Interferon conjugates.

I

INTERFERON CONJUGATES DESCRIPTION OF THE INVENTION

Interferon, in particular interferon a2a, is a pharmaceutically active protein that has antiviral and antiproliferative activity. For example, interferon is used to treat hair cell leukemia and Kaposi's sarcoma and is active against hepatitis. In order to increase its 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 half-life, thus preventing them from developing their maximum clinical potency. In recent years, PEG-conjugated biomolecules have been shown to possess useful clinical 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, Advanced Drug

Delivery Systems 10, 91 (1993)). Among these, they have better physical and thermal stability, protection against susceptibility to enzymatic degradation, greater solubility, a longer period of circulating half-life in vivo, less clearance and higher potency. Branched PEG conjugates have been reported to have 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. Other

REF: 24605

-1..ί PEGylation effect of certain proteins may be reduced in vitro activity accompanied by enhanced in vivo activity. This has been observed in G-CSF (Satakeishikawa et al., Cell Structure and Function 17, 157-160 (1992)), IL-2 (Katre et al., Proc. Nati. Acad. Sci. USA 84, 1487 ( 1987))_t TNP-oi (Tsutsumi et al., Japan J. Cancer Res. 85, (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, PEGylation reduces antiviral activity in vitro but increases antiproliferative activity of human tumor cells. However, the new 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 PEG interferon conjugates. Although the antiproliferative activity of the conjugate is greatly increased over other PEG interferon-α conjugates, the reduction in antiviral activity is still similar. Furthermore, the PEG interferon-α conjugate of this invention is non-immunogenic, virtually inducing antibody formation. In contrast, 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. Branched PEG has the advantage that it allows the binding of 2 linear PEG molecules in a

-2 individual site, thus doubling the mass of bound PEG without the need for multiple pegylation sites.

Compared to unmodified IFNa (i.e. IFNa without bound PEG), the conjugate has a longer period of circulating half-life and a longer plasma residence time, lower immunogenicity, lower clearance and higher antiproliferative activity, simultaneously with lower antiviral activity in vitro. Compared to other PEGIFNa conjugates, the conjugate of this invention has much greater antiproliferative activity, disproportionate to the increase or decrease that occurs with its other characteristics, and virtually no immunogenicity.

The physiologically active PEG-IFNa conjugate species of the present invention have the formula:

O II

ROCH₂CH₂(OCH₂CH₂) n — O — C - NH <1 (CH₂)₄

L

ROCH₂CH₂(OCH₂CH₂) n'— O — C — Nhf —X— ^IFN<*

OO

The conjugate of the present invention has the same uses as IFNa, for example, in antiproliferative applications. In particular, the PEG interferon-α conjugates of the present invention are useful for immunomodulatory treatment of disorders such as neoplastic diseases, for example, capillary cell leukemia, CML, sarcoma of

-3Kaposi, and infectious diseases, the different IFNa {especially IFNa2a) are used in the same way 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. Furthermore, these conjugates have an antiproliferative activity that is superior to that of IFNof. Also as stated, 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 unwanted activity. For example, if an unwanted side effect is associated with antiviral activity, elimination of this activity would eliminate the side effect, while maintaining antiproliferative activity. Therefore, the present invention also encompasses pharmaceutical compositions made from compounds of formula I or their salts and the methods of producing them.

The pharmaceutical compositions of the present invention employed for disease control or prevention comprise an interferon conjugate of general formula I and a therapeutically inert, non-toxic and therapeutically acceptable filler material. Pharmaceutical compositions

5 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

-4 method of administration and other factors known to specialists.

The claimed conjugate is the physiologically active PEG-IFNa conjugate, of the formula:

OR

II

ROCH₂CH₂(OCH₂CH₂) n — O “C” NH i

(CH₂)₄

R'OCH₂CH₂(OCH₂CH₂) n'— O — C — NH

O —XIFNce where R and R 'are independently of each other lower alkyl; X is NH or O (X is at least one of the functional groups of the selected IFNor molecule of NH₃ or OH); n and n 'are integers whose sum is from 600 to 1500; and the average molecular weight of the polyethylene glycol units in said conjugate is approximately 26,000 daltons to approximately 66,000 daltons. The conjugate of formula I has a branched structure in which two PEG units are linked to the protein by a single link.

The numbers nyn 'are selected such that the resulting conjugate of formula I has a physiological activity of IFNa, which activity may represent the same as, rather than, or a part of, the corresponding activity of the unmodified IFNa, nyn' ( n and n 'can be the same number or different numbers) represent the number of ethylene glycol units in the PEG. An individual unit of

-5PEG from OCH₂CH₂, has a molecular weight of approximately 44 daltons. The molecular weight of the conjugate (excluding the molecular weight of the IFNa) depends on the numbers n and n '. The sum of nyn 'for the conjugate of formula I is 600 to 1500 obtaining a conjugate having a total average molecular weight of PEG units from about 26,000 to

66,000, preferably from approximately 35,000 to 45,000 daltons, and more especially from approximately 39,000 to

45,000 daltons, with 40,000 daltons being the most especially 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 be individually 420 or 520 or both can be 420 or 520, or both they can be 455. The preferred ratio of nyn 'is about 0.5 to 1.5, with an especially preferred ratio of about 0.8 to about 1.2. A molecular weight of approximately "a certain number means that it fluctuates within a reasonable range of this number, determined by conventional analysis techniques.

Also preferred is a conjugate of formula I where IFNa is IFNa2a, a conjugate where R and R 'are methyl, a conjugate where X is NH, and a conjugate where nyn' are individually or both, or 420 or 520. A conjugate having all the characteristics mentioned above is especially preferred.

R and R 'can be any lower alkyl, understanding as such an alkyl group having from one to six atoms of

-6carbon 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 'can be the same or different.

By IFNa (interferon a) and its lFNa2a species, it is meant the natural or recombinant protein, preferably human, that 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 classes of modified proteins, is included. Obtaining and separating IFNa 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 IFNa activities. Such activity in a conjugate can be determined by tests well known in the art, for example the tests described below.

-7 (see also Rubinstein et al ,, J. Virol. 37, 755 {1981}, *

Borden et al., Cañe. Res. 42, 4948 (1982)). Part of this invention is a conjugate of formula I that has higher antiproliferative activity and lower antiviral activity than unmodified IFNa.

The conjugate of formula I is obtained by covalently binding IFNa to PEG which has been activated by replacing the hydroxyl of PEG with a binding group, forming a reagent 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 bonding takes place via an amide or ester bond. In a preferred conjugate, binding occurs via an amide bond (X is

NH). Part of this invention is a method of increasing the antiproliferative activity of IFNa while reducing the antiviral activity of IFNa, by binding IFNa as described above to a reagent of formula II to give a PEG-IFN conjugate.

X represents the binding site on IFNa whereby the PEG reagent of formula II is covalently linked to IFNa. The reagents bind to primary amino groups (XH = NHJ on eg lysine or the N-terminus of IFNa. The reagents can also bind to a hydroxyl (XH = OH) on eg serine.

-8O

YOU

ROCH2CH 2 (OCH₂CH₂) π —Oc — NH (CH₂)₄

R'OCH₂CH₂(OCH₂CH₂)_n O — C — ríhf

II

OR

0 'or — ν

<img file="MX9704012A_D0001.tif" />

+ XH-IFN0C or II

ROCH₂CH₂(OCH₂CH₂)_n —O — C - NH: h₂)₄

-Nh &lt;L ί

R'OCH₂CH₂(OCH₂CH₂)_n_OR—C — NH —Χθ and&gt;

q + HON or

IFNa

<img file="MX9704012A_D0002.tif" />

<img file="MX9704012A_D0003.tif" />

The reagent of formula II (PEG2-NHS), in which a total of 2 mono-methoxy PEG chains (m-PEG) are attached to Usin, each to the ct and amino groups by means of carbamate (urethane) linkages and having the carboxyl group of lysine activated 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 preferred mean MW (molecular weight) of the PEG obtained is approximately 20,000

-910 daltons, which provides a total PEG mass of approximately 40,000 daltons in PEG2-NHS (other MW can be obtained by varying n for the PEGalcohol starting materials 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 XX primarily reacts with one or more primary amino groups (eg, the N-terminus and lysine side chains) of IFNa (eg, IDNa2a) to form an amide bond between IFNa and the basic polymer backbone of the PEG. The pegylation reaction can also take place between the PEG2-NHS and the free hydroxyl groups (if any) (eg serine) of IFNa to form an ester bond. The reaction mechanism is shown above. The reaction conditions are conventional for a skilled person and are described in detail below. The PEG reagent is combined with IFNa under slightly basic conditions at low temperature under conditions suitable for a nucleophilic substitution that will produce the conjugate of formula I. This is also shown in the reaction mechanism above.

The binding of the reagents to the IFNa can be carried out by conventional methods. PEGs of any selected PM of this invention can be used. 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 IFNa and is conjugated with more than one bound.

-1011 molecule of reagent 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 purified, unmodified IFNa-free conjugate of formula I and conjugates with more than one bound reagent is identified accordingly. In addition, the reagents of formula II release one lysine per reagent ^ during acid hydrolysis, so that the number of lysines in the hydrolysis indicates the number of PEGs bound to the protein, thereby determining the number of reagent molecules bound to the conjugate.

The following examples are described for the purpose of illustrating the invention and are not limiting in any way. IFNa2a is used in these examples. Other IFNoi species can also be conjugated to PEG, by the methods described in the examples.

DESCRIPTION OF THE DRAWINGS

Figure 1: PEG2-IFNalpha2a antitumor activity in immunosuppressed mice implanted subcutaneously with human kidney A498 cells. All animals received a 2 x 1Q subcutaneous implant^S human kidney A498 cells the

-1112 day 33 of the study. On day 0 of the study, treatment with PEG-IFNalfa2a was started. The indicated amount (30, 60, 120, or 300 pg) of PEG2-IFNalfa2a was administered subcutaneously under the side opposite the tumor, 1 time per week for a period of four weeks.

Figure 2: Antitumor activity of IFNalfa2a in the immunosuppressed mouse implanted subcutaneously with human kidney A498 cells. All animals received a 2 x 10 subcutaneous implant^and human kidney A498 cells on study day 33. On day 0 of the study, treatment with lFNalfa2a was started. The stated amount (10, 20, 40 or 100 pg} of

IFNalfa2a was administered subcutaneously under the side opposite the tumor, 3 times a week for a period of four weeks.

Figure 3: Antitumor activity of PEG2-IFNalpha2a in immunosuppressed mice implanted subcutaneously with human kidney ACHN cells. All animals received a 2 χ 10 subcutaneous implant⁶ human kidney ACHN cells on day 25 of the study. On day 0 of the study, treatment with PEG2-IFNalfa2a was started. The indicated amount (30, 60, 120, or 300 / ig) of PEG2-IFNalfa2a was administered subcutaneously below the non-tumor flank, 1 time per week over a period of five weeks.

Figure 4: Antitumor activity of IFNalfa2a in immunosuppressed mice implanted subcutaneously with human kidney ACHN cells. All animals received a 2 x 10 subcutaneous implant^and human kidney ACHN cells on day 25 of the study. On day 0 of the study, treatment was started

-1213 with IFNalfa2a. The indicated amount (10, 20, 40, or 100 of IFNalfa2a was administered subcutaneously under the side opposite the tumor, 3 times per week for a period of five weeks.

Figure 5: PEG2-IFNalpha2a antitumor activity in immunosuppressed mice implanted subcutaneously with human kidney G402 cells. All animals received a 2x10 subcutaneous implant^and human kidney G402 cells on day 45 of the study. On day 0 of the study, treatment with PEG2-IFNalfa2a was started. The indicated amount (30, 60, 120, or 300 gg) of PEG2-IFNalfa2a was administered subcutaneously under the side opposite the tumor, 1 time per week for a period of five weeks.

Figure 6; Antitumor activity of IFNalfa2a in immunosuppressed mice implanted subcutaneously with human kidney G402 cells. All animals received a 2x10 subcutaneous implant^s human kidney G402 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 gg) of

IFNalfa2a was administered subcutaneously under the side opposite the tumor, 3 times per week for a period of five weeks.

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.

-13L (Huntsville, Ala). Fractogel® EMD CM 650 (S) resin, with 25-40 gm size particles, was supplied by EM Separations (Gibbstown, MA). Concentrated Saline Buffered Phosphate (10X) (PBS), pH 7.3, was purchased from BioWhittaker (Walkersville, MD). Prefabricated sodium dodecyl (lauryl) sulfate / polyacrylamide gel electrophoresis gels (SDS-PAGE) and electrophoresis units were purchased from NOVEX (San Diego, CA). Concentrated Fast Stain 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. (Woods Hole, MA). The rest of the reagents used were of the highest quality available on the market. Cannulated rats in 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 gmoles) of the reagent of formula II (mean MW of 40,000 daltons) were added to 50 mg (2.6 gmoles) 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)

-14 of Fractogel EMD CM 650 (S), with a flow rate of 20 mi / minute. The column was previously equilibrated with 10 Mm ammonium acetate, Ph 4.5. The column effluent was monitored by UV absorbance at 2 80 nm. The column was then washed with the equilibration buffer until the UV absorbance returned to the baseline. 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 NaCI in buffer ammonium acetate 40 Mjn. The unmodified IFN that remained on the column was eluted with 0.5M NaCI in the same buffer. The column was regenerated by washing with 1.0M NaCI followed by washing with equilibration buffer. The pooled fractions of the conjugate of formula I were concentrated in a shaking cellular Amicon concentrator equipped with a YM10 membrane at approximately a concentration of 1 mg / ml.

The cation exchange resin, Fractogel CM 650 (S) used for purification, effectively adsorbed PEG and unmodified IFN. The adsorption strength depended on the degree of pegylation. The conjugate bound less tightly than the unmodified IFN. PEG-IFN oligomers were eluted with 40 Mm ammonium acetate, whereas the conjugate of formula I eluted with 0.12 M NaCI. Unmodified IFN eluted with 0.5 M NaCI. All preparations contained &lt;5 EU / mg of endotoxins. The resulting preparation contained&gt; 9 9% of conjugate of formula I and was free of unmodified IFN.

-1516

B. Large-scale preparation of the conjugate of formula I.

Six thousand two hundred and forty milligrams (156 gmoles) of the reagent of formula II (mean molecular weight of 40,000 daltons) were dissolved in 63 ml of 1mM HC1 at 4 ° C and quickly added to 125 ml of a solution containing 1000 mg ( 52 emoles) of interferon in borate buffer 50 (nM, 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. 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 to 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 speed of 1 , 3 cm / min. The column was washed with the equilibration buffer followed by 10mM NaCl to remove excess reagent, reaction side products, and PEG-IFN oligomers. The conjugate of formula I was eluted with equilibration buffer containing 200 mM NaCl. 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 into the final formulation buffer, 20mM sodium acetate, pH 5.0, containing 150mM NaCl, El total yield of the conjugate of formula I was 40-45%.

The PEG-IFN purified from the large-scale preparation consists of&gt; 99% conjugate of formula I. The molecular weight

-1617 Average conjugate of formula I in this example is 62,000 daltons, including the molecular weight of lFNa2a which is 19,241 daltons, and the average molecular weight of the reagent which is between 40,000 and 45,000 daltons, about 43,000 daltons.

Example 2

Characterization of the conjugate of formula I

Protein determination

Protein concentrations were determined using an A value._2B0 1.0 for a 1 mg / ml solution of IFNa «2a.

SDS-PAGE analysis

The conjugate was analyzed by sodium dodecyl (lauryl) sulfate / polyacrylamide gel electrophoresis (8-16%), under reducing conditions, according to the methods of Laemmli (Nature 227, 680 (1970)). SDS-PAGE analyzes containing protein-looking PEG conjugates were performed using Fast Stain (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 the preparations contained &lt;5 EU / mg of endotoxins.

Example 3.

In vitro bio-activities of the conjugate of formula I

Antiviral activity in bovine kidney cells

The in vitro antiviral activity of IFNa2a and the conjugate of formula I was determined as prepared in the example.

lA, in a cell culture bioassay using cells

-1718 Madin-Darby bovine kidney (MDBK) activated with vesicular stomatitis virus (Rubinstein et al., See above). Antiviral activities are summarized in Table 1, along with their corresponding residual activities, as a percentage of the starting IFN.

Table, l

Antiviral activities

<td>Samples</td><td>PEG Kind</td><td>Total mass from PEG (kDa)</td><td># Lys modified</td><td>Exercise specific (U / mg)</td><td>Exercise residual (%)</td>

<td>IFNa2a</td><td> —</td><td> —</td><td> —</td><td>2.00 x 10 *</td><td> 100</td>

<td>Conjugate of formula I</td><td>Branched</td><td> 40</td><td> 1</td><td>1.40 x 10 '</td><td> 7</td>

In vitro antiproliferative activity in human tumor cells.

In vitro antiproliferative activities were tested in 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 2mM glutamine CGrand Island Biologicals, Grand Island, NY). The cells were explored and determined to be free of mycoplasma. Cells were added (2 x 10⁴) to wells of microtiter plates (Costar, MA) in 100 μΐ of medium. Various concentrations of IFN and the conjugate of formula I prepared according to Example Ι.Α. were added to the wells in a volume of 100 µΐ. Plates were incubated at 37'C in 5% C0₂ during

-1819 hours. Cells were pulsed with 0.25 / Ci / well of³Htimidine (New England Nuclear, Boston, MA), sixteen hours prior to cell harvest. Cells were collected on glass filters and counted in a liquid scintillation counter. The results were expressed as% inhibition, calculated using 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 mean of all cases. Experiments were performed at least twice with comparable results.

Antiproliferative activities (IC_S0) of IFN and the conjugate are listed in Table 2. The data indicates that there is a 28-fold increase in antiproliterative activity for the conjugate of formula I, relative to that of IFN.

labia 2,

Activate intra-iterative iprol in_vitro in human Paudi cell lines (Burkitt lymphoma)

Shows

IFNce2a

Conjugate of formula I

IC50 (ng / ml) AntipTOliferátivQ

0,56

0,02

Exercise

Increase

28x

-19ι

Example 4 EaCTÍM, pG¿aéfc.i.ga

Female Sprague Dawley rats, surgically implanted with jugular cannulas, with an average body weight of 240-260 g, with free access to food and water, were individually housed and kept on a 12 hour dark light cycle. Jugular cannulas were washed with PBS within 4-6 hours after arrival. The next day, after washing with 0.15-0.2 ml of PBS, 2x10 * units of IFNa in 0.2-0.4 ml of PBS were injected, 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 drawn at 5, 15 and 30 minutes, as well as at 1, 3, 5, 12 and 24 hours after the injection of IFN and, the conjugate of formula I. At all times indicated, After discarding the first 0.15 - 0.2 ml of blood, an aliquot of 0.5 ml of blood was drawn using a new syringe through the jugular cannula. The samples were poured separately, into serum tubes at room temperature. After all the samples were collected at the indicated times, the tubes were centrifuged at 14,000 xg in an Eppendorf refrigerated centrifuge for 10 minutes. The serum was transferred separately to 1.5 microfuge tubes and frozen at -80 ° C, until used for the bioassay. Serum samples were appropriately diluted and antiviral activity was determined in each

-2021 one of the indicated times, as described. From the graph of time versus activity, the terminal half-lives of the conjugate of formula I and IFNa were determined and reported in Table 3, which also includes plasma residence times.

Table 3

Terminal half-lives (t_1/2) and average plasma residence time

Residence time

Shows £ .1 / 3 (hacas ?. in Plasma (hours)

IFNa2a 2.1 1.0

Conjugate of formula I 15.0 20.0 t_1/2 terminal estimated by linear logarithmic regression.

Example 5 Immunoaenicity

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 aggregated form of IFNa2a which is more immunogenic than the monomeric form. Blood samples were drawn 19 days after the last injection, and the serum was evaluated for the presence of neutralizing antibodies.

As seen in Table 4, IFNa2a injected mice produced neutralizing antibodies, and this response was greatly increased in aggregate injected mice.

-2122 interferon. Antibodies were not detected in most animals injected with the conjugate of this invention.

Table 4

Immunogaaigidád

Antibody (UNI / mi) *

Median Treatment

IFNa2a 2,400

IFNa2a Aggregates 42,667

Conjugate of formula I 0 * Units / ml interferon neutralizers Example 6

Antitumor activity · in vivo

Margin 217 - 8,533

8.000 - 768.000

- 1,133

The in vivo antitumor activity of a conjugate of formula I (PEG-IFNalfa2a) and IFNalfa2a was evaluated, by determining their 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 (Harían) received a subcutaneous implant under the left rear side of 2 x 10® A498 human kidney cells (Figures 1 and 2), ACHN human kidney cells (Figures 3 and 4), or G402 human kidney cells (figures 5 and 6). 3 to 6 weeks were allowed for the tumors to establish, as indicated. The size criteria for acceptance in the study was from 0.05 to 0.50 cubic centimeters. Mice were treated with weekly total doses of unmodified PEG2-IFNalfa2a or IFNalfa2a of 30, 60, 120, or 300 gg. If

-2223 PEG2-IFNalfa2a mice were treated once a week (Monday) with 30, 60, 120 or 300 gg of PEG2-IFNalfa2a per treatment. In the case of unmodified IFNalfa2a, 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 from 4 to 5 weeks depending on the aggressiveness of the tumor. Tumor volumes were measured every Monday before treatments.

Results: PEG2-IFNalfa2a showed a marked reduction in tumor size A498 compared to unmodified IFNalfa2a at all weekly dosage levels tested, at 7 days, 14 days, 21 days, and 28 days after starting treatment (Figures 1 and 2) . The treatment continued for four weeks. Seven days after treatment was discontinued, three mice from each group were sacrificed. In the three mice treated with PEG2-IFNalfa2a, no residual tumor was observed. In the mice treated with lFNalfa2a without modifying the A498 tumor weight it was 1.28 grams, 0.62 grams and 1.60 grams respectively in each of the three mice. A498 tumor weight was 2.3-2 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 existence or not of tumors was determined by palpation in seven mice. All seven mice were found to be free of tumor tissue by palpation.

PEG2-IFNalfa2a showed a significant reduction in ACHN tumor size compared to IFNalfa2a without

-2324 modify, for weekly dosage levels of 60, 120, and 300 pg, at 14 days, 21 days, 28 days, and 35 days (Figures 3 and 4).

PEG2-XFNalfa2a showed a significant reduction in G402 tumor size by 5 compared to unmodified IFNalfa2a, for weekly dosing levels of 60 and 120 gg, at 14 days, 21 days, 28 days, and 35 days (Figures 5 and 6).

*****

It is noted that in relation to this date, the best method known by the applicant to put the aforementioned invention into practice is the one that is clear from the present description of the invention.

Having described the invention as above, the property contained in the following is claimed as property:

-2425