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Article , Tissue Distribution, Plasma Binding Studies of 10-Dehydroxyl-12-Demethoxy-Conophylline, a Novel Anti-Tumor Candidate, in Rats

Chengjun Jiang 1,†, Jie Li 1,2,†, Xianghai Cai 3, Nini Li 1, Yan Guo 1 and Dianlei Wang 1,*

1 School of , Anhui University of Chinese , Hefei 230012, China; [email protected] (C.J.); [email protected] (J.L.); [email protected] (N.L.); [email protected] (Y.G.) 2 School of Environmental and Chemical Engineering, Zhaoqing University, Zhaoqing 526061, China 3 State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China; [email protected] * Correspondence: [email protected]; Tel.: +86-0551-68129153; Fax: +86-0551-68129153 † These authors contributed equally to this work.

 Received: 10 December 2018; Accepted: 10 January 2019; Published: 14 January 2019 

Abstract: 10-Dehydroxyl-12-demethoxy-conophylline is a natural anticancer candidate. The motivation of this study was to explore the pharmacokinetic profiles, tissue distribution, and plasma protein binding of 10-dehydroxyl-12-demethoxy-conophylline in Sprague Dawley rats. A rapid, sensitive, and specific ultra-performance liquid chromatography (UPLC) system with a fluorescence (FLR) detection method was developed for the determination of 10-dehydroxyl-12-demethoxy-conophylline in different rat biological samples. After intravenous (i.v.) dosing of 10-dehydroxyl-12-demethoxy-conophylline at different levels (4, 8, and 12 mg/kg), the half-life t1/2α of intravenous administration was about 7 min and the t1/2β was about 68 min. The AUC0→∞ increased in a dose-proportional manner from 68.478 µg/L·min for 4 mg/kg to 305.616 mg/L·min for 12 mg/kg. After intragastrical (i.g.) dosing of 20 mg/kg, plasma levels of 10-dehydroxyl-12-demethoxy-conophylline peaked at about 90 min. 10-dehydroxyl-12-demethoxy-conophyllinea absolute oral was only 15.79%. The pharmacokinetics process of the was fit to a two-room model. Following a single i.v. dose (8 mg/kg), 10-dehydroxyl-12-demethoxy-conophylline was detected in all examined tissues with the highest in kidney, liver, and lung. Equilibrium dialysis was used to evaluate plasma protein binding of 10-dehydroxyl-12-demethoxy-conophylline at three concentrations (1.00, 2.50, and 5.00 µg/mL). Results indicated a very high protein binding degree (over 80%), reducing substantially the free fraction of the compound.

Keywords: 10-dehydroxyl-12-demethoxy-conophylline; pharmacokinetics; tissue distribution; protein binding rate

1. Introduction 10-Dehydroxyl-12-demethoxy-conophylline (Figure1) isolated from Melodinus tenuicaudatus is an indole alkaloid consisting of two pentacyclic aspidosperma skeletons. Aspidosperma alkaloids, the largest alkaloid in the monoterpenoid indole alkaloid (MIA) family, can be extracted from a variety of plants, with approximately 250 family members [1]. Monoterpenoid indole alkaloids (MIAs) provide a rich source of medicinal products. Examples include the anticancer compounds vinblastine and from Catharanthus roseus (Madagascar periwinkle) and camptothecin from Camptotheca acuminata [2]. Monoterpenoid indole alkaloids were brought to the attention

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Molecules 2018, 23, x FOR PEER REVIEW 2 of 10 of many researchers due to the MIAs’ complicated structures and potent biological activities [3,4]. AspidospermaAspidosperma alkaloid alkaloid bases bases are anti-parasitic, are anti‐parasitic, including including Plasmodium, Plasmodium, Leishmania, andLeishmania, Trypanosoma, and andTrypanosoma, have potential and cytotoxicityhave potential that cytotoxicity can be used that to can develop be used anti-tumor to develop effects anti‐ [tumor5]. Researchers effects [5]. CaiResearchers et al. [6, 7Cai] found et al. strong[6,7] found anti-tumor strong cytotoxicanti‐tumor components cytotoxic components in the study in the of indole study alkaloidof indole anti-tumoralkaloid anti activity‐tumor molecules. activity molecules. Based on Based the structure–activity on the structure– relationshipactivity relationship analysis, analysis, it was found it was thatfound aspidospermine that aspidospermine and 10-dehydroxyl-12-demethoxy-conophylline and 10‐dehydroxyl‐12‐demethoxy‐conophylline represent represent one of one the of basic the skeletonsbasic skeletons of anti-tumor of anti‐tumor activity activity in active in active monoterpene monoterpene alkaloids alkaloids [8]. [8]. At At present, present, the the research research on on 10-dehydroxyl-12-demethoxyconophylline10‐dehydroxyl‐12‐demethoxyconophylline focusesfocuses onon chemical chemical composition composition and and pharmacological pharmacological activityactivity [ 6[6,7,9].,7,9]. It It has has significant significant inhibitory inhibitory activity activity on on five five human human cancer cancer cell cell lines, lines, namely, namely, the the HL-60, HL‐ SMMC-7721,60, SMMC‐7721, A-549, A‐549, MCF-7, MCF and‐7, and SW480 SW480 tumor tumor cell cell lines lines [10 [10].]. Further Further mechanism mechanism studies studies showed showed thatthat the the active active ingredient ingredient of of 10-dehydroxyl-12-demethoxy-conophylline 10‐dehydroxyl‐12‐demethoxy‐conophylline exerts exerts its its anti-tumor anti‐tumor activity activity through the inhibition of the Wnt signaling pathway (IC50 = 0.21, 0.45 μm) [11]. This is completely through the inhibition of the Wnt signaling pathway (IC50 = 0.21, 0.45 µm) [11]. This is completely differentdifferent from from the the structure structure and and mechanism mechanism of of the the tubulin tubulin inhibitor inhibitor vinblastine vinblastine components components [ 12[12,13],13] suggestingsuggesting better better selectivity selectivity and and less less toxic toxic effects effects on on tumor tumor cells. cells. Among Among the the candidate candidate compounds compounds of theof newthe drug,new drug, and based and on based pharmacodynamic on pharmacodynamic and and characteristics, toxicity characteristics, it is of great significanceit is of great to thoroughlysignificance study to thoroughly the pharmacokinetic study the propertiespharmacokinetic of 10-dehydroxyl-12-demethoxy-conophylline. properties of 10‐dehydroxyl‐12‐demethoxy‐ conophylline.

Figure 1. Chemical structure of 10-dehydroxyl-12-demethoxy-conophylline. Figure 1. Chemical structure of 10‐dehydroxyl‐12‐demethoxy‐conophylline. According to previous pharmacodynamic and toxicity results [14], the candidate compound has the aboveAccording two characteristics, to previous and pharmacodynamic is suitable as a candidate and toxicity compound results for [14], further the pharmacokinetic candidate compound study, has in orderthe above to conduct two characteristics, pharmacokinetic and screening is suitable and as evaluation. a candidate To compound our knowledge, for further no ultra-performance pharmacokinetic liquidstudy, chromatography–fluorescence in order to conduct pharmacokinetic (UPLC-FLR) screening method and for evaluation. the quantification To our knowledge, of pharmacokinetic no ultra‐ profilesperformance and distribution liquid chromatography characteristics– offluorescence 10-dehydroxyl-12-demethoxy-conophylline (UPLC‐FLR) method for the quantification in rats has been of performed.pharmacokinetic Therefore, profiles in this and study, distribution the plasma characteristics pharmacokinetic of parameters,10‐dehydroxyl tissue‐12 distribution,‐demethoxy‐ andconophylline protein binding in rats rate has in rat been and performed. human plasmas Therefore, and in rats in werethis study, studied the using plasma the UPLC-FLR pharmacokinetic method. parameters, tissue distribution, and protein binding rate in rat and human plasmas and in rats were 2.studied Results using and Discussionthe UPLC‐FLR method.

2.1.2. Results Validation and of Discussion Analytical Method The method was validated in terms of selectivity, linearity, precision, accuracy, recovery, and2.1. stabilityValidation according of Analytical to the European Method Agency’s guidelines [15]. The calibration curve of 10-dehydroxyl-12-demethoxy-conophyllineThe method was validated in terms of in selectivity, rat blood sampleslinearity, was precision, linear, ranging accuracy, from recovery, 39 to 6000 and ng/mLstability (39, according 78, 156, to 310, the 1250, European 2500, 5000,Medicines 6000 ng/mL)Agency’s with guidelines a lower [15]. limit The of quantificationcalibration curve (LLOQ) of 10‐ ofdehydroxyl 39 ng/mL.‐12 R2‐demethoxyfor all standard‐conophylline curves was in 0.9973.rat blood The samples relative was standard linear, deviation ranging of from precision 39 to was6000 4.51%,ng/mL 8.29%, (39, 78, 2.55%, 156, 310, and 1250, 1.46% 2500, at 39, 5000, 156, 1250,6000 andng/mL) 4000 with ng/mL, a lower respectively. limit of quantification Accuracies determined (LLOQ) of for39 intra-dayng/mL. R and2 for inter-day all standard were curves all within was 100% 0.9973.± 15%The ofrelative the actual standard values. deviation Moreover, of noprecision significant was matrix4.51%, effect8.29%, was 2.55%, observed and 1.46% after at dilution. 39, 156, 1250, Thus, and this 4000 optimized ng/mL, respectively. UPLC method Accuracies was proven determined to be for intra‐day and inter‐day were all within 100% ± 15% of the actual values. Moreover, no significant matrix effect was observed after dilution. Thus, this optimized UPLC method was proven to be sensitive, selective, and rapid for the determination of 10‐dehydroxyl‐12‐demethoxyconophylline in rat plasma.

Molecules 2019, 24, 283 3 of 10 sensitive, selective, and rapid for the determination of 10-dehydroxyl-12-demethoxyconophylline in Moleculesrat plasma. 2018, 23, x FOR PEER REVIEW 3 of 10 The calibration curve of 10-dehydroxyl-12-demethoxy-conophylline were prepared by spiking the standardThe calibration working curve stock of solutions 10‐dehydroxyl with 10‐12µL‐demethoxy of the different‐conophylline concentration, were prepared and 120µ Lby blank spiking rat thetissue standard homogenate working sample stock in solutions a 1.5 mL with centrifuge 10μL of tube. the different The calibration concentration, standards and were 120μ preparedL blank rat at tissueconcentrations homogenate of 0.292, sample 0.586, in 1.172,a 1.5 mL 2.343, centrifuge 4.688, 9.375, tube. and The 18.751 calibrationµg/mL standards for the tissue were sample. prepared With at concentrationsthe concentration of 0.292, (C) as 0.586, the abscissa, 1.172, 2.343, the peak 4.688, area 9.375, of the and analyte 18.751 μ wasg/mL used for as the the tissue ordinate sample. for linear With theregression. concentration The lower (C) as limit the abscissa, of quantitation the peak was area determined of the analyte according was used to as the the signal-to-noise ordinate for linear ratio regression.(S/N) of the The chromatogram, lower limit of and quantitation the lower limit was of determined detection was according determined to the according signal‐to to‐noise the (S/N) ratio (S/N)of 3. We of the used chromatogram, the weighted and least the squares lower method limit of todetection perform was the determined regression operation according to to obtain the (S/N) the oflinear 3. We regression used the equation, weighted which least issquares the standard method curve. to perform The calibration the regression curves, operation correlation to coefficients, obtain the linearand linear regression ranges ofequation, 10-dehydroxyl-12-demethoxy-conophylline which is the standard curve. The in plasmacalibration and curves, tissue are correlation shown in coefficients,Table1. and linear ranges of 10‐dehydroxyl‐12‐demethoxy‐conophylline in plasma and tissue are shown in Table 1. Table 1. The calibration curves, correlation coefficients, and linear ranges of 10-dehydroxyl-12- Tabledemethoxy-conophylline 1. The calibration incurves, plasma correlation and tissue coefficients, are shown in and the table.linear ranges of 10‐dehydroxyl‐12‐ demethoxy‐conophylline in plasma and tissue are shown in the table. Sample Calibration Curve R2 Linear Range (µg/mL) Sample Calibration Curve R2 Linear Range (µg/mL) Plasma y = 1.3661 + 0.008 R2 = 0.9973 0.039–6.000 2 PlasmaHeart y = 8577.8xy = 1.3661− 1701.100 + 0.008 R2 = 0.9977R = 0.9973 0.29–18.7000.039–6.000 2 HeartLiver y =y 7997.6x = 8577.8x− 3192.600 − 1701.100 R2 = 0.9956R = 0.9977 0.292–9.3750.29–18.700 LiverSpleen y =y 8203.4x = 7997.6x− − 164.7903192.600 R2 = 0.9992R2 = 0.9956 0.292–18.7800.292–9.375 SpleenLung y =y 7602.5x = 8203.4x + 911.940 − 164.790 R2 = 0.9910R2 = 0.9992 0.292–9.3750.292–18.780 LungKidney y =y 8413.3x = 7602.5x− 194.040 + 911.940 R2 = 0.9970R2 = 0.9910 0.292–18.7000.292–9.375 KidneyBrain y = 9592.8xy = 8413.3x− 2367.300 − 194.040 R2 = 0.9943R2 = 0.9970 0.292–9.3750.292–18.700 Brain y = 9592.8x − 2367.300 R2 = 0.9943 0.292–9.375 2.2. Pharmacokinetics Study 2.2. Pharmacokinetics Study The plasma concentration–time data were fitted using the DAS 2.0 program. The concentration– timeThe curves plasma of 10-dehydroxyl-12-demethoxy-conophylline concentration–time data were fitted using the following DAS 2.0 intragastrical program. The and concentration intravenous– timeadministration curves of are10‐dehydroxyl shown in Figures‐12‐demethoxy2 and3 and‐conophylline Table2. following intragastrical and intravenous administration are shown in Figures 2 and 3 and Table 2.

Figure 2. Mean plasma concentration–time (mean ± SD) profiles of 10-dehydroxyl-12-demethoxyconophylline Figure 2. Mean plasma concentration–time (mean ± SD) profiles of 10‐dehydroxyl‐12‐ in rats after receiving a single intravenous (i.v.) dose at different concentration levels (4, 8, and 12 mg/kg, demethoxyconophylline in rats after receiving a single intravenous (i.v.) dose at different n = 6). concentration levels (4, 8, and 12 mg/kg, n = 6).

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Figure 3. Mean plasma concentration–time (mean ± SD) profiles of 10‐dehydroxyl‐12‐ demethoxyconophylline following an intragastrical (i.g.) dose of 20 mg/kg in rats (n = 6).

FigureTable 3. 2.Mean Profiles plasma of 10 concentration–time‐dehydroxyl‐12‐demethoxy (mean ± SD)‐conophylline profiles of 10-dehydroxyl-12-demethoxyconophylline after oral administration of 20 mg/kg Figure 3. Mean plasma concentration–time (mean ± SD) profiles of 10‐dehydroxyl‐12‐ followingand intravenous an intragastrical administration (i.g.) dose of 204, 8, mg/kg and 12 in mg/kg rats (n =in 6). rats (mean ± SD, n = 6). demethoxyconophylline following an intragastrical (i.g.) dose of 20 mg/kg in rats (n = 6). Table 2. Profiles of 10-dehydroxyl-12-demethoxy-conophylline4 mg/kg 8 mg/kg after12 oral mg/kg administration of20 20 mg/kg mg/kg Parameter andTable intravenous 2. Profiles administration of 10‐dehydroxyl(i.v.) of 4,‐12 8,‐ anddemethoxy 12 mg/kg(i.v.)‐conophylline in rats (mean after±(i.v.) SD,oral n administration= 6). of(i.g.) 20 mg/kg

andt 1/2intravenousα (min) administration6.983 ± 2.299 of 4, 8, and7.108 12 mg/kg ± 1.908 in rats (mean7.046 ± SD,± 2.050 n = 6). 52.415 ± 1.920 4 mg/kg 8 mg/kg 12 mg/kg 20 mg/kg Parameter t1/2β (min) 68.5294 mg/kg ± 3.290 66.4658 mg/kg ± 4.551 69.31512 mg/kg ± 4.324 69.94220 mg/kg ± 2.680 Parameter (i.v.) (i.v.) (i.v.) (i.g.) Tmax (min) (i.v.)2 ± 0 (i.v.)2 ± 0 (i.v.)2 ± 0 90(i.g.) ± 0 t1/2α (min) 6.983 ± 2.299 7.108 ± 1.908 7.046 ± 2.050 52.415 ± 1.920 CLt1/2 (mL/min)α (min) 0.041 ± 0.070 0.039 ± 0.120 0.041 ± 0.050 0.046 ± 0.013 t1/2β (min) 6.983 68.529 ± 2.299± 3.290 7.108 66.465 ± 1.908± 4.551 7.046 69.315 ± 2.050± 4.324 52.415 69.942 ± 1.9202.680 V (L/kg) 2.096 ± 0.0465 1.906 ± 0.012 2.014 ± 0.019 4.881 ± 2.390 Ttmax1/2β (min)(min) 68.529 2 ±± 3.2900 66.465 ± 2 4.551± 0 69.315 ± 2 4.324± 0 69.942 90 ± 2.6800 CLMRT (mL/min) (min) 91.444 0.041 ±± 4.4280.070 85.105 0.039 ± 1.882± 0.120 81.871 0.041 ± ±5.1570.050 256.76 0.046 ± ± 0.0133.510 Tmax (min) 2 ± 0 2 ± 0 2 ± 0 90 ± 0 V (L/kg) 2.096 ± 0.0465 1.906 ± 0.012 2.014 ± 0.019 4.881 ± 2.390 AUC0–t (μg∙min/mL) 59.256 ± 6.910 128.938 ± 8.871 234.524 ± 12.070 52.048 ± 4.671 CLMRT (mL/min) (min) 0.041 91.444 ± 0.070± 4.428 0.039 85.105 ± 0.120± 1.882 0.041 81.871 ± 0.050± 5.157 0.046 256.76 ±± 0.0133.510 AUC0–∞ (μg∙min/mL) 68.478 ± 8.167 131.438 ± 9.392 305.616 ± 17.432 74.307 ± 16.548 AUC0–tV( µ(L/kg)g·min/mL) 2.096 59.256 ± 0.0465± 6.910 1.906 128.938 ± 0.012± 8.871 2.014 234.524 ± 0.019± 12.070 4.881 52.048 ±± 2.3904.671 AUC0–MRT∞ (µg (min)·min/mL) 91.444 68.478 ± ±4.4288.167 85.105 131.438 ± 1.882± 9.392 81.871 305.616 ± 5.157± 17.432 256.76 74.307 ±± 16.5483.510 After i.v. administration of 10‐dehydroxyl‐12‐demethoxy‐conophyllinein in rats, the elimination AUC0–t (μg∙min/mL) 59.256 ± 6.910 128.938 ± 8.871 234.524 ± 12.070 52.048 ± 4.671 half‐life was approximately 7 min and the t1/2β was about 68 min. AUC0→∞ increased in a dose‐ AUCAfter0–∞ i.v. (μg administration∙min/mL) 68.478 of 10-dehydroxyl-12-demethoxy-conophyllinein ± 8.167 131.438 ± 9.392 305.616 ± 17.432 in rats,74.307 the elimination± 16.548 disproportional manner from 68.478 μg/L∙min for 4 mg/kg to 305.616 mg/L∙min for 12 mg/kg. After half-life was approximately 7 min and the t1/2β was about 68 min. AUC0→∞ increased in a dose-proportionali.g. dosingAfter i.v. at 20 administration mg/kg, manner plasma from of 68.478levels10‐dehydroxyl ofµg/L 10‐dehydroxyl·min‐12 for‐demethoxy 4 mg/kg‐12‐demethoxy‐conophyllinein to 305.616‐conophylline mg/L in· minrats, peaked forthe 12elimination mg/kg. at about 90 min. 10‐Dehydroxyl‐12‐demethoxy‐conophylline absolute oral bioavailability was only 15.79%. Afterhalf‐ i.g.life dosingwas approximately at 20 mg/kg, 7 plasma min and levels the of t1/2 10-dehydroxyl-12-demethoxy-conophyllineβ was about 68 min. AUC0→∞ increased in peaked a dose‐ The experimental results show a good linear correlation between AUC0→t and the dose studied (r = atdisproportional about 90 min. manner 10-Dehydroxyl-12-demethoxy-conophylline from 68.478 μg/L∙min for 4 mg/kg to 305.616 absolute mg/L oral∙min bioavailability for 12 mg/kg. wasAfter 0.9821), as shown in Figure 4, suggesting that the in vivo process of 10‐dehydroxyl‐12‐demethoxy‐ onlyi.g. dosing 15.79%. at 20 The mg/kg, experimental plasma levels results of 10 show‐dehydroxyl a good‐12 linear‐demethoxy correlation‐conophylline between peaked AUC0→ att aboutand the90conophylline dosemin. 10 studied‐Dehydroxyl conforms (r = 0.9821),‐ 12to‐ demethoxylinear as kinetics shown‐conophylline over in Figure the dose absolute4, suggesting range oral studied. bioavailability that Characteristics, the in vivowas onlyprocess analysis 15.79%. of of variance for t1/2, CL, and V were not significantly different (p > 0.05). 10-dehydroxyl-12-demethoxy-conophyllineThe experimental results show a good linear conforms correlation to linear between kinetics AUC over0→t and the the dose dose range studied studied. (r = Characteristics,0.9821), as shown analysis in Figure of variance 4, suggesting for t1/2, that CL, andthe in V werevivo process not significantly of 10‐dehydroxyl different‐ (12p >‐demethoxy 0.05). ‐ conophylline conforms to linear kinetics over the dose range studied. Characteristics, analysis of variance for t1/2, CL, and V were not significantly different (p > 0.05).

Figure 4. Dose positive correlation of single dose (4, 8, and 12 mg/kg) AUC0→t (n = 6).

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Figure 4. Dose positive correlation of single dose (4, 8, and 12 mg/kg) AUC0→t (n = 6). At present, there are no studies on the pharmacokinetics of 10-dehydroxyl-12- At present, there are no studies on the pharmacokinetics of 10‐dehydroxyl‐12‐ demethoxyconophylline in China. The pharmacokinetic profiles of a single injection experiment demethoxyconophylline in China. The pharmacokinetic profiles of a single injection experiment in in the tail vein of rats can be found initially, and the drug is consistent with a two-compartment the tail vein of rats can be found initially, and the drug is consistent with a two‐compartment pharmacokinetic model. According to the characteristics of the two-compartment model drug, pharmacokinetic model. According to the characteristics of the two‐compartment model drug, the the drug has the fastest distribution in the central chamber with high blood perfusion and a slower drug has the fastest distribution in the central chamber with high blood perfusion and a slower entry entry into the outer tissue compartment. The pharmacokinetic profiles show that t1/2α is only into the outer tissue compartment. The pharmacokinetic profiles show that t1/2α is only about 7 min about 7 min and t1/2β is about 69 min, which means that the elimination is very fast in the central and t1/2β is about 69 min, which means that the elimination is very fast in the central compartment compartment and the elimination of the tissue is relatively slow. and the elimination of the tissue is relatively slow. 2.3. Tissue Distribution Study 2.3. Tissue Distribution Study Tissue distribution was assessed at three different time points (5, 20, and 45 min) after intravenous administrationTissue distribution of 8 mg/kg was 10-dehydroxyl-12-demethoxy-conophylline assessed at three different time points (5, in 20, rats. and The 45 resultsmin) after are presentedintravenous schematically administration in Figure of 85 .mg/kg 10‐dehydroxyl‐12‐demethoxy‐conophylline in rats. The results are presented schematically in Figure 5.

Figure 5. Concentration of 10-dehydroxyl-12-demethoxy-conophylline in rat tissues at 5, 20, and 45 min Figure 5. Concentration of 10‐dehydroxyl‐12‐demethoxy‐conophylline in rat tissues at 5, 20, and 45 after receiving a single i.v. dose of 10-dehydroxyl-12-demethoxy-conophylline at 8 mg/kg (mean ± SD, min after receiving a single i.v. dose of 10‐dehydroxyl‐12‐demethoxy‐conophylline at 8 mg/kg (mean n = 6). ± SD, n = 6). The results demonstrated that 10-dehydroxyl-12-demethoxy-conophylline underwent a rapid and wideThe distribution results demonstrated to all the examined that 10 tissues.‐dehydroxyl Levels‐12 of‐ 10-dehydroxyl-12-demethoxyconophyllineindemethoxy‐conophylline underwent a rapid inand the wide heart anddistribution kidney were to markedlyall the higherexamined than intissues. other tissuesLevels at theof 510 min‐dehydroxyl time points.‐12‐ Thedemethoxyconophyllinein concentration of the drug in the in heart tissues and with kidney high were blood markedly flow may higher be affected than in by other the rate tissues of blood at the perfusion.5 min time Atpoints. the equilibrium The concentration time of of 20 the min, drug the in concentration tissues with high of drug blood mainly flow may distributed be affected in the by liver,the rate kidney, of blood and lungperfusion. tissues At gradually the equilibrium decreased, time and of the 20 drugmin, concentrationthe concentration in the of lungdrug reached mainly thedistributed highest in at the this liver, time, kidney, which and may lung be related tissues to gradually the accumulation decreased, of and the drug in the concentration lungs. At the in eliminationthe lung reached period the of highest 45 min, at it mainlythis time, distributed which may in be the related liver and to the kidney accumulation tissues, and of thedrugs drugs in the in eachlungs. tissue At the gradually elimination decreased period to of a very45 min, low it concentration. mainly distributed It can in be the seen liver from and the kidney data that tissues, the drug and isthe mainly drugs distributed in each tissue in tissuesgradually with decreased large blood to a flow,very low and concentration. the distribution It incan the be kidney seen from and the lung data is thethat highest, the drug indicating is mainly thatdistributed the kidney in tissues and the with lung large are blood the target flow, organs and the for distribution the drug concentration in the kidney ofand the lung drug is [the16]. highest, During indicating the course that of drug the kidney administration and the fromlung tailare veinthe target to elimination, organs for the the mean drug tissue-to-plasmaconcentration of ratiosthe drug (Kp) [16]. of 10-dehydroxyl-12-demethoxy-conophyllineDuring the course of drug administration from increased tail vein at to first elimination, and then decreased,the mean tissue which‐to was‐plasma consistent ratios with (Kp) the of 10 trend‐dehydroxyl of tissue‐ concentration,12‐demethoxy‐ indicatingconophylline that increased the distribution at first ofand drugs then in decreased, tissues was which mainly was affected consistent by bloodwith the flow trend (Figure of tissue6). The concentration, concentration indicating of plasma that in the the braindistribution was kept of to drugs a small in amount,tissues was which mainly may beaffected affected by by blood blood–brain flow (Figure barrier 6). factors, The concentration which provide of aplasma great research in the brain direction was kept and to value a small for amount, the practical which applicationmay be affected of the by drug. blood– Therefore,brain barrier the factors, tissue distributionwhich provide experiment a great provides research a referencedirection forand the value study for of thethein practical vivo distribution application characteristics of the drug. of drugsTherefore, and providesthe tissue a theoreticaldistribution basis experiment for the development provides a of reference drugs in thefor laterthe study stage. of the in vivo distribution characteristics of drugs and provides a theoretical basis for the development of drugs in the later stage.

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Figure 6. Ratio of of tissue tissue-to-plasma‐to‐plasma concentrations concentrations (Kp) (Kp) at at 5, 5, 20, 20, and and 45 45 min min after after receiving receiving a a single single i.v. i.v. Figure 6. Ratio of tissue‐to‐plasma concentrations (Kp) at 5, 20, and 45 min after receiving a single i.v. Figuredose of 6. 10-dehydroxyl-12-demethoxy-conophylline10 Ratio‐dehydroxyl of tissue‐‐12to‐‐demethoxyplasma concentrations‐conophylline (Kp) at 8at mg/kg 5, 20, and (mean (mean 45 min±± SD,SD, after nn == receiving6). 6). a single i.v. dosedose of of 10 10‐dehydroxyl‐dehydroxyl‐12‐12‐demethoxy‐demethoxy‐conophylline‐conophylline at at8 mg/kg8 mg/kg (mean (mean ± SD,± SD, n =n 6).= 6). 2.4. Protein Binding Binding Study Study 2.4.2.4. Protein Protein Binding Binding Study Study Equilibrium dialysis is considered a reference method [[17]17] for assessing plasma protein–drugprotein–drug Equilibrium dialysis is considered a reference method [17] for assessing plasma protein–drug bindingEquilibrium rate where dialysis non non-specific‐specific is considered binding a has reference no impact method on on the the [17] determination determination for assessing of of plasma the the free free protein fraction fraction–drug [18]. [18]. binding rate where non‐specific binding has no impact on the determination of the free fraction [18]. bindingProtein binding rate where degrees degrees non‐ specificevaluated evaluated binding using using hasthree three no concentration impact concentration on the levels determination levels (1.00, (1.00, 2.50, 2.50, of and the and 5.00 free 5.00 μ fractiong/mL)µg/mL) of [18]. 10 of‐ Protein binding degrees evaluated using three concentration levels (1.00, 2.50, and 5.00 μg/mL) of 10‐ Protein10-dehydroxyl-12-demethoxy-conophyllinedehydroxyl binding‐12‐ demethoxydegrees evaluated‐conophylline using three were were concentration 81.86% 81.86% ± 4.2%,± 4.2%, levels 82.61% 82.61% (1.00, ± 3.1%,2.50,± 3.1%, andand and 5.00 81.25% 81.25% μg/mL) ± 2.9%,± of2.9%, 10 in‐ dehydroxyl‐12‐demethoxy‐conophylline were 81.86% ± 4.2%, 82.61% ± 3.1%, and 81.25% ± 2.9%, in dehydroxylratin rat plasma plasma (Figure‐12‐ (Figuredemethoxy 7), 7and), and ‐wereconophylline were 86.88% 86.88% ± were 7.3%,± 7.3%,81.86% 84.10% 84.10% ± ± 4.2%, 2.7%,± 82.61% and2.7%, 84.65% and ± 3.1%, 84.65% ± 6.1%, and± 81.25%in6.1%, human ± in 2.9%, plasma human in rat plasma (Figure 7), and were 86.88% ± 7.3%, 84.10% ± 2.7%, and 84.65% ± 6.1%, in human plasma ratplasma(Figure plasma (Figure8), respectively.(Figure8), respectively. 7), and were 86.88% ± 7.3%, 84.10% ± 2.7%, and 84.65% ± 6.1%, in human plasma (Figure(Figure 8), 8), respectively. respectively.

Figure 7. Protein binding ratio of 10‐dehydroxyl‐12‐demethoxy‐conophylline in rat plasma. Figure 7. Protein binding ratio of 10-dehydroxyl-12-demethoxy-conophylline in rat plasma. FigureFigure 7. 7.Protein Protein binding binding ratio ratio of of 10 10‐dehydroxyl‐dehydroxyl‐12‐12‐demethoxy‐demethoxy‐conophylline‐conophylline in in rat rat plasma. plasma.

Figure 8. Protein binding ratio of 10-dehydroxyl-12-demethoxy-conophylline in human plasma. Figure 8. Protein binding ratio of 10‐dehydroxyl‐12‐demethoxy‐conophylline in human plasma. FigureFigure 8. 8.Protein Protein binding binding ratio ratio of of 10 10‐dehydroxyl‐dehydroxyl‐12‐12‐demethoxy‐demethoxy‐conophylline‐conophylline in in human human plasma. plasma.

Molecules 2019, 24, 283 7 of 10

This experimental study shows that 10-dehydroxyl-12-demethoxy-conophylline is more significantly combined with the protein to form a binding drug in the plasma, and there is no significant difference in the protein binding rate between the rat and the human, after the drug enters the circulatory system. When 10-dehydroxyl-12-demethoxy-conophylline is present at the same time as a drug with a competitive binding protein, it may cause an increase in the concentration of free drug in the plasma to cause a poisoning reaction. It is necessary to consider this factor in subsequent drug interaction study experiments, in order to ensure an effective drug concentration.

3. Materials and Methods

3.1. Chemicals and Reagents 10-Dehydroxyl-12-demethoxy-conophylline (purity >99.0%) was provided by the Kunming Institute of Botany, Chinese Academy of Sciences. Internal standard 11-methoxymelodinine (purity >99.0%) was provided by the Kunming Institute of Botany, Chinese Academy of Sciences. Methanol (chromatographically pure) was purchased from OCEANPAK Chemicals (Baotian Technology Biological Co., Ltd., Hefei, China) Normal saline was purchased from Beijing Jiajia Kangtai Trading Co., Ltd. (Beijing, China). All other reagents and chemicals were analytical reagents and did not require further purification.

3.2. Animal Experimentation Sprague Dawley rats (half male and female) with a body mass 180–220 g were provided by the Laboratory Animal Center of Medical University of Anhui (Hefei, China), Certificate number: SCXK(wan) 2015-002. Animals were housed under controlled conditions (temperature 22 ± 2 ◦C, humidity 50% ± 5%, 12 h dark/light cycle) with standardized diet and acclimatized for 7 days, prior to the experiments. All protocols and care of the rats were performed in strict compliance with the Guidelines for the Use of Laboratory Animals (National Research Council) and authorized by the Animal Care and Use Committee of Anhui University of Chinese Medicine.

3.3. UPLC-FLR Analysis Chromatographic separation was performed using a UPLC system (Waters ACQUITY-CLASS. US) with an analytical column Kinetex (C18, 1.7 µm, 2.1 mm × 100 mm; Phenomenex; California; CA, USA) at 25 ◦C. Water and methanol (75:25) were used as the isocratic elution, running at a flow rate of 0.2 mL/min. The detector was used with fluorescence (FLR) detection with a fluorescence excitation wavelength of 330 nm and a fluorescence emission wavelength of 439 nm.

3.4. Pharmacokinetic Studies The rat intravenous doses were determined as 4, 8, and 12 mg/kg, and the intragastric administration dose was 20 mg/kg. These were selected to investigate whether the pharmacokinetics of the drug was linear over the range of doses used. In all, 18 (SD) rats (half male and female) were randomly divided into three groups, namely, high-, medium- and low-dose groups. The rats fasted 12 h be before the administration of 10-dehydroxyl-12-demethoxy-conophylline but with free access to water. 10-dehydroxyl-12-demethoxy-conophylline was dissolved in normal saline and added with 5% ethanol. Amounts of 4, 8, and 12 mg/kg (0.5 mL/100 g) were injected into the tail vein of the rat, and 0.3 mL of blood was taken from the eyelid before administration (0 min) and 2, 5, 15, 30, 60, 90, 120, 180, 240, and 300 min after intravenous administration. Another 6 rats (half male and female) were administered an intragastrical (i.g.) dose of 20 mg/kg. Blood samples (0.3 mL) were collected by retro-orbital bleeding into heparinized 1.5mL tubes before administration (0 min) and 15, 30, 60, 90, 120, 240, 480, and 600 min after intragastric administration. Immediately after collection, the blood samples were centrifuged at 3500 rpm for 10 min to obtain plasma, and originating supernatants were also stored at −70 ◦C until analysis. Molecules 2019, 24, 283 8 of 10

Plasma samples (90 µL) were spiked with 10 µL internal standard (100 ng/mL) and extracted with 300 µL of methanol by vortexing for 10 min. The plasma and aqueous phases were separated by centrifugation at 14,000 rpm for 10 min. The supernatants were transferred to another tube and evaporated to dryness at 50 ◦C. The residue was dissolved in 100 µL methanol and vortexed. A 2 µL aliquot of the solution was injected into the UPLC system for analysis.

3.5. Tissue Distribution Study For the tissue concentration study of 10-dehydroxyl-12-demethoxy-conophylline, 18 (SD) rats (half male and female) were randomly divided into three groups. The rats were administered intravenously at a dose of 8 mg/kg of 10-dehydroxyl-12-demethoxy-conophylline, and the rats were euthanized at 5, 20, and 45 min after administration. Samples of their tissues and blood were carefully collected and washed with sodium phosphate buffer (pH 7.4) to remove blood. Each tissue (0.2 g) was homogenized in normal saline (1 mL). Subsequently, an amount 120 µL of the homogenate was pipetted into 480 µL of methanol and vortexed for 3 min, and then centrifuged at 14,000 rpm for 10 min. The supernatant was then centrifuged again at 14,000 rpm for 10 min and stored at −70 ◦C until analysis. The preparation process for analysis was the same as described above for plasma.

3.6. Plasma Protein Binding Test Protein binding was determined in vitro by equilibrium dialysis of plasma of rats or . Blank dialysate, internal dialysate, and external dialysate were prepared separately. The internal dialysate was prepared by taking 0.4 mL of blank plasma and adding 100 µL of the 10-dehydroxyl-12-demethoxy-conophylline series standard solution to prepare the equivalent of 10-dehydroxyl-12-demethoxy-conophylline plasma concentrations of 0.078, 0.156, 0.312, 0.625, 1.250, 2.500, 5.000, and 20.000 µg/mL simulating biological samples. The external dialysate was prepared by taking 0.4 mL of blank dialysate and adding 100 µL of the 10-dehydroxyl-12-demethoxy-conophylline series standard solution to prepare the equivalent of 10-dehydroxyl-12-demethoxyconophylline plasma concentrations of 0.078, 0.156, 0.312, 0.625, 1.250, 2.500, 5.000, and 20.000 µL/mL simulating dialysate samples. The protein binding rate of 10-dehydroxyl-12-demethoxy-conophylline in plasma was measured using the equilibrium dialysis method in this experiment. The concentration of unbound compound (B) and bound compound to protein (A) were calculated as follows:

F = (A − B)/A, (1) where F is the bounded fraction, A is the compound concentration in the plasma compartment, and B is the concentration of the compound in the phosphate buffer compartment.

3.7. Pharmacokinetic and Statistical Analysis The pharmacokinetic parameters were calculated using the pharmacokinetic software DAS 2.0 (Chinese Pharmacological Association, Beijing, China) by non-compartmental and compartmental analysis method. The elimination half-life (t1/2) was determined by linear regression of the terminal portion (last the points) of the plasma concentration–time curve. The area under the plasma concentration–time curve from zero to the last measurable plasma concentration point (AUC0-t) was calculated using the linear trapezoidal method. Extrapolation from time zero to infinity (AUC0-∞) was calculated as follows: AUC0-∞ = AUC0-t + Ct/ke, (2) where Ct is the last measurable plasma concentration and ke is the terminal elimination rate constant. Absolute oral bioavailability (F) of 10-dehydroxyl-12-demethoxy-conophylline was calculated according to the following equation: Molecules 2019, 24, 283 9 of 10

AUC(ig)/Dose(ig) F (%) = × 100, (3) AUC(iv)/Dose(iv) where AUCig and AUCiv are the AUC values after intragastric and intravenous administration of 10-dehydroxyl-12-demethoxy-conophylline, respectively, and Div and Dig are the doses for intravenous and intragastric administration of 10-dehydroxyl-12-demethoxyconophylline, respectively. Data were presented as means with their standard deviation (mean ± SD).

4. Conclusions Solid pharmacokinetic knowledge of natural compounds can play a critical role in proper design and choice of in vitro pharmacological models to evaluate the molecular mechanisms in a kinetically relevant manner [19–21]. In this paper, a rapid, sensitive, and specific UPLC-FLR method was developed for the determination of 10-dehydroxyl-12-demethoxy-conophylline in different rat biological sample. To the best of our knowledge, this paper is the first report of a thorough study of the pharmacokinetic profiles, distribution characteristics, and protein binding rate of 10-dehydroxyl-12-demethoxyconophylline in rats. This work lays a theoretical foundation for further pharmacological research and has certain new drug development value.

Author Contributions: The experiments were conceived and designed by D.W. The experiments were supervised by X.C. The experiments were equally performed by C.J. and J.L., N.L. Chromatographic analysis of plasma and biosamples; Y.G. analysis of pharmacokinetic behavior of 10-dehydroxyl-12-demethoxy-conophylline. All authors read and approved the final manuscript. Funding: This project was financially supported by the State Key Laboratory of Phytochemistry and PlantResources in West China (No. P2015-KF06). Acknowledgments: We are very grateful to Peng Huang for making a major contribution to the revision of the article. The authors are grateful to every person who helped in this work. Conflicts of Interest: The authors declare no conflict of interest

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Sample Availability: Samples of the compounds are not available from the authors.

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