Flurbiprofen Derivatives in Alzheimer's Disease: Synthesis

Since cyclooxygenase-2 inducible isoform (COX-2), one of the target enzymes .... The Boc protective group was finally removed with 10% (v/v) solution ...
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Bioconjugate Chem. 2008, 19, 349–357

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Flurbiprofen Derivatives in Alzheimer’s Disease: Synthesis, Pharmacokinetic and Biological Assessment of Lipoamino Acid Prodrugs Rosario Pignatello,*,† Valentina Pantò,† Stefano Salmaso,§ Sara Bersani,§ Venerando Pistarà,‡ Vladimir Kepe,| Jorge R. Barrio,| and Giovanni Puglisi† Dipartimento di Scienze Farmaceutiche and Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale A. Doria, 6 - 95125 Catania, Italy, Dipartimento di Scienze Farmaceutiche, Università degli Studi di Padova, Via F. Marzolo, 5 - 35131 Padova, Italy, and Department of Molecular and Medical Pharmacology, The David Geffen School of Medicine at UCLA, Los Angeles, California 90095. Received August 18, 2007; Revised Manuscript Received October 27, 2007

Flurbiprofen (FLU) lipophilic prodrugs with lipoamino acids (LAA) 6a-e were synthesized for brain delivery. Chemical and plasmatic stability of prodrugs 6a-e as well as pharmacokinetic distribution studies for the prodrugs 6b and 6d were carried out. FLU prodrugs 6a-e were compared to the parent drug for their ability to inhibit binding of [F-18]FDDNP to in Vitro formed β-amyloid protein (Aβ fibrils). FLU-LAA conjugates showed a typical prodrug stability profile, being stable in PBS at pH 7.4 and releasing the active drug in plasma. Compound 6d yielded a slow accumulation of FLU in the brain. In the in Vitro inhibition assay, all prodrugs except for the prodrug with the longest alkyl side chain (6e) were effective as inhibitors of [F-18]FDDNP binding to Aβ fibrils with EC50 values in the 10-300 nM range. The different brain accumulation kinetics shown by FLU and its LAA conjugate 6d suggested a possible slow-releasing activity of FLU by these prodrugs in the brain or a differential pharmacological effect deserving further, detailed studies on their biodistribution and pharmacological profile.

INTRODUCTION Brain inflammation is one of the biochemical processes contributing to neurodegeneration in Alzheimer’s disease (AD) (1). Recent studies have demonstrated that mediators of inflammatory reaction in AD, such as cytokines, free radicals, adhesion molecules, and prostaglandins, were neurotoxic in experimental models (2). These compounds were hypothesized to act as extracellular signals promoting neuronal degeneration (3, 4). The formation of extracellular senile plaques, neuropathological fibrillar deposits of β-amyloid protein (Aβ), has been associated with a variety of proteins which may also be potential propagators of inflammation (5). A possible role of inflammation in AD pathogenesis and development is also supported by clinical evidence indicating that administration of nonsteroidal anti-inflammatory agents (NSAIDs) to AD patients slows deterioration of cognitive functions (6), reduces the incidence of AD (7), and exerts a neuroprotective effect (8, 9). Direct NSAID interaction with fibrillar β-amyloid protein (Aβ) has also been shown in Vitro (10). Since cyclooxygenase-2 inducible isoform (COX-2), one of the target enzymes of NSAIDs, has been detected at high levels in the brain of AD patients (11), it has been hypothesized that NSAIDs primarily exert their neuroprotective effect by inhibition of COX-2 mediated synthesis of pro-inflammatory prostanoids (8). Experimental evidence further suggests that inhibition of high expression levels of both COX-1 and COX-2 isoforms produces beneficial effects against brain inflammation (12). * Corresponding author. Prof. Rosario Pignatello, Phone: +39 0957384021; Fax: +39 095222239; email: [email protected]. † Dipartimento di Scienze Farmaceutiche, Università degli Studi di Catania. ‡ Dipartimento di Scienze Chimiche, Università degli Studi di Catania. § Università degli Studi di Padova. | The David Geffen School of Medicine at UCLA.

Some NSAIDs have been shown to decrease the production of Aβ42, the major component of senile plaques of AD brain, and counteract the progression of Aβ42 pathology in transgenic mouse models of AD (13). The proposed mechanism for this activity is an allosteric modulation of γ-secretase activity, the enzyme responsible for the formation of β-amyloid. The inhibition of Aβ42 production is independent from the antiCOX activity and is related to the chemical structure of the compounds, with some NSAIDs being active (ibuprofen, sulindac, flurbiprofen, indomethacin, diclofenac) and others not (naproxen, aspirin, celecoxib) (14, 15). NSAID availability in the brain is crucial to the efficiency of these drugs as neuroprotective agents. In order to enhance brain availability of NSAIDs, we investigated amphiphilic conjugates with lipoamino acids (LAA). In this report, we focus our interest on flurbiprofen (FLU, 1) a drug that, because of its anti-inflammatory and analgesic activities attributed to the inhibition of both COX isoforms, has been considered as a potential neuroprotective agent in AD therapy (16–21). Furthermore, FLU reduced the secretion of both Aβ42 and Aβ40 peptides in Neuro-2a cells and rat primary cortical neurons (12, 22) as well as in the brain of tg2576 β-amyloid transgenic mice (13, 14). FLU was modified with LAA residues; this was aimed at increasing its availability in the brain. LAA are R-amino acids bearing alkyl side chains, whose length and structure can be modified to achieve the desired physicochemical properties (23). Because of the presence of an alkyl chain and a polar amino acid head, LAA conjugation yields amphiphilic derivatives, with a membrane-like character that can favor their interaction with and penetration through biological membranes and barriers (24–26). Recent pharmacological studies have focused attention on the R-enantiomer of FLU (R-FLU) as a possible agent for AD treatment. R-FLU, in fact, lacks the side effects of NSAIDs mediated by their inhibition of COX. However, both S- and R-FLU have been shown to be equipotent in lowering Aβ42 levels in mouse brain and share the same mechanism of

10.1021/bc700312y CCC: $40.75  2008 American Chemical Society Published on Web 12/12/2007

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interaction with the γ-secretase complex (13). In this first study, we thus chose to perform the chemical derivatization using the racemic form of FLU; a further exploitation of the potentiality of FLU-LAA prodrugs could be made using pure R-FLU. LAA with different alkyl side chains ranging from 4 to 12 carbon atoms were linked to FLU through an ethanolamine spacer via an ester bond between FLU and the spacer and an amide bond between the LAAs and the spacer. The chemical stability profiles of all synthesized conjugates (6a-e) were determined in pH 7.4 phoshate buffer, while the pharmacokinetic and biodistribution patterns were evaluated for 6b and 6d after intravenous administration to mice. The capacity of FLU and prodrug 6 to interact with the insoluble fibrillar aggregates of β-amyloid (1–40) protein was also tested using radiofluorinated [F-18]FDDNP in a competition assay with in Vitro formed β-amyloid (1–40) fibrils. [F-18] FDDNP (2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile) is a positron-emitting radiofluorinated molecular imaging probe with binding affinity for neuropathological lesions found in AD, β-amyloid senile plaques, and neurofibrillary tangles, used for in ViVo positron emission tomography (PET) imaging of the neuropathology load in AD patients’ brains (27).

EXPERIMENTAL PROCEDURES Materials. FLU (racemic form), (()N-Boc-2-aminohexanoic acid (4a), (()2-aminoalkanoic acids, and the other reactants and solvents were purchased from Sigma-Aldrich Chimica Srl (Milan, Italy). The 2-aminoalkanoic acids were Boc-protected before use following a published procedure (28) to give compounds 4b-e. All solvents were dried by distillation according to standard methods (29) and stored over 4 Å molecular sieves activated at 400 °C for at least 24 h. [F-18]FDDNP was prepared by tosyloxy-to-fluorine nucleophilic substitution from cyclotron-produced [F-18]fluoride anion and 2-{[6-(2,2-dicyano-1-methylvinyl)-2-naphthyl](methyl) amino}ethyl 4-methylbenzenesulfonate as described elsewhere (30) and immediately used for competitive binding assays with FLU and prodrugs 6a-e. The male Balb/c mice weighing 22 ( 2 g used for the in ViVo studies had free access to food and were housed at the Department of Pharmaceutical Sciences, University of Padua. Animal experiments were performed in accordance with Italian law (D.L. 116/92) and European guidelines (EEC n. 86/609). Instrumentation. TLC analysis was used to check the purity of compounds using silica gel aluminum plates (Merck F254–366); the spots were detected by spraying the plate with ninhydrin or an acid–base reactant. Elemental analysis was carried out on a Carlo Erba 1106 analyzer; samples were kept under vacuum for 24 h over P2O5 before analysis. FT-IR spectra were recorded as nujol mulls with a Perkin-Elmer 1600 spectrophotometer. 1 H NMR spectra were obtained in CDCl3 at 200 MHz using a Varian Innova instrument. Chemical shifts are reported in ppm, using TMS as internal standard. Mass spectra were measured with a triple quadripole instrument (IPE sciex API 3000) operating in SIM mode with positive ion electrospray. The partition and distribution coefficients (cLog P and cLog D7.4) of FLU and prodrugs were calculated using the ACD/LogP 5.15 software (Advanced Chemistry Development Inc., Toronto, Canada) and the Pallas 3.0 software package (CompuDrug International, Inc., Sedona, AZ), respectively (Table 1). Synthesis of FLU-EA Intermediate (3). FLU (1) (0.82 mmol, 200 mg) was dissolved in 20 mL of anhydrous dichloromethane (DCM), and 1-hydroxybenzotriazole (HOBt) (0.82 mmol, 123 mg) and triethylamine (TEA) (1.64 mmol, 227 µL) were added. The solution was treated with 1.64 mmol (232 mg) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-

Pignatello et al. Table 1. General Structure and Properties of FLU-EA-LAA Conjugates 6a-e

HPLC method

compound n 6a 6b 6c 6d 6e FLU 3 b

3 5 7 9 11 – –

cLog Pa 4.82 ( 0.56 5.88 ( 0.56 6.94 ( 0.56 8.01 ( 0.56 9.07 ( 0.56 4.11 ( 0.37 –

cLog Pb cLog D7.4b 4.67 5.69 6.71 7.73 8.75 4.24 –

3.88 4.83 5.78 6.80 7.74 0.18 –

eluent (ACN/water, RT % v/v) (min) 60:40 60:40 75:25 90:10 90:10 75:25 75:25

7.1 13.3 8.1 9.6 15.8 1.3 5.9

a Predictions were made using the ACD LogP 5.15 software. Calculated using the Pallas 3.0 software.

ride (EDAC) and the mixture was kept at 0 °C in an ice–water bath for 2 h. N-Boc-ethanolamine [tert-butyl-N-(2-hydroxyethyl)carbamate] (190 µL, 1.23 mmol) in 10 mL of DCM was added to the reaction mixture and stirred at 0 °C for 2 h. The mixture was brought to room temperature and stirred for an additional 4–6 h. The solvent was removed under vacuum, and the oily residue was dissolved in 20 mL DCM, washed twice with 20 mL aliquots of water, followed by 5% NaHCO3 solution, 5% aqueous acetic acid, and finally brine. The organic phase was separated, dried over anhydrous sodium sulfate, and evaporated. The final residue was purified by flash chromatography on silica gel with ethyl acetate/cyclohexane, 40:60 (v/v) to obtain compound 2. The Boc protective group was finally removed with 10% (v/ v) solution of TFA in DCM for 30 min at room temperature. The solvents were evaporated under a nitrogen flow, and the residue was treated with 20 mL of 5% sodium hydrogen carbonate and extracted with 20 mL DCM. The organic phase was separated, dried as described above, and evaporated to dryness to give FLU-EA [3; 2-(2-fluoro-4-biphenyl)propionic acid 2-aminoethyl ester] (Scheme 1). The oily compound was kept refrigerated until use. Elemental analysis for C17H18FNO2, % found (theoretical): C 70.70 (71.06), H 6.22 (6.31), N 4.86 (4.87). IR (cm-1): 1733.7 (ester C)O), 3302.2 (amine); MS (m/z): 288 [M + 1]+ (100%); 1H NMR (ppm): 7.49–7.30 (m, 6H, aromatic), 7.08 (m, 1H, ∅-H), 7.04 (s, 1H, ∅-H), 4.26 (t, 2H, OCH2), 3.68–3.54 (q, 1H, CH-CH3), 3.08 (m, 2H, CH2NH2), 1.39 (d, 3H, CH3). Synthesis of FLU-EA-LAA Derivatives (6a-e): General Procedure (Scheme 1). A Boc-LAA derivative (4a-e, 0.25 mmol) was dissolved in 10 mL DCM, and HOBt (0.16 mmol, 24.5 mg) and TEA (0.32 mmoles, 44 µL) were added. The chosen LAA was then activated by adding EDAC (0.32 mmol, 45.4 mg) and stirring the mixture for 2 h in an ice–water bath. A solution of 3 (0.25 mmol, 68.9 mg) in 10 mL DCM was then added, and the mixture was kept at 0 °C for 2 h and then at room temperature for 18–24 h. Upon completion of the conjugation (TLC), the mixture was extracted as reported above, and the waxy residue was purified by column chromatography, using an ethyl acetate/cyclohexane gradient from 50:50 to 60: 40 (v/v) to isolate compounds 5a-e. The obtained product was deprotected by 20% TFA in DCM, and the residue was purified by semipreparative TLC (0.5 mm layer silica gel plates, SIL G-50, Macherey-Nagel GmbH & Co., Düren, Germany) eluted with an 80:20 DCM/methanol mixture. Compounds 6 were stored in closed amber glass containers at 4 °C.

Lipoamino Acid Prodrugs of Flurbiprofen

Analytical Characterization of Compounds 6a-e. 6a (n ) 3): 2-(2-fluoro-4-biphenyl)propionic acid 2-(2-aminohexanoyl)aminoethyl ester. Elemental analysis for C23H29FN2O3, % found (theoretical): C 69.21 (68.98), H 7.31 (7.30), N 7.00 (6.99). IR (cm-1): 1678.9 (amide C)O), 1750.1 (ester C)O). MS (m/z, %): 401.6 [M + H]+ (100), 313 (13), 288 (7). 1H NMR (ppm): 7.99 (broad, 1H, NH), 7.76–7.40 (m, 6H, aromatic), 7.22 (m, 1H, ∅-H), 7.15 (s, 1H, ∅-H), 4.41 (m, 2H, OCH2), 3.72 (q, 1H, CH-CH3), 3.42 (m, 2H, CH2-NH2), 1.79 (m, 2H, CH2), 1.61 (d, 3H, CH3), 1.35–1.24 (t, 4H, CH2), 0.85 (t, 3H, ω-CH3). 6b (n ) 5): 2-(2-fluoro-4-biphenyl)propionic acid 2-(2aminooctanoyl)aminoethyl ester. Elemental analysis for C25H33FN2O3, % found (theoretical): C 69.80 (70.07), H 7.77 (7.76), N 6.50 (6.54). IR (cm-1): 1679.8 (amide C)O), 1741.8 (ester C)O). MS (m/z, %): 429.5 [M + H]+ (100), 368 (4), 340 (5). 1H NMR (ppm): 7.97 (broad, 1H, NH), 7.52–7.34 (m, 5H, aromatic), 7.26 (s, 1H, ∅-H), 7.11 (m, 1H, ∅-H), 7.05 (s, 1H, ∅-H), 4.10 (m, 2H, OCH2), 3.72 (q, 1H, CH-CH3), 3.45 (m, 2H, CH2-NH2), 1.77 (m, 2H, CH2), 1.48 (d, 3H, CH3), 1.25–1.21 (t, 8H, CH2), 0.82 (t, 3H, ω-CH3). 6c (n ) 7): 2-(2-fluoro-4-biphenyl)propionic acid 2-(2aminodecanoyl)aminoethyl ester. Elemental analysis for C27H37FN2O3, % found (theoretical): C 70.80 (71.02), H 8.16 (8.17), N 6.11 (6.14). IR (cm-1): 1679.6 (amide C)O), 1739.3 (ester C)O). MS (m/z, %): 457.7 [M + H]+ (100), 396 (8), 368 (7). 1H NMR (ppm): 8.04 (broad, 1H, NH), 7.62–7.38 (m, 5H, aromatic), 7.56 (s, 1H, ∅-H), 7.09 (m, 1H, ∅-H), 7.02 (s, 1H, ∅-H), 4.30 (broad m, 2H, OCH2), 3.90 (q, 1H, CH-CH3), 3.49 (m, 2H, CH2-NH2), 1.66 (m, 2H, CH2), 1.56 (d, 3H, CH3), 1.32–1.17 (t, 12H, CH2), 0.88 (t, 3H, ω-CH3). 6d (n ) 9): 2-(2-fluoro-4-biphenyl)propionic acid 2-(2aminododecanoyl)aminoethyl ester. Elemental analysis for C29H41FN2O3, % found (theoretical): C 71.59 (71.87), H 8.56 (8.53), N 5.81 (5.78). IR (cm-1): 1667.1 (amide C)O), 1734.1 (ester C)O). MS (m/z, %): 485.6 [M + H]+ (100), 486 (35), 396 (9). 1H NMR (ppm): 7.98 (broad, 1H, NH), 7.54–7.39 (m, 6H, aromatic), 7.21 (m, 1H, ∅-H), 6.88 (s, 1H, ∅-H), 4.34 (m, 2H, OCH2), 3.76 (q, 1H, CH-CH3), 3.48 (m, 2H, CH2-NH2), 1.77 (m, 2H, CH2), 1.63 (d, 3H, CH3), 1.32–1.21 (m, 16H, CH2), 0.86 (t, 3H, ω-CH3). 6e (n ) 11): 2-(2-fluoro-4-biphenyl)propionic acid 2-(2aminotetradecanoyl)aminoethyl ester. Elemental analysis for C31H45FN2O3, % found (theoretical): C 72.88 (72.62), H 8.88 (8.85), N 5.45 (5.46). IR (cm-1): 1664 (amide C)O), 1728 (ester C)O). MS (m/z, %): 513.9 [M + H]+ (100), 485 (55), 457 (20), 420 (12). 1H NMR (ppm): 8.04 (broad, 1H, NH), 7.50–7.26 (m, 5H, aromatic), 7.46 (s, 1H, ∅-H), 7.12 (m, 1H, ∅-H), 6.97 (s, 1H, ∅-H), 4.24 (m, 2H, OCH2), 3.75 (q, 1H, CH-CH3), 3.46 (m, 2H, CH2-NH2), 1.79 (m, 2H, CH2), 1.60 (d, 3H, CH3), 1.30–1.22 (t, 20H, CH2), 0.88 (t, 3H, ω-CH3). Chemical Stability Assay. Each prodrug 6a-e (1 mg) was dissolved in DMSO (100 µL) and diluted to 1 mL with isotonic phosphate buffer (PBS, pH 7.4). The mixture was incubated at 37 ( 1 °C, and 100 µL aliquots were withdrawn at regular time intervals (every 7–8 days). HPLC analysis was performed to determine the amount of intact prodrugs as well as the amounts of released FLU (1) and FLU-EA (3), products of hydrolytic cleavage of the FLU-EA ester bond or the EA-LAA amide bond, respectively. A Varian ProStar chromatograph equipped with a Varian 410 autosampler and a Waters C18 Symmetry column (5 µm, 4.6 × 150 mm), connected to a Waters C18 Symmetry precolumn, was used for the analysis. The UV detector was set at 247 nm. 10 µL aliquots were injected by means of a Varian 410 autosampler and the compounds eluted at 1 mL/min under isocratic conditions with different water/ acetonitrile (ACN) mixtures, as summarized in Table 1.

Bioconjugate Chem., Vol. 19, No. 1, 2008 351 Scheme 1. General Procedure for the Synthesis of FLU-EA-LAA Prodrugs

Plasma Stability Evaluation. Three heparinized mouse whole blood samples (300 µL) were mixed with 150 µL of 50:50 water/ACN containing 9 µg of either FLU, 6b or 6d, and kept at 37 °C. At scheduled times (30, 60, 120, 180, 240, and 300 min), 50 µL of the samples were withdrawn and added of 100 µL of ACN to precipitate proteins. The samples were centrifuged for 5 min at 4 °C and 14 000 rpm, and the supernatant was analyzed according to the procedure reported below. The drug and prodrug contents were referred to the standard titration curves. Analytical Procedure. The content of FLU, FLU-EA (3), and prodrugs 6b and 6d in the tissue extraction samples was evaluated by reverse-phase HPLC using an analytical C-18 column (Luna, Phenomenex, 250 × 4.6 mm). NAP standard solution (50 µL) was added to the samples as an internal standard. The column was eluted with a gradient of water (eluent A) and ACN (eluent B), both containing 0.05% TFA, according to the following gradient scheme: 40% eluent B for 3 min, up to 60% eluent B in 20 min, to 90% eluent B in 2 min, and back to the initial conditions in 2 min. The UV detector was set at 247 nm. FLU, 6b, 6d, and NAP retention times were 20.5, 14.7, 25.5, and 13.9 min, respectively. Standard curves were calculated for each compound in the range 0.08–40 µg/ mL with r2 ) 0.996 – 0.999. The amounts of the compounds 6b and 6d and their hydrolysis products FLU and FLU-EA were determined from the standard curves using the corresponding peak areas. In the case of solid tissues, the blood contribution was evaluated and subtracted according to the procedure reported in the literature (31). Extraction Tests from Plasma and Organs. The recovery of compounds 1, 6b, and 6d from mouse liver, kidney, brain, spleen, and plasma was evaluated. 100 µL of heparinized whole blood was withdrawn from mice by retrobulbar puncture and added to 100 µL of 50:50 (v/v) ACN/water mixture containing different amounts (1, 2, and 3 µg) of FLU or FLU equivalent amounts of prodrugs, and 50 µL of naproxen standard solution (NAP) (0.1 µg/µL in the same solvent mixture). The samples were mixed and centrifuged for 3 min at 3000 rpm. Aliquots of 50 µL of clear supernatant were

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withdrawn, mixed with 100 µL of ACN, vortex-mixed, and finally centrifuged for 3 min at 14 000 rpm. The supernatants containing the extracted compounds were analyzed by HPLC as reported above. Liver, kidney, brain, and spleen taken from the same mouse were washed with physiological solution and gently dried on paper. Different volumes (10, 20, 30, 40, and 50 µL) of ACN solutions containing 50 ng/µL of each compound were added to 50 mg of liver, kidney, brain, and spleen samples. The samples were mixed with 50 µL of NAP standard solution (0.1 µg/µL) and 1 mL of 50:50 (v/v) ACN/water mixture. The samples were homogenized and then centrifuged for 5 min at 4000 rpm. The supernatants were collected by suction and the pellets treated three times with 1 mL of ACN/water mixture and once with 1 mL of ACN. All five supernatants were combined and centrifuged for 3 min at 14 000 rpm. The final volume was lyophilized, and the residue was dissolved in 200 µL of 50:50 (v/v) ACN/water mixture, centrifuged for 5 min at 14 000 rpm, and finally analyzed by reverse-phase HPLC according to the gradient elution reported above. Pharmacokinetic Assay. Solutions containing 4 mg of FLU (1) or FLU equivalent compounds 6b or 6d in 1 mL of DMSO were diluted with 3 mL of phosphate-buffered saline (0.15 M NaCl, 20 mM phosphate buffer), pH 7.4. 110 mice were divided into 3 groups of 35 animals (test groups) and 1 group of 5 animals (control group). Each group received one of the above solutions; the animals were injected via the tail vein with 100 µL of the respective solution. The 5 animals in the control group were intravenously injected with 100 µL of the vehicle (1:3 DMSO/saline solution). At scheduled times, 50 µL of blood samples were randomly withdrawn by retrobulbar puncture; at least three animals were used per each time point. The blood samples were immediately mixed with 50 µL of a NAP standard solution (0.1 µg/µL). Aliquots of the supernatant (50 µL) were mixed with 50 µL of ACN, centrifuged for 5 min at 14 000 rpm to precipitate proteins, and analyzed by reverse-phase HPLC as described above. At scheduled times (30, 60, 180, 420, and 560 min) seven animals/group were bled and sacrificed and liver, spleen, kidney, and brain were taken. The collected blood was processed as described above. The whole organs were washed in saline and gently dried with absorbent paper, weighed, spiked with 50 µL of the above standard NAP solution, and processed according to the procedure previously reported. The amounts of recovered FLU-EA-LAA prodrugs, FLU-EA (3), and free FLU were determined by HPLC analysis as described above. Radioactive [F-18]FDDNP Competitive Binding Assay with Aβ(1–40) Fibrils. Aβ(1–40) Fibril Formation. β-amyloid(1–40) fibrils were prepared in Vitro from the commercially available Aβ(1–40) peptide (Biosource, Camarillo, CA) according to published methods (30). Briefly, 1.0 mg of Aβ(1–40) peptide was dissolved in 1 mL of phosphate buffered saline (PBS), pH 7.4, and stirred with a magnetic stirring bar for 3–4 days at 37 °C, after which a visibly cloudy solution was obtained. This mixture was further diluted to 10 mL with PBS, pH 7.4, and used as such for competitive binding assays of FLU and prodrugs 6a-e against [F-18]FDDNP. The fibril formation was confirmed using Congo red (32) and Thioflavine T (33) UV spectrophotometric assays. Fibrils were used immediately after their production was confirmed. Competition Binding Assays. The methodology published for determination of competition binding assays with NAP and ibuprofen (IBU) was used (10). Fresh 5 mM ethanol solutions of FLU and of all prodrugs were prepared for each competitive binding assay. Solutions of each compound in 1% ethanol/PBS, pH 7.4, in the range from 0.1 pM to 300 nM were incubated with 9.25 MBq/mL of [F-18]FDDNP (specific activity >74

Pignatello et al.

Figure 1. Degradation profiles of compounds 6a-e in isotonic phosphate buffered saline at pH 7.4.

GBq/µCi) and with 0.875 µg/mL of Aβ(1–40) fibrils for 60 min. The fibrils were isolated by filtration through Anodisc 25 alumina membrane filters (0.2 µm particle retention; Whatman International Ltd., Maidstone, England) in a 1225 sampling manifold (Millipore, Bradford, MA) that was modified with stainless steel support screens (Millipore) and glass sample chambers. Each filter was washed twice with 3 mL of PBS, pH 7.4. The radioactivity retained by the filters was measured and decay-corrected to a common reference time with a Packard Cobra II Auto-Gamma gamma counter (Packard, Meriden, CT). The binding of [F-18]FDDNP to Aβ(1–40) fibrils without a competitor determined 100% specific binding. Competition results were analyzed and Ki values then calculated using the ligand binding module for SigmaPlot 2001 (SPSS, Chicago, IL). The comparison of one- and two-site models for binding involved calculating the F statistic with a significance level of P < 0.05. All radioactive binding assays were performed in triplicate.

RESULTS AND DISCUSSION Chemistry and Stability Tests. The lipophilic prodrugs of FLU were synthesized according to a procedure used for the preparation of other LAA conjugates, using water-soluble carbodiimide-assisted coupling reactions (34–36). Compounds 6a-e were obtained in 50–80% yield and with high purity using semipreparative TLC. No attempt was made at this level to separate the eventual diastereomers formed by using racemic FLU and Boc-LAA as starting compounds. Spectroscopic and mass analyses confirmed the assigned structure; in particular, IR spectroscopy showed the disappearance of the stretching peak of the FLU carboxyl group (at 1697 cm-1 in nujol) and appearance of two new peaks, around 1720–1750 cm-1, due to the FLU-EA ester bond, and 1665–1680 cm-1, related to the EA-LAA amide bond. The increasing lipophilicity of prodrugs with the increasing length of the side alkyl chain of the LAA residue is shown by the calculated Log P and Log D7.4 values (Table 1). The chemical stability of compounds 6a-e was tested in phoshate buffered saline at 37 °C and pH 7.4 (Figure 1). Lengthening of the LAA alkyl side chain, and therefore of compound lipophilicity, was associated with an increasing stability to chemical hydrolysis, except for compound 6b, which showed the highest stability. Hydrolytic cleavage of all prodrugs was completed after 7 weeks. The HPLC analysis showed that all the prodrugs 6 were hydrolyzed predominantly into FLU

Lipoamino Acid Prodrugs of Flurbiprofen

Figure 2. Plasma FLU levels after i.v. administration of the free drug (2) or conjugates 6b (O) and 6d (•) (100 µg FLU equivalent/mouse). In the case of 6b and 6d, FLU levels refer to the amount of injected FLU equivalents.

from the earliest incubation times on, since the intermediate FLU-EA (3) was detected only at very low concentrations. Compound 3 was also very susceptible to hydrolysis in PBS, and a very rapid conversion into FLU was observed within 24 h of incubation (data not shown). As expected, the ester bond in the prodrugs 6a-e was more susceptible to hydrolysis than the amide link between the spacer and the LAA residue. The sensitivity to chemical hydrolysis of the ester bond in the prodrugs was verified by incubating compounds 6a-e in a 3:1 (v/v) 2 M KOH/methanol mixture at 37 °C. After 15 min, the complete hydrolysis of the prodrugs into the parent FLU was observed (HPLC) (data not shown). In ViWo Biodistribution Tests. Based on their alkyl side chain length, prodrugs 6b and 6d were chosen as representatives of the short- and long-chain LAA conjugates to carry out pharmacokinetic and biodistribution tests. Preliminary validation studies demonstrated the reliability of the extraction and analytical procedures adopted in the experimental protocols. The recovery of FLU and prodrugs 6b and 6d from blood spiked with these compounds was 89.7 ( 4.3%, 91.5 ( 6.8%, and 94.9 ( 4.2%, respectively. The recovery from the homogenized tissues was in the range 86–94% for all analyzed organs (liver, kidney, brain, lungs, and spleen). These values were used in the elaboration of the pharmacokinetic data to normalize the value of recovered products. Neither FLU nor FLU-EA were detected in blood and homogenized organ samples supplemented with 6b and 6d, indicating that these prodrugs were not hydrolyzed during the extraction procedure. Figure 2 shows the time-course FLU levels in plasma after i.v. administration of the parent drug or prodrugs 6b and 6d to mice. In the case of the latter compounds, no intact prodrug was detected in blood 2 min after injection. After the same amount of time, low levels of free FLU were observed after administration of both 6b and 6d. The fast disappearance of the prodrugs (Figure 2) can be only partially ascribed to their plasma degradation, since preliminary studies demonstrated that both conjugates are relatively stable upon in Vitro incubation in plasma (about 25% degradation after 2 h and 40% degradation after 6 h) (Figure 3). The absence of the prodrug in plasma and the low FLU levels in liver and kidneys can thus be ascribed to a quick partition into peripheral compartments, namely, endothelial tissue, from which the following hydrolysis by endothelial lipases justifies the prolonged and stable drug levels observed in the circulation. A more likely alternative is the liver release of the hydrophobic compounds via the bile, release into the small intestine, and reabsorption prior to hydrolysis via endothelial lipases. A detailed pharmacokinetic or radiolabeled prodrug study would answer the question and is planned as a further step of this research.

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Figure 3. Stability of FLU or prodrugs 6b and 6d after incubation in mouse plasma at 37 °C.

In contrast, after i.v. injection, free FLU did not undergo a rapid partition into the peripheral compartments and maintained prolonged high plasma drug levels compared with the drug levels reached after the administration of prodrugs 6b and 6d. To get information about the in ViVo fate of 6b and 6d, prodrug and free FLU contents in the main organs were measured. The experiments were carried out for 9 h, since prodrug degradation made longer time evaluations difficult. Figure 4 shows the extracellular accumulation of FLU in the main organs after FLU, 6b, or 6d i.v. administration to mice. After i.v. administration, free FLU rapidly distributed to the various tissues with concentration distributions in the order liver > brain > kidney > spleen. FLU concentration in these organs was almost constant up to 3 h, follow by a slow decline. Only negligible amounts of the drug were detected after 7 h. In the case of prodrugs 6b and 6d, only the released parent FLU was observed. The intact prodrugs could not be detected in the homogenized tissues, similarly to what was observed in the blood. The two prodrugs, however, displayed a markedly different behavior with regard to FLU availability to various organs. From the first times after administration of 6b, only small amounts of FLU were detected in the liver, kidneys, and spleen, while negligible disposition was observed in the brain. Conversely, the higher homologue 6d led to accumulation of higher amounts of released FLU in the various organs. In general, after 6d administration the maximal FLU concentration in the main tissues was reached later than after free FLU injection. The 6d derivative yielded maximal FLU accumulation in the liver and spleen after 1 and 3 h after administration, respectively. The FLU levels in the kidneys also increased throughout 9 h, indicating that the drug was constantly released from the peripheral compartments and possibly eliminated by glomerular filtration or distributed to other kidney tissues. Again, more detailed studies are required to determine the exact elimination routes of the drug after i.v. administration of these prodrugs, for instance, by determining their concentration vs time in urine or feces. Concerning the presence of FLU in the brain, injection of the parent drug led to constant brain levels for 3 h; therafter, the drug rapidly disappeared from this area. Such a pattern conforms to the levels of FLU in plasma, where the drug was detectable only for 3 h (Figure 2). After injection of compound 6d, the released FLU appeared in the brain after 1 h, and the intracerebral FLU levels continued to rise up to the ninth hour, reaching higher values than those observed in the first 3 h after the injection of free FLU. The maximum brain/liver ratio (about 12% brain/plasma concentration) was reached 1 h after injection. These pharmacokinetic findings suggest that the prodrugs dispersed rapidly and massively into a peripheral compartment. In the case of 6b, which was found to be more stable than the upper homologues in plasma, the parent drug was not released into the brain; with 6d, free FLU was released and accumulated

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Pignatello et al.

Figure 4. FLU concentrations in liver, brain, kidneys, and spleen after i.v. administration (100 µg FLU equiv/mouse) of the free drug (white) or conjugates 6b (black) and 6d (gray). In the case of the latter compounds, FLU levels were referred to the amount of FLU released from the prodrugs in ViVo.

preferentially in the brain tissue; actually, the brain/plasma FLU concentration obtained with 6d increased over time to reach about 30% 420 min from injection. The different tissue distribution obtained with the various FLU derivatives is probably due to their dissimilar in ViVo pathway/ metabolism, such as the previously mentioned enterohepatic reticulation or association with vessel endothelium. However, a profound characterization of the pharmacokinetic profile of prodrugs 6 was beyond the intent of this paper; thus, further in ViVo pharmacological studies will be started to elucidate the fate of these compounds. The stark differences observed in the in ViVo behavior between the two prodrugs highlight the effect that the LAA residue can exert on their biopharmaceutical profiles. In particular, a relatively small difference in the chain length (four methylene groups) in the side alkyl chain of the LAA promoiety of 6b and 6d led to a considerably different distribution profile. The combined analysis of the chemical and plasmatic stability tests confirmed the validity of these conjugates as possible prodrugs for FLU. Compounds 6, in fact, showed good stability in aqueous buffer and a slow release of the active drug in plasma. [F-18]FDDNP Binding Assay. IBU and NAP showed competitive inhibition of [F-18]FDDNP binding with in Vitro β-amyloid fibrils, with weak binding for IBU (Ki ) 11.3 ( 5.2 µM for S-IBU and 44.4 ( 17.4 µM for R-IBU) and with 3 orders of magnitude higher binding affinity for NAP (Ki ) 2.76 ( 0.95 nM for S-NAP and Ki ) 5.71 ( 1.31 nM for R-NAP) (10, 30, 37). In contrast to NAP and IBU, FLU proved to be ineffective in blocking [F-18]FDDNP from binding to its two specific binding sites on in Vitro formed Aβ(1–40) fibrils in the concentration range of competitors used (0.1 pM to 300 nM). This apparent lack of binding can be better understood if structural factors are taken into consideration. The structure of FLU is related to that of IBU, i.e., they both have a central phenyl ring and propanoic acid residue. However, while IBU possesses an isobutyl group in the para position to the alkyl carboxilic acid

chain, FLU has a phenyl ring in the para position and a fluorine meta substitution. Such an arrangement of substituents in FLU most likely pushes both phenyl rings out of plane due to steric and electrostatic interactions between the para phenyl ring and the meta fluorine atom. The consequent lack of planarity may then decrease the probability of FLU interaction with the [F-18]FDDNP binding site. FLU-EA, the intermediate FLU ester with 2-aminoethanol, also showed no apparent competition in the in Vitro assays. The FLU prodrugs 6a (C4 alkyl chain), 6b (C6 alkyl chain), 6c (C8 alkyl chain), and 6d (C10 alkyl chain), on the other hand, demonstrated competitive inhibition of [F-18]FDDNP binding to β-amyloid(1–40) fibrils. Prodrug 6e (C12 alkyl chain) did not compete with [F-18]FDDNP binding. Prodrugs 6a, 6b, and 6d competed with [F-18]FDDNP for low-affinity binding (Ki values: 6a 0.386 µM; 6b 3.65 µM; and 6d 0.017 µM), prodrug 6c, on the other hand, showed competition for both high- and low-affinity binding sites with Ki ) 25.3 pM and 0.396 µM, respectively. The results are shown in Figure 5. Conjugation of the LAA residues to FLU changes the nature of its interaction with the Aβ(1–40) fibril surface. Hydrophobic interaction between the alkyl side chain with the hydrophobic areas on the surface of the fibrils becomes important and may overcome the effects of steric hindrance allowing the prodrugs to bind to the [F-18]FDDNP binding site(s). We also cannot underestimate interactions of the free amino group with the anionic side chains on the fibril. There are several aspartate and glutamate residues in the structure of the Aβ protein which can form strong electrostatic interactions and anchor the prodrugs 6a-d on the surface. The capacity of prodrug 6c to bind to both [F-18]FDDNP binding sites on the fibrils and also the complete absence of competition suggested different hypotheses. The processes involved may include micellization of the hydrophobic prodrugs at higher concentrations (high nanomolar) under the conditions used for the competition assays (1% ethanol in PBS) and interactions of the micelles with the fibril surface. Formation

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Bioconjugate Chem., Vol. 19, No. 1, 2008 355

Figure 5. Prodrugs 6a-d inhibition [F-18]FDDNP binding profile to the in Vitro formed β-amyloid(1–40) fibrils. Compounds 6a (A), 6b (B), and 6d (D) competed with [F-18]FDDNP for one binding site. Compound 6c (C) blocked [F-18]FDDNP from both binding sites.

of micelles or other types of lipophilic aggregates can also be taken into consideration to explain the pharmacokinetic profile observed for prodrugs 6 and discussed previously. This statement implies that the amount injected in these studies is large and the pharmacokinetics would be altered by a mass effect. The amount administered for the present studies had to be reasonably high to permit chemical analysis, but in ViVo studies using radiolabeled derivatives of these compounds (e.g., subnanogram/ kg injected) may produce different and more reliable (e.g., mass independent) results.

CONCLUSIONS The combined analysis of chemical and plasmatic stability data indicate these conjugates as possible prodrugs for FLU. They showed, in fact, good stability in buffer solution and a gradual release of the active drug in plasma, most probably upon enzymatic intervention. Compound 6d, in particular, behaved as a slow-release FLU prodrug of FLU. Even though their physicochemical properties were not optimized, it is possible to speculate that selected structural changes, e.g., the kind of spacer between the drug and the LAA promoiety, could lead to novel, more selective compounds for brain delivery of FLU. The inhibitory activity of [F-18]FDDNP binding to in Vitro formed β-amyloid protein fibrils confered a structure-binding affinity relationship to these compounds; this study demonstrates that, if any differential pharmacological effect was observed with respect to FLU, it is not because of

direct binding to the fibrils, but rather to other mechanisms (e.g., anti-inflammatory mechanisms), suggesting FLU-EA-LAA derivatives as potential prodrugs for AD treatment. Experimental results showed that only FLU was found in specific organs even when prodrugs 6b and 6d were injected. A possible explanation is that the lipophilic prodrugs accumulated in a specific peripheral compartment, e.g., bile and following enterohepatic recirculation, from where FLU was released; thereafter, it entered the brain and was slowly eliminated by kidneys and liver. In addition, compounds 6b and 6d possess different lipophilic/hydrophilic balance and structure that may affect their pharmacokinetic properties and stability, in particular, the high stability in buffer that seems unusual for an ester linkage. Similarly, the different distribution kinetics recorded in the brain after the administration of either the prodrugs or parent FLU deserve a detailed pharmacological study, for instance, by using radiolabeled forms of FLU-LAA conjugates. As discussed above, the probable development of this study will be to apply the same synthetic strategy to R-FLU, e.g., the drug enantiomer that is currently under clinical trial as a potential agent in AD therapy. The issue of FLU-LAA prodrug stereochemistry, in fact, deserves attention, since their stability in plasma and organs can also be affected by stereochemical differences related to the enzymes involved in the metabolism and/or bioconversion to FLU.

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ACKNOWLEDGMENT Professor Istvan Toth, at the School of Pharmacy, University of Queensland, Brisbane (Australia), is gratefully acknowledged for mass analysis. We would like to thank Gerald Timbol at the David Geffen School of Medicine at UCLA, Los Angeles (USA), for performing the Aβ fibril binding assays and Professor Paolo Caliceti, at the University of Padua (Italy), for his helpful discussion in the manuscript preparation. This work was partially supported by the USA Department of Energy grant DE-FC0302ER63420.

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