Prodrugs of scutellarin: Ethyl, benzyl and N,N-diethylglycolamide ester synthesis, physicochemical properties, intestinal metabolism and oral bioavailability in the rats

Article abstract of DOI:10.1016/j.ejps.2006.07.007

In an effort to enhance the oral bioavailability of scutellarin, ethyl, benzyl and N,N-diethylglycolamide ester of scutellarin were synthesized. The hydrolysis of the prodrugs follows first-order kinetics in aqueous solution, and produced a V-shaped pH profile. The N,N-diethylglycolamide ester is highly susceptible to enzymatic hydrolysis in human plasma (t_1/2 ≈ 7 min) with a high stability in aqueous solution (t_1/2 ≈ 16 day, pH 4.2). Compared with the solubility of scutellarin, the solubility of glycolamide ester was about ten times in pH 4.0 buffer, and about thirty five times in water. Its apparent partition coefficient increased significantly from -2.56 to 1.48. Glycolamide ester of scutellarin was chosen to investigate the intestinal metabolism and in vivo bioavailability. Degradation studies in the intestinal tract content and homogenates indicated intestinal metabolism before absorption was a crucial obstacle for the prodrug. N,N-Diethylglycolamide ester can be protected from the degradation in the intestinal lumen by an emulsion. A significant increase in the plasma AUC and C_max of the prodrug emulsion was observed in rats, compared with that of the scutellarin-cyclodextrin complex (P < 0.01). The emulsion of N,N-diethylglycolamide ester produces a 1.58-fold enhancement in apparent bioavailability and 1.4-fold increase in the absolute bioavailability compared to the scutallarin-cyclodextrin complex.

Full text of DOI:10.1016/j.ejps.2006.07.007

european journal of pharmaceutical sciences 2 9 ( 2 0 0 6 ) 385–393
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Prodrugs of scutellarin: Ethyl, benzyl and
N,N-diethylglycolamide ester synthesis, physicochemical
properties, intestinal metabolism and oral bioavailability
in the rats
Feng Cao , Jian-Xin Guo , Qi-Neng Ping , Zheng-Gen Liaob
a
a
a,∗
a
Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
Key Laboratory of Modern Preparation of TCM, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to enhance the oral bioavailability of scutellarin, ethyl, benzyl and N,N-
diethylglycolamide ester of scutellarin were synthesized. The hydrolysis of the prodrugs
follows first-order kinetics in aqueous solution, and produced a V-shaped pH profile. The
N,N-diethylglycolamide ester is highly susceptible to enzymatic hydrolysis in human plasma
Received 7 October 2005
Received in revised form 4 July 2006
Accepted 28 July 2006
Published on line 7 August 2006
(t1/2 ≈ 7 min) with a high stability in aqueous solution (t ≈ 16 day, pH 4.2). Compared with
1/2
the solubility of scutellarin, the solubility of glycolamide ester was about ten times in pH
4
.0 buffer, and about thirty five times in water. Its apparent partition coefficient increased
Keywords:
Scutellarin
significantly from −2.56 to 1.48. Glycolamide ester of scutellarin was chosen to investigate
the intestinal metabolism and in vivo bioavailability. Degradation studies in the intestinal
tract content and homogenates indicated intestinal metabolism before absorption was a
crucial obstacle for the prodrug. N,N-Diethylglycolamide ester can be protected from the
degradation in the intestinal lumen by an emulsion. A significant increase in the plasma
AUC and Cmax of the prodrug emulsion was observed in rats, compared with that of the
scutellarin–cyclodextrin complex (P < 0.01). The emulsion of N,N-diethylglycolamide ester
produces a 1.58-fold enhancement in apparent bioavailability and 1.4-fold increase in the
absolute bioavailability compared to the scutallarin–cyclodextrin complex.
Prodrugs
Glycolamide ester
Intestinal metabolism
Emulsion
Oral absorption
Bioavailability
©
2006 Published by Elsevier B.V.
1
.
Introduction
0.4% in beagle dogs (Ge et al., 2003). Its poor ability to pen-
etrate cell membranes has long been a major impediment to
its overall effectiveness as an oral drug.
Recently, considerable attention has been focused on the
development of prodrugs, the bioreversible derivatives of the
active compound, to mask polar or ionizable groups within
a molecule. With respect to prodrugs of a higher lipophilic-
ity derived from active compounds with carboxylic functions,
ester formation represents a well-known approach. Strategies
Scutellarin is the major flavonoid component of traditional
Chinese medicine Erigeron breviscapus. It has been proved to be
effective in dilating blood vessels, impoving homodynamics,
decreasing the viscosity of blood, reducing the blood platelet
count and preventing platelet conglomeration (Liu et al., 2003;
Chen and He, 1998). Because of its poor aqueous solubility and
low lipophilicity, the oral bioavailability was approximately
∗
Corresponding author. Tel.: +86 25 8327 1098; fax: +86 25 8330 1606.
E-mail address: pingqn@cpu.edu.cn (Q.-N. Ping).
0928-0987/$ – see front matter © 2006 Published by Elsevier B.V.
doi:10.1016/j.ejps.2006.07.007

3
86
european journal of pharmaceutical sciences 2 9 ( 2 0 0 6 ) 385–393
to produce ester prodrugs include the esterification of gan-
ciclovir with the amino acid valine (Sugawara et al., 2000),
the formation of bisphosphonate prodrugs (Niemi et al., 2000)
or the formation of ester prodrugs of ␤-lactam antibiotics
ization (positive ion mode)–mass spectrometry [ESI (+)–MS]
1
was recorded on a HP1100 LC/MSD instrument. Proton ( H)
1
nuclear magnetic resonance spectra ( H NMR) were recorded
on an ACF-500BRUK spectrometer. Samples were dissolved in
an appropriated deuterated solvent, and chemical shifts were
reported as parts per million (ı) relative to tetramethylsilane
(0.00) which served as an internal standard. Elemental analy-
ses were performed on an Elementar Vario EL III instrument.
The progress of various reactions was followed by thin layer
chromatography (TLC). TLC was performed on EM Reagents
DC aluminum foil plates coated to a thickness of 0.2 mm with
silica gel (60 mesh). All solvents and chemicals were of reagent
grade.
(Mizen and Burton, 1998). This increases the overall lipophilic-
ity of the molecule and promotes membrane permeability
and oral absorption. However, the increase in lipophilicity
produced by a prodrug approach does not always lead to
major improvements in oral bioavailability. This is due to
the multiple barriers towards oral delivery. It is well known
that efflux transporters are present in the enterocyte mem-
brane, which are capable of intercepting drugs during mem-
brane passage and placing them back into the gut lumen.
Enterocytes are metabolically competent cells that express a
wide range of drug metabolising enzymes including esterases,
cytochrome P450 isoforms and UDP-glucuronyl transferases
2.2.1. Sodium scutellarin
A mixture of scutellarin (5.00 g, 0.011 mol), NaHCO3 (0.89 g,
0.011 mol) and 50 ml water was heated with a water bath
(Prueksaritanont et al., 1996). Ester-type drugs and prodrugs
◦
are hydrolyzed by esterases present in the intestinal tract
content (Crauste-Manciet et al., 1998) and mucosa (Cook et
al., 1995). A few studies have paid much attention to the
metabolism of ester prodrug in the intestinal tract content
and mucosa (Hirayama et al., 2000; Annaert et al., 2000). Oil-
in-water (o/w) submicron emulsion was previously found to
be effective in protecting the ester prodrug from enzymatic
attack in the intestinal lumen and improving its oral absorp-
tion (Nicolaos et al., 2003).
The present work was initiated to develop prodrugs of
scutellarin, possessing a high enzymatic bioconversion rate
and favorable physicochemical properties. Three ester pro-
drugs were synthesized and their physicochemical properties
were evaluated. N,N-Diethylglycolamide ester of scutellarin
was selected for the systemic study of intestinal degradation.
Furthermore, the protective effect and oral bioavailability of
microemulsion were investigated to gain insight of the effi-
ciency of the ester prodrug.
maintained at 50 C for 1 h. Upon cooling, the brown-colored
solution was filtered off and evaporated under reduced pres-
sure. The product was dried to yield sodium scutellarin (5.12 g),
yield: 96%.
ꢀ
2.2.2. Ethyl-4 ,5,6-trihydroxyflavone-7-glucuronide ester
(D1)
Sodium scutellarin (7.00 g, 0.015 mol) was suspended in 30 ml
N,N-dimethylformamide. The suspension was admixed with
ethyl bromine (3.37 ml, 0.045 mol) and catalytic amount of KI
◦
(0.1 g, 0.6 mmol). The reaction mixture was heated at 120 C
while monitoring by TLC (eluent: 6% water/0.1% glacial AcOH
in methanol). After about 3 h, the reaction mixture was poured
onto ice (50 g) and then extracted with ethyl acetate (4 × 50 ml).
The combined extracts were washed with a 2% aqueous
solution of sodium thiosulphate and water saturated with
sodium chloride. After drying over anhydrous sodium sul-
phate, the ethyl acetate was removed under reduced pres-
sure to give D1 (1.4 g) which was purified by recrystallization
from methanol–ethyl acetate, yield: 19.8%; mp 257–259 C; 1H
◦
2
.
Materials and methods
NMR (DMSO-d6): ı 12.73 (s, 1H, 5-OH), 10.38 (s, 1H, 6-OH),
ꢀ
ꢀ
ꢀ
8
.60 (s,1H, 4 -OH), 7.92–7.96 (m, 2H, 2 -H and 6 -H), 7.00 (s,
ꢀ
ꢀ
2
.1.
Materials
1H, 8-H), 6.86–6.95 (d, J = 7.4 Hz, 2H, 3 -H and 5 -H), 6.82 (s,1H,
3
ꢀꢀ ꢀꢀ
-H), 5.47–5.53 (m, 2H, 1 -H and 5 -H), 5.28 (m,2H, –CH2–),
ꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
Scutellarin, 4 ,5,6-trihydroxyflavone-7-glucuronide (purity
4.12–4.18 (m, 3H, 2 -H, 3 -H and 4 -H), 2.50–3.46 (m, 3H, other
>
97%, lot no. HB 20031203) was purchased from Yunnan
sugar protons), 1.21 (t, J = 4.4 Hz, 3H, –CH3–); ESI(−)–MS (m/z):
−
phytopharmaceutical Co. Ltd. (China). Soya lecithin was
purchased from Shanghai Taiwei pharmaceutical Co. Ltd.
489.1[M − H] , calculated molecular weight: 490.4. Anal. calcd.
for C23H22O12: C, 56.33; H, 4.52. Found: C, 55.88; H, 4.75. HPLC
analysis showed a single major peak accounting for 98% of the
total peak area.
(
Shanghai, China, PC% >92%). Labrafac@CC (caprylic/capric
triglycerides) and Labrasol (caprylocaproyl macrogolglyc-
erides) were kindly presented by Gattefosse Co. (France).
Transport medium consisted of Hanks’ balanced salt solution
ꢀ
2.2.3. Benzyl-4 ,5,6-trihydroxyflavone-7-glucuronide ester
(
HBSS) containing 25 mM glucose and 10 mM Hepes. HBSS
(D2)
contained 0.40 g/l KCl, 0.06 g/l KH2PO4, 8.00 g/l NaCl, 0.047 g/l
Na2HPO4 (anhydrous), 1.00 g/l d-glucose, 0.35 g/l NaHCO3,
Sodium scutellarin (4.84 g, 0.01 mol) was suspended in 20 ml
N,N-dimethylformamide. The mixture was added with benzyl
bromide (1.20 ml, 0.01 mol) and then heated at 120 C while
◦
0
.01 g/l phenol red and was adjusted to pH 7.0. Doubly
distilled water (ddwater) was used.
monitoring by TLC (eluent: 25% acetone in petroleum ether).
After about 1.5 h, the reaction mixture was poured onto ice
(50 g) and then extracted with ethyl acetate (4 × 50 ml). The
combined extracts were washed with water saturated with
sodium chloride. After drying over anhydrous sodium sul-
phate and filtering, the ethyl acetate was removed under
2
.2.
Synthesis
Conventional procedures were used for the synthesis of the
novel derivatives. Melting points were determined on a melt-
ing point apparatus and were uncorrected. Electrospray ion-
◦
reduced pressure to give D2 (1.6 g), yield: 30.0%; mp 223–226 C;

european journal of pharmaceutical sciences 2 9 ( 2 0 0 6 ) 385–393
387
1H NMR (DMSO-d6): ı 12.73 (s, 1H, 5-OH), 10.36 (s, 1H, 6-OH),
tion (IPBS) following the method described by Bundgaard and
Nielsen (1988).
ꢀ
ꢀ
ꢀ
8
.59 (s, 1H, 4 -OH), 7.88–7.59 (m, 2H, 2 -H and 6 -H), 7.27–7.38
ꢀ
(
m, 5H, -ph), 6.99 (s, 1H, 8-H), 6.91–6.94 (d, J = 8.8 Hz, 2H, 3 -H
ꢀ
ꢀꢀ
ꢀꢀ
and 5 -H), 6.81 (s, 1H, 3-H), 5.52–5.54 (m, 2H, 1 -H and 5 -H),
5
2.3.3. Solubility determinations
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
.21–5.30 (m, 3H, 2 -H, 3 -H and 4 -H), 4.25–5.17 (d, J = 9.6 Hz,
Solubility determinations were carried out in phthalate buffer
(pH 4.2) where the prodrugs may exhibit maximal stability,
and deionized water as the method described by Dias et al.
(2001). Parallel experiments were performed to determine the
solubility of scutellarin in the same media.
2
H, –CH2–), 3.33–3.46 (m, 3H, other sugar protons); ESI(−)–MS
−
(m/z): 551.2[M − H] , calculated molecular weight: 552.4. Anal.
calcd. for C28H24O12: C, 60.87; H, 4.38. Found: C, 60.47; H, 4.42.
HPLC analysis showed a single major peak accounting for 98%
of the total peak area.
2.3.4. Apparent partition coefficient determination
2
.2.4. N,N-Diethyl-2-chloroacetamide
Partitioning studies were performed with n-octanol and IPBS
following standard procedures (Dias et al., 2001). Parallel
experiments were performed to determine the apparent par-
tition coefficient of scutellarin.
A stirred solution of diethylamine (11.9 ml, 0.11 mol) and ben-
zene (20 ml) was cooled to 0 C with a drying tube over the con-
denser and chloracetyl chloride (7.9 ml, 0.11 mol) was added
dropwise, maintaining the temperature below 10 C. After 0.5 h
◦
◦
at room temperature, 15 ml water was added twice. The com-
bined organic extracts were dried over anhydrous sodium sul-
phate, filtered and the solvent removed under reduced pres-
sure to give a brown oil, which was used direct in the following
procedure.
2.4.
Degradation in intestinal tract
2.4.1. Preparation of intestinal tract contents and
intestinal homogenates of rat
All the animal studies reported here adhere to the Princi-
ples of Laboratory Animal Care. According to the method of
Crauste-Manciet et al. (1997) intestinal tract contents (pH 6.8)
were prepared. The intestinal homogenates (pH 7.0) were pre-
pared as the method described by Augustijns et al. (1998).
Protein content of the intestinal tract contents and intesti-
nal homogenates were determined according to the method
of Lowry et al. (1951) using bovine serum albumin as a stan-
dard.
ꢀ
2
.2.5. N,N-Diethylglycolamide-4 ,5,6-trihydroxyflavone-7-
glucuronide ester (D3)
The procedure was based on the general method of Bundgaard
and Nielsen (1988). In brief, to a solution of the scutel-
larin (5 g, 0.11 mol) in N,N-dimethylformamide (20 ml) was
added triethylamine (1.2 ml), KI (0.15 g) and N,N-diethyl-
2
-chloroacetamide (1.79 g, 0.012 mol). The reaction mixture
◦
was heated to 120 C while monitoring by TLC (eluent: 5%
water/0.1% glacial AcOH in methanol). After about 3 h, the
reaction mixture was poured onto ice (50 g) and then extracted
with ethyl acetate (4 × 50 ml). The combined extracts were
washed with a 2% aqueous solution of sodium thiosulphate,
2.4.2. Metabolism studies in intestinal tract contents and
intestinal homogenates
After optimization of the incubation conditions in preliminary
experiments, protein content for each sample was adapted
to balance between sensitivity of the analytical method and
linearity of the esterase activity of the preparations. Pro-
tein contents of the intestinal tract content and intestinal
homogenates were adapted to 0.6 and 0.1 mg protein ml incu-
2
% sodium bicarbonate and water saturated with sodium
chloride. After drying over anhydrous sodium sulphate, the
ethyl acetate was removed under reduced pressure to give
D3 (4.52 g), yield: 72.7%; mp 145–147 C; 1H NMR (DMSO-d6):
◦
ꢀ
−1
ı 12.73 (s, 1H, 5-OH), 10.34 (s, 1H, 6-OH), 8.58 (s, 1H, 4 -OH),
bation solution , respectively. The enzymatic preparations
(2 ml) were pre-incubated at 37 C for 5 min. The reactions
ꢀ
ꢀ
◦
7
(
.93–7.98 (m, 2H, 2 -H and 6 -H), 7.02 (s, 1H, 8-H), 6.86–6.96
ꢀ
ꢀ
d, J = 7.4 Hz, 2H, 3 -H and 5 -H), 6.81 (s, 1H, 3-H), 5.54 (s, 2H,
O–CH2–CO), 5.27–5.30 (m, 2H, 1 -H and 5 -H), 4.33–5.02 (m,
H, 2 -H, 3 -H and 4 -H), 3.43 (m, 3H, other sugar protons),
.25–3.30 (m, 4H, –CH2– and –CH2–), 0.98–1.14 (t, J = 7.1 Hz,
were started by the addition of 15 ␮l of the D3 stock solu-
tion in DMSO to make a final concentration of 13 ␮mol. At
predetermined time, the reactions were stopped by adding
100 ␮l of the incubation solution to 200 ␮l of ice-cold acetoni-
trile. The samples were centrifuged at 12,000 rpm for 10 min
and the amount of D3 in the supernatant was measured by
HPLC.
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
3
3
6
+
H, –CH3– and –CH3–); ESI(+)–MS (m/z): 576.1[M + H] , calcu-
lated molecular weight: 575.5. Anal. calcd. for C27H29NO13: C,
6.35; H, 5.08. Found: C, 56.15; H, 5.12. HPLC analysis showed
5
a single major peak accounting for 98% of the total peak
area.
The protective effect of D3 formulation was evaluated
by replacing the stock solution of D3 in DMSO with the o/w
emulsion (which was prepared as the following section) and
following the above-mentioned methodology. Controlled
experiments were made by directly adding 15 ␮l of D3 stock
solution to 2 ml of transport medium. All experiments were
conducted in triplicate.
2
.3.
Physicochemical properties studies
2
.3.1. Degradation kinetics in aqueous solutions
The degradation of prodrugs was investigated in different
buffer solutions (0.02 M, ꢀ = 0.5) in the pH range 1.2–9.0 fol-
lowing standard procedures (Dias et al., 2001).
2.5.
Bioavailability studies in the rats
2
.3.2. Degradation studies in human plasma
2.5.1. Formulation preparation
The hydrolysis of the esters was studied in human plasma
diluted to 80% with pH 7.4 isotonic phosphate buffer solu-
2.5.1.1. Parenteral solution. Scutellarin (15 mg, 0.032 mol) and
diethanolamine (3.4 mg, 0.032 mol) were dissolved in 10 ml of

3
88
european journal of pharmaceutical sciences 2 9 ( 2 0 0 6 ) 385–393
an aqueous solution containing 0.9% of sodium chloride. The
2.5.3. Treatment of plasma samples
solution was sterilized though 0.22 ␮m filter.
Fifty microliters of 1% phosphoric acid solution was added to
200 ␮l of plasma thawed. The mixture was vortexed for 3 min
2
.5.1.2. Microemulsions. The emulsion was prepared accord-
and kept for 5 min. Methanol (0.4 ml) was added and the mix-
ture was vortexed for 3 min prior to centrifugation for 10 min at
12,000 rpm. The organic phase was transferred into new tubes
and the contents evaporated to dryness under a stream of air
ing to a standard procedure (Benita and Levy, 1993) and the
method of Nicolaos (2003). D3 (50 mg) was dissolved in a mix-
ture of Labrafac@CC (4.0 g, 11.9%, w/w), Labrasol (1.0 g, 3.0%,
◦
◦
w/w) and soya lecithin (0.8 g, 2.4%, w/w) at 80 C under nitro-
at approximately 40 C. The dried extracts were reconstituted
gen gas protection (the solubility of D3 in the oil phase was
with 150 ␮l of mobile phase solution, vortexed at high speed
for 3 min, and centrifuged again for 10 min at 12,000 rpm. The
entire volume of the reconstituted material (150 ␮l) was trans-
ferred to autosampler vials and 50 ␮l injected onto the HPLC.
◦
1
5 mg/ml at 80 C.). The aqueous phase was made by a mix-
ture of polysorbate 20 (0.2 g, 0.6%, w/w) and water (27.5 g).
◦
Both phases were then mixed at 80 C by a phase inversion
method using a high shear mixer and a coarse o/w emul-
sion was made. The coarse emulsion was rapidly cooled and
homogenized using high-pressure homogenizer (EmulsiFlex-
C5, Avestin Inc., Ottawa, Canada). The pH of the emulsion was
then adjusted to pH 4.5. The finished emulsion was purged
with nitrogen gas and stoppered. The mean droplet size and
2.5.4. Pharmacokinetics and statistical analysis
The plasma concentration–time data of scutellarin were fitted
by 3P87 Pharmacokinetics Program (The Section of Mathemat-
ical Pharmacology of Chinese Mathematical Pharmacological
Society) and the pharmacokinetic parameters were calculated.
The area under the concentration–time curve (AUC0–t) was
ꢁ
-potential value of submicron emulsion were determined by
a laser diffraction particle size analyzer (Zetasizer3000HSA,
Malvern, Orsay, France). Each emulsion sample was diluted in
ddwater (1:200) to an appropriate concentration before mea-
determined with trapezium method. AUC
t–∞
was calculated
by dividing the last plasma concentration over the elimina-
tion rate constant. Cmax and Tmax were determined through
the observation of individual animal concentration versus
time curses. According to the definition of Krise et al. (1999),
the apparent bioavailability (Fapp) of the scutellarin following
administration of the prodrug was calculated by dividing the
scutellarin AUC following prodrug dosing by that from scutel-
larin dosing. The absolute bioavailability (Fabs) of scutellarin
◦
surement at 25 C. The mean droplet size and ꢁ-potential
value of the emulsion were 189.1 ± 20.1 nm and −23.7 ± 4.3 mV,
respectively. These results were mean ± S.D. error bar of three
samples. D3 in the emulsion preparation process was stable,
which was checked by quantitative analysis of D3 in the emul-
sion. Results from the study of the stability of the emulsion
◦
revealed that D3 in the emulsion was stable at 4 C for at least
was estimated as the AUC0 ratio of the oral preparations to
–∞
1
year.
the parenteral solution.
To compare the main parameters of different dosages, a
two-sided unpaired t-test was conducted with Microsoft Excel.
Statistical significance was indicated with P < 0.01.
2
.5.1.3. Scutellarin complex. Scutellarin–cyclodextrin tetra-
component complex was prepared as the described in our
paper (Cao et al., 2005). Oral solution was prepared by
dissolving 44.7 mg of scutellarin–cyclodextrin complex in
2.6.
Analytical procedures
4
ml of ddwater to make a concentration 1.5 mg/ml of the
scutellarin.
All samples obtained from the various studies were assayed
by using HPLC. The HPLC system consisted of a pump (Model
LC-10A, Shimadzu, Japan), a shim-pack CLC-ODS column
2
.5.2. Rat experiment
◦
Male Sprague–Dawley rats (weight range 280–300 g) were
fasted for 24 h prior to experiment. A random experimental
design was used. Animals (six rats for each formulation)
were randomly assigned to one of the three groups. The oral
solution of scutellarin complex and the emulsion of D3 were
performed by means of a steel probe with a length of 6 cm,
whereas the i.v. dosing was carried out by injecting the par-
enteral solution through the tail vein. According to the clinical
scutellarin dosage (120 mg/day 60 kg for adult), the dosage of
(150 mm × 6 mm i.d., Shimadzu) maintained at 30 C, an UV
detector (Model SPD-10A, Shimadzu) at 334 nm and a data sta-
tion (Model SCL-10A, Shimadzu).
2.6.1. HPLC assay I
This method was used to analyze the samples obtained
from the studies in vitro. The composition of the mobile
phase was methanol–water–phosphoric acid–triethylamine
(53:47:0.1:0.1, v:v:v:v). The mobile phase was delivered at a flow
rate of 1 ml/min. The injection volume was 20 ␮l and the rela-
tive retention time was found to be about 10.0 min. The corre-
lation coefficients of the standard curve for the prodrugs were
all above 0.9998. Precision assay showed that the averages of
the relative standard deviations (R.S.D.) within 1 day were all
below 2.4% and among every other day were all below 4.0%.
1
0.8 mg/day kg for rat was used whatever the administration
route. The dosage of prodrug D3 was 13.5 mg/day kg for rat,
which was equivalent to 10.8 mg/day kg of scutellarin (on a
molar basis). Before blood sampling, the animals were anes-
thetized with diethyl ether. Blood samples of 0.4 ml were taken
from the ophthalmic venus plexus and put into heparinized
tubes at 5, 10, 15, 30, 45, 60, 120, 240, 360 and 420 min for oral
formulations. For parenteral solution, blood was collected
at 0.5, 2, 5, 10, 15, 30, 60, 240, 360, 720 min. The blood was
immediately centrifuged for 10 min at 4000 rpm and 200 ␮l of
2.6.2. HPLC assay II
This method was used to analyze plasma samples of
scutellarin. The composition of the mobile phase was
methanol–water–phosphoric acid (40:60:0.1, v:v:v). The
mobile phase was delivered at a flow rate of 1 ml/min. The
◦
plasma was then removed and stored at −20 C until assay for
scutellarin.

european journal of pharmaceutical sciences 2 9 ( 2 0 0 6 ) 385–393
389
◦
◦
◦
◦
Scheme 1 – Synthesis of the prodrugs. (a) H2O, 50 C, 1 h; (b) DMF, KI, 120 C, 3 h; (c) DMF, 120 C, 1.5 h; (d) benzene, 0 C, 0.5 h;
◦
(
e) TEA, KI, 120 C, 3 h.
injection volume was 50 ␮l and the relative retention time was
found to be 8.0 min. The regression equation for scutellarin
content (ng/ml) in the plasma ranging from 20 to 400 ng/ml
was A = 114.62C − 554.97 (r = 0.9992). The plasma assay was
acid- and base-catalyzed hydrolysis together with a water-
catalyzed hydrolysis. Lipophilicity of the prodrug (Table 1) does
not appear to correlate with the stability of the prodrug. These
esters exhibit maximum stability between pH 4.0 and 4.5 and
moderate stability at neutral pH. Considering this fact the sol-
ubility studies were carried out at pH 4.2. With pH in that range
2
validated by analysis of n = 3 quality control samples con-
taining 20, 100, and 400 ng/ml scutellarin in blank plasma.
The recoveries were found to be accurate to 89.4%, 84.2%,
and 80.7% and precise to ± 6.0%, 3.1%, and 5.1% of 20, 100,
and 400 ng/ml, respectively. Precision assay showed that the
average of the R.S.D. within 1 day was 3.4% and among every
other day was 5.0%.
◦
and at 37 C the half-lives of approximately 13, 8 and 16 days
were calculated for D1, D2 and D3 in an aqueous solution,
respectively. It is possible that the benzyl group in the ester
D2 can stabilize the carbonium ion intermediate in the course
of hydrolysis and increased the rate of hydrolysis. The half-
lives of D3 in the buffer (0.02 M, ꢀ = 0.5) with pH 2.5, 5.6, 7.4, 9.0
are 3.37 days, 19.9 days, 1.9 h and 1.14 h, respectively.
3
.
Results and discussion
3.2.2. Degradation in human plasma
3
.1.
Synthesis of the prodrugs
The susceptibility of the prodrugs to enzymatic hydrolysis
was studied in 80% human plasma in IPBS solution. The
degradation equation of D1 and D2 in the human plasma
The general procedures for the synthesis of scutellarin pro-
drugs are described as Scheme 1. Various methods have been
used to synthesis ester prodrug from carboxylic compounds
2
was log C = −0.0011t + 0.5189 (r = 0.9732) and log C = −0.0036t +
2
0.9957 (r = 0.9030), respectively. The observed degradation
(Yano et al., 2002; Leisen et al., 2003). Here a mild synthe-
rate was higher than that in the buffer solution. t1/2 value
sis condition was found to protect from the instabilization of
flavonoid and sodium scutellarin successfully reacted with the
corresponding bromide in DMF. To making the process more
smoothly, ethyl bromine and sodium scutellarin should be
separately excessive for the preparation of D1 and D2. Accord-
ing to the procedure of Bundgaard and Nielsen (1988), D3 was
prepared in high yield.
3
.2.
Physicochemical properties of the prodrugs
3
.2.1. Aqueous stability
In most cases the degradation was followed for several half-
lives. However, in cases where the degradation was very slow,
the half-lives of degradation were determined using the initial
rate method. The influence of pH on the overall rate of hydroly-
◦
sis of the three prodrugs was investigated at 37 C (Fig. 1). From
Fig. 1 – pH-rate profiles for the degradation of the prodrugs
at 37 C. Results are mean of three determinations.
the figure is seen that the compounds are subjected to specific
◦

3
90
european journal of pharmaceutical sciences 2 9 ( 2 0 0 6 ) 385–393
Table 1 – The physio-chemical properties for scutellarin and its prodrugs
Compound
Solubility in pH 4.2 buffer (␮g/ml)
Solubility in ddwater (␮g/ml)
log Papp
Scutallarin
42.66 ± 0.80
12.40 ± 0.56
10.30 ± 0.45
420 ± 30
14.44 ± 0.67
13.06 ± 0.74
ND
500 ± 20
−2.56 ± 0.04
2.37 ± 0.10
ND
1.48 ± 0.09
D1
D2
D3
Values are the mean ± S.D. of three determinations. ND: not determined. Apparent partition coefficient values (Papp) were carried out in n-
◦
octanol/phosphate buffer (pH 7.4) at 37 C.
of D1 was 273 min and D2 88 min. From the time course for
the disappearance of D3 in 80% human plasma at 37 C, the
the method described by Nicolaos et al. (2003). The degrada-
tion of D3 in intestinal lumen is shown in Fig. 2. At 90 min,
the remaining percentage of D3 in the incubation mixture
of the emulsion, stock solution and control was 79.35 ± 1.2%,
44.96 ± 4.9% and 61.44 ± 6.5%, respectively. The stock solution
had a significantly (P < 0.01) lower D3 remaining than that of
the control solution. The amount of D3 remaining was sig-
nificantly (P < 0.01) higher in the emulsion than in the stock
solution and the control. The half-life of the prodrug D3 with
pH 6.8 was estimated from Fig. 1 to be about 2.03 h, which
was very similar to the half-life of 2.0 h of the prodrug D3 in
intestinal contents with pH 6.8. These results indicated that
emulsion exhibited a protective effect against enzyme attack
and chemical degradation in rat intestinal lumen. The pro-
tective effect is consistent with previous finding with other
esters (Nicolaos et al., 2003). On the other side, polysorbate 20
in the emulsion as emulsifying agent may show the esterase
intrinsic inhibitory effect (Crauste-Manciet et al., 1998). How-
ever, the protective effect of the prodrug emulsion would be
decreased because the prodrug D3 improves both aqueous sol-
ubility and lipophilicity of the parent drug, which could also
be located in the aqueous phase or in the interfacial film and
attacked easily by enzyme.
As esterase-mediated degradation of an ester prodrug
may be a limiting factor for its trans-epithelial transport.
The esterase activity in homogenates from various intestinal
segments was investigated in order to identify a possible
absorption window with low esterase activity and hence
increased ester prodrug absorption. The time-dependent
degradation of D3 in homogenates from several segments of
rat intestine is represented in Fig. 3. The in vitro hydrolysis rate
of D3 in duodenum mucosa was the fastest and D3 remaining
after 30 min incubation was 32.26 ± 7.70%. The mean remain-
◦
apparent half-life of hydrolysis of D3 is estimated to be 10 min.
These results strongly suggest that these prodrugs are sus-
ceptible to serum esterase and the liberation of scutallarin
from the prodrugs satisfies with the prerequisite in prodrug
design. Furthermore, in this case, simple alkyl ester, D1, has
the longest half-life, aryl ester, D2, has a moderate half-life and
N,N-disubstituted glycolamide ester shows to be cleared with
remarkable speed in human plasma. The result of the low-
est half-life for the glycolamide esters is in agreement with
Bundgaard (1991).
3
.2.3. Solubility and partition coefficient
Aqueous solubility of the prodrugs and parent drug was deter-
mined at pH 4.2 (phthalate buffer) and ddwater after 24 h
equilibration period. The values of solubility and apparent par-
tition coefficient are provided in Table 1. The solubility of D2 in
ddwater and its log Papp could not be found due to the insta-
bility. As the substitute group changes from aliphatic group
to aromatic group, the solubility of D1 and D2 in the buffer
decreases and the lipophilicity of the prodrugs increases to
a larger extent, compared with the parent drug. In compar-
ison, the solubility of D3 increases about 10-fold in buffer,
and about thirty five times in ddwater. Partition coefficient
values increases from −2.56 of scutellarin to 1.48 upon gly-
colamide substitution. It seems that not only the solubility
but also the lipophilicity are significantly enhanced by D3.
The dual enhancement was also shown for morpholinoalkyl
ester prodrugs of indomethacin and naproxen by Tammara
et al. (1993). It was, therefore, expected that the enhanced
lipophilicity and solubility may increase oral bioavailability.
Combining with the characteristics of high susceptibility to
enzymatic hydrolysis in human plasma and the high stabil-
ity in aqueous solution, D3 was selected as the candidate for
further studies.
3
.3.
Metabolism in intestinal contents and intestinal
homogenates
Chen et al. (2003) indicated that the intestinal disposition con-
tributed more to the poor bioavailabilities than hepatic dispo-
sition for flavonoids. Beaumont et al. (2003) also suggested that
an ideal ester prodrug for oral delivery should exhibit the prop-
erty of resistance to hydrolysis during the absorption phase.
It is most important to study the degradation properties of
the ester prodrug in the intestine, so as to protect the prodrug
from the metabolism in the intestine. For this purpose, an oil-
in water (o/w) submicron emulsion was prepared according to
Fig. 2 – Time-dependent degradation of D3 in rat intestinal
lumen, represented as the percentage of the initial
concentration of 13 ␮mol. The amount of protein in the
incubation mixture was 0.6 mg/ml. Results are mean + S.D.
error bar of three determinations.

european journal of pharmaceutical sciences 2 9 ( 2 0 0 6 ) 385–393
391
Fig. 4 – Scutellarin plasma concentration after
Fig. 3 – Time-dependent degradation of D3 in homogenates
from several segments of rat intestinal, represented as
percentage of initial concentration of 26 ␮mol. The amount
of protein in the reaction mixture was 0.1 mg/ml. Results
are mean + S.D. error bar of three determinations.
administration of emulsion (ꢀ) (n = 6), and scutellarin–␤-CD
complex (ꢁ) (n = 6).
in Table 2. Pharmacokinetic parameters obtained after par-
enteral administration of scutellarin (n = 6) show a constant
of Cmax of 175.07 ± 13.12 ␮g/ml, Tmax of 0.50 min, a MRT of
ing percentage of D3 after 60 min incubation in jejunum,
40.16 ± 0.56 min and a AUC
of 1622.69 ␮g min/ml. C
max
of
0
–∞
ileum and colon mucosa was 24.95 ± 3.4%, 21.02 ± 3.80% and
the emulsion (335.29 ng/ml) is higher than that of the com-
plex (139.29 ng/ml) (P < 0.01). This difference may be due to fast
dissolution and absorption of the prodrug in the emulsion,
a rapid and quantitative breakdown to yield high circulat-
ing concentrations once absorbed. On the other hand, Tmax
is significantly lower for the emulsions (9.17 min) in compar-
ison to the complex (15 min) (P < 0.05). There is some differ-
ence between the AUC of D3 emulsion (32,941.84 ng min/ml)
and the complex (20,858.89 ng min/ml) (P < 0.01). The apparent
bioavailability of scutellarin following D3 administration was
found to be 158%. Furthermore, the absolute bioavailability of
D3 emulsion increased by 1.4-fold, compared to the complex.
5
7.25 ± 3.40%, respectively. A significant difference (P < 0.01)
was observed among the activity of duodenum mucosa with
that of other mucosa. The degradation rates of D3 was in
the order duodenum > ileum ≥ jejunum > colon. The rank is
similar to those obtained by Gelder et al. (2000) and Cook et
al. (1995). This order might be due to the lipid composition
in the intestinal membrane, resulting in different cellular
responses and enzyme activities (Ungell et al., 1998). From
the site-specific degradation findings, a strategy of colon
targeting may reduce esterase-mediated degradation of D3
and possibly increase its oral absorption.
A
double-peak phenomenon is observed in plasma
3
.4.
Bioavailability studies in the rats
concentration–time profiles of scutellarin via oral adminis-
tration. Double peaks have also been observed with other
drugs (Wang, 1999), including flavonoid compound (Lesser
et al., 2004). An “intraintestinal” recycling process proposed
by Liu and Hu (2002) may explain the second peak. Because
scutellarin is a drug of glucuronidated isoflavone and poorly
permeable, it is expected to be reconverted by the intestinal
microflora into their aglycone forms in the colon, which could
then be easier reabsorbed and re-metabolized to scutellarin.
Probably due to the esterase-mediated and chemical degra-
dation, the oral aborption of scutellarin prodrug is only little
bit better than that of scutellarin–cyclodextrin complex. It is
expected that a prodrug of scutellarin with higher log P value
may exhibit higher membrane permeability and bioavailabil-
ity, but more important in the case, to slow down the rate
of presystematic metabolism may much more enhance the
Because of the low lipophilicity of scutellarin, it was dif-
ficult to make a stable emulsion (o/w) of scutellarin and
scutellarin–cyclodextrin tetracomponent complex was used
as control, which had a 1.25 ± 0.05-fold enhancement in
the apparent bioavailability of the rats orally compared to
the scutellarin suspension (data not shown). In all studies,
equimolar doses of drug and prodrug were administered and
plasma concentrations of parent drug were recorded as a func-
tion of time. As the half-life of D3 in the blood of rats was less
than 0.50 min, the concentration of D3 was lower than detec-
tion limit at 5 min after oral administration (data not shown).
The plasma concentration–time profiles of scutellarin are
shown in Fig. 4. The mean pharmacokinetic parameters
derived from a non-compartmental analysis are presented
Table 2 – Pharmacokinetic parameters and bioavailability of oral formulations
Cmax (ng/ml)
Tmax (min)
AUC0–420 (ng min/ml)a
MRT0-420 (min)
Fapp (%)
Fabs (%)
Scutellarin (complex)
D3 (emulsion)
139.77 ± 27.46
335.29 ± 186.22
15.00
9.17 ± 2.04
20858.89 ± 3498.91
207.08 ± 33.10
172.29 ± 24.74
100
158
1.9
2.6
*
*
*
**
32941.84 ± 9113.03
Results are mean ± S.D. of six determinations.
a
AUC is for scutellarin.
∗
Significantly different (P < 0.05) from scutellarin complex.
∗
Significantly different (P < 0.01) from scutellarin complex.
∗

3
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european journal of pharmaceutical sciences 2 9 ( 2 0 0 6 ) 385–393
bioavailability. The species differences in esterase activity of
intestinal were observed by Gelder et al. (2000), the general
order of decreasing activity being: rat > man > pig. Yoshigae et
al. (1998) observed that the hydrolase activities of rat intestinal
mucosa for an ester prodrug were significantly high than those
of dog (50–260-fold). Esterase activity in rat whole blood was
approximately 100 and 400 times higher than that in dog and
human whole blood, respectively (Minagawa et al., 1995) and
the similar results were also observed in this paper. Other ani-
mals with lower esterase activity in the intestine and blood are
expected to have a better enhancement effect on absorption.
The assumption needs to be validated in our further studies
of D3 in other animals.
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4
.
Conclusions
In conclusion, the glycolamide ester prodrug of scutellarin
has the highest susceptibility to enzymatic hydrolysis in
human plasma (t1/2 ≈ 7 min), a higher stability in aqueous
solution, a good enhancement of apparent partition coeffi-
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