Solubilization of dipropofol, an antibacterial agent, using saccharide and ascorbic acid

Article abstract of DOI:10.1248/bpb.30.1565

Dipropofol has a strong antibacterial activity against Gram-positive bacteria. However, it lacked the solubility in water and this property was supposed to limit its efficacy. We tried to improve the solubility and found a new solubilization method of dipropofol in water by the addition of a monosaccharide or ascorbic acid.

Full text of DOI:10.1248/bpb.30.1565

August 2007
Notes
Biol. Pharm. Bull. 30(8) 1565—1568 (2007)
1565
Solubilization of Dipropofol, an Antibacterial Agent,
Using Saccharide and Ascorbic Acid
Masahiro OGATA, ,a,c Kentaro OKA,b Masako SEKI,b Midori HOSHI,b Hirokatsu TAKATSU,c
*
b
Tadahiko MASHINO,b Shiro URANO,c and Toyoshige ENDO
a Aomori University; 2–3–1 Kohbata, Aomori, Aomori 030–0943, Japan: b Kyoritsu University of Pharmacy; 1–5–30
Shiba-koen, Minato-ku, Tokyo 105–8512, Japan: and c Shibaura Institute of Technology; 3–9–14 Shibaura, Minato-ku,
Tokyo 108–8548, Japan. Received November 8, 2006; accepted June 7, 2007; published online June 7, 2007
Dipropofol has a strong antibacterial activity against Gram-positive bacteria. However, it lacked the solubil-
ity in water and this property was supposed to limit its efficacy. We tried to improve the solubility and found a
new solubilization method of dipropofol in water by the addition of a monosaccharide or ascorbic acid.
Key words dipropofol; monosaccharide; ascorbic acid; solubilization
For more than 30 years, vancomycin has been a reliable was applied to determine the antimicrobial activity of the test
treatment for Gram-positive bacterial infection. Injectable compounds. The test organisms were grown overnight at
forms of vancomycin were introduced, and have been used 37 °C by shaking in a TSB medium and the aliquots (100 ml)
exclusively for methicillin-resistant Staphylococcus aureus were streaked onto trypticase soy agar plates. A dipropofol
(MRSA) infection. Recently, the MRSA strain, which con- solution (1 mg/ml in methanol) was suspended with saccha-
tains the vanA gene, with high resistance to vancomycin ride (1 g) or ascorbic acid (1 g). The perfect removal of
(MIC ꢀ128 mg/ml) has been reported in clinical isolates.1,2) methanol produced a dipropofol complex with saccharide or
The emergence of vancomycin resistant bacterial strains is a ascorbic acid, which dissolved in distilled water (5 ml). For
very serious public health problem. Therefore, a new anti- plate diffusion tests, 50 ml of the test compound was dropped
MRSA antibiotic is clinically of interest.
on paper disks (diameter 8 mm), which were dried under ster-
Dipropofol (Fig. 1) was recognized as an oxidative dimer- ile conditions and put on and an agar plate inoculated with
ization metabolite of propofol (2,6-diisopropylphenol), a the test organism. The plates were cultivated at 37 °C for
novel sedative and anesthetic agent having antioxidant activ- 24 h.
ity, but not antibacterial activity.3,4) Dipropofol was investi-
Combination Study and Synergism Determination
gated to have a higher antioxidant activity than that of propo- The in vitro combinational effect of the dipropofol with
fol, and to show potent antibacterial activity against Gram- ascorbic acid was examined by the checkerboard titration
positive strains including MRSA and vancomycin resistant method. Overnight a culture of S. aureus, grown in 10 ml of
Enterococci (VRE).4,5)
Mueller–Hinton broth at 37 °C, was diluted 103-fold with
However, the insolubility of dipropofol in water could be fresh Mueller–Hinton broth, followed by the addition of S.
plausible for its efficacy and application fields. Consequently, aureus (about 5ꢃ104 CFU) onto 20 ml Mueller–Hinton agar
the solubilization of dipropofol in water was a next target. To layers which contained dipropofol (0—6.25 mg/ml) and
obtain the water-solubility of the compounds, many re- ascorbic acid (0—12.5 mg/ml). After 18 h of incubation at
searchers have used synthetic approaches and selected suit- 37 °C, the MIC value was determined for each fraction. The
able derivatives.6,7) However, we selected another method of fractional inhibitory concentration (FIC) for each compo-
adding other types of compounds, expecting complex forma- nent was calculated based on the following formula: FIC
tions and synergistic activity with the additions.
indexꢄ(MIC of drug A, tested in combination)/(MIC of drug
A, tested alone)ꢂ(MIC of drug B, tested in combination)/
(MIC of drug B, tested alone). The interaction was defined as
synergistic if the FIC index was ꢅ0.5, additive if the FIC
MATERIALS AND METHODS
Chemicals Dextrose anhydrous, saccharose, ascorbic index was ꢀ0.5, and antagonistic if the FIC index was ꢀ4.8)
acid and lecithin (from egg) were purchased from the Wako
Oxidation Reduction Potential The oxidation reduction
Pure Chemical Co. (Osaka, Japan). D(ꢁ)-Fructose, D(ꢂ)- potential of dipropofol and ascorbic acid were estimated
mannose, D(ꢂ)-xylose, maltose monohydrate, and D-sorbitol using cyclic voltammetry methodology.9) The cyclic voltam-
were purchased from Sigma-Aldrich Japan K.K. (Tokyo, meter used for these studies was obtained from POTENTIO-
Japan). Dipropofol was synthesized by the method reported STAT/GALVANOSTAT HA-301 (Hokuto Denko Co., Ltd.,
previously.4)
Japan). A three-electrode system was employed with
Antimicrobial Susceptibility The disk-diffusion test, Ag/AgCl as the reference electrode, a glassy carbon elec-
using S. aureus 209P and E. coli JCM5491 (ATCC25922) trode as the working electrode and a platinum wire as a
counter electrode. The cell contains 0.5 mM sample and N,N-
dimethylformamide (contain 60 mM tetra-n-butylammonium
hexafluorophosphate): 41.75 mM MES buffer (pH 7.4)
(ꢄ6 : 4). The cyclic voltammetry tracings were recorded
from ꢁ0.2 V to 1.5 V at a scan rate of 50 mV/s.
Fig. 1. Structure of Dipropofol
Synthesis of 3,3ꢀ,5,5ꢀ-Tetraisopropyldiphenoquinone
∗ To whom correspondence should be addressed. e-mail: ogata-ms@aomori-u.ac.jp
© 2007 Pharmaceutical Society of Japan

1566
Vol. 30, No. 8
(Dipropofolquinone) Propofol (1 g) was dissolved in
CH2Cl2 (10 ml) and stirred with CuCl (OH)·tetramethyleth-
ylenediamin (TMEDA) (16 mg) for 5 h at room temperature.
The reaction product was extracted with AcOEt, after re-
moval of the solvent, and crystallized from hexane to give
the dipropofolquinone (990 mg, 99%) as reddish violet rods.
1
FAB-MS m/z: 353 (MꢂH)ꢂ. H-MNR (300 MHz, CDCl3, d,
ppm): 1.20 (24H, d, Jꢄ6.7 Hz, 8ꢃCH3), 3.20 (4H, m,
Jꢄ6.7 Hz, 4ꢃCH), 7.62 (4H, s, H-2, 2ꢆ, 6, 6ꢆ). mp 225 °C.
Reduction of Dipropofolquinone with Ascorbic Acid or
Saccharides Dipropofolquinone (20 mg) was dissolved in
ethanol (99 ml) and stirred with ascorbic acid (200 mg,
20 mg, or 2 mg) (dipropofolquinone : ascorbic acidꢄ1 : 10,
1 : 1, 10 : 1) for 4 h at room temperature. Glucose (2 g), fruc-
tose (2 g), mannose (2 g), xylose (2 g), or sorbitol (2 g) were
dissolved in water (1 ml) and stirred with dipropofolquinone
in the same manner and the plausible reaction was monitored
with TLC. The reaction product was extracted with AcOEt
and the solvent was evaporated. The reactant was analyzed
with TLC (hexane : AcOEtꢄ10 : 1) and 1H-MNR.
Fig. 2. An Outline of This Experiment
Stability of Dipropofol Dipropofol (10 mg) was dis-
solved in ethanol (9 ml), added to water (pH 7, 1 ml), water
(pH 2, 1 ml), or ascorbic acid (10 mg), glucose (10 mg), fruc-
tose (10 mg), mannose (10 mg), xylose (10 mg) or sorbitol
(10 mg) were dissolved in water (1 ml). Those mixture solu-
tions were irradiated with UV-C (254 nm, 4.9 W) for 24 h at
room temperature. The reaction product was extracted with
AcOEt, after the removal of the solvent, the reactant was ana-
lyzed with TLC (hexane : AcOEtꢄ10 : 1) and 1H-MNR.
RESULTS AND DISCUSSIONS
For the solubilization of dipropofol, several procedures
were examined. However, the salt formation with alkaline
metals was found to decrease its activity, and an addition of
dipropofol solution (1 mg/ml in methanol) into distilled
water or saccharide solution caused the liberation of the
dipropofol. But as shown in Fig. 2, the direct addition of sac-
charide powder to a dipropofol solution gave a complex in-
cluding dipropofol and saccharide after the perfect removal
of methanol which could dissolve in distilled water (the same
Fig. 3. The Antibacterial Activity of the Dipropofol Complex
(A) Dipropofol–mannose complex dissolved in water, (a) direct application of the
complex solution, (b) the solution (a) was adsorbed on paper disks, (c) dipropofol was
adsorbed on a paper disk from a methanol solution, or (d) mannose solution adsorbed
on a paper disk. (B) Dipropofol complex with other saccharides, (1) glucose, (2) fruc-
tose, (3) xylose, (4) sorbitol, (5) mannose, (6) sucrose, or (7) maltose. Left and center
were dipropofol–sugar complexes dissolved in water and adsorbed on paper disks.
procedures were used for glucose, sucrose, fructose, man- Right was the direct application complex of solution.
nose, xylose, maltose, and sorbitol).
To determine the antibacterial activity of the complex so-
Also, when dipropofol is dissolved in a surface-active
lution, Staphylococcus aureus 209P, grown in a Trypticase agent, such as Tween 20 and Tween 80, emulsion (including
soy (TS) broth at 37 °C overnight, was applied to the surface 1.2% lecithin and 10% soybean oil), which is utilized for the
of the TS agar dishes, and these dishes were incubated at medical preparation of propofol and cyclodextrin, these solu-
37 °C overnight (Fig. 3A, (a) 15 ml sample solution together tions had no antibacterial activity (data not shown). These
with one drop each of a 50 mM solution and (b—d) a paper dipropofol complexes (disaccharides, surface-active agent,
disk impregnated with sample solutions). The antibacterial emulsion or cyclodextrin) were supposed to form micell, and
activity was evaluated from the diameters of the inhibition hold the active site inside of the micell.
zone. As shown in Figs. 3A and B, when a dipropofol–mono-
Another problem with decreasing the activity of the
saccharide (glucose, fructose, mannose, xylose and sorbitol) dipropofol was air oxidation to form dipropofolquinone,
complex was directly applied onto the plates, a clear antibac- which has no antioxidant and antibacterial activities. It is
terial activity was observed. The paper disk did not show any well known that ascorbic acid is a kind of sugar and works
zonal inhibition, however, clear growth inhibition was also as a reducing agent of a-tocopherol in vivo, which is a
observed under the disk. This phenomenon might suggest the lipophilic antioxidant.10) Similarly, it could be expected to the
higher ability complex formation of saccharides, which also show synergistic effect by the combined use of dipropofol
induced the lack of diffusivity of the dipropofol–saccharide and ascorbic acid.
complex. Disaccharides, such as sucrose and maltose, have
no antibacterial activity (Figs. 3B (6), (7)).
As shown in Table 1, the minimum inhibitory concentra-
tions (MICs) of dipropofol and ascorbic acid were 3.12

August 2007
1567
mg/ml and 6.25 mg/ml against S. aureus 209P, respectively. paper disk applications, and their inhibition zones were
The MIC of dipropofol declined to 0.78 mg/ml, when com- larger than those of the ascorbic acid alone. This phenome-
bined with ascorbic acid. A synergistic effect was obtained non showed a diffusible property of dipropofol into water in
by the combined use of dipropofol and ascorbic acid. The the presence of ascorbic acid. Moreover, the dipropofol–
data were analyzed using the fractional inhibitory concentra- ascorbic acid complex inhibited the growth of E. coli
tion (FIC) index (FIC index was ꢅ0.5). The FIC index is the JMC5491, a Gram-negative bacteria of less susceptible to
most frequently used method to determine the interactions many antibiotics and insensitive to dipropofol by their cell
between antimicrobial drugs.9) The reactant of dipropofol- surface structure (Fig. 5, right). Growth inhibition was ob-
1
quinone and ascorbic acid was analyzed using TLC and H- served by direct application onto the plates, but it was also
1
NMR. The H-NMR identified the product of the Rf value observed under the paper disk (data not shown).
0.21 as dipropofol (data not shown)5) and dipropofolquinone
Combinational administration could be an alternative to
(Rf value 0.54) was a result of recovering dipropofol (Rf the development of new classes of agents11—14) and we have
value 0.21) with ascorbic acid (dipropofolquinone : ascorbic studied the combined effect between dipropofol and other
acidꢄ1 : 10, 1 : 1) (Fig. 4A-1). It could be reasonable to as- classes of antibiotics. The synergy against VRE was only
sume the ascorbate reduction action in relation with this syn-
ergism. On the other hand, the addition of saccharide did not
stimulate the dipropofol formation reaction. The oxidation
potential of dipropofol and ascorbic acid were 401 mV and
303 mV, respectively, using cyclic voltammetry. This result is
also supported by the ascorbate reduction action.
In addition, the stability of dipropofol was investigated by
UV (254 nm) irradiation (Fig. 4A-2). In a solution containing
ascorbic acid, the dipropofol was stable after 24 h, but the so-
lution of added water (pH 2) or glucose was not stable and
dipropofolquinone was generated. We considered that the
ascorbic acid worked as a reducing agent of dipropofol as is
the case of a-tocopherol and ascorbic acid (Fig. 4B).
In Fig. 5, a complex formation experiment was carried out
using ascorbic acid instead of the saccharides. In this case of
S. aureus 209P, a Gram-positive bacteria, zonal growth inhi-
bition was observed by direct application as well as by the
Table 1. MICs (mg/ml) of Dipropofol and in Combination with Ascorbic
Acid
A.A (mg/ml)
DP
(mg/ml)
12.5
6.25
3.12
1.56
0.78
0.39
0.19
0.09
0
6.25
3.12
1.56
0.78
0.39
0.19
0
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢂ
ꢂ
ꢂ
ꢁ
ꢁ
ꢁ
ꢁa)
ꢂ
ꢂ
ꢂ
ꢁ
ꢁ
ꢁ
ꢁa)
ꢂ
ꢂ
ꢂ
ꢁ
ꢁ
ꢁ
ꢁa)
ꢂ
ꢂ
ꢂ
ꢁ
ꢁ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢁ
ꢁ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢁ
ꢁ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
Fig. 4. Antibacterial Activity of the Dipropofol–Ascorbic Acid Complex
(A-1) Dipropofolquinone reductive reaction with ascorbic acid or sugar, a: dipropo-
fol, b: dipropofolquinone : ascorbic acid (1 : 10), c: dipropofolquinone : ascorbic acid
(1 : 1), d: dipropofolquinone : ascorbic acid (10 : 1), e: dipropofolquinone : glucose
(1 : 100), f: dipropofolquinone. (A-2) Stability of dipropofol. a: dipropofol only, b:
dipropofolꢂwater (pH 2), c: dipropofolꢂascorbic acid, d: dipropofolꢂglucose, e:
dipropofol (not irradiated), f: dipropofolquinone (not irradiated). (B) The scheme of
the oxidation-reduction reaction of dipropofol and ascorbic acid.
DP: dipropofol, A.A: ascorbic acid. ꢂ: growth, ꢁ: inhibition, a) synergism.
Fig. 5. Dipropofol–Ascorbic Acid Solution in Water, Left; S. aureus 209P, Right; E. coli JCM5491
(a) and (b) were paper disk with dipropofol–ascorbic acid complex solutions, (c) direct application of the dipropofol–ascorbic acid complex solution, (d) and (e) were paper disks
with ascorbic acid alone, and (f) direct application of ascorbic acid alone in the medium.

1568
Vol. 30, No. 8
3) Kvam C., Granese D., Flaibani A., Pollesello P., Paolletti S., Biochem.
Biophys. Res. Commum., 193, 927—933 (1993).
4) Ogata M., Sato T. S., Kunikane T., Oka K., Seki M., Urano S., Hira-
matsu K., Endo T., Biol. Pharm. Bull., 28, 1120—1122 (2005).
5) Ogata M., Shin-ya K., Urano S., Endo T., Chem. Pharm. Bull., 53,
344—346 (2005).
6) Yoshida O., Yasukata T., Sumino Y., Munekage T., Narukawa Y.,
Nishitani Y., Bioorg. Med. Chem. Lett., 12, 3027—3031 (2002).
7) Rubenstein M. S., Baichwal V., Beckmann H., Clark D. L.,
Frankmoelle W., Roche D., Santha E., Schwender S., Thoolen M., Ye
Q. J., Jaen J. C., J. Med. Chem., 44, 3599—3605 (2001).
8) Bouanchaud H. D., J. Antimicrob. Chemother., 30 (Suppl. A), 95—99
(1992).
9) Chevion S., Roberts M., Chevion M., Free Radic. Biol. Med., 47,
1019—1027 (2000).
10) Sies H., Murphy M. E., J. Photochem. Photobiol. B, 8, 211—218
(1991).
11) Zhao W. H., Hu Z. Q., Okubo S., Hara Y., Shimaura T., Antimicrob.
Agents Chemother., 45, 1737—1742 (2001).
12) Rand K. H., Houck H. J., Antimicrob. Agents Chemother., 48, 2871—
2875 (2004).
confirmed with the combination between dipropofol and ri-
fampicin.15) To our knowledge, this is the first study describ-
ing the enhancing activity of ascorbic acid to the antimicro-
bial compound, and in the presence of ascorbic acid dipropo-
fol was solubilized in water, stable to air oxidation and
showed synergistic potency against Gram-positive and Gram-
negative bacteria. In conclusion, we could offer a new route
for the development of solubilzing lipophilic compounds,
which was abandoned due to strong lipohilicity. Detailed
studies of the antibacterial activity in vivo are now under in-
vestigation.
Acknowledgments We thank Miss Masako Hirano and
Mr. Yosuke Tosa for their helpful assistance. This work was
supported in part by a research grant from the KANPO Sci-
ence Foundation, Tokyo, Japan.
REFERENCES
13) Deshpande L. M., Jones R. N., Clin. Microbial. Infect., 9, 1120—1124
(2003).
1) Centers for Disease Control and Prevention, MMWR, 51, 565—567
(2002).
2) Centers for Disease Control and Prevention, MMWR, 51, 902 (2002).
14) Walsh C., Nature Rev., 1, 67—70 (2003).
15) Ogata M., Kunikane T., Seki M., Oka K., Urano S., Seki S., Seki Y.,
Endo T., Biol. Pharm. Bull., 28, 1773—1775 (2005).