Poly(ester amide) - American Chemical Society

Chapter 2

Downloaded by COLUMBIA UNIV on August 5, 2012 | http://pubs.acs.org Publication Date: March 28, 2008 | doi: 10.1021/bk-2008-0977.ch002

From Drug-Eluting Stents to Biopharmaceuticals: Poly(ester amide) a Versatile New Bioabsorbable Biopolymer 1

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Zaza Gomurashvili , Huashi Zhang , Jane Da , Turner D. Jenkins , Jonathan Hughes , Mark Wu , Leanne Lambert , Kathryn A. Grako , Kristin M . DeFife , Kassandra MacPherson , Vassil Vassilev , Ramaz Katsarava , and William G. Turnell 1

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MediVas L L C , 6275 Nancy Ridge Drive, San Diego, CA 92121 Center for Medical Polymers and Biomaterials, Georgian Technical University, 69, Kostava Strasse, 0175 Tbilisi, Georgia

New biodegradable and tissue-resorbable co-poly(ester amides) (PEAs) useful for biomedical applications were prepared using a versatile Active PolyCondensation (APC) method, which involves di-p-toluenesulfonic acid salts of bis(L-α-amino acid)-α,ω-alkylene diesters and active diesters of dicarboxylic acids as monomers. A P C reactions were carried out at mild temperatures (40-60°C) and allowed the synthesis of regular, linear, polyfunctional PEAs with high molecular weights. The physical properties of PEAs are critically dependent upon the structure of the polymer backbone. A wide range of mechanical properties and biodegradation rates can be achieved by varying the three components in the backbone: α-amino acids, diols and dicarboxylic acids. Indeed, there is a growing need for wider variations of biocompatible P E A compositions and methods for the delivery of different therapeutic molecules at controlled rates, while affording enhanced mechanical and physical properties. Therefore, various types of new non-toxic building blocks based upon bulky diols (isosorbide, 17β-estradiol), unsaturated (fumaric) and aromatic diacids (hydroxycinnamic acid, 1,3-bis(4carboxyphenoxy)-propane) were developed and successfully 10

© 2008 American Chemical Society In Polymers for Biomedical Applications; Mahapatro, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

11 incorporated into the main backbone of PEA. In vitro biodegradation tests with enzymes have shown that changes in the polymer backbone and functional groups resulted in a wide range of degradation rates that further exemplify the usefulness of these compositions in biomedical applications.


Introduction a-Amino acids-based poly(ester-amide)s, (PEAs), belong to the class of AABB type heterochain polymers. This relatively new class of amino acid based PEAs have been extensively studied (1-4) because they combine favorable properties of both polyesters and polyamides, such as good thermal stability, tensile strength and modulus, increased hydrophilicity, biodegradation and biocompatibility. The amino acid rich compositions give these polymers natural protein-like qualities, resulting in a high capacity for hydrogen bonding between polymer chains, between polymer and loaded therapeutic, and/or between polymer and water. Entirely composed of nontoxic building blocks, such as essential a-amino acids, aliphatic dicarboxylic acids and aliphatic a,co-diols, AABB type PEAs have proved to be promising biodegradable materials for various biomedical applications. PEAs have been successfully tested in animals and humans for cardiovascular applications (5) and ex-vivo cell based assays have strongly supported recent human trial data indicating that PEAs are blood and tissue compatible, with advantageous properties for implantation (6). The PEAs reported in this work were prepared in a simple way by solution or interfacial polycondensation, where di-p-toluenesulfonic acid salts of bis-(aamino acid)-a,co-alkylene diesters react with chlorides of dicarboxylic acids (interfacial polycondensation) or their active diesters (Active Polycondensation, APC). The APC method involves the condensation of two partners: (I) biselectrophilic, activated dicarboxylic acids, and (II) bis-nucleophilic, acid salts of bis-(a-amino acid)-a,co-alkylene diesters in combination with di-ptoluenesulfonic acid salts of L-lysine benzyl ester. This reaction proceeds under mild conditions in common organic solvents and leads to polymer of high molecular weight (up to 300 KDa). A detailed review of the APC method has been recently summarized by Katsarava (7). In addition to the functionalizable end-groups, assuming one amino and one carboxylic (or p-nitrophenyl ester) end group, the PEA polymer backbone can accommodate pendant functional groups (charged or uncharged, but polar) introduced on multifunctional monomers, for example C-protected L-lysine, that are useful for covalent conjugation of biologically active compounds. Novel polyelectrolyte type PEAs with polar pending groups along the chain will significantly change the physico-chemical and biological properties of the polymer, such as the ability to diffuse through cell membranes.

In Polymers for Biomedical Applications; Mahapatro, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


12 The tremendous value that PEAs have in the health science industry relates directly to the polymers' compatibility in biological systems and the mechanism by which they degrade and loaded drugs are released. PEA's mechanism of biodegradation and drug release is by surface erosion and primarily follows zero order kinetics (8,9). These unique material properties have shown that PEAs have a multiuse potential as a new family of biodegradable biomaterials useful as drug delivery platforms or as components of resorbable surgical implants. Extensive research at MediVas has lead to a vast assortment of PEA copolymers confirming that the APC method has virtually unlimited possibilities for designing new polymeric materials with a wide variety of mechanical, physico-chemical and biological properties that are easily achieved by varying the three component building blocks. In addition, because these polymers have a significant number of lateral functional groups, fiinctionalized PEAs can be subjected to subsequent chemical attachment of bioactive agents, thereby making this group of polymers effective for stabilizing biologies in addition to serving as platforms for drug delivery. These new polyfiinctional non-toxic, bio compatible compounds with improved mechanical properties are suitable for specific clinical applications, such as coating materials for drug-eluting stents, stabilization and oral delivery of biologies, targeted delivery of drugs to the front and back of the eye, topical or transdermal drug delivery, and therapeutic wound dressings for surgical procedures.

Experimental Materials All starting materials and solvents were obtained from commercial sources (Sigma-Aldrich, Fisher Scientific Int.). Active di-esters (of Formula I): di-pnitrophenyl adipate, 1.1 (I), di-p-nitrophenyl sebacate, 1.2 (I), di-p-nitrophenyl fumarate, 1.3 (10), and bis-nucleophilic monomers: (of general Formula II) dip-toluenesulfonic acid salts of bis(a-aminoacyl)-ot,co-alkylene diesters (1, 2), and di-p-toluenesulfonic acid salt of L-lysine benzyl ester (11,12) were prepared as described previously.

Monomer Synthesis Preparation of Di-/?-Nitrophenyl propane (1.4)

Ester of l,3-Bis(4-carboxyphenoxy)

Precursor diacid l,3-bis(4-carboxyphenoxy) propane (CPP) was prepared by refluxing 2.1 mol. eq. of 4-hydroxybenzoic acid with 1 mol. eq. of 1,3-


13 dibromopropane in alkaline water solution for 8-12 h. Solution was then poured into 700 mL of 1M HC1 and filtered. An off-white solid obtained was suspended in water/ethanol 1:1 mixture, re-filtered and dried under vacuum which gave 62 % yield. Product was recrystallizedfromDMF:water, (1:1 v/v), mp. 310 °C. ,

0 N--NO;2 J

(1.4)

7.9 g. (25 mmol) of CPP, 7.3 g (52.5 mmol) of p-nitrophenol and a few drops of DMF were suspended in 100 mL of dry chlorobenzene under nitrogen. Then a diluted solution of 4 mL (54.8 mmol) thionyl chloride in 10 mL of chlorobenzene was added drop-wise to reaction mixture at ambient temperature for 20 min, heated to 75°C while a slow stream of nitrogen was introduced to evacuate formed gases. After 8 h, the reaction mixture became homogenous. The cooled solution was diluted with 120 mL hexane and left over night at 0°C. Yellow crystals of compound 1.4 were collected, washed with hexane and dried in vacuum over night at 45°C. Yield was 10.3 g, (74 %.). Recrystallization from acetone yielded pale yellow crystals, mp 161.6°C; U NMR, (DMSO-d , 500 MHz) 5: 8.33 (d, 4H, Ar), 8.10 (d, 4H, Ar), 7.58 (d, 4H, Ar), 7.17 (d, 4H, Ar), 4.29 (t, 4H, -0-CH -), 2.27 (m, 2H, -CH -CH -). l

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Preparation of Di-p-Toluenesulfonic Acid Salts of Bis-(oc-amino acid)-a,coAlkylene Diester Monomer (Formula II) General procedure: 2 eq. of a-amino acid and 1 eq. of diol were suspended and refluxed in toluene in the presence of 2.2 eq. of TsOH monohydrate, with the resulting water seized in a Dean-Stark condenser. Monomers were thoroughly purified by multiple recrystallizations (1).

Preparation of (Compound 11.5)

Di-TFA

Salt

of

Bis-(L-Leu)-estradiol-3,17p-diester

bis(Boc-L-leucine)estradiol-3,17p-diester, (IL5): 1.5 g (5.51 mmol) of 17,p-estradiol, 3.43 g (13.77 mmol) Boc-L-leucine monohydrate and 0.055 g (0.28 mmol) of p-toluenesulfonic acid monohydrate were dissolved into 20 mL of dry N,N-dimethylformamide at room temperature under argon; 0.067 g of DMAP and 5.4g (26.17 mmol) of DCC were introduced and stirring was continued for 6 h. At the end of reaction, 1 mL of acetic acid was added to destroy the excess of DCC. Precipitated urea was filtered off and filtrate poured into 80 mL of water. Product was extracted three times with 30 mL of ethylacetate, dried over sodium sulfate, solvent evaporated, and remainings


14 subjected to chromatography on a column (7:3 hexanes: ethylacetate). A colorless glassy solid of analytically pure compound (II.5a) obtained a 2.85 g, 74% yield. De-protection of (II.5a) Boc-protected monomer proceeded quantitatively in 10 mL of dry dichloromethane and 4 mL of dry TFA. After 2 h of stirring at room temperature, a homogenous solution was diluted with 300 mL of anhydrous ether and left at 0°C over night. White crystals were collected, washed twice with ether, and dried in a vacuum oven at 45 °C. Yield 2.67 g, (90%). Mp= 187.5°C.


Co-PEA Synthesis by Solution Active Polycondensation This method involves equimolar amounts of (I) and (II), and organic base as the acceptor of liberated p-toluenesulfonic acid. Polymers with high molecular weights, and yields frorp 68 to 95% (depending on mol. weights) were characterized by GPC, H NMR spectroscopy and DSC. Their enzymatic hydrolysis (lipase, a- chymotrypsin) was studied. A general procedure is described for preparation of copolymer 4-(Leu-6) .5oLys(Bz) .5o-. A 0.1 mole of a bis-nucleophile monomer II (here, a mixture of 0.05 mol. of L-Leu-6 and 0.05 mol. of II.3), 0.1 mol of di-p-nitrophenyl adipate (monomer I) stirred in 52 mL of N,N-dimethylformamide (DMF) at 60°C and dry triethylamine (NEt ) added (30.8 mL, 0.22 mol.) corresponded to total monomer concentration 1.2 mol/L. The reaction was conducted over 24 h and crude samples monitored by GPC. The purification of co-PEAs was achieved by multiple precipitation from solvent (ethanol, dichloromethane) into non-solvent (water or cold ethylacetate) until no /?-nitrophenol was detected in the UV spectrum. New co-PEAs were labeled in the following way: y-IAA-xJm -[AA-x j , where AA stands for amino acid, y, diacid fragment (or number of methylene group in diacid) in monomer I; and x is the diol fragment (or the number of methylene group in linear aliphatic diol) in monomer II. Therefore, in the above example polymer is composed of adipic acid, bis-(L-Leu)-l,6-hexanediol diester and benzylated L-Lysine. Scheme 3 shows the chemical structure of some representative ^o-PEAs synthesized. Due to the equal reactivity of diamines, integration of H NMR signals of copolymers display in most cases exactly the same monomer feed ratios as in the starting mixture. 0

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Deprotection of Polymeric Benzyl Esters Catalytic hydrogenolysis of co-PEA(Bz) was conducted under dry H in combination with Pd/C catalyst as described (13). The deprotected co-PEA was designated co-PEA(H). The covalent conjugation of nitroxyl radicals (4-amino2,2,6,6-tetramethylpiperidine-l-oxyl) onto co-PEA(H) was performed according to the published and patented method (Chu et al.,12,13). 2


15 In Vitro Enzyme Catalyzed Biodegradation Studies


Biodegradation studies by weight loss were performed for four PEAs (fig 2). Circular PEA films were cast (ethanol) on stainless steel discs of 11 mm diameter with ca. 16.8 mg film weight on each. Films were placed into glass vessels containing 2 mL of Dulbecco's phosphate buffered saline solution at pH 7.0 containing 8.5 U/mL concentration of enzyme (oc-chymotrypsin). Glass vessels were maintained at 37°C and gently shaken. Enzyme solution was renewed twice weekly. Discs were taken out at 7, 14, 21 and 28 day time points, washed three times with DI water and dried up to constant weight at 50°C (about another 24 hr). Weight loss (%) was calculated as: % weight loss = [polymer start weight - polymer end weight] / [polymer starting weight x 100] Comparison of Endothelial Cell Viability in the Presence of Monocytemediated Co-PEA and Therapeutic PEA Degradation Products PEA monocyte degradation experiments were conducted as follows: polymer film was cast into glass wells, dried and sterilized. Freshly isolated human monocytes were plated into the polymer coated wells (2xl0 cells per well) in 2 mL of cell growth media (RPM1 media with autologous serum). Plates were placed at 37°C in a C 0 incubator and media replaced every third day for two weeks. The collected media with degradation products (PEA 8-Leu(6) 5Lys(Bz) .25 or co-PEA 17(J supernatants) were used to grow human coronary artery endothelial cells (ECs). ATP assays were conducted using a ViaLight Plus assay kit (Cambrex) at 24 and 48 hour time points to determine the percent viability of the ECs grown in the two polymer degradation product supernatants. 6

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Results and Discussion Monomer Synthesis The following amino acids were selected for new co-PEA designs: L-Lys for the purpose of incorporating free lateral carboxylate groups into the polymeric backbones; L-Leu - for imparting elastomeric properties, based upon prior studies (1,4,8)', and L-Phe for enhancing mechanical properties of the resulting PEAs. Amino acid-based bis-nucleophilic monomers (II) are represented in scheme 1.


16 O O HOTos.H N-HC-C-0-R -0-C-CH--NH2.TosOH R R 2

(II)

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where R = (CH ) , designated as "3", for 1,3-propanediol (CH ) , designated as "6", for 1,6-hexanediol Isosorbide, designated as DAS; 2

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R = CH CH(CH ) (Leu, L-leucine) CH Ph (Phe, L-Phenylalanine) 3


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HOTos.H N-CH-(CH ) -NH .TosOH ; 2

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COOCH C H 2

( Lys(Bz))

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CT t)H Isosorbide

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Scheme 1 Other bis-electrophilic monomers prepared have the general structure shown in scheme 2, where y represents a dicarboxylic acid.

o

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N

- 0 "

o

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y

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~ ^ ~ 0 " Scheme 2

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Synthesis of PEA Copolymers The co-PEAs were synthesized by solution polycondensation with equal molar amounts of diamine salts II and active diesters I in an amide type solvent (DMA) under optimal reaction conditions previously described for homo-PEAs (1). The close chemical nature of the salts II and di-p-nitrophenyl esters I easily allowed us to carry out the polycondensation with combinations of multiple monomers (minimum three), which resulted in copolymers with randomly distributed components with the same ratio as in the starting reaction mixture. Synthesized co-PEAs achieved high yields (up to 90 %) and high weight average molecular weights from 85 000 Da to 300 000 Da. Complete incorporation of monomers into polymer backbone was confirmed by *H NMR spectroscopy, which showed the same unit content in the main backbone as in the starting reaction mixture. The co-PEAs obtained have a reduced viscosity (^ ) as high as 1.50 dL/g (8-Ieu(6) .5o - lys(Bz) o) and excellent film-forming properties, an indication of relatively high molecular weights of the co-PEAs synthesized. red

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17 Polydispersity indices estimated by GPC ranged from 1.24 to 1.9, which is typical for PEAs prepared by active polycondensation (1,2). Sample copolymer structures are depicted in scheme 3. Mechanical Properties of the Co-PEAs Table I shows tensile properties of selected co-PEAs, all containing Lys(Bz) units at 25% of total di-amine monomers. The tensile property data showed that 8-Leu(6)o. -Lys(Bz) .25 was the weakest of the co-PEA samples tested. Decrease in the length (y) of polymethylene groups in the diacid monomer I, or the incorporation of phenylalanine increased the tensile strength of the resulting co-PEAs. Following a general trend, shortening the polymethylene chain in the diol or diacid caused a decrease of film elasticity and increased the Young's modulus (2.5 GPa). The tensile strength can be further increased by processing conditions that induce molecular orientation Introduction of bulky aliphatic fragments of isosorbide contributed significantly to a higher Tg and an increased Young's modulus whilst maintaining biocompatibility and solubility in ethanol. From the data in Table I, we observe that the monomer bis(L-Leu)-isosorbide-diester (Leu(DAS)) contributed higher Tg; whereas the monomer bis(L-Leu)-hexanediol-diester, (Leu(6)) led to increased elongation. Incorporation of both monomers in various feed ratios resulted in new PEAs with different combinations of mechanical properties and glass transitions (Figure 1).


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In Vitro Enzyme Catalyzed Biodegradation The effect of co-PEA composition on enzymatic biodegradation was studied in enzyme phosphate buffer solution. Figure 2 summarizes lipase-catalyzed weight loss data from four different types of co-PEA films: a) 4-Leu(6) 5Lys(Bz) . , b) 8-Leu(6)o.75-Lys(Bz)o. , c) 4-Leu-(6)o.5-Phe(6)o. -Lys(Bz) . , d) 8-Leu(6)o.5-Phe(6)o. 5-Lys(Bz) . 5. There was virtually no weight loss in the PBS control during the same testing period. Several of these co-PEAs show close to zero order kinetics, a property important for sustained and controlled release of drugs. As illustrated in Figure 2, an increase of polymethylene chain in diacid y from 4 (samples a and c) to 8 (samples b and d) significantly decreased weight loss rates. The effect of ct-amino acids on enzyme-catalyzed biodegradation is evident when comparing sample a with sample c, or sample b with sample d: The replacement of the L-leucine component with L-phenylalanine led to a sharp increase in weight loss, particularly in the shorter y acids (samples a and c). The increase in the biodegradation rate due to the L-Phe is consistent with the data of previous studies (1,8,9). The solution viscosity (77^) measurements before and after 120 h of biodegradation showed no changes, an indication of a surface erosion mechanism, without compromising bulk properties (8,9). 07

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0 25


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a75

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02S

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- Leu(6)

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1 5

II

O

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O 2

1.5"

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0 5

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r

o

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o i

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CH CH(CH )2

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O

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NH-

— \

/

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Poly(ester amide) - American Chemical Society

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