Olmesartan medoxomil-loaded mixed micelles: Preparation, characterization and in-vitro evaluation

Olmesartan medoxomil-loaded mixed micelles: Preparation, characterization and in-vitro evaluation

Accepted Manuscript Olmesartan Medoxomil-loaded mixed micelles: Preparation, characterization and invitro evaluation Mohamed A. El-Gendy, Mona I.A. El...

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Accepted Manuscript Olmesartan Medoxomil-loaded mixed micelles: Preparation, characterization and invitro evaluation Mohamed A. El-Gendy, Mona I.A. El-Assal, Mina Ibrahim Tadros, Omaima N. ElGazayerly PII:

S2314-7245(16)30184-4

DOI:

10.1016/j.fjps.2017.04.001

Reference:

FJPS 32

To appear in:

Future Journal of Pharmaceutical sciences

Received Date: 21 December 2016 Revised Date:

30 March 2017

Accepted Date: 3 April 2017

Please cite this article as: El-Gendy MA, El-Assal MIA, Tadros MI, El-Gazayerly ON, Olmesartan Medoxomil-loaded mixed micelles: Preparation, characterization and in-vitro evaluation, Future Journal of Pharmaceutical sciences (2017), doi: 10.1016/j.fjps.2017.04.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Olmesartan Medoxomil-loaded mixed micelles: Preparation, characterization and in-vitro evaluation Mohamed A. El-Gendy1, Mona I. A. El-Assal1, Mina Ibrahim Tadros2, Omaima N. El-Gazayerly2 1 Pharmaceutics and pharmaceutical technology department, faculty of pharmaceutical sciences and pharmaceutical industries, Future University, 11835 Cairo, Egypt. 2 Department of pharmaceutics and industrial pharmacy, faculty of pharmacy, Cairo University, Kasr El-Aini Street, 11562 Cairo, Egypt.

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Solubility of OLM

Oral bioavailability of OLM

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Schematic illustration of preparation of OLM-loaded mixed micelles composed of Pluronic F127® and Pluronic P123®

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• USP dissolution apparatus II

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• The paddles were rotated at 100 rpm • The dissolution vessel contained 900 mL of Sorensen’s phosphate buffer (pH 6.8) as dissolution medium maintained at 37 ± 0.5 C0 • A dialysis bag containing 5 mL sample was withdrawn at 15, 30, 45 and 60 minutes

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Abstract: Olmesartan medoxomil (OLM) is highly lipophilic in nature (log p=4.31) which attributes to its low aqueous solubility contributing to its low bioavailability 25.6%. OLM was loaded into mixed micelles carriers in a trial to enhance its solubility, thus improving its oral bioavailability. OLM-loaded mixed micelles were prepared, using a Pluronic® mixture of F127 and P123, adopting the thin-film hydration method. Three drug: Pluronic® mixture ratios (1:40, 1:50and 1: 60) and various F127: P123 ratios were prepared. OLM Loaded mixed micelles showed stability up to 12 hours. The particle size of the systems varied from 364.00 nm (F3) to 13.73 nm (F18) with accepted Poly dispersity index (PDI) values. The in-vitro release studies of OLM from mixed micelles versus drug aqueous suspension were assessed using the reverse dialysis technique in a USP Dissolution tester apparatus (type II). The highest RE% (43%) was achieved with OLM-loaded mixed micelles (F8) when compared to (35%) of drug suspension.

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(Keywords: Olmesartan medoxomil, mixed micelles, thin film hydration, reverse dialysis)

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1. Introduction Hypertension is known as a chronic elevation of arterial blood pressure higher than 140 mmHg systolic blood pressure (SBP) over 90 mmHg diastolic blood pressure (DBP) [1]. Hypertension is as a prevalent disease all over the world, yet remains under-diagnosed and under-treated. It is also considered as the most predominant and influential contributor for cardiovascular (CV) diseases globally[2]. It has a direct relationship with morbidity and mortality and it is necessary for reduction of blood pressure to avoid consequence risks [3, 4]. Recently it is recommended that hypertensive patients achieve a goal of 140 mmHg SBP and 90 mmHg DBP, but patients suffering from diabetes mellitus or an elevated CV risk should target a BP goal of 130 mmHg SBP over 80 mmHg DBP[5]. The risk of sudden stroke can be reduced to 35% by lowering SBP by 10 mmHg or DBP by 5 mmHg. By the age of 56 a reduction of ischemic heart disease also can be reduced by 25 % [2, 6, 7]. Angiotensin II receptor blockers (ARBs) are conventionally used for hypertension treatment. These group proved reduction in morbidity and mortality with patients that suffering from hypertension and are recommended as first-line drugs for the treatment[8]. Nowadays bioavailability problems due to poor aqueous solubility cause the rejection of about 40% of newly developed active pharmaceutical ingredients[9]. Beside, up to 70% of synthesized drug molecules found to have solubility problems. Olmesartan medoxomil (OLM) is an antihypertensive drug from ARBs family. This drug has low aqueous solubility due to high lipophilic nature (log p=4.31). OLM has a low bioavailability 25.6% due to the poor aqueous solubility and the efflux of hydrophobic therapeutic agents by means of drug resistance pump in GIT [10, 11]. Polymeric micelles (PMs) are promising vehicle for poorly water soluble drugs. PMs are generally spherical in shape (when the hydrophilic part is longer than the core block) with narrow size distribution but may change under certain conditions. Conversely, increasing the length of the core part beyond that of the shell-forming chains may generate various nonspherical structures, including rods and lamellae [12]. They are self-assemblies of amphiphilic block copolymers in aqueous media forming a micelle structure above their critical micelle concentration (CMC). PMs consist of a core and shell configuration; the inner core is the hydrophobic part of the block copolymer, which encapsulates the poorly watersoluble drug, whereas the outer protects the drug from the aqueous medium. PMs well increase the water solubility of such agents by 10-to 5000-fold[13].

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2. Materials and methods 2.1. materials Olmesartan Medoxomil (GVK Biosciences, Hyderabad, India), Pluronic® F-127 and Pluronic® P123 (Poly (ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) triblock copolymers (Sigma Aldrich, St. Louis, Missouri, USA), Absolute ethyl alcohol, Sodium dihydrogen orthophosphate-1-hydrate and Disodium hydrogen orthophosphate-1-hydrate (El Nasr Pharmaceutical Chemicals, Abuzaabal, Egypt), Dialysis tubing cellulose membrane (M.wt cut-off 12,000-14,000) (Sigma Aldrich, St. Louis, Missouri, USA). 2.2. Methods 2.2.1. Preparation of OLM-loaded mixed micelles OLM-loaded mixed micelles were prepared, using a Pluronic® mixture of F127 and P123, adopting the thin-film hydration method using rotavapor (Büchi® Rotavapor® RII evaporator, Flawil, Switzerland)[14]. Three drug: Pluronic® mixture ratios (1:40, 1:50and 1: 60) and various F127: P123 ratios were investigated in an attempt to optimize

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ACCEPTED MANUSCRIPT the design of these di-functional triblock copolymer micelles. The composition of all systems (F1–F18) is shown in table (1).

Table 1

1

F2

1 1:40

1

P123

0

30

10

20

20

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40

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F1

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Composition of the prepared OLM-loaded mixed micelles systems Drug : Polymer Drug F127 System code ratio Weight ratio

1

10

30

1

0

40

1

50

00

1

40

10

1

30

20

1

20

30

1

10

40

1

0

50

1

60

0

1

50

10

1

40

20

1

30

30

F16

1

20

40

F17

1

10

50

F18

1

0

60

F4 F5 F6

F8 1:50 F9

F12 F13 F14 F15

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F7

1:60

2.2.2. Characterization of OLM-loaded mixed micelles

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Physical stability

The clarity / turbidity of the developed mixed micelles were observed visually, at 25 ± 0.5◦C for the next 12 hours following the preparation. The time since the preparation of a clear solution of the mixed micelles to turbidity is measured for each system[15].

Determination of drug content

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2.2.2.2.

A unit dose of OLM-loaded mixed micelles was dissolved in absolute ethyl alcohol and the solution was measured spectrophotometrically at 257 nm using spectrophotometer (UV-1800 Shimadzu, Kyoto, Japan).. Determination of particle size, PDI and zeta potential

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2.2.2.3.

2.2.2.4.

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The mean size (z-ave) and the polydispersity index (PDI) of OLM-loaded mixed micelles were determined by photon correlation spectroscopy (PCS) that analyses the fluctuations in light scattering due to the Brownian motion of particles. To avoid the multi-scattering phenomena, each system was diluted (10 times) with de-ionized water and was placed into a quartz cuvette. All measurements were performed, in triplicate, at 25 ± 0.5◦C, at 90◦to the incident beam using a zetasizer nano series (Malvern, Worcester-shire, UK)[16]. PDI values ranging from 0.1 -0.3 are considered optimum since they indicate uniform particle size distribution. The zeta potential of OLM-loaded mixed micelles was determined according to electrophoretic light scattering technology using laser Doppler Anemometer coupled with Zetasizer Nano ZS. Measurements were carried out in triplicate, at 25± 0.5 0C, using the Helmholtz- Smoluchouski equation built into software.

Morphological examination

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The morphological aspects of OLM-loaded mixed micelles were evaluated using transmission electron microscopy (TEM). One drop of each system was placed on a copper grid and the excess was removed using a filter paper. Following, one drop of phosphotungstic acid aqueous solution (2%, w/v) was added and the excess was similarly removed. Finally, the grid was examined under a transmission electron microscope (JEOL JSM 5200 )[17].

2.2.2.5.

In-vitro drug release studies

The in-vitro release studies of OLM from mixed micelles, drug aqueous suspension were assessed, in triplicate, using the reverse dialysis technique in a USP Dissolution tester apparatus type II (Vision® G2 Elite 8TM, Hanson, California, USA) at 37 ± 0.5◦C[18]. Based on the dissolution method provided by United States Food and Drug Administration (FDA) for OLM oral tablets, the release studies of OLM-loaded mixed micelles were performed in Sorensen phosphate buffer (pH 6.8, 900 ml) to ensure creation of perfect sink conditions. One dialysis bag containing 5 ml of the dissolution medium was withdrawn at 15, 30, 45 and 60 minutes. The withdrawn volume was replenished with 5 mL of fresh blank medium. The withdrawn samples were analysed for the drug spectrophotometrically at 257 nm. Mean drug released percentage was plotted against time to compare the release profiles

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Where y is the percentage drug released at time t.

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of these formulations. Release efficiency percentage (RE %) was estimated to evaluate the release enhancement by mixed micelles by calculating the area under the release curve – values (AUC) at 60 minutes using the trapezoidal rule. It is expressed as percentage of the area of the rectangle corresponding to 100% release, for the same total time (60 minutes), according to the following equation:

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3. Results and discussion 3.1. Preparation of OLM-loaded mixed micelles Polymeric micelles consist of a core and shell structure; the inner core is the hydrophobic part of the block copolymer, which encapsulates the poorly water-soluble drug, whereas the outer shell of the hydrophilic block of the copolymer protects the drug from the aqueous environment. The outer shell is attributed to the hydrophilic PEO in Pluronic® P123 and F127, while the inner core related to hydrophobic PPO. OLM was dispersed in the core of mixed polymeric micelles[19].

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3.2. Characterization of OLM-loaded mixed micelles 3.2.1. Physical stability The physical stability of the investigated micelles was assessed following storage at room temperature over 12 hours. Once precipitation was visually observed, the micelles were considered phase separated[20]. Therefore, the onset of precipitation was estimated following the first appearance of flakes. As shown in Table (2), at a drug: mixed polymers ratio of 1: 40 (F1-F5) the micelles got turbid rapidly. It was clear that drug: mixed polymers ratio of 1: 40 was not sufficient to improve their kinetic stability. Increasing the drug: mixed polymers ratio to 1:50 or 1:60 has improved the kinetic stability of the mixed micelles up to12 hours. An optimum Pluronic® F127:P123 content and ratio is needed so that P123 could exhibit a higher solubilizing capacity for poorly water soluble drugs and provide higher kinetic stability while F127 with a higher ratio of EO/PO could be used to solubilize the micelles in aqueous medium. The lack of P123 content in F6 and F12 could account for the poor kinetic stability of these systems. Table 2

Physical stability of OLM-loaded mixed micelles at room temperature Formula Code

Stability (h)

F1

0

F2

0

F3

0.5

5

1

F5

1.5

F6

1

F7

10

F8

12

F9

10

F10

12

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F13

12

F14

10

F15

10

F17 F18

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3.2.2. Determination of drug content The results in table (3) show the drug content of OLM in OLM-loaded mixed micelles. The original amount of OLM used during the preparation of OLM loaded mixed micelles was 20 mg. The drug content reached up to 84 %, this loss is suggested to occur during the preparation process. Table 3 Drug content values of OLM-loaded mixed micelles Formula Code

Drug Content (mg)

Drug Content (%)

F7

12.5

62.5

F8

14.9

74.5

F9

11.7

58.5

F10

16.8

84

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56.5

F13

15.2

76

F14

13.8

69

F15

15.4

77

F16

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F17

11.5

F18

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F11

55

57.5

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29.5

Table 6

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3.2.3. Determination of particle size, PDI and zeta potential As shown in table (4), the particle size of the systems varied from 364.00 nm (F3) to 13.73 nm (F18), where it was difficult to determine the particle size, PDI and zeta potential of F1 and F2 because they got turbid immediately. With exception of F3 which had a PDI of 0.88, the remaining systems showed acceptable values ranging from 0.37 (F4) to 0.09 (F9). High PDI values could indicate the tendency for agglomeration of micelles and consequently phase separation. Higher kinetic stability was achieved at high drug: mixed polymer ratio. The zeta potential values of all systems were negatively charged. Yet, the values were not high enough to ensure stability for longer periods of time, as they ranged from -4.31 mV to -12.10 mV.

Particle size, PDI and zeta potential values of OLM-loaded mixed micelles. Formula Zeta potential Particle size (nm) PDI Code (mV) -

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-

-

-

364.00 ± 30.41

0.88

-11.40 ± 0.21

F4

287.88 ± 5.09

0.37

-7.41 ± 0.14

F5

248.52 ± 0.39

0.21

-8.78 ± 1.30

F6

217.00 ± 12.59

0.26

-6.00 ± 0.33

F7

101.60 ± 61.70

0.31

-5.05 ± 0.03

F8

61.30 ± 2.67

0.11

-6.38 ± 0.05

F9

59.74 ± 6.23

0.09

-8.00 ± 0.27

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F1

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20.00 ± 0.28

0.22

-6.42 ± 2.59

F11

18.00 ± 0.09

0.23

-7.24 ± 2.16

F12

280.00 ± 51.97

0.34

-12.10 ± 0.42

F13

43.90 ± 1.50

0.35

-5.66 ± 2.21

F14

35.76 ± 6.79

0.18

-6.18 ± 0.31

F15

23.00 ± 0.30

0.17

F16

19.28 ± 0.12

0.12

F17

16.34 ± 0.12

0.11

F18

13.73 ± 0.02

0.15

-8.32 ± 0.20

-5.88 ± 0.38 -5.42 ± 0.26 -4.31 ± 0.18

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3.2.4. Morphological examination Representative photomicrographs of OLM-loaded mixed micelles(F8) were illustrated in Fig. (1), it is clear that the developed micelles were fairly dispersed in aqueous media and formed homogeneous small-sized spherical structures with a smooth surface.

Fig. 1 TEM photomicrographs of OLM-loaded mixed micelles (F8) 3.2.5. In-vitro drug release studies The in-vitro release profiles of OLM from OLM aqueous suspension (20mg) and OLMloaded mixed micelles are shown in figures (2-3). The percentage of drug released after 1 hour from the micellar systems ranged from 34 % for (F17) to 70 % for (F8) which exhibited higher release when compared to 35% for drug aqueous suspension. The release efficiency percentage are graphically illustrated in figure (4). The release efficiency of the micellar systems ranged from 26 % (F17) to 43% (F8) which exhibited higher release efficiency when compared to 20% for aqueous suspension. The superiority of OLM-loaded mixed micelles (F8) could be explained in correlation to the increase in PEO length by Pluronic F127

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80.0 F7

60.0

F8

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Drug Released (%)

100.0

F9

40.0

F10 F11

20.0

Plain Drug

0

10

20

30

40

Time (min)

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0.0 50

60

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F14

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Drug Released (%)

Fig. 2. In-vitro drug release profiles of OLM-loaded mixed micelles prepared at a Drug: mixed polymers ratio (1:50) in Sorensen’s phosphate buffer (pH 6.8) at 37 + 0.5 ◦C (mean + S.D., n=3)

20

F15 F16 F17 F18 Plain Drug

30

40

50

60

Time (min)

Fig. 3. In-vitro drug release profiles of OLM-loaded mixed micelles prepared in a ratio of Drug: mixed polymers ratio (1:60) in Sorensen’s phosphate buffer (pH 6.8) at 37 + 0.5 ◦C (mean + S.D., n=3)

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Fig. 4. Release efficiency (%) of OLM-loaded mixed

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Declaration of interest

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Conclusion The Mixed Micelle Technique was adopted in the current work to enhance the aqueous solubility of OLM using various drug: Pluronics F127 /P123 ratios. Low drug: Pluronics F127/P123 ratios (1:10, 1:20 and1:30) failed to solubilize OLM, while increasing the Pluronics F127/P123 to drug ratio (1:40) showed a low stability profile at R.T. Increasing the Pluronics F127/P123 to drug ratios (1:50 and 1:60) enhanced OLM solubility and maintained good stability. The mean particle size of the systems varied from 364 nm (F3) to 13.73 nm (F18). The best achieved system (F8) was prepared using drug: Pluronics® F127/P123 ratio of 1:50 at a Pluronic® F127: Pluronic® P123 ratio of 30:20. It showed a good stability > 12 h at R.T., the highest rate of drug release and the highest release efficiency percentage compared with other systems and the drug suspension.

The authors do not have declaration of interest. References

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WB. Kannel, Blood pressure as a cardiovascular risk factor: prevention and [1] treatment, JAMA 275 (1996) 1571-1576. [2] S. MacMahon, R. Peto, R. Collins, J. Godwin, J. Cutler, P. Sorlie, Blood pressure, stroke, and coronary heart disease: part 1, prolonged differences in blood pressure: prospective observational studies corrected for the regression dilution bias. The Lancet 335 (1990) 765-774. [3] AV. Chobanian, GL. Bakris, HR. Black, WC. Cushman, LA. Green, JL. Izzo, Seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure, Hypertension 42 (2003) 1206-1252. [4] L. Hansson, A. Zanchetti, SG. Carruthers, B. Dahlöf, D. Elmfeldt, S. Julius, Effects of intensive blood-pressure lowering and low-dose aspirin in patients with hypertension: principal results of the Hypertension Optimal Treatment (HOT) randomised trial. The Lancet 351 (1998) 1755-1762. [5] G. Mancia, G. De Backer, A. Dominiczak, R. Cifkova, R. Fagard, G. Germano, 2007 Guidelines for the management of arterial hypertension, Eur Heart J. 28 (2007) 1462-1536. [6] S. Lewington, R. Clarke, N. Qizilbash, R. Peto, R. Collins, Prospective studies collaboration. Age-specific relevance of usual blood pressure to vascular mortality: a meta-

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EP

TE D

M AN U

SC

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analysis of individual data for one million adults in 61 prospective studies. The Lancet 360 (2002) 1903-1913. [7] APCS. Collaboration, Blood pressure and cardiovascular disease in the Asia Pacific region, J Hypertens. 21 (2003) 707-716. [8] DH.Smith, Comparison of angiotensin II type 1 receptor antagonists in the treatment of essential hypertension, Drugs 68 (2008) 1207-1225. [9] AN. Lukyanov, VP. Torchilin, Micelles from lipid derivatives of water-soluble polymers as delivery systems for poorly soluble drugs. Advanced drug delivery reviews 56 (2004) 1273-1289. [10] M. Wehling, Can the pharmacokinetic characteristics of olmesartan medoxomil contribute to the improvement of blood pressure control? Clin Ther. 26 (2004) 7-21. [11] S. Matsushima, K. Maeda, C. Kondo, M. Hirano, M. Sasaki, H. Suzuki, Identification of the hepatic efflux transporters of organic anions using double-transfected Madin-Darby canine kidney II cells expressing human organic anion-transporting polypeptide 1B1 (OATP1B1)/multidrug resistance-associated protein 2, OATP1B1/multidrug resistance 1, and OATP1B1/breast cancer resistance protein, J Pharmacol Exp Ther. 314 (2005) 1059-1067. [12] L. Zhang, A. Eisenberg. Multiple morphologies and characteristics of “crew-cut” micellelike aggregates of polystyrene-b-poly (acrylic acid) diblock copolymers in aqueous solutions, J Am Chem Soc. 118 (1996) 3168-3181. [13] R. Savić, A. Eisenberg, D. Maysinger, Block copolymer micelles as delivery vehicles of hydrophobic drugs: micelle–cell interactions, J Drug Target. 14 (2006) 343-355. [14] S. Varona, Á. Martín, MaJ.Cocero, Liposomal incorporation of lavandin essential oil by a thin-film hydration method and by particles from gas-saturated solutions, Ind & Eng Chem Research 50 (2011) 2088-2097. [15] S. Kulthe, N. Inamdar, Y. Choudhari, S. Shirolikar, L. Borde, V.Mourya, Mixed micelle formation with hydrophobic and hydrophilic Pluronic block copolymers: implications for controlled and targeted drug delivery. Colloids and Surfaces B: Biointerfaces. 88 (2011) 691696. [16] GA. Abdelbary, MI. Tadros, Brain targeting of olanzapine via intranasal delivery of core–shell difunctional block copolymer mixed nanomicellar carriers: in vitro characterization, ex vivo estimation of nasal toxicity and in vivo biodistribution studies. Int J Pharm. 452 (2013) 300-310. [17] HP. Thakkar, BV. Patel, SP.Thakkar, Development and characterization of nanosuspensions of olmesartan medoxomil for bioavailability enhancement, J Pharm. and Bioallied Sciences 3 (2011) 426-429. [18] M. Levy, S. Benita, Drug release from submicronized o/w emulsion: a new in vitro kinetic evaluation model. Int J Pharm. 66 (1990) 29-37. [19] M. Yokoyama, T. Okano, Y. Sakurai, S. Suwa, K. Kataoka, Introduction of cisplatin into polymeric micelle, J Controlled Release. 39 (1996) 351-356. [20] Oh. KT, TK. Bronich, AV.Kabanov, Micellar formulations for drug delivery based on mixtures of hydrophobic and hydrophilic Pluronic® block copolymers, J Controlled Release. 94 (2004) 411-422. [21] VY. Erukova, OO. Krylova, YN. Antonenko, NS. Melik-Nubarov, Effect of ethylene oxide and propylene oxide block copolymers on the permeability of bilayer lipid membranes to small solutes including doxorubicin, Biochimica et Biophysica Acta (BBA)-Biomembranes. 1468 (2000) 73-86.

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