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Nanomemulsion
of
megestrol
acetate
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bioavailability and reduced food effect
for
improved
oral
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Yixian Li, Chung Kil Song, Min-Kyoung Kim, Hyosang Lim, Qingbo Shen,
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Don Haeng Lee, Su-Geun Yang*
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* Corresponding to Su-Geun Yang, Ph.D. (Sugeun.Yang@Inha.ac.kr)
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The solubility studies of MGA in each composition
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The components for nanoemulsion were determined by the solubility of MGA in oils,
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surfactants and co-surfactants. The solubility studies of MGA were conducted by placing an
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excess amount of MGA in 1 mL of each vehicle, and solubilized on a vortex mixer. The
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mixtures were kept at room temperature in a shaking water bath for 72 hrs to reach
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equilibrium. The samples were centrifuged at 10,000 rpm for 10 min. The supernatant was
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taken, filtered through 0.45 μm membrane filter and diluted with solvent if necessary for
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analysis. The concentration of MGA in each vehicle was determined by Agilent 1200 series
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high performance liquid chromatography (HPLC) system. Based on the solubility study
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(Table S1), MCT oil, Cremophore RH40 and propylene carbonate were selected as oil,
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surfactant and cosurfactant, respectively, due to higher solubility of MGA than other vehicles.
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Table S1. The solubility of MGA in various vehicles at ambient condition for 3 days (n=3)
Oils
Surfactants
Co-surfactants
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Vehicles
Solubility (mg/g)
Triacetin
9.56±0.60
OMEGA 3 fatty acid
7.25±0.33
MCT oil
7.28±0.34
Solutol 15
8.37±0.29
Cremophor EL
7.60±0.26
Cremophore RH40
8.58±0.27
Tween 20
6.65±0.30
Tween 80
6.36±0.30
Ethanol
11.67±1.60
PEG 400
5.01±0.12
Transcutol
18.61±2.10
Propylene glycol
3.85±0.02
Lutrol 400
5.09±0.07
Propylene carbonate
40.07±1.12
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Determination of composition for MGA NE
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On the result of the solubility studies of MGA, medium-chain triglyceride (MCT oil) was
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selected as the oil phase for the development of nanoemulsion. In previous solubility study,
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triacetin showed higher solubility than MCT oil. Triacetin is triglyceride of short chain fatty
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acid (acetic acid). Even though triacetin shows higher solubility than other oils, it has some
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shortage as a core forming oil material. Triacetin is water-miscible oil, leaks out from oil
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droplets and eventually cause precipitation of drug in media (data is not shown). Cremophore
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RH40 was used as surfactant along with propylene carbonate as cosurfactant. The
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compositional ratio of each component for nanoemulsion was determined based on the phase
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diagram following previous our study. The selected composition of nanoemulsion
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preconcentrate was 53% Cermophor RH 40 (surfactant), 23% propylence carbonate (co-
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surfactant), 21% MCT (oil). The loading of MGA in this composition, determined using
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HPLC as described in above section “The solubility studies of MGA in each composition”,
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was 25±1.2 mg/g. MGA was dissolved in the nanoemulsion preconcentrate at the
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concentration of 20 mg/g (loading efficiency was 100 %). Then water can be added by ratio
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of 1:5 to 10. The optimal concentration of MGA which can sustain the stable nanoemulsion
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without precipitation was determined by droplet size analysis and microscopic observation.
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Figure S1. MGA NE (A) and MGA MS (B).
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Figure S2. Dissolution of MGA NE (○; MGA Nanoemuslion, ●; MGA microcrystal
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suspension) using a USP 37 dissolution apparatus II (708-DS Dissolution Apparatus, Agilent
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Technologies, CA) with 900 mL of simulated gastric fluids without pepsin (0.2% sodium
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chloride in 0.7% hydrochloric acid) at 37 ± 0.5°C with a paddle speed of 50 rpm. Dissolution
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samples were dropped on the media, and media samples (5 ml) were taken, filtered (0.45 μm)
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and introduced to HPLC for analysis.
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a)
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b)
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c)
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Figure S3. LC-MS/MS spectra of (a) blank plasma (b) MGA-spiked plasma (c) MGA & IS
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(medroxyprogesterone acetate , 50 ng/ml)-spiked plasma.
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Table S2. Intra-day (n=6) and inter-day (n=6) precision and assay accuracy of quality control
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samples for the determination of megestrol acetate at different concentrations (5, 50 and 100
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ng/mL) in plasma samples.
Intra-day
Inter-day
Nominal
concentration
(ng/mL)
Measured
concentration
(mean ± SD)
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Precision
Accuracy
(CV, %)
(bias, %)
Measured
concentration
(mean ± SD)
Precision
Accuracy
(CV, %)
(bias, %)
5
4.95 ± 0.46
9.32
99.09
5.14 ± 0.61
11.87
102.8
50
51.05 ± 2.05
6.74
102.10
50.18 ± 2.77
5.52
100.37
100
100.79 ± 1.78
4.26
100.79
101.58 ± 2.19
2.15
101.53
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The enhancement bioavailability of MGA NE in the rat
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Sprague-Dawley rats (Orient Bio Co., Seoul, Korea) were used to confirm the improvement
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of oral bioavailability after treatment of MGA NE. The animals were fasted for 24 h for the
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oral administration of MGA MS and MGA NE. They were randomly divided into two groups.
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The femoral artery and vein of the rats were cannulated with a polyethylene tube (PE-50;
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Clay Adams, Parsippany, NJ) after anaesthetization. The rats were administrated with MGA
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suspension or MGA NE containing same amount (10 mg/kg) of MGA. 300 µL blood was
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obtained through femoral artery at pre-determination time (0, 30, 60, 90, 120, 180, 240, 360,
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480, 600, and 720 min) after oral administration. These samples were immediately
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centrifuged at 10,000 rpm for 5 min and stored at -70°C until drug analysis was carried out
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using HPLC-MS-MS.
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Table S3. Pharmacokinetic parameters of MGA MS and MGA NE in SD rats. The data
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represent the mean ± S.D. (n=4) and paired student t-test was performed.
Parameter
MGA MS (A)
MGA NE (B)
Ratio (B/A)
AUCall (ng·min/mL)
100427.3±52058.6
648949.9±275852.9**
6.46
Cmax (ng/mL)
423.0±371.7
3890.8±2587.9**
9.19
Tmax (min)
375.0±278.7
52.5±28.7
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T1/2, terminal (min)
-*
210.1±55.4
-
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* Parameter couldn’t be obtained because of large variation of data at terminal phase.
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** p < 0.001 (n=4)
Conc. of megestrol (ng/ml)
5000
Suspension
Nanoemulsion
4000
3000
2000
1000
0
0
2
4
6
8
10
12
Time (hrs)
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Figure S4. Mean plasma concentration profile of MGA MS (●) and MGA NE (○) in SD rats.
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The data represent the mean ± S.D. (n=4)
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Figure S5. Box plots summarizing the inter-distribution of AUC (A) and Cmax (B) of MGA
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NE and MGA MS in fed and fasting conditions. The data represent the mean ± S.D. (n=6).