1131
layer was sequentially extracted by 1 HCl twice, 10% Female Wistar rats (150±200 g) were purchased from the
Naproxen polymeric prodrug
m
w w of NaHCO3 twice, deionized water once and a satu- National Laboratory Animal Breeding and Research
}
rated solution of NaCl once. The extracted solution was Center (Taipei, Taiwan). Rats were divided into three
dried over MgSO4 and the solvent was removed by a rotary groups of 8 each. Group I served as a control group
vacuum evaporator. The yellow liquid was puri®ed by without using drugs. Groups II and III received naproxen
1
column chromatography (70±130 mesh silica gel purchased (7 0 mg kg ) and the polymeric prodrug (42 mg kg 1),
-
-
.
from Merckas a stationary phase and ethyl acetate±hexane, respectively, where the dose was molecularly equivalent to
}
1:4 v v, as a mobile phase). The ®nal product was a white the free drug. Drugs were administered as a homogeneous
solid with a yield of 46% with a mass spectrum of 274 microsuspension in an aqueous solution of Tween-80 (10%
+
(molecular ion), 185 (M -CO2CH2CH2OH). Analyses: w v) intraperitoneally. Thirty minutes after administra-
}
calculated for C16O4H18 : C, 70 06; H, 6 61; found : C, tion, each rat received in its right hind paw a subplantar
.
.
69 87; H, 6 64. IR (NaCl, cm ): 2976 (sp C-H), 1726 injection of a 0 8% carrageenan in normal saline ( -
1
3
-
.
.
.
(C?O), 1607 (ring C?C), 1197 (C>O). 1H NMR (CDCl3, carrageenan, type IV, Sigma, 0 1 mL rat). The measure-
}
): 1 60 (d, 3H, - CHCH ), 3 75 (m, 3H, CHCH and ment of the hind-paw volume was carried out using a Ugo
.
.
CH2OH), 3 90 (s, 3H, -OCH3), 4 20 (m, 2H, CO2CH2), Basile Plethysmometer model 7150, before any treatment
.
U
U
3
3
.
.
7 10±7 75 (m, 6H, ArH). UV VIS (dioxane):
}
.
245 nm.
.
¯
(Vo) and in any intervals (Vt) after the injection of the
³
max
drugs. All results were expressed as means s.e.m. Stat-
istical evaluations were performed using analysis of vari-
ance followed by the Newman±Keul’s test for subgroup
Copolymerization
!
P
.
0 01).
comparison (level of signi®cance
The 1:3 molar ratio of HN±methacrylic acid in feed was
}
used for copolymerization. The 33% w w monomer con-
centration in dioxane was prepared. After deoxygenating
by alternate connection of the polymerization vessel to
Results and Discussion
Synthesis of monomer and polymeric prodrug
}
vacuum and nitrogen gas, 2% w w of AIBN to the total
weight of monomers was added. Copolymerization was
°
carried out for 3 h at 65 C under nitrogen. HN±methacrylic
acid copolymer was precipitated into methanol±water,
}
1:2 v v. The copolymer was ®ltered and dried with a
40% yield.
In the HN synthesis, the white precipitate was isolated
when DCC was added into the drug-contained solution.
This precipitate was assigned to be a proton-ionized DCC
1
intermediate by H NMR spectroscopy. Since it was di -
cult to remove the cyclohexylurea from the ®nal product
HN by simple recrystallization, column chromatography
was used for puri®cation. The same method was adapted to
synthesize Nap-EtOH, but the naproxen was linked to OH
groups of ethylene glycol at two ends (i.e., Nap-Et-Nap).
The Nap-EtOH was thus prepared by modifying the re-
ported method (Weber & Meyer-Trumpener 1994). The
acyl derivative of naproxen was prepared and then esteri-
®cation with ethylene glycol was added in the presence of
triethylamine. The Nap-EtOH was obtained with a quan-
titative yield comparable with that reported in the litera-
ture. However, the excess ethylene glycol was required to
prevent the disubstitution of naproxen on the two-hydroxyl
groups. Also, the reverse addition of equal molar ratio of
ethylene glycol into acyl naproxen led to the formation of
disubstituted drug compound.
Polymeric prodrug hydrolysis without enzyme
. m
Each sample (5±30 mg) was dissolved in 10 mL of 0 1 pH
.
°
7 4 phosphate buŒer solution at 37 C. At certain intervals,
the solvent was removed by freeze-drying under vacuum.
The drug and drug-EtOH were extracted from the polymer
residue with 20 mL of acetone. The supernatant acetone
solution was then withdrawn and evaporated using a rotary
vapor evaporator to produce a white solid. The white solid
was dissolved in 1 mL of ethanol and analysed by HPLC.
Quantities of the hydrolysates in the solution were deter-
mined by comparing the HPLC areas with the calibration
curves, which were obtained from known concentrations
of standards. The correlation coe cients of standard
C
curves were 0 999.
.
The HN molar percent in polymeric prodrug was calcu-
1
lated from H NMR spectral data, as shown in Figure 1.
The ratio of the peaks around 7±8 ppm, corresponding to
Polymeric prodrug hydrolysis with enzymes
.
six aromatic protons from HN to the total area between 0 4
and 2 2 ppm, which were attributed to 8 protons in HN
.
The prodrug hydrolysis with enzymes was conducted using
a procedure similar to that stated above, except that the
enzyme was added at a concentration of 50 L containing
23 U esterase, or 1 mg of lipase containing 61 U activities.
and ®ve protons in methacrylic acid (marked by an asterisk
in the structural formula as depicted in Figure 1). The
molar percent of HN can be calculated from the following
equations :
}
Ia Ib 6x (8x 5y)
}
¯
-
(1)
(2)
Anti-in ammatory activity
-
¯
1
x
y
The polymeric drug anti-in¯ ammatory activity was evalu-
ated using carrageenan-induced oedema test on rat paws where Ia is the area of aromatic protons, Ib is the area of
according to the technique reported by Winter et al (1962). aliphatic protons marked by an asterisk in Figure 1, x is
Wang
