Interferon conjugates

The invention relates to a physiologically active conjugate of interferon alpha (IFNα) with branched polyethylene glycol (PEG), a process for the preparation of conjugates and pharmaceutical preparations containing a conjugate (Int. Cl. 7: Α61Κ 47/48).

CONDITION ΊΈΗΝ1Κ.Ε

Interferon, more precisely interferon 2a, is a pharmacologically active prolcin with antiviral and antiproliferative effects. Interferon is used, for example, in the treatment of hair cell leukemia and Kaposi's sarcoma, and has also been shown to be effective in hepatitis. In order to improve stability and solubility as well as reduce immunogenicity, pharmaceutically active proteins such as interferon can bind to polyethylene polyethylene glycol (PEG).

The bioavailability of prosthetic drugs is often limited by the short plasma half-life, which prevents them from achieving maximum climactic activity. In recent years, PEG-conjugated biomolecules have been shown to possess beneficial properties from the aspect of clinical use (Inada et al., J. Bioact. And Compatible Polviners 5, 343 (1990); Delgrado et al., Critical Reviews in Therapeutic Drug Carrier Systems). 9, 249 (1992); Kaire, Advanced Drug Delivery Systems 10, 91 (1993)). These include better physical and thermal stability, protection against susceptibility from enzymatic degradation, increased solubility, longer in vivo circulating half-lives, reduced release, and improved elasticity. It has been observed that branched PEG conjugates have an increased pH value and thermal lability and increased stability towards proteolytic digestion compared to linear PEG conjugates. (EP-A 0 400 472 and Monfardini et al., Bioconjugate Chem., B, 62 (1995)). Remaining properties of PEG proteins are reduced immunogenicity and antigenicity, as well as reduced toxicity. This has been observed, inter alia, in G-CSF (SatakeIshikavva et al., Cell structure and Function, 17, 157-160 (1992)), IL-2 (Katre et al.,

-*)

49533 Β

Proc. Natl. Acad. Sci. USA 84, 1487 (1987), TNF-α. (Tsutsumi 1 et al., Jpn. J. Cancer Res., 85, 9 (1994), IL-6 (Inoue et al., J. Lab. Clin. Med. 124, 529, (1994)) and CD <- l_gG (Chamovv et al., Bioconj. Chem. 5, 133 (1994)).

DETAILED DESCRIPTION OF THE INVENTION

It has been observed that in the case of interferon as well, binding to PEG decreases antiviral activity in vitro but increases antiproliferative activity against human tumor cells. Thus, the invention of this novel PEG interferon conjugate has unexpected antiproliferative properties, so that it acts more intensely than interferon itself and other PEG interferon conjugates. In addition to the fact that the antiproliferative activity was significantly improved with PEG-interferon α conjugates, the reduction of antiviral activity remained the same. In addition, the subject of the present invention, the PEGinterferon-α conjugate is non-immugenic, i. does not stimulate antibody production.

Accordingly, the invention relates to a new class of PEG interferon derivatives (IFNα). As will be seen in the section below, the conjugate encompassed by the present invention has a branched PEG structure. The advantage of branched PEG is that it allows the binding of 2 linear PEG molecules to one site, thus increasing the mass of bound PEG without increasing the binding site of PEGylation.

Compared with unmodified IFNα (pF-free IFNα), the conjugate has a prolonged circulating half-life and plasma retention time, reduced immunogenicity, decreased release, and increased antiproliferative activity accompanied by decreased in vivo antiviral activity. Compared to other PEGIFNa-conjugates, the conjugate of the present invention has a much higher antiproliferative activity, which is not proportional to an increase or decrease in the intensity of the properties, and has virtually no immunogenicity.

The physiologically active PEG-IFNα conjugate encompassed by the present invention has the formula:

49533 Β

ROGHaCHz / OCHzCH ^ n — 0

Ο II

<img file="RS49533B_D0001.tif" />

ROCH₂CH₂(OCH₂CH2) n ·

<img file="RS49533B_D0002.tif" />

IFNoc

The conjugate of the present invention has the same application as IFNα, e.g. antiprotective.

In particular, the PEG interferon-α conjugates of the present invention are useful for treating diseases associated with immunomodulatory disorders such as non-plasma such as hair cell leukemia, CML, Kaposi's sarcoma and infectious diseases, in the same way that IFNα (especially IFNα2a) is used. for the treatment of these diseases. In all of this, the conjugate of the present invention has improved properties including exceptional laxity, increased solubility, prolonged circulating half-life and extended plasma residence time. In addition, the antiproliferative activity of these conjugates is superior to that of JFNα. As already mentioned, the conjugates have different antiviral and antiproliferative effects. This feature was additionally used 73. improving the desired activity of the conjugate and at the same time reducing or eliminating side effects. If for example. an undesirable side effect associated with antiviral activity, its elimination will also eliminate the side effect while the antiproliferative activity is retained. Therefore, the present invention contains pharmaceutical preparations based on substances or their salts having the structure of the formula and methods for their preparation.

The pharmaceutical compositions of the present invention are for the treatment and prevention of disease and comprise interferon conjugates of general formula I and therapeutically inert or acceptable, non-toxic excipients. The pharmaceutical preparations to be used are formulated and dosed in a manner based on practical medical experience, the characteristics of the disorder, the general condition of the patient, the route of administration and prescribing of the protein conjugate and other facts known to clinicians.

the phrase conjugate is a physiologically active conjugate of PEG-IFNci of general formula:

49533 Β

Ο

Η ROCHgCHgiOCH ^ H ^ n- ~ Ο * —S * ~ ΝΗ

L / GM »\

.Η

ΗΌΟ ^ ΟΗ ^ ΟΟΡ ^ ΟΗ ^ η'— ° —C - Α —X IFNa Ο 0 where R and R 'each represent C |._halkyl; X is NH or Ο (X represents at least one of the functional groups in the molecule ll-'Να selected from NH-₂ or OH); η 1 n 'iziiosc from 600 to 1500; and wherein the average molecular weight of the pdiethylene glycol chain in said conjugate is from 26,000 to 66,000 daltons. The conjugate of formula I has a branched structure because the two PEG chains are linked to the protein by a single bond.

VrcJnosii /.a ι »ι n 'are selected so that the resulting conjugate of formula 1. ipa iiziological Lk! Iviiusi ll'Nlit. and / .razenu and isioj or to a greater extent, or in vicIli f'rakcije corresponding to activity / a petoJ1G1ko \ api ΙΕΝα. η in '(nor n' can be l.sli i'i ra / .liciti) picdslav lja | ii number of units ciilengiikola in PI'G. One PEG unit. OCl RC! F has a molecular weight of 44 daltons. The molecular weight of the conjugate (due to the molecular weight of IFNα) depends on the^:. The sum of n 'in the conjugate of formula 1 is nd 600 to 1 500. which produces conjugates with a total average molecular weight of 1Ί · 4 ϊ units from 26000 to .66000 and best from 35000 to 45000 daltons, and ordered 39000 to 45000 daltons. gdc is particularly preferred to be 40,000 daltons. The preferred sum for nin 'is from 800 to 1200, with an average sum of 850 to 1000, where the preferred value of the sum is 910. Either n or n' may be 420 or 520, or both may be 420 or 520. or 455. The preferred ratio for nin 'is from 0.5 to 1.5 with a particularly preferred ratio from 0.8 to 1.2. The molecular weight ud ”about where a certain number means to be within a suitable range of a log number as determined by conventional analytical techniques,

Also preferred is a conjugate of formula I wherein the IFNcc may be lb'NctZa, a conjugate wherein R 1 and R 2^! methyl, a conjugate wherein X represents NH, and a conjugate wherein nin 'each for itself or both may be 420 or 520. Such a conjugate is especially preferred which has all of the above characteristics.

49533 Β

1 'ι II' can be any alkyl. wherein the alkyl moiety may have from one to six aioms such as rnetyl, ethyl. isopropyl. etc. Forked aikils are covered. Pož.eiini alkyl jc melyl. In terms of the PEG groups of formula I, R and R 'may have the same or different values.

li'No. (inififeron ') and its subtypes IFNa2a means a natural or recombinant protein, preferably human, obtained from any conventional source such as tissues, protein synthesis, cell culture with natural or recobinant cells. Included is any of the proteins that have activity for IFNα, such as muteins or other modified proteins. Obtaining and isolating 1FNa from a natural or ecombinanyl source is well known (Pestka, Arch. Biochem. Biophvs. 221. 1, (1 683)). The preferred IFNα is! FNcx2a. which is given here, is obtained by the known [-n'.smpciina (Pestka. Sci. Am. 249. 36 (1983); European Patent bt. 43 980)

Both the isiologically active conjugate of ibrmula I and the activity for ll'Ncc under which pudiazuiiicva bull&gt; which traction or a large number of known activities of IFNct. as determined by various experiments known to the state of the art. The conjugates of the present invention possess IFNct activity as demonstrated by the antiproliferative activity of preunion cells and antiviral activity against virus-infected cells. Onc represent the known activities of IFNct This activity of the conjugate inoz.e on receipt ·. to be determined by the experiments described below which are well known to those skilled in the art (see also Rubinstein et al., J. Virol. 37, 755 (1981): Borden et al. Canc. Res. 42, 4948 (1982)). Dco of the invention relates to a conjugate of formula I having higher anliproliferative activity and less antiviral activity than for unmodified ΙΡ'Να.

The iomiyl conjugate is obtained by covalently binding IFNct to PEG which is activated by replacing the PEG hydroxyl with a linking group. by reacting a reagent that is a PEG derivative of an ester of N-hydroxy succinamide (especially monomethoxy PEG) ibrmnle P. Reagents can be obtained by conventional methods (Mondafini et al., As above). Binding is performed via an amide or ester bond. In one preferred conjugate, coupling is performed with a rhecoamide bond (Hjs NH). Dco of the present invention provides a method of increasing the antiproliferative activity of IFNα, thereby reducing the antiviral activity of IFNα. by binding IFNci as described above to the reagent of formula II to give the PEG-IFN conjugate.

X represents a binding site on IFNα at which the PEG rcagens of formula II are covalently bound to IFNα. Reagents bind to primary amino groups (HN = NH₂) lysine or for the N-terminal end of IFNα. Reagents can also be attached to the hydroxyl group (HN = OH) of serine.

v

VOSNgSNHOSNgSNgJp —O — C — ΝΗ

ROCHaCHsfOCHaCH ₂) _η—

<img file="RS49533B_D0003.tif" />

+ ΓΡΝ-ΓΡΝ «

<img file="RS49533B_D0004.tif" />

<img file="RS49533B_D0005.tif" />

ROCH₂CH₂(OCH₂CH₂)_n--ο — C

II ο

A reagent of formula II (PEG2-NHS) in which 2 of the total number of monomethoxy group chains (m-PEG) are bound to lysine. each for α and ε amino groups via bonds on carbamates (urethane) and with the carboxyl group of lysine activated on succinimidyl ester, can be obtained by conventional methods, based on known methods (Monfardini et al., see above) which can be. applied to a reagent with an R1 group representing lower alkyl, and having the desired value for n. The reagent is available from Shearwater Polymcrs Inc., (Huntsville, Alabama). The preferred mean value for the molecular weight of PEG is 20,000 daltons. relative to a total PEG weight of 40,000 daltons in PEG2-NHS (other molecular weights can be obtained

49533 Β The usual procedure is by varying the values ​​for PEG alcohols which are the starting materials for the reagent of form II).

Reagents of formula II can be coupled to IFNα by conventional methods. Rcagens i'oniiule 11 first reacts with one or more primary amino groups (e.g., at the terminal end and lysine side chains) of IFNα (e.g., IFNα2a) to form a bond between both IFNα and the PEG polymer chain. The pegylation reaction takes place between PEG2-NHS and (if any) free hydroxyl groups (e.g. serine) of IFNa to form an ester bond. The reaction mechanisms are shown below. The PEG reagent is combined with IFNs. at a moderately basic core at low temperature under conditions suitable for nucleophilic substitution to give the conjugate of formula I. This is also shown in the above-mentioned reaction mechanism.

Reagent binding for IFNo. it can be performed by conventional procedures. PEGs of any molecular weight encompassed by the present invention may be used. The reaction conditions are selected so as to obtain the desired conjugate with the reagent that binds to it. Conjugate f'orniule 1. having one reactant forrnule II. It is separated by methods from unmodified IFNa and conjugates to which more than one reagent molecule is attached. Purification methods, such as cayon ion-derived chromatographs, can be used to separate conjugates based on charge differences and are therefore effective for separating conjugates according to nolculus masses. The content of the fractions obtained by cation ion exchange hyinatography can be identified on the basis of molecular weight using conventional methods. for example, mass spectroscopy, SDS-PAGE electrophoresis. or other methods for separating molecules from molecular inasaines. In this way, the fraction containing the conjugate of formula I released from the uncanodified IFα and the conjugate by acid hydrolysis is identified, so that the number of lysine molecules in the hydrolysis indicates the number of PEGs bound to the protein. and thus the number of molecules of reagents bound to the conjugate can be determined.

The application which serves as a further illustration of the present invention. IFNct2ase is used in these examples. Other types of IFNa can also be bound to PEG supplements supported herein by appropriate examples.

Antitumor activity for REO2-! R1^noYes! 1a2z. code

HliSv Va

DESCRIPTION OF FIGURES human A498 kidney cells are subcutaneously implanted. On the 33rd day from the start of the experiment, all animals were subcutaneously implanted with 2 χ 10⁶ human A498 kidney cells. On day zero of the study, treatment with IFNaIfa2a was started. A certain amount (10, 20, 40 or 100 [mu] g) of IFN alpha 2a was administered subcutaneously to the side of the loin opposite to that on which the tumor was located, once a week for a period of four weeks.

Figure 2: Anti-tumor activity for PEG2-1FNalla2a in hairless niches to which human A498 kidney cells were subcutaneously implanted. On the 33rd day from the beginning of the inspection. pigs were implanted subcutaneously in animals 2 χ 10⁶ human A498 kidney cells. On the zero day of the examination, the treatment with H began^?Nalfa2a. A certain amount (10. 20. 40 or 100 [mu] g) of IkN alpha 2a was administered subcutaneously to the side of the loin opposite to that on which the tumor was located. 3 times a week for a period of four weeks.

Figure 3: Antitumor activity for PEG2-1FNalpha2a in hairless mouse mice are subcutaneously implanted human renal ACHN cells. On the 25th day from the beginning of the experiment,

ΤΊ

V 011PJ ίΐ 'ΙιίΙ

<img file="RS49533B_D0006.tif" />

vClljd UUUlCgd.

On day zero of the study, treatment with IFNalpha2a was started. A certain amount (30. 60. 1 20 or 300 pg) of PEG2-IFN alta 2a was administered subcutaneously to the side of the loin opposite to the turner, once a week for a period of four weeks.

Figure 4: Antitumor activity for! FNalfa2a in hairless yin horses were subcutaneously implanted with human ACHN kidney cells. On the 25th day from the beginning of the trial. all animals were implanted subcutaneously for 2 h 10⁶ human A498 kidney cells. On day zero of the study, treatment was started with IFNalpha2a. A certain amount (10. 20. 40 or 100 gg) of IFN alpha 2a was administered subcutaneously to the side of the loin opposite to that on which the tumor is located. iri times a week for a period of five weeks.

Figure 5: Antilumar activity for PEG2-IFNalpha2a in hairless mice subcutaneously implanted with human renal G402 cells. 45th day from the beginning of the experiment,

49? J3 Β all animals were subcutaneously implanted 2 x 10^ft human G402 kidney cells. On the first day of testing, treatment with IFNalpha2a was started. A certain amount (30. 60, 120 or 300 pg) of IFN alpha 2a administered subcutaneously to the side of the loin is opposite to that on which the tumor is, once a week for a period of five weeks.

Figure 6: Antitumor activity for IFNalpha2a in hairless mice subcutaneously implanted with human G402 kidney cells. 45th day from the start of the trial. pigs were implanted subcutaneously for 2 h 10⁶ human G402 kidney cells. On day zero of the study, treatment with IFNalpha2a was started. A certain amount (10. 20. 40 or 100 [mu] g) of IFN alpha 2a was administered subcutaneously to the side of the loin opposite to that on which the tumor is, three times a week for a period of five weeks.

Example 1

Preparation of Formula 1 conjugates

M alcn i al i

Interferon «2a was prepared using known melodies (Peslka, supra). Polyethylene glycol (PEG) reagent of formula II was purchased from Shearwater Water Polyers, Inc. (Hunisville. Ala). Fractogel® EMD CM 650 (S) resin, with a particle size of 25 - 40 μm, was obtained from EM Scparations (Gibbstown, MA). Concentrated (10x) phosphate buffer solution (PBS), pH 7.3, purchased from BioWhittaker (Walkersville, MD). Already pouring sodium dodecyl (laurel) sulfate / polyacrylamide gel electrophoresis (SSC-PAGE) gels and electrophoresis kits were purchased from NOVEX (San Diego, CA). The concentrated 'Fast Stain' dye for staining PEG conjugate proteins on SDS-PAGE is kiipijeii from Ζ, υίοη Kesearch, Inc. (Newion, MA). LAL kit. endotoxin test js purchased from Associaies of Cape cod, lnc. (Wuods hole, MA). All other reagents used were of the highest possible quality. Jugular vem cannula rats and BDF-1 mice were purchased from Charles River Laboratories (Wilmington, ML).

Experimental procedures? SMALL SCALE preparation of conjugates of Formula I

49533 Β

An ounce of eight milligrams (5.2 μηιοί) of the Formula II reagent (average MW of 40,000 daltons) was added to 50 mg (2.6 μηιοί) of IFNa in 10 ml of 100 mM borate, pH 8.0. The final protein to reagent ratio was 1: 2. The reaction mixture was stirred at 4'C for two hours. The reaction was stopped by adjusting the pH to 4.5 with glacial acetic acid.

The reaction mixture was diluted 50 times with water, passed through a 0.2 μ filler and applied to an Amicon column packed with 100 ml (3.2x13 cm) of Fractogel EMD CM 650 (S) at a flow rate of 20 ml / min. The column was previously equilibrated with 10 mM ammonium acetate, pH 4.5. The column eluate was monitored by measuring UV absorbance at 280 nm. The column was then washed with equilibration buffer until UV absorbance was reduced to baseline. PEG-IFN conjugates with more than one reagent l'orinule 11 (PEG-IFN oligomers) were eluted with 40 mM ammonium acetate. pH 4.5 and the conjugate of Formula 1 was 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 pulc.ru. The column was recovered by washing with 1.0 M NaCl. and the washed equilibration flushes with pulp. The pooled fractions of Forinule 1 conjugates were concentrated in Amicon cells for mixing with stirring, over ΥΜ10 membranes. to a concentration of lmg / mi.

The fractional resin Fractogel CM 650 (8) used for purification adsorbed PEG and unmodified IFN. The strength of adsorption depended on the degree of l'Egylation. The conjugates bound less tightly than the unmodified IFbi. PEGli'N oligomers were eluted with 40 mM ammonium acetate, while the conjugate of Formula I was eluted with 0.12 M NaCl. Unmodified IFN was eluted with 0.5 M NaCl. All preparations contained &lt;5EU / mg endotoxin. The final preparation contained&gt; 99% of the conjugates of Formula I and did not contain unmodified IFN.

B. LARGE-SCALE preparation of a conjugate of Formula I

Six thousand two hundred and forty milligrams (156 pmol) of Forinule II reagents (average molecular weight 40,000 daltons) were dissolved in 63 ml of 1 mM IICL at 4 ° C and rapidly added to 125 ml of a solution containing 1000 mg (52 μηιοί) of interferon in 50 mM borate buffer, pH 9.0.

49533 The final protein ratio was 1: 3 and the final protein concentration of the reaction mixture was 5.3 mg / ml. The reaction mixture was stirred at 4 ”C for 2 hours. The reaction was quenched by adjusting the pH to 4.5 with glacial acetic acid.

The reaction mixture was diluted with 10 pools of water and applied to a column packed with 600 [mu] l 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 equilibration buffer followed by 10 mM NaCl to remove excess reagent. reaction by-products and PEG-IFN oligomers. The conjugate of Formula 1 was eluted with equilibration buffer with 200 mM NaCl. Unmodified interferon still adsorbed on the column was removed by washing with 0.75 M NaCl in equilibration buffer. The Fonnule 1 conjugate, eluted at 0.3-0.5 mg / ml, was further concentrated and passed through a diafilter into the final formulation buffer - 20 mM sodium acetate. pll 5.0. which contained 150 mM NaCl. The total yield of the Forniula I conjugate was 4045% and the purified PEG-IFN from the LARGE-SCALE preparation was coated with&gt; 99% of the conjugate of Formula 1. The average molecular weight of the conjugate of Formula I from. of this example is 62,000 daltons, taking into account the molecular weight of IFNa2a of 19,241 daltons. and an average reagent molecular weight of between 40,000 and 45,000 dallons. about 43,000 daltnna.

Pi imer 2

Characterization of Formula 1 conjugates

Determination of protein

Protein concentration was determined by A₂8o values ​​1.0 for 1 mg / ml rasivor Il-Na ci2a.

SDS-PAGE analysis

Conjugai was analyzed by sodium dodecyl (lauryl) sulfab'polyacryamide (8-16%) gel dechirophoresis. under reducing conditions, according to the method of Laemmli (Nature 227,

680 (1970)). SSC-PAGE with PEG-conjugates was stained with the protein bojoin Fast

Siain (Zoion Research. Lnc.) According to the manufacturer's instructions for use.

Determination of endotoxin levels

Endotoxin levels were determined by the LAL method, according to the manufacturer's instructions for

Lipotrebu. All preparations contained &lt;5 EU / mg endotoxin.

Example 3

In I 'itro bioactivity of Formula 1 conjugates

Antiviral activity in bovine renal cells and viro antiviral activities of lFNa2a and the conjugate of Formula 1 prepared as in Example 1A were determined in tesla in cell culture. where Madin-Darbv bovine (MDBK) renal cells infected with vesicular stoniatitis virus (Rubinslein et al. supra) were used. The values ​​of anliviral activity are given in Table 1 together with their corresponding residual activities as a percentage of the initial 1FN.

l'abc'la 1

<td colspan="6">Anti-viral activity</td>

<td>Samples</td><td>Vrsla PEG</td><td>Total mass of PEG (kDa)</td><td># Lys modified</td><td>Specific activity (U / mg)</td><td>Residual aklivnosr (%)</td>

<td>lFNa2a</td><td> -</td><td> -</td><td> -</td><td>2.00 x 10⁸</td><td> 100</td>

<td>Conjugate of formula I</td><td>With branching</td><td> 40</td><td> 1.</td><td>1.40 x 10⁷</td><td> 7</td>

in viiro aniiprolifcrativna activity in human lumor cells

49533 In vitro antiproliferative activity was tested on human Daudi (Burkitt limibm) cells. as described by Borden et al. Human Daudi cells were stored as stationary suspension cultures in RPMI 1540 enriched in 10% fetal bovine scrumon) and 2 mM glutamine (Grand Island Biologicals, Grand Island, NY). Screening of cells for mycoplasma was performed and its absence was established. Cells (2 χ 10⁴) were added to wells on microtiar plates (Costar, (Α) in 100 μΐ medium. Different concentrations of IFN'a and the conjugate of Formula 1 prepared as in Example JA were added to wells in a volume of 100 μΐ. The plates were incubated for 72 hours at 37 ° C in 5% CCR. Using a pulse of electric current, the cells were treated with 0.25 pCi / well Ήliniidinoin (Nc \ v England nuclear. Boston, MA). sixteen hours prc žeive cells. (The cells were collected on glass filters and measured with liquid scintillation bioaccumulator. Rczullali were expressed as% inhibition calculated using the formula:

% mhibicijc [[Α-Β'Ά] h 100.

gdc:

A - cpm ii conliol culture (cells incubated sanio in mcdium)

B ~ cpm ii experimental culture

U / .urci were analyzed in quadriplicate and the standard deviation was less than 20% of the mean value of all cases. The experiments were performed at least twice with comparable ie / .ultaiinia.

The antiproliferative activities (IC50) of IFN and conjugates are listed in Table 2. The results show that there is a 28-fold increase in antiproliferative activity for the conjugate of Formula I compared to that of IFN.

Table 2

<td colspan="4">In Vitro antiproliferative activity in human Daudi (Burkitt's lymphoma) cell lines</td>

<td></td><td>Antiprol and ferrata vni (ng / ml)</td><td>IC50</td><td>Increased activity</td>

<td>! FNa2a</td><td> 0,56</td><td></td><td>lx</td>

<td>Conjugate of Formula I</td><td> 0,02</td><td></td><td>28x</td>

49533 Β

Example 4

I'armacokinetics

Female Sprague-Dawley rats, with surgically inserted jugular cannula. with an average weight of 240-260 g. with separate residence. they had free access to lyre and \ odi and lived in a 12-hour cycle of light and darkness. 12 about 4-6 hours from arrival. jugular cannulas were flushed with PBS. The next day. after washing with 0.1 to 50.2 ml of PBS. 2x10 ”units of IFNa were injected into 0.2-0.4 ml of PBS. which is injected with 0.15-0.2 ml of PBS to ensure that a complete amount of the drug is introduced into the animal. Thus, each animal received a dose of 8x10 ”IF \ a icđmica / kg current weight.

Ij / blood samples were taken after 5. 15 and 30 minuia, as well as i. 3. 5. 12 and 24 halls after the injection of II'Na and conjugala l'orinule 1. At all time lačkania. After the first 0.15 - 0.2 ml of blood has been removed. an aliquot of 0.5 ml of blood was taken using a new needle through. jugular kaniiu. Samples were transferred to scium separation tubes at room temperature. When all time point samples were collected, the tubes were centrifuged on a 14,000 h chilled Eppendorf 10 niinuia centrifuge. The separated scrum was transferred to 1.5 [mu] l plastic microfuge tubes and frozen at 8 [FC. Until use in the bioassay test. The serum samples were diluted appropriately. The terminal half-lives of the conjugates l'ormule 1 and IFNα, which are listed in Table 3 and which also include plasma residence times, were determined from the time versus activity graph.

Table 3

<td colspan="3">Terminal half-life (ίι /₂&gt; and mean plasma residence time</td>

<td>Sample</td><td>you /₂(time)</td><td>Plasma residence time</td>

<td>IFNa2a Conjugate of Formula I</td><td> 2,1 15,0</td><td> 1,0 20,0</td>

<td colspan="3">Terminal you /₂ estimated using log linear regression</td>

Example 5 linonogenos

Nonnain BDFs and injections (ten per group) were injected intraperitoneally once a day five times a week with various interferon preparations with 300,000 units of antiviral activity. Some mice were also injected with an aggregated form of lFNa2a. which is a greater immunogen than the monomeric form. Blood samples were taken 19 days after the last injection and serum was tested for neutralizing antibodies.

As shown in Table 4, mice injected with lF \ 'u2a developed iieuializing antibodies and this response was significantly increased in mice injected with interieron aggregates. No antibodies were detected in most of the animals injected with the conjugates of the present invention.

Table 4

<td colspan="3">Iniunogenosi</td>

<td rowspan="2">Trciman</td><td colspan="2">Aniitelo (INU / ml) *</td>

<td>Medium</td><td>Scope</td>

<td>II · N (t2a</td><td> 2 400</td><td> 217-8 533</td>

<td>Agrcgati lFNctZa</td><td> 42 667</td><td> 8 000-768 000</td>

<td>Conjugal 1'ormule i</td><td> 0</td><td> 0-1 133</td>

Rgjpit 6

Antitumor activity In ΙΊνο

The in vivo antitumor activity of the conjugates of Formula I (PEG2-IFNalpha2a) and uninodified PEG2-IFNalpha2a were evaluated by determining their ability to reduce the size of various human tumor cells subquay implanted in mice. The results are shown in Figures 1-6.

Procedure: Aumic hairless mice (Harlan) received an implant of 2 χ 10 human renal A498 cells below the left posterior loin (Figures 1 and 2), human renal zACHN cells (Figures 3 and 4), or human renal G402 cells (Figures 5 and 6). ). 3 to 6 weeks were allowed to establish the tumor, as indicated. Size criterion for

49533 Β The acceptance in the study was 0.05 to 0.5 cubic centimeters. Mice were given typical weekly doses of PEG2-1FNalpha2a or unmodified IFNalphade of 30-60 and 20 or 300 ug. In the case of PEG2-IFNalpha2a, mice were treated once a week and Monday) with 30, 60, 120 and 300 gg of PEG2-IFNalpha2a per treatment, respectively. In the case of modified FNalpha2a, mice were treated three times a week (Mondays, Wednesdays and Fridays) with 10. 20. 40 or with 100 pg lFNalfa2a per treatment. The duration of treatment was 4 to 5 weeks, depending on the aggressiveness of the tumor. Lumor volumes were measured every Monday before treatment.

Rc / .uhaii: PI: G2-1FNalfa2a showed a significant reduction in A498 tumor size and comparison with unmodified IFNalfa2a for all levels of listed weekly doses. scdnii'g. of the fourteenth. twenty-first and twenty-eighth days after the onset of irciman (Fig. 112). Ict ictnian sc na.stavvao four weeks. Scdam days after discontinuation of treatment per ui mouse \ / s \ ake groups were 'oila lived. In iri mice treated with Pl.i (i211-Nal fa2a, no tumor residues were present. In mice treated with unmodified IIalia2a, the weight of A498 tumors was 1.28 g, 0.62 g, and 1.60 g, respectively, in each of the three mice. The L498 tumor weights in the three conirole mice were 2.32 g. 2.37 g. ι 1.04 g. After 80 days from the end of the four-week treatment, the existence of the tumor was determined by palpation in seven mice. In all seven mice, palpation revealed the absence of tumor tissue.

PEG2-1FNalpha2a showed a significant reduction in the size of the AC'HN tumor compared to unmodified IFNalpha2a for weekly doses of 60, 120, and 300 Lig, at 14 days. 21 days. 28 days and .35 days (Figures 3 and 4).

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