Synthesis and biopharmaceutical characterisation of new poly(hydroxyethylaspartamide) copolymers as drug carriers

Biochimica et Biophysica Acta 1528 (2001) 177^186

www.bba-direct.com

Synthesis and biopharmaceutical characterisation of new poly(hydroxyethylaspartamide) copolymers as drug carriers Paolo Caliceti a , Santina Maria Quarta a , Francesco Maria Veronese a , Gennara Cavallaro b , Elisa Pedone b , Gaetano Giammona b; * b

a Dipartimento di Scienze Farmaceutiche, Universita© degli Studi di Padova, Via Marzolo 5, 35131 Padua, Italy Dipartimento di Chimica e Tecnologie Farmaceutiche, Universita© degli Studi di Palermo, Via Archira¢ 32, 90123 Palermo, Italy

Received 20 March 2001 ; received in revised form 19 July 2001; accepted 25 July 2001

Abstract Four new poly(hydroxyethylaspartamide)-based copolymers bearing (a) poly(ethylene glycol) 2000, (b) poly(ethylene glycol) 5000, (c) poly(ethylene glycol) 2000 and hexadecylalkyl, (d) poly(ethylene glycol) 5000 and hexadecylalkyle, as pendant groups were synthesised. The copolymers were obtained by partial aminolysis of polysuccinimide with poly(ethylene glycol) and hexadecylalkyl amino derivatives followed by reaction with ethanolamine. Naked polyhydroxyaspartamide was obtained by polysuccinimide reaction with ethanolamine. The nuclear magnetic resonance, infrared, light scattering and elemental analysis allowed for the extensive physico-chemical characterisation of the carriers. The molecular mass of all the polymers was in the range of 27 000^34 000 Da, and the polydispersivity was in the range of 1.5^ 1.7. By intravenous injection to mice bearing a solid tumour, all the polymeric carriers displayed a bi-compartmental pharmacokinetic behaviour. Both the poly(ethylene glycol) and the hexadecylalkyle conjugation prolonged and enhanced the distribution phase of poly(hydroxyethylaspartamide). The poly(ethylene glycol) conjugation was found to promote the carrier elimination by kidney ultrafiltration and to prevent partially the accumulation in the spleen and in the liver. The poly(ethylene glycol)/hexadecylalkyle conjugates localised preferentially in the liver were over 30% of the dose/g of tissue was determined after 144 h from administration. In the tumour all the polymers displayed a relevant accumulation that significantly increased throughout the time to reach high concentrations after 24 h. In particular, the poly(ethylene glycol)/hexadecylalkyle conjugates achieved a concentration of 15^25% of the dose/g of tissue after 24 h from administration that was maintained up to 144 h. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Polyaspartamide derivative ; Amphiphilic copolymer; Polymeric carrier ; Pharmacokinetics; Biodistribution

1. Introduction In the past two decades there has been an increasing interest in the development of prolonged or controlled drug delivery systems (DDS) and intensive e¡orts have been making to design therapeutic systems able to deliver e¤ciently drugs to the target sites [1]. The coupling of biologically active compounds to soluble polymeric carriers is a fascinating approach to advanced drug delivery. In this regard an advanced bioconjugation technology has been developing to increase the drug solubility and stability, to prolong its permanence in the body, to promote the targeting to the disease site and ¢nally to enhance the therapeutic index [2].

* Corresponding author. Fax: +39-91-617-7333. E-mail address : [email protected] (G. Giammona).

Since the polymer is frequently the main component of polymeric bioconjugates, it is clear that its rational design represents a key issue to achieve derivatives having the desired physico-chemical and biopharmaceutical properties. Therefore, the polymer chemical composition and molecular mass are fundamental parameters in determining the conjugate properties ([3] and notes therein). Furthermore, the introduction of speci¢c pendant groups in the polymer backbone is often carried out to modify deeply the carrier properties. Examples are the conjugation of directing functions such as glycosides, folic acid, hormones, peptides and monoclonal antibody fragments. Besides, a suitable balance between hydrophilic and hydrophobic moieties allows one to obtain macromolecules that can arrange into supramolocular structures, `polymeric micelles', with long permanence in the circulation and low liver uptake [4^6]. The approach of tailoring polymeric micelles as drug

0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 1 9 1 - X

BBAGEN 25230 18-10-01

178

P. Caliceti et al. / Biochimica et Biophysica Acta 1528 (2001) 177^186

vehicles was initiated by Ringsdorf et al. along with the concept of mimicking lipoprotein structures [4]. Polymeric micelles can o¡er, over several types of particle drug carriers, some advantages such as wide applicability to drugs and normally small particle size [7] that can result on one side in escaping the capture by the reticuloendothelial system [11] and, on the other, in promoting the localisation into tumour mass by enhanced permeability retention (EPR) e¡ect [3]. In this case drugs can be carried by polymeric micelles either via chemical conjugation or physical entrapment [8^10]. To date, several poly(amino acid) derivatives have been largely investigated as drug delivery systems [12,13]. Among them K,L-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) is one of the most promising drug carriers since it was demonstrated to possess suitable physico-chemical characteristics for development of macromolecular prodrugs, such as biodegradability, high water solubility, multi-point drug attachment, excellent biocompatibility and low cost [14^16]. In vitro and in vivo studies, performed with di¡erent therapeutic molecules, demonstrated that the conjugation to PHEA can improve their therapeutic potential allowing for a sustained drug release in the blood [17]. In addition, PHEA was also proposed to promote drug release into the disease site [18]. In order to exploit the advantages of PHEA as drug carrier, new PHEA derivatives have been synthesised in our laboratories by introduction of hydrophilic or hydrophilic/hydrophobic pendant groups into its polymeric structure. The aim of the research is the preparation of new drug carriers possessing the advantageous properties of the starting polymer and peculiar performances in drug delivery. In this paper the synthesis and the physico-chemical characterisation of PHEA copolymers bearing PEG 2000 (PHEA^PEG2000 ), PEG 5000 (PHEA^PEG5000 ), PEG 2000 and hexadecylalkyle (PHEA^PEG2000 ^C16 ), PEG 5000 and hexadecylalkyle (PHEA^PEG5000 ^C16 ), is reported. Aimed at investigating the in£uence of the pendant groups on the pharmacokinetic properties of the carrier an in vivo study was carried out using mice bearing solid tumour. The carrier distribution in the main tissues was determined and their potential application in tumour targeting was discussed.

chased from Fluka (Italy). Instagel, Soluene 350 and Hionic Fluor were furnished by Canberra Packard (Groningen, The Netherlands). All the other reagents were of technical grade and were supplied by Sigma (St. Louis, MO, USA). The male NCL mice weighing 23^25 g used for in vivo studies were furnished from the Department of Pharmaceutical Sciences (University of Padua). Animal treatments were performed according to the Italian law (DL no. 116/ 92) and the institutional European guidelines (EEC no. 86/ 609). Polysuccinimide (PSI) was prepared in a large yield by polycondensation of DL-aspartic acid according to the method reported in literature [20]. K,L-Poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) was prepared by complete aminolysis of PSI in DMF solution keeping reaction temperature between 22³C and 26³C [20].

2. Materials and methods

2.3.1. PSI^PEG copolymers A solution of O-(2-aminoethyl)-OP-methylpolyethylene (PEG-NH2 , Mr 2000 or 5000) in DMF (1.5U1034 mol/5 ml) was added dropwise at room temperature to a solution of PSI in DMF (4U1033 mol of repeating units/5 ml). The reaction mixture was maintained at 60³C for 15 or 30 h, respectively, for PEG2000 -NH2 and PEG5000 -NH2 under argon and continuous stirring ; afterwards it was precipitated with ethyl ether, washed several times with methanol and ¢nally dried under vacuum for several hours.

2.1. Chemicals Hexadecylamine was purchased from Aldrich (Steinheim, Germany). [3 H]Ethanolamine was purchased from Amersham International (Amersham, UK). DL-Aspartic acid and O-(2-aminoethyl)-OP-methylpolyethylene glycol 2000 (90.4 mmol NH2 /g) and O-(2-aminoethyl)-OP-methylpolyethylene glycol 5000 (90.17 mmol NH2 /g) were pur-

2.2. Apparatus Elemental analysis (C, H, N) was carried out on a Carlo Erba model 1106 analyser. Infrared (IR) spectra were recorded using a Perkin Elmer 1720 IR Fourier Transform spectrophotometer in potassium bromide discs. The 1 H-nuclear magnetic resonance (NMR) spectra were obtained with a Bruker AC-250 instrument operating at 250.13 MHz. The molecular characterisation of PHEA and PHEA copolymers samples was performed by a multidetector SEC system. The system consisted of an Alliance 2690 separation module, a single capillary viscosimeter (SCV), a di¡erential refractometer (DRI) from Waters (Milford, MA, USA) and an additional multi-angle light scattering (MALS) photometer from Wyatt (Santa Barbara, CA, USA). The experimental conditions used are described elsewhere [19]. Dynamic light scattering (DLS) experiments were made using a Brookhaven Instrument 2030-AT 128-channel digital correlator and an ILT 550 argon laser tuned at 514.5 nm. For the determination of the di¡usion coe¤cients of polymer and micelles, was employed the CONTIN Program. 2.3. Polymer synthesis

BBAGEN 25230 18-10-01

P. Caliceti et al. / Biochimica et Biophysica Acta 1528 (2001) 177^186

2.3.2. PSI^PEG^C16 copolymers A solution of hexadecylamine (C16 ) in DMF (3.6U1034 mol/5 ml) was added dropwise at 60³C to a solution of PSI^PEG in DMF (4U1033 mol of repeating units/5 ml). The reaction mixture was maintained at 60³C for 7 h under argon and continuous stirring; afterwards it was precipitated with ethyl ether, washed several times with ethyl ether and ¢nally dried under vacuum for several hours. 2.3.3. PHEA^PEG and PHEA^PEG^C16 copolymers Ethanolamine (1U1032 mol) was added dropwise to DMF solutions of PSI^PEG or PSI^PEG^C16 (4.12U1033 mol/3.2 ml) maintaining the reaction temperature between 22³C and 26³C under stirring for 3 h. The polymer was precipitated with ethyl ether, washed several times with acetone to neutrality and ¢nally dried under vacuum. The polymer was dispersed in water and puri¢ed by exhaustive dialysis running distilled water using Visking Dialysis Tubing with a molecular mass cut-o¡ of 12 000^ 14 000 Da. The solution was lyophilised and the puri¢ed products, obtained with a 94^97% yield (as based on starting PSI), were characterised by elemental analysis, IR spectrophotometry and 1 H-NMR analysis. The analytical and spectral data of PHEA derivatives were in agreement with attributed structures: PHEA^PEG2000 : calculated per C7:18 H12:35 N2:25 O3:58 (related to 1.25 mol%) : C, 46.00; H, 6.59; N, 16.81; found: C, 45.88; H, 6.47; N, 17.02. PHEA^PEG5000 : calculated per C10:18 H18:28 N2:03 O5:08 (related to 1.81 mol%) : C, 48.83; H, 7.30; N, 11.36; found: C, 48.78; H, 7.47; N, 12.02. IR spectra of PHEA^PEG2000 and PHEA^PEG5000 (KBr) showed bands at: 3312 cm31 (OH ; -NH-), 1657 cm31 (amide I) and 1542 cm31 (amide II) belonging to PHEA and a band at 953 cm31 attributable to ether C^O stretching of PEG. 1 H-NMR of PHEA^PEG2000 (D2 O): N 2.80 (m, 2 H, -CO-CH-CH2 -CO-NH-), 3.36 (m, 2 H, -NH-CH2 -CH2 OH), 3.63 (m, 2 H, -NH-CH2 -CH2 -OH), 3.69 (s, 178 H, -CH2 -CH2 -O-), 4.74 (m, 1 H, -NH-CH(CO)CH2 ). 1 H-NMR of PHEA^PEG2000 (D2 O): N 2.78 (m, 2 H, -CO-CH-CH2 -CO-NH-), 3.34 (m, 2 H, -NH-CH2 -CH2 OH), 3.64 (m, 2 H, -NH-CH2 -CH2 -OH), 3.69 (s, 454 H, -CH2 -CH2 -O-), 4.72 (m, 1 H, -NH-CH(CO)CH2 ). PHEA^PEG2000 ^C16 : calculated per C8:16 H14:21 N2:35 O3:68 (related to 1.35 mol% of PEG2000 and to 4.9 mol% of -C16 ): C, 48.02; H, 6.97; N, 16.13; found: C, 47.98; H, 6.87; N, 16.02. PHEA^PEG5000 ^C16 : calculated per C11:5 H9:26 N2:16 O2:21 (related to 1.73 mol% of PEG5000 and to 6.6 mol% of C16 ): C, 61.50; H, 9.26; N, 13.47; found C, 61.35; H, 9.47; N, 13.09. IR spectra of PHEA^PEG2000 ^C16 and PHEA^ PEG5000 ^C16 (KBr) showed bands at: 3312 cm31 (OH; -NH-), 2985 cm31 and 2854 cm31 (C^H stretching of

179

C16 chains), 1657 cm31 (amide I), 1542 cm31 (amide II) belonging to PHEA, and a band at 953 cm31 attributable to ether C^O stretching of PEG. 1 H-NMR of PHEA^PEG2000 ^C16 (D2 O): N 0.87 (t, 3 H, -CH2 -CH3 ), 1.28 (m, 28 H, -CH2 -CH2 -CH2 -), 2.80 (m, 2 H, -CO-CH-CH2 -CO-NH-), 3.36 (m, 2 H, -NH-CH2 CH2 -OH), 3.63 (m, 2 H, -NH-CH2 -CH2 -OH), 3.69 (s, 178 H, -CH2 -CH2 -O-), 4.74 (m, 1 H, -NH-CH(CO)CH2 ). 1 H-NMR of PHEA^PEG5000 ^C16 (D2 O): N 0.87 (t, 3 H, -CH2 -CH3 ), 1.28 (m, 28 H, -CH2 -CH2 -CH2 -), 2.78 (m, 2 H, -CO-CH-CH2 -CO-NH-), 3.34 (m, 2 H, -NH-CH2 -CH2 OH), 3.64 (m, 2 H, -NH-CH2 -CH2 -OH), 3.69 (s, 454 H, -CH2 -CH2 -O-), 4.72 (m, 1 H, -NH-CH(CO)CH2 ). 2.4. [3 H]PHEA and [3 H]PHEA derivatives preparation One hundred mg of lyophilised polymer (PHEA or PHEA^PEG2000 or PHEA^PEG5000 or PHEA^PEG2000 ^ C16 or PHEA^PEG5000 ^C16 ) was dissolved in 2.2 ml of anhydrous N-methylpyrrolidone/pyridine (3.6:1 v/v) and added to 228 mg of p-nitrophenylchloroformate and 21 mg of dimethylaminopyridine. The solution was maintained under stirring for 1 h at 0³C and then for 1 h at room temperature. The activated polymer was precipitated from the reaction solution by dropwise addition of anhydrous diethyl ether/acetone (6:4 v/v) and the precipitate was recovered by centrifugation for 5 min at 4³C at 3000 rpm, addition of 10 ml of the diethyl ether/acetone mixture and separated by centrifugation. The washing procedure was repeated ¢ve times and then the precipitate was desiccated under vacuum, dissolved in 4.2 ml of anhydrous dimethylformamide/pyridine mixture (9:1 v/v) and added of 20 Wl of [3 H]ethanolamine. The mixture was maintained under stirring at room temperature for 24 h, then the polymer was precipitated by diethyl ether/acetone mixture and washed ¢ve times with the same solvent mixture. The product was recovered by centrifugation and desiccated under vacuum. The polymer was ¢nally dissolved in 10 ml of water and ultra¢ltered using a cut-o¡ 10 000 Da membrane until complete elimination of the free [3 H]ethanolamine. 2.5. In vivo studies One-millimeter diameter fragments of Erlich solid tumour were subcutaneously implanted into 240 NCL mice. The tumour was left to develop for 8 days to reach a weight in the range of 0.1^0.2 g. The mice were randomly divided into ¢ve groups of 48 animals each. One hundred Wl of a 2 mg/ml [3 H]polymer solution in 0.02 M phosphate bu¡er, 0.15 M NaCl, pH 7.2 was injected into the tail vein of the animals. At scheduled times the animals were bled and euthanised and heart, lungs, kidneys, spleen, liver and tumour were taken. The blood samples were put into heparinised vials, centrifuged and the polymer content in the plasma was esti-

BBAGEN 25230 18-10-01

180

P. Caliceti et al. / Biochimica et Biophysica Acta 1528 (2001) 177^186

mated by radioactivity evaluation after addition of 3 ml of Instagel cocktail to 20 Wl of plasma. The tissues were carefully washed in saline solution, dried on paper and weighed. Soluene 350 was added to the organs (10 ml/g tissue) and steeped at 60³C until complete dissolution. Two hundred and ¢fty Wl of samples were added to 2.75 ml of Ionic Fluor and the radioactivity was evaluated by L-counter. One hundred Wl [3 H]mouse serum albumin solution (800 Wg/ml in 0.02 M phosphate bu¡er, 0.15 M NaCl, pH 7.2) was injected into ten tumour-bearing animals. At scheduled times the animals were killed and the blood, heart, lungs, kidney, spleen, liver and tumour were taken and processed as described above to estimate the plasma residue. 2.6. Data elaboration The plasma concentration means and the standard deviations ( þ S.D.) of the naked and derivatised PHEA were estimated on the basis of the radioactivity obtained from the plasma samples. The pharmacokinetic parameters were determined by computer elaboration of the polymer concentrations in plasma obtained at the scheduled times according to the equation: C…t† ˆ Ae3K t ‡ Be3 L t The concentration means and the standard deviations ( þ S.D.) of the naked and derivatised PHEAs in the various tissues were determined by the elaboration of the radioactivity values obtained from the tissue samples. The residual plasma volume in each organ was estimated by the [3 H]MSA levels and the obtained data were used to correct the polymer disposition concentrations in the tissues [21]. 3. Results and discussion Aimed at developing new soluble polymeric drug carriers with peculiar physico-chemical and biological properties, PHEA derivatives bearing PEGs (Mr 2000 or 5000 Da) or PEGs and hexadecylalkyle pendant molecules were prepared. PEG was selected because, by virtue of its high hydrophilic character, has been using largely in the preparation of `stealth' polymeric drug carriers. Indeed, PEG conjugation was demonstrated to convey to drug carriers as well as to proteins suitable properties such as limited uptake by the reticuloendothelial system, prolonged circulation time and improved immunological character [22^ 24]. The hexadecylalkyle was co-substituted at the polymeric backbone in the preparation of PHEA^PEG2000 ^C16 and PHEA^PEG5000 ^C16 to obtain new macromolecular carriers that one side can escape the phagocytic system and on the other can arrange into supramolecular struc-

tures, namely polymeric micelles, that can entrap physically lipophilic drugs. Furthermore, these conjugates should possess the suitable properties of PHEA such as biodegradability, solubility and biocompatibility. PHEA is obtained from PSI, a linear polyimide which can be cheaply and readily prepared by thermal polycondensation of aspartic acid. The preparation of PHEA involves a simple chemistry that yields, under controlled conditions, a polymer with low dispersivity and high degree of purity [20]. 3.1. Synthesis of PHEA^PEG copolymers PEG2000 or PEG5000 -K,L-poly(N-hydroxyethyl)-DL-aspartamide copolymers (PHEA^PEG2000 and PHEA^ PEG5000 ) were synthesised by a two-step procedure reported in Fig. 1: 1. Partial aminolysis of PSI by O-(2-aminoethyl)-OP-methylpolyethylene with Mw 2000 or 5000 to obtain PSI^ PEG copolymers; 2. Total aminolysis of PSI^PEG copolymers by ethanolamine to obtain PHEA^PEG copolymers.

The puri¢ed products were characterised by IR spectrophotometry, 1 H-NMR and elemental analysis. The IR spectra revealed the presence of a band at 953 cm31 attributable to ether C-O stretching of PEG. 1 HNMR analysis revealed a peak at 3.69 N that corresponds to -(OCH2 CH2 )n - of PEG chains. The degree of derivatisation (DD) by PEGs was determined by 1 H-NMR and was calculated by the following ratio: DD = (polyethyleneglycol groups/polymer repeating unit)U100 (mol). The DD was calculated by comparing the integral of the peak corresponding to protons at 3.69 N assigned to -(OCH2 CH2 )n - that belong to linked PEG with the integral of the peak related to protons at 4.74 N assigned to -NH-CH(CO)-CH2 - (belonging to PHEA). The DD values reported in Table 1 show that 1.25 mol% and 1.81 mol% (PEG mol/PHEA repeating unit mol) copolymers are obtained with PHEA^PEG2000 and PHEA^PEG5000 , respectively. The PEG conjugation degree was con¢rmed by the elemental analysis where a fair correspondence between the theoretical and the experimental data was found. The di¡erences in PEG conjugation degree observed with the two derivatives could be attributable to the di¡erent mol wt of the PEG. Indeed, the coupling reaction rate of PSI is not a simple function of the basicity of amine derivative but it depends greatly on the secondary structure of the polymer, i.e., ethanolamine reacts very quickly, whereas the reaction of comparatively basic amines (histamine) is unexpectedly slow [25,26]. To note that the strict control of the reaction conditions was found to be of primary relevance to obtain derivatives with proper DD.

BBAGEN 25230 18-10-01

P. Caliceti et al. / Biochimica et Biophysica Acta 1528 (2001) 177^186

181

Fig. 1. Scheme of PHEA^PEG copolymer synthesis.

Among the various parameters temperature, in particular, was found to determine the conjugation degree since runs carried out at 35³C and the same molar ratio yielded a product with a lower DD value (in the range of 0.6^0.63 mol%). 3.2. Synthesis of PHEA^PEG^C16 copolymers In order to obtain PHEA derivatives able to form polymeric micelles composed by a hydrated outer shell and a hydrophobic inner core that can incorporate poor watersoluble drugs, we prepared PHEA derivatives bearing PEG 2000 and hexadecylalkyl functions and PEG 5000 and hexadecylalkyl functions.

PHEA^PEG2000 ^C16 and PHEA^PEG5000 ^C16 were prepared starting from PSI by the three step procedure depicted in Fig. 2: 1. Partial aminolysis of PSI by O-(2-aminoethyl)-OP-methylpolyethylene with Mw 2000 or 5000 to obtain PSI^ PEG copolymers 2. Partial aminolysis of PSI^PEG copolymers by hexadecylamine to obtain PSI^PEG^C16 copolymers 3. Total aminolysis of PSI^PEG^C16 copolymers by ethanolamine to obtain PHEA^PEG^C16 copolymers

The IR spectroscopic analysis performed with the puri¢ed products revealed the presence of a band at 2854 cm31 that

BBAGEN 25230 18-10-01

182

P. Caliceti et al. / Biochimica et Biophysica Acta 1528 (2001) 177^186

Table 1 Main physico-chemical properties of PHEA, PHEA^PEG2000 , PHEA^PEG5000 , PHEA^PEG2000 ^C16 , and PHEA^PEG5000 ^C16 PHEA PHEA^PEG2000 PHEA^PEG5000 PHEA^PEG2000 ^C16 PHEA^PEG5000 ^C16

PEG DD (mol%)

C16 DD (mol%)

Yield %

Mw

Polydispersity index

^ 1.25 1.81 1.35 1.73

^ ^ ^ 4.9 6.6

^ 94 97 92 96

31 300 26 950 33 010 27 350 34 220

1.71 1.45 1.54 1.49 1.61

corresponds to C^H stretching of C16 chains and a band at 953 cm31 attributable to ether C^O stretching of PEG. 1 H-NMR analysis revealed a peak at 3.69 N that corresponds to -(OCH2 CH2 )n - of PEG chains and peaks at 0.87 and 1.28 N assigned, respectively, to -CH2 CH3 and -CH2 CH2 -CH2 - that belong to linked C16 . The degree of derivatisation (DD) by PEGs, determined by 1 H-NMR as above reported, indicated a substitution yield in the range of 1.35^1.73 mol%. The degree of derivatisation of C16 was also determined by 1 H-NMR and was calculated by the following ratio: DD = (hexadecyl groups/polymer repeating unit)U100 (mol). The DD was calculated by comparing the integral of the peak related to protons at 0.87 N assigned to -CH2 CH3 (or the integral of the peak related to protons at 1.28 N assigned to -CH2 -CH2 -CH2 -) that belong to linked C16 with the integral of the peak related to protons at 4.74 N assigned to -NH-CH(CO)-CH2 - (belonging to PHEA). The DD values of C16 reported in Table 1 (4.9 and 6.6 mol% for PHEA^PEG2000 ^C16 and PHEA^PEG5000 ^C16 , respectively) were con¢rmed by the elemental analysis and indicate that a higher substitution degree is obtained as compared to PEG. This further underlines the in£uence of the substituent physico-chemical properties on the bioconjugation process. 3.3. Pharmacokinetic studies In order to evaluate the potential of the synthesised carriers in the tumour drug delivery, the in vivo studies were carried out using mice bearing a subcutaneous solid tumour. For the study the polymers were 3 H-radiolabelled by conjugation of [3 H]ethanolamine to the polymeric backbone. This labelling procedure was chosen because it allowed for obtaining high radioactive compounds by conjugation of few [3 H]ethanolamine molecules. Furthermore, due to the the low molecular mass and the hydroxyl function of this radioactive compound, the labelling method allowed for the substantial preservation of the physicochemical properties of the carrier. Fig. 3 reports the time course pro¢les of PHEA and PHEA derivatives in plasma obtained by intravenous injection of the various carriers in mice. All of the pharmacokinetic behaviours ¢tted a bi-compartmental model indicating that the polymers undergo peripheral distribution.

Despite the polymers display apparently similar pharmacokinetic pro¢les, the pharmacokinetic parameters reported in Table 2 point out signi¢cant di¡erences in the in vivo performance of the various carriers. The PHEA distribution half time (t1=2 K) changes by both PEG and hexadecylalkyl conjugation. In particular, the distribution time increases as the PEG molecular mass increases while the distribution phase of the PEG/C16 derivatives is about two times longer with respect to the PEGylated counterparts. The k12 (central to peripheral compartment constant rate) values further con¢rm that the distribution rate of the various carriers to the peripheral compartment follows the rank order: PHEA 6 PHEA^PEG2000 6 PHEA^ PEG5000 6 PHEA^PEG2000 ^C16 6 PHEA^PEG5000 ^C16 . The high and similar k12 and k21 (peripheral to central compartment constant rate) values obtained with the naked PHEA point out that a rapid equilibrium between the central and peripheral compartment takes place. Instead, the lower k12 and k21 values of the PHEA derivatives indicate that the conjugates undergo a slower equilibrium during the distribution phase as compared to the plain polymer. To note that the PEG derivatives, and the PHEA^PEG5000 in particular, possess lower k21 as compared to k12 , indicating that these carriers are retained e¤ciently in the peripheral compartment. On the contrary, the PEG/C16 derivatives were found to accumulate in the peripheral compartment more slowly and are released more rapidly to the central one. All the polymers present higher distribution volumes (VdL ) with respect to the calculated central compartment volume (Vc ), indicating that a remarkable localisation in the peripheral compartment occurs. The VdL obtained with PHEA^PEG2000 and PHEA^PEG5000 , 150% and 300% of the plain PHEA one, respectively, points out that the PEG conjugation promotes the carrier disposition in the peripheral tissue and that the extent of accumulation increases as the PEG molecular mass increases. According to the k12 / k21 values, the distribution volumes of the PHEA^PEG^ C16 derivatives were found to be lower than the ones obtained with the PHEA^PEG copolymers, con¢rming their lower retention in the peripheral compartment. The di¡erent behaviour in plasma displayed by the various carriers can be ascribed to their uneven physicochemical character, namely the hydrophilic/hydrophobic balance and the structural arrangement of the macromolecules. In this regard, a preliminary study carried out with

BBAGEN 25230 18-10-01

P. Caliceti et al. / Biochimica et Biophysica Acta 1528 (2001) 177^186

183

Fig. 2. Scheme of PHEA^PEG^C16 copolymer synthesis.

Sudan Black demonstrated that the PEG/C16 derivatives solubilise hydrophobic molecules whereas this does not occur with the naked and the PEG-modi¢ed PHEAs. This evidence suggests that the conjugation a¡ects the structural conformation of the polymer. It is reasonable to think that PHEA, by virtue of its high hydrophilic character, assumes an extended and £exible conformation. PEG, which presents a high hydrodynamic volume [27], should further enhance this conformation. On the contrary the alkyl conjugation can induce the coiled arrangement of the macromolecules to micelle-like structures constituted by a hydrophobic core and the exposition of the PEG

function on the supramolecular surface. These structural di¡erences, together the unequal hydrophobic/hydrophilic balance, can dictate the pharmacokinetic properties of the carriers, namely rate and extent of the peripheral compartment distribution and elimination. Moreover, some preliminary information on the size of both micelle-assembled and unassembled copolymer macromolecules in aqueous medium have been obtained by light-scattering techniques so that the radius of unassembled PHEA^PEG5000 ^C16 and PHEA^PEG2000 ^C16 copolymers was evaluated to be equal to about to 4 and 3 nm, respectively, while the radius of micelle-assembled PHEA^PEG5000 ^C16 and

BBAGEN 25230 18-10-01

184

P. Caliceti et al. / Biochimica et Biophysica Acta 1528 (2001) 177^186

Fig. 3. Time course pro¢les of PHEA, PHEA^PEG2000 , PHEA^PEG5000 , PHEA^PEG2000 ^C16 , and PHEA^PEG5000 ^C16 in plasma. The standard deviations ( þ S.D.) were calculated on the basis of eight animals/point.

PHEA^PEG2000 ^C16 copolymers, evaluated at polymer concentrations above 0.001% w/v, was estimated to be equal to about 115 and 80 nm, respectively. Further studies are on-going on PHEA^PEG^C16 derivatives in order to con¢rm the possibility of their arrangement in micelle-like structures. 3.4. Tissue distribution studies In Fig. 4 the distribution pro¢les of the various carriers in liver, spleen, kidneys and solid tumour are reported. In lungs and heart all carriers displayed a negligible accumulation. The accumulation time courses in the kidneys show that both the PEG derivatives quickly disperse in this organ even though the polymer concentration drops from about 15% of the dose obtained after 15 min from administration to about 2% determined at 144 h. This behaviour

indicates that the PEG promotes the kidney elimination of the carrier probably because of the high hydrophilic character and the favourable extended conformation conferred by the PEG. On the contrary, the naked and the PEG/C16 derivatives slowly accumulate in this organ throughout the time suggesting that these macromolecules, despite their molecular mass not exceeding 65 000 Da usually considered the renal ultra¢ltration limit, are slowly eliminated by renal ultra¢ltration and are slightly entrapped in this tissue. In the spleen, liver and solid tumour, all polymers accumulated throughout the time to reach remarkable concentrations after 144 h from the administration. Among the carriers, the naked PHEA was the one displaying the highest disposition in the spleen (over 20% of the dose after 144 h) whilst no signi¢cant di¡erences were observed among the conjugates. The lower accumulation of the PEG and PEG/C16 derivatives as relative to the

Table 2 Main pharmacokinetic parameters obtained by intravenous injection to NCL mice of PHEA, PHEA^PEG2000 , PHEA^PEG5000 , PHEA^PEG2000 ^C16 , and PHEA^PEG5000 ^C16 A B K L t1=2K (min) t1=2L (min) AUC0ÿr (Wg min/ml) C0 (Wg/ml) CL (min31 ) Vc (ml/g) VdL (ml/g) k12 k21

PHEA

PHEA^PEG2000

PHEA^PEG5000

PHEA^PEG

204.98 168.4 0.049 4.9U1034 13.99 1410.4 3.5U10‡5 373.4 3.8U1035 0.036 0.078 0.026 0.022

199.85 126.6 0.025 4.9U1034 27.73 1420.7 2.7U10‡5 326.5 5.8U1035 0.047 0.12 0.014 0.009

238.9017 46.9980 0.0119 7.324U1034 58.3 946.2 8.4U10‡4 285.9 1.7U1034 0.0504 0.2333 0.0066 0.0025

135.04 199.3 0.011 8.7U1034 64.3 795.4 2.4U10‡5 334.4 6.1U1035 0.04 0.07 0.0035 0.0069

2000 ^C16

PHEA^PEG

5000 ^C16

162.05 196.2 5.7U1033 6.7U1034 121.05 1029.6 3.2U10‡5 358.3 4.8U1035 0.043 0.072 0.0018 0.0034

t1=2K , distribution phase half time; t1=2L , elimination phase half time; AUC0ÿr , area under the curve; C0 , concentration in plasma at time zero ; CL, clearance; Vc , central compartment volume ; VdL , distribution volume ; k12 , central to peripheral compartment constant rate; k21 : peripheral to central compartment constant rate.

BBAGEN 25230 18-10-01

P. Caliceti et al. / Biochimica et Biophysica Acta 1528 (2001) 177^186

185

Fig. 4. Distribution pro¢les of PHEA, PHEA^PEG2000 , PHEA^PEG5000 , PHEA^PEG2000 ^C16 , and PHEA^PEG5000 ^C16 in kidneys, spleen, liver and solid tumour. The standard deviations ( þ S.D.) were calculated on the basis of eight animals/point.

naked polymer can be attributable to the `stealth' properties of PEG which prevent the phagocytic uptake occurring in reticuloendothelial cell-rich organs. In the liver all the macromolecules accumulated remarkably throughout the experimental time. However, the PEG and C16 conjugation determines signi¢cant di¡erences in the carrier disposition behaviour. The two PEG derivatives display the lower localisation as compared to the other polymers. This is in agreement with the stealth properties of PEG and the structural £exibility of the carrier. High accumulation was instead observed with the two PEG/C16 conjugates that reach the level of 40% of the dose after 144 h. In this case the macromolecular arrangement into globular forms and the lipophilic character conferred by the alkyl functions to the carrier could promote the liver uptake. In this regard we must bear in mind that lipophilic as well as globular structures (liposomes and polymeric nanoparticles) are easily taken up by this organ. Interesting results were obtained as far as the disposition in the tumour is concerned. In all cases the accumulation reached the maximal concentration after 24 h from the injection and these tissue levels were maintained up to 144 h. The two PHEA^PEG^C16 derivatives were found to disperse into the neoplastic tissue more e¤ciently than the naked PHEA and the PHEA^PEG derivatives. Also in

this case, the di¡erent molecular conformation and hydrophilic/hydrophobic balance can be advocated to explain the di¡erent behaviour obtained with the various carriers. Indeed, it is known that the passive accumulation of macromolecules in solid tumours takes place by EPR e¡ect which takes advantage of the rich and permeable vasculature of this tissue. Data reported in the literature demonstrate that the vehicle architecture plays a relevant role in EPR since high molecular mass loosely coiled polymers can undergo extravasation and be retained in the tumour mass [28]. By such means, the particular conformation assumed by the PEG/C16 derivatives could promote the polymer localisation in this tissue. 4. Conclusions The design of polymeric drug carriers with speci¢c physico-chemical and biological properties is a challenging goal presented by many research groups working on controlled and prolonged delivery ¢elds. A suitable strategy to prepare tailor-made soluble macromolecules is preparation of graft-copolymers of macromolecules that on their own possess the requisites for drug delivery. In the case of PHEA, derivatives with interesting properties for drug de-

BBAGEN 25230 18-10-01

186

P. Caliceti et al. / Biochimica et Biophysica Acta 1528 (2001) 177^186

livery could be obtained by grafting at the polymeric backbone of selected hydrophilic and hydrophobic moieties. This allows obtaining macromolecules that on one side retain the suitable properties of PHEA, namely biodegradability, solubility, multifunctionality and biocompatibility, whereas on the other, the nature of the substituents conveys new physico-chemical and biopharmaceutical properties. The results reported in this paper show that stealth PHEA forms can be prepared by PHEA derivatisation with PEGs. Indeed, PEG conjugation allows, similarly to what was observed with liposomes and nanoparticles, to limit strongly the polymer uptake by macrophages of the immune system so prolonging time circulation of drug and improving its in vivo performance. Interestingly, the co-substitution of the hydrophilic PEG and the hydrophobic hexadecylalkyl yielded macromolecules that could arrange to supramolecular structures. This peculiar structural conformation permits the physical entrapment of hydrophobic molecules and can provide for the drug delivery by avoiding the chemical conjugation to the macromolecule. Furthermore, the architecture of these carriers was found to promote the delivery to tumour tissues by a passive mechanism. Both of these properties can be successfully exploited in the preparation of a suitable drug delivery in the treatment of cancer. Acknowledgements This research was supported by MURST and CNR grant programs. The authors thank Dr. Maria Rosalia Mangione of the CNR of Palermo for contribution. References [1] E. Mathiowitz (Ed.), Encyclopedia of Controlled Drug Delivery, Wiley, New York, 1999. [2] S. Brocchini, R. Duncan, Pendent drugs, release from polymers, in: E. Mathiowitz (Ed.), Encyclopedia of Controlled Drug Delivery, Wiley, New York, 1999. [3] K. Kataoka, Targetable polymeric drugs, in: K. Park (Ed.), Controlled Drug Delivery : Challenges and Strategies, Am. Chem. Soc., 1997, pp. 49^71 [4] H. Bader, H. Ringsdorf, B. Schmidt, Water soluble polymers in medicine, Angew. Makromol. Chem. 123/124 (1984) 457^485. [5] G.S. Kwon, K. Kataoka, Block copolymer micelles as long-circulating drug vehicles, Adv. Drug Deliv. Rev. 16 (1995) 295^309. [6] G.S. Kwon, T. Okano, Polymeric micelles as new drug carriers, Adv. Drug Deliv. Rev. 21 (1996) 107^116. [7] M. Yokoyama, A. Satoh, Y. Sakurai, T. Okano, Y. Matsumura, T. Kakizoe, K. Kataoka, Incorporation of water-insoluble anticancer drug into polymeric micelles and control of their particle size, J. Controlled Release 55 (1998) 219^229. [8] G.S. Kwon, M. Naito, K. Kataoka, M. Yokoyama, Y. Sakurai, T. Okano, Block copolymer micelles as vehicles for hydrophobic drugs, Colloid Surf. B Biointerfaces 2 (1994) 429^434.

[9] S.B. La, T. Okano, K. Kataoka, Preparation and characterization of the micelle-forming polymeric drug indomethacin-incorporated poly(ethylene oxide)-poly(L-benzyl-L-aspartate) block copolymer micelles, J. Pharm. Sci. 85 (1996) 85^90. [10] K. Kataoka, H. Togawa, A. Harada, K. Yasugi, T. Matsumoto, S. Katayose, Spontaneous formation of polyion complex micelles with narrow distribution from antisense oligonucleotide and cationic block copolymer in physiological saline, Macromolecules 29 (1996) 8556^ 8557. [11] V.P. Torchilin, Structure and design of polymeric surfactant-based drug delivery systems, J. Controlled Release 73 (2001) 137^172. [12] S.E. Matthews, C.W. Pouton, M.D. Threagill, Macromolecular systems for chemotherapy and resonance imaging, Adv. Drug Deliv. Rev. 18 (1996) 219^267. [13] R. Duncan, Drug polymer conjugates; potential for improved chemotherapy, Anticancer Drugs 3 (1992) 175^210. [14] G. Giammona, G. Puglisi, B. Carlisi, R. Pignatello, A. Spadaro, A. Caruso, Polymeric prodrugs: K,L-poly(N-2-hydroxyethyl)-DL-aspartamide as a macromolecular carrier for some non-steroidal antiin£ammatory agents, Int. J. Pharm. 57 (1989) 55^62. [15] G. Giammona, B. Carlisi, G. Pitarresi, G. Fontana, Hydrophilic and hydrophobic polymeric derivatives of antiin£ammatory agents such as Alclofenac, Ketoprofen and Ibuprofen, J. Bioact. Compat. Polym. 6 (1991) 129^141. [16] R. Duncan, D. Starling, F. Rypacek, J. Drobnic, J.B. Lloyd, Pinocytosis of poly-K,L-(N-2-hydroxyethyl)-DL-aspartamide, Biochim. Biophys. Acta 717 (1982) 248^254. [17] G. Giammona, G. Puglisi, G. Cavallaro, G. Spadaro, G. Pitarresi, Chemical stability and bioavailability of acyclovir coupled to K,Lpoly(N-2-hydroxyethyl)-DL-aspartamide, J. Controlled Release 33 (1995) 261^271. [18] G. Cavallaro, G. Pitarresi, M. Licciardi, G. Giammona, Polymeric prodrug for release of an antitumoral agent by speci¢c enzymes, Bioconjug. Chem. 12 (2001) 143^151. [19] R. Mendichi, G. Giammona, G. Cavallaro, A. Giacometti Schieroni, Molecular characterisation of a K,L-poly[(hydroxyethyl)-DL-aspartamide] by light scattering and viscosimetric studies, Polymer 41 (2000) 8649^8657. [20] G. Giammona, B. Carlisi, S. Palazzo, Reaction of K,L-poly(N-2-hydroxyethyl)-DL-aspartamide with derivatives of carboxylic acid, J. Polym. Sci. Polym. Chem. Ed. 25 (1987) 2813^2818. [21] T. Yamaoka, Y. Tabata, Y. Ikada, Comparison of body distribution of poly(vinyl alcohol) with other water soluble polymer after intravenous administration, J. Pharm. Pharmacol. 50 (1995) 349^354. [22] C. Monfardini, F.M. Veronese, Stabilisation of substances in circulation, Bioconjug. Chem. 9 (1998) 418^450. [23] R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubezkoy, V. Torchilin, R. Langer, Biodegradable long-circulating polymeric nanospheres, Science 263 (1994) 1600^1603. [24] D. Bazile, C. Prud'homme, M.T. Bassoulet, M. Mazlard, G. Spenlehauer, M. Veillard, Stealth MePEG-PLA nanoparticles avoid uptake by the mononuclear phagocyte system, Int. J. Pharm. 29 (1995) 53^ 65. [25] J. Vlasak, F. Rypacek, J. Drobnik, V. Saudek, Properties and reactivity of polysuccinimide, J. Polym. Sci. Polym. Symp. 66 (1979) 59^ 64. [26] G. Giammona, L.I. Giannola, B. Carlisi, M.L. Bajardi, Synthesis of macromolecular prodrug of Procaine, Histamine and Isoniazid, Chem. Pharm. Bull. 37 (1989) 2245^2247. [27] J.M. Harris, S. Zalipsky (Eds.), Poly(ethylene glycol). Chemistry and Biological Applications, ACS, Washington, DC, 1997. [28] R. Duncan, Polymer conjugates for tumour targeting and intracytoplasmatic delivery. The EPR e¡ect as a common gateway, Res. Focus Rev. 2 (1999) 441^449.

BBAGEN 25230 18-10-01