Synthesis and in vitro Antimicrobial Activity of Chloramphenicol- Conjugated Copolymers

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  • http://jbc.sagepub.com/Compatible Polymers

    Journal of Bioactive and

    http://jbc.sagepub.com/content/5/3/283The online version of this article can be found at:

    DOI: 10.1177/088391159000500304

    1990 5: 283Journal of Bioactive and Compatible PolymersMark D. Franko, James E. Gates and Raphael M. Ottenbrite

    Conjugated CopolymersSynthesis and in vitro Antimicrobial Activity of Chloramphenicol-

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    Synthesis and in vitro AntimicrobialActivity of Chloramphenicol-

    Conjugated Copolymers

    MARK D. FRANKO AND JAMES E. GATES*

    Department of BiologyVirginia Commonwealth University

    Richmond, VA 23284, USA

    RAPHAEL M. OTTENBRITE

    Department of ChemistryVirginia Commonwealth University

    Richmond, VA 23284, USA

    ABSTRACT: Two copolymers of maleic anhydride, 10-undecendyl chloride-co-maleic anhydride and acryloyl chloride-co-maleic anhydride, were synthesized,esterified with chloramphenicol, and tested for antimicrobial activity viaKirby-Bauer disk sensitivity and broth dilution methods. Both polymer-drugconjugates exhibited antimicrobial activities of at least 10% of that exhibitedby unconjugated chloramphenicol. As maleic anhydride copolymers are takenup selectively by macrophages, they should be effective in concentrating the an-tibiotic in these cells allowing for its use at lower dosage. Such agents mightprove effective in treating diseases such as legionellosis with fewer toxic side-effects such as aplastic anemia.

    INTRODUCTION

    Antibiotics and other chemotherapeutics have been instrumentalAin the treatment of microbial infection. Generally, such agents aregiven systemically although the pathogen may be localized in specificcells or tissues. Legionella pneumophila, Mycobacterium tuberculosis

    *Please correspond with: James E Gates, Department of Biology, 816 Park Ave.,Richmond, VA 23284 (804) 367-1562

    Journal of BIOACTIVE AND COMPATIBLE POLYMERS, Vol 5-July 1990

    0883-9115/90/03 0283-10 $4.50/0@ 1990 Technomic Pubhshmg Co., Inc.

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    and Chlamydia psittaci are a few examples of pathogens which in thecourse of infection become intracellular parasites of macrophages [1]. Ifchemotherapeutics could be targeted to those infected cells, it is likelythat their effectiveness could be increased and toxic side-effects couldbe reduced.Several studies have demonstrated that polyanions such as maleic

    anhydride copolymers are taken up by and stimulate macrophagesthereby enhancing the immune response [2-8]. By attaching an appro-priate chemotherapeutic to a maleic anhydride copolymer it may bepossible to deliver the chemotherapeutic preferentially to macrophagesto aid in the destruction of pathogens taken up by these phagocyticcells. Additional applications include cross-linking the copolymer afterattachment of a chemotherapeutic to form a gel to be used as a topicalwound dressing. For example, chloramphenicol has been attached toBiozan-R and used as an ophthalmic ointment; the antibiotic-conjugatewas less irritating and more water soluble [9].Although a number of maleic anhydride copolymers exist already,

    none would be expected to readily add chloramphenicol without consid-erable deactivation of the antibiotic [10-13]. Copolymers of maleicanhydride with 10-undecendyl chloride (UCMA) and acryloyl chloride(ACMA) were therefore synthesized. As greater activity has beenobserved when the pharmacon is not attached directly to the backboneof the polymer, these differed in the length of the side-arm linking theantibiotic to the polymer backbone (Figure 1). They both contain anacid chloride group which reacted readily with the primary alcohol ofchloramphenicol forming an ester linkage. These copolymer-chlor-amphenicol conjugates were tested to determine if the antibiotic re-tained its in vitro antimicrobial activity.

    MATERIALS AND METHODS

    Reagents

    All reagents were obtained from Aldrich Chemical Co. except for 10-undecendyl chloride which was obtained from Fluka Chemical Co.They were purified further as follows: pyridine, acryloyl chloride, and10-undecendyl chloride were distilled under reduced pressure; maleicanhydride was recrystallized from chloroform; acetone was dried overK2C03, decanted and distilled with a small amount of permanganate;the azobisisobutylnitrile (AIBN) was recrystallized from methanol.Chloramphenicol, anhydrous ethyl ether and chloroform were used asreceived. Bacteria and culture media were obtained from Difco.

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    Figure 1. Structure of 10-undecendyl chloride-co-maleic anhydride (UCMA) and acryloyl Ichloride-co-maleic anhydride (ACMA).

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    Preparation of 10-Undecendyl Chloride-co-Maleic Anhydride (UCMA)and Acryloyl Chloride-co-Maleic Anhydride (ACMA)

    Maleic anhydride (10 g, 0.1 Mol) and a mole equivalent of 10-undecendyl chloride (21.9 mL) were dissolved in 34 mL acetone to forma 90% (w/v) concentration, to which 0.6 g AIBN initiator were added.Dry N2 was bubbled into this solution for 10-15 min, after which thepolymerization flask was sealed and placed in a constant temperaturebath at 75 C for 17 h. The UCMA was recovered as a precipitate afteradding the reaction mixture drop-wise to 300 mL dried ethyl ether withrapid stirring. As small amounts of precipitate formed, they were col-lected to prevent the polymer from conglomerating into large clumpswhich are difficult to break apart. The polymer was washed in chloro-form and dried in vacuo at room temperature.Acryloyl chloride-co-maleic anhydride (ACMA) was prepared by a

    similar procedure: 18.1 g (0.18 Mol) maleic anhydride and 15 mL acry-loyl chloride were dissolved in 39 mL acetone (90% w/v mixture) and0.87 g AIBN were added

    Chloramphenicol Esterification

    To prepare the UCMA-chloramphenicol conjugate (UCMA-C), 5 mLacetone, 0.6 mL pyridine, and 2.15 g chloramphenicol were mixed andcooled in an ice bath. A mole equivalent (2.0 g) of UCMA in 10 mL ofacetone was added slowly. This mixture was stirred at room tempera-ture for 2 h and then heated at 45 for another 2 h. The solution wasconcentrated by rotoevaporation, and the product was precipitated byadding the reaction mixture drop-wise to a 70/30 mixture of benzeneand chloroform. The precipitate was collected by filtration, washedwith chloroform, and dried in vacuo at room temperature.ACMA-chloramphenicol (ACMA-C) was prepared in a similar manner

    mixing 0.5 mL pyridine and 2.0 g chloramphenicol in 5 mL acetone andadding 1.17 g ACMA.

    Characterization of the Polymers

    Nuclear magnetic resonance (NMR) and infra-red (IR) spectroscopywere performed on the UCMA, ACMA, and polymer-chloramphenicolconjugates. Samples were dissolved in an appropriate deuterated sol-vent (CD3COCD3 or CCI3D) and analyzed using an EM-360 60 MHzNMR Spectrometer. IR samples were prepared by making translucentdisks with each compound and Bad (1:100, respectively) and analyz-ing with a Perkin-Elmer Model 283 IR Spectrophotometer.

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    Samples of UCMA, ACMA, UCMA-C, and ACMA-C were purified bywashing with CHCIs, dried under reduced pressure and analyzed forpercent C, H and N. Theoretical percentages of C, H, and N werecalculated for the polymer-antibiotic conjugates (ACMA-C and UCMA-C) with 25%, 50%, 75% and 100% antibiotic loading. Standard curves of% chloramphenicol loading vs. % N were generated and the actual %loading was extrapolated from the curve.ACMA-C and UCMA-C were water solubilized by stirring 0.1 g in

    1 mL 0.01 M NaHC03 forming the sodium salt. Molecular weightswere determined using BioRad P-6 polyacrylamide gel column chro-matography. Blue dextran (2 x 101 Daltons) was used to determinethe void volume. Rose bengal (1,017 Daltons) was used as a standardmarker.

    Antimicrobial Testing

    Antimicrobial activities were determined by both Kirby-Bauer diskdiffusion [14] and by minimum inhibitory concentration (MIC) [15].Copolymers and copolymer-chloramphenicol conjugates were dissolvedin 0.01 M NaHCO3 (10% w/v). Unconjugated chloramphenicol was dis-solved in 95% ethanol (10% w/v). ACMA-C, UCMA-C, and chloram-phenicol (C) were diluted in distilled H20 to obtain concentrations of 0,0.001, 0.005, 0.01, 0.05, 0.1, 0.5, and 1.0 mg chloramphenicol/10 gL,filter sterilized (0.2 gm) and adsorbed on sterile 1/4&dquo; diameter paperdiscs (Difco). Control discs were prepared with ACMA, UCMA, a mix-ture of either ACMA or UCMA and chloramphenicol (ACMA + C orUCMA + C), ethanol and NaHCO3 at appropriate concentrations.Escherichia coli (ATCC 35218) and Staphylococcus aureus (ATCC

    25923) cultures were reconstituted from Difco BacterolTM discs as perinstructions and spread onto Muller Hinton agar plates using sterilecotton swabs. The discs were applied aseptically after 15 min. Zones ofinhibition were measured to the nearest mm after 20 h at 35 C. Each

    plate was replicated in triplicate. The zones of inhibition around anygiven disc did not vary measurably from plate to plate.MICs were determined by diluting serially the various polymers and

    polymer drug conjugates in Tryptic soy broth (Difco) in 96-well plates.Wells were inoculated with 1:2000 dilutions of log phase cultures ofeither microorganism and incubated at 35 C for 12 h. Individual wellswere scored as turbid, slightly turbid, or clear, and the lowest concen-tration of chloramphenicol giving a slightly turbid or clear cell was de-termined for each replicate. MIC was determined as the mean of 3 ormore such replicates.

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    RESULTS AND DISCUSSION

    The H NMR spectra of ACMA and UCMA were both dominated byone broad high peak between 2 and 3 ppm. The protons responsible forthis peak are on the polymers backbone. The H NMR spectrum ofchloramphenicol had a multiplet centered on 8 ppm due to the protonsof the aromatic ring. The proton on the carbon between the carbonylcarbon and the dichloride gave a sharp peak at ca. 6.1 ppm and theamino proton appeared as a sharp peak at 5.1 ppm. The hydroxyl pro-tons and those of adjacent carbons formed a multiplet centered at ca.3.5 ppm.After conjugation of chloramphenicol to ACMA or UCMA, these char-

    acteristic peaks for the individual polymers or antibiotic were observedin the spectra of the polymer-antibiotic conjugates. The large peak ofthe hydrocarbon polymer backbone at 2-3 ppm and the multiplet ofchloramphenicols ring at 8 ppm were present on spectra of the conju-gates. As these conjugates were purified in solvents in which the indi-vidual polymers and antibiotic were soluble, these spectra providestrong evidence for conjugation. Similar evidence of conjugation wasprovided by IR spectra.Theoretically, at 100% loading of chloramphenicol to UCMA (1:1

    ratio of chloramphenicol to monomer) one would expect 53.2% C, 5.5%H, and 4.8% N. We observed 52.5% C, 5.8% H and 4.6% N indicating a91% loading of antibiotic in UCMA-C. ACMA-C at 100% loading wouldbe expected to contain 45.5% C, 3.4% H and 5.9% N. It contained 47.4%C, 4.4% H and 5.6% N indicating an 88% loading of antibiotic inACMA-C. These % loading values were used to determine the amountof chloramphenicol per unit weight of UCMA-C or ACMA-C in the an-timicrobial activity assays. P-6 gel chromatography indicated that thepolymer-chloramphenicol conjugates had a MW of approximately 3,200Daltons suggesting the presence of about 6 subunits.Neither ACMA nor UCMA (0.002 mg-2.000 mg per disc) had any in-

    trinsic antimicrobial activity (data not shown), nor was there much evi-dence of synergism when free antibiotic was mixed with the polymers(Figures 2 and 3: ACMA + C, UCMA + C). Esterification of the chloram-phenicol to ACMA or UCMA resulted generally in a less than 10-foldreduction in antimicrobial activity in disk sensitivity studies. For ex-ample, 1 mg of chloramphenicol conjugated to either ACMA or UCMAprovided a zone of inhibition against either microorganism equal to orslightly greater than that of 0.1 mg of pure chloramphenicol. Likewise,0.5 mg and 0.1 mg of chloramphenicol as ACMA-C or UCMA-C exhib-ited an inhibitory effect equal to or greater than 0.05 mg and 0.01 mg

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  • 291

    of free chloramphenicol, respectively (Figures 2 and 3: ACMA-C orUCMA-C vs. C). UCMA-C, the polymer with the longer spacer arm be-tween the polymer backbone and the antibiotic, generally was more in-hibitory than ACMA-C.Minimum inhibitory concentration (MIC) data were similar, with an

    8-fold or less reduction of activity being observed. The MIC of ACMA-Cagainst S. aureus was 16 jig chloramphenicol/mL culture medium;whereas, that of unconjugated chloramphenicol was 2 Ag/mL (8-fold re-duction of activity). Against E. coli, the MIC of ACMA-C was 256 wgchloramphenicol/mL compared to 64 gg/mL for unconjugated chloram-phenicol (4-fold reduction). UCMA-C exhibited a MIC of 8 gg chloram-phenicol/mL culture medium against S. aureus compared to 2 ttg/mLfor unconjugated chloramphenicol (4-fold reduction) and 256 /tgchloramphenicol/mL culture medium against E. coli compared to 64gg/mL for unconjugated chloramphenicol (4-fold reduction).That these maleic anhydride-chloramphenicol copolymers are at

    least 10% as effective as free chloramphenicol in vitro is promising.Chloramphenicol is a highly effective antibiotic. Its use has beenlimited primarily because of its toxic side-effects. By conjugating thisantibiotic to a target-specific delivery system such as ACMA or UCMA,lower dosages might prove effective in treating those diseases wherethe pathogen infects the macrophage for all or at least part of its infec-tious cycle. Also, conjugation might reduce the potential for toxic side-effects such as aplastic anemia even at higher dosages as reticulocytewould come in contact with less free chloramphenicol.The potential use of these polymers in the development of drug

    delivery systems warrants further study. Such a maleic anhydridecopolymer system is highly flexible, as one can vary the functionalgroups present for addition of a wide variety of drugs and additionaltargeting moieties. Also, an infinite variety of different sized polymerscan be readily synthesized. Studies are planned to test these agents inan animal model system.

    ACKNOWLEDGEMENTS

    We thank Joseph G. Smith, Department of Chemistry, Virginia Com-monwealth University, who performed the NMR and IR spectroscopy,Anthony Curtis and Richard Mills, Biology Department, VirginiaCommonwealth University for determining the molecular weights ofthese polymers and the A. H. Robbins Co., Richmond, VA for perform-ing the elemental analyses.

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    REFERENCES

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    2. Adams, D. O., W. J. Johnson, P. A. Marino and J. A. Dean. 1983. CancerResearch, 43:3663-3667.

    3. Baird, A. and A. Kaplan. 1980. In: Anionic Polymeric Drugs, pp. 185-210.New York: John Wiley & Sons.

    4. Butler, G., Y. Xing, G. Gifford and D. Flick. 1985. Ann. N.Y. Acad. Sci.,446:149-159.

    5. Kuus, K., R. M. Ottenbrite and A. Kaplan. 1985. J. Bio. Resp. Mod.,4:46-59.

    6. Kusser, W, K. Zimmer and F. Fielder. 1985. Eur. J. Biochem., 151:601-605.7. Oda, T., T. Morinaga and H. Msaeda. 1986. Proc. Soc. Exper. Bio. & Med.,

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    8. Pucceti, P. and A. Giampietri. 1978. Pharm. Res. Comm., 10:489-501.9. Simionescu, C., M. Popa and S. Dumitriu. 1986. Farmacia (Bucharest),

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    10. Hansch, C., E. Kutter and A. Leo. 1969. J. Med. Chem., 12:746-749.11. Hansch, C., K. Nakmoto, M. Gorin, P. Denisevich, E. Garrett, S. M.

    Heman-Ackah and C. H. Won. 1973. J. Med. Chem., 16:917-922.12. Manyan, D., G. K. Arimura and A. Yunis. 1975. Molec. Pharm.,

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    13. Shemyakin, M. M., M. N. Kolosov, M. M. Levitov, K. I. Germanova, M. G.Karapetyan, Y. B. Shetsov and E. M. Bamdas. 1956. J. Gen. Chem. USSR.,26:885-893.

    14. Bauer, A. W, M. M. Kirby, J. C. Sheris and M. Truck. 1966. Amer. J. Clin.Path., 45:493-496.

    15. Barry, A. L. 1976. The Antimicrobic Susceptibility Test: Principles andPractices. Ch. 1, 4 and 6. London: Lea & Febiger.

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