumor A498 was 2.32 grams, 2.37 grams

And 1.94 grams in each of the three compared mice. At 80 days after the end of the four-week treatment period, the presence of tumors is determined by manual examination in the seven periods. All seven mice were free of tumor tissue by manual inspection.

PEG2-IFNalpha2a showed a clear decrease in the size of ACHN tumors compared to PEG2-IFNalpha2a

Unadjusted IFNalpha2a for weekly dose levels of 60, 120, and 300 micrograms, at 14 days, 21 days, 28 days, and 35 days (Figures 3 and 4).

PEG2-IFNalpha2a showed a clear decrease in G402 tumor size compared to PEG2-IFNalpha2a

Unadjusted IFNalpha2a for weekly dose levels of 60 and 120 micrograms, at 14 days, 21 days, 28 days, and 35 days (Figures 5 and 6).