Polymeric micelles as drug stabilizers: the camptothecin and simvastatin cases

J. DRUG DEL. SCI. TECH., 20 (4) 249-257 2010

Polymeric micelles as drug stabilizers: the camptothecin and simvastatin cases C. Alvarez-Lorenzo, A. Concheiro* Departamento de Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782-Santiago de Compostela, Spain *Correspondence: [email protected] Polymeric micelles are appealing drug nanocarriers due to their role as solubilizers of hydrophobic drugs and to their suitability for tuning drug bioavailability and biodistribution. Although implicit in their ability to host drugs, the usefulness of polymeric micelles as protective agents has been less studied. In this review, the potential of diverse amphiphilic block copolymers for creating microenvironments in which labile drugs are protected against adverse agents is analyzed. Particularly, examples of the success of polymeric micelles in the prevention of the hydrolysis of the lactone rings of camptothecin and simvastatin are presented. The maintenance of the lactone form of camptothecin is essential for the antitumor effect and the safety of the treatment. The lactone form of simvastatin is a prodrug required for efficient intestinal absorption. Amphiphilic block copolymers that lead to relatively hydrophobic cores or that create acidic surroundings are singled out as promising components of micelles in which the chemical stability of the drug is favored. Key words: Polymeric micelles – Anticancer agents – Statins – Pluronic – Tetronic – Lactone ring stability – Drug hydrolysis – PEO-PPO block copolymers – Poloxamer – Poloxamine.

external stimulus is applied are also useful for a fine control of the site and the rate of the delivery [14-19]. Such a gathering of features points out polymeric micelles as drug carriers capable of modifying drug disposition and of increasing the efficacy and the safety of the treatments [20]. Comprehensive reviews about the potential of polymeric micelles as components of drug formulations have been recently published, and significant improvements in oral bioavailability of drugs included in polymeric micelles have been reported [11, 20, 21]. Among other polymeric surfactants, those bearing poly(ethylene oxide) (PEO) (also known as poly(ethylene glycol), PEG) blocks are particularly appealing excipients for drug delivery and comprehensive reviews can be found elsewhere [6, 22, 23]. These hydrophilic blocks provide stealth shells that contribute to the physical stability of the micelles in the body fluids and prevent premature opsonization. The capability of poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO) block copolymers to self-assemble and form polymeric micelles and also to undergo sol-gel transitions upon heating give them the quality of “smart” or “intelligent” materials [24]. According to the structure of the main chain, PEO-PPO block copolymers can be classified into two main families: (i) linear PEO-PPO-PEO triblocks or poloxamers available under the trade names Pluronic or Lutrol, and (ii) X-shaped derivatives known as poloxamines or Tetronic, which are made of an ethylenediamine central group bonded to four chains of PPO-PEO blocks [25-27] (Figure 1). Poloxamers and poloxamines are commercially available in a wide range of varieties differing in molecular weights and block ratios; some poloxamers being already approved by regulatory agencies (such as FDA and EMEA) as components of drug dosage forms. In general, PEO-PPO block copolymers show good cell compatibility and, although they are not biodegradable, those varieties with molecular weights below 10-15 kDa are easily cleared from the body by renal filtration [28]. PEO-PPO amphiphiles show unique concentration-dependent aggregation behavior in water, exhibiting two critical concentration values [29]. The first governs the transition from unimolecular (unimer) to multimolecular (micelle) aggregates and determines the CMC. The temperature-responsiveness of the amphiphiles causes the

I. Polymeric micelles: general aspects

Polymeric micelles have attracted enormous attention as drug carriers due to their advantages compared to micelles of common surfactants and to other nanocarriers [1, 2]. In general, amphiphilic copolymers are prone to self-assemble at relatively low concentrations rendering core-shell micelles. The core results from hydrophobic interactions among the less polar segments of amphiphilic chains, while in the shell the hydrophilic regions of the copolymer create an adequate interface to deal with the surrounding aqueous environment. Polymeric micelles are endowed with a great thermodynamic and kinetic stability, i.e., the critical micellar concentration (CMC) of amphiphilic copolymers is low and, therefore, polymeric micelles may withstand dilution better than conventional micelles. Even when the dilution entails the amphiphilic copolymer concentration to be below the CMC, the disassembly occurs relatively slowly (on the minutes to hours scale compared to the seconds scale of conventional micelles) due to the entanglement of the polymeric chains in the micelle [3]. These features, namely low CMC and high physical stability, explain the ability of the polymeric micelles to remarkably enhance the apparent aqueous solubility of hydrophobic drugs in aqueous medium by hosting them in the hydrophobic cores. Furthermore, once a polymeric micelle-based formulation is administered to the body, the physical stability of the micelles prevents a too fast disassembly that could lead to drug precipitation in the body fluids [4, 5]. The copolymer architecture determines the blood circulation time and the capability of the polymeric micelles to control drug release rate and site [6-9]. Drug-loaded polymeric micelles are passively accumulated in pathological sites with affected and leaky vasculature (tumors, inflammations, and infarcted areas) via the enhanced permeability and retention (EPR) effect [10, 11]. Active targeting of polymeric micelles to specific tissues or cells can be achieved by anchoring specific ligand molecules, such as antibodies, to the shell [12, 13]. Stimuli-responsive amphiphilic block copolymers can enable the micelles to switch drug release on/off as a function of intrinsic or extrinsic body variables. Micelles that undergo changes in drug permeability when the microenvironmental conditions change or an 249

J. DRUG DEL. SCI. TECH., 20 (4) 249-257 2010

Polymeric micelles as drug stabilizers: the camptothecin and simvastatin cases C. Alvarez-Lorenzo, A. Concheiro

lactone form and the fast non-specific distribution to the whole body [38, 39]. CPT inhibits the enzyme DNA topoisomerase I, initially by noncovalent binding and subsequently by stabilization of the complex through a nucleophilic attack by the enzyme at the acyl position of the CPT lactone ring [40]. However, the high chemical reactivity of the α-hydroxycarbonyl group in the lactone ring, which is absolutely required for cytotoxic activity, induces a rapid equilibration under physiological conditions between the lactone form and the ring-opened carboxylate form. The lactone is converted to the carboxylate in a pHdependent equilibrium (Figure 2) and according to pseudo-first-order kinetics [41]. Moreover, the carboxylate form of CPT binds with human serum albumin (HSA) with extraordinary efficiency, and thus the presence of HSA in blood or serum promotes the lactone hydrolysis and thus shifts the equilibrium toward the pharmacologically ineffective carboxylate form [42, 43]. Under physiological conditions, most CPT molecules exist in the carboxylate form, which is responsible for severe collateral effects (such as myelosuppresion, haemorrhagic cystitis and diarrhea). In addition to the synthesis of new derivatives and pro-drug products [44, 45], the development of adequate carriers for the delivery of CPT lactone form has been the subject of great interest. Polymeric micelles have been postulated as relevant CPT carriers able to both solubilize and protect the lactone form and, even, able to site-specific delivery by possessing targeting elements or stimuli-induced responsiveness [46]. Research on CPT stabilization has been intensively carried out using polymeric micelles consisting of PEG-poly(aspartate) block copolymers, generically designed as PEG-P(Asp), or Pluronics covalently conjugated with poly(acrylic acid) (Pluronic-PAA) (Figure 3). The results are summarized in Table I.

Sequential poloxamer HOCH 2-CH 2-(O-CH 2-CH 2)a-1- (CH-CH 2-O)b-(CH 2-CH 2-O)a-1-CH 2-CHOH CH 3

Sequential poloxamine

CH3

CH3

(CH-CH2-O)b-(CH2-CH2-O)a-1-CH2-CHOH

HOCH2-CH2-(O-CH2-CH2)a-1 -(O-CH2-CH)b N-CH2-CH2-N HOCH2-CH2-(O-CH2-CH2)a-1 -(O-CH2-CH)b

(CH-CH2-O)b-(CH2-CH2-O)a-1-CH2-CHOH

CH3

CH3

Reverse poloxamine CH3

CH3

CH3

CH3

(CH2-CH2-O)a-(CH-CH2-O)b-1-CH-CH2OH

HOCH2-CH-(O-CH2-CH)b-1 -(O-CH2-CH2)a N-CH2-CH2-N HOCH2-CH-(O-CH2-CH)b-1 -(O-CH2-CH2)a CH3

(CH2-CH2-O)a-(CH-CH2-O)b-1-CH-CH2OH CH3

CH3

Methylated poloxamine CH

CH3

3

HOCH2-CH2-(O-CH2-CH2)a-1 -(O-CH2-CH)b

CH3

CH3

(CH-CH2-O)b-(CH2-CH2-O)a-1-CH2-CHOH

+

N-CH2-CH2-N HOCH2-CH2-(O-CH2-CH2)a-1 -(O-CH2-CH)b CH3

Cl-

(CH-CH2-O)b-(CH2-CH2-O)a-1-CH2-CHOH CH3

Figure 1 - Structure of a sequential poloxamer and a sequential, a reverse-sequential and a methylated sequential poloxamine. Sequential poloxamines are synthesized by the reaction of the acceptor ethylenediamine molecule first with propylene oxide (PO) and then with ethylene oxide (EO) precursors, resulting in a four-arm PEO-terminated molecular structure. In the case of reverse-sequential poloxamines, the acceptor is primarily reacted with EO and afterwards with PO, leading to tetra-functional block copolymers displaying PPO terminal segments.

N

CMC values to decrease as temperature increases. Varieties possessing high molecular weight and small EO/PO ratio have the lowest CMC values [24, 30, 31]. The second critical concentration is the minimal concentration required for gel formation upon heating. In general, micellization is observed at much lower concentrations (< 1 wt %) than those required for gel formation (> 15 wt %) [32]. Gelation involves the formation of a packed micelle-based physical network [33, 34]. For a given concentration, the temperature at which the sol-gel transition takes places depends on the EO/PO ratio and molecular weight of the copolymer. Thus, gel formation can be fine-tuned by changing the concentration or the temperature of the solution. Materials showing sol-to-gel temperature around 37 ºC are especially attractive, not only as solubilizers of hydrophobic drugs, but also as components of in situ gelling systems for the sustained release of drugs. In addition to the temperature-responsiveness, several families of block copolymers have been designed to be multi-stimuli responsive, enabling precise tuning of micellar performance [17, 19]. There are numerous references in literature to the successful enhancement of drug solubility in biological fluids, and simultaneous modulation of the circulation time, release profile, and cellular internalization of drugs when loaded in polymeric micelles [36, 37]. By contrast, the chemical stabilization of drug labile groups by hosting in polymeric micelles has received less attention. In this regard, the next two sections summarize the results already obtained for two particularly challenging drugs, camptothecin and simvastatin, both bearing a lactone group that determines not only drug solubility but also therapeutic efficiency.

O

N

OH

N

N

Lactone

O

O-

O C2H5

C2H5 O OH

Carboxylate

O OH

Figure 2 - The conversion of the lactone form to the carboxylate form of camptothecin occurs under neutral and basic conditions and is largely dependent on hydroxide ion. The conversion of the carboxylate to the lactone occurs under neutral and acidic conditions.

a) PEG-P(Asp(R))

α

β

H3C-(OCH2CH2)n -CH2-NH-(COCHNH)x -(COCH2CHNH)y-H R= H, benzyl, n-butyl, or lauryl b) Pluronic-PAA

CH3

CH2COOR

COOR

CH3

HO-(CH2CH2O) -(CH2C-O)-(CH2CHO) -(CH2CH2O)-H x x y-1 CH O HC OH n

CH3

CH3

HO-(CH2CHO)-(CH2CH2O) -(CH2C-O)-(CH2CHO) -(CH2CH2O) -H x x-1 y-1 CH HC

O OH

m

...

II. THE CASE OF CAMPTOTHECIN

Camptothecin (CPT) and its derivatives are very potent antitumor agents, although their therapeutic use is strongly limited by the lack of water-solubility, instability of the pharmacologically active

Figure 3 - Structure of a) esterified block copolymer PEG-P(Asp(R)) made from poly-(ethylene glycol)-poly(aspartic acid) block copolymer and b) a copolymer of Pluronic and poly(acrylic acid), Pluronic-PAA.

250

Polymeric micelles as drug stabilizers: the camptothecin and simvastatin cases C. Alvarez-Lorenzo, A. Concheiro

J. DRUG DEL. SCI. TECH., 20 (4) 249-257 2010

Table I - Micellar systems tested as CPT carriers: loading efficiency and protection of the lactone form. Copolymer

Micelles preparation method

Loading efficiency

Percentage of lactone in the CPT released at 24 h

Ref.

PEG-P(Asp) with several ester chains

Dialysis (5 mg copolymer + 0.5 mg CPT) Emulsion (5 mg copolymer + 0.5 mg CPT) Evaporation (5 mg copolymer + 0.5 mg CPT)

1-2% 20-30% 50-100%

-

47

PEG-P(Asp) with benzyl groups

Evaporation (CPT 5-40% of the weight of the copolymer)

20-90%

85% in PBS pH 7.4, 72% in serum

48

PEG-P(Asp) with butyl groups PEG-P(Asp) with lauryl groups

Evaporation (CPT 5-40% of the weight of the copolymer) Evaporation (CPT 5-40% of the weight of the copolymer)

20-50% 50%

N-phthaloyl chitosan-grafted PEG

Dialysis (CPT 5-40% of the weight of the copolymer)

30-90%

80% in PBS pH 7.4, 57% in serum, 43% in 4% HSA

52

Cholic acid chitosan-grafted PEG methyl ether

Dialysis (CPT 5-40% of the weight of the copolymer) Emulsion (CPT 5-40% of the weight of the copolymer)

15-35% 45-70%

53 81% in PBS, 70% in serum, 33% in 4% HSA

Evaporation (CPT 5-40% of the weight of the copolymer)

30-65%

Methoxy PEG-bpoly(valerolactone)

Evaporation (1 mg CPT and 10 mg of copolymer) Lyophilization (1 mg CPT and 10 mg of copolymer)

2-65% 3-96%

10-60% in PBS 10-60% in PBS

54

Pluronic-g-PAA

Equilibrium solubilization (0.225 mg in 10 ml of 1-4% copolymer solution)

0.4 g/g

3-67% in PBS, 8% in serum

55

Okano et al. [47] analyzed the dependence of incorporation efficiency and stability of CPT in PEG-P(Asp) micelles on the hydrophobicity of the micellar core and the incorporation method. Block copolymers bearing benzyl and methylnaphtyl ester groups led to micelles that hosted more drug in the lactone form (quantified by reverse-phase HPLC analysis), compared to micelles made of block copolymers bearing n-butyl or lauryl ester groups. The results suggest that location of CPT molecules deeply inserted in a hydrophobic core can notably contribute to stabilize the lactone form of CPT in the micellar core. These authors tested three incorporation methods. The dialysis method involved the preparation of a block copolymer and CPT solution in dimethyl sulfoxide. The solution was dialyzed against water and then collected and filtrated. This approach rendered clear micellar solution with low yields of CPT incorporation (1-2 %). The emulsion method consisted in mixing a block copolymer and CPT solution in methylene chloride with distilled water. The methylene chloride was then evaporated and the remaining aqueous solution filtered. The emulsion method resulted in cloudy solutions due to the presence of aggregates larger than polymeric micelles, with loading yield of 20-30 %. The evaporation method involved the evaporation of chloroform from a block copolymer and CPT solution. Distilled water was then added to the residue and the system was sonicated and, finally, filtered or centrifuged. The resultant micellar solution was clear and the yield of CPT incorporation ranged from 50 to 100 % [47]. This approach was therefore used by this research group for further experiments. Studies with PEG-P(Asp) copolymers possessing different contents in benzyl ester revealed that the in vitro release rate of CPT from the micelles decreased as the benzyl content increased, enabling sustained delivery for as long as several days [48]. Drug stability into the micelles was found to be dependent on the chemical structure of the hydrophobic chain of the block copolymer, drug content and chain lengths. The block copolymer composed of one PEG block of 5,000 Da and one P(Asp) block possessing 27 units of aspartic acid (69  % esterified with benzyl ester groups) showed the best protective performance. CPT-loaded polymeric micelles incubated in PBS buffer at pH 7.4 or in fetal bovine serum preserved the lactone form of CPT to an extent of 85 % in PBS and 72 % in serum after 24 h. By contrast, free CPT dissolved in PBS or in serum significantly exhibited ring opening. Only 20 % and 35 % of the lactone rings remained intact after 2.3 h in serum and in PBS, respectively. Regarding CPT stability, partially benzylesterified PEG-P(Asp)

copolymers possessing α-amide and β-amides (see Figure 3) were found to stabilize the lactone form of CPT better than copolymers possessing α-amide only bonds. These findings suggest that an adequate balance between the hydrophobicity and the rigidity of the micellar core or between the hydrophobicity and the steric configuration of the hydrophobic block chains is required for the protection of CPT [49]. Intravenous administration of CPT-loaded micelles in mice evidenced that the stability of the micelles in vivo depends on the proportion of benzyl ester groups and the length of PEG in the polymers to a greater degree than micelles in vitro. Again the most stable formulation of CPT-loaded micelles was obtained using PEG-P(Asp) with PEG of 5000 Da, 27 units of aspartic acid, and 57-75 % benzyl esterification of the aspartic residue. These CPT-loaded micelles showed about a 17-fold lower blood clearance value than unstable micelles [50]. Furthermore, a single i.v. injection of CPT-loaded micelles (dose 1530 mg/kg) inhibited tumor growth in mice subcutaneously transplated by colon 26 tumor cells. Such improved antitumor activity confirms that the polymeric micelles maintain the active CPT lactone form even in the presence of serum. Biodistribution studies indicated that CPT-delivery using polymeric micelles results in nearly 8 times higher accumulation in tumors (approximately 1.3 % of injected dose per gram of tissue) compared with CPT in solution (Figure 4). Plasma levels of CPT-loaded micelles (1.1 % of injected dose) were approximately 150 times higher than CPT solution. Elevated pulmonary CPT levels

Figure 4 - CPT biodistribution in mice bearing colon 26 tumor 24 h after i.v. injection of CPT-loaded polymeric micelles and CPT solution at a dose of 2.5 mg/kg. Each value represents the mean ± SD (n = 3). *a < 0.05, **a < 0.01, compared with CPT solution (Students' t-test). Reprinted from Kawano et al. [51], with permission of Elsevier.

251

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Polymeric micelles as drug stabilizers: the camptothecin and simvastatin cases C. Alvarez-Lorenzo, A. Concheiro

achieved with CPT solution were attributed to embolization of lung capillaries arising from drug precipitation [51]. Following a similar approach, Opanasopit et al. [52] synthesized N-phthaloylchitosan-grafted poly (ethylene glycol) methyl ether (mPEG) (PLC-g-mPEG) which self-assembles as sphere-like particles able to host CPT. CPT was incorporated into polymeric micelles using a dialysis method starting from 5 mg of PLC-g-mPEG polymer and CPT (5-40 % of polymer) solution in 2 mL of DMSO or DMF. The incorporation efficiency increased ranging from 30 to 90 % with an increase in the initial CPT loading from 5 to 40 %. To elucidate the effects of polymeric micelles on the lactone-carboxylate hydrolysis over time, CPT-loaded polymeric micelles were incubated in pH 7.4 PBS buffer, in 50 % fetal bovine serum and in 4 % HSA at a CPT concentration of 55 µg/mL (Figure 5). As determined by reverse-phase HPLC, the lactone form of CPT was preserved in the inner core of micelles to an extent of 80 % in pH 7.4 PBS, 57 % in serum, and 43 % in HSA, after 24 h (Figure 5b). In the case of CPT solutions, only 32, 20 and 5 % of the lactone ring remained in pH 7.4 PBS, in serum, and in HSA, respectively, after 3 h (Figure 5a). This indicates that the incorporation of CPT into the hydrophobic inner core of micelles was advantageous for the preservation of the active lactone form at a high concentration over a long period of time. Therefore, the chitosan derivative PLC-g-mPEG can form polymeric micellar systems able to incorporate and stabilize CPT in considerably high yields and to control drug release in vitro for several hours. Similarly, polymeric micelles prepared from cholic acid chitosan-grafted poly(ethylene glycol)methyl ether (CS-mPEG-CA) and loaded with CPT using the emulsion method were able to prevent the hydrolysis of the lactone group of the drug as efficiently as those made of the chitosan derivative PLC-g-mPEG [53]. Following the reasoning that hydrophobic cores protect CPT better from hydrolysis, polymeric micelles based on methoxy poly(ethylene glycol)-b-poly(valerolactone) (mPEG-b-PVL) copolymers have been also evaluated. PVL was chosen as the hydrophobic segment due to its slower degradation rate and greater hydrophobicity than PLGA, PLA or PGA polymer [54]. The CMC of the copolymers in water decreased with increasing molecular weight of hydrophobic segment. The micellar solutions maintained their sizes at 37ºC for six weeks without aggregation or dissociation. A lyophilization method (dissolution of 10 mg of copolymer and 1 mg of CPT in dimethylsulfoxide, followed by freeze-drying and addition of water) yielded higher CPT loading efficiency and lower aggregation than the evaporation method (dichloromethane/methanol solvent). The loading efficiency of CPT could be greater than 96 % and a steady release rate of CPT was kept for 26 days. Moreover, the mPEG-b-PVL polymeric micelles offered a 2- to 3-fold increase in the stability of CPT lactone form at 37 ºC in PBS buffer. A very different approach for protecting CPT lactone form was

developed by our group in collaboration with Bromberg et al. [55]. Polymeric micelles capable of creating an acidic microenvironment were considered suitable for avoiding or delaying the lactone hydrolysis. Thus, a family of Pluronics covalently conjugated with poly(acrylic acid) (Pluronic-PAA) that combines the solubilization capability of the PEO-PPO-PEO copolymer and the pH-sensitivity of the polyelectrolyte was prepared via C-C bonding between the polyether and PAA (see Figure 3) [56-60]. Pluronic-PAA possesses the ability to self-assemble into micelle-like aggregates at certain concentrations, temperature, and pH [61-63]. These micelles are capable of solubilizing hydrophobic compounds, which become protected from the hostile aqueous environment and can be released at very low rates [64-67]. The two Pluronic-PAA copolymers and the respective parent Pluronic copolymers that were tested as CPT micellar carriers differ considerably in the content of PO groups. The L92-PAA possess a 2-fold higher weight content of the PO groups (~ 36 wt %) than F127-PAA (~ 15 wt %). The PPO segments can strongly affect the hydrophobicity and the surface activity of the Pluronic-PAA copolymers and therefore their capacity to interact with CPT and to prevent the hydrolysis of its lactone group. In fact, L92-PAA copolymers showed lower CMC values than F127-PAA (2 × 10-3 vs. 4 × 10-3 g/mL, respectively). On the other hand, the intrinsic dissociation constants (pK0) were found by potentiometric titration to be 6.27, 4.95, 4.67 and 4.56 for L92-PAA, F127-PAA, uncross-linked poly(acrylic acid) and acrylic acid, respectively [68, 69]. At pH 5, where the lactone form of the compound is stable yet water-insoluble, a significant increase in solubility was observed for polymer concentrations above the respective CMCs. Equilibrium loading of 1 % solution of each polymer was ca. 1 µmol CPT/g polymer. For the greatest polymer concentration analyzed (4 %), CPT solubility was 3- to 4-fold higher than that in water at pH 5. If these solubilization data are expressed as µmol of CPT per effective content of PPO in the copolymer, the loadings of 4 % F127-PAA and L92-PAA solutions (3.77 ± 0.24 and 1.35 ± 0.04 µmol/g PPO, respectively) were significantly higher than those in the solutions of the parent Pluronic F127 and L92 (1.51 ± 0.02 and 0.62 ± 0.01 µmol/g PPO, respectively). This finding indicates that the CPT loading can occur both via solubilization into hydrophobic PPO cores of the micelles and via the drug entrapment into the interfacial layers between PPO and more hydrophilic POE-PAA layers. The disappearance of the lactone and the appearance of the carboxylate forms of CPT in aqueous medium were monitored by the decrease and increase in absorbance at 354 and 385 nm, respectively [70, 71]. After reaching CPT equilibrium solubilization in 4 % polymer solutions at pH 5.0 and 25 ºC, the filtered solutions were stabilized at 15 ºC to avoid interference of the turbidity that appears in the solutions of L92 and L92-PAA polymers. Then the pH was quenched to 8.0 by the addition of a concentrated TRIS solution, at which point the lactone hydrolysis began. The evolution of the absorbance at each wavelength was recorded for 2-3 h. Pseudo-first-order rate constants of the lactone-carboxylate conversion were calculated from the slope of the plot of the natural logarithmic concentration of the lactone form vs. time. Figure 6 shows the effect of the polymers on the stability of the lactone form. In the absence of the amphiphilic polymers, quenching of pH from 5 to 8 induced a quick conversion of the lactone form to the carboxylate form. The half-life value obtained in water agreed well with that previously reported of 20-50 min in phosphate buffer [72, 73]. F127 and L92 micelles considerably lowered the hydrolysis rate, particularly the last ones. The more hydrophobic core of the L92 micelles minimizes the contact of the alkaline aqueous medium with the drug, delaying the hydrolysis process. Furthermore, the solubilization of CPT into the Pluronic-PAA micelles considerably enhanced the stability of the lactone group of the drug (Table II). This is related to the hydrophobicity of the cores and the presence of PAA chains, which can locally counteract the outer alkaline pH and increase the microviscosity around the cores as the PAA chains become ionized.

Figure 5 - Rate of lactone ring opening for (a) camptothecin (CPT) and (b) camptothecin (CPT)-loaded polymeric micelles forming from N-phthaloylchitosan-grafted mPEG polymer in the presence of (n) pH 7.4 PBS (control); (s) 50 % FBS; (l) 4 % HAS (n = 3); *significant differences compared with the control (a < 0.05). Reprinted from Opanasopit et al. [52] with permission of Elsevier. 252

Polymeric micelles as drug stabilizers: the camptothecin and simvastatin cases C. Alvarez-Lorenzo, A. Concheiro

1.5

In the particular case of CPT, the tablets were prepared by either direct compression of the drug (4 mg)/F127-PAA (196 mg) physical blend or of particles obtained by suspension in ethanol followed by evaporation, or compression after kneading the components in the presence of ethanol [74]. Porosity and water uptake rate were strongly dependent on the fabrication procedure, ranking in the order: direct compression of physical blend > compression after suspension/evaporation in ethanol > compression after kneading. Tablets prepared with the physical mixture showed the slowest release pattern (Figure 7). These tablets, despite having a greater total porosity, require more time to complete the disintegration than those obtained by kneading and suspension-evaporation. Despite the tablet disintegration, the CPT release was sustained for at least 6 h, which was attributed to the ability of the Pluronic-PAA copolymers to form micellar aggregates at the hydrated surface of the particles. The release patterns may be explained as being the result of two concomitant effects: i) rapid formation of a stiff gel layer in the surface of the most porous tablets, which controls the release of the drug and obstructs deeper layers of the tablet from swelling/dissolution; the formed gel consists of closely packed swollen particles additionally stabilized by micellar aggregates of the PPO segments of Pluronic, which delay drug release from the matrices; and ii) possible changes in the drug structure that can occur during the processing with the polymer, before and/or during compression. Physical mixing did not alter the fraction of CPT being in the pharmaceutically active lactone form, whilst the preparation of the tablets by the other two methods caused a significant reduction in the lactone form content (Figure 8). Such an increase in the carboxylate

L92-PAA

Ln (CPT lactone)

1.0 F127-PAA

0.5

L92

0.0 F127

-0.5

Control

-1.0 -1.5 0

30

60

90 120 Time (min)

150

180

Figure 6 - Effect of Pluronic F127, Pluronic L92, F127-PAA, and L92-PAA micelles on the stability in aqueous environment of the lactone form of camptothecin at pH 8 and 15 ºC. Control experiments were carried out in the absence of polymer. Reprinted from Barreiro-Iglesias et al. [55] with permission of Elsevier. Table II - Rate constants of the hydrolysis process and half-life (t1/2) values of the lactone form of CPT in water and 4 wt% aqueous polymer solutions at pH 8.0. Reprinted from [55] with permission from Elsevier. Medium

Hydrolysis rate (x 103 min-1 ± SD)

Half-life (h)

Water F127 L92 F127-PAA L92-PAA

22.15 ± 0.82 11.09 ± 1.13 7.37 ± 0.22 2.40 ± 0.19 0.27 ± 0.10

0.52 1.05 1.57 4.80 42.77

J. DRUG DEL. SCI. TECH., 20 (4) 249-257 2010

Further experiments were conducted to evaluate the enhancement of CPT stability in human serum by an HPLC method. CPT solubilization into Pluronic-PAA micelles proved to be an effective barrier against the drug decomposition. The parameter t1/2 was estimated to be 0.16, 1.1, and 1.7 h for free CPT and for CPT in F127-PAA and L92-PAA micelles, respectively. This means that CPT is about 10-fold more stable when encapsulated into the polymeric micelles. Therefore, the internalization of the drug in the micelles of Pluronic-PAA notably hinders the hydrolytic opening of the lactone ring in both alkaline medium and human serum [55]. The potential interest of Pluronic-PAA copolymers for the development of CPT dosage forms prompted us to prepare self-micellizable solid dosage forms. Pluronic polymers are seldom used to prepare tablets owing to their low melting point, which hinders the compression and other common technological processes. By contrast, Pluronic-PAA copolymers proved to be suitable excipients for direct compression [74]. The Pluronic-PAA matrices are expected to form a gel layer with a complex structure in which micellar aggregates can be formed and drugs can be solubilized into or released from. This swelling/micellization behavior is pH-dependent; Pluronic-PAA copolymers show a more pH-responsive behavior than PAA microgels (e.g. Carbopol) and can hold the drug tighter at lower (stomach) pH due to hydrophobic interactions not available with PAA microgels. The known ability of Pluronics and derivatives to enhance permeability through biological membranes increases the potential interest of Pluronic-PAA as oral excipient [21]. For example, Pluronic-PAA microgels loaded with megestrol acetate showed significantly enhanced bioavailability of this hydrophobic drug due to the retention of the microgels in the gastrointestinal tract of a rat model and promotion of absorption [75]. On the other hand, theophylline, hydrochlorothiazide or nitrofurantoin tablets prepared using Pluronic-PAA as the main excipient proved useful for achieving a specific delivery in the intestine [76].

Figure 7 - Cumulative release of camptothecin in water (pH 5.5) from tablets containing 4 mg of drug and 196 mg of Pluronic-PAA, and prepared by physical blending (circles), suspension/evaporation (squares) and kneading (triangles). Reprinted from Bromberg et al. [74] with permission of Taylor and Francis Ltd.

Figure 8 - Lactone content in tablets prepared by various procedures (physical blending PB, suspension/evaporation SE, and kneading K) relative to the CPT control. Reprinted from Bromberg et al. [74] with permission of Taylor and Francis Ltd. 253

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Polymeric micelles as drug stabilizers: the camptothecin and simvastatin cases C. Alvarez-Lorenzo, A. Concheiro

form may promote, to some extent, the dissolution rate of the drug inside the tablet and, as a consequence, its release.

HO

III. THE CASE OF SIMVASTATIN

O

O

Statins represent the first-choice drug group for the management of hypercholesterolemia and for reducing the morbidity and mortality associated with coronary heart disease [77]. Simvastatin, like other statins, is a polycyclic compound with a pH-dependent structure and solubility, being practically insoluble at acidic pH [78, 79]. Following oral administration, the lipophilic simvastatin lactone form is passively absorbed in the intestine. Then, the lactone form is reversibly converted into the enterocyte and in plasma to its corresponding α-hydroxy acid, which is a potent inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, an essential enzyme involved in the biosynthesis of cholesterol. Simvastatin acid, but not lactone, is a substrate of the P-glycoprotein efflux transporters. In the enterocyte, the fraction converted to simvastatin acid could be subject to P-glycoproteinmediated excretion back into the gut lumen [80]. Therefore, prevention of premature hydrolysis of simvastatin lactone form is considered an essential key point to ensure the quality of its pharmaceutical dosage forms [81]. At intermediate pH values (pH ~ 5), simvastatin predominantly exists in the lactonic form (Figure 9). However, it can be reversibly hydrolyzed to the hydroxy acid form at both very low and alkaline pH. Formulation of the poorly soluble simvastatin lactone form in micellar systems may enable both improvement of solubility and delay of hydrolysis, resulting in greater oral bioavailability [82, 83]. In this context, we investigated the interaction of simvastatin with poloxamers (Pluronics) and poloxamines (Tetronics) of various PO/EO ratios and its incidence on the solubility and the stability of simvastatin lactone form (Table II). Three varieties of Pluronic (F87, F127 and P123), seven varieties of conventional sequential poloxamines (Tetronic 304, 901, 904, 908, 1107, 1301 and 1307), a reverse-sequential variety (Tetronic 150R1) and a chemically modified derivative, Nmethylated Tetronic 1107 were chosen for the study. Although only a few compared to poloxamers, the studies carried out with poloxamines showed their potential as components of transdermal formulations [84], tissue scaffolds [85-88] and nanoparticle components [89]. Extensive reviews about poloxamine features and potential as drug carriers have been published elsewhere [27, 90]. The two tertiary amine central groups play an essential role conferring thermodynamical stability and pH sensitivity and enabling further chemical modifications (e.g. methylation of the amine group; see Figure 1) in order to attain additional performances [91]. The incidence of structural features and the physical-chemical conditions of the medium, particularly the pH and the ionic strength on poloxamine micellization have been recently reported [31, 92-94]. The capability of poloxamine micelles to host relatively hydrophobic drugs and to increase their apparent solubility and/or to protect them against chemical or biological degradation has been demonstrated for relevant therapeutic agents, such as antifungal or antiviral drugs [26, 95-97]. According to the HLB values (Table III), the poloxamers and poloxamines evaluated as simvastatin solubilizers can be classified in three groups: a) highly hydrophilic (F87, F127, T908, T1107 and T1307); b) medium hydrophilic (T304, T904 and T1304) and c) highly hydrophobic (P123, T701, T901, T1301, T90R4 and T150R1). HCl 10 mM was chosen as the solvent medium since it mimics the gastric conditions, it is the most unfavorable for simvastatin solubilization and it induces the hydrolysis of lactone form. In fact, simvastatin solubility was remarkably enhanced in micellar medium of P123, T1301 and T150R1, showing partition coefficients of up to 210, 100 and 47.5, respectively. This finding is in agreement with the greater hydrophobicity of P123, T1301 and T150R1 compared to the other Pluronic and Tetronic varieties, and suggests that intense PO-PO hydrophobic interactions are required to form micelles capable of hosting

O CH3

H

H

H3C H3C

H

O CH3

CH3

H3C

O OH

O

H

H3C H3C

HO

O

H

H CH3

H3C

Figure 9 - Structures of the lactone and hydroxy acid forms of simvastatin. Table III - Structural characteristics of Pluronics F87, F127 and P123, sequential Tetronics (T304, T901, T904, T908, T1107, T1301 and T1307), reverse Tetronic (T150R1) and methylated-T1107, and ratio of lactone to hydroxy acid (L/H) forms of simvastatin in drug-saturated 10% w/w copolymer solutions in HCl 10 mM (pH 2.5). Data taken from [31, 98]. Copolymer

EO units

PO units

EO/ PO ratio

MW (Da)

HLB

L/H ratio

F87 F127 P123 T304 T901 T904 T908 T1107 Met-T1107 T1301 T1307 T150R1

122 200 39 15 11 60 456 240 240 16 288 20

40 65 69 18 73 68 84 80 80 104 92 116

1.50 1.43 0.28 0.83 0.15 0.88 5.43 3 3 0.15 3.13 0.17

7700 12600 5750 1650 4700 6700 25,000 15,000 15,030 6800 18,000 7900

24 18-23 8 12-18 1-7 12-18 > 24 18-23

0.8:1 1.2:1 1.2:1 0:1 0.2:1 0:1 0.2:1 0:1 1.5:1 20:1 8:1 7.5:1

1-7 > 24 1-7

the drug [98]. In quantitative terms, micellar solutions of 10 % T901, T904, T908, T1107, met-T1107, T1301, T1307 and T150R1 raised the apparent solubility of simvastatin by a factor of 3.7, 8.5, 2.4, 4.7, 8.3, 391, 21, and 152, respectively [31, 98]. The lactone and carboxylate forms of simvastatin in the polymeric micelles (drug-saturated 10 % w/w Pluronic or Tetronic solutions) were analyzed by HPLC after 15 and 60 days of adding the drug to the copolymer solutions. A solution of drug in acetonitrile was used to estimate the lactone/carboxylate ratio in simvastatin as supplied. Compared to poloxamines, Pluronic F87, F127 and P123 showed a greater protection capability than T304, T901, T904, T908, T1107, but less than T1301, T1307 and T150R1 (Table II). This suggests that the stabilization of the lactone form is positively correlated to the number of the PO units, while no correlation could be found with the number of EO units. Therefore, strong hydrophobic cores are more suitable for forming micelles that host the lactone species and protect them efficiently against the hydrolysis caused by the surrounding acid aqueous environment. These results, together with a detailed analysis of simvastatin-copolymer interactions at the air-water interface, suggest that simvastatin molecules are preferably located in the core-shell interface, close to the PPO core but without interfering in its arrangement. On the other hand, the positive effect of the methylation of T1107 indicates a higher drug/micellar core affinity [31, 98]. A relevant aspect related to the in vivo performance of polymeric micelles refers to their kinetic stability, which enables the micelles to remain assembled for long time after dilution [99]. Recording the absorbance of simvastatin-loaded micelles (10 % poloxamine in HCl 10 mM) when subjected to a sudden 26-fold dilution, three differentiated responses were evidenced: a) one fastly disintegrating group (T901, T904, T908 and T1107), b) one intermediate group (Pluronic 254

Polymeric micelles as drug stabilizers: the camptothecin and simvastatin cases C. Alvarez-Lorenzo, A. Concheiro

F87 and poloxamines T304, T1301 and T150R1) that still maintain solubilized nearly 60 % drug after 1 h, and c) one very stable group formed by Pluronics F127 and P123 and poloxamines T1307 and metT1107. Therefore, copolymers with long PPO and PEO blocks are the most suitable carriers for protecting the lactone form and preventing premature drug leakage once diluted in the body fluids [31, 98].

15. 16. 17.

*

18.

Appropriate design of the architecture of block copolymers obtains micelles capable of providing microenvironments at the core that are highly suitable for hosting and protecting drugs against adverse agents. In the case of camptothecin, micelles of PEG-P(Asp), PCL-mPEG or mPEG-b-PVL with very hydrophobic cores or of Pluronic-PAA that can create an acidic environment have proved capable of considerably delaying or even impeding the hydrolysis of the lactone ring, which is vital for the therapeutic usefulness of this drug. Poloxamers and poloxamines possessing long PO blocks appear as suitable components of micelles that efficiently load simvastatin lactone form, prevent premature formation of the α-hydroxy acid form, and that can withstand sudden, intense dilutions. Therefore, polymeric micelles appear as an attractive nanocarrier for the formulation of poorly-soluble drugs bearing labile groups.

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Acknowledgements This work was financed by the Ministerio de Ciencia e Innovación (SAF2008-01679), FEDER, and Xunta de Galicia (PGIDIT07CSA002203PR), Spain. The authors would like to thank Lev Bromberg (Massachusetts Institute of Technology) and Alejandro Sosnik (CONICET-Universidad de Buenos Aires) for their collaboration and support over the years.

Manuscript Received 20 January 2010, accepted for publication 6 April 2010.

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