54
`
`Bloconjugate Chem. 1993, 4, 54-62
`
`Copolymers of Lysine and Polyethylene Glycol: A New Family of
`Functionalized Drug Carriers
`
`Aruna Nathan, Samuel Zalipsky,*r+ Sylvie I. Ertel, Spiro N. Agathos,t Martin L. Yarmush,a and
`Joachim Kohn’,ll
`Department of Chemistry, Rutgers-The State University of New Jersey, New Brunswick, New Jersey 08903.
`Received August 14, 1992
`
`~~
`
`~
`
`Poly(PEG-Lys), a new, water-soluble poly(ether urethane), derived from L-lysine and poly(ethy1ene
`glycol) was investigated as a precursor for the preparation of polymeric drug conjugates. To facilitate
`a wide variety of coupling chemistries, the pendent carboxyl groups of poly(PEG-Lys) were converted
`to other reactive functional groups (amino, hydroxyl, active ester, and aldehyde) in high yield. These
`reactive pendent chains were then used as anchors for the covalent attachment of penicillin V and
`cephradine, two clinically used antimicrobial agents. Coupling to the carrier was achieved in good
`yields and the chemical versatility of this system was demonstrated by the preparation of conjugates
`having antibiotic ligands linked via biostable or biodegradable linkages to the carrier, either directly
`or via a spacer. Conjugate 4, poly(PEG-Lys-penicillin V ester), was obtained by linking penicillin V
`to the polymer backbone via hydrolytically labile ester bonds. This conjugate exhibited activity similar
`to that of the parent drug against three clinically important strains of bacteria. Drug activity coincided
`with the release of the drug from the carrier. Hydrolytically stable cephradine-containing conjugates
`were prepared by three different coupling methods but showed no antibiotic activity. 14C-labeled
`poly(PEG-Lys) was injected into mice and its biodistribution was monitored for 48 h. The carrier
`showed no preferential uptake by liver, spleen, or kidney. No signs of acute toxicity were evident in
`mice or rats when poly(PEG-Lys) was administered iv and ip at doses up to 10 g/kg. These results
`indicate that poly(PEG-Lys) is a promising precursor for the preparation of soluble drug conjugates.
`
`INTRODUCTION
`Attachment of a drug to macromolecular carriers can
`be used to manipulate biodistribution, permeability
`through biological barriers, and pharmacokinetics (1).
`Since attachment of a small drug to a macromolecule will
`slow the rate of drug excretion, drug-polymer conjugates
`can be used not only for drug targeting but also for the
`sustained release of a drug over time. Numerous polymers,
`such as poly(amino acids), polysaccharides, poly(viny1
`alcohols), polyvinylpyrrolidinone, polyphosphazenes, and
`poly(acry1ic acid) derivatives have been explored as
`potential carriers for pharmacologically active agents ( I ) .
`Due to its recognized biocompatibility, PEG’ is one of
`the most widely investigated drug carriers (2,3). Attach-
`ment of PEG to various drugs, proteins, and liposomes
`has been reported to delay clearance by the reticuloen-
`dothelial system (4-6). However, PEG has only two
`
`+ To whom correspondence should be addressed at Liposome
`Technology, Inc., 1050 Hamilton Ct, Menlo Park, CA 94025.
`* Waksman Institute of Microbiology and Department of
`Chemical and Biochemical Engineering, Rutgers University.
`Department of Chemical and Biochemical Engineering,
`Rutgers University.
`1 To whom correspondence should be addressed at Department
`of Chemistry, Rutgers University, P.O. Box 939, Piscataway, NJ
`08855.
`Abbreviations used: PEG, polyethylene glycol; BSC-PEG,
`bis(succinimidy1 carbonate) of PEG; HOSu, N-hydroxysuccin-
`imide; EDC, 1-ethyl-3- [3-(dimethylamino)propyl]carbodiimide;
`DCC, dicyclohexylcarbodiimide; DCU, dicyclohexyl urea; GPC,
`gel permeation chromatography; y- ABA, y-aminobutyric acid;
`DMAP, 4-(dimethylamino)pyridine; MIC, minimum inhibitory
`concentration. A schematic notation is used throughout the text
`to refer to polymeric products. Similar notations were used in
`previous pub1ications;see for example ref 19. Figures 1-5 provide
`the exact chemical structures of all polymeric products.
`
`reactive groups (the hydroxyl groups at the ends of the
`chain) that can be used either directly (5, 7-9) or after
`suitable functionalization (2,5, 7,8) for covalent attach-
`ment of ligands. This severely limits not only the drug
`“loading” of such conjugates but also the design options
`for PEG-based drug-carrier systems. To circumvent this
`limitation, PEG-containing polymers were prepared that
`bear reactive pendent groups along the polymer backbone.
`However, the properties of such functionalized polymers
`are often quite different from those of PEG itself (10,II).
`In a different approach, copolymers of PEG and amino
`acids were explored as biomaterials. These materials were
`usually prepared by the polymerization of a-amino acid
`N-carboxy anhydrides of y-benzyl-L-glutamate (12), p-ben-
`zyl-L-aspartate (1 31, Ne- (benzyloxycarbonyl)-L-lysine (14),
`proline (151, and phenylalanine (16) using amino-PEG as
`an initiator. This approach led to block copolymers of
`the general formula ABA (12,15,16) or AB (13,14), where
`the A block is composed of homopolypeptide and PEG is
`the B block. In some instances such block copolymers
`were further polymerized by enzymatic (16) and chemical
`( 1 7) methods yielding multiblock copolymers of higher
`molecular weights. Potentially useful drug carriers, ob-
`tained by oligocondensations of PEG-diamine or PEG-
`disuccinate with N,N’-bis(L-phenylalany1)hexamethyl-
`enediamine or N,N’-adipoylbis(L-phenylalanine), were
`described by Ulbrich et al. (18).
`In contrast to the previously known PEG/amino acid
`block copolymers, the polymers described here are strictly
`alternating polymers of the general structure: -(PEG-
`Lys),-, in which PEG chains of variable molecular weights
`are linked to the cy- and €-amino groups of lysine through
`stable urethane linkages (2). Such copolymers retain the
`desirable properties of PEG, while providing reactive
`pendent groups (the carboxylic acid groups of lysine) at
`strictly controlled, predetermined intervals along the
`
`7 043-1 802/93/2904-0054$04.00f 0
`
`0 1993 American Chemical Society
`
`SANOFI-AVENTIS Exhibit 1023 - Page 54
`
`IPR for Patent No. 8,951,962
`
`

`
`Copolymers of Lyslne and Polyethylene Glycol
`
`BSC-PEG
`A
`
`H-LyS-OR
`
`poly(PEG-LyS-OR)
`
`H2N-NH2
`
`o**
`
`1
`
`0 - R
`
`L
`
`H
`0 - R
`
`1
`
`B
`1 R=CHzCH3
`2 R = H
`Figure 1. Preparation of poly(PEG-Lys) copolymers: (A)
`schematic representation, (B) detailed chemical structures.
`
`polymer chain. The reactive pendent groups can be used
`for derivatization, cross-linking, or conjugation with other
`molecules. Recently we reported on the usefulness of this
`new class of polymers for preparation of hydrogels (19).
`The present paper describes some of the biological
`properties of poly(PEG-Lys) and explores the synthesis
`of polymeric conjugates with pharmacologically active
`substances.
`
`RESULTS AND DISCUSSION
`Polymer Synthesis. Succinimidyl carbonates (SC) of
`PEG were recently used by Zalipsky et al. (20, 21) for
`covalent modification and cross-linking of proteins. The
`high reactivity of SC-activated polymers toward amines
`as well as their ease of preparation and excellent storage
`stability prompted our use of bis(succinimidy1 carbonates)
`of PEG (BSC-PEG) as starting materials for the prepa-
`ration of alternating PEG-lysine polyurethanes (Figure
`1). While the previously described interfacial polymer-
`ization of BSC-PEG with L-lysine ethyl ester yielded high
`molecular weight polymers (1) (191, the aqueous solution
`polymerization of BSC-PEG and lysine produced polymers
`(2) of molecular weights suitable for use as drug carriers
`(M, I 50 000). The solution polymerization procedure
`was also conveniently applied for the preparation of
`radiolabeled carriers using [14C]lysine.
`Functionalization. The amount of free carboxyl
`groups present on 2 or obtained after hydrolysis of the
`ethyl ester groups of 1 was determined quantitatively by
`titration with sodium methoxide. In both cases the content
`of free carboxyl groups was essentially as expected from
`calculations of the polymer composition. Although car-
`boxyl groups can be used directly for the covalent
`attachment of drug molecules via amide and ester linkages,
`transformations of the carboxyl groups to other reactive
`functional groups were also investigated (Figure 2).
`First, we explored the attachment of ligands via amide
`bonds, using glycine ethyl ester as a simple model
`compound and EDC as the condensation reagent. Amino
`acid analysis of conjugate 3 (Figure 3) revealed a G1y:Lys
`ratio of 0.9511, indicating that about 95% of all free
`carboxyl groups had reacted with glycine ethyl ester. The
`
`Bloconlugete Chem., Vol. 4, No. 1, lQQ3 55
`loading of glycine was calculated to be 4.4~10-~ equivlg of
`polymer. Similarly, activation of the carboxyl groups with
`EDC or pivaloyl chloride was used for attachment of
`ethanolamine and ethylene diamine residues to the
`polymer, thus introducing primary hydroxyl and amino
`In these
`groups, respectively (derivatives 4 and 5).
`reactions cross-linking is possible. When a diamine was
`added to the pivaloyl chloride-activated polymer, an
`insoluble gel was obtained. However, when the order of
`addition was reversed, e.g., the activated polymer was
`added into a large excess of diamine (see Experimental
`Section), the obtained product remained soluble.
`The reaction of poly(PEG-Lye) with DCC and HOSu in
`methylene chloride was found to be the most convenient
`way for the introduction of active esters into the polymeric
`backbone. Quantitation of active acyl groups (22) showed
`that over 95% of the carboxyl groups could be converted
`into active HOSu ester residues by this reaction. The
`polymeric active ester derivative (6) proved most useful
`for direct attachment of primary-amine-containing drugs
`or spacer residues such as y-aminobutyric acid (derivative
`7).
`The derivative having reactive aldehyde groups (9) was
`prepared using a modification of the method of Vandoorne
`et al. (23). In the first step, derivative 8 was obtained via
`reaction of 3-amino-1,2-propanediol with the HOSu ester
`6. Alternatively, 8 could be prepared by the EDC-mediated
`coupling of 3-amino-1,2-propanediol to 2. In the second
`step, the mild oxidation of the 1,2-diol moieties with
`periodate produced aldehyde groups. Derivative 9 had a
`tendency to form an insoluble polymer during attempts
`to isolate and dry the material. Most likely, cross-links
`were formed due to aldol condensation or formation of
`acetals as a result of the reaction between newly introduced
`aldehyde groups (-70 % of the repeat units) and remaining
`diol residues (-30% of the repeat units). Derivative 9
`was therefore kept in an aqueous solution at 4 "C, which
`was assayed for aldehyde content with 2,4-dinitrophe-
`nylhydrazine (24) and used directly for drug-attachment
`reactions.
`Attachment of Drugs. Two antimicrobial agents
`(penicillin V and cephradine) were chosen for the initial
`evaluation of poly(PEG-Lys) as a drug carrier. Previously,
`a number of macromolecular derivatives of &lactam
`antibiotics had been prepared and evaluated (25). Here,
`penicillin V was linked to the hydroxyl groups of 4 via
`hydrolytically labile ester bonds yielding derivative 10
`using DCCIDMAP according to a previously reported
`procedure (Figure 4) (8, 9).
`The extent of drug incorporation was 66 % as determined
`by NMR and elemental analysis of sulfur. In another
`series of experiments, cephradine was linked to the poly-
`(PEG-Lys) backbone via three different attachment
`strategies leading to conjugates 11-13 (Figure 5). In the
`first conjugate (ll), the drug was attached to the carrier
`directly via an amide bond. In conjugate 12, similarly
`obtained from an HOSu ester intermediate, cephradine
`was positioned at a 5-atom distance from the polymer
`backbone. In an alternative coupling scheme, cephradine
`was attached to the polymer via stable secondary amine
`linkages, derived from the reaction of the aldehyde pendent
`chains of derivative 9 with the primary amine of the drug
`under reducing conditions. This scheme led to the
`attachment of cephradine at =40% of the repeat units.
`Considering that only -70% of lysine residues of the
`polymer 9 were bearing aldehyde groups, the efficiency of
`the reductive amination step was comparable to the
`formation of amide bonds in derivatives 11 and 12. The
`
`SANOFI-AVENTIS Exhibit 1023 - Page 55
`
`IPR for Patent No. 8,951,962
`
`

`
`50 Bioconjugate Chem., Vol. 4, No. 1, 1993
`
`Nathan et el.
`
`4: poly(PEG-Lys ethanolamide) R = OH
`5: poly(PEG-Lys ethylene diamine) R = NH2
`
`H
`
`8: poly(PEG-Lys-aminopropanediol)
`
`~
`
`~
`
`~
`H
`
`~
`
`T
`
`~
`
`-
`
`~
`
`~
`
`9: poly( PEG-Lys-aldehyde)
`
`OH
`
`H
`
`Figure 2. Detailed chemical structures for derivatives of poly(PEG-Lys). Starting from free pendent carboxylic acid groups, derivatives
`were prepared with pendent hydroxyl groups (4), pendent amino groups (5), pendent N-hydroxysuccinimide active ester groups (6),
`and pendent diol groups (8). Further derivatization reactions yielded the y-aminobutyric acid (y-ABA) derivative (7) and the aldehyde
`derivative (9). See Experimental Procedures for details.
`
`1
`
`I
`
`r
`
`H
`
`
`
`H
`
`L
`
`\
`.NH
`CH: )- o - CH, -CH,
`
`0
`
`bility of linking chemically sensitive drugs to the polymer
`backbone via a variety of mild coupling protocols.
`In the attachment reaction of penicillin V, the carboxyl
`groups of penicillin, which are essential for antimicrobial
`activity, were tied up by ester bonds. Penicillin bound in
`this way should be biologically inactive. However, the
`gradual hydrolysis of the ester bonds under physiological
`conditions, should result in the release of penicillin V in
`biologically active form. In order to test this hypothesis,
`the activity of poly(PEG-Lys-penicillin V ester) (10) was
`tested against clinically important strains of bacteria
`(Table I). During these experiments, the release of free
`penicillin from 10 was monitored spectrophotometrically.
`Since 95 70 of all bound penicillin V was released from the
`conjugate within 24 h, we concluded that the activity of
`10 was indeed due to the presence of free penicillin V
`(Figure 6). On the other hand, none of the cephradine
`containing conjugates (1 1-13) inhibited the growth of
`cephradine-sensitive microorganisms, even when applied
`at doses up to 10 times the MIC of free cephradine. This
`unexpected observation is currently under further inves-
`tigation.
`
`poly(PEG-Ly~)
`I
`Gly-OEt
`3
`Figure 3. Detailed chemical structure of the glycine ethyl ester
`(Gly-OEt) conjugate of poly(PEG-Lys).
`advantage of coupling via reductive amination is that
`conjugate 13 preserved the positive charge of the amino
`group of cephradine, considered to be of importance for
`its antimicrobial activity (26). In all penicillin and
`cephradine conjugates, the integrity of the labile B-lactam
`unit was confirmed by a selective, quantitative iodometric
`assay (27). These model reactions established the feasi-
`
`SANOFI-AVENTIS Exhibit 1023 - Page 56
`
`IPR for Patent No. 8,951,962
`
`

`
`Copolymers of Lysine and Polyethylene Glycol
`
`Bioconjvgwte chem., VOI. 4, NO. I, lQQ3 57
`
`poly(PEG-Lys ethanolamide)
`4
`
`Penicillin V
`
`DCCIDMAP
`
`c
`
`H
`
`H
`
`1 0
`Figure 4. Preparation of the penicillin V conjugate (10) from the hydroxyl derivative poly(PEG-Lys-ethanolamide) (4). See Experimental
`Procedures for details. This scheme resulted in the attachment of penicillin V via a hydrolyzable ester linkage to the ethanolamide
`spacer. Cleavage of the ester bond (marked by an arrow) was shown to restore biologically active, free penicillin V.
`
`1
`
`poly( PEG-LYS-OSU)
`6
`
`Cephradine
`
`t
`
`A
`
`poly( PEG-LyS-YABA)
`7
`
`DCC/HOSu
`
`Cephradine
`
`H
`H
`N ~ ; y * ( C H z c H Z O ) . -
`0
`
`i" c=o
`
`-1
`
`B
`
`i
`
`1 2
`
`COZH
`
`-
`
`poly( PEG-Lys-aldeh yde)
`9
`
`Cephradine
`NaCNBH3
`
`r u
`
`U
`
`NH
`
`C
`
`Figure 5. Preparation and chemical structures of different conjugates of cephradine: (A) attachment of cephradine via the
`N-hydroxysuccinimide active ester, poly(PEG-Lys-OSu), through an amide linkage (arrow); (B) attachment of cephradine via the
`y-aminobutyric acid derivative, poly(PEG-Lys-y-ABA), through an amide linkage (arrow); (C) attachment of cephradine via the
`aldehyde derivative, poly(PEG-Lys-aldehyde), through a secondary amine linkage (arrow). See Experimental Procedures for details.
`
`Biological Properties of Poly(PEG-Lys). The sta-
`bility of 14C-radiolabeled poly(PEG-Lys) over a period of
`
`48 h was determined by incubation in human plasma at
`37 OC, followed by GPC analysis of the polymer molecular
`
`SANOFI-AVENTIS Exhibit 1023 - Page 57
`
`IPR for Patent No. 8,951,962
`
`

`
`so Blooon/agate Chem, Vol. 4. No. 1. 1993
`
`Natmn et al.
`
`Table I. Growth Inhibition by Free Penicillin V and Polymer-Bound Penicillin V
`
`control experiments”
`
`microorganism
`(MIC")
`E. coli (>200)
`K. pneumoniae (>200)
`S. aureus (3)
`E. faecalis (30)
`S. pyogenes (50)
`
`neat
`growth medium
`+
`+
`+
`+
`+
`
`PEG
`+
`+
`+
`+
`+
`
`PEG-Lys
`+
`+
`+
`+
`+
`
`PEG-Lys-spacer
`
`+++++
`
`Free Pen Vb
`at
`10 X MIC
`X
`X
`
`at MIC
`X
`X
`
`poly(PEG-Lys-Pen-V)°
`at
`10 X MIC
`
`at MIC
`
`vllxx
`
`nulxx
`
`“ MIC, the minimum inhibitory concentration of penicillin V in ug/mL, is given in parentheses for each microorganism. ” Growth index:
`+, growth; —, no growth; X, these strains are resistant to penicillin V at 200 pg/mL, the highest concentration tested.
`120
`
`Nolan('5)
`
`100
`
`80
`
`20
`
`60
`
`40
`
`O
`
` 96Doulgrnm
`
` \\\\\\\\\““\\%
`
`L‘ —:¢a:!:_:5
`§§§§;§§
`gggé
`Figure 7. Biodistribution of poly(PEG-“C-Lys) in CD—1 male
`mice.
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`Tlme
`
`(h)
`
`Figure 6. Release of penicillin V from poly(PEG-Lys-penicillin
`V) in physiological phosphate buffer at 25 °C. The amount of
`penicillin V released was determined spectrophotometrically ()\,,,,,,,
`= 260 nm).
`
`weight at predetermined intervals of time. Since the
`distribution of radioactivity in the analyzed aliquots did
`not change, we concluded that the backbone of poly(PEG-
`Lys) is stable in human plasma for at least 48 h. Similarly,
`when poly(PEG-Lys) was incubated in phosphate buffer,
`no change in molecular weight was detected by GPC
`analysis of polymer aliquots extracted from the phosphate
`buffer solution over up to 48 h. These observations are
`in agreement with the previously reported stability of
`aliphatic urethanes toward acidic, basic, and enzymatic
`hydrolysis (28, 29).
`The in vivo distribution of the radiolabeled polymer
`was determined in CD1 male mice in the usual fashion.
`Briefly, a sample of poly(PEG-“C-Lys) was injected and
`the polymer content of selected body fluids and tissues
`was measured at various time intervals by quantitative
`B-counting. These experiments showed that most of the
`radioactivity is located in the circulating blood and is
`uniformly distributed throughout the different tissues
`(Figure 7). Specifically, when the results were calculated
`in terms ofdose per organ, no excess uptake of radioactivity
`by the liver, spleen, or kidney was observed. Excretion of
`the polymer appears to be primarily through the kidney
`and the GI tract.
`
`The parent polymer carrying nonreactive ethyl ester
`pendent chains (1) was subjected to acute toxicity tests in
`mice and rats. Using both iv and ip injections of polymer
`solutions in physiological saline, up to 10 g/ kg body weight
`of 1, were administered. Even at this high dose level, no
`signs of acute toxicity or histopathological changes in liver
`or kidney were observed and polymer 1 was classified as
`nontoxic under the conditions of this test assay.
`
`S ummary and Conclusions. In this paper we describe
`the functionalization of poly(PEG-Lys), a new, water-
`soluble poly(ether urethane). This polymer retains many
`of the favorable properties of PEG, while providing
`multiple attachment sites along the polymeric backbone
`at equal distances from each other. Several pathways for
`the introduction of reactive functional groups onto the
`polymer backbone were developed. The corresponding,
`functionalized derivatives were used for the convenient
`attachment of various ligands, either directly or via an
`appropriate spacer. The resulting conjugates had both
`biologically stable or labile bonds. The distance between
`neighboring attachment sites can be readily controlled by
`the length of the PEG monomer used in the synthesis of
`the poly(PEG-Lys) chain, while the final length of the
`poly(PEG-Lys) chain can be controlled by the polymer-
`ization conditions such as reaction time, concentration of
`monomers, and stirring rate (1 9). The chemical versatility
`of this new class of polymers for preparation of various
`drug conjugates represents a significant advantage over
`previously reported drug-carrier systems. Preliminary
`studies of the acute toxicity, plasma stability and biodis-
`tribution suggest a wide range of potential applications
`for PEG-Lys-derived polymeric drugs.
`
`EXPERIMENTAL PROCEDURES
`
`Materials and Methods. Cephradine and penicillin
`V were obtained from Sigma; [“C]lysine was from NEN
`Research Products; PEG-2000 and phosgene solution
`(20%) in toluene (Fluka) were used for preparation of
`BSC-PEG according to the previously published procedure
`(19-21). Additional chemicals from common commercial
`sources were used without purification. HPLC-grade
`solvents were used for synthesis and for GPC. ‘H and “C
`NMR spectra were recorded on a Varian XL-200 spec-
`trometer at room temperature using CDCI3 and D20 as
`solvents. FT-IR spectra were recorded on a Mattson
`Cygnus 100 spectrophotometer. UV/vis spectra were
`recorded on a Perkin-Elmer Lambda 3B spectrophotom-
`eter. GPC (relative to PEG standards) (30) was performed
`using an HPLC system consisting of a Perkin-Elmer series
`
`SANOFI-AVENTIS Exhibit 1023 - Page 58
`IPR for Patent No. 8,951,962
`
`

`
`Copolymers of Lysine and Polyethylene Glycol
`410 LC pump, a Waters 410 RI detector, and a Perkin
`Elmer DEC 3000 data station. DMF containing 0.1 % (w/
`v) of LiBr was used as the mobile phase, at a flow rate of
`1 mL/min on two PL-gel columns (300 mm X 7.7 mm;
`particle size, 5 pm; pore size, lo3 and lo5 A, respectively)
`connected in series. For aqueous GPC, 0.1 M acetate buffer
`(pH 5.4) was used as the mobile phase at a flow rate of 0.7
`mL/min and TSK Gel-2000 and -4000 columns were used
`in series. The amount of free carboxylic acid groups in
`poly(PEG-Lys) was determined by nonaqueous titration
`with sodium methoxide in methanol/ toluene using thymol
`blue as the indicator. The amount of active ester in poly-
`(PEG-Lys-OSu) was determined by a modification of the
`published titration method (22). @-Lactam content in the
`polymer-antibiotic conjugates was determined by iodo-
`metric titrations as described by Alicino (27). Amino acid
`analysis was performed according to the procedure of
`Meltzer (31). Dialysis against distilled water using a
`Spectrapor membrane with molecular weight cutoff of
`12000-14000 was routinely used for removal of low
`molecular weight compounds, including free drugs, from
`polymer conjugates.
`Poly(PEG-Lys-OEt) (1) was prepared by interfacial
`polymerization (methylene chloride/aqueous solution at
`pH 8) as described in detail elsewhere (19).
`Preparation of Poly(PEG-Lys) (2). L-Lysine (0.032
`g, 0.22 mmol) was dissolved in 5 mL of 0.1 M borate buffer
`(pH = 9.3). To this solution was added BSC-PEG (0.5 g,
`0.22 mequiv). The reaction mixture was stirred at room
`temperature for 15 min and then dialyzed against distilled
`water. The product was recovered by lyophilization to
`yield 0.36 g (72%). M, = 47 000 Da, was determined by
`GPC (in 0.1 M acetate buffer, TSK columns, relative to
`PEG standards). FT-IR (film on NaC1, cm-'1: v 2870 (CH),
`1719 (C=O of urethane), shoulder at 1740 (C=O of acid),
`1105 ((2-0). 'H NMR (DzO): 6 4.06 (4 H, t, terminal
`CH2 of PEG), 3.56 (173 H, PEG overlapping with
`a-CHNH), 2.98 (2 H, m, c-CH~NH), 1.15-1.76 (6 H, br m,
`CH2 of Lys) ppm. l3C NMR (CDC13): 6 173.45 (C=O of
`acid), 156.50 (C=O of a-NH urethane), 155.93 (C=O of
`e-NH urethane), 63.90-70.48 (CH2 of PEG), 53.44, 40.39,
`31.91, 29.17, 21.98 (CH2 of Lys) ppm. An identical
`procedure was used for the preparation of 14C-labeled poly-
`(PEG-Lys) using 14C-Lys (250 pCi in 0.032 g). Alterna-
`tively 2 was obtained by mild alkaline hydrolysis of 1 (19).
`Preparation of Poly(PEG-Lys-Gly-OEt) (3). Poly-
`(PEG-Lys) (0.4 g, 0.18 mequiv) was dissolved in HzO (40
`mL). To this solution was added Gly-OEt-HC1 (5.58 g,
`0.04 mol). The pH was adjusted to 4.8 and EDC (0.77 g,
`4.0 mmol) was added. The pH was maintained at 4.8 by
`the addition of 0.1 N HC1. After 3 h the reaction mixture
`was acidified with 0.1 N HC1 to pH 4 and the product was
`extracted with methylene chloride (3 X 50 mL). The
`combined extracts were washed with saturated brine, dried
`over anhydrous MgSO4, concentrated to 10 mL, and poured
`into cold ether (50 mL). The polymer was collected by
`filtration, washed with cold ether, and dried in vacuo. It
`was further purified by dialysis followed by lyophilization.
`Yield: 0.3 g (73%). 1H NMR (CDCl3): similar to that of
`poly(PEG-Lys) except that additional absorptions at 6
`1.25 (3 H, t, CH3), 3.98 (2 H, d, CH2 of glycine), 4.09-4.27
`(6 H, terminal CH2 of PEG + CHZCH~), 6.76 (1 H, m,
`CONHCH2) ppm were observed. Amino acid analysis
`[according to the procedure of Meltzer (31)l of the product
`showed a lysine to glycine ratio of 1:0.95, indicating that
`95% of the side chains had reacted with Gly-OEt.
`Preparation of Poly(PEG-Lys-ethanolamide) (4).
`Poly(PEG-Lys) (1 g, 0.45 mequiv) was dissolved in
`
`Bioconjugate Chem., Vol. 4, No. 1, 1993 59
`methylene chloride (10 mL). To this solution was added
`triethylamine (0.09 mL, 0.68mmol). The flask was cooled
`in an ice-water bath and pivaloyl chloride (0.08 g, 0.68
`mmol) was added. The reaction mixture was stirred for
`2 h and cooled again to 0 "C and dry ethanolamine (0.08
`g, 1.36 mmol) was added. After stirring for 24 h,
`triethylamine hydrochloride was removed by filtration and
`the filtrate was added into ether (50 mL). The crude
`material was collected by filtration and purified by
`recrystallization from 2-propanol. Yield: 0.82 g (80.5 % ).
`FT-IR (film on NaC1, cm-l): v 2888 (CH), 1720 (C-0 of
`urethane), 1671 (C=O of amide), 1109 (C-0).
`lH NMR
`(CDC13): 6 6.96 (1 H, m, CONH), 5.76 (1 H, d, a-NH of
`Lys), 5.25 (1 H, m, c-NH of Lys), 4.17 (4 H, terminal CH2
`of PEG), 3.61 (175 H, PEG overlapping with a-CHNH
`and HCHzOH), 3.37 (2 H, m, CHzNHCO), 3.12 (2 H, m,
`e-CHzNH), 1.25-1.91 (6 H, br m, CH2 of Lys) ppm.
`Preparation of Poly(PEG-Lys-ethylenediamine)
`(5). Poly(PEG-Lys) (0.8 g, 0.36 mequiv) was dissolved in
`methylene chloride (15 mL). To this solution was added
`triethylamine (0.08 mL, 0.63 mmol). The flask was cooled
`in an ice-water bath and pivaloyl chloride (0.074 g, 0.63
`mmol) was added. The reaction mixture was stirred for
`2 h, cooled again to 0 "C and added to dry ethylenediamine
`(0.216 g, 3.6 mmol) in methylene chloride (5 mL) cooled
`in an ice bath. After stirring for 24 h, triethylamine
`hydrochloride was removed by filtration and the filtrate
`was concentrated and added into ether (50 mL). After
`stirring for 2 h, the ether layer was decanted and fresh
`ether was added to remove unreacted impurities. The
`precipitate was then collected by filtration and further
`purified by recrystallization from 2-propanol. Yield: 0.61
`g (75%). FT-IR (film on NaC1, cm-l): v 2888 (CH), 1720
`(C=O of urethane), 1672 (C=O of amide), 1109 (C-0).
`lH NMR (CDC13): 6 7.15 (1 H, m, CONH), 5.75 (1 H, d,
`a-NH of Lys), 5.37 (1 H, m, c-NH of Lye), 4.13 (4 H,
`terminalCH2 of PEG), 3.58 (173 H, PEG overlapping with
`a-CH-NH), 3.30 (2 H, m, CHZNHCO), 3.10 (2 H, m, E-CHZ-
`NH), 2.75 (2 H, m, CHZNHZ), 1.21-1.91 (6 H, br m, CH2
`of Lys) ppm.
`Preparation of poly(PEG-Lys-OSu) (6) was as pre-
`viously reported (19).
`Preparation of Poly(PEG-Lys-r ABA) (7). y-Amino
`butyric acid (0.25 g, 2.43 mmol) was dissolved in phosphate
`buffer (10 mL, pH = 7.4). To this solution was added 6
`(0.98 g, 0.43 mequiv). The reaction mixture was stirred
`at room temperature for 0.5 h and dialyzed against distilled
`water. The product was isolated by lyophilization. TLC
`(toluene-acetic acid-water 5:5:1) showed absence of free
`y-ABA (visualization using ninhydrin). Yield: 0.85 g
`(85%). 'H NMR (D20): 6 4.07 (4 H, t, terminal CH2 of
`PEG), 3.57 (173 H, PEG overlapping with a-CHNH), 2.91-
`3.10 (4 H, m, c-CH2NH overlappingwithCH2NHCO), 1.12-
`1.82 (8 H, br m, CH2 of Lys overlapping with CHZCHZ-
`COOH) ppm.
`Preparation of Poly(PEG-Lys-aminopropanediol)
`(8). Poly(PEG-Lys-OSu) (2 g, 0.87 mequiv) was dissolved
`in methylene chloride (20 mL). To this solution was added
`3-amino-1,2-propanediol(O.29 g, 4.36 mmol). The reaction
`mixture was stirred at room temperature for 2 h and the
`product precipitated in ether (100 mL). After cooling to
`4 OC the crude product was collected by filtration, dissolved
`in HzO, and purified further by dialysis. The product was
`recovered by lyophilization. Yield: 1.7 g (80%). FT-IR
`(filmonNaC1,cm-9: v2885 (CH),1719 (Mofurethane),
`1674 (C=O of amide), 1110 (C-0).
`'H NMR (CDCl3):
`6 7.14 (1 H, m, CONH), 5.80 (1 H, d, a-NH of Lys), 5.26
`(1 H, m, c-NH of Lys), 4.16 (4 H, terminal CH2 of PEG),
`
`SANOFI-AVENTIS Exhibit 1023 - Page 59
`
`IPR for Patent No. 8,951,962
`
`

`
`60 Bioconlugete Chem., Vol. 4, No. 1, 1993
`3.60 (176 H, PEG overlapping with a-CHNH, CHOH, and
`CHzOH), 3.37 (2 H, m, CHzNHCO), 3.13 (2 H, m, e-CH2-
`NH), 1.26-1.94 (6 H, br m, CH2 of Lys) ppm.
`Preparation of Poly(PEG-Lys-aldehyde) (9). Poly-
`(PEG-Lys-aminopropanediol) (1.2 g, 0.53 mequiv) was
`dissolved in water (15 mL) and treated for 1 h with sodium
`periodate (0.11 g, 0.53 mmol) at 4 "C, in the dark. The
`reaction mixture was dialyzed against distilled water. The
`aldehyde content of the product determined with 2,4-
`dinitrophenylhydrazine (24) was 0.32 mmol/g, corre-
`sponding to 68% of the maximal theoretical value. The
`aqueous solution of the product was stored at 4 "C and
`used directly in conjugation reactions.
`Preparation of Poly(PEG-Lys-penicillin V) (10).
`Penicillin V (0.094 g, 0.267 mmol) and 4-(dimethylamino)-
`pyridine (0.008 g, 0.065 mmol) were added to a solution
`of 4 (0.400 g, 0.178 mequiv) in methylene chloride (4 mL).
`The solution was cooled and treated with DCC (0.048 g,
`0.232 mmol). The reaction mixture was stirred at 4 "C,
`for 4 days. The DCU was removed by filtration and the
`product precipitated with cold ether. The crude product
`(0.25 g) was further purified on a Sephadex LH-20 column
`using methanol as the eluant. TLC (methanol) showed
`absence of free drug in the product. FT-IR (film on NaC1,
`cm-l): u 2882 (CH), 1785 (C=O of &lactam), 1706 (C=O
`of urethane), shoulder at 1630 (C=O of amide), 1110
`lH NMR (CDC13): 6 6.82-7.1 (3 H, m, meta and
`(C-0).
`para H's of the phenyl group), 7.2-7.4 (2 H, m, ortho H's
`of the phenyl group), 5.59 (1 H, m, COCHNH), 5.41 (1 H,
`d, a-NH of Lys), 5.3 (1 H, d, CHNH), 5.09 (1 H, m, e-NH
`of LYS), 4.53 (2 H, 8, OCH&O), 4.45 (1 H, 8, CHCOOH),
`4.16 (4 H, t, terminal CHZ of PEG), 3.61 (173 H, PEG
`overlapping with a-CH-NH), 3.28 (2 H, m, CHzNHCO),
`3.15 (2 H, m, c-CH~NH), 1.31-1.91 (12 H, br peak due to
`CH2 of Lys) overlapping with 1.46 and 1.49 (2 s, CH3 of
`Pen V) ppm. Anal. Calcd for poly (PEG-Lys-penicillin
`V): S, 1.15 (for drug attachment to 100% of the side
`chains). Found S, 0.87 (corresponding to drug attachment
`to 66% of the side chains).
`Preparation of Poly(PEG-Lys-cephradine) (1 1).
`Cephradine (0.15 g, 0.43 mmol) was dissolved in a mixture
`of water (4.5 mL) and dioxane (2 mL). To this solution
`was added 6 (0.50 g, 0.22 mequiv) followed by a dropwise
`addition of NaHC03 solution (0.055 g in 1 mL of H20).
`
`The solution (pH - 7.5) was stirred at 25 "C for 1 h,
`acidified to pH - 6.5 and the product was extracted with
`
`methylene chloride (3 X 25 mL). The extracts were
`combined and washed with brine, dried (MgSO,), and
`concentrated to 10 mL. The concentrated solution was
`added slowly into cold ether (50 mL), causing the drug-
`polymer conjugate to precipitate. After cooling to 4 "C
`for several hours the product was collected by filtration,
`washed with cold ether, and dried in vacuo, yielding 0.36
`g (71%). The product was further purified by dialysis
`against distilled water and recovered by lyophilization (0.27
`g). TLC (methanol) showed absence of free drug (visu-
`alization using UV and ninhydrin). FT-IR (film on NaC1,
`cm-l): v 2882 (CH), 1782 (C=O of 8-lactam), 1719 (C=O
`of urethane), shoulder at 1670 (C=O of both amides),
`lH NMR (DzO): 6 5.75 (1 H, m, CH=C),