Synthesis and characterization of polyester conjugates of ciprofloxacin

European Journal of Medicinal Chemistry 45 (2010) 3844e3849

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Original article

Synthesis and characterization of polyester conjugates of ciprofloxacin Marcin Sobczak* Medical University of Warsaw, Faculty of Pharmacy, Department of Inorganic and Analytical Chemistry, ul. Banacha 1, 02-097 Warsaw, Poland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 April 2010 Received in revised form 15 May 2010 Accepted 18 May 2010 Available online 24 May 2010

Two-, three-, four and six-arm, star-shaped poly(3-caprolactone) and polylactide homopolymers were synthesized via ring-opening polymerization of cyclic esters in the presence of glycerol, penthaerythritol, dipentaerythritol and poly(ethylene glycol) as initiators and stannous octoate as a catalyst. Thus obtained oligomers were successfully used in the synthesis of novel polyester conjugates of ciprofloxacin. The structures of the polymers and conjugates were determined by means of GPC, MALDI-TOF MS, NMR and IR studies. The in vitro release of ciprofloxacin from obtained conjugates was investigated. Ó 2010 Elsevier Masson SAS. All rights reserved.

Keywords: Macromolecular prodrugs Macromolecular conjugates Aliphatic polyesters Fluoroquinolones Ciprofloxacin Controlled release

1. Introduction Polymeric materials are used in therapeutic applications, such as active macromolecular pharmaceutical substances, blood substitutes, tissue regeneration, auxiliary materials and excipients, in production of macromolecular prodrugs, polymeric drug delivery systems, therapeutic systems, etc. Controlled drug delivery technology represents one of the most rapidly advancing areas of science. The polymeric prodrugs, drug delivery systems and therapeutic systems exhibit unique pharmacokinetics, body distribution and pharmacological efficacy [1e13]. Biodegradable polymers like polylactide (PLA), poly(3-caprolactone) (PCL) and copolymers of lactides (LA) and 3-caprolactone (CL) are very often used as drug delivery systems. Aliphatic polyesters are usually prepared by ring-opening polymerization (ROP) of the corresponding cyclic monomers, that is DLLA, LLA or CL. PLA and PCL can be efficiently obtained by ROP in the presence of ionic initiators as well as coordinative and enzymatic catalysts [14e28]. Recently, the novel polyurethane conjugates of norfloxacin were obtained in our laboratory [29]. The polyester prodrugs of norfloxacin were obtained by us, too [30]. In this paper, we describe the synthesis of a series of polyester conjugates of ciprofloxacin. Ciprofloxacin is the most prominent member of fluoroquinolone antibiotics. It is frequently used as

a wide-spectrum antibiotic to treat and prevent infections caused by bacteria in human bodies [31]. The present paper is the continuation of our previous works [29,30]. We believe that the obtained polyester can find practical applications as effective drug delivery systems transporting active substances to specific body locations at the required rate. 2. Experimental 2.1. Materials

3-Caprolactone (CL, 2-oxepanone, Aldrich 99%) was dried and distilled before use over CaH2 at reduced pressure. 3,6-Dimethyl1,4-dioxane-2,5-dione (DLLA and LLA, rac-lactide and L-lactide, Aldrich 98%) was crystallized from a mixture of dry toluene with hexane and dried under vacuum. Poly(ethylene glycol) (PEG) (Mn ¼ 400 Da, Serva Feinbiochemica), pentaerythritol (PET) (Aldrich 99%), glycerol (GL) (Aldrich 99%), dipentaerythritol (DPET) (Aldrich, technical grade), ciprofloxacin (CIP) (Aldrich 99%) were exhaustively dried under vacuum prior use. Stannous octoate (SnOct2, tin (II) 2-ethylhexanoate) (Aldrich 95%), pyridine (Aldrich 99%), dichloromethane and anhydrous methanol were used as received. 2.2. Measurements

* Tel.: þ48 22 572 07 55; fax: þ48 22 572 07 84. E-mail addresses: [email protected], [email protected] 0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2010.05.037

The polymerization products were characterized by means of 1H and 13C NMR (Varian 300 MHz), and FTIR spectroscopy (Spectrum

M. Sobczak / European Journal of Medicinal Chemistry 45 (2010) 3844e3849 Table 1 Polymerization of CL, LLA and DLLA in the presence diols and SnOct2. Yielda (%)

Monomer/ Initiator

M/I/Cat

CL/PEG CL/GL CL/PET CL/DPET LLA/PEG LLA/GL LLA/PET LLA/DPET DLLA/PEG DLLA/GL DLLA/PET DLLA/DPET

200:1:0.5 z100 300:1:0.66 96 400:1:0.5 85 600:1:0.33 79 200:1:0.5 91 300:1:0.66 82 400:1:0.5 65 600:1:0.33 61 200:1:0.5 89 300:1:0.66 77 400:1:0.5 72 600:1:0.33 63

O O

Mnb (Da)

PDc Mnd (Da)

PDe hinhf (dL/ PF g)

9400 10300 11000 9900 10000 9100 8700 8100 9600 7600 10200 8400

1.1 1.2 1.2 1.3 1.2 1.2 1.3 1.3 1.2 1.2 1.3 1.3

1.2 e e e 1.2 e e e 1.2 e e e

6600 e e e 4900 e e e 4100 e e e

0.45 0.41 0.44 0.47 0.37 0.33 0.30 0.31 0.40 0.29 0.39 0.37

3845

ss ss ss ss vl vl vl vl vl vl vl vl

Reaction conditions: temp. of 120  C, time 48 h, monomers e 0.05 mol. PF e physical form, ss e sticky solid, vl e viscous liquid. a calculated by the weight method. b Mn e number-average molecular weight determined by GPC. c PD e polydispersity determined by GPC. d Mn e number-average molecular weight determined by MALDI-TOF. e PD e polydispersity determined by MALDI-TOF. f hinh e measured at 20  C with C ¼ 2 g/L in CH2Cl2.

1000, Perkin Elmer). The NMR spectra were recorded in CDCl3 or DMSO-d6. The IR spectra were recorded from KBr pellets. Relative molecular mass and molecular mass distributions were determined by MALDI-TOF MS and GCP techniques. The MALDI-TOF spectra were measured in the linear mode on a Kompact MALDI 4

R(OH) x

+

O SnOct2

x .y

R O C

CH2CH2CH2CH2CH2 OH y x

O

R(OH)x

+

O

H 3C x .y O

SnOct2 CH3

O CH3 R O C

O CH3

CH O C

CH OH y

x

O

x = 2 (for PEG), 3 (for GL), 4 (for PET) and 6 (for DPET) Scheme 1. Synthesis scheme for two-, three-, four- and six-arm, star-shaped polyesters.

Kratos analytical spectrometer using a nitrogen gas laser with 2[(4-hydroxyphenyl)diazenyl] benzoic acid (HABA) as a matrix. GPC measurements were made at 25  C in the tetrahydrofuran solution using Shimadzu C-R4 Chromatopac apparatus. The molecular weights were calibrated with polystyrene standards. Polymer viscosity was measured in chloroform (at 20  C) using an Ubbelohde viscometer. The amount of released CIP was determined by a UVeVis spectrophotometry (UV-1202 Shimadzu) at the adsorption maximum of the free drug in aqueous buffered solutions (lmax ¼ 277 nm) using a 1 cm quartz cell [32,33].

Table 2 IR and NMR data. CIP: 13C NMR (DMSO-d6, d, ppm): 1.25e1.4 (8) I (7), 2.73 (1), 2.82 (2), 3.88 (6), 7.62 (3), 7.92 (4), 8.83 (5), FTIR (KBr, cm1): 3470 (yOH), 1710 (yCO), 1624 (yC]C i C]N), 1452 (dCH2 i uCH2), 1194 (dCH, gCH i yCeO), 1102 (rings), 801 (yCeN i dCH2). PCL-PEG: 1H NMR (CDCl3, d, ppm): 1.26 (2H, m, eOCH2CH2CH2CH2CH2C(O)Oe), 1.67 (4H, m, ‑OCH2CH2CH2CH2CH2C(O)Oe), 2.32 (2H, t, eOCH2CH2CH2CH2CH2C(O)Oe), 3.68 (2H, t, eOCH2CH2Oe), 3.72 (2H, t, ‑CH2OH, end group), 4.13 (2H, t, eOCH2CH2CH2CH2CH2C(O)Oe), 4.21 (2H, CLeOCH2CH2Oe); 13C NMR (CDCl3, d, ppm): 24.8 (‑OCH2CH2CH2CH2CH2C(O)Oe), 25.7 (eOCH2CH2CH2CH2CH2C(O)Oe), 28.3 (‑OCH2CH2CH2CH2 CH2C(O)Oe), 33.7 (eOCH2CH2CH2CH2CH2C(O)Oe), 63.8 (eCH2C(O)OH, end group), 64.0 (CLeOCH2CH2Oe), 64.4 (‑OCH2CH2CH2CH2CH2C(O)Oe), 70.0 (eOCH2CH2Oe), 173.7 (eOCH2CH2CH2CH2CH2C(O)Oe); FTIR (KBr, cm1): 2945 (nasCH2), 2868 (nasCH3), 1723 (nC]O), 1245 (nCeO). PCL-GL: 1H NMR (CDCl3, d, ppm): 5.25 (1H, p, ]CHeOe), 4.29 (2H, d, eCH2eOe), 4.05 (2H, t, eCH2CH2OC(O)e), 3.64 (2H, t, eCH2CH2OH, end group), 2.29 (2H, t, eCH2CH2COOe), 1.63 (4H, m, eCH2CH2COOe), 1.36 (2H, m, eCH2CH2CH2CH2CH2e); 13C NMR (CDCl3, d, ppm): 173.1 (eC(O)Oe), 63.6 (eCH2CH2OC(O)e), 62.0 (eCH2eOe), 33.5 (eCH2CH2COOe), 27.8 (eCH2CH2OC(O)e), 25.2 (eCH2CH2COOe), 24.0 (eCH2CH2CH2CH2CH2e); FTIR (KBr, cm1): 2948 (yasCH2), 2864 (ysCH2), 1726 (yC]O), 1292 (CeO and CeC), 1240 (yasCOC), 1190 (yOCeO), 1170 (ysCOC). PCL-PET: 1H NMR (CDCl3, d, ppm): 4.01 (2H, t, eCH2CH2OC(O)e), 3.60 (2H, t, eCH2CH2OH, end group), 2.24 (2H, t, eCH2CH2COOe), 1.58 (4H, m, eCH2CH2COOe), 1.33 (2H, m, eCH2CH2CH2CH2CH2e); 13C NMR (CDCl3, d, ppm): 173.2 (eC(O)Oe), 63.8 (eCH2CH2OC(O)e), 63.5 (CeCH2Oe), 33.7 (eCH2CH2COOe), 33.4 (CeCH2Oe), 27.9 (eCH2CH2OC(O)e), 25.2 (eCH2CH2COOe), 24.2 (eCH2CH2CH2CH2CH2e); FTIR: 2949 (yasCH2), 2865 (ysCH2), 1727 (yC]O), 1294 (CeO and CeC), 1239 (yasCOC), 1189 (yOCeO), 1170 (ysCOC). PCL-DPET: 1H NMR (CDCl3, d, ppm): 1.26 (2H, m, eOCH2CH2CH2CH2CH2C(O)Oe), 1.67 (4H, m, ‑OCH2CH2CH2CH2CH2C(O)Oe), 2.32 (2H, t, eOCH2CH2CH2CH2CH2C(O)Oe), 3.66 (2H, t, ‑CH2OH, end group), 4.12 (2H, t, eOCH2CH2CH2CH2CH2C(O)Oe); 13C NMR (CDCl3, d, ppm): 24.8 (‑OCH2CH2CH2CH2CH2C(O)Oe), 25.7 (eOCH2CH2CH2CH2CH2C(O)Oe), 28.3 (‑OCH2CH2CH2CH2 CH2C(O)Oe), 33.2 (CeCH2Oe), 33.7 (eOCH2CH2CH2CH2CH2C(O)Oe), 63.3 (CeCH2Oe), 63.8 (eCH2C(O)OH, end group), 64.3 (eOCH2CH2CH2CH2CH2C(O)Oe), 173.7 (eOCH2CH2CH2CH2CH2C(O)Oe); FTIR (KBr, cm1): 2945 (nasCH2), 2868 (nasCH3), 1723 (nC]O), 1245 (nCeO). PLA-PEG: 1H NMR (CDCl3, d, ppm): 5.16 (1H, q, eCH(CH3)e), 4.35 (1H, q, eCH(CH3)OH, end group), 4.24 (2H, LAeOCH2CH2Oe), 3.61 (2H, t, eOCH2CH2Oe), 1.57 (3H, d, eCH3); 13C NMR (CDCl3, d, ppm): 169.9 (eC(O)Oe), 69.3 (eCH(CH3)e), 16.9 (eCH3); FTIR: 2998 (yasCH3), 2948 (ysCH3), 2883 (yCH), 1760 (yC]O), 1453 (dasCH3), 1345e1388 (dsCH3), 1365e1360 (d1CH þ dsCH3), 1315e1300 (d2CH), 1270 (dCH þ yCOC), 1215e1185 (yasCOC þ rasCH3), 1131 (rasCH3), 1100e1090 (ysCOC), 1045 (yCeCH3), 960e950 (rCH3 þ yCC), 875e860 (yCeCOO), 760e740 (dC]0), 715e695 (gC]O), 515 (d1CeCH3 þ dCCO), 415 (dCCO), 350 (d2CeCH3 þ dCOC), 300e295 (dCOC þ d2CeCH3), 240 (sCC). PLA-GL: 1H NMR (CDCl3, d, ppm): 5.23 (1H, p, ]CHeOe), 5.15 (1H, q, eCH(CH3)e), 4.34 (1H, q, eCH(CH3)OH, end group), 4.26 (2H, d, eCH2eOe), 1.58 (3H, d, eCH3); 13C NMR (CDCl3, d, ppm): 169.8 (eC(O)Oe), 69.2 (eCH(CH3)e), 62.3 (eCH2eOe), 16.8 (eCH3); FTIR: 2996 (yasCH3), 2946 (ysCH3), 2883 (yCH), 1761 (yC]O), 1453 (dasCH3), 1348e1388 (dsCH3), 1368e1360 (d1CH þ dsCH3), 1315e1300 (d2CH), 1270 (dCH þ yCOC), 1215e1185 (yasCOC þ rasCH3), 1130 (rasCH3), 1100e1090 (ysCOC), 1045 (yCeCH3), 960-950 (rCH3 þ yCC), 875e860 (yCeCOO), 760e740 (dC]0), 715e695 (gC]O), 515 (d1CeCH3 þ dCCO), 415 (dCCO), 350 (d2CeCH3 þ dCOC), 300e295 (dCOC þ d2CeCH3), 240 (sCC). PLA-PET: 1H NMR (CDCl3, d, ppm): 5.19 (1H, q, eCH(CH3)e), 4.38 (1H, q, eCH(CH3)OH, end group), 1.62 (3H, d, eCH3); 13C NMR (CDCl3, d, ppm): 169.5 (eC(O)Oe), 69.0 (eCH(CH3)e), 63.4 (CeCH2Oe), 33.3 (CeCH2Oe), 16.6 (eCH3); FTIR: 2997 (yasCH3), 2946 (ysCH3), 2882 (yCH), 1759 (yC]O), 1452 (dasCH3), 1348e1388 (dsCH3), 1368e1360 (d1CHþdsCH3), 1315e1300 (d2CH), 1270 (dCH þ yCOC), 1215e1185 (yasCOC þ rasCH3), 1132 (rasCH3), 1100e1090 (ysCOC), 1046 (yCeCH3),. 960e950 (rCH3 þ yCC), 875e860 (yCeCOO), 760e740 (dC]0), 715e695 (gC]O), 517 (d1CeCH3 þ dCCO), 414 (dCCO), 352 (d2CeCH3 þ dCOC), 300e295 (dCOC þ d2C-CH3), 244 (sCC). PLA-DPET: 1H NMR (CDCl3, d, ppm): 1.51 (3H, q, eCH(CH3)C(O)Oe), 4.40 (1H, q, eCH(CH3)OH, end group), 5.18 (1H, q, eOCH(CH3)C(O)Oe); 13C NMR (CDCl3, d, ppm): 17.2 (eOCH(CH3)C(O)Oe), 20.9 (eCH(CH3)C(O)OH), 33.5 (CeCH2Oe), 63.6 (CeCH2Oe), 67.2 (eCH(CH3)OH, end group), 69.1 (eOCH(CH3)C(O)Oe), 169.7 (eC(O)Oe); FTIR (KBr, cm1): 2996 (yasCH3), 2946 (ysCH3), 2881 (yCH), 1761 (yC]O), 1451 (dasCH3), 1348e1388 (dsCH3), 1368e1360 (d1CH þ dsCH3), 1315e1300 (d2CH), 1270 (dCH þ yCOC), 1215e1185 (yasCOC þ rasCH3), 1130 (rasCH3), 1100e1090 (ysCOC), 1045 (yCeCH3), 960e950 (rCH3 þ yCC), 875e860 (yCeCOO), 760e740 (dC]O), 715e695 (gC]O), 515 (d1CeCH3 þ dCCO), 415 (dCCO), 350 (d2CeCH3 þ dCOC), 300e295 (dCOC þ d2CeCH3), 240 (sCC). PCL-PEG e poly(3-caprolactone) obtained in the presence of poly(ethylene glycol), PLA-GL e polylactide obtained in the presence of glycerol, PLA-PET polylactide obtained in the presence of pentaerythritol, etc.

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M. Sobczak / European Journal of Medicinal Chemistry 45 (2010) 3844e3849

Fig. 1. The MALDI-TOF spectrum of the product of the CL polymerization in the presence of PEG.

2.3. Polymerization procedure Monomers (CL, DLLA, LLA), initiators (PEG, PET, GL, DPET) and the SnOct2 catalyst were placed in a 10 mL glass ampule under nitrogen atmosphere. The reaction vessel was then kept standing in a thermostated oil bath at 120  C over 48 h (Table 1). When the reaction time was completed, the cold reaction product was dissolved in CH2Cl2, precipitated from cold methanol with diluted hydrochloric acid (to wash out the catalyst residue) and dried under vacuum for 72 h. The precipitation was repeated three times. 2.4. Macromolecular conjugates synthesis The conjugates were prepared under nitrogen atmosphere at room temperature immediately before use. The polyesters were dissolved in CH2Cl2 (1 g/150 mL) and this solution was placed in a 500 mL three-necked flask equipped with a stirrer and addition funnel. Next, pyridine (5 ml) was added. A solution of CIP in CH2Cl2 (20 g L1) was placed in the funnel and added drop wise into the reactor, while the reaction mixture was vigorously stirred. After the addition procedure was completed, the reaction mixture was left stirring for an additional 8 h, then it was washed with dilute hydrochloric acid and water. The washing was continued for about

1 h. The conjugates isolated from the solution (organic phase) were kept under vacuum at room temperature for no more than one week. 2.5. Biodegradation of polyester conjugates Dried polymer (1 g) was poured into aqueous buffered solution (100 mL, pH 1, 4 and 7) at 37  C. The mixture was stirred and a 5 mL sample was removed at selected intervals and 5 mL of buffer was replaced. The quantity of released drug was analyzed by means of UV spectrophotometer determined from the calibration curve obtained previously under the same conditions. 3. Results and discussion 3.1. Ring-opening polymerization of cyclic esters The aim was to obtain a low-molecular weight polyesters which can be subsequently used as macromolecular conjugates of ciprofloxacin. The polymerization reactions of CL, DLLA and LLA were carried out in the presence of initiators like poly(ethylene glycol) (PEG), glycerol (GL), penthaerythritol (PET) or dipentaerythritol (DPET), and stannous octoate (SnOct2) as catalyst. Reaction

M. Sobczak / European Journal of Medicinal Chemistry 45 (2010) 3844e3849

3847

Table 3 Synthesis of polyester conjugates of CIP. Code

Polyester conjugates

Drug contenta

CON-1 CON-2 CON-3 CON-4 CON-5 CON-6 CON-7 CON-8 CON-9 CON-10 CON-11 CON-12

CL/PEG/CIP CL/GL/CIP CL/PET/CIP CL/DPET/CIP LLA/PEG/CIP LLA/GL/CIP LLA/PET/CIP LLA/DPET/CIP DLLA/PEG/CIP DLLA/GL/CIP DLLA/PET/CIP DLLA/DPET/CIP

2.3 3.0 3.5 5.5 2.8 4.2 5.6 8.6 3.0 5.4 4.8 8.2

a CIP units content in polyester conjugates (% mol), calculated by 1H NMR. CL/PEG/ CIP e poly(3-caprolactone)-poly(ethylene glycol)-ciprofloxacin conjugate, LLA/GL/ CIP e polylactide-glycerol-ciprofloxacin conjugate, DLLA/PET/CIP e polylactidepenthaerythritol-ciprofloxacin conjugate, etc.

conditions, yields and average molecular weight of obtained polyesters are summarized in Table 1. Under these conditions, cyclic monomers underwent ROP and the low-molecular weight polyesters with chain end hydroxyl groups were obtained (Scheme 1). The synthesized PCL and PLA polymers had two-, three-, four- and six-arm star shapes. Chemical structures of the synthesized polymers have been confirmed using 1H and 13C solution NMR and FTIR spectroscopy. Molecular weights of polymers have been determined using gel permeation chromatography (GPC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and a viscosity method. The reaction yields were in the 89e100, 77e96, 65e85 and 61e79% range for two-, three-, four- and six-arm star-shaped polyesters, respectively. The number-average molecular weights determined from GPC for CL oligomers lie in the 9.400e11.000 Da range, and the polydispersity indexes in the 1.1e1.3 range. For LLA and D,LLA oligomers the Mn values are 7.600e10.200 and the Mw/Mn values lie in the 1.2e1.3 range. The IR and NMR data were shown in Table 2. Fig. 1 shows typical MALDI-TOF spectrum of the obtained PCLPEG. The MALDI-TOF spectrum of PCL comprises two series of peaks. The most prominent series of peaks is characterized by a mass increment of 114 Da, which is equal to the mass of the repeating unit in the PCL polymer (Fig. 1). This series is assigned to PCL terminated with a hydroxyl group and detected as the Naþ adduct (residual mass: RM ¼ 41 Da). The second series of the peaks is also from PCL terminated with a hydroxyl group, but corresponds to the Kþ adduct (RM ¼ 57 Da). The MALDI-TOF spectrum of PLA comprises two series of peaks, too. The main series comes from PLA terminated with a hydroxyl group and corresponds to the Naþ adduct (RM ¼ 42 Da), while the second series of smaller peaks is also from PLA terminated with

Fig. 2. The 1H NMR spectrum of CIP (in DMSO).

a hydroxyl group, but corresponds to the Kþ adduct (RM ¼ 57 Da). In the MALDI-TOF spectrum of PLA both populations of chains of even and odd number of lactic acid m.u. can be observed. The odd number of acid m.u. shows that under the process conditions the polymer chain undergoes intermolecular transesterification (leading to an exchange of segments), which is a typical phenomenon for the polymerization of lactides. Formation of PCL and PLA macrocycles was not observed. 3.2. Synthesis of polyester conjugates of ciprofloxacin The macromolecular conjugates were obtained from the reactions of the two-, three-, four, six-arm, star-shaped PCL and PLA with ciprofloxacin (CIP) (Table 3) (Scheme 2). The chemical structures of the prepared conjugates were confirmed by 13C, 1H NMR and IR studies. Typical proton NMR spectra of pure CIP and of the reaction products of the two-armed PCL with CIP are shown in Figs. 2e4, respectively. All the conjugate spectra have revealed characteristic peaks of CIP, indicating successful preparation of the polyester-CIP conjugate. The CIP content in the PCL or PLA conjugates calculated by 1H NMR. The signal intensity of the 3 and the signal intensity of the D (for PCL) or G (for PLA) has been compared. The drug content in the

O F

O

COOH +

N

N

HO

R

F _

C O

H2O N

HN HN

R – oligoester segment Scheme 2. Synthesis of the polyester conjugates of ciprofloxacin.

O

N

R

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M. Sobczak / European Journal of Medicinal Chemistry 45 (2010) 3844e3849

Fig. 5. Release of CIP from CL/PEG/CIP, CL/GL/CIP, CL/PET/CIP, CL/DPET/CIP conjugates at pH 1, 4 and 7.

Fig. 3. The 1H NMR spectrum of the conjugate e PCL-CIP (CON-1) (in DMSO).

conjugates amounts to 2.3e8.6 mol %. The one is dependent on the structure of the polyester. The CIP content in the macromolecular conjugates increased, when the number of arms were raised as follows: two < three < four < six-arm. 3.3. Drug release from polyester conjugates Hydrolysis studies were carried out to evaluate the CIP release from the macromolecular conjugates under different conditions. The study was performed by incubation of macromolecular conjugates in buffer solution at pH 1, 4 and 7.4. The in vitro release profiles of CIP from the different polyesters are shown in Figs. 5e7. The PLA and PDLA conjugates underwent to faster CIP release as compared to the PCL conjugates. 17% of linked

Fig. 4. The 1H NMR spectrum of the conjugate e PLA-CIP (CON-5) (in DMSO).

drug was release from CL/PEG/CIP conjugate (CON-1) within 35 days at pH 7, 22% from LLA/PEG/CIP (CON-5) conjugate and 29% from DLLA/PEG/CIP conjugate (CON-9), for example (Figs. 5e7). It was found that the rate of CIP release from obtained conjugates depends on the structure of the polyester (kind of glycol segment) and the order of hydrolysis is as follows: DLLA/PEG/CIP (CON-9) (37% drug released within 35 days at pH 1) < DLLA/GL/CIP (CON-10) (35%) < DLLA/PET/CIP (CON-11) (33%) < DLLA/DPET/CIP (CON-12) (29%) (Fig. 7). The results suggest a higher stability of obtained polyester conjugates to chemical hydrolysis at pH 7 than 4 or 1. Percentage of the released CIP after 35 h incubation was about 37% from DLLA/ PEG/CIP (CON-9) at pH 1 and 32% from DLLA/PEG/CIP (CON-9) at pH 4 and 2 from DLLA/PEG/CIP (CON-9) at pH 7 (Fig. 7). After 35 h incubation the CIP release was 23% from CL/PEG/CIP (CON-1) at pH 1 and 19% from CL/PEG/CIP (CON-1) at pH 4 and 17% from CL/PEG/ CIP (CON-1) at pH 7 (Fig. 5). The rates of CIP release for the PLA conjugates appear similar but are slower than the PDLA conjugates. Percentage of the released CIP after 35 h incubation was about 37% from DLLA/PEG/CIP (CON-9), 35% from DLLA/GL/CIP (CON-10), 33% from DLLA/PEG/CIP (CON-11), 29% from DLLA/PEG/CIP (CON-11) at pH 1 (Fig. 7). However, after 35 h incubation the CIP release was 30% from LLA/PEG/CIP (CON-5), 29% from LLA/GL/CIP (CON-6), 26% from LLA/PET/CIP (CON-7), 25% from LLA/DPET/CIP (CON-8) at pH 1 (Fig. 6). The difference in release rates observed between PLA and PDLA conjugates can be attributed to the difference in their crystallinity. PCL has a high degree of crystallinity and thus its biodegradation rate is very slow. When PCL was copolymerized with poly(ethylene glycol) the conjugate have a higher biodegradation rate. PEG is a hydrophilic, water soluble and fully biodegradable polymer. The hydrolytic degradation of polymer involves the chemical scission at an ester linkage by a water molecule. If the polymer is hydrophilic, water molecule can easily penetrate into the structure and hydrolyze the

Fig. 6. Release of CIP from LLA/PEG/CIP, LLA/GL/CIP, LLA/PET/CIP, LLA/DPET/CIP conjugates at pH 1, 4 and 7.

M. Sobczak / European Journal of Medicinal Chemistry 45 (2010) 3844e3849

Fig. 7. Release of CIP from DLLA/PEG/CIP, DLLA/GL/CIP, DLLA/PET/CIP, DLLA/DPET/CIP conjugates at pH 1, 4 and 7.

ester bond. Preliminary results show the rate of ciprofloxacin release from polyester conjugates depends on kind of glycol unit in the polymer chain and the order of hydrolysis is as follows: PEG > GL > PET > DEPT. In conclusion, the release rate is seen to be highly dependent on the kind of ester or glycol segment in the polyester and pH of the medium. Kinetics of the biodegradation polyester conjugates and the cytotoxic tests are still under study. Final results will be presented in the next paper and our discussion will be completely. 4. Conclusions We reported a novel macromolecular conjugates of CIP. The CIP was covalently connected to the chain end of the two-, three-, four- and six-arm, star-shaped PCL and PLA via an ester linkage. The synthesis of those conjugates was done in two steps. First, the ring-opening polymerization of ECL, LLA and DLLA in the presence of initiators PEG, GL, PET, DPET and SnOct2 as a catalyst was carried out. In the second step, the reaction of the polyester with CIP was performed. Our method of the synthesis is simple and effective. The investigation of the CIP release indicated that the release rate of the drug depends on the structure of polyesters (kind of ester or glycol segments) and could be effectively controlled by altering the pH values of the environment. The possibility of using the obtained conjugates as prodrugs of CIP is currently in progress. We believe that the obtained polyester conjugates of CIP are good potential candidates for carriers in drug delivery systems. Acknowledgement I gratefully acknowledge the financial support of the Warsaw Medical University. References [1] J. Jagur-Grodzinski, Biomedical application of functional polymers. React. Funct. Polym. 39 (1999) 99e138. [2] K.E. Uhrich, S.M. Cannizzaro, R.S. Langer, K.M. Shakesheff, Polymeric systems for controlled drug release. Chem. Rev. 99 (1999) 3181e3198. [3] F.M. Veronese, M. Morpurgo, Bioconjugation in pharmaceutical chemistry. Farmaco 54 (1999) 497e516.

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