CorrespondenceR. S. BhattaPharmacokinetics & Metabolism Division CSIR- Central Drug Research Institute CSIR Sector 10 Jankipurum Ext. Lucknow-226031 India Tel.: + 91/522/2772 974 Fax: + 91/522/2771 942 [email protected][email protected]
Key words●▶ Rohitukine●▶ Dysoxylum binectariferum●▶ pharmacokinetics●▶ HPLC-UV●▶ plasma protein binding
Pharmacokinetics, Tissue Distribution and Plasma Protein Binding Studies of Rohitukine: A Potent Anti-hyperlipidemic Agent
Authors Y. S. Chhonker1, 2, H. Chandasana1, 2, A. Kumar3, D. Kumar1, T. S. Laxman1, 2, S. K. Mishra4, V. M. Balaramnavar4, S. Srivastava4, A. K. Saxena4, R. S. Bhatta1, 2, 3
Affiliations Affiliation addresses are listed at the end of the article
Introduction▼Traditional Indian medicine has been used for thousands of years in the Indian subcontinent, involving a variety of herbs and plants collec-tively beneath an experiential formula. The Dys-oxylum richii and Dysoxylum binectariferum is an Indian plant, conventially used to treat rigid limbs, skin irritations, facial distortion in chil-dren, nervous system depression and inflamma-tory symptoms [1, 2]. Numerous chemical components have been reported in D. binectar-iferum, such as chromone alkaloids, triterpene and saponin [3].Rohitukine (RH, ●▶ Fig. 1a), the foremost chr-omone alkaloid isolated from the stem bark of D.binectariferum (Family-Meliaceae), has been identified as 5,7-dihydroxy-8-((3S,4R)-3-hy-droxy-1-methylpiperidin-4-yl)-2-methyl-4H-chromen-4-one [4–7]. RH has assorted pharmacological activities counting anti-cancer, anti-inflammatory, immune-modulatory, anti-leishmanial, anti-ulcer, CNS depressant, anti-fer-tility, anti-implantation [4, 8–13]. RH is an important precursor for the semi-synthetic derivatives flavopiridol and P-276-00. Currently,
flavopiridol and P-276-00, the first clinically evaluated cyclin dependant inhibitors are in phase IIb clinical trials [14–20].At present, RH is in preclinical development phase as novel antidyslipidemic agent. RH (50 mg/kg) significantly decreased the plasma total cholesterol (24 %) and accompanied with an increase in high density lipoprotein (21 %) [21]. RH also inhibits adipogenic differentiation in vitro as well as inhibit development of dys-lipi-demia in HFD fed hamsters in vivo [22]. Despite intensive research on pharmacodynamics of RH in previous decades, limited pharmacokinetics data is existing [23].The major hurdle of a new chemical entity that is sought to be developed as a drug candidate is attributed to their undesira-ble pharmacokinetic profile. In order to evaluate pharmacokinetics of the RH, it needs appropriate understanding of absorption, distribution, metabolism, and excretion (ADME) studies.Therefore, aim of our study was to investigate ADME properties of RH including solubility, pH dependent stability, pharmacokinetics, tissue distribution, and plasma protein binding using rapid HPLC-UV method for quantification. Further these finding in correlation to biological
Abstract▼Rohitukine (RH) is a chromone alkaloid consid-ered as one of the major active component of Dysoxylum binectariferum, exhibiting diverse pharmacological activities such as anti-hyperlipi-demic, anti-cancer, anti-inflammatory, immuno-modulatory, anti-leishmanial, anti ulcer and anti-fertility. There’s still a lack of information of RH, inclusive of pharmacokinetics, tissue distri-bution and excretion, in vivo studies in experi-mental animals, such as hamster and rats. In this study, a selective and sensitive bioanalytical method was developed and validated using
HPLC-UV system. The assay was applied to esti-mate pharmacokinetics, tissue distribution and excretion of RH in hamster at 50 mg/kg oral dose. It rapidly reached systemic circulation and distributed to various tissues, and highest con-centration was observed in liver. The pharma-cokinetic parameters such as clearance (CL/F) was 3.95 ± 0.9 L/h/kg, volume of distribution (Vd/F) was 17.34 ± 11.34 L/kg and elimination half-life was 2.62 ± 1.34 h. RH shows moderate protein binding ~ 60 % and found stable in gastro-intestinal fluid, a property that favors oral admin-istration.
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response will be useful for safe and efficacious therapeutic use of RH.
Experimental▼Materials and methodsPlant materialThe stem bark of D. binectariferum was collected from Sind-huburg, Maharashtra, India, and identified by the Botany Divi-sion of CSIR-Central Drug Research Institute, Lucknow, India. A voucher specimen (number 4032) has been kept in the herbar-ium of the Institute.
Extraction, fractionation and isolation of compoundsAir dried, ground stem bark (2.0 kg) was successively extracted ( ●▶ Fig. 2) with 95 % ethanol (4 × 5.0 lit.) by cold percolation and the ethanolic extract was filtered and concentrated under reduced pressure below 50 °C to yield a dark brown viscous mass (crude extract, 44 g). This ethanolic extract (35 g) was fraction-ated into 4 fractions, i. e., hexane soluble (F1, 2.0 g), chloroform soluble (F2, 12.8 g), n-butanol soluble (F3, 4.2 g) and n-butanol insoluble (F4, 15.8 g) fractions. The most active fraction F2 was repeatedly chromatographed over a silica gel (100–200 mesh)
column and successively eluted with hexane, chloroform, and chloroform–methanol mixtures (19:1, 9:1, 4:1 and 7:3, v:v), while chloroform and chloroform-methanol mixture 19:1 yielded a brown powdered compound as major constituent (1.2 g). It was crystallized in methanol and characterised as rohitukine [11, 12] by MS, 1H and 13C NMR spectra and compar-ing with the reported literature
ChemicalsRH (> 98 %) was extracted by previously described method at Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute (CDRI) India. Phenacetin, methanol (HPLC grade) and DSC-18 cartridges (Discovery Supelco Cat. no. 52602-U), activated charcoal, pepsin, pancreatin were purchased from Sigma (India) Pvt. Ltd. Glacial acetic acid AR, ammonium acetate, dimethyl sulphoxide (DMSO) AR, potassium phosphate mono basic and sodium hydroxide were purchased from E Merck Lim-ited (Mumbai, India). Sodium acetate was purchased from S.D. fine-chem Pvt. Ltd. (India). Heparin sodium injection. IP (1 000 IU/mL) was purchased from Biologicals E. Ltd. (Hyderabad, India).
AnimalsGolden Syrian hamsters (150 ± 20 g) were obtained from the Labo-ratory Animal Division of CSIR-CDRI (Lucknow, India). The animals were kept in an environmentally controlled breeding room (at 25 ± 1 °C, humidity of 55 ± 5 %, and a 12/12 h light/dark cycle) for at least one-week acclimatization before experiment. Hamsters were fasted for 12 h before experiment. All the procedures were carried out in strict accordance with the approved Institutional Animal Ethics Committee (IAEC approval no IAEC/2012/91 Nb).
Fig. 1 Chemical structures of a Rohitukine, b Phenacetin.
Fig. 2 Extraction procedure of RH from stem bark of Dysoxylum binectariferum.
3DrugRes/2014-06-0757/4.9.2014/MPS Original Article
Chhonker YS et al. Rohitukine Pharmacokinetics in Hamster … Drug Res 2014; 64: 1–8
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HPLC-UV conditionsThe Waters HPLC system, Milford USA consisted of a quaternary pump (600), auto sampler (717) and UV detector (2496). The resolution and better peak shape of analytes were achieved on a Phenomenex C18 column (4.6 × 250 mm, particle size 5 µm) pro-tected with a Phenomenex C18 guard column. A 50 µL aliquot of each sample was injected into the HPLC system. The system was analysed in isocratic mode with a mobile phase consisting of 10 mM sodium acetate buffer (pH 5.5): methanol (62: 38, v/v) at a flow rate of 1.0 mL/min. Mobile phase was filtered through 0.22 µm membrane filter (Millipore, USA) and degassed ultrasoni-cally for 15 min prior to use. The absorption wavelength was set to 257 nm and 240 nm for RH and internal standard (IS), respectively.
Preparation of standard stock solution and calibration curveIndividual stock solutions of 1 mg/mL concentration for RH and IS were prepared in DMSO and methanol, respectively. The working stocks of RH were prepared by step-wise dilution. The calibration standards were prepared by parallel dilution, briefly 290 μL of plasma or tissue homogenate spiked with 10 μL of appropriate RH standard solution. Phenacetin ( ●▶ Fig. 1b) was used as an IS at final concentration of 5 µg/mL. The dynamic range of the calibration curve in plasma was 0.032–8 µg/mL. The liver, kidney and lung tissues were homogenized in tris buffer (pH 7.4) at 1:2 w/v, spleen tissue at 1:5 w/v. The dynamic range of the calibration curve in tissues was 0.032–8 µg/mL. Plasma and tissues samples were then extracted as described in the “sample preparation” section. Quality control (QC) samples at four different concentrations (0.032, 0.125, 2 and 8.0 µg/mL were prepared separately in five replicates, independent of the calibration standards. Test samples and quality control samples were interpolated from the calibration curve to obtain the con-centrations of the analyte.
Plasma and tissue samples preparationThe plasma and tissue samples were extracted by solid phase extraction (SPE) method. A simple SPE method was followed for extraction of RH and IS from plasma and tissue homogenate. SPE was carried out using 1 cc, C-18 (DSC-18, Supelco) cartridge. Cartridges were conditioned with 2 mL methanol followed by 2 mL sodium acetate buffer (10 mM, pH 5.5) (SAB). Plasma sam-ples (300 µL) were diluted to 700 µL with SAB and loaded into the cartridges. Then cartridges were washed with 2.0 mL of SAB and analytes were eluted with 2 mL of methanol. The eluents were collected in glass tubes and evaporated to dryness under nitro-gen in water bath set at 40 °C. The dry residues were finally reconstituted in 100 µL methanol and supernatant was injected to HPLC for analysis.
Assay validationThe complete HPLC method validation of RH in plasma and tis-sue homogenate was determined as per the FDA guidelines. The method was validated for specificity, selectivity, linearity, preci-sion, accuracy, recovery and stability [24].Specificity and selectivity of the method was determined by analyzing the chromatogram of blank hamster plasma or tissue homogenate with that of RH and IS spiked plasma/tissue homogenate sample. The standard curves were prepared by analysis of calibration standard bio-samples and plot of peak area ratios of RH and IS v/s corresponding RH concentration. Intra- and inter-day accuracy and precision were determined by
analyzing QC samples at lower limit of quantitation (LLOQ), low, medium and high concentration. The extraction recoveries of RH at three QC levels were determined by comparing the peak area of RH in pre-treated bio-samples with those obtained by directly determining identical RH standard solutions. The LLOQ was defined as the lowest RH determinable concentration with mean value deviation and coefficient of variation < 20 % using five bio-samples. The stability of RH in bio-samples were evalu-ated at − 80 °C for 30 days and after 3 freeze-thaw ( − 80 °C/room temperature) cycles using QC samples at two concentration lev-els (0.125 and 8 µg/mL). In addition, the autosampler stability of the reconstituted solution was studied by placing QC samples at two concentrations at room temperature for 24 h.
Application of the methodSolubility of RHAqueous solubility of any drug candidate in drug discovery is crucial parameter which influences pharmacokinetic parame-ters in terms of oral absorption and distribution of drug. Solubil-ity assay in drug discovery research is often kinetic solubility based on detection of the concentration at which compound form precipitate in aqueous solution. Kinetic solubility was per-formed by spiking RH at a concentration 1, 5, 10, 20, 40, 50, 80 µg/mL in 50 mM tris buffer (pH 7.4). Spiked microfuge tubes were kept at room temperature for 2 h and after incubation each microfuge tube was centrifuged at 13 000 rpm for 15 min. The supernatant solution containing dissolved RH was analyzed. Concentration at which RH precipitate out was considered as upper level of soluble concentration.
Stability studies of RH in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF)SGF and SIF were prepared as described in the 26th United Stated Pharmacopeia. Briefly, SGF (0.2 g NaCl, 3.2 g pepsin, 7 mL HCl and H2O upto 1 000 mL, pH 1.2) and SIF (6.805 g KH2 PO4, 0.896 g NaOH, 10.0 g pancreatin and H2O upto 1 000 mL, pH 6.86). RH (10 µg/mL) was added to SGF and SIF and incubated at 37 °C on a shaking water bath. Samples (200 µL) were collected at 0, 15, 30, 60, 120 min and two volumes of methanol containing IS was added immediately after sample collection to stop reaction. All these samples were analyzed on HPLC to estimate the percent-age loss with respect to 0 min time point. The percentage of par-ent compound remaining at each time point relative to the 0 min sample is calculated from peak area ratios.
Plasma protein binding studyThe determination of the unbound fraction (fu) of a drug in plasma was performed by modified charcoal adsorption method [25, 26]. The modified charcoal adsorption assay operated under non-equilibrium conditions and involves measuring the time course of decline of the concentration of bound drug when the free drug is being removed by charcoal adsorption. This method avoids the problem of nonspecific adsorption and liquid parti-tioning which are often encountered in the traditional methods. This method is relatively fast, economic and easy to perform. The charcoal suspension was incubated at room temperature for 30 min on a shaking water bath. The study was carried out in triplicate at 10 µg/mL concentration of RH. The spiked plasma was allowed to equilibrate for 15 min prior to study. Charcoal suspension (5.0 mL) was transferred into a 30 mL glass tube, cen-trifuged at 3 000 g for 15 min at 25 °C, and the supernatant was carefully decanted off. Then spiked plasma (5.0 mL) was added
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onto the charcoal pellet under continuous stirring at 37 ± 1 °C. Samples (350 µl) were withdrawn at 0, 3, 5, 10, 15, 30, 45, 60, 90 and 120 min and then centrifuged (to separate plasma) at 12 000 rpm for 2 min and immediately transferred supernatant plasma into 2.0 mL microcentrifuge tubes. Plasma was stored at − 80 °C till analysis. The plasma samples were then processed by SPE method described in plasma sample preparation section.Percent RH remaining in the supernatant hamster plasma v/s time data was fitted to a two-compartment model, intravenous bolus non-linear regression program on Phoenix WinNonlin 6.3. The model is described by the following biexponentially equation:
Bt = A1 e − αt + A2 e − βt
Where B(t) is % bound at time t, A1 and A2 are Y intercepts, α and β are distribution and disposition rate constants for the 2 phases, respectively.
In vivo pharmacokinetic studyThe in vivo pharmacokinetics study was performed in male hamster to demonstrate the applicability of developed and vali-dated bioanalytical method. Hamsters were divided in three groups each containing six animals and sparse sampling tech-nique was carried out during blood. The RH was administered per oral (PO) at 50 mg/kg via oral gavage collection, as 0.5 % w/v carboxy methyl cellulose suspension. Blood samples were col-lected by sparse sampling from the retro orbital pluxes into micro-centrifuge tubes containing heparin (20 IU/mL) as anti-coagulant at 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 6.0, 7.0, 8.0, 10.0, 12.0, 18.0, 24.0 30.0 and 48.0 h post dosing. Plasma was har-vested by centrifuging the blood samples at 2 000 × g for 5 min and stored at − 70 ± 10 °C till analysis.
Tissue distribution studyHamsters were divided in three groups each containing five ani-mals and sparse sampling technique was carried out during tis-sue distribution study. The hamsters were sacrificed by decapitation at 0.5, 1, 2, 4 and 6 h post-dosing. The organs such as liver, spleen, lung, and kidney were excised, trimmed of extra-neous fat, residual muscle, connective tissue and rinsed with normal saline, then blotted dry and immediately froze at − 20 °C, prior to sample processing for RP-HPLC analysis. Each tissue sample was minced (sample weight was 0.5 g, except for spleen: 0.25–0.3 g) and individually homogenized with tris buffer in 1:2 ratio by a tissue homogenizer (Tissues Tearor, Biospec product,
Inc. Bartlesville, USA). The homogenized tissue samples were processed as described above and a 50 µL aliquot of supernatant was injected into the RP-HPLC system. As described above, spe-cific calibration curve was separately prepared for each tissue sample obtained from drug free hamster and partially validated. The concentration of RH in each sample was expressed in terms of µg/g tissue and calculated by equation: Ct = CsVs /P, where Ct represents the tissue concentration (µg/g), Cs is the supernatant concentration, Vs is the supernatant volume and P is the weight of the sample [27].
Fig. 3 Percentage remaining of RH after incubating with SGF and SIF at 37 °C, (Mean ± S.D. n = 3). (Color figure available online only).
Fig. 4 Representative HPLC-PDA overlay chromatograms of a AS blank and AS 1 µg/mL compound RH spiked in acetonitrile, b CS blank hamster plasma and CS 1 µg/mL compound RH spiked in hamster plasma and c RH spiked in hamster plasma at LLOQ level.
5DrugRes/2014-06-0757/4.9.2014/MPS Original Article
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Pharmacokinetic data analysisThe pharmacokinetic parameters were determined by using extra-vascular noncompartmental analysis module of Phoenix WinNonlin (version 6.3, Pharsight, MountainView, CA). The elimination t1/2 was obtained from the formula of 0.693/K. The area under the curve (AUC0–∞) was estimated with linear trape-zoidal method from 0–tlast and extrapolation from tlast to infinity based on the observed concentration at the last time point divided by the terminal elimination rate constant (K). For oral administration, clearance (CL/F) and the apparent volume of dis-tribution of the elimination phase (Vd/F) were calculated as Dose/AUC0–∞ and Dose/K.AUC0–∞, respectively. The mean resi-dence time (MRT) was calculated as AUMC0–∞/AUC0–∞.
Results and Discussion▼Method developmentTo optimize chromatographic conditions, different analytical columns, mobile phase compositions and injection solvents were evaluated. A number of columns (Cyano, C-8 and C-18) were evaluated and Phenomenex C18 column (4.6 × 250 mm, par-ticle size 5 µm) resulted in better chromatographic resolution and selectivity. The selection of mobile phase components were critical factor to achieve fine chromatographic peak shapes and sensitivity. Water, ammonium acetate, and sodium acetate were investigated for the optimal chromatographic condition. Change of mobile phase pH impacted the retention time and peak shape. Acetonitrile and methanol as organic solvent in the mobile phase were also compared. Methanol was found to be a better solvent than acetonitrile. The results showed that adding 0.1 % formic acid in the aqueous portion could sufficiently construct the chromatographic peaks sharp and symmetric, in order to attain higher sensitivity for RH and IS. It is necessary to screen a suitable IS to track the analyte in the in vivo quantitative study. Structure analog is usually selected as IS. Due to unavailability of structure analogue of RH, phenacetin was used as IS. It was found that phenacetin had similar chromatographic behavior, extraction and recovery with RH. The mobile phase consisting of methanol: 10 mM sodium acetate buffer pH 5.5 (38:62, v/v) with flow rate of 1.0 mL/min was found suitable during chromato-graphic optimization. Under these optimum conditions, RH and IS were free of interference from endogenous substances. The observed retention times were ~7.8 min and ~14.3 min for RH and IS, respectively.Plasma and tissue homogenates are a complex mixture of pro-teins, glycoproteins, lipids and salts which can interfere with HPLC analysis. We tried protein precipitation, liquid-liquid extraction and solid phase extraction methods and the sensitiv-ity for first two methods was not high enough for the pharma-cokinetic studies. Finally, the solid phase extraction was found best suited method. During this process, samples were not diluted but washed so as to get cleaner sample. Initial addition of 700 µL aqueous 10 mM SAB solution into plasma had resulted in two advantages; (i) it increased the volume and dilution of sam-ples, became advantageous in handling and reduce the analyte loss during transfer to SPE cartridges; (ii) alteration of pH resulted in strong interaction with SPE cartridges and better cleaning of water soluble components. Finally, we concentrated the extract to fall in the detectable range as required for phar-macokinetic applications.
SpecificityandselectivityThe RP-HPLC chromatograms of blank plasma and RH spiked blank plasma are shown in ●▶ Fig. 4. The relevant chromatograms of hamster plasma and tissues were also obtained under the same HPLC conditions (figures not shown). The retention time (Tr) of RH in plasma was 7.8 min. The retention time of the RH and IS showed less variability with a relative standard deviation (R.S.D.) well within the acceptable limit of ± 0.5 %. There were no interfering peaks within the elution times for either reference standard and tested samples.
Linearity and sensitivityThe linear regressions of RH in hamster plasma and tissues were obtained between the peak area ratio and concentration of standard solutions. The linearity of calibration curves were demonstrated by correlation coefficients (r2) values obtained for the regression line. The calibration curves were obtained over the concentration range of 0.032–8 µg/mL for RH in hamster plasma and different tissues showed r2 = 0.997 ( ●▶ Table 1). The LLOQ was found 32 ng/mL with R.S.D. < 20 % and LOD was 10 ng/mL.
Accuracy and precisionThe assay performance data for the determination of independ-ent QC samples of RH in plasma and tissues are presented in ●▶ Table 2. The deviation from nominal concentration (accu-racy) ranged from 8.7 to − 2.37 % intra and inter-day. The preci-sion around the mean value was never greater than ± 15 % at any of the concentrations studied, indicating an acceptable precision and accuracy of the present method.
RecoveryThe extraction recovery of RH from hamster plasma and tissue homogenate was obtained from five replicate analyses of the QC samples at high, medium and low concentrations. The mean extraction recoveries of RH ranging from 85.70 to 94.45 %, in hamster plasma and tissue homogenate, indicated satisfactory reliability and validity of the present method. In addition, the extraction recovery of of the IS was 69.23 ± 4.7 %.
StabilityThe stability of RH in hamster plasma and tissues was assayed according to the procedures described above. The data for rat plasma were summarized in ●▶ Table 3 and indicated that results obtained were well within the acceptable limits. Freeze-thaw stability after three cycles was verified and no significant differ-ences were observed. RH spiked plasma samples were stable for at least 24 h at room temperature. The results of long-term sta-bility study indicated that the spiked samples were stable at − 80 °C over 15 days.
Table 1 Standard curves and correlation coefficients of RH in plasma and tissue matrices.
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Application of the methodSolubility of RHSolubility of RH was found up to 80 µg/mL in aqueous tris buffer (pH 7.4). The rationale of using pH 7.4 buffer was to ensure opti-mum solubility of drug at physiological condition and the results will be useful for conducting in-vitro assays where solubility of the compound is of major concern.
SGF and SIF stability of RHThe stability data for RH in SGF and SIF is shown in ●▶ Fig. 3. The RH found stable in SGF and SIF media containing pepsin and pancreatin as an enzymes. The stability of RH in the SIF/SGF favors oral administration.
Plasma protein binding studyThe percentage protein binding in male hamster plasma was found to be 59.97 ± 2.30 %. The percentage binding was estimated from the decline of percentage of the drug remaining in the supernatant after addition of charcoal. ●▶ Fig. 5 depicts the per-
centage of RH remaining (mean ± SD, n = 3) vs. time after the addition of hamster plasma containing 10 µg/mL of RH onto the charcoal pellet. It is fact that only free drug concentration in plasma (unbound drug) can reach the active site and exhibit pharmacological activity. Therefore, determination of the con-centration of unbound drug in the plasma is important for phar-macokinetic and pharmacodynamic studies.
Pharmacokinetics studyThe mean plasma concentration-time curves of RH in male hamster after oral administration was shown in ●▶ Fig. 6. The developed method was suitable for estimation of RH at terminal phase of plasma concentration profile upto 48 h. The sensitivity of the method was sufficient for accurate prediction of terminal slope with extrapolation less than 15 %. The concentration of RH in plasma achieved the peak less than 0.5 h, decreased sharply to the valley near 4 h, then slightly increased to the second peak near 8 h and then declined slowly to the detection limit around 48 h after oral administration. The main pharmacokinetic parameters of oral and intravenous administrations were sum-marized in ●▶ Table 4. The tmax (h) after oral dosing was 0.5 h, suggesting that RH could be quickly absorbed. The compound reached a maximum concentration (Cmax) 6.32 ± 0.32 µg/mL. The clearance (Cl/F) and volume of distribution (Vd/F) of RH were found to be 3.95 ± 0.9 L/h/kg and 17.34 ± 11.34 L/kg, respectively. There is no significant interspecies difference observed in ham-ster and rat pharmacokinetic. Our previous publication has reported rat pharmacokinetic data [23]. The pharmacokinetic data of RH reported here suggest that it could be detected up to 48 h which might be useful for the prolong duration of treat-ment.
Tissue distributionAll tissue samples were processed as described earlier and assayed. ●▶ Fig. 7 shows the concentration of RH in each tissue after PO administration to hamster. The detection of RH in tis-sues indicated the extensive distribution into the extravascular system of hamster. Significantly, highest levels were detected in liver and kidney (7.51 ± 0.25 µg/g and 3.09 ± 1.11 µg/g, respec-tively), followed by spleen (1.78 ± 0.29 µg/g) and lung (1.72 ± 0.35 µg/g) with less RH concentrations.Such widespread distribution of RH appeared to be a supporting evidence for extensively distributed into tissues and organs with abundant blood flow such as lungs, liver, kidneys and spleen. The differed disposition properties among tested tissues can be explained by the following facts: (i) the hepatic clearance and renal excretion (extra hepatic clearance) may be the major routes for RH elimination due to the significantly highest and
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Chhonker YS et al. Rohitukine Pharmacokinetics in Hamster … Drug Res 2014; 64: 1–8
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relatively high RH levels in liver and kidneys, respectively; (ii) dense blood-vessel network with high blood flow in the liver, lungs and kidney may have a relevance for the noteworthy dis-tribution in these organs.
ConclusionIn the evaluation of pharmacokinetic behavior in hamster, RH was found rapidly absorbed within 15 min after oral dose. This study is first to describes the detailed pharmacokinetic, tissue distribution and plasma protein binding of RH. The high RH dis-position observed in liver is representative of its usefulness in hyperlipidmia. Plasma protein binding is an imperative study of drug development for correlation of PK-PD data as well as guid-
ing for first in human (FIH) dose. Protein binding data reveals that there was moderate binding [~59 % ] in hamster plasma. The pH dependent stability studies of RH could guide for further in vitro pharmacokinetic characterization and it might be the probable reasons for favorable fast absorption. The developed and validated HPLC-UV method can be utilized for preclinical studies of RH such as dose escalation, metabolite identification and transporters involved in the absorption. This method can also be useful for future clinical investigations as RH has shown potential to be used in human after complete preclinical safety and efficacy studies. RH was observed upto 48 h in plasma, which indicated prolong efficacy following single dose adminis-tration. The results reported in this study will positively provide a basis for its selection to be developed as an oral antihyperlipi-demic agent and also offer important clues for further structural modification.
Acknowledgements▼We are also thankful to THUNDER and HOPE project for partial funding. Authors Y.S.C. and V.M.B.are also thankful to the Indian Council of Medical Research (ICMR) and H.C. for CSIR for finan-cial support. CSIR-CDRI communication no. 8773.
Conflict of Interest▼There is no potential conflict of interest.
Affiliations1 Pharmacokinetics & Metabolism Div., CSIR- Central Drug Research Institute,
Lucknow, India2 Academy of Scientific and Innovative Research (AcSIR), New Delhi, India3 Department of Pharmaceutics, National Institute of Pharmaceutical
Education and Research (NIPER), Raebareli, India4 Medicinal & Process Chemistry Division, CSIR- Central Drug Research
Institute, Lucknow, India
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