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School of Health and Allied Sciences Pokhara University Study on pharmacokinetics and tissue distribution of the isocorydine derivative (AICD) in rats by HPLC-DAD method Laxmi N. Neupane Sixth Semester, B. Pharm. School of Health and Allied Sciences Pokhara University, Lekhanath-12, Kaski Nepal 06/28/2022 Pharmaceutical Seminar 5 1
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LNN 6th sem

Apr 15, 2017

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Page 1: LNN 6th sem

School of Health and Allied Sciences

Pokhara University

1

Study on pharmacokinetics and tissue distribution of the isocorydine

derivative (AICD) in rats by HPLC-DAD method

Laxmi N. NeupaneSixth Semester, B. Pharm.

School of Health and Allied SciencesPokhara University, Lekhanath-12, Kaski Nepal

05/03/2023 Pharmaceutical Seminar 5

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Contents

• Introduction• Material and Methods• Results• Discussion• Conclusion• Reference

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Introduction

HPLC couple with DAD is a technology used to the seprate and quantitate mixture of substances in a solution.

HPLC-DAD is a ideal for analyzing ICD in a biological matrix because it is simple, economical, applicable, and provide an acceptable sensitivity and stability.

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Cont…

Isocorydine(ICD) Is a derivative of Aporphine alkaloid. Wide range of pharmacological and biological activities. Mainly: antioxidant, antiprotozoal, cytotoxic and anti-

Parkinson’s disease. FDA: approved listed as prescription drug as well as

medicare drugs to cure endogenous pain.

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Limitation to development a Novel chemotherapy: short half life, rapid elimination, low tolerated dose(32.2 mg/kg in mice).

Modified structure: 8-Amino-isocorydine possesses a better anticancer activity than ICD.

8-Amino-isocorydine : it is unstable in aqueous solution at room temperature.

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Fig: Chemical structures of 8-acetamino-isocorydine (AICD) and isocorydine (ICD).

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Cont…

For synthesized AICD: 8-acetoamino-isocorydine was synthesized through acetylating 8-Amino-isocorydine with acetylcholine is a stable compound.

AICD: Good pharmacological activity, thus selected for a new drug development.

In vivo: useful to determine the pharmacokinetic properties and tissue distribution.

HPLC-DAD is a ideal analysis than HPLC-UV.

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Materials and methods

1. Chemicals and reagents D.leptopodum (plant) Referance compound: ICD(identified by NMR) AICD: synthesized in laboratory Purity: both 98% Reagent: methanol, dichloromethane, ethyl acetate and

ammonia are HPLC grade.

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2. Instrumentation An Aligent 1200 series liquid chromatography system

equipped with a G1312A binary pump. A G1315B diode array detector performing wavelength

190 nm-950 nm Software: Data Analysis System (DAS) 2.0

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Cont…

3. Animal Wister rat: Male 200-220 g. Breed: breeding in room temperature at 25◦ C Humidity: 50%±10% Before experiment: access of food is limited.

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Cont…

4. Preparation of standard solution and quality control (QC) sample

Stock solution AICD: with methanol and final concentration of 1.0mg/ml.

Series of standard solution: 0.1, 0.5, 5.0, 15.0, 40.0, 120.0, 360.0, 1100.0 μg/mL further dilution with stock solution.

Standard solution of IS with methanol: 100.0 μg/mL. All solution kept: -20oC. Prepare standard calibration sample: 10 μL solution

evaporated by a gentle steam of nitrogen.

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Cont…

Drug free rat plasma solution added: 100 μL The final standard plasma and tissues concentrations were

0.01, 0.05, 0.5, 1.5, 4.0, 12.0, 36.0, and110.0 μg/mL. QC samples containing AICD in plasma had concentrations

of 0.5, 5.0, 50.0 and 100.0 μg/mL (very low, low, middle and high, respectively).

All of these solutions were prepared fresh before use

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Cont…5. Sample pretreatment liquid-liquid extraction (LLE) method was used to extract the

AICD from biological samples( plasma, tissue homogenates). Samples were kept at room temperature. 100 μL plasma sample was spiked with 10 μL IS standard

solution (100 μg/mL). 400 μL of dichloromethane was added and mixed by vortex for

1min. sample was centrifuged at 10,000 rpm for 10 min . The residue was reconstituted with 100 μL of the mobile phase,

and 20 μL was injected to the HPLC system.

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Cont…

6. Chromatographic conditions The mobile phase consisted of methanol with 0.02%

aqueous ammonia (pH7.3,70:30, v/v) with a flow rate of 1mL/min.

Isocratic elution was used for all processes. Chromatograms were monitored at 270 nm and the

temperature of column was kept at 30oC.

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Cont…

7. Method validation All methods were validated the guidelines of the US Food

and Drug Administration (FDA) Validation parameters consist: selectivity, linearity,

accuracy, precision, recovery and stability.

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Cont…8. Pharmacokinetical assay of AICD Wistar rats (n=12,male) were randomly divided into two

equal groups. One group was administrated AICD (100mg/kg, i.v.)

through the tail vein. Other group was administered oral AICD (200 mg/kg). Dose AICD was prepared in 0.9% sterile saline to contain

20mg/mL or 40mg/mL of AICD for i.v. or oral administration.

All rats were fasted 12 h before administration.

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Cont…

Rats were bled from the retro-orbital sinus by capillary tubes at the following time points:

IV administration:0 (pre-dose), 0.033, 0.083, 0.25, 0.5, 0.83, 1.33, 2, 3, 4, 6 and 8h.

Oral administration:time extent to 10 and 12 h. Each blood sample (300 μL) was collected in heparinized

tube sand centrifuged at 4000 rpm for 15 min to obtain the plasma.

Plasma was frozen at -20oC before the assay.

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Cont…9. Tissue distributed assay of AICD Rats (n=24) were randomly divided into four equal groups, and

each rat was administrated AICD (100mg/kg) through the tail vein.

Four groups of rats were euthanized 10 min, 1 h, 3 h or 6 h after administration.

Dissected organs and tissues included the heart, liver, brain, lungs and kidneys from each rat. Cleaned with physiological saline, which was absorbed by filter paper.

Tissues : weighed and homogenized in normal saline solution (250mg/mL).

Homogenates tissue stored:- 20oC until analysis.

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Cont…

10. Calculations All data was calculated using Microsoft Excel 2007

software. The concentration-time curves and pharmacokinetic

parameters of AICD were obtained through the DAS 2.0 software.

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Results

1. Optimization of HPLC conditions Methanol with 0.02% aqueous ammonia (pH 7.3, 70:30, v/v)

was used as mobile phase and the analytic column was a SinoChrom ODS-BP C18 column.

Chromatograms were monitored at 270 nm. Isocratic elution was used for all processes. Reverse phase(RP)–HPLC is the main tool used for the analysis

of alkaloids. pH of mobile phase: important factor, RP-HPLC for alkaloid

compounds, because the retention behavior of alkaloids is affected by the pH of the mobile phase.

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Cont…

2. Optimization of the sample preparation Protein precipitation and LLE were used for the

pretreatment of biological samples. Solvents: dichloromethane, ethyl ether, ethyl acetate, and

dichloromethane. dichloromethane as the best extraction solvent: few

impurities in the chromatographic peaks or interference peaks.

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Cont…

3. Validation of the HPLC assay Selectivity of the method was evaluated by comparing the

blank plasma and spiked plasma. Samples kept at room temperature for 4 h Thus, sample storage at -20oC was feasible and suitable for

further pharmacokinetic and tissue distribution.

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Cont…

Figure 2 Representative chromatograms for the pharmacokinetic investigation of AICD: (a) blank plasma sample; (b) standard solution in blank plasma including AICD and IS (ICD); (c) real plasma sample processed 50 min after oral administration of AICD (200mg/kg); (d) real plasma sample processed 50 min after i.v. administration of AICD (100 mg/kg).Mobile phase: methanol with 0.02% aqueous ammonia adjusted top H7.3 (70:30, v/v) with a flow rate of 1mL/min; column: SinoChrom ODS-BP C18 (250mm4.6 mm,i.d.5 μm); detection wavelength: 270nm; retention time: AICD at 4.0min and ICD at 6.8 min.

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Cont…4. Pharmacokinetics The pharmacokinetic profiles were determined from a rat plasma

concentration-time course after oral or i.v. administration of AICD.

Figure 3 Plasma concentration-time profiles of AICD in rats by oral administration (200mg/kg) and i.v. administration (100mg/kg).

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Cont…5. Tissue distribution Tissue distribution was performed at 10min, 1h, 3h, and 6h after i.v.

administration at a single dosage at 100mg/kg. The lungs, kidneys and liver homogenate had high concentration of

AICD at the four time points, suggesting that AICD easily enters these organs but not in BBB.

Figure :4 Statistical results of the tissue distribution of AICD in rats after i.v. administration (100mg/kg).

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Discussion

Modifications at the C-8 position in the D ring of Isocorydine were the focus of our screening as antitumor agents.

Hydrogen atom at C-8 possesses high chemical reactivity and is easily lost.

So 8-amino-isocorydine has been obtained which was proved to have a good antitumor effect in vitro but was not stable in aqueous solution.

So, under the pro-drug theory, AICD was synthesized through acetylating 8-amino-isocorydine with acetyl chlorine

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Cont…

The result of the pharmacokinetics and tissue distribution investigation about the AICD supported that t1/2 of AICD in rats after i.v. and oral administration were 2.2 h and 2.0 h, respectively.

Oral administration of AICD could fast elimination than i.v. because AICD remained in rats for a relatively long time.

AICD did not pass through the BBB, unlike its parent compound ICD mainly because introducing an acetamino in the D-ring C-8 site of ICD enhanced its polarity and hydrophilicity.

Side effect may be reducing by AICD at the time of tissue distribution.

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Conclusion

A simple and effective HPLC-DAD method coupled with an LLE method was developed for determining the metabolic behavior and tissue distribution of AICD.

AICD is a modifying structure of ICD, which is selected as a candidate for a new drug development.

The tissue distribution was performed lung, kidney and liver homogenate had high concentration thus it may be effective in the treatment of lung, kidney and liver cancer.

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Reference

Chen Y, Yan Q, Zhong M, Liu J, Zhao Q, Di D and Liu J (2015) Study on pharmacokinetics and tissue distribution of the isocorydine derivative in rats by HPLC-DAD method, Acta Pharmaceutica Sinica B, 5(3); 238-245.

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