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Vamshi K. Manda, Bharathi Avula, Zulfiqar Ali, Ikhlas A. Khan,Larry A. Walker, Shabana I. Khan

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Evaluation of In Vitro Absorption,Distribution, Metabolism, andExcretion (ADME) Properties ofMitragynine, 7-Hydroxymitragynine,and Mitraphylline

DOI 10.1055/s-0034-1368444Planta Med 2014; 80: 568–576

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Abstract!

Mitragyna speciosa (kratom) is a popular herb inSoutheast Asia, which is traditionally used to treatwithdrawal symptoms associated with opiate ad-diction. Mitragynine, 7-hydroxymitragynine, andmitraphylline are reported to be the central ner-vous system active alkaloids which bind to theopiate receptors. Mitraphylline is also present inthe bark of Uncaria tomentosa (catʼs claw). Severaltherapeutic properties have been reported forthese compounds but limited information is avail-able on the absorption and distribution proper-ties. This study focuses on evaluating the absorp-tion, distribution, metabolism, and excretion(ADME) properties of these compounds and theireffect on major efflux transporter P-glycoprotein,using in vitro methods. Quantitative analysis wasperformed by the Q‑TOF LC‑MS system. Mitragy-nine was unstable in simulated gastric fluid with26% degradation but stable in simulated intesti-nal fluid. 7-Hydroxymitragynine degraded up to27% in simulated gastric fluid, which could ac-count for its conversion to mitragynine (23%),while only 6% degradation was seen in simulatedintestinal fluid. Mitraphylline was stable in simu-lated gastric fluid but unstable in simulated intes-tinal fluid (13.6% degradation). Mitragynine and7-hydroxymitragynine showed moderate perme-ability across Caco-2 andMDR-MDCKmonolayerswith no significant efflux. However, mitraphyllinewas subjected to efflux mediated by P-glycopro-tein in both Caco-2 and MDR-MDCK monolayers.Mitragynine was found to be metabolically stablein both human liver microsomes and S9 fractions.In contrast, both 7-hydroxymitragynine and mi-traphylline were metabolized by human liver mi-crosomes with half-lives of 24 and 50min, re-

spectively. All three compounds exhibited highplasma protein binding (> 90%) determined byequilibrium dialysis. Mitragynine and 7-hydroxy-mitragynine inhibited P-glycoprotein with EC50values of 18.2 ± 3.6 µM and 32.4 ± 1.9 µM, respec-tively, determined by the calcein-AM fluorescentassay, while no inhibition was seen with mitra-phylline. These data indicate the possibility of adrug interaction if mitragynine and 7-hydroxy-mitragynine are coadministered with drugs thatare P-glycoprotein substrates.

Abbreviations!

ABC: ATP-binding cassetteADME: absorption, distribution, metabolism,

and excretionATCC: American Type Culture CollectionBBB: blood-brain barrierCaco-2: human colonic adenocarcinomaCL’int: intrinsic clearanceCNS: central nervous systemHBSS: Hanks balanced salt solutionHLM: human liver microsomesLy: lucifer yellowMRP: multidrug-resistant proteinPapp: apparent permeability coefficientP‑gp: P-glycoproteinRD: relative differenceSGF: simulated gastric fluidSIF: simulated intestinal fluidT1/2: half-lifeTEER: transepithelial electrical resistance

Supporting information available online athttp://www.thieme-connect.de/products

Evaluation of In Vitro Absorption, Distribution,Metabolism, and Excretion (ADME) Propertiesof Mitragynine, 7-Hydroxymitragynine, andMitraphylline

Authors Vamshi K. Manda1, Bharathi Avula1, Zulfiqar Ali1, Ikhlas A. Khan1,2, Larry A. Walker1,3, Shabana I. Khan1,2

Affiliations 1 National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS, USA2 Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS, USA3 Department of Pharmacology, School of Pharmacy, The University of Mississippi, University, MS, USA

Key wordsl" Mitragyna speciosal" Uncaria tomentosal" Rubiaceael" intestinal transportl" blood brain barrierl" P‑gp

received January 20, 2014revised March 31, 2014accepted April 2, 2014

BibliographyDOI http://dx.doi.org/10.1055/s-0034-1368444Planta Med 2014; 80: 568–576© Georg Thieme Verlag KGStuttgart · New York ·ISSN 0032‑0943

CorrespondenceShabana I. KhanNational Center for NaturalProducts ResearchSchool of PharmacyUniversity of MississippiUniversity, MS 38677USAPhone: + 16629151041Fax: + 16629157062skhan@olemiss.edu

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Fig. 1 Chemical structures of mitragynine (A),7-hydroxymitragynine (B), and mitraphylline (C).

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Introduction!

Mitragyna speciosa Korth. (kratom; Rubiaceae), a traditional herbin Southeast Asia, is widely known for its psychoactive propertiesdue to the presence of several alkaloids present in the leaves ofthis plant. These alkaloids act as powerful opioid agonists and il-licit responses similar to opioid analogs such as morphine. Tradi-tionally, the leaves of kratom, which are consumed mainly bychewing, smoking, or in the form of a tea, are used in treatingthe withdrawal symptoms associated with opiate addiction andsometimes as an opioid substitute due to their euphoric effectsin the brain [1]. Additionally, the leaves of kratom are also usedas a detoxifying agent as well as in the treatment of other ail-ments such as chronic pain, gastrointestinal discomfort, depres-sion, anxiety, and hypertension [2]. Of the several alkaloidspresent, mitragynine, 7-hydroxymitragynine, and mitraphyllineare considered to be the pharmacologically active constituentsin kratom [3]. Mitragynine is the most abundant, while 7-hy-droxymitragynine and mitraphylline are present in minor quan-tities [4]. Several preclinical studies have implicated the use ofmitragynine (indole alkaloid; l" Fig. 1A) as an analgesic, anti-in-flammatory, and potent opioid agonist [5–7]. However, 7-hy-droxymitragynine (indole alkaloid; l" Fig. 1B) was found to bethe most potent opioid agonist of all the alkaloids present in kra-tom and exhibited antinociceptive and analgesic effects withgreater potency than morphine [8]. Mitraphylline (pentacyclicoxindole alkaloid;l" Fig. 1C) is also present in the bark of Uncariatomentosa (Willd.) DC. (catʼs claw; Rubiaceae) and has beenshown to possess anti-inflammatory, anticancer, and potentialanti-Alzheimer properties [9–11]. Mitragynine and 7-hydroxy-mitragynine pharmacokinetics and tissue distribution have beenreported in vivo in rodent models [12–14]. However, there are noreports about protein binding, intestinal transport mechanisms,or metabolic properties for these two compounds. Further, thereare no ADME studies reported on mitraphylline despite its widespectrum of reported potential uses. Considering that kratom is

mainly consumed by the oral route and the three alkaloids havesignificant CNS effects, it is important to study the mechanismsinvolved in intestinal absorption and BBB transport.The current study aimed to evaluate the ADME properties of mi-tragynine, 7-hydroxymitragynine, and mitraphylline. The stabili-ty in gastric and intestinal fluids was evaluated because it deter-mines how much compound is degraded before intestinal ab-sorption. To predict intestinal absorption and BBB penetration,bidirectional transport across Caco-2 andMDR-MDCK cell mono-layers was measured, respectively [15,16]. Phase I and Phase IImetabolic stability was determined by incubating compoundswith either HLM or S9 fractions. Plasma protein binding was de-termined by equilibrium dialysis. A recent report has indicatedthat the efflux pump P‑gp is involved in conferring resistance toseveral clinically used opioid drugs [17]. Accordingly, we studiedthe potential interactions of mitragynine, mitraphylline, and 7-hydroxymitragynine with P‑gp, using the calcein-AM uptake as-say, due to their opioid activity.

Results and Discussion!

As part of our ongoing research on the authentication, validation,safety, and biological evaluation of dietary supplements [18], mi-tragynine, 7-hydroxymitragynine, and mitraphylline were se-lected for evaluating ADME properties in the current study. Afteroral administration, a drug has to encounter fluids of the stomachand intestine before it reaches the luminal side of the intestine forabsorption. The amount of drug available for intestinal absorp-tion is mainly dependent on its stability in gastric and intestinalfluids. Upon 2 h incubation in SGF (pH 1.2), 27% of 7-hydroxymi-tragyninewas degraded (l" Table 1). This may be attributed to thedehydroxylation and protonation of 7-hydroxymitragynine atpH 1.2, with 73% still available for intestinal absorption. Mitragy-nine and mitraphylline degraded up to 26.0% and 3.96%, respec-tively, in SGF (l" Table 1). In SIF (pH 6.8), 7-hydorxymitragynine

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Table 1 Stability of mitragynine, 7-hydroxymitragynine, and mitraphylline in simulated gastric fluid at pH 1.2. Data are expressed as mean ± SD of triplicate sam-ples in one experiment; RD = relative difference.

Incubation

time

(min)

7-Hydroxymitragynine Mitragynine Mitraphylline

Concentration in

SGF (µg/mL)

Formation of

mitragynine with

time (%)

% RD Concentration in SGF

(µg/mL)

% RD Concentration in SGF

(µg/mL)

% RD

0 4.81 ± 0.15 – – 4.33 ± 0.10 – 4.52 ± 0.12 –

30 4.40 ± 0.11 4.2 7.6 4.00 ± 0.09 8.9 4.40 ± 0.14 0.98

60 4.10 ± 0.07 12.0 15.7 3.52 ± 0.25 20.0 4.32 ± 0.11 2.84

120 3.52 ± 0.08 23.4 27.0 3.21 ± 0.23 26.0 4.34 ± 0.09 3.96

Table 2 Stability of mitragynine, 7-hydroxymitragynine, and mitraphylline in simulated intestinal fluid at pH 6.8. Data are expressed as mean ± SD of triplicatesamples in one experiment; RD = relative difference.

Incubation time

(min)

7-Hydroxymitragynine Mitragynine Mitraphylline

Concentration in

SIF (µg/mL)

% RD Concentration in

SIF (µg/mL)

% RD Concentration in

SIF (µg/mL)

% RD

0 4.60 ± 0.10 – 4.52 ± 0.15 – 4.71 ± 0.06 –

30 4.53 ± 0.15 1.23 4.40 ± 0.13 0.96 4.60 ± 0.10 3.6

60 4.42 ± 0.18 3.88 4.41 ± 0.10 2.67 4.23 ± 0.12 11.6

180 4.32 ± 0.14 6.08 4.34 ± 0.08 3.56 4.12 ± 0.14 13.6

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showed 6% degradation, while mitragynine degraded up to3.56% after 2 h incubation (l" Table 2). However, mitraphyllineshowed greater degradation (13.6%; l" Table 2) in SIF comparedto the other two alkaloids. The biopharmaceutical classificationsystem suggests that compounds with degradation greater than5% in SGF and SIF after 2 h incubation time may have stabilityproblems that contribute to lower bioavailability [19]. On this ba-sis, poor stability of 7-hydroxymitragynine and mitragynine inSGF and mitraphylline in SIF may impact the overall systemicavailability of the three alkaloids. Low oral bioavailability of mi-tragynine in a recent pharmacokinetic study [13] can be ex-plained in terms of its high degradation in SGF (26%).The second step in the oral bioavailability of a compound is theabsorption across the luminal and abluminal sides of the intes-tine. We used Caco-2 model (21-day culture) to predict the intes-tinal absorption of these alkaloids. Caco-2 cells, originally derivedfrom human colorectal adenocarcinoma epithelial cells, formphysiologically and morphologically differentiated cell mono-layers, which exhibit similar functional characteristics as humanintestinal enterocytes. The Papp values calculated from the Caco-2bidirectional transport experiment showed an excellent correla-tion with in vivo human intestinal absorption [16]. The transportof all three compounds was linear for 2 h and concentration-de-pendent (l" Fig. 2A–C). The Papp values of mitragynine in theabsorptive direction (apical to basolateral) were 24.2 ± 2.6 ×10−6 cm/s and 25.3 ± 2.2 × 10−6 cm/s, while in the secretory direc-tion (basolateral to apical), Papp values were 26.3 ± 2.7 × 10−6 cm/sand 29.1 ± 2.0 × 10−6 cm/s at 5 µM and 10 µM, respectively, asshown in l" Table 3. 7-Hydroxymitragynine was less permeablethan mitragynine, with Papp values of 16.1 ± 1.2 × 10−6 cm/s(5 µM) and 17.4 ± 1.5 × 10−6 cm/s (10 µM) in the absorptive direc-tion and 19.2 ± 1.0 × 10−6 cm/s (5 µM) and 21.2 ± 2.7 × 10−6 cm/s(10 µM) in the secretory direction (l" Table 2). The permeabilityof mitraphyllinewasmuch lower in the absorptive directionwithPapp values of 6.3 ± 1.9 × 10−6 cm/s (5 µM) and 7.2 ± 2.1 × 10−6 cm/s(10 µM) than in the secretory direction with Papp values of21.2 ± 1.1 × 10−6 cm/s (5 µM) and 24.3 ± 2.0 × 10−6 cm/s (10 µM)

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(l" Table 2). The Papp values of all three alkaloids were signifi-cantly higher than the low permeability marker compound ate-nolol (2.0 ± 0.6 × 10−6 cm/s), but lower than the high permeabilitymarker compound propranolol (34.2 ± 2.6 × 10−6 cm/s). Based onPapp values, these alkaloids can be categorized as compounds ex-hibiting intermediate intestinal absorption. The differences inpermeability among these three alkaloids could be explained bydifferences in their lipophilicity. The calculated logP (pH 7.4;ChemDraw) values of mitragynine, 7-hydroxymitragynine, andmitraphylline are 3.4, 2.2, and 1.1, respectively. Previous reportssuggest that lipophilicity (logP in the range of 1–4) is directlyproportional to the intestinal absorption for passively diffusedcompounds with no efflux [20]. Accordingly, in our study, Pappvalues of mitragynine in the absorptive direction were highercompared to 7-hydroxymitragynine and mitraphylline (l" Table3). The other parameter which is important in delineating thetransport mechanisms of a drug is the efflux ratio (Papp B–A/PappA–B). Drugs with an efflux ratio close to unity are considered tobe transported by passive diffusion, while a higher efflux ratio(> 2) indicates their efflux by transporters present at the luminalwall of the intestine [21]. Based on these criteria, our study dem-onstrates that both mitragynine and 7-hydroxymitragynine withefflux ratios of 1 and 1.2 (l" Table 3 and Fig. 3), respectively, aretransported by passive diffusion. In contrast, mitraphyllineshowed an efflux ratio of 3.4 (l" Table 3), which suggests that ef-flux transporters are involved in its transport, which is evidentby its lower Papp values in the absorptive direction. Further, weinvestigated the involvement of efflux transporters P‑gp, MRP 1,and MRP 2 in the bidirectional transport of mitraphylline. In thepresence of a P‑gp inhibitor (verapamil), the absorptive Papp wasincreased and the secretory Papp was decreased, while there wasno effect on the permeability by the MRP 1 and 2 inhibitor(MK571) (l" Fig. 4A). Furthermore, the efflux ratio of mitraphyl-line reached closed to unity with the addition of a P‑gp inhibitor(l" Fig. 4B). These results suggest that P‑gp is the primary effluxtransporter that limits mitraphylline intestinal absorption and

Table 3 Apparent permeability, % transport, and efflux ratio of mitragynine, 7-hydroxymitragynine, and mitraphylline across Caco-2 cell monolayers. Atenololand propranolol were used as low and high permeability markers, respectively, in each experiment. Data are expressed as mean ± SD of 3 independent experi-ments. Papp values in the absorptive direction (A–B) were compared with Papp values in the secretory direction (B–A) by Studentʼs t-test. *** P values < 0.001 wereconsidered to be significant. Efflux ratio = Papp B–A/Papp A–B.

Compound Papp × 10−6 cm/sec % Transport Efflux

ratioAbsorptive

direction (A–B)

Secretory

direction (B–A)

Absorptive

direction (A–B)

Secretory

direction (A–B)

Mitragynine (5 µM) 24.2 ± 2.6 26.3 ± 2.7 10.0 ± 0.3 11.2 ± 1.3 1.0

Mitragynine (10 µM) 25.3 ± 2.2 29.1 ± 2.0 11.4 ± 0.8 12.3 ± 1.0 1.1

7-Hydroxymitragynine (5 µM) 16.1 ± 1.2 19.2 ± 1.0 7.3 ± 0.5 8.4 ± 0.6 1.2

7-Hydroxymitragynine (10 µM) 17.4 ± 1.5 21.3 ± 2.7 7.8 ± 0.8 8.2 ± 0.8 1.2

Mitraphylline (5 µM) 6.3 ± 1.9 21.2 ± 1.1*** 3.0 ± 0.2 10.0 ± 0.6*** 3.3

Mitraphylline (10 µM) 7.2 ± 2.1 24.3 ± 2.0*** 3.5 ± 0.1 11.2 ± 0.8*** 3.4

Atenolol (200 µM) 2.0 ± 0.6 2.1 ± 0.4 2.0 ± 1.1 2.1 ± 0.8 1.0

Propranolol (200 µM) 34.2 ± 2.6 35.4 ± 1.9 17.3 ± 1.4 19.4 ± 2.6 1.0

Fig. 2 Bidirectional transport and efflux ratio (D)of mitragynine (A), 7-hydroxymitragynine (B), andmitraphylline (C) across Caco-2 cell monolayers for2 h. The transport experiment was performed asdescribed in Materials and Methods. The data arerepresented as mean ± SD of 3 independent experi-ments (n = 1 in each experiment).Efflux ratio = Papp B–A/Papp A–B.

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may, in turn, decrease its systemic availability when adminis-tered orally.Given the pharmacological effects reported in the CNS, we deter-mined the in vitro BBB permeability of three alkaloids using anMDR-MDCK cell monolayer model. Endothelial cells lining the

BBB are less permissive than those in the periphery, due to thepresence of tight junctions with no fenestrations between cells.Moreover, ABC transporters actively efflux drugs from these en-dothelial cells back into peripheral circulation [22]. While, sev-eral in vitro models exist for predicting BBB permeability, we

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Fig. 3 Bidirectional transport and efflux ratio (D)of mitragynine (A), 7-hydroxymitragynine (B), andmitraphylline (C) across MDR-MDCK cell mono-layers for 2 h. The transport experiment was per-formed as described in Materials and Methods. Thedata are represented as mean ± SD of 3 indepen-dent experiments (n = 1 in each experiment). Effluxratio = Papp B–A/Papp A–B.

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chose the MDR-MDCK cell model due to its high TEER values,which are comparable to human brain endothelial cells. Addi-tionally, permeability values obtained from the MDR-MDCKbidirectional transport study show an excellent correlation withthe permeability values from in vivo animal studies. According toWang et al. [15], compounds which show absorptive Papp values> 3.0 × 10−6 cm/s are considered to possess significant BBB pene-tration, whereas compounds with Papp values < 1.0 × 10−6 cm/sare considered not to permeate the BBB. Similar to Caco-2 trans-port, all three compounds showed linear transport across MDR-MDCK cell monolayers, which was concentration-dependent(l" Fig. 3A–C). Mitragynine (5–10 µM) showed Papp values in therange of 15.3 ± 1.7 × 10−6 cm/s and 16.2 ± 1.8 × 10−6 cm/s in the ab-sorptive direction indicating significant BBB permeation (l" Table4). In the secretory direction, the Papp values were in the range of17.2 ± 1.6 × 10−6 cm/s and 18.1 ± 1.7 × 10−6 cm/s. Similarly, 7-hy-droxymitragynine (5–10 µM) showed significant permeationacross MDR-MDCK cells in the absorptive direction with Papp val-ues of 12.4 ± 0.9 × 10−6 cm/s and 13.2 ± 1.2 × 10−6 cm/s. The Pappvalues in the secretory direction were 15.3 ± 1.4 × 10−6 cm/s –

16.1 ± 1.6 × 10−6 cm/s (l" Table 4). The efflux ratios of 1.1 and 1.2suggested a passive diffusion with no efflux involved for bothcompounds across MDR-MDCK monolayers (l" Table 4, Fig. 3D).Mitraphylline showed significantly lower permeability than mi-tragynine and 7-hydroxymitragynine. However, it could still be

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categorized as a BBB permeating drug as the Papp values in theabsorptive direction (3.3 ± 0.3 × 10−6 cm/s at 5 µM and 3.4 ± 0.2 ×10−6 cm/s at 10 µM) (l" Table 4) are significantly higher than ate-nolol (Papp 1.1 ± 0.2 × 10−6 cm/s), which is a non-CNS penetratingdrug. However, the Papp values of mitraphylline in the secretorydirection were 22 ± 2.3 × 10−6 cm/s (5 µM) and 20 ± 1.0 × 10−6 cm/s (10 µM) (l" Table 4) with a high efflux ratio of 6.6 (l" Table 4,Fig. 3D). This indicates the involvement of efflux transporters inthe membrane permeability of mitraphylline across MDR-MDCKcell monolayers. In the presence of a P‑gp inhibitor, the efflux ra-tio was reduced to 1 (l" Fig. 4D) and there was an increase in ab-sorptive Papp values with a proportional decrease in the secretoryPapp values as shown in l" Fig. 4C. A similar phenomenon wasseen for mitraphylline in the Caco-2 model. All the Papp valueswere lower than the highly CNS penetrating drug caffeine whichshowed an absorptive Papp value of 23.2 ± 2.8 × 10−6 cm/s, whichwas similar to a previously published report [23]. It should benoted that the MDR-MDCK model only provides an initial insightof a compoundʼs ability to penetrate the BBB. Further in vivostudies, such as in situ brain perfusion, are needed to determinethe concentrations in the brain of these three CNS active alka-loids. Furthermore, the monolayer integrity was maintained dur-ing transport experiments, which was confirmed by TEER mea-surements and Ly permeability.

Table 4 Apparent permeability, % transport, and efflux ratio of mitragynine, 7-hydroxymitragynine, and mitraphylline across MDR-MDCK cell monolayers. Ate-nolol and caffeine were used as low and high permeability markers, respectively, in each experiment. Data are expressed as mean ± SD of 3 independent experi-ments. Papp values in the absorptive direction (A–B) were compared with Papp values in the secretory direction (B–A) by Studentʼs t-test. ***P values < 0.001 wereconsidered to be significant. Efflux ratio = Papp B–A/Papp A–B.

Compound Papp × 10−6 cm/sec % Transport Efflux ratio

Absorptive

direction (A–B)

Secretory

direction (B–A)

Absorptive

direction (A–B)

Secretory

direction (A–B)

Mitragynine (5 µM) 15.3 ± 1.7 17.2 ± 1.6 7.4 ± 0.7 7.5 ± 0.8 1.1

Mitragynine (10 µM) 16.2 ± 1.8 18.1 ± 1.7 6.8 ± 0.6 7.3 ± 1.1 1.1

7-Hydroxymitragynine (5 µM) 12.4 ± 0.9 15.3 ± 1.4 5.2 ± 0.7 6.6 ± 0.5 1.2

7-Hydroxymitragynine (10 µM) 13.2 ± 1.2 16.1 ± 1.6 5.6 ± 0.9 7.0 ± 1.0 1.2

Mitraphylline (5 µM) 3.3 ± 0.3 22.1 ± 2.3*** 1.7 ± 0.1 8.4 ± 2.4*** 6.6

Mitraphylline (10 µM) 3.4 ± 0.2 20.2 ± 1.0*** 1.9 ± 0.1 10.0 ± 0.8*** 5.9

Atenolol (200 µM) 1.1 ± 0.2 1.2 ± 0.1 1.2 ± 0.7 1.7 ± 0.6 1.0

Caffeine (100 µM) 23.2 ± 2.8 24.3 ± 1.5 19.1 ± 1.8 20.4 ± 3.3 1.0

Fig. 4 Apparent permeability (A) and efflux ratios(B) of mitraphylline across Caco-2 monolayer withand without the presence of P‑gp (Verapamil) andMRP (MK 571) inhibitors in absorptive (A–B) andsecretory (B–A) directions. Mitraphylline Papp andefflux ratio across the MDR-MDCK monolayer in thepresence and absence of a P‑gp inhibitor (verapa-mil) are represented in (C) and (D), respectively.* P < 0.05, ** p < 0.01, and *** p < 0.001 (A–C)were determined by one-way ANOVA, followed byBonferroniʼs multiple comparison tests.*** P < 0.001 (D) was determined by Studentʼs t-test. All the data are expressed as mean ± SD of 3independent experiments (n = 1 in each experi-ment).

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Additionally, binding to plasma proteins such as albumin andglobulins plays a crucial role in BBB penetration, as only the freedrug in the plasma is available for transport across endothelialcells into brain tissue [24]. We employed equilibrium dialysis toevaluate the plasma protein binding of all three alkaloids. After24 h equilibration, the % free drug concentrations of mitragynine,7-hydroxymitragynine, and mitraphylline at 5 and 15 µM werebetween 5–6%, 9.–10%, and 6–7%, respectively, as shown in Table1S, Supporting Information. This means > 90% of each was boundto plasma proteins. The extent of protein binding is directly cor-

related to the lipophilicity of a compound [24]. Our data is inagreement with this as the calculated clogP values of mitragy-nine, 7-hydroxymitragynine, and mitraphylline were 3.4, 2.2,and 1.1, respectively. High protein binding may not necessarilyaffect the activity of a compound as other factors such as associa-tion constant (binding capacity to protein), time to reach equilib-rium with the brain, and BBB permeability rates, which also playa crucial role in determining the extent of BBB penetration [25].Further in vivo studies are required to study these parameters.

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Fig. 5 Time-depen-dent metabolic deple-tion of mitragynine,7-hydroxymitragynine,and mitraphylline inpooled human liver mi-crosomes (Phase I me-tabolism; A) and S9fractions (Phase II me-tabolism; B). Testoster-one (A) and 7-hydroxy-coumarin (B) were usedas positive controls forPhase I and Phase IImetabolism, respec-tively. The data are ex-pressed asmean ± SD of3 independent experi-ments (n = 1 in each ex-periment).

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After intestinal absorption, liver metabolism is an important fac-tor that determines the systemic availability of a compound. Theinvolvement of Phase I and Phase II metabolismwas evaluated inHLM and S9 fractions using NADPH and UDPGA as cofactors, re-spectively. CL’int (mL/min/kg) was estimated by determining thein vitro microsomal T1/2, which was measured by quantifyingthe loss of a parent compound upon incubation with liver micro-somes. In HLM, mitragynine was found to be stable with morethan 70% of the drug still remaining after 2 h incubation(l" Fig. 5A). There was more depletion of mitragynine (55%) inS9 fractions upon 2 h incubation (l" Fig. 5B). These results indi-cate that mitragynine is metabolically stable, which could ac-count for the long elimination T1/2 seen in an in vivo pharmacoki-netic study in rats [13]. In contrast, 7-hydroxymitragynine wasconverted to mitragynine (45%) in the presence of HLM with aT1/2 of 24min and a CL’int of 43.2 ± 3.5mL/min/kg, while in S9fractions, it was more stable with 60% of the drug still remainingafter 2 h incubation (l" Fig. 5A and B). This is the first report ofquantifying the metabolic conversion of 7-hydroxymitragynineinto mitragynine. Further in vivo studies are needed to evaluateif this conversion has any pharmacological effects when studiesare done with 7-hydroxymitragynine, considering the differentactivities and affinities on the opioid receptors of these two com-pounds. Mitraphylline was metabolized in the HLM fraction to agreater extent (80% disappearance) with a T1/2 of 50min and aCL’int of 89.7 ± 2.8mL/min/kg, while in the S9 fraction, 65% of thedrug still remain unmetabolized (l" Fig. 5A and B). This demon-strates that mitraphylline is primarily metabolized by Phase Imetabolic enzymes and may be rapidly eliminated due to its highclearance rate.Mitragynine and 7-hydroxymitragynine are potent opioid recep-tor agonists and illicit effects similar to opioid drugs. Several pre-

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clinical studies have indicated that currently used opioid drugsare substrates for P‑gp that affects their bioavailability and distri-bution [26]. We investigated if mitragynine, 7-hydroxymitragy-nine, and mitraphylline had any inhibition potential towardsP‑gp by using the widely used calcein-AM uptake assay. Mitragy-nine and 7-hydroxymitragynine showed a dose-dependent in-crease in calcein-AM uptake in MDR-MDCK cells (Fig. 1S, Sup-porting Information), while mitraphylline had no effect on cal-cein-AM uptake. The EC50 values of the P‑gp inhibition of mitra-gynine (18.2 ± 3.6 µM) and 7-hydroxymitragynine (32.4 ± 1.9 µM)were comparable to the positive control verapamil (22.3 ±1.4 µM) as shown in Fig. 1S, Supporting Information. Based onthis in vitro data, mitragynine and 7-hydroxymitragynineshowed significant P‑gp inhibition, which could lead to potentialdrug-drug interactions when coadministered with drugs that areP‑gp substrates.In conclusion, mitragynine showed moderate permeabilityacross Caco-2 andMDR-MDCKmonolayers. However, its instabil-ity in SGF may limit its overall systemic availability. While it wasfound to be metabolically stable in liver microsomes, mitragy-nine showed high plasma protein binding, which can explain itslonger T1/2 and volume of distribution observed in previous phar-macokinetic studies. 7-Hydroxymitragynine also exhibited mod-erate intestinal and BBB permeability with a significant conver-sion to mitragynine in both SGF (23%) and HLM (45%). Further,it was rapidly metabolized by Phase I metabolic enzymes with ashort T1/2 and high clearance that may lead to low bioavailabilityand distribution to tissues, while it showed a high binding toplasma proteins. Mitraphylline was found to be a P‑gp substrate,which could be responsible for its limited intestinal and BBB per-meability. Like the other two alkaloids, mitragynine was highlybound to plasma proteins. Mitragynine and 7-hydroxymitragy-nine showed P‑gp inhibition similar to verapamil, which suggeststhe possibility of drug interactions. This in vitro study providesinformation related to important ADME parameters that couldhelp in designing future pharmacokinetic and pharmacodynamicstudies with mitragynine, 7-hydroxymitragynine, and mitra-phylline.

Materials and Methods!

Caco-2 cells were purchased from ATCC. DMEM, MEM, HBSS,HEPES, trypsin EDTA, penicillin-streptomycin, and sodium pyru-vate were obtained from GIBCO BRL Invitrogen Corporation. FBSwas from Hyclone Lab, Inc. CYPremes™ HLM and S9 fractions(pooled mixed sex) were from In Vitro Technologies, Inc. Trans-well® plates (12-well, 1.12mm diameter, 0.4 µM pore size) werefrom Costar Corp. G-6-PDH, G-6-P, NADP+, UDPGA, atenolol, andall other chemicals were from Sigma Chem. Co. The purity of allthe positive controls used in this study was ≥ 98%.Mitragynine was isolated as described earlier [27] and its puritywas confirmed by chromatographic analysis to be ≥ 95%. Mitra-phylline and 7-hydroxymitragynine (purity > 90%) were pur-chased from ChromaDex®. All test compounds and positive con-trols were dissolved in DMSO, the concentration of which did notexceed 1% during experiments.

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Quantitative analysis of mitragynine, 7-hydroxy-mitragynine, and mitraphylline stability in simulatedgastric fluid and simulated intestinal fluidSGF and SIF were prepared as described earlier [28]. All three testcompounds were first dissolved in SGF or SIF (5mg/mL) and thenfurther diluted to 5 µg/mL. Aliquots (1mL) of 5 µg/mL of eachcompound were pipetted into glass tubes and incubated at 37°Cin a shaking water bath (120 rpm) for 0, 30, 60, and 120 minutesfor SGF and 0, 60, 120, and 180 minutes for SIF (in triplicate). Ateach time point, 100 µL of the sample was taken out and added to200 µL of ice-cold methanol and mixed well. Samples were cen-trifuged at 12000 rpm for 5min and the supernatant was usedfor Q‑ToF UHPLC analysis. RD, which determines the % of drug re-maining at the end of the incubation period compared to the ini-tial amount, was calculated using the following equation:

RD ¼Ci � Cf

Ci� 100%

where Ci is the initial concentration of the drug at zero time andCf is the concentration at the end of the incubation period.

Cell culture and bidirectional transport assay inCaco-2 and MDR-MDCK modelsCaco-2 and MDR-MDCK cells were maintained in similar condi-tions as described earlier [29]. For transport assays, cells betweenpassage numbers 30–42 were seeded in Transwell plates. Physio-logically and morphologically developed Caco-2 cell monolayerswith TEER values greater than 400 ohm/cm2 were used for intes-tinal transport assays. MDR-MDCK monolayers with TEER valuesgreater than 1100 ohm/cm2 were used for the BBB transport ex-periment [30].HBSSwith 10mMHEPES at pH 7.4 was used as the transport buf-fer in both cell lines. Prior to the transport experiment, the cellswere preincubated with the transport buffer for 30min. Afterpreincubation, the transport buffer was aspirated from both sidesand replaced with test compounds diluted in transport buffer (5and 10 µM). Test compoundswere added to the apical side for de-termining apical to basolateral transport (A–B; absorptive direc-tion) and to the basal side for determining basolateral to apicaltransport (B–A; secretory direction). For inhibition studies, vera-pamil and MK571 were added to both compartments. The vol-umes of apical and basolateral chambers were 0.6 and 1.5mL, re-spectively. Aliquots of 200 µL were taken out from the basolateral(for A–B transport) or from the apical (for B–A transport) cham-ber at 30, 45, 60, 90, and 120 minutes and replaced with an equalvolume of transport buffer. At the end of the experiment, an ali-quot was also taken out from the donor side for analysis of thedrug. After extracting with an equal volume of methanol, thesamples were analyzed by Q‑ToF UHPLC for quantitation. Papp(cm/sec) was calculated from the following equation:

Papp = (dq/dt) × 1/Co × 1/A

where dq/dt is the rate of transport, Co is the initial test concen-tration in the donor compartment, and A is the surface area of thefilter. To quantify the rate of transport, a cumulative amount ofthe test compounds was plotted against time (min) and the slopewas determined.

Assay for metabolic stability in human livermicrosomes and S9 fractionsPhase I and Phase II metabolic stability of mitragynine, 7-hy-droxymitragynine, and mitraphylline were evaluated in HLM(NADPH as a cofactor) and S9 fractions (UDPGA as a cofactor), re-spectively. The assay conditions and reaction mixtures were sim-ilar as described previously [23]. Testosterone and 7-hydroxy-coumarin were used as positive controls for Phase I and Phase IImetabolism, respectively. The reaction mixture was primed for 5minutes at 37°C and then the test compound (5 µM) or positivecontrol (10 µM) was added to initiate the reaction. Aliquots of100 µL were collected from reaction volumes at predeterminedtime points of 0, 15, 30, 60, 90, and 120 minutes and extractedwith 200 µL of ice-cold methanol. After centrifuging for 15 min-utes at 12000 rpm (4°C), the supernatants were analyzed byQ‑ToF UHPLC. T1/2 and CL’int (mL/min/kg) were calculated as re-ported previously [28]:

T1/2 = 0.693/k

where k is the slope of the line obtained by plotting the naturallogarithmic percentage (Ln %) of mitragynine, 7-hydroxymitra-gynine, and mitraphylline remaining in the reaction mixture ver-sus incubation time (minutes).

CL0 int¼0:693

in vitro T1=2� mL incubationmg microsomes

� 45mg microsomesg liver

� 20g liverkg bw

Analytical methodAll samples and controls were analyzed using Agilent LC-quadru-pole time-of-flight (QToF) (model 6530)-mass spectrometry (Agi-lent Technologies). The UHPLC system consisted of a binarypump, autosampler, degasser, and a column compartment. Themass detector was equippedwith an electron spray ionization in-terface that was connected to Agilent MassHunter Work Station(B.05.00). Mitragynine, 7-hydroxymitragynine, and mitraphyl-line were analyzed using an Agilent SB‑C8 RRHD column(100mm× 2.1mm I.D., 1.8 µm). A column temperature of 35°Cand sample temperature of 15°C were maintained during analy-sis. The mobile phase consisted of water (A) and acetonitrile (B),both containing 0.1% formic acid at a flow rate of 0.25mL/min,which were applied in the following gradient elution: 85%A:15%B to 63%A:37%B in 10min, 0%A:100% B in next 2 minutes,and a re-equilibration period of 3.0 minutes. The total run timefor analysis was 12min. The injection volumewas 5 µL. The efflu-ent from the LC column was directed into the ESI probe. Massspectrometer conditions were optimized to obtain maximal sen-sitivity (drying gas [N2] flow rate 9.0 L/min; drying gas tempera-ture, 250°C; nebulizer, 35 psig, sheath gas temperature, 325°C;sheath gas flow, 10 L/min; capillary, 3500 V; skimmer, 65 V andfragmentor voltage 125 V). Compounds were confirmed in thepositive ion mode with m/z = 399.22, 415.22, and 369.18 for mi-tragynine, 7-hydroxymitragynine, and mitraphylline, respective-ly. The lower limit of quantification and detection of mitragyninewas 5 pg/mL, and 50 pg/mL for 7-hydroxymitragynine andmitra-phylline, respectively. The analysis of atenolol, propranolol, caf-feine, testosterone, and 7-hydroxycoumarin was done as de-scribed earlier [30].

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Statistical methodsAll values are represented as mean ± SEM (n = 3). Papp values inthe B–A direction were compared with Papp values in the A–B di-rection by Studentʼs t-test. Nonparametric data were analyzed byone-way ANOVA, followed by Bonferroniʼs multiple comparisontests using GraphPad Prism Version 5. P < 0.05 was considered tobe statistically significant.

Supporting informationDetails about the assays for plasma protein binding and P‑gp in-hibition, data about the protein binding of mitragynine, 7-hy-droxymitragynine, and mitraphylline in human plasma by equi-librium dialysis, and a figure showing dose-response curves ofP‑gp inhibition by mitragynine and 7-hydroxymitragynine inMDR-MDCK cells can be found in Supporting Information

Acknowledgements!

The United States Department of Agriculture, Agricultural Re-search Service, Specific Cooperative Agreement No. 58–6408–1–603 and the FDA “Science Based Authentication of Dietary Sup-plements” award number 1 U01 FD004246–02 are acknowledgedfor partial support of this work.

Conflict of Interest!

The authors declare no conflict of interest.

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