196
Forrest et al.
(t, J = 8.7 Hz, 1H, H13), 7.11 (d, J = 9 Hz, 1H, NH), 7.33–8.16 yielding 0.5-mM PEG-b-PCL micelles after removing the
(m, 15H).
7-palmitate-paclitaxel 4c. To a solution of 3 (25 mg,
volatile organic solvent.
The incorporation of prodrugs into PEG-b-PCL micelles
was verified by equivalent retention times in ultraviolet (UV)
and refractive index (RI) chromatographs from gel perme-
ation chromatography. PEG-b-PCL micelles were injected on
an OHpak SB-806M GPC column (20-ml injections, 0.5-mM
PEG-b-PCL, 0.75 ml/min of ddH2O, 10-C; Shodex, Kawasaki,
Japan) and detected by refractive index (RI) and UV absor-
bance (228 nm). Prodrug loading into PEG-b-PCL micelles
was quantitatively determined by reverse-phase HPLC (All-
tech Econosphere 3-mm 4.6 ꢀ 50 mm) using a 0.01% (v/v)
trifluoroacetic acid (TFA)—acetonitrile (ACN) gradient (40–
100% ACN, 50-C, 228-nm detection and trans-stilbene as
internal standard). Hydrodynamic diameters of PEG-b-PCL
micelles were determined by dynamic light scattering (DLS;
NICOMP 380 ZLS, Particle Sizing Systems, Santa Barbara,
CA). Data were analyzed by intensity-weighted Gaussian dis-
tribution fitting (NICOMP version 1.76). Measurements were
made for a minimum of 10 min or at least 100 ꢀ 105 counts in
channel 1.
0.211 mmol) in 1 ml of THF, 5 drops of 1-M TBAF (tetrabu-
tylamoniumfloride) in THF was added. The reaction mixture
was stirred at room temperature for 1 h. The resulting solution
was reduced to dryness in vaccuo, redissolved in 2 ml CH2Cl2,
washed with water (5 ml ꢀ 1), and the organic layer dried over
Na2SO4. Removal of solvent followed by preparatory TLC on
silica (1: 1 EtOAc:hexane) provided 4c as a white solid (20 mg,
1
90% yield). H NMR (400 MHz, CDCl3) d 0.88 (t, 3H, CH3),
1.10 (s, 3H, H17), 1.22 (s, 3H, H16), 1.76 (s, 3H, H19), 1.93 (s,
3H, H18), 1.92–2.14 (m, 2H, H6), 2.3 and 2.56 (m, 2H, H14),
2.58 (s, 3H, 4-Ac), 3.91 (d, J = 6.9 Hz, 1H, H3), 4.23 (d, J = 8.1
Hz, 1H, H20), 4.30 (d, J = 1.8 Hz, 1H, 10-OH), 4.35 (d, J = 8.1
Hz, 1H, H20), 4.42 (dd, J = 6.6 and 10.8 Hz, 1H, H7), 4.68 (d,
J = 2.1 Hz, 1H, H2_), 4.98 (dd, J = 1.5 and 9.3 Hz, 1H, H5), 5.13
(d, J = 1.8 Hz, 1H, H10), 5.69 (d, J = 6.9 Hz, 1H, H2), 5.73 (dd,
J = 1.8 and 9 Hz, 1H, H3_), 6.34 (t, J = 8.7 Hz, 1H, H13), 7.11
(d, J = 9 Hz, 1H, NH), 7.33–8.16 (m, 15H).
Synthesis of 2-palmitate-paclitaxel 5c. The method for
synthesis of 2-palmitate-paclitaxel 5c is described below. Syn-
thesis of 5a–b were according to the same procedure, with
substitution of the appropriate fatty anhydride.
PEG-b-PCL Micelle Prodrug Release Studies
Release experiments were based on the methodology of
Eisenberg and coworkers [17] with modifications for temper-
ature and pH control. Micelle prodrug solutions were pre-
pared at 0.5 mM (PEG-b-PCL basis) with 20% w/w prodrug
as above, and 0.5 ml of each solution was diluted to 2.5 ml
with ddH2O and injected into 10,000 MWCO dialysis
cassettes (Pierce, Rockford, IL; n = 4). As a control, an equal
quantity of prodrug dissolved in 100 ml of EtOH was injected
into separate cassettes (n = 3). Dialysis cassettes were placed
in a well-mixed temperature controlled water bath at 37-C,
overflowed with ddH2O so that the bath volume was ref-
reshed every 15 to 20 min. Peristaltic pumps under computer
control separately injected 50 g/l solutions of tribasic and
monobasic phosphate to maintain pH at 7.4 T 0.05 (apparatus
built in-house). At fixed time points, dialysis cassette
volumes were made up to 2.5 ml with ddH2O, 100-ml aliquots
l aliquots withdrawn, and prodrug concentrations determined
by reverse-phase HPLC (see supra).
2-palmitate-paclitaxel 5c. To a solution of paclitaxel 1
(100 mg, 0.117 mmol) in 1.5 ml dry toluene was added
palmitic anhydride (115.79 mg, 0.234 mmol) and DMAP
(11.435 mg, 0.0936 mmol). The reaction mixture was stirred
at room temperature for 12 h. The resulting solution was
washed with a 1-M HCl (5 ml ꢀ 1) and water (5 ml ꢀ 1), and
the organic layer was dried over Na2SO4. Removal of solvent
followed by preparatory TLC (1:1 EtOAc:hexane) provided
1
5c as a white solid (60 mg, 47% yield). H NMR (400 MHz,
CDCl3) d 0.87 (t, 3H, CH3), 1.10 (s, 3H, H17), 1.22 (s, 3H,
H16), 1.76 (s, 3H, H19), 1.93 (s, 3H, H18), 1.92–2.14 (m, 2H,
H6), 2.3 and 2.56 (m, 2H, H14), 2.58 (s, 3H, 4-Ac), 3.91 (d,
J = 6.9 Hz, 1H, H3), 4.23 (d, J = 8.1 Hz, 1H, H20), 4.30 (d,
J = 1.8 Hz, 1H, 10-OH), 4.35 (d, J = 8.1 Hz, 1H, H20), 4.42
(dd, J = 6.6 and 10.8 Hz, 1H, H7), 4.68 (d, J = 2.1 Hz, 1H,
H2_), 4.98 (dd, J = 1.5 and 9.3 Hz, 1H, H5), 5.13 (d, J = 1.8 Hz,
1H, H10), 5.69 (d, J = 6.9 Hz, 1H, H2), 5.73 (dd, J = 1.8 and
9 Hz, 1H, H3_), 6.34 (t, J = 8.7 Hz, 1H, H13), 7.11 (d, J = 9 Hz,
1H, NH), 7.33–8.16 (m, 15H).
Octanol-Water Partition Coefficients
Preparation and Characterization
of Prodrug Loaded PEG-b-PCL Micelles
Octanol-water partition coefficients (log Po/w) of pacli-
taxel prodrugs were determined indirectly by microemulsion
electrokinetic chromatography (MEEKC) based on the tech-
Paclitaxel prodrug loaded PEG-b-PCL micelles were nique of Klotz et al. [18]. Running buffer was prepared by
prepared by dissolving PEG-b-PCL (5000:10500, Mw/Mn 1.11, titration of 25-mM sodium phosphate monobasic with 50-mM
JCS Biopolytech Inc., Toronto, Ontario Canada) and pro- sodium tetraborate to pH 7.00, and 1.44 g of sodium dodecyl
drug in a minimum volume of acetone and adding drop-wise sulfate, 6.49 g of 1-butanol, and 0.82 g of heptane were made
to vigorously stirred ddH2O using a syringe pump. The up to 100 ml with phosphate-borate buffer. The running buffer
organic solvent was then removed by stirring under an air was ultrasonicated for 30 min in a closed 250-ml flask in ice
purge. Where stated, samples were further concentrated by water (G112SP1 Special Ultrasonic Cleaner, Laboratory
prolonged evaporation under an air purge. After removing Supplies Company Inc., Hicksville, NY). Longer times may
the organic solvent, PEG-b-PCL micelles were passed be required to obtain a stable emulsion with lower power
through a 0.22-mm polyestersulfone filter to remove insoluble ultrasonicators. Compounds and standards (n = 3) were dis-
material and unincorporated drug [8]. In a typical experi- solved in the running buffer (0.05 mg/ml) with 0.5 ml/ml of
ment, 1 mM of PEG-b-PCL was dissolved in 0.75 ml of dry nitromethane and 0.5 ml/ml of 1-phenyldodecane by ultra-
acetone and added dropwise (50 ml/min) to 2 ml of ddH2O sonication (10 min) in a closed tube and centrifuged