Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing water-soluble prodrugs of paclitaxel

Journal of Controlled Release 63 (2000) 141–153 www.elsevier.com / locate / jconrel

Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing water-soluble prodrugs of paclitaxel Maurizio Ceruti, Paola Crosasso, Paola Brusa, Silvia Arpicco, Franco Dosio, Luigi Cattel* Dipartimento di Scienza e Tecnologia del Farmaco, Universita` di Torino, Via Pietro Giuria 9, 10125, Torino, Italy Received 28 May 1999; accepted 19 August 1999

Abstract Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, used clinically for the treatment of ovarian and breast cancer. Due to its aqueous insolubility it is administered dissolved in ethanol and Cremophor EL (polyethoxylated castor oil), which has serious side effects. In order to eliminate this vehicle, in previous work we entrapped paclitaxel in conventional and in polyethylene glycol coated liposomes. However, in neither formulation did we obtain satisfactory entrapment efficiency. In this study we increased the paclitaxel concentration entrapped in liposomes by incorporating different water-soluble prodrugs, such as the 29-succinyl, 29-methylpyridinium acetate and 29-mPEG ester paclitaxel derivatives, in the lipid vesicles. Liposomes containing 29-mPEG (5000)–paclitaxel showed the best performance in terms of stability, entrapment efficiency and drug concentration (6.5 mg ml 21 ). The in vitro cytotoxic activity of this liposomal prodrug was similar to that of the parent drug. The pharmacokinetic parameters for the free and for the liposomal prodrugs fitted a bi-exponential plasma disposition. The most important change in pharmacokinetic values of the prodrug vs. the free drug liposomal formulations was t 1 / 2 b, plasma lifetime, which was longer in liposomes containing 29-mPEG (5000)– paclitaxel.  2000 Elsevier Science B.V. All rights reserved. Keywords: Paclitaxel prodrugs; Liposomes; Pharmacokinetics

1. Introduction Paclitaxel (Taxol), a diterpenoid derived from the needles and bark of the Pacific yew tree Taxus brevifolia [1], is an antimitotic agent with a unique action mechanism; it binds polymerized tubulin, inhibiting depolymerisation [2–4]. Clinical trials have shown that paclitaxel has *Corresponding author. Tel.: 139-11-670-7693; fax: 139-11670-7695. E-mail address: [email protected] (L. Cattel)

antitumoral activity particularly against ovarian carcinoma, breast cancer, head and neck cancers and non-small cell lung cancer [5–7]. In view of its aqueous insolubility, paclitaxel has to be dissolved in a mixture of 50% ethanol and 50% Cremophor EL (a polyoxyethylated castor oil). Cremophor has been associated with a number of side effects, including hypersensitivity and neurotoxicity [8–14]. The use of prophylactic steroids and histamine receptor antagonists as anti-allergic pre-medication may decrease the incidence and severity of acute hypersensitivity reactions. However, milder reactions have still been

0168-3659 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00198-4

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found to occur in 5–30% of patients. While the impressive efficacy of paclitaxel has driven clinical usage of the drug forward despite these shortcomings, a water-soluble formulation of paclitaxel could obviate these problems and improve its pharmacological profile. Numerous attempts have been made to improve the solubility and pharmacological properties of paclitaxel. To improve its solubility, complexes with cyclodextrins [15–17], mixed-micellar formulations [18], emulsions [19] and polymeric micellar paclitaxel formulations using amphiphilic block copolymers have been investigated [20,21]. Prodrug synthesis has also been extensively studied, to increase the aqueous solubility of Paclitaxel. The preferred position for the preparation of prodrugs of paclitaxel is the 29 position, since many 29-acyl– paclitaxel derivatives hydrolyze fairly rapidly back to paclitaxel in the blood compartment [22]. The preparation of 29-succinylpaclitaxel has been described [23] and the first approach thus consisted in the preparation of succinylamide derivatives [24]. Since the configuration of the C-7 hydroxyl group does not seem to be a factor in determining cytotoxicity, C-7 prodrug esters have also been synthesized [23]. Nicholau designed Taxol ester with strong electron withdrawing substituents, such as alkoxy in the a position of the ester, in order to accelerate hydrolytic cleavage [25]. In vitro these prodrugs have cytotoxic properties against tumor cell lines comparable to those of paclitaxel; in addition, human plasma catalyses the release of active paclitaxel. Water-soluble paclitaxel–sialyl derivatives have also been developed [26]. 29-methylpyridinium acetate–paclitaxel (29-MPA–paclitaxel), a stable prodrug very soluble in water, rapidly releases paclitaxel in human plasma with antitumoral properties similar to those of parent drug [27]. In addition, 29-MPA–paclitaxel, combining a highly hydrophobic segment (Taxol skeleton) with a polar head (pyridinium moiety) showed the characteristic features of a self-aggregating molecule. Polyethylene glycol (PEG) is an amphiphilic macromolecule that, in the molecular weight range 2–12 kDa, imparts greater aqueous solubility to conjugates of hydrophobic organic compounds or proteins, augmenting circulating half-time and decreasing immunogenicity [28]. A prodrug strategy employing PEG as a solubilising agent has been successfully demonstrated in the case of paclitaxel

[29,30]. PEG has been entrapped in liposomes to improve the pharmacokinetics. The PEG prodrug of paclitaxel, simply dissolved in water, delivers paclitaxel as effectively in vivo as the current Cremophor EL formulation. An alternative approach to improving water solubility of hydrophobic drugs involves conjugation to a stable macromolecular drug carrier. We therefore prepared a paclitaxel–albumin conjugate in which the drug was covalently linked to human serum albumin through a succinyl spacer [31]. Liposomes have also been used to formulate a variety of poorly soluble drugs [32–35]. Different liposome-based formulations containing paclitaxel have been developed, with good in vitro and in vivo antitumoral activity, similar or only slightly less than that of the parent drug [18,36–40]. We recently compared conventional paclitaxel-loaded liposomes with a PEGylated liposomal formulation, in terms of in vitro drug release rate, cytotoxic activity, pharmacokinetic and biodistribution behavior [41]. This research aimed to improve solubilisation and targeting of paclitaxel, using different water-soluble prodrugs entrapped in liposomes. It was seen that this approach increases drug entrapment, maintaining good stability and cytotoxic activity, as well as enhancing plasma stability of paclitaxel.

2. Materials and methods

2.1. Chemicals Paclitaxel (compound 1) was donated by Indena (Milan, Italy). Egg yolk phosphatidylcholine (PC), phosphatidylglycerol (PG transesterified from egg PC), cholesterol (CHOL), dipalmitoylphosphatidylcholine (DPPC), stearylamine (SA), succinic anhydride, 2-fluoro-1-methylpyridinium tosylate and methoxypoly (ethylene glycol) acid (mPEG acid 5 kDa) were from Sigma Chemical (Milan, Italy). The purity of the phospholipids was .99%.

2.2. Synthesis of 29 -succinyl–paclitaxel (2) Paclitaxel (1) (33 mg, 0.0386 mmol) was dissolved in 0.5 ml of dry pyridine to which 7.7 mg of succinic anhydride (0.0772 mmol) and 0.5 mg

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(0.00386 mmol) of 4-dimethylaminopyridine were added [31]. The resulting solution was stirred for 3 h at room temperature. The product was purified by chromatography on a silica-gel 60 column to give 34.6 mg of compound 2. Rf was 0.42 in chloroform– methanol (90:10). Yield: 94%. 1 H-NMR (CDCl 3 ): d 1.11 (s, 3H, C17–H), 1.19 (s, 3H, C16–H), 1.62 (s, 3H, C19–H), 1.76 (s, 3H, C18–H), 2.2 (m, 2H, C14–H), 2.22 (s, 3H, C10–OAc), 2.43 (s, 3H, C4– OAc), 2.6 (m, 14H, 16H), 2.22 (s, OAc), 2.43 (s, OAc), 2.6 (m, 4H, CH 2 CH 2 ), 3.34 (d, 1H, C3–H), 4.17 (d, 1H, C20–H), 4.48 (d, 1H, C7–H), 4.96 (dd, 1H, C5–H), 5.51 (d, 1H, C29–H), 5.67 (d, 1H, C2–H), 6.21 (t, 1H, C13–H), 6.27 (s, 1H, C10–H), 7.07 (d, 1H, NH), 7.3 (m, 39-Ph), 7.4 (m, 39-NBz), 7.5 (m, 2-OBz), 7.73 (d, 39-OBz), 8.1 (d, 2-OBz).

2.3. Synthesis of 29 -methylpyridiniumacetate– paclitaxel (3) Paclitaxel (10 mg, 0.012 mmol) was dissolved in dichloromethane (0.4 ml) and treated with triethylamine (2 ml, 1.3 equiv.) and 2-fluoro-1methylpyridinium tosylate (4 mg, 1.2 equiv.), under dry argon [27]. After stirring at room temperature for 30 min, the reaction was complete, as shown by HPLC analysis (Waters, Symmetry, C 18 column, 10 mM ammonium acetate in 20% water–methanol→methanol over 30 min, 0.8 ml min 21 , lmax 5 254 nm, 280 nm. R: 1, 25.1, 3: 21.5). The crude product was purified by HPLC, and solvent was removed under vacuum to give 10 mg of compound 3. Yield: 80%. 1 H-NMR (CDCl 3 ): d 1.11 (s, 3H, C17–H), 1.2 (s, 3H, C16–H), 1.57(s, 3H, C19–H), 1.67 (s, 3H, C18–H), 2.2 (m, 2H, C14–H), 2.22 (s, 3H, C10–OAc), 2.3 (s, 3H, C4–OAc), 2.24 (m, 14H, 16H), 2.32 (s, OAc), 2.43 (s, OAc), 2.7 (m, 4H, CH 2 CH 2 ), 3.9 (d, 1H, C3–H), 4.17 (d, 1H, C20–H), 4.48 (d, 1H, C7–H), 5.2 (dd, 1H, C5–H), 5.51 (d, 1H, C29–H), 5.67 (d, 1H, C2–H), 6.21 (t, 1H, C13–H), 6.27 (s, 1H, C10–H), 7.07 (d, 1H, NH), 7.3 (m, 39-Ph), 7.4 (m, 39-NBz), 7.5 (m, 2-OBz), 7.73 (d, 39-OBz), 8.1 (d, 2-OBz), 8.41 (t, 1H, Pyr–H), 10.48 (d, 1H, Pyr–H).

2.4. Synthesis of 29 -mPEG–paclitaxel (4) 86 mg of mPEG acid (0.0172 mmol) were dis-

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solved in 10 ml of anhydrous methylene chloride, and to this solution, at 08C, 4.38 mg (0.0398 mmol) of dimethylaminopyridine (DMAP), 3.66 ml (0.023 mmol) of diisopropylcarbodiimide (DIPC) and 20 mg (0.023 mmol) of paclitaxel were added [29]. The resulting solution was allowed to warm at room temperature and left for 16 h. The reaction mixture was washed with 0.1 N HCl, dried and evaporated in vacuum to yield the product as a white solid, which was dissolved in methylene chloride and crystallized from diethyl ether to obtain 90.6 mg of compound 4. Yield: 90%. 1 H-NMR (CDCl 3 ): d 1.11 (s, 3H, C17– H), 1.19 (s, 3H, C16–H), 1.62 (s, 3H, C19–H), 1.76 (s, 3H, C18–H), 2.2 (m, 2H, C14–H), 2.22 (s, 3H, C10–OAc), 2.43 (s, 3H, C4–OAc), 2.6 (m, 14H, 16H), 2.22 (s, OAc), 2.43 (s, OAc), 2.6 (m, 4H, CH 2 CH 2 ), 3.30 (d, 1H, C3–H), 3.59–3.71 (br s, PEG) 4.17 (d, 1H, C20–H), 4.48 (d, 1H, C7–H), 4.96 (dd, 1H, C5–H), 5.51 (d, 1H, C29–H), 5.67 (d, 1H, C2–H), 6.21 (t, 1H, C13–H), 6.27 (s, 1H, C10–H), 7.07 (d, 1H, NH), 7.3 (m, 39-Ph), 7.4 (m, 39-NBz), 7.5 (m, 2-OBz), 7.73 (d, 39-OBz), 8.1 (d, 2-OBz).

2.5. Chemical stability of paclitaxel prodrugs 1 mg of compound 2, 3 or 4 was dissolved in 50 ml of dimethyl sulfoxide and diluted with phosphate buffer solutions at pH 5.8 and 7.4 to a final volume of 1 ml. After incubation at 48C, aliquots of 100 ml were removed at different times and diluted by adding water and acetonitrile to a total volume of 0.5 ml; the paclitaxel released from the prodrug was extracted using the liquid-phase extraction method and analyzed using HPLC (Merck Hitachi HPLC system). Paclitaxel was extracted by adding 4.0 ml of tert-butyl methyl ether and vortex-mixing the sample for 30 s. The mixture was then centrifuged for 15 min at 1500 rpm (ALC 4226 centrifuge, Italy) after which 3.0 ml of organic layer was transferred and evaporated to dryness under nitrogen. The residue was reconstituted with 100 ml of an acetonitrile– water mixture (60 / 40). 50 ml of the solution was injected into a Symmetry C 18 column, 5 mm (Waters, Milan, Italy), equipped with a C 18 column guard. The column was eluted with acetonitrile–water (60:40). Detection was performed using UV adsorption measurement at 227 nm (flow-rate 1

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Table 1 29-succinyl paclitaxel (2) liposomes a Phospholipidic composition

Initial drug–lipid molar ratio b

Final drug–lipid molar ratio b

Paclitaxel concentration c (mg ml 21 )

Entrapment efficiency d (%)

PC–PG 9:1 (A) PC–PG–CHOL 9:1:2 (B) PC–PG–CHOL 9:1:5 (C) PC–SA 9:1 (D) PC–SA–CHOL 9:1:2 (E) PC–SA–CHOL 9:1:5 (F)

1:25 1:21 1:21 1:21 1:21 1:28

1:33 1:42 1:55 1:20 1:37 1:48

1.0 0.8 0.6 1.8 0.9 0.7

75 51 38 100 57 58

a

Values are means of three experiments. S.D. values (not reported) were below 5% of the mean values in all cases. Drugs and lipids were evaluated as described in Section 2. c Drug concentration was determined by HPLC analysis (RP18 ) after liquid-phase extraction and was expressed as paclitaxel concentration. d Entrapment efficiency was expressed as the ratio between the final drug-to-lipid ratio and the initial drug-to-lipid ratio. b

ml min 21 ). To calculate peak areas, the detector was interfaced to an electronic integrator (Perkin Elmer LCI-100). The drug concentration was calculated from standard curves. The assay was linear over the concentration range tested (20–800 ng) [31].

2.6. Preparation and characterization of liposomes Several kinds of phospholipid compositions were used as shown in Tables 1 and 2. Briefly, lipid films of specific phospholipid composition, with or without cholesterol, containing paclitaxel or its prodrugs, were hydrated with 5 mM Hepes buffer (pH 7.4) containing 145 mM NaCl (buffer 1), by shaking at room temperature, or at 418C for liposomes containing DPPC, followed by 10 cycles of extrusion through Nucleopore filters with 0.1 mm pores. After extrusion, the preparations were dialyzed for 16 h at 48C, to remove unentrapped drug. Phospholipid

phosphorus was assessed in each liposome preparation by phosphate assay after destruction with perchloric acid [42]. Size distribution of liposomes was monitored by photon correlation spectroscopy using a Coulter Model N4SD submicron particle analyzer (Coulter Electronics, FL, US). The amount of prodrug entrapped in the liposomes was determined by HPLC (Merck Hitachi HPLC system) as follows. 10 ml of the liposomal suspension was destroyed by adding 200 ml of acetonitrile. After sonication and centrifugation for 15 min at 10 000 rpm (ALC 4224 centrifuge, Italy), 40 ml of the solution was injected into a Symmetry C 18 column, 5 mm (Waters, Milan, Italy). Different elution mixtures were used for paclitaxel and the three prodrugs. Paclitaxel was eluted with acetonitrile–water 60:40, compound 2 with acetonitrile–water–phosphoric acid 60:40 pH 2.8, compound 3 with an eluent gradient A–B over 30 min where A is 20% 10 mM

Table 2 29-MPA paclitaxel (3) liposomes a Phospholipidic composition

Initial drug–lipid molar ratio b

Final drug–lipid molar ratio b

Paclitaxel concentration c (mg ml 21 )

Entrapment efficiency d (%)

PC–PG 9:1 (G) PC–PG–CHOL 9:1:5 (H)

1:7 1:7

1:8.5 1:9.5

4.0 3.5

80 71

a

Values are means of three experiments. S.D. values (not reported) were below 5% of the mean values in all cases. Drugs and lipids were evaluated as described in Section 2. c Drug concentration was determined by HPLC analysis (RP18 ) after liquid-phase extraction and was expressed as paclitaxel concentration. d Entrapment efficiency was expressed as the ratio between the final drug-to-lipid ratio and the initial drug-to-lipid ratio. b

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ammonium acetate (pH 6) and B is 100% acetonitrile, while compound 4 was eluted with 75:25 methanol–water. Detection was performed using UV adsorption measurement at 227 nm for compounds 1, 2 and 4, and at 280 nm for compound 3. To calculate peak areas, the detector was interfaced to an electronic integrator (Perkin Elmer LCI-100). Drug concentrations were calculated from standard curves on a daily basis. The assays were linear over the concentration range tested (5–30 ng).

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adhesion. Various dilutions (100 ml) of substances were added in triplicate and incubated for 72 h. The supernatants were removed and the cells washed and incubated for 16 h with 200 ml of medium, containing 1 mCi of [ 3 H]-leucine (58 Ci mmole 21 ) (Amersham, Bucks, UK). The cells were harvested (Skatron cell harvester, Norway) and the incorporated radioactivity b-counted. The results were expressed as the percentage of [ 3 H]-leucine incorporation, with respect to control values, background values being subtracted.

2.7. Physical and chemical stability of liposomes 2.10. Pharmacokinetic studies Formulations D, G and N (Table 4) were evaluated for physical and chemical stability in the storage condition (buffer 1) at 48C. Drug leakage from liposomes was determined by removing portions of liposomes from a pool stored at pH 7.4 and 48C, at various times (24 h, 1 week, 2 months). The liposomes containing paclitaxel prodrugs were subjected to centrifugation at 12 000 rpm (ALC 4226 centrifuge) for 30 min; in these conditions they remained suspended and paclitaxel hydrolyzed from the prodrug precipitated [43]. The formulations were then dialyzed for 16 h at 48C to remove the released prodrug. After separation, liposomes were re-analyzed for drug amount as described above. A change in the amount of entrapped drug was interpreted as an indication of liposomal instability. Changes in mean diameter were also monitored (not reported).

2.8. Tumor cell lines The cell lines used were HT-29, a human colorectal adenocarcinoma, and MeWo, a human melanoma (donated by Dr. S. Canevari, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy). Both cell lines were maintained in RPMI 1640 medium containing 10% fetal calf serum and 0.1% antibiotics (penicillin, streptomycin, gentamycin), in a 5% CO 2 humidified atmosphere at 378C [44].

2.9. Inhibition of cellular protein synthesis The method used was essentially as described elsewhere [44] with minor modifications. The cells, maintained as described above, were seeded in microtiter plates and incubated for 3 h to allow cell

Pharmacokinetics studies were performed as described elsewhere [45], using female Balb / c mice (1 month old, 18–20 g, Charles River Italia, Milan, Italy). The care and handling of animals were in accordance with the provisions of the European Economic Community Council Directive 86 / 209, recognized and adopted by the Italian Government (approval decree D.M. No. 230 / 95-B). The animals were treated with 100 ml of 29-PEG–paclitaxel dissolved in physiological solution or with 100 ml of 29-PEG–paclitaxel liposomes, both at a dose of 0.3 mg / mouse (15 mg kg 21 ) of paclitaxel. For comparison, a pharmacokinetic evaluation of paclitaxel was performed. Paclitaxel (15 mg kg 21 ) was dissolved in Diluent 12 (50:50 Cremophor–ethanol) at 7 mg ml 21 and then diluted with 5% glucose to 3 mg ml 21 prior to injection. The different formulations were injected through the tail vein (groups of 3–5 animals each). Blood samples were taken from the retro-orbital plexus at various times (0, 30, 60, 240, 1440 min), and centrifuged immediately (5 min, 10 000g). Plasma samples were stored at 2208C and subsequently analyzed for paclitaxel. Each experiment was repeated three times. All samples (50 ml) were buffered with 0.4 ml of 0.2 M ammonium acetate (pH 5.0) and 50 ml of acetonitrile. Paclitaxel was extracted from plasma by solid-phase extraction [46] on 1 ml Sep Pac Vac CN Cartridges (Waters, Milan, Italia). The columns were first conditioned with 2 ml of methanol followed by 2 ml of 0.01 M ammonium acetate (pH 5); they were not allowed to dry. The samples were loaded into individual columns and washed with 2 ml of 0.01 M ammonium acetate (pH 5.0) and 1 ml of n-hexane. The columns

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were dried under vacuum for 1 min. Paclitaxel was eluted from the columns into collection tubes using two 1 ml volumes of 0.1% triethylamine in acetonitrile. The eluents were evaporated to dryness under nitrogen. The residues were reconstituted in 200 ml of 60:40 acetonitrile–water, and 50 ml were injected into the HPLC column as described above. The following pharmacokinetic parameters were determined using Kinetica 2.00.200 software (InnaPhase): area under the curve (AUC); mean residence time (MRT); total body clearance (Cl); volume of distribution at steady state (Vss ); plasma half-life for the distribution and elimination phases (t 1 / 2 a, t 1 / 2 b).

3. Results

3.1. Synthesis of paclitaxel prodrugs Paclitaxel has a very limited loading efficiency in liposomes. Inclusion of cholesterol, or increasing the paclitaxel–lipid ratio above 1:33, leads to a dramatic decrease in loading efficiency and physical stability of the formulation, so that the highest paclitaxel concentration achievable in the liposomal formulation is 1–1.5 mg ml 21 [36,37,41].

To increase drug entrapment in the liposomal formulation, maintaining good stability and cytotoxic activity, we derivatized paclitaxel with hydrophilic groups (negative, positive or neutral charged) in order to obtain more water-soluble compounds with reduced interaction with the liposomal bilayer and increased entrapment efficiency in the aqueous compartment [41]. Three paclitaxel prodrugs were prepared (Fig. 1): 29-succinyl–paclitaxel (compound 2) was prepared by esterification of the 29-hydroxyl group, using succinic anhydride in the presence of 4-dimethylamino pyridine in dry pyridine [23]. 29methylpyridinium acetate–paclitaxel (compound 3) was obtained by reacting paclitaxel with 2-fluoro-1methylpyridinium tosylate, in the presence of triethylamine in dichloromethane [27]. Purification by reversed-phase high-pressure liquid chromatography followed by ion exchange gave pure 29-MPA paclitaxel. 29-PEG–paclitaxel (4) was prepared by condensation of the 29-hydroxyl group of paclitaxel with the carboxylic group of mPEG acid in presence of diisopropylcarbodiimide (DIPC) as condensing agent and dimethylaminopyridine (DMAP) as base [29]. The isolated prodrugs were fully characterized by

Fig. 1. Paclitaxel prodrugs preparation.

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NMR. Crystallization of compound 4 gave it a 97% purity, the remaining 3% being free paclitaxel.

3.2. Stability of paclitaxel prodrugs Compounds 2, 3 and 4 were incubated at 4 or 378C in PBS buffer and at pH 5.8 or 7.4, to determine their stability to hydrolysis (Figs. 2 and 3). The ester linkage of compound 2 was very stable at 48C, either at pH 5.8 or 7.4, for up to 6 days of storage, while at 378C the percentage of paclitaxel released from the prodrug increased after incubation from pH 5.8 to pH 7.4 (4% to 12%). Compound 3 showed good stability at 48C in buffer solution at the two pH values tested. After 6 days at 48C, only 4% of paclitaxel had been released from the prodrug. When incubated at 378C, the amount of paclitaxel released increased after 24 h to the value of 30–35%. For compound 4, the best storage conditions were pH 5.8 and 48C. In these conditions the prodrug released only 4.5% of paclitaxel after 6 days of incubation. The same results were observed during

Fig. 3. Paclitaxel release at 378C from 29-PEG–paclitaxel (circles), 29-succinyl–paclitaxel (triangles pointing down) and 29MPA–paclitaxel (hexagons) in PBS buffer pH 7.4, 29-PEG–paclitaxel (squares), 29-succinyl–paclitaxel (triangles pointing up) and 29-MPA–paclitaxel (diamonds) in PBS buffer pH 5.8. The values are arithmetic means of three experiments. S.D. values not reported (less than 6%).

incubation at 378C; after 24 h at pH 7.4, 94% of paclitaxel was released, while at pH 5.8 the release was markedly slower (15%). In addition, in human plasma at 378C, 29-PEG–paclitaxel promptly released the parent drug (t 1 / 2 hydrolysis, 30 min) showing the lability of the ester derivative.

3.3. Preparation of liposomes

Fig. 2. Paclitaxel release at 48C from 29-PEG–paclitaxel (circles), 29-succinyl–paclitaxel (triangles pointing up) and 29-MPA–paclitaxel (triangles pointing down) in PBS buffer pH 5.8, 29-PEG– paclitaxel (squares), 29-succinyl–paclitaxel (triangles pointing up) and 29-MPA–paclitaxel (triangles pointint down) in PBS buffer pH 7.4. The values are arithmetic means of three experiments. S.D. values not reported (less than 6%).

Different liposomal formulations were used to entrap paclitaxel–prodrugs, varying liposome properties such as charge and membrane fluidity. As shown in Table 1, which gives data for compound 2, using a negatively charged membrane, phospholipid composition A PC–PG 9:1, containing the prodrug and lipids in the molar ratio of 1:33 (drug–lipid), was found to have good entrapment efficiency (75%) and limited drug concentration in the liposomal formulation (maximum 1 mg ml 21 ); the same was observed elsewhere using unmodified paclitaxel [41]. The best results (1.8 mg ml 21 ) were obtained using positively charged liposomes including 10% of SA in

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Table 3 29-mPEG–paclitaxel (4) liposomes a Phospholipidic composition

Initial drug–lipid molar ratio b

Final drug–lipid molar ratio b

Paclitaxel concentration c (mg ml 21 )

Entrapment efficiency d (%)

PC–PG 9:1 (I) PC–PG–CHOL 9:1:5 (L) PC–PG–CHOL 9:1:5 (M) PC–PG–CHOL 9:1:5 (N)

1:12.6 1:12.6 1:6.3 1:3.9

1:15 1:16.5 1:8.25 1:5.10

2.0 2.0 4.0 6.5

84 77 78 78

a

Values are means of three experiments. S.D. values (not reported) were below 5% of the mean values in all cases. Drugs and lipids were evaluated as described in Section 2. c Drug concentration was determined by HPLC analysis (RP18 ) after liquid-phase extraction and was expressed as paclitaxel concentration. d Entrapment efficiency was expressed as the ratio between the final drug-to-lipid ratio and the initial drug-to-lipid ratio. b

the phosphatidylcholine bilayer (D). Moreover, it was impossible to increase paclitaxel concentration above 1.8 mg ml 21 or drug-to-lipid ratio above 20. Thus, introduction of the succinic group in the hydrophobic structure of paclitaxel may be insufficient to increase its hydrophilicity and reduce molecular interaction with the phospholipidic bilayer. Furthermore, analogously to paclitaxel liposomes, inclusion of 20 or 50% of cholesterol in the negatively or positively charged membrane (B, C, E, F), dramatically decreased both entrapment efficiency and physical stability of the formulation [41]. Compound 3 (Table 2) was well entrapped in negatively charged liposomes. Formulation G afforded good loading efficiency and a paclitaxel concentration of 4 mg ml 21 , and the inclusion of 50% cholesterol only resulted in a small reduction in entrapment efficiency (3.5 mg ml 21 ). The neutral and highly water-soluble PEGylated paclitaxel (4) was entrapped in different liposomal formulations (Table 3). Formulation N, composed of

PC–PG–CHOL 9:1:5, afforded good loading efficiency, increasing paclitaxel prodrug concentration in the liposomes, 6.5 times compared to unmodified paclitaxel. Furthermore, using PEGylated paclitaxel it was possible to introduce 50% of cholesterol into the formulation without losing stability or entrapment efficiency. This formulation was found to be chemically and physically stable under physiological conditions for at least 2 months. No difference in mean diameter occurred during this period, confirming the absence of aggregation or fusion events (data not shown).

3.4. Stability of liposomes containing prodrugs For each prodrug, the most suitable liposomal formulation, in terms of entrapment efficiency and drug concentration, was chosen to assess stability during storage (Table 4). Formulation D, containing 29-succinyl–paclitaxel and composed of PC–SA 9:1, showed slow drug release from liposomes after 2

Table 4 Stability during storage a of the most suitable prodrug paclitaxel liposomes b Formulation

29-succinyl-paclitaxel liposomes (D) 29-MPA–paclitaxel liposomes (G) 29-PEG–paclitaxel liposomes (N)

Phospholipid

Encapsulated drug (% of initial)

composition

24 h

1 week

2 months

PC–SA 9:1 PC–PG 9:1 PC–PG–CHOL 9:1:5

90.2 83.5 95.5

85.4 54.7 93.2

75.7 Not determined 90.2

a Stability of liposomes was expressed as a percentage of the initial drug remaining in liposomes after 24 h, 1 week and 2 months in storage conditions. b Values are means of three experiments. S.D. values (not reported) were below 5% of the mean values in all cases.

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months. On the contrary, formulation G, containing 29-MPA–paclitaxel and composed of PC–PG 9:1, was unstable during storage: after 1 week, 45.3% of initial entrapped drug was released. Formulation N, containing 29-PEG–paclitaxel, was very stable for at least 2 months, 90% of the entrapped drug still being present.

3.5. Cytotoxic activity The cytotoxic activity of liposomes encapsulating paclitaxel and its prodrugs was tested through the inhibition of protein synthesis in HT-29 and MeWO cells; the cytotoxicity of compounds 1, 2 and 3 in free form was also evaluated. In Table 5, the IC 50 values of the drugs are summarized: Compound 2 had reduced cytotoxic activity, but still retained an IC 50 in the 80–90 nM range. On the contrary, compounds 3 and 4 had IC 50 values slightly reduced with respect to unmodified paclitaxel for both paclitaxel-sensitive cell lines (6–60 nM), indicating that paclitaxel had been released from the prodrug. All the liposomal formulations showed a similar cytotoxicity as the free prodrug.

3.6. Pharmacokinetic evaluation The next step was to assess the pharmacokinetic behaviour of the prodrug 29-PEG–paclitaxel, free or encapsulated in liposomes, compared to paclitaxel dissolved in Cremophor EL or in a liposomal formulation. 29-PEG–paclitaxel was the prodrug chosen for the pharmacokinetic study, being most

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suitable because of its high incorporation efficiency in liposomes (N), its high drug concentration and its good stability. Moreover, this formulation showed good cytotoxic activity on cell lines sensitive to paclitaxel. The pharmacokinetic behaviour was evaluated in Balb / c mice after iv injection of 29-PEG–paclitaxel, either free or encapsulated in liposomes at the paclitaxel dose of 15 mg kg 21 (0.3 mg / mouse). The pharmacokinetic parameters for the free prodrug or liposomal formulation were obtained from bi-exponential fitting of plasma data and are summarized in Table 6. Fig. 4 shows the plasma paclitaxel concentration vs. time profiles of free paclitaxel administered in Cremophor EL, conventional paclitaxel liposomes composed of PC–PG 9:1 (41), PEGylated paclitaxel dissolved in physiological solution and liposomal formulation (N) containing the PEGylated prodrug. After bolus administration, all the tested formulations follow a biphasic pattern with a rapid distribution phase (t 1 / 2 a 8.22 min, 5.07 min, 5.04 min and 15.16 min, respectively). The terminal elimination phase (t 1 / 2 b) was rapid for the parent drug (t 1 / 2 b 1.31 h) (Table 6) but coating paclitaxel with mPEG increased the b half-life of paclitaxel (t 1 / 2 b 9.27 h). Furthermore, the area under the plasma concentration vs. time curve (AUC) showed a marked increase with the free PEGylated prodrug, with respect to the parent drug, comparable to that observed using conventional paclitaxel liposomes. Entrapment of 29-PEG–paclitaxel in liposomes produced a marked increase in the a and b half-lives

Table 5 Cytotoxic activity a in human cell lines Substance

IC 50 for HT 29 cells

IC 50 for MeWo cells

Paclitaxel b (1) Paclitaxel–liposome 29-succinyl–paclitaxel (2) 29-succinyl–paclitaxel–liposomes 29-MPA–paclitaxel (3) 29-MPA–paclitaxel–liposomes 29-PEG–paclitaxel (4) 29-PEG–paclitaxel–liposomes

4.060.8 6.561 80.065 40.063 6.062 8.064 15.066 18.062

70.061 50.0 62 90.067 100.069 50.064 60.065 38.068 43.068

a b

The IC 50 values are expressed in nM. The values are arithmetic means of nine determinations. Stock solution 2310 23 M in dimethylsulfoxide, then diluted to 2310 27 M with 0.9% NaCl.

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Table 6 Pharmacokinetic parameters a

AUC (mg ml 21 min) t 1 / 2 a (min) t 1 / 2 b (h) Cmax (mg ml 21 ) MRT (h) Vss (ml) Cl (ml h 21 )

Paclitaxel

29-mPEG– paclitaxel

Paclitaxel liposomes

29-mPEG–paclitaxel liposomes

0.0014 8.22 1.31 0.149 1.13 4.07 3.59

0.66 5.07 27.69 0.0030 7.55 286.00 27.00

0.70 5.04 9.27 0.052 7.81 54.745 7.27

3.92 15.16 47.16 0.0037 62.46 559.00 4.58

a The pharmacokinetic parameters for free drugs or liposomal formulations were obtained from bi-exponential fitting of plasma data using Kinetica software. The values are arithmetic means of three experiments (n53–5 animals for group). S.D. values (not reported) were below 5% of the mean values in all cases.

(t 1 / 2 a 15.16 min and t 1 / 2 b 47.16 h) and in the AUC (3.92 mg ml 21 min), and a reduced clearance (4.58 ml h 21 ), maintaining a constant blood level and slow release of paclitaxel.

4. Discussion In order to reduce the systemic toxicity of paclitaxel (neurotoxicity, cardiotoxicity) and to avoid

Fig. 4. Pharmacokinetic behaviour of paclitaxel in Cremophor EL (circles), paclitaxel in conventional liposomes (PC–PG 9:1) (squares), 29-PEG–paclitaxel in buffer (triangles pointing down) and 29-PEG–paclitaxel liposomes (Formulation N) (triangles pointing up). Plasma paclitaxel concentrations were evaluated after solid-phase extraction (CN) by HPLC analysis (RP18 ). The values are arithmetic means of three experiments (n53–5 animals for group). S.D. values not reported (less than 4%).

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using toxic excipients such as Cremophor EL, which causes severe hypersensitive reactions [10–12], we tested some new formulations, i.e. conventional or PEGylated liposomes [41]. We found in vitro antitumoral activity similar to that of the free drug and pharmacokinetic behavior (t 1 / 2 , Cl, AUC) markedly superior to that shown of paclitaxel in Cremophor. In particular, PEGylated liposomes were in some ways similar to stealth liposomes, i.e. long plasma circulation time and a considerable decrease in drug uptake in RES organs. However, in neither paclitaxel liposomal formulation did we obtain a satisfactory drug concentration inside the lipid vesicles. Thus, in preliminary results obtained by in vivo evaluation of the antitumoral activity of liposome-loaded paclitaxel, we hardly reached the minimum therapeutic concentration (data not published) [34,40]. In the current study we aimed to increase the paclitaxel concentration inside liposomes, by evaluating the incorporation of different water-soluble prodrugs of paclitaxel: the 29-succinyl (2), 29methyl-pyridinium acetate (3) and 29-PEG ester (4). By varying the liposomal formulation as well as physical properties such as diameter, charge and membrane fluidity we obtained the best liposomal formulation for each prodrug. Among these, the liposome-containing 29-mPEG–paclitaxel, composed of PC–PG–CHOL 9:1:5, showed stability over 2 months, good entrapment efficiency (78%) and high paclitaxel concentration (6.5 mg ml 21 ), whereas liposomes loaded with 2 or 3 were less stable in storage and contained a much lower quantity of drug (1.8 mg ml 21 and 4 mg ml 21 , respectively). The addition of cholesterol, which has a bilayer– rigidifying effect, limited the formation of phaseseparated lamellae [18], contributing to the stability of PEGylated liposomes [47–49]. Similarly in our case, for liposomal 29-PEG–paclitaxel, the introduction of 50% cholesterol in the formulation enhanced the physical and chemical stability. The presence of cholesterol may also prevent fusion or aggregation between vesicles induced by the presence of PEG molecules [18]. The amphiphilicity of the paclitaxel prodrugs apparently improves the association with the lipid bilayer, increasing the entrapment efficiency. The in vitro antitumoral activity of liposomes entrapped with paclitaxel prodrugs was tested on two

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human carcinoma cell lines, HT-29 and MeWo, the former being more sensitive to paclitaxel than the latter. The liposomal formulations containing 3 or 4 maintained the cytotoxicity of the free drug, since the two prodrugs were promptly hydrolyzed, giving the parent drug after 48 h of incubation with tumor cells. Liposomes loaded with paclitaxel–29succinyl ester were much less cytotoxic, probably because this prodrug was more resistant to hydrolysis, as shown by its relative stability in buffers at different temperatures and / or pHs. To assess the pharmacokinetic behavior of the most efficacious paclitaxel–liposomal formulation, 29-PEG–paclitaxel entrapped in liposomes, administered by iv injection in mice, was compared with free or liposome-incorporated paclitaxel. The pharmacokinetic parameters for the free prodrug or liposomal formulation followed a bi-exponential plasma disposition. Entrapment of a paclitaxel watersoluble prodrug, such as 29-PEG–paclitaxel, produced marked differences, in terms of the pharmacokinetic parameters calculated for free paclitaxel. In particular, the values of t 1 / 2 a, t 1 / 2 b, AUC, MRT and Vss were found to be much higher for 29-PEG– paclitaxel vectored in liposomes than for liposomal paclitaxel or free drug. The most important change in the pharmacokinetic value on going from prodrug to free drug liposomal formulation was in the late plasma t 1 / 2 b value, which was very similar to that found for paclitaxel entrapped in stealth liposomes [41].

5. Conclusion In conclusion we successfully obtained liposomes, containing high paclitaxel concentrations inside the lipid bilayer, by using a water-soluble prodrug such as 29-mPEG (5000)–paclitaxel ester. This formulation was very stable in storage conditions and was able to release the free drug promptly, as shown by the high in vitro antitumoral activity, comparable to that of the parent drug. More interestingly, this formulation has the additional advantage of avoiding the use of the toxic Cremophor EL, used in the commercial formulation of paclitaxel. Additional work is needed to verify the in vivo antitumoral

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activity of liposomal 29-PEG–paclitaxel in different conditions. [16]

Acknowledgements [17]

Mr. Daniele Zonari’s excellent technical assistance is appreciated. This work was supported by MURST (60%) and Progetto Nazionale ‘Tecnologie Farmaceutiche’ grants, and by the Azienda Ospedaliera San Giovanni Battista.

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