Preparation of Evodiamine Solid Dispersion & It’s Pharmacokinetics

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Preparation of Evodiamine Solid Dispersion & It’s Pharmacokinetics: 

Preparation of Evodiamine Solid Dispersion & It’s Pharmacokinetics H. XU , T. ZHANG , H. YANG , X. XIOA , Y. BIAN, D. SI AND C. LIU Indian Journal Of Pharmaceutical Science Issue – May June-2011 1 Matushree V. B. Manvar College Of Pharmacy, Dumiyani Presented By :- Alpesh Gajjar Accepted – 15 th May, 2011 Revised – 11 th May, 2011 Received – 8 th February, 2010 Guided By :- Dr. H. M. Tank Mr. Sandip Patel

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 2 Contents Abstracts Introduction Materials And Methods Evaluation Parameter Result & Discussion Acknowledgement References

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 3 Abstracts In order to increase the dissolution rate and bioavailability, solid dispersion of evodiamine in PVP K 30 with different enriched samples of evodiamine to PVP K 30 ratios were prepared by solvent method. Our study shows that the dissolution rate of evodiamine was significantly higher in the solid dispersion system in comparison with that in enriched samples of evodiamine or physical mixtures. The increase of the dissolution rate was evidently related to the ratio of evodiamine to PVP K 30 .

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 4 The solid dispersion system (enriched sample of evodiamine/PVP K 30 =1/6, w/w) gave the highest dissolution rate: about 27.7-fold higher than that of enriched samples of evodiamine in hard capsules. Powder X-Ray diffraction study showed that enriched samples of evodiamine presented a total chemical stability after it’s preparation as solid dispersion. In-vivo administration studies indicated that solid dispersion of evodiamine in hard capsules had a higher C max and shorter T max than those of physical mixtures in hard capsules, and the difference of C max and T max between them were significant.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 5 These result suggest that solid dispersion of evodiamine in hard capsules has a notably faster and greater absorption rate than enriched samples of evodiamine in physical mixture hard capsule and corresponds with the in-vitro dissolution. Keywords Dissolution rate, evodiamine, pharmacokinetics, solid dispersions, X – ray powder diffraction

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 6 Introduction Evodiamine, is an indolequinoline alkaloids, is an major component in the fruit of Evodia rutaecarpa , widely used for a long time to treat abdominalgia, hernia and menorrhalgia . Further studies demonstrated that evodiamine had anti tumor potential by inhibiting proliferation, inducing apoptosis and reducing invasion and metastasis of a wide variety of tumor cells. More importantly, evodiamine not also shows little toxicity to normal human peripheral blood cells.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 7 It is necessary to increase the drug solubility in the g.i.t., thus increasing the oral absorption of poorly water - soluble drug. The solid dispersion technique, which has been widely used to improve the dissolution rate, solubility and oral absorption of poorly water – soluble drugs and thus improve bioavailability.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 8 Materials And Methods PVP K 30 ( Tianjin Tiantai Fine Chemicla Co., Ltd., Tianjin, China) Evodiamine ( The National Institute For The Control Of Pharmaceutical & Biological Products, Beijing, China) Evodiae rutaecarpa (Pharmaceutical Company, Hebei, China) HPLC Grade Methanol (Tianjin Concord Technology Co., Ltd) Deionized Water ( Milli – Q water system, Millipore Bedford, MA, USA) – For preparation of sample and buffer solution. The other materials were of analytical reagent grade.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 9 1. Extraction and purification of EV from Evodia rutaecarpa (Juss). Benth: Amount Of Evodia Rutaecarpa is 8 times higher than the 70% Ethanol Extracted solution is filtered and dried under reduced pressure Extract + 24 times Water of pH 3 in the water precipitation process The above sediment put in aluminum oxide column Chromatographic Separation Enriched Sample of EV were acquired (content of Evodiamine is 11.5%) Distilling 3 times & 2hr each time

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 10 The Chromatography condition, Loading amount – 0.4 gm/ml Eluant – Acetoacetate/Dichloromethane – 70:30 Loading volume – 5 bed volume(BV) Eluant Flow Rate – 2 BV/ hr 2. Preparation of physical mixtures and solid dispersion Solid dispersion of EV was prepared with ESEV:PVP K 30 in 1:2, 1:4, 1:6, 1:8, 1:10 weight ratios by solvent method.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 11 For example, Weigh 2gm of ESEV + 4gm of PVP K 30 Dissolve in 200ml of alcohol Placed in lyophilizer at 60 ° C under vacuum Sample is pulverized, sieved Fraction ≤187.5 µm were selected For Physical mixtures: Prepared by grinding ESEV and PVP K 30 in a mortar ( ESEV/PVP K 30 = 1:2, 1:4, 1:6, 1:8, 1:10). The fraction ≤187.5 µm were selected for further investigation.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 12 3. Preparation of enteric capsules: For dissolution and animal experiments the following 3 types of capsules were prepared by filling the powders into hard capsules, SDEV hard capsules (SDEV - HC) ESEV in physical mixture hard capsules (PMEV - HC) ESEV hard capsules (EV - HC) Each hard capsules contained 6.25mg of EV.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 13 Evaluation Parameter Dissolution Studies: According to Chinese Pharmacopoeia 2005 apparatus no. 2 (oar method) with RCZ-5A dissolution apparatus(Tianjin, China) Solubility of evodiamine in pH 6.8 phosphate buffer is 3.8 µg/ml at 37±0.5°C according to the equilibrium method. Volume of dissolution medium = 900ml Temperature = 37±0.5°C Rotation speed = 100rpm Volume of sample to be withdrawn = 5ml Filter through 0.45 µm membrane filter

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 14 Concentration of drug was determined by a HPLC – MS/MS. 2. X – ray powder diffraction: Performed using a Rigaku D/max 2500v/pc X-ray diffract meter equipped with a high frequency 18 kW X – ray generator (Rigaku Corp., Japan) Data was processed using DMSNT software (version 1.37, Scintag Inc.) X – ray source = copper filament x – ray tube operated at 40 kv and 200 mA Sample were scanned at a rate of 1° 2ø/min over a range of 3-50 degrees.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 15 In vivo administration studies: 8 beagle dogs The Experimental Group (EG) The Reference Group(RG) After an overnight fast of 12 hrs Give 8 SDEV – HC with 150 ml water Give 8 PMEV – HC with 150 ml water Empty Stomach Food and drug were not allowed during the following 4 hr period of test. The cross over test was performed 1 week later after the first administration.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 16 For pharmacokinetic analysis, 3ml venous blood samples were collected in heparin tubes before 30 min of administration and 10, 30, 60, 90, 120, 180, 240, 360, 480, 720 and 1440 min after administration. The blood samples were centrifuged at 2500 rpm for 10 min at room temperature to obtain plasma. Plasma Transfer to 2 ml tube Frozen at -20 o C for 1hr Analyze It

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 17 HPLC - MS/MS: By using a Finnigan HPLC instrument (Finnigan, San Jose CA) Consisting of a Surveyor autosampler (Thermo Finnigan, San Jose, USA) and a TSQ Quantum Discovery Max TM triple quadruple mass spectrometer (Thermo Finnigan, San Jose, USA). Xcalibur software (version 1.4, Thermo Finnigan, San Jose, USA) was used to control the instruments, and for data acquisition and processing. HPLC Condition: Mobile Phase: Methanol:Ammonium Acetate (including 1% formic acid) = 80:20(v/v)

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 18 Flow rate: 0.4ml/min Chromatographic Column: Symmetry C18 (5 µm, 4.6 × 100mm, Serial No. 186002616) Injection Volume: 10 µl Finnigan TSQ Quantum Discovery MAX (Thermo Finnigan, San Jose, USA) Operated with an electrospary ionization (ESI) under following condition: Positive mode Ion spray voltage: 4000 V Capillary temperature: 280 o Sheath gas pressure: 40 psi Auxiliary gas pressure : 10 psi

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 19 Quantization was achieved using selective reaction monitoring (SRM) based on m/z=304.1 160.8 for Ev with the collision energy of 20 V. 200 µl of plasma + 100 µl of methanol containing glibenclamide (500ng/ml) + EtoAc Mix & Centrifuged (2000 rpm) for 10 min Supernatant liquid formed Evaporate under nitrogen flow at 40 O Residue Formed Dissolve in 100 µl of mobile phase HPLC –MS/MS

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 20 Pharmacokinetic analysis: The AUC (0-24 h) and T 1/2 were calculated using a two compartmental approach with 3P87 software. The C max and T max were obtained directly from the observed data AUC was calculated by trapezoidal method. Statistical analysis: Differences in the pharmacokinetic parameter between the two groups were compared by analysis of variance. A P – Value of less than 0.05 was considered statistically significant.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 21 Results And Discussion Dissolution studies were performed under sink condition in a pH 6.8 phosphate buffer. PMEV – HC and SDEV – HC were tested for dissolution properties and compared with that of EV – HC. The results shows in figure 1(a – e). It shows that release rate of EV from physical mixture is slightly increases than the EV – HC, and the release rate of SDEV – HC is remarkable increase than the PMEV – HC and EV – HC. The release rate of EV from SDEV – HC is depend on the ESEV – PVP K 30 ratios and reached maximum at ratio 1:6.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 22 Fig. 1 Dissolution profile of different ESEV – PVP K 30 weight ratios PMEV – HC and SDEV – HC.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 23 Pandit and khakurel suggested that, Dissolution rate of solid dispersion is decreases at higher proportion of polymer due to leaching out of carrier during dissolution so it form a concentrated layer of solution around drug particle, therefore the migration of released particle was slowed down. Drug Release, Time SDEV – HC (ESEV/PVP K30 = 1/6) PMEV – HC (ESEV/PVP K 30 = 1/10) EV - HC At 20 min 64.6% 3.3% 2.72% At 40 min 88.6% 3.6% 3.2%

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 24 The X – ray diffraction patterns corresponding to ESEV, PVP K 30 , Solid dispersion (ESEV/PVP K 30 = 1/6), Physical mixture (ESEV/PVP K 30 = 1/6) are shown in fig 2 (a - d) The above observation proved that ESEV remain unalterable after it’s manufacturing as solid Fig. 2 - X – Ray diffraction pattern of ESEV, PVP K 30 , PMEV – HC, SDEV – HC.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 25 dispersion and that crystallization of PVP K 30 does not modify the crystalline structure of the drug, so it shows that there is no drug interaction between the ESEV and PVP K 30 . Fig. 3 - Plasma concentration – time curve in beagle dogs after oral administration of 8 SDEV – HC and PMEV – HC containing 50 mg of Evodiamine.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 26 Drugs C max (ng/ml) (means ± SD) T max (h) (means ± SD) AUC ( 0-24 h) (ng/ml) (means ± SD) T 1/2 (means ± SD) SDEV – HC 27.85 ± 13.78 0.57 ± 0.19 51.22 ± 38.01 0.25 ± 0.25 PMEV – HC 10.48 ± 7.28 2.18 ± 0.88 34.31 ± 28.07 2.18 ± 0.88 The above result suggest that the absorption rate of SDEV – HC is notably faster and greater than that of PMEV – HC and the bioavailability of the former is notably faster and greater than that of the latter. The cross over test was used to avoid the influences of individual differences in the beagles.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 27 Acknowledgement This project was supported by National Key Technologies R and D Program of China in the 11 th Five year Plan under Grant No. 2007BAI4106 and National Basic Research Program of China, No. 2006CB933303.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 28 References Papers: Yoshizumi M, Houchi H, Ishimura Y, Hirose M, Kitagawa T, Tsuchiya K, et al. Effect of evodiamine on catecholamine secretion from bovine adrenal medulla. J Med Invest 1997;44:79 – 82. Chiou WF, Sung YJ, Liao JF, Shum AY, Chen CF. Inhibitory effect of dehydroevodiamine and evodiamine on nitric oxide production in cultured murine macrophages. J Nat Prod 1997;60:708 – 11. King CL, Kong YC, Wong NS, Yeung HW, Fong HH, Sankawa U. Uterotonic effect of Evodia rutaecarpa alkaloids. J Nat Prod 1980;43:577 – 82.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 29 Chiou WF, Chou CJ, Shum AY, Chen EF. The vasorelaxant effect of evodiamine in rat isolated mesentric arteries: Mode of action. Eur J Pharmacol 1992;215:277 – 83. Ogasawara M, Matsubara T, Suzuki H. Screening of natural compounds for inhibitory activity on colon cancer cell migration. Biol Pharm Bull 2001;24:720 – 3. Kan SF, Yu CH, Pu HF, Hsu JM, Chen MJ, Wang PS. Anti – proliferative effect of evodiamine on human prostate cancer cell lines DU145 and PC3. J Cell Biochem 2007;101:44 – 56. Kang SF, Haung WJ, Lin LC, Wang PS, Inhibitory effect of evodiamine on the growth of human prostate cancer cell line LNCaP. Int J Cancer 2004;110:641 – 51.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 30 Books: Pharmacopoeia of People’s Republic of China, (in Chinese). Vol. 1. Beijing: Chemical Industry Press;2002.

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Matushree V. B. Manvar College Of Pharmacy, Dumiyani 31 T h a n k Y o u