o
`
`ilmted States Patent [191
`Davis et al.
`_
`a
`
`[11]
`[45]
`
`.
`
`4,179,337
`_ Dec. 18, 1979
`
`[54] NON-IMMUNOGENIC POLYPEP'I'IDES
`[76] Inventors: Frank F. Davis, 19 Farmingdale Rd.,
`East Brunswick, NJ. 08816;
`Theodorus Van Es, 313 Overbrook
`Rd,, Piscataway, NJ, 08854;
`Nicholas C_ palczuk’ 45 w_ Franklin
`
`st" Bound Bwok’ NJ‘ 08805
`
`.
`[21] Appl' No" 819331
`[22] Filed:
`Jul. 28, 1977
`
`1
`[63
`
`Related US. Application Data
`Continuation-impart of Ser. No. 596,931, Jul. 17, 1975,
`abandoned, which is a continuation-in-part of Ser. No.
`381,191, JUL 20’ 1973, abandonm
`,
`[51] Int. Cl.2 ....................... .. C07G 7/00; C07G 7/02;
`A61K 37/26; A61K 37/48
`[52] US. Cl. ........................... .. 435/181; 260/ 112.5 R;
`260/112.7; 424/78; 424/94; 424/177; 424/ 178;
`-
`'
`435/180
`[58] Field of Search ................. .. 195/63, 68, DIG. ll;
`424/94, 177, 178, 78; 260/ll2.5, 112 R, 6, 8,
`
`112.7 _
`
`[56]
`
`References Cited
`U'S' PATENT DOCUMENTS
`9/1971
`Ziffer et a1. .......................... .. 195/63
`3,607,653
`3,619,371 1l/1971 Crook et a1. .......... ..
`.. 195/63
`3,639,213
`2/1972 Ginger et a1. ..... ..
`195/63
`3,645,852
`2/1972 Axon et a1. ....... ..
`.. 195/68
`3,788,948
`1/ 1974 Kagedal et a1. .
`..... .. 195/68
`3,959,080
`5/1976 Orth et a]. .............. .. 195/68 x
`4,002,531
`1/1977 Royer ....... ..
`195/DIG. ll
`4,055,635 10/1977 Green et a1. ............... .. 195/DIG. 11
`Primary Examiner—David M. Naff
`Attorney, Agent, or Firm-Omri M. Behr
`[57]
`ABSTRACI.
`.
`.
`.
`Polypeptides such as enzymes and msuhn are coupled. to
`polyethylene glycol or polypropropylene glycol havmg
`a molecular weight of 500 to 20,000 daltons to provide
`a physiologically active non-immunogenic water solu
`ble polypeptide composition. The polyethylene glycol
`or polypropylene glycol protect the polypeptide from
`loss of activity and the composition can be injected into
`the mammalian circulatory system with substantially no
`immunogenic response.
`
`27 Claims, N0 Drawings -
`
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`Page 1 of 12
`
`

`
`1
`
`NON-IMMUNOGENIC POLYPEPTIDES
`
`RELATED APPLICATIONS
`This application is a continuation-in-part of our co
`pending applicationfser. No. 596,931 ?led July 17;v
`1975, which, ‘in turn, is’a continuation-in-part of our
`then co-pending' application, Ser. No. 381, 19-1,“ ?lediJu‘ly'
`20, 1973, both now‘ abandoned.
`I‘
`‘
`‘
`‘
`'
`
`'
`
`BACKGROUND OF THE INVENTION
`‘1. Field’ of the Invention
`Non-im'munogenic polypeptides.
`' 2'. Description of the Prior Art
`The use of polypeptides in circulatory systems for the
`purpose of engendering a particular physiological re
`sponse is well known in the medicinal arts. Among the
`best known polypeptides utilized for>this purpose is
`insulin which is used in the treatment of diabetes. An
`other group of polypeptides to which great'therapeutic
`potential has been attributed are enzymes of the various
`classes. The principal factor which has severely limited
`the use in therapeutics of polypeptides in particular,
`enzymes, has been the fact that most of these com
`pounds elicit an immunogenic response in the circula
`tory system. This response being the production of
`antibodies to the polypeptides by the circulatory system
`into which they‘ are injected. This effect has one or both
`of two secondary consequences. The ?rst being the
`destruction of polypeptides by the antibodies so called
`forth, or, the second more seriously, the appearance of
`an allergic response.
`‘
`The destruction of the polypeptide by the antibodies
`is believed to’be responsible for the rather low residence
`time of insulin in the human circulatory system, hence,
`persons af?icted with diabetes are forced to inject them
`selves fairly frequently with fresh doses of insulin. In
`the case of enzymes not only is there a problem of de
`struction of the polypeptide and the subsequent nega
`tion of its physiological activity but also the most unde
`sired elicitation of an allergic reaction.
`If it were found possible to so modify polypeptides
`that their desired physiological activity was maintained
`totally or at least in substantial proportion and at the
`same time ‘no immunogenic response was generated
`within the circulatory system, then it would be possible
`to utilize thesemost valuable compounds in the mam
`malian circulatory system without the aforementioned
`__disadvanta'ges and in the far ‘smaller amounts than has
`heretofore been’ possible. "‘
`The ‘problems set ‘farm hereinabove are well‘recog
`'ni’zed‘and various approaches have been taken in at
`tempts to solve them. The attachment of enzymes to
`insoluble supports has been the subject of a great deal of
`work. Reviewsdealing'with this subject will be found
`55
`in Silman and‘Katchalski, Ann. Rev. Biochem., 35, 873
`(1966), and Goldstein, Fermentation Advances, Aca
`demic Press, New York (1969) page 391. This approach
`however while‘ of academic interest does not provide
`injectable long-life polypeptides. Another approach
`which has been taken to‘ provide polypeptides of length
`ened‘ in vivov life has been the micro incapsulation of
`‘ enzymes which‘ha‘s'been‘ discussed in numerous articles
`vby Chang and co-workers, namely, Science, '146, 524
`v (1964); Trans. Am.‘So'c.',=l2’,'l3 (1966); Nature, 218, 243
`(1968); Can. J. "PhysiolQPharmacoL, 47, 1043 (1969);
`Canad. ‘J. Physiol. PharmacoL, 45,‘ 705 (1967). A further
`approach lay‘ in the heat “stabilization of enzymesby
`
`5
`
`10
`
`4,179,337
`2
`attaching carboxy methylcellulose to an enzyme such as
`Trypsin (Mitz‘and Summaria, Nature, 189, 576 (1961)
`and the'atta'chment of proteases to hydrophilic carriers
`(Brummer, et al, Eur. J. Biochem., 25, 129 (1972). These
`approaches however do not provide the polypeptides in
`a soluble forrnf-which is the most desirable for injection
`and dosage‘control of injectable materials. A further
`approach. has been the attachment of synthetic poly
`mers'to polypeptidal proteins. A review of this work
`will~be found in.Sela, “Advances in Immunology”, 5,
`30, (1966) Academic Press, New York. In this work, it
`has been shown that while homopolymers of amino
`acids are nearly all non-immunogenic, when these poly
`mers are attached to immunogenic proteins the immu
`nogenic activity is not masked and antibodies are pro
`duced in test circulatory systems. For example, while
`polyglycine itself is non-immunogenic, when attached
`to a protein that conjugated protein becomes a hapten.
`Similarly while dextran itself is slightly immunogenic
`when coupled to insulin the insulin-dextran coupled
`material is believed to become substantially immuno
`genic.
`‘
`
`SUMMARY OF THE INVENTION
`There are provided by this invention peptides and
`polypeptides coupled to polymers which are substan
`tially non-immunogenic and retain a substantial propor
`tion of the desired physiological activity of the base
`polypeptide.
`,
`In the process of this invention a substantially straight
`chain polymer is modi?ed, suitably at one end thereof,
`either by the alteration of the terminal group or by the
`addition thereto of a coupling group having activity vis
`a vis a polypeptide and reacting said activated polymer
`with‘the polypeptide. Thev protected polypeptides of the
`present invention are injectable in aqueous solution into
`the mammalian circulatory system or intramuscular and
`call forth substantially no‘ immunogenic response.
`DESCRIPTION ‘OF THE PREFERRED
`EMBODIMENTS
`' The polymers u‘tilized‘fo'r protection purposes in the
`procedures of the present invention possess a substan
`tially linear ethereal or carbon carbon backbone. It has
`been found that utilizing branch’chain polymers will
`give rise to substances which do generate an immuno
`genic‘response. Nevertheless a certain amount of substi
`tution in the backbone is permissible. For example, the
`backbone may be ‘substituted by alkyl groups or alkoxy
`groups provided that said alkyl or alkoxy groups con
`tainiless than 5, preferably 2 or less carbon atoms.
`Among the polymers of choice may be mentioned poly
`‘ethylene glycol, and polypropylene glycol, of these
`polyethylene glycol is particularly preferred.
`' v“his also preferred to operate in a polymer chain
`length area of "between 500 andv 20,000 daltons, about
`750 to 5,000 d‘altons being especially preferred.
`Several‘ modes‘oficouplingthe polymer with the
`polypeptide are available and will be ‘discussed in fur
`ther' ‘detail hereinbelow. It should be borne in mind
`however that the portions of any given polypeptide
`moiety which has a desirable physiological action may
`vary from peptide to peptide. Thus, in certain enzymes
`one or two amino acid residues may be principally re
`sponsible for this desirable physiological activity. In
`choosing a coupling agent to-couple a polymer to the
`polypeptide it would be desirable to utilize coupling
`
`35
`
`40
`
`45
`
`50
`
`65
`
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`Page 2 of 12
`
`

`
`A A
`CO,C
`
`’
`
`5
`
`5
`
`5
`
`is the carbon atorn of a labile COOH group. _While the
`foregoing exempli?cation shows the peptide moiety
`being coupled through an amino group, and this is be
`lieved to be the most likely coupling point, the inven
`tion is not limited thereto as other labile moieties, i.e.
`thial might'also provide'a' coupling locus.
`In the ?rst category there maybe mentioned as a
`suitable coupling groupycyanuric chloride or. ?uoride.
`In this procedure polyethylene glycol (hereinafter
`PEG) is taken up in a suitable reaction inert solvent
`such as a hydrocarbon solvent suitably anhydrous ben
`zene containing a small amount of a weak base such as
`sodium carbonate, and cyanuric chloride added thereto.
`The reaction mixture is- then quenched with water,
`insoluble material removed, followed by removal of the
`solvent, suitably under reduced pressure to yield 2
`PEG-4-hydroxy-6-chloro-1,3,5-triazine.
`The thus produced activated polymers are then re
`acted with a solution of polypeptide in a suitable buffer.
`After reaction is complete the unreacted activated poly
`mer is removed, suitably by contacting with an gel
`permeation resin= such as Sephadex 6-50 and the pro
`tected polypeptide removed and puri?ed in the usual
`manner. Since these products are to be considered as
`polypeptides'care must be taken during the puri?cation
`procedure that they not be denatured. Therefore, it is
`desirable either to permit them to remain in buffered
`aqueous solution or, if it is deemed essential, to isolate
`them in the solid state that such isolation be carried out
`by the recognized procedure such as lyophilization of
`the protected peptide.
`Thus, as the partial structure, say,
`
`4,179,337
`4
`3
`is the residual nitrogen of a labile primary amino moiety
`agents which do not have an af?nity for these particular
`on the peptide, and ‘in
`active moieties. While this is a desirable goal it is not
`always possible to comply absolutely with it. It is there
`fore necessary in individual cases to effect a compro
`mise, that is to say, sacri?ce a certain amount of activity
`maintenance for the granting of a substantial amount of
`non-immunogenicity. The ?nal results obtained will
`depend not only on the coupling agent used but also the
`proportions of reagents and molecular weight ofthe
`polymer. Nevertheless, it has been found practical with
`most polypeptides to utilize between 10 and 100, suit
`ably between 15 and 50 moles of polymer per mole of
`polypeptide. Utilizing these proportions it has been
`found that at least 15% of the physiological activity has
`been maintained. While the scope of the invention
`should not be considered limited thereto, it is generally
`preferred to provide conditions wherein at least 40% of
`the physiological activity is preserved.
`The procedures of the present invention are generally
`20
`applicable to peptides and polypeptides, that are of
`particular interest in applications involving enzymes
`and peptide hormones. Among the enzyme categories
`which may be used may be mentioned:
`Oxidoreductases such as: Urate: oxygen oxidoreduc
`tase (1.7.3.3; “uricase”); Hydrogen-peroxide: hydrogen
`peroxide oxidoreductase (1.11.1.6; “catalase”); Choles
`terol, reduced - NADP: oxygen oxidoreductase (20-8
`hydroxylating) (1.14.1.9; “Cholesterol 20-hydroxy
`lase”).
`Transferases such as: UDP glucuronate glucuronyl
`transferase , (acceptor unspeci?c) (2.4.1.17; “UDP
`glucuronyltransferase”); UDP glucose: a-D-Galactose
`l-phosphate uridylyltransferase 2.7.7.12).
`Hydrolases such as: Mucopeptide N-acetylmuramyl
`hydrolase (3.2.1.17; lysozyme); . Trypsin (3.4.4.4); L
`Asparagine aminohydrolase (3.5.1.1; “Asparaginase”).
`Lyases such as: Fructose-1,6-diphosphate D
`“aldo
`’ glyceraldehyde-3-phosphate¢lyase
`(4. l .2. 12:
`lase”).
`Isomerases such as: ‘ D-Xylose ketol-isomerase
`(5.3.1.5; xylose isomerase) and‘
`Ligases such \as: l_.-Citrulline:v L-aspartate ligase
`(AMP) (6.3.4.5).
`,
`Arnongthe peptide hormones may be mentioned are
`insulin, ,ACTH, Glucagon, Somatostatin, Somatotropin,
`Thymosin, Parathyroid ‘hormone, ,Pigmentary hor
`mones, Somatomedin, Etythropoietin,Luteinizing hor
`mone, Chorionic Gonadotropin, Hypothalmic releasing
`factors, Antidiuretic hormones, Thyroid stimulating
`hormone and Pr‘olactin..
`_
`The resultant products from these two categories of
`coupling are exempli?ed hereinbelow. Polyethylene
`glycol (hereinafter PEG) has beenselected as the prey
`ferred embodiment of the polymer used, but has been
`employed solely for purposes of illustration and not
`limitation. Similar products would be obtained with any
`of the other polymers utilized in the scope of the present
`invention- PEG—O— ‘is the terminal portion of a
`polymer chain where 5 is the residual oxygen from the
`terminal hydroxyl, similwarly in PEG-NH,N is the
`residual terminal nitrogen of the amino group replacing
`the terminal hydrogen. Again with respect to the pep
`tide moiety which is representationally. ‘illustrated
`below as
`
`45
`
`PING
`Another suitable terminal group is the acyl azide
`terminaligroup. In the production thereof, the terminal
`hydroxyl of the polymer is reacted with chloroacetic
`anhydride and subsequently with diazomethane to yield
`the methyl ester of the carbomethoxyether. Treatment
`with hydrazine gives‘ the corresponding hydrazide
`which on treatment with nitrous acid yields the desired
`
`acyl azide." _ The azido moiety of the thus produced acyl azide will ' . ' _. I ‘
`
`
`
`
`
`
`
`react with, say, a labile aminomoiety on the peptide to
`provide a protected peptide of the partial structure, say,
`
`PEG--O-CH2 . co. sir-1MP
`
`‘ In place of treatment withhydrazine there may be
`utilized N-ethoxy carbonyl-,2-ethoxy 1,2-dihydroquino
`line, EEDQ. The quinoline .is eliminated to give the
`corresponding mixed carbonicvanhydride which is a
`“disappearing-l’ coupling grouphaving as the effective
`couplingzmoiety, say, the O-PEG-methyl carbonyl moi
`ety- which attachesitself tofree amino groups on the
`polypeptide, to provide-aresulting product which has
`
`65
`
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`

`
`5
`the same structure as that shown to be obtained in the
`preceeding paragraph hereof.
`The succinate group may also be used as a coupling
`moiety. In this modi?cation the glycol, for example,
`PEG or PPG, is taken up in a suitable reaction inert
`solvent in the presence of a mild base, such as sodium
`bicarbonate and treated with a dihalosuccinic anhy
`dride such as dibromosuccinic anhydride. The thus
`produced, say, PEG-dihalo succinate is then available
`for reaction with a polypeptide yielding a protected
`peptide of partial structure
`
`4,179,337
`6
`The carboxyamino or thiocarbonylamino benzyl link-‘
`age between the terminal oxygen of the glycol such as
`PEG and a nitrogen of a free amino group on the poly
`peptide is prepared from the p-amino-benzyl ether of
`the glycol. The p-amino benzyl ether is treated with
`phosgene or thiophosgene to yield the corresponding
`amino acid chloride (or amino thioacid chloride) which
`is then reacted with the free amino group of a polypep
`tide, to yield a protected peptide having the partial
`structure
`
`The p-diazo benzyl group is a suitable coupling agent.
`P-nitrobenzyl chloride is reacted with the glycol, suit
`ably PEG in the presence of base, suitably an alkali in an
`anhydrous non-hydroxylic medium, preferably under
`re?ux to produce the corresponding PEG p-nitrobenzyl
`ether which is then reduced to the corresponding amine
`by catalytic hydrogenation followed by diazotization to
`yield the desired O-PEG-p-diazonium benzyl ether
`which is then available for coupling with the polypep
`tide, to yield a protected peptide having the partial
`structure
`
`,1:
`PEG-O-Cl-lz
`
`NH . cm or syiu-rww
`
`The 2-(hydroxy-3-carboxy)propyl linkage group,
`which is attached to the terminal oxygen of the glycol
`at the 2 position of the propyl group.
`1,3-O-isopropylidene 2-bromo propane-1,3-diol is
`reacted with the glycol, say PEG in anhydrous medium
`in the presence of a base, suitably an alkali, preferably
`with heating. The resulting Z-glyceryl glycol ether is
`treated with cyanogen bromide at high pH and low
`temperature. The polypeptide is added thereto and the
`coupling occurs to leave the above group between the
`terminal oxygen of the glycol and a nitrogen of a pri
`mary amine group on the polypeptide, to yield a pro
`tected peptide having the partial structure
`:
`
`20
`
`25
`
`30
`
`The 3-(p-diazophenyloxy)-2-hydroxy propyloxy
`group is prepared by treating the glycol in the presence
`of alkali at moderately elevated temperatures in an
`aqueous medium with glycidyl p-nitrophenyl ether to
`form the corresponding 3-(p-nitrophenyloxy) 2-hydrox
`ypropyl ether of PEG. The nitro group is reduced to
`the corresponding amino group, preferably by aqueous
`titanous chloride in dilute mineral acid and the resulting
`amine diazotized to provide the diazonium ether which
`may then be reacted with the polypeptide, to yield a
`protected peptide having the partial structure
`
`45
`
`'
`
`PEG-O-CHZ . CH . (OH) . CHr'O
`
`50
`
`55
`
`The l-glycydoxy-4-(2'-hydroxy-3'-propyl) butane
`group is attached to the terminal oxygen group of the
`glycol and is reacted with free amino group or a poly
`peptide.
`1,4-butanedioldiglycidyl ether is reacted. with the
`glycol, suitably PEG in the‘presence of an alkali and a
`reducing agent such as sodium borohydride. The reac
`tion takes place at room temperature and yields the
`desired PEG ether with an oxirane group at the end of
`the side chain. This oxirane group reacts with a free
`amino group to form a C-N linkage, on the polypep
`tide to yield a protected peptide having the partial
`
`structure
`
`~
`
`65
`
`Also available is the anthranilate moiety. In this mod
`i?cation the glycol is again taken up in a reaction inert
`solvent and treated with isatoic anhydride to yield the
`anthranilate ester which is used without further puri?
`cation in the next stage which comprises diazotization.
`The diazotization is carried out in the usual manner, for
`example, the anthranilate ester is taken up in water, the
`solution acidi?ed, suitably with glacial acetic acid,
`cooled, and sodium nitrite added thereto. The diazo
`nium salt thus formed is available for reaction with
`polypeptides.
`An interesting alternative modi?cation using azido
`groups involves the photochemical attachment of an
`azide coupling group. For example, the glycol in a
`buffer is treated with 4-?uoro-S-nitrophenylazide, the
`unreacted azide is then removed, suitably by dialysis.
`The enzyme in question, for example lysozyme, is
`taken up in water, treated with the reagent and again,
`irradiated, suitably at reduced temperatures to yield, for
`example, PEG-Z-nitrophenyl-lysozyme.
`In the foregoing discussion, the carbon atom of the
`polymer to which the coupling moiety is attached is the
`carbon bearing the terminal hydroxy group. In the case
`of, say, PPG and PEG, two terminal hydroxy groups
`are present per moiety. Thus, the possibility exists of
`cross linking betweenv the polypeptide moieties. Such
`cross linking is undesirable but may be readily avoided.
`One method of cross linking avoidance is to carry out
`the reactions using a large excess of polymer either at
`the stage of combination with the coupling moiety or at
`the coupling state itself.
`
`NOVARTIS EXHIBIT 2099
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`

`
`4,179,337
`7
`Alternatively, and preferably, one end of the polymer
`is pre-blocked. Such polymers, for example, alkylated
`PEGs such as methoxy polyethelene glycol (MPEG),
`are commercially available. Such polymers, of course,
`have only one labile group per polymer moiety.
`The terminal hydroxy group may be converted into
`an amino group. In this procedure the polymer is re
`acted at its terminal hydroxyl group either with a sulfo
`nating agent such as toluene sulfonyl chloride or with a
`halogenating agent such as triphenyl phosphine in car
`bon tetrachloride or triphenyl phosphine and a suitable
`N-halosuccimide. The thus produced halide or tosylate
`is then treated with sodium azide and reduced with
`lithium aluminum hydride to give the corresponding
`terminal amino compound.
`The polymer bearing the terminal amino group is
`then coupled with a carboxy group of the polypeptide
`using methods well known in the art. The use of a water
`soluble carbodiimide such as l-cyclohexyl-3-(2-mor
`pholino ethyl) carbodiimide, metho-p-toluene sulfonate
`being especially preferred. There is thus produced an
`amide linkage comprising the nitrogen of the polymer
`and the appropriate carbonyl group of the peptide of
`the general structure
`
`0
`
`25
`
`8
`PREPARATION I
`Preparation of Z-O-Methoxy Polyethylene
`Glycol-4,é-dichloro-s-triazine (“Activated MPEG”)
`Cyanuric chloride (5.5 g; 0.03 mole) was dissolved in
`400 ml anhydrous benzene containing 10 g anhydrous
`sodium carbonate. MPEG-1900 (19 g; 0.01 mole) was
`added and the mixture was stirred overnight at room
`temperature. The solution was ?ltered, and 1600 ml
`petroleum ether (boiling range, 35° C.-60° C.) was
`added slowly with stirring. The ?nely divided precipi
`tate was collected on a ?lter and redissolved in 400 ml
`benzene. The precipitation and ?ltration process was
`repeated several times until the petroleum ether was
`free of residual cyanuric chloride as determined by high
`pressure liquid chromatography on a column of 5 pm
`“LiChrosorb” (E. Merck), 250x 3.2 mm, developed
`with hexane, and detected with an ultraviolet detector.
`Titration of activated MPEG-1900 with silver nitrate
`after overnight hydrolysis in aqueous buffer at pH 10.0,
`room temperature, gave a value of 1.7 moles of chloride
`liberated per mole of MPEG.
`In accordance with the above procedure but, where
`in place of MPEG-1900, MPEG 5000 is used, activated
`MPEG-5000 was similarly prepared using a 1:3 molar
`ratio of MPEG and cyanuric chloride.
`
`In place of direct coupling of the amino group to
`form an amido linkage with the polypeptide, the cou
`pling may take place via a maleimide group. In this
`modi?cation w-amino PEG is reacted with maleic anhy
`dride and the resultant N-PEG-maleimide reacted with
`the desired polypeptide yield a protected peptide of the
`structure
`
`v
`O-(IOW
`
`o—c0~v\~
`
`69
`PEG-N
`
`I-Iereinabove
`
`c)\ \
`
`//
`o
`
`69
`—N
`
`35
`
`45
`
`is the terminal nitrogen on the polymer moiety where
`the terminal hydroxyl has been replaced by a primary
`amino group.
`Hereinafter the suffix number (i.e., PEG 750) signi?es
`the molecular weight in daltons of the polymer in ques
`tron.
`In an especially preferred embodiment of this inven
`tion, alkoxy, suitably methoxy polyethylene glycols,
`have been attached covalently to the polypeptides.
`
`EXPERIMENTAL
`SOURCES OF MATERIALS
`Trinitrobenzene sulfonic acid was purchased from
`Nutritional Biochemicals Company. Cyanuric chloride
`(2,4,6-trichloro-s-triazine) was obtained from Aldrich
`Chemicals and was recrystallized twice from benzene
`immediately before use. Methoxy polyethylene glycols
`of 1900 and 5000 daltons (MPEG-1900 and MPEG
`5000) were supplied by Union Carbide
`
`60
`
`65
`
`EXAMPLE I
`Urate: Oxygen Oxidoreductase (Uricase)
`(1.7.3.3)-PEG-Carbomethyl Conjugate
`(a) Preparation of PEG
`(i) Preparation of PEG-Methyl Carbomethoxy Ester
`PEG 750 (2.0 g) is dissolved in liquid ammonia (30
`ml.) and the solution treated with sodium until the blue
`color persists for 5 minutes. The ammonia is allowed to
`, evaporate on a stream of dry nitrogen. The residue is
`treated with methyl chloroacetate (5 ml.) and the mix
`ture allowed to stand overnight at room temperature
`and ?nally heated to 100° for 1 hour. The excess reagent
`is removed under reduced pressure to provide PEG
`methyl carbomethoxy ester.
`(ii) Preparation of PEG-Methoxy carbohydrazide
`PEG-methyl carbomethoxy ester (2.0 g), methanol
`(300 ml.) and hydrazine hydrate (15 ml.) are re?uxed
`overnight and the solution is evaporated under reduced
`pressure to yield PEG-methoxy carbohydrazide.
`(iii) Preparation of PEG-Carboxymethyl Azide
`The above hydrazide (1.0 g) is dissolved in 2% hy
`drochloric acid (150 ml.) and 5% sodium nitrite solution
`(9 ml.), slowly added with stirring and allowed to stand
`for 20 minutes at room temperature to yield a solution
`of PEG-carboxymethyl azide which is used in the cou
`pling stage.
`(b) Coupling of Urate: Oxygen Oxidoreductase with
`PEG-Carboxymethyl Azide
`The solution containing the azide (16 ml., as pro
`duced in part (iii) supra) is adjusted to pH 8.7 by the
`addition of sodium phosphate. Uricase (25 mg) is added
`and the solution is stirred for 2 hours at room tempera
`ture. The solution is dialyzed and the modi?ed enzyme
`isolated by chromatography on Sephadex G-50. If de
`sired lyophilization yields the protected enzyme in dry
`form.
`In accordance with the foregoing procedures, but
`utilizing asparaginase or insulin in place of uricase there
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`EXAMPLE IV
`UDP glucuronate glycuronyltransferase (acceptor
`unspeci?c) (2.4.1.17; “UDP glycuronyltransferase”)
`PEG-succinato conjugate
`(a) Preparation of PEG-dibromo succinate
`PEG (1.0 g) is dissolved in dry benzene (10 ml.) con
`taining sodium bicarbonate (1.0 g). Dibrorno succinic
`anhydride (0.5 g) is added and the solution stirred over
`night. The solution is ?ltered and the ?ltrate concen
`trated under reduced pressure to yield PEG-dibromo
`succinate.
`In accordance with the foregoing procedure, but
`using diodo succinic anhydride in place of dibromo
`succinic anhydride, there is obtained the corresponding
`PEG-diodo succinate.
`'(b) PEG succinato UDP Glucuronyl transferase
`UDP glucuronyl transferase (50 mg) in buffer solu
`tion (10 ml., pH 7.0) is slowly treated with PEG
`dibromo succinate (100 mg) in water (5 ml) at room
`temperature. The pH is maintained between 7—8..'IIhe
`desired PEG-enzyme conjugate is isolated by chroma
`tography, on Sephadex G-50. If desired, the product
`may be isolated by lyophilization.
`In accordance with the foregoing procedure but
`where in place of UDP glucuronyltransferase there is
`used insulin, there is obtained PEG succinato insulin.
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`4,179,337
`10
`yield a solution of the enzyme-PEG conjugate which
`is obtained the corresponding PEG-asparaginase or
`PEG-insulin conjugate. Similarly, there may beused
`may, if desired, be lyophilized.
`PPG in place of PEG to provide the corresponding
`In accordance with the above procedure, but where
`PPG-uricase and asparaginase conjugates.
`in place of cholesterole 20 hydroxylase, insulin is used
`there is obtained the corresponding PEG-N-maleimido
`EXAMPLE II
`insulin conjugate.
`Hydrogen Peroxide: Hydrogen-Peroxide
`Oxidoreductase (1.1 1.1.6;
`.
`“Catalase")-2-PEG-4-Hydroxy-1,3,5-Triazin-6-yl
`Conjugate
`(a) Preparation of
`PEG-4-hydroxy-6-chloro-1,3,5-triazine
`PEG 750 (30 g., 0.04 mole) or PEG 2,000 (80 g., 0.04
`mole) is dissolved in 150 ml. anhydrous benzene con
`taining 8 g. Na2CO3. The solution is cooled to 10° and
`cyanuric chloride (7.38 g., 0.04 mole) is added. The
`solution is stirred overnight at 10". Water (5 ml.) is
`added, and the solution then is brought to room temper
`ature for several hours, followed by heating at 40° over
`night. Insoluble material is centrifuged off, and solvent
`is removed by reduced pressure in a rotary evaporator
`at 40°. A small amount of precipitate which sometimes
`appears during concentration is removed by the addi
`tion of a small amount of benzene to lower the viscosity,
`25
`followed by centrifugation and reconcentration. The
`PEG-4-hydroxy-6-chloro-1,3,5-triazine, a viscous liquid
`at 40", is stored in the freezer.
`(b) Preparation of PEG-HTA-Catalase Conjugate
`Catalase (60 mg; 8.7x 10*7 moles) is dissolved in 3
`ml. 0.05 M borate buffer, pH 9.0. PEG 750 (470 mg) is
`added. After 3 hours the pH is readjusted to 9.0 with
`sodium hydroxide and the solution left at room temper
`ature overnight. The pH is again adjusted to 9.0. Unre
`acted PEG is removed by passing the solution through
`a column of Sephadex 6-50. The PEG-HTA-catalase
`conjugate is concentrated on a rotary evaporator to 1
`mg. protein/ml. and stored in the freezer. HTA E -4
`hydroxy-1,3,5-triazin-6-yl).
`‘
`In accordance with the foregoing procedure, but
`using Carbowax 2000 a similar coupled product is ob
`tained. Similarly, in accordance with the above proce
`dure, but in place of catalase, D-Xylose ketol isomerase
`(xylose isomerase) ‘or insulin is used, there are obtained
`the corresponding PEG-HTA-xylose isomerase and
`PEG-HTA-insulin conjugates.
`EXAMPLE n1
`Cholesterol, reduced - NADP: Oxygen Oxidoreductase
`"
`(ZO-B-hydroxylating) (1.14.1.9; cholesterol
`20-hydroxylase)-PEG-N-Maleimido Conjugate
`(a) Formation of N-PEG-maleimide
`Maleic anhydride (1.0 gal/100 mole), benzene (50'
`ml.) and w-amino-PEG (1/200 mole) are re?uxed for 2
`hours. The solution is evaporated under reduced pres
`sure and heated at 200° in a stream of dry nitrogen for
`2 hours.
`(b) Reaction of N-PEG maleimide with cholesterol
`20-hydroxylase
`.
`Cholesterol 20-hydroxylase (25 mg) is added to a
`solution of N-PEG-maleimide (70 mg) in 0.1 M phos
`phate buffer (pH 7.0, 10 ml.). The solution is allowed to
`stand at room temperature for 1 hour. The solution is
`dialyzed and the desired product is isolated from the
`dialysate by chromatography'on Sephadex G-50 to
`
`EXAMPLE V
`UDP Glucose: a-D-galactose-l-phosphate
`‘
`uridylytransferase (2.7.7.12)
`_
`(U DP-GPU-transferase)-PEG-anthranilate conjugate
`(a) Preparation of PEG anthranilate ester
`PEG (1.0 g) is dissolved in dry benzene (10 ml.) con
`taining sodium bicarbonate (1.0 g). Isatoic anhydride
`(0.25 g) is added and the solution stirred overnight. The
`solution is ?ltered and the ?ltrate evaporated at 40° C.
`under reduced pressure to yield PEG-anthranilate ester
`which is used in the next step without further puri?ca
`tion.
`(i) Preparation of PEG anthranilate ester diazonium salt
`The PEG-anthranilate ester produced as above is
`dissolved in water (5.0 ml.) and glacial acetic acid (0.5
`ml.) added, the solution is cooled to 0°—2° and a concen
`trated solution of sodium nitrite added dropwise with
`stirring. Addition of sodium nitrite is stopped when
`nitrous acid is present.
`(b) Coupling of UDP glucose:
`a-D-galactose-l-phosphate uridylyltransferase with
`PEG-anthranilate ester diazonium salt
`UDP-GPU transferase (25 mg) is dissolved in buffer
`solution (5 ml., pH 7—7.5) and the solution cooled to
`0°—2". This solution is added dropwise to the diazotized
`solution prepared as above. The pH is maintained at
`7-7.5. After 2 hours, the solution is allowed to come to
`room temperature and ?ltered and the desired com
`pound is isolated by Sephadex chromatography. If de
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`4,179,337
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`sired, the conjugate may be isolated in solid form by
`lyophilization.
`.
`In accordance with the above procedure but where in
`place of UDP-GPU transferase there is employed insu
`lin, the corresponding insulin - PEG anthranilate ester is
`obtained.
`
`bromide which in turn is similarly converted into PEG
`
`m-azide.
`
`.i
`
`..
`
`.
`
`EXAMPLE VI
`Mucopeptide N-acetylmuramylhydrolase
`(3.2. l. l7)-4-azido-2-nitro-phenyl PEG
`(a) 4-azido-2-nitrophenyl PEG
`PEG (1.0 g) is dissolved in borate buffer, pH 9.8 (100
`ml.) and the solution treated with 4-?uoro-3-nitrophe
`nyl azide (1.0 g) in acetone (10 ml.). The reaction mix
`ture is stirred at 40° overnight and ?ltered. The ?ltrate
`is dialyzed against water, ?ltered and freeze dried.
`(b) Photochemical li