LNN 6th sem

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.
Pokhara University, Lekhanath-12, Kaski Nepal
12/10/2016 Pharmaceutical Seminar 5 1

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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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Cont…
 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.
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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Cont…
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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