'9 \

wonua INTELLECTUAL PROPERTY ORGANIZATION

INTERNATIONAL APPLICATION PUBLISHED UNDER TI-IE PATENT COOPERATION TREATY (PCT) g

Intematlonal Bureau P“?

(51) In¢er1mfi0n=lPa*¢n¢ Classifiwfion 5 = (11) International Publication Number: W0 94/15625A1

l A61K 35/14’ C08G 71/00’ C07K 3/06 (43) International Publication Date: 21 July 1994 (2l_.07.94)

1 (30) Priority Data:

‘ Piscataway, NJ 08854 (US).

(21) International Application Number: PCT/US94/00552 i (81) Designated States: AU, BG, B5 3 1 N

; (22) International Filing Date: 13 January 1994 (13.01.94) SK, UA, European patent (A

(71) Applicant: ENZON, INC. [US/US]; 40 Kingsbridge Road,

(12) Inventors: HAILAHAN, Terrence, w.; s2 Hazelwood Av I1 enue, Metuchen, NJ 08840 (US). GILBERT, Carl, W.; 26

Hampton Court, Basking Ridge, NJ 07920 (US). .

‘ (74) Agents: MERCANTI, Michael, N. et a1.; Enzon, Inc., 40 .; Kingsbridge Road, Piscataway, NJ 08854 (US).

.0?“ .5?Pa .321BEEa asKR,LK,MG MN MW

-.:-155$8 U:11 Urs 5"»? .51 GB, GR, IE, IT, LU, MC, NL

I 08/003,985 15 January 1993 (15.01.93) US i PublishedI With international search report.

I (57) Abstract

Conjugates containing a substance withcoagulant activity, such as recombinant fac-

‘ tor VIII, and non-antigenic polymers, such as3 poIy(ethylene glycol), are disclosed (as shown

in the figure). Also disclosed are methods of3 forming the novel conjugates of this invention.

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C(54) Title: FACTOR vm - POLYMERIC c CONJUGATES K C I I 1 P O P

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FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCI' on the front pages of pamphlets publishing internationalapplications under the PCT.

AT Austria GBAU Australia GEBB Barbados GNBE Belgium GR GreeceBF Burkina Faso BU Hungary NOBG Bulgaria IE Ireland NZBJ Benin Italy PLBR Brazil Japan PTBY Belarus Kenya R0

Canada KG Kyrgystan RUCentral African Republic KP Democratic People's Rqaublic SDCongo of Korea SESwitzerland Republic of Korea SICote d‘Ivoire Kazakhstan SK

CM Carnemon Liechtenstein SNCN China Sri Lanka TDCS Czechoslovakia Luxembourg TGCZ Czech Rqiublic LV Latvia TJDE Germany MC Monaco ‘ITDK Denmark MD Republic of Moldova UAES Spain MG Madagascar USFI finland ML Mali UZFR France MN Mongolia VNGA Gabon

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umwa KingdomGwrsiaGuinea

98809

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MauritaniaMalawiNigerNetherlandsNorwayNew ZcalandPolandPortugalRomaniaRussian FederationSudanSwedenSloveniaSlovakiaSenegalChadTogoTajikistanTrinidad and TobagoUkraineUnited States of AmericaUzbekistanViet Nam

W0 94/15625 PCT/US94/00552

l

FACTOR VIII - POLYMERIC CONJUGATE

The present invention is directed to methods ofmodifying substances having Factor VIII activity, such asthe pro-coagulant glycoprotein Factor VIII and, moreparticularly, to methods for the modification of suchsubstances with non-antigenic polymers, such aspolyethylene glycol, and the resultant conjugates.

Background of the Invention

The process of blood clotting typically beginswith trauma to a blood vessel. The damaged vessel wallinitiates hemostasis by causing adherence and accumulationof platelets at the injury site and by activating plasmaproteins which initiate the coagulation process. A seriesof proteins, including Factor VIII, are activatedsequentially by specific _proteolytic cleavages andconformational changes leading to the deposition ofinsoluble fibrin which curtails the blood flow.

In addition to forming a platelet plug, theplatelets release vasoactive amines which cause arteriolarvasoconstriction causing locally reduced blood pressurethat lasts for hours. This, in combination with theplatelet plug, effectively controls bleeding.

Hemophilia is a bleeding disorder caused by thelack of an essential blood factor. Hemophilia A (classichemophilia) is the most common and is caused by a geneticdeficiency or abnormality of Factor VIII. Hemophilia B

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(Christmas disease) is caused by a deficiency orabnormality of Factor IX. Hemophilia is an X-linkedgenetic abnormality and therefore primarily affects males.Approximately 10-20/100,000 males have hemophilia A and1/100,000 have hemophilia B. L

Hemophilia is a heterogeneous disorder whoseseverity of bleeding tendency is determined by the specificpoint mutations. Mutant Factor VIII proteins can haveclotting activities that vary from inear normal to severelydeficient. The clinical features of hemophilia A and B areidentical. Less ‘than 1% clotting factor activity isdefined as severe hemophilia, and is accompanied byspontaneous bleeding in the muscles and larger joints.Repeated bleeding in joints causes arthropathy which is amajor chronic complication. Hemarthroses are worsened bymuscle atrophy due to muscle bleedings. This affects bothposture and gait of the older patient. In about one-halfof all hemophiliacs, repeated hemarthroses result ineventual deformity and crippling. These patients usuallyhave Factor ‘VIII levels of <5% of normal. In. mildhemophilia (5-40% FVIII or FIX), bleeding does not occurexcept after trauma. Moderately severe hemophilia (1-5%FVIII or FIX) has clinical features between the severe andmild hemophilia.

Conventional treatment of hemophilia consists ofreplacement of the Factor VIII from pooled donor plasmincluding fresh, frozen plasma or Factor VIII concentratesand, more recently, recombinant Factor VIII products. ‘Hometherapy, surgery and prolonged treatment of hemophiliacshave been eased by the availability of Factor VIII

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preparations. With the availability of replacement bloodfactors, the life expectancy for hemophiliacs is almostnormal. However with the increased use of pooled donorpreparations, there has been a higher transmission of viraldiseases such as hepatitis (eg. B, delta, non A non B) andHIV. It has been estimated that 50% of hemophilia patientsare either hepatitis positive or HIV positive. These viralinfections are now the major cause of morbidity andmortality in patients with hemophilia.

Ttadecrease the possibility of transmitting viralinfections through blood factor preparations, firms haveincreased purification techniques to reduce the virus load.These measures can include pasteurization,detergent/solvent disruption of viral membranes, andmonoclonal antibody purification. Blood banks nowroutinely screen all donor blood for AIDS virus andhepatitis. Yet none of these measures can guaranteefreedom from viral contamination.

Further disadvantages of jpooled. donor' plasmainclude the cost and availability of the purified bloodfactors. With increased purification steps, the cost ofblood factor therapy has increased. Availability of theblood factors is also a concern. Theoretically, thesefactors should be administered prophylactically in manycases to avoid the sequelae of uncontrolled bleeding (eg.development of joint disorders). However, cost,availability and the pharmacokinetics make an effectiveprophylactic therapy unfeasible.

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With the advent of recombinant DNA technology,researchers have now cloned and are testing a number ofblood factors, including rh-factor VIII:c in patients withhemophilia. While recombinant technology may overcome theproblems of viral contamination and availability, it doesnot affect the pharmacokinetics of the factors nor theformation of inhibitors (antibodies) in patients. It isestimated that 10-15% of all patients with hemophilia Awill develop IgG antibodies that will nullify the value ofreplacement therapy. Inhibitor development occursprimarily in jpatients with severe hemophilia althoughantibodies to Factor VIII in mild hemophilia have beenreported. Approximately 5-15% of patients with severehemophilia have antibodies to Factor VIII or IX. It hasbeen estimated that the actual risk of developingneutralizing antibodies by age 20 is as high as 15-24%.Joint bleedings often cannot be controlled and adequatelytreated and many of these patients are severelyhandicapped. ~

To overcome the neutralizing effect of theantibodies, physicians can be forced to increase the dosageto the factor. However, there is often a decreasedresponse to the replacement therapy despite increases indosage. Care in administering the factors as well as theadministration. of steroids and other" immunosuppressiveagents such as azathioprine, cyclophosphamide and high-dosei.v. gammaglobulin G is often required to prevent or limitthe development of antibodies and hypersensitivityreactions. Antibody depletion through plasmapheresis hasbeen used to decrease the inhibitor titers in circulation.Interferon a2a has been also used successfully to treat

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cases of postpartum acquired inhibition to Factor VIII:c.However, such immunosuppressive techniques have been onlypartially successful and raise the risk that the patientwill be more vulnerable to opportunistic infections, eg.HIV or hepatitis.

In light of the complications and risks inherentin the conventional treatment of classic hemophilia, it isdesirable to provide a modified Factor VIII which is lesslikely to cause the formation of inhibitor antibodies. Inlight of the high costs of recombinant Factor VIII, it isalso highly desirable to increase the clearance time forFactor VIII activity. The terms "disappearance time" and"clearance time" are used herein to denote the time takenfor Factor VIII activity to decrease to 50% of its maximumlevel.

Summary of the Invention

The various embodiments of the present inventionprovide Factor VIII conjugates having prolonged circulatinglife and activity in mammals. In addition, there are alsoprovided methods for the modification of Factor VIII withnon-antigenic polymeric materials such as, polyethyleneglycol (PEG). The Factor VIII fraction included in theconjugates preferably comprises a protein having FactorVIII activity which has been formed using recombinanttechnology. The Factor VIII fraction may also be derivedfrom human or animal plasma sources, such as bovine orporcine plasma. Transgenic sources are also contemplated.As used herein, the term "Factor VIII fraction" means anysubstance which demonstrates the ability in vivo to

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function as mammalian Factor VIII, i.e. activate Factor Xand continue the intrinsic clotting cascade. The FactorVIII fraction can also comprise other proteins and reagentssuch as Von Willebrands' factor, as well as other serumproteins including albumin, fibrin, fibrinogen, etc.

According to one embodiment of _the presentinvention, Factor VIII preparations are reacted with molarexcesses of a suitable activated polyalkalene oxide such asmethoxypo1y(ethylene glycol)-N-succinimidyl carbonate (SC-PEG) under conditions sufficient to effect conjugationwhile maintaining at least a portion of the Factor VIIIactivity. Such conditions include reacting thesubstituents at temperatures of up to about 27°C and inpharmaceutically acceptable buffer systems. As usedherein, the term "molar excess" is meant to indicate theratio of the number of moles of polymeric substance to thenumber of moles of Factor VIII. The reaction is thenterminated by adding a molar excess of a compound thatreacts very quickly with any free SC-PEG, such as glycine.The resulting" ‘modified Factor’ ‘VIII may' then beadvantageously stabilized with human serum albumin (HSA)and sterile filtered. This method has been found toprovide a long acting modified Factor VIII conjugate whichis less susceptible to antibody inhibitors and retains alarge percentage of the protein's original activity. Theconjugates are substantially resistant to in vivohydrolysis and thus provide prolonged -activity afteradministration.

These and other embodiments of the presentinvention are described in detail below.

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Brief Description of the Drawings

Figure 1 is a graph indicating the activity of aPEG-modified Factor VIII, prepared according to oneembodiment of the present invention, as a function of totalprotein concentration.

Detailed Description

The various embodiments of the present inventionadvantageously provide methods for modifying substanceshaving Factor VIII activity with substantially non-antigenic polymeric substances under relatively mildconditions, for example- at a pH of about 6.5 - 8,preferably of about 6.5 - 7.5, and at temperatures which donot exceed 27°C, and are preferably in the range of from 2- 10°C. Those skilled in the art will appreciate thatFactor VIII is a relatively large and sensitiveglycoprotein. The present invention provides methods formodifying Factor VIII without subjecting the protein toharsh conditions which could eliminate its activity.

The substantially non-antigenic polymersubstances included in the conjugates are preferablypoly(alkylene oxides). ‘Within.this group of substances arealpha-substituted polyalkylene oxide derivatives such asmethoxypolyethylene glycols or other suitable alkyl-substituted derivatives such as Cf%§ alkyl groups. It ispreferred, however, that the non-antigenic material be amonomethyl-substituted PEG homopolymer. Alternativepolymers such as other polyethylene glycol homopolymers,

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polypropylene glycol homopolymers, other alkyl-polyethyleneoxides, bis-polyethylene oxides and co-polymers or blockco-polymers of poly(alkylene oxides) are also useful. Inthose aspects of the invention where PEG-based polymers areused, it is preferred that they have molecular weights offrom about 1,000 to about 10,000. Molecular weights ofabout 2,000 to 7,000 are preferred and.molecular weights ofabout 5,000 are particularly preferred.

As stated above, covalent modification of theprotein material is preferred to provide a hydrolysis-resistant conjugate. The covalent modification reactionincludes reacting a substance having the desired FactorVIII activity with a substantially non-antigenic polymericsubstance under conditions sufficient to effectconjugations while maintaining at least a portion of theFactor VIII activity. W

The polymers may be activated in order to effectthe desired linkage with the protein substance. . Byactivation, it is understood by those of ordinary skill inthe art that the polymer is functionalized to include adesired reactive group. Examples of such activation aredisclosed in U.S. patents 4,179,337 and 5,122,614, whichare hereby incorporated by reference. In the disclosuresof these patents, the hydroxyl end groups of polyalkyleneglycols are converted into reactive functional groups andthus activated.

According to one preferred embodiment, a FactorVIII fraction is modified with SC-PEG such as disclosed inthe '614 patent, supra. This particularly preferred

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activated form of PEG for use in the present invention ispoly(ethylene glycol)-N-succinimide carbonate. Thisactivated polymer forms stable, hydrolysis-resistantcarbamate (urethane) linkages with amino groups of theprotein. Isocyanate-activated PEG's are also of use.While the references incorporated herein describe epsilonamino group modifications of lysine, other conjugationmethods are also contemplated. Carbohydrate and/or acidgroup or other amino acid.modifications are also within thescope of the present invention. Covalent linkage by anyatom between the protein and polymer is possible.Moreover, non-covalent conjugation such as lipophilic orhydrophilic interactions are also contemplated.

In order to prepare the Factor VIII fraction forthe polymeric modification, the pH of the Factor VIIIfraction is preferably adjusted to about 6.5 - 8, mostpreferably to about 6.5 - 7.5. The Factor VIII may beadversely affected by a pH» above about 8 so that rangeshould be avoided. The pH of the Factor VIII fraction ispreferably modified through a vigorous buffer exchange bydialyzing the Factor VIII against an appropriate saltbuffer system. For example, the buffer exchange can beconducted by placing the Factor VIII fraction.in a dialysisbag suspended in a salt buffer and changing the buffer withfresh solution several times. The salt buffer may, forexample, comprise 50 mM sodium phosphate and 100 mM sodiumchloride per liter at a pH of 7. Such a buffer exchange isalso useful in removing undesirable low molecular weightcomponents which may be present in commercially availableFactor VIII fractions.

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The process of the present invention includespreparing or providing the activated polymer and,thereafter, reacting it with the Factor VIII substance. Areaction mixture is prepared by adding a solutioncontaining the activated.polymer, preferably SC-PEG, to theFactor VIII fraction. The SC-PEG solution is preferablyprepared with the same buffer and pH, i.e. 6.5 - 8, as thesalt buffer utilized in the buffer exchange. In thereaction mixture, the protein is reacted with anappropriate amount of the activated polymer, which istypically present in a several-fold molar excess over theenzymatic-like substance. The polymeric excess will rangefrom about 5 to about 125 fold molar excess and preferablyfrom about 15 to about 50 fold molar excess of the polymerto the Factor VIII protein. The reaction is carried out attemperatures of from about 2 to 27°C, and preferably attemperatures of from 2 - 10°C over time periods rangingfrom a few minutes to as long as 12 hours. Depending uponthe reaction conditions, the artisan can tailor the profileof the resultant conjugate. For example, large molarexcesses of polymer reacted with the protein result inconjugates having relatively long circulating times in vivobut somewhat less activity than unmodified or slightlymodified proteins. The inverse of the foregoing is alsotrue. Smaller molar excesses reacted with the proteinprovide conjugates with higher activity and somewhatshorter circulating life. In all instances, the conjugatehas significantly prolonged circulating life in vivo overthe unmodified protein.

One of the advantages of the present modificationprocess is that it can be carried out at relatively mild

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reaction conditions which will not adversely effect theprotein. The reaction is then stopped by adding a molarexcess of a compound which reacts quickly with the SC-PEG.For example, a 250-fold molar excess of glycine issufficient to terminate the reaction between.the SC-PEG andthe Factor VIII.

Following the conjugation reaction, the desiredproduct is recovered using known techniques and purifiedusing column chromatography or similar apparatus ifnecessary. For example, excess reagents can then beremoved from the reaction mixture by the same dialysisprocedure described above. The modified-Factor VIIIfraction is then preferably formulated with a stabilizer.For example, human serum albumin (HSA), which acts as acarrier protein and protects the modified Factor VIII fromproteolytic cleavages. A final concentration of about 0.5mg/ml to about 5 mg/ml HSA, e.g. about 1 mg/ml HSA, andabout 0.5 mg/ml to about 5 mg/ml, e.g. about 1 mg/ml, ofmodified Factor VIII is suitable. The stabilized modified-Factor VIII is then preferably gravity filtered through asterile filter, e.g. a 0.45 micron filter. Gravityfiltration is preferred to other forms of filtration, suchas syringe filtration, since Factor VIII is a shearsensitive molecule whose activity can be adversely affectedby the shear forces encountered during a more rigorousfiltration.

After stabilization and filtration, the modified-Factor VIII may also be lyophilized. Tests have shown thatFactor VIII modified by the above process have retainedlarge percentages of their activity even after ;being

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lyophilized and reconstituted.

Another aspect of the present invention providesmethods of treatment for hemophilia. The method includesadministering, in a pharmaceutically acceptable vehicle,such as a parenteral solution, an effective amount of thecompositions described herein to alleviate clottingdeficiencies. Those of ordinary skill in the art willrealize that the amount of the conjugate used in the methodof the present invention will vary somewhat from patient topatient, however, conjugates capable of delivering fromabout 15 IU/kg to about 100 IU/kg per administration or anamount sufficient to nmintain a level greater than 0.01IU/ml blood are preferred. The optimal dosing of theconjugate can be determined from clinical experience.

Example 1

Modification of Factor VIII with Polyethylene Glycol(PEG) Purified Factor VIII (5 mg), obtained from AlphaTherapeutics, Los Angeles, CA, was dialyzed against 50 mMsodium phosphate pH 7.0, 100 mM NaCl (4 X 1L) overnight.To four 1 mg aliquots was added a 0, 25, 50 and 100-foldmolar excesses of methoxypoly(ethylene glycol)-N-succinimidyl carbonate (SC-PEG), respectively. The SC-PEGwas added as a 10 mg/ml solution in the above buffer andthe reaction mixtures were allowed to set on ice for 2hours with occasional stirring, at which time the reactionswere stopped by the addition of a 250-fold molar excess ofglycine. Excess reagents were then removed by dialysis asdescribed above. -

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Factor ‘VIII activity ‘was determined. using achromogenic assay. Even after treatment with up to 100-fold excess SC-PEG, 45% of the original activity remainedas illustrated in Figure 1. Figure 1 is a graph of theactivities, measured by a DADE Factor VIII chromogenicassay kit LAMQ obtained from Baxter Healthcare, Deerfield,Illinois, of the modified Factor VIII proteins formed inExample 1. .As indicated in the legend, the control "NativeFactor VIII" which underwent all reaction steps without anySC-PEG had the highest activity. This graph indicates thathigh activities are retained by the Factor VIII even afterextensive modification with SC-PEG.

Example 2

Reaction mixtures containing 0, 25, and 50-foldmolar excesses of SC-PEG, respectively, with Factor VIIIfractions were prepared as above but were then formulatedwith 1 mg/ml HSA prior to gravity filtration through a 0.45micron sterile filter. Chromogenic assays of filteredsamples gave the following results:

SPECIFIC ACTIVITYSample Ugmg F.VIII , 5

CONTROLACTIVITY

O-PEG 44.9 100%25-PEG 40.7 91%50-PEG 43.7 97%

Aliquots (1 ml) of the samples were thenlyophilized and vacuum sealed in serum vials using an FTSshelf lyophilizer. The samples were then reconstitutedwith. 1 ml water and compared ‘with the unlyophilized

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controls in the chromogenic assay. Activity before _andafter lyophilization were as follows:

SPECIFIC ACTIVITY

Sample Ulmg F.VIII % GJHFOLBefore After ACTIVITY

0-PEG 44.9 23.4 52%25-PEG 40.7 16.4 36%50-PEG 43.7 35.5 79%

Example 3

Factor VIII was modified in this example withpoly(ethylene glycol) succinoyl-N-hydroxysuccinimide ester(SS-PEG). The Factor VIII was dialyzed against so mMsodium phosphate pH 7.0, 100 mM NaCl overnight. Three, 500mg reactions were set up with 25, 50 and 100 fold excesses,respectively, of SS-PEG. The SS-PEG was added as a 10mg/ml solution in the above buffer. The reactionsproceeded on ice for 2 hours with occasional stirring andwere then quenched by the addition of a 100 fold excess ofglycine. HSA was added to 5% (w/w) and all samples weredialyzed overnight in 59 mM sodium phosphate pH 7.0, 100 mMNaCl. The results of the chromogenic assay for Factor VIIIactivity were as follows.

SAMPLE % CONTROL ACTIVITY

UNMODIFIED F. VIII 10025-PEG F. VIII 6850-PEG F. VIII 52100-PEG F. VIII 35

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Example 4The circulating half-life of PEG-modified

Factor VIII of the present invention was determined.Three mice were injected.in ‘the tail vein.with 100 Unitsof Factor VIII obtained from.Alpha-Therapeutics and setsof three mice each were similarly injected with PEG-Factor VIII (SC-PEG) prepared in the manner describedabove. The mouse plasma was collected after 4 hours, 8hours, 24 hours, 48 hours, 72 hours and 7 days viaorbital eye bleed. At the indicated times, plasma wasobtained from the mice and stored at 4°C until assay.Pooled plasma samples from each time point were assayedfor clotting activity using the Factor VIII chromogenicassayu The data was graphed and the T%'s"were calculatedfrom the slopes.

The average T% for the samples is given in thefollowing table. Each of the PEG-modified Factor VIIIsamples had a significantly longer T.,2 than did theunmodified Factor VIII control. Additionally, the PEG-Factor VIII preparations were absorbed much more rapidlyinto the blood stream than the unmodified Factor VIII.

HALF-LIVES OF FACTOR-VIII IN MICE

COMPOUND ACTIVITY _T%

Unmodified Factor VIII 100% Iihs.PEG-Factor VIIIfi 81% ' $.hs.PEG-Factor VIIIw 88% Eihs.PEG-Factor VIIINO 79% 55 hcs.

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The various embodiments of the presentinvention, therefore, provide conjugates which retainsignificant levels of Factor VIII activity whileincreasing circulating half-life and having less of atendency to cause the formation of inhibitor antibodies.

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WE CLAIM : 4

1. A biologically active conjugate comprising afirst substance having Factor VIII activity bound to asubstantially non-antigenic polymeric substance with acarbamate (urethane) linkage.

2. The conjugate of claim 1 wherein said firstsubstance comprises factor VIII.

3. The conjugate of claim 2 wherein said factorVIII is of recombinant origin.

4. The conjugate of claim 2 wherein said factorVIII is of mammalian origin.

5. The conjugate of claim 2 wherein said factorVIII is of transgenic origin.

6. The conjugate of claim 1 wherein said firstsubstance comprises fractions of the protein Factor VIII.

7. The conjugate of claim 1 wherein said polymericsubstance comprises a poly(alkylene oxide).

8. The conjugate of claim 7 wherein said polymericsubstance comprises an alpha-substituted polyalkyleneoxide derivative.

9. The conjugate of claim 7 wherein said polymericsubstance is selected from "the. group <consisting ofpolyethylene glycol homopolymers, polypropylene glycol

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homopolymers, alkyl-capped. polyethylene oxides, bis-polyethylene oxides and copolymers or block copolymers ofpoly(alkylene oxides).

10. The conjugate of claim 7 wherein said polymericsubstance comprises poly(ethylene glycol).

11. The conjugate of claim 7 wherein said polymericsubstance has a molecular weight of about 1,000 to about10,000.

12. The conjugate of claim 11 wherein saidpolymeric substance has a molecular weight of about 2,000to about 7,500. .

13. The conjugate of claim 11 wherein saidpolymeric substance has a molecular weight of M about5,000.

14. A method of preparing a conjugate having -FactorVIII activity‘ comprising' reacting‘ a zfirst substancehaving Factor VIII activity with a substantially non-antigenic polymeric substance under conditions sufficientto effect conjugation of said first substance and saidpolymeric substance with a carbamate (urethane) linkagewhile maintaining at least a portion of the activity ofthe first substance.

15. The method of claim 14 wherein said polymer isa poly(alkylene oxide).

16. The ‘method of claim 14 ‘wherein said

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polyalkylene oxide is an alpha-substituted polyalkyleneoxide derivative.

17. The ‘method. of claim. 16 ‘wherein saidpoly(alkylene oxide) is a polyethylene glycol.

18. The method of Claim 14 wherein said reactingstep comprises providing a molar excess of saidsubstantially non-antigenicjpolymeric substance relativeto said first substance.

19. The method of Claim 18 wherein said reactingstep comprises providing about a 15 - 50 fold molarexcess of said substantially non-antigenic polymericsubstance relative to said first substance. '

20. _ The method of Claim 14 wherein said reactingstep is conducted at temperatures of up to about 27°C.

21. The method of Claim 20 wherein said reactingstep is conducted at temperatures of about 2 - 10°C.

22. The method of Claim 14 wherein said firstsubstance comprises Factor VIII.

23. A method of treating hemophilia comprisingadministering a therapeutically effective amount of theconjugate of claim 7.

24. A method of treating hemophilia comprisingadministering a therapeutically effective amount of theconjugate of claim 1.

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INTERNATIONAL SEARCH REPORT International lPP1i°l5°fl‘N°

A. CLASSIFICATION OF SUBJECT MATTERIPC(5) :A61K 35l14;C08G 71IM;C07K 3/06US CL :530!383,402,406;514I12,21

According to International Patent Classification (IPC) or to both national elalaificafion and IPC

FIELDS SEARCHED

PCTlUS94l00552

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Minimum documentation aeuebed (classification system bllowed by elaaaification symbols)

U.S. . $30I3I3,402,406;514I12,21

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Blectronicdatabaaeoonauledduringtlreinternalionalleareb(nameofdalabaaeand,wherepn.eticable,aarebter|nsuaed)CAS online, medline, aps, biosis,embase,dementsearch terma:Factor Vlll, coniugateamrethane, carbamatemolyethyleneglycol, polyalkylene oxide.

C DOCUMENTS CONSIDERED T0 IE RELEVANT

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US,A, 4,970,300 (Fulton et al.l 13 November 1990, col. 11, 1-18, 20-24

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W0,A, 92116555 (Enzon, Inc.) 01 October 1992, pages 8, 1-2411, 13, lines 33-38, claims 1-2, 5, 7, 10-16, 24, page 1,lines 15-17.

US, A, 5,122,614 (Zalipsky) 16 June 1992, col. 1, 2 and 3, 1-24FIGS. 1-3.

US, A, 5,219,564 (Zalipsky) 15 June 1993,whole document. 1-24

US, A, 5,234,903 _(Nho et al.l 10 August 1993, whole 1-24document.

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Date of the actual complexion of the nnternauonal search Date of mailing of the intemational aeareh report

04 March 1994 2 1 ‘N and mailin add f the ISAIUS Authorized ffieer'#......-» ..=....."='.:.°-.......... 0 w,,,,, .,.'°"""" c SAYALAWailing!-on. D.C. 202.31 '

Facsimile No. NOT APPLICABLE Tel neNo. 70 308-0196Form PCT/ISAIZIO (second aheet)(.Iuly 1992)-a

INTERNATIONAL SEARCH REPORT International application NoPCT!US94I(IJ552

C(Cont1nuat|on) DOCUMENTS CONSIDERED TO BE RELEVANT

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Acla Medica et Biologica, vol. 36, number 1, issued 1988, 1-24Sakuragawa et 21., "Studies on the Stability of Factor.V1IImodified by polyethylene glycol‘ , pages 1-5 . V

Form PCTIISA/210 (continuation of second |heet)(July 1992):