Pharmacokinetic and Pharmacodynamic Consequences of Cytochrome P450 3A Inhibition on Mitragynine Metabolism in Rats

Abstract

Mitragynine, an opioidergic alkaloid present in Mitragyna speciosa (kratom), is metabolized by cytochrome P450 3A (CYP3A) to 7-hydroxymitragynine, a more potent opioid receptor agonist. The extent to which conversion to 7-hydroxymitragynine mediates the in vivo effects of mitragynine is unclear. The current study examined how CYP3A inhibition (ketoconazole) modifies the pharmacokinetics of mitragynine in rat liver microsomes in vitro. The study further examined how ketoconazole modifies the discriminative stimulus and antinociceptive effects of mitragynine in rats. Ketoconazole [30 mg/kg, oral gavage (o.g.)] increased systemic exposure to mitragynine (13.3 mg/kg, o.g.) by 120% and 7-hydroxymitragynine exposure by 130%. The unexpected increase in exposure to 7-hydroxymitragynine suggested that ketoconazole inhibits metabolism of both mitragynine and 7-hydroxymitragynine, a finding confirmed in rat liver microsomes. In rats discriminating 3.2 mg/kg morphine from vehicle under a fixed-ratio schedule of food delivery, ketoconazole pretreatment increased the potency of both mitragynine (4.7-fold) and 7-hydroxymitragynine (9.7-fold). Ketoconazole did not affect morphine’s potency. Ketoconazole increased the antinociceptive potency of 7-hydroxymitragynine by 4.1-fold. Mitragynine (up to 56 mg/kg, i.p.) lacked antinociceptive effects both in the presence and absence of ketoconazole. These results suggest that both mitragynine and 7-hydroxymitragynine are cleared via CYP3A and that 7-hydroxymitragynine is formed as a metabolite of mitragynine by other routes. These results have implications for kratom use in combination with numerous medications and citrus juices that inhibit CYP3A.

SIGNIFICANCE STATEMENT

Mitragynine is an abundant kratom alkaloid that exhibits low efficacy at the μ-opioid receptor (MOR). Its metabolite, 7-hydroxymitragynine, is also an MOR agonist but with higher affinity and efficacy than mitragynine. Our results in rats demonstrate that cytochrome P450 3A (CYP3A) inhibition can increase the systematic exposure of both mitragynine and 7-hydroxymitragynine and their potency to produce MOR-mediated behavioral effects. These data highlight potential interactions between kratom and CYP3A inhibitors, which include numerous medications and citrus juices.

Introduction

Mitragyna speciosa (kratom) is a tree indigenous to Southeast Asia that is locally known as ketum, biak, or krathom and belongs to the Rubiaceae family (Hassan et al., 2013; Hemby et al., 2019; Prozialeck et al., 2019). Kratom leaves have been used by natives for centuries for their stimulant- and opioid-like effects and to treat common ailments including pain (Jansen and Prast, 1988; Warner et al., 2016; Singh et al., 2017; Swogger and Walsh, 2018). In recent years, kratom has gained popularity in the United States (US) for mood elevation, pain relief, and mitigation of opioid withdrawal symptoms (Swogger et al., 2015; Warner et al., 2016; Prozialeck et al., 2019; Grundmann et al., 2022). Kratom is not currently regulated in the US, and is relatively easy to access at herbal stores, tobacco/smoke shops, and on the internet (Veltri and Grundmann, 2019). Although kratom products are consumed to treat numerous conditions, its effects have not been rigorously evaluated in controlled clinical studies (Kruegel et al., 2019; Veltri and Grundmann, 2019). A recent clinical pharmacokinetic study demonstrated exposure of kratom alkaloids after an oral dose of kratom tea; in that study mitragynine showed fast absorption [time to reach C_max (T_max) = 1 hour] with an elimination half-life of 45.3 hours (Tanna et al., 2022).

The opioidergic activity of kratom is often attributed to its most abundant bioactive alkaloid mitragynine and its metabolite, 7-hydroxymitragynine (7HMG). Mitragynine is reported to be 66% of the total alkaloidal fraction of kratom, whereas 7HMG analyzed in freshly prepared extracts are below the lower limit of quantification (0.01% w/w), suggesting that 7HMG is not biosynthesized in Mitragyna leaves (Hassan et al., 2013; Singh et al., 2020; Tanna et al., 2022). Instead, 7HMG appears to be an oxidative metabolite of mitragynine, at least in human liver microsomes, where the conversion is catalyzed by the cytochrome P450 3A (CYP3A) enzyme (Kamble et al., 2019). 7HMG is also found as a circulating metabolite after mitragynine administration in mice, rats, dogs, and humans (Hiranita et al., 2020; Maxwell et al., 2020; Berthold et al., 2022; Tanna et al., 2022). Mitragynine binds to a number of receptors, including the μ-opioid receptor (MOR) to which 7HMG also binds (Obeng et al., 2020). 7HMG exhibits higher potency and efficacy than its parent as a MOR agonist (Kruegel et al., 2016; Obeng et al., 2020). In the electrically stimulated guinea pig ileum assay, 7HMG is 46-fold more potent than mitragynine and 13-fold more potent than morphine (Takayama et al., 2002; Matsumoto et al., 2004). The higher potency and MOR efficacy of 7HMG compared with mitragynine is often assumed to indicate that 7HMG is a driver of kratom’s MOR activity (Kruegel et al., 2019). However, a recent study in mice reported that 7HMG administered at a dose sufficient to achieve plasma concentrations similar to those resulting from an oral mitragynine dose does not induce similar levels of antinociception (Berthold et al., 2022). This result suggests that 7HMG formation does not contribute to the effects of mitragynine. 7HMG is only one of several metabolites of mitragynine (Kamble et al., 2019), and several of these metabolites have been shown to produce MOR-mediated effects (Matsumoto et al., 2006; Chakraborty et al., 2021). Thus, it is important to understand how changes in the systemic exposure of mitragynine or its CYP3A-mediated metabolites, as a result of altered systemic clearance of mitragynine, impact pharmacodynamic responses.

The present study investigated in rats the pharmacokinetics and pharmacodynamics of mitragynine and the potential importance of its formation to 7HMG by pretreatment with a known CYP3A inhibitor, ketoconazole (Eagling et al., 1998; Mandlekar et al., 2007). It was hypothesized that ketoconazole pretreatment inhibits mitragynine metabolism, which might help differentiate the pharmacological activity of mitragynine itself versus formation of CYP3A-mediated metabolites. Drug discrimination and hot plate assays were used in the pharmacodynamic studies (Obeng et al., 2021). Two potential outcomes were evaluated. First, if the opioidergic effects were primarily driven by mitragynine itself as opposed to formation of 7HMG, then ketoconazole was expected to increase the potency of mitragynine. Second, if 7HMG mediated the effects of mitragynine through CYP3A-mediated formation, then ketoconazole was expected to decrease the potency of mitragynine. Morphine is not metabolized via CYP3A (De Gregori et al., 2012), and therefore ketoconazole was not expected to modify the effects of morphine. A radioligand binding assay was used to assess possible affinity of ketoconazole at MOR.

Materials and Methods

Chemicals and Reagents

Mitragynine was isolated from the dried leaves of Mitragyna speciosa obtained from Pure Land Ethnobotanicals (Madison, WI) using protocols previously described (Ponglux et al., 1994; Ali et al., 2014) and prepared in our laboratory as either the hydrochloride or sulfate salt (purity >98%). 7HMG (purity ≥98%) was semisynthesized in our laboratory as previously described (Takayama et al., 2002). The chemical purity and structural characterization of mitragynine and 7HMG were determined by high-performance liquid chromatography photodiode array detection (HPLC-PDA), high-resolution quadrupole time of flight (Q-TOF) mass spectrometry (HR-MS), and nuclear magnetic resonance proton and carbon (¹H or ¹³C NMR) spectroscopy (Sharma et al., 2019). Nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt (NADPH) was obtained from MP Biomedicals (Solon, OH). Ketoconazole, verapamil, and phenacetin were purchased from Sigma (St. Louis, MO). Male Sprague-Dawley rat liver microsomes (RLMs) were purchased from XenoTech, LLC (Lenexa, KS). Liquid chromatography–mass spectrometry (LC-MS)–grade acetonitrile, formic acid, ammonium acetate, analytical grade potassium dihydrogen phosphate, carboxymethyl cellulose sodium salt (Na-CMC), and dipotassium hydrogen phosphate were purchased from Fisher Scientific (Fair Lawn, NJ). [³H][D-Ala2, D-Leu5]-enkephalin ([³H]DADLE) (PerkinElmer, Boston, MA), [³H][D-Ala2, N-MePhe4, Gly-ol]-enkephalin ([³H]DAMGO) (PerkinElmer), [³H]U69,593 (PerkinElmer), DAMGO (Tocris Bioscience, Bristol, UK), and morphine sulfate pentahydrate (National Institute on Drug Abuse Drug Supply Program, Rockville, MD) were used for the in vitro radioligand and in vivo functional assays. For the in vitro radioligand and in vivo functional assays, dose/concentration is expressed as the weight of the salt form listed above or as the base if no salt form is noted. The compounds were dissolved in dimethyl sulfoxide (Sigma, St. Louis, MO) to form stock concentrations of 10 mM for the in vitro radioligand binding study and a vehicle consisting of sterile water containing 5% Tween 80 (polyoxyethylenesorbitanmonooleate, obtained from Sigma) and 5% propylene glycol (Sigma) for the in vivo functional study. For the in vivo functional study, each solution was filtered with a 0.2-μm pore size syringe filter (Millex-LG, 0.20 μm, SLLG025SS), and compounds and vehicle were administered using 25-gauge needles at a volume of 1.0 ml/kg body weight except mitragynine and 7HMG, which were administered at volumes of 1.0–10 ml/kg due to limited solubility. Mitragynine was tested up to 56 mg/kg; two higher doses of mitragynine (100 and 178 mg/kg) were lethal (Obeng et al., 2022). Mitragynine [intraperitoneally (i.p.)] and ketoconazole [by oral gavage (o.g.)] were administered 30 minutes before sessions; other compounds were administered i.p. 15 minutes before sessions. The dose and pretreatment time were based on previous studies (Hiranita et al., 2019; Obeng et al., 2021). Because the potency of mitragynine to produce discriminative stimulus effects is 3-fold greater after i.p. compared with o.g. administration, the i.p. route was chosen to generate the in vivo dose-effect functions.

In Vitro Metabolism of Mitragynine in RLM

The metabolism of mitragynine in male RLMs was investigated using a modification of a previously reported method (Kamble et al., 2019, 2020). Mitragynine (1.0 μM) was incubated with RLMs at 1.0 mg/ml of protein concentration supplemented with NADPH (1.0 mM) in the presence or absence of ketoconazole (3.0 μM) at 37°C for 60 minutes in a 50-mM phosphate buffer (pH 7.4). The volume of the reaction was adjusted with the phosphate buffer at pH 7.4 to a total of 200 μl, and the organic content of the reaction was kept below 0.5% v/v (Patil et al., 2015; Shah et al., 2015). An aliquot of 25 μl was withdrawn at 0, 5, 10, 15, 30, and 60 minutes and mixed with 125 μl of acetonitrile containing verapamil (25 ng/ml) as an internal standard (IS). The samples were filtered using a MultiScreen Solvinert 96 well 0.45 μm pore size filter plate (Millipore, Burlington, MA) under centrifugation at 2000 × g for 5 minutes. The filtrate was then subjected to ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) analysis. The details of the UPLC-MS/MS method are provided in Supplemental Methods section 1.1.

In Vivo Pharmacokinetics of Mitragynine in Rats

Eight male Sprague-Dawley rats weighing 225 ± 25 g and implanted with a catheter into the right external jugular vein were obtained from Envigo (Indianapolis, IN). The animals were singly housed in ventilated cages in a temperature- (22 ± 2.0°C) and humidity-controlled (53% ± 14%) vivarium with a 12-hour light/dark cycle (lights on at 07:00) during which food (2918 Teklad global 18% protein rodent diets; Envigo, Frenchtown, NJ) and reverse osmosis water were available at all times. Animals were acclimated no less than 72 hours prior to testing. Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida and were in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The rats were fasted for 12 hours before the oral administration of ketoconazole or mitragynine. All of the rats were randomly separated into two groups (n = 4). Rats in group 1 received 30 mg/kg of ketoconazole o.g. as a suspension prepared in 1% w/v carboxymethyl cellulose sodium salt (Na-CMC) (Chen et al., 2016). After 30 minutes, mitragynine (13.3 mg/kg, solution formulation prepared by dissolving an equivalent amount of mitragynine sulfate salt in water) was administered o.g. to these rats. The 13.3-mg/kg dose of mitragynine was chosen because it was the ED₅₀ value of mitragynine (o.g., 30 minutes prior to session) in our pilot study to produce discriminative stimulus effects in rats discriminating morphine (3.2 mg/kg). The animals were then transferred to the BASi-Culex automated blood drawing metabolic cages (West Lafayette, IN) for 24 hours. Each cage was equipped with a tether system for automated blood collection and a refrigerated compartment for the storage of collected blood samples. Blood samples were collected at the following time points: predose and 0.08, 0.25, 0.5, 0.75, 1, 2, 4, 8, and 12 hours postdose. On a separate day, group 2 rats received a single dose of mitragynine only (13.3 mg/kg, o.g.), and blood samples were obtained at each time point similarly to group 1 using the Culex automated blood drawing system. The blood samples were centrifuged at 2000 × g for 10 minutes, and plasma samples were separated. The plasma samples were stored at −20°C until UPLC-MS/MS analysis (Supplemental Methods section 1.1).

In Vivo Functional Assays

Animals

Adult female and male Sprague-Dawley rats (n = 8 per sex; Taconic Biosciences, Germantown, NY) weighing approximately 250 and 300 g upon arrival, respectively, were used. Animals were housed as described in the In Vivo Pharmacokinetics of Mitragynine in Rats section. After the acclimation period of no less than 72 hours, body weights were maintained individually by adjusting daily food rations at no less than 85% of the free-feeding body weight based on a growth function provided by the vendor (available up to 16 weeks old) and on a subsequent growth estimate in the present laboratory, with access to food (Dustless Precision Pellets Grain-Based Rodent Diet; Bio-Serv, Frenchtown, NJ) in their home cages approximately 30 minutes after daily experimental sessions, in addition to 45-mg sucrose pellets (Dustless Precision Pellets 45 mg, Sucrose; Bio-Serv) available during experimental sessions as described below. Water was available at all times in the vivarium. Experimental protocols were approved by University of Florida IACUC.

Apparatus

Hot plate

A hard black anodized aluminum plate built-in digital thermometer (Hot Plate Analgesia Meter, 1440 Analgesia Hot Plate with RS-232 Port and Software; Columbus Instruments, Columbus, OH) was 33.1 cm long, 28.8 cm wide, and 9.9 cm high. A clear acrylic cage (26.7 cm long, 26.7 cm wide, and 31.0 cm high) surrounded the square plate to confine an animal on the plate (total height: 40.9 cm). The size of the heated surface was 25.4 cm long, 25.4 cm wide, and 1.9 cm high. The temperature accuracy was ±0.1°C.

Drug discrimination

Eight operant conditioning chambers (Model ENV-203; Med Associates Inc., Fairfax, VT) measuring 25 cm long × 25 cm wide × 31 cm high per chamber were used. Each of these chambers was enclosed within a sound-attenuating cubicle equipped with a fan for ventilation and white noise to mask extraneous sounds. On the front wall of each chamber were two retractable, 5-cm–long response levers, 5 cm from the midline and 9 cm above the grid floor. A downward displacement of each lever with a force approximating 0.20 newtons (N) defined a response and resulted in no audible “feedback” click. Two amber light-emitting diodes (LEDs) as stimulus lights were located in a row above levers (one LED per lever). A receptacle lever for the delivery of 45-mg sucrose pellets (Dustless Precision Pellets 45 mg, Sucrose; Bio-Serv) via a pellet dispenser (Model ENV-203-20; Med Associates Inc.) was mounted on the midline of the front wall between the two levers and 2 cm above the floor. A house light was centrally mounted on the upper wall facing the wall equipped with the retractable levers. Each operant conditioning chamber was connected to a Dell desktop computer (Intel Core i7-7700 3.60 GHz processor, 16.0 GB of RAM, Microsoft Windows 10) through an interface (MED-SYST-8; Med Associates Inc.). Med-PC software version V (Med Associates Inc.) controlled experimental events and provided a record of responses.

Experimental Procedures

The temperature, humidity, and light/dark cycle in an experimental room were equivalent to those in the vivarium. All experiments were conducted in the light cycle (08:00 to 17:00 hours). The in vivo MOR activity of mitragynine and 7HMG were tested in the hot plate (high-efficacy demand) assay and drug discrimination (low-efficacy demand) procedures (Obeng et al., 2021) using the same rats. The sample size of each experiment group per treatment was eight using a within-subject design (n = 4 per sex). Prior to commencement of each experiment, body weights of subjects were measured.

Hot plate

Antinociception was assessed in the hot plate test as previously described (Obeng et al., 2021). Rats were placed on the heated (52.0 ± 0.1°C) hot plate apparatus and the latency to respond (jump, lick, or paw-shake) was determined manually using a stopwatch (Martin Stopwatch; Martin Sports, Carlstadt, NJ) by trained and experimentally blinded raters. The maximum latency was set to 60 seconds to avoid tissue damage. Before drug discrimination test sessions, baseline latency was measured. After the determination of the baseline, each rat received a test compound or vehicle consisting of sterile water containing 5% Tween 80 and 5% propylene glycol administered i.p. and was returned to its respective home cage. After the drug discrimination test sessions, each rat was placed on the heated plate, and postsession hot plate latency was measured. Morphine was administered i.p. because of its poor oral bioavailability (Katagiri et al., 1988).

Drug discrimination procedures

A) Lever-response shaping

After the acclimation period to the vivarium, each experimental session commenced by placing the subject in an individually assigned chamber (Med Associates, Fairfax, VT) daily up to 120 minutes at the same time each day, during the light period, 7 days per week. Each rat was fed in their home cage 30 minutes after the experimental session. Each session started with the presentation of one of the retractable levers and the illumination of the LED above the presented lever. Each downward deflection of the lever turned off the LEDs and activated the pellet dispenser for 0.1 seconds [fixed-ratio (FR) 1 schedule] followed by a 0.1-second time-out period during which LEDs were turned off, the house light was illuminated, and responding had no scheduled consequences; the retractable levers remained presented during the time-out. The right- versus left-assignment of lever presentation was switched daily (i.e., right-left-right-left). After 50 reinforcers per session were delivered within 20 minutes for four consecutive sessions under the FR1 and FR3 schedule of reinforcement and for two consecutive sessions under the FR5 schedule of reinforcement, the response requirement was increased to FR10. After 50 reinforcers per session were presented within 20 minutes for two consecutive sessions under the FR10 schedule of reinforcement, drug discrimination training commenced.

B) Drug discrimination training

Experimental subjects were divided into two groups per training drug (n = 4 per sex per training drug): morphine (3.2 mg/kg, i.p., administered 15 minutes before sessions) for one group and mitragynine (32 mg/kg, i.p., administered 30 minutes before sessions) for the second group (Obeng et al., 2021).

Immediately after training dose or vehicle, each subject was returned to their respective home cage and then placed in the operant conditioning chamber after the pretreatment interval. Each training session started with the presentation of both levers and the illumination of the LEDs above each lever under the FR1 schedule of reinforcement. Each downward deflection of the correct lever, determined by administration of the training drug or vehicle, turned off both LEDs and activated the pellet dispenser delivering the sucrose pellet for 0.1 seconds followed by a 0.1-second time-out period during which LEDs were turned off, the house light was illuminated, and responding had no scheduled consequences; the retractable levers remained presented during this time-out time. Responses on the incorrect lever had no programmed consequence. Each training session lasted for up to 20 minutes or until a maximum of 50 pellets was delivered, whichever occurred first. For half of the rats, the right lever was correct after administration of a training drug and the left lever was correct after administration of vehicle; the opposite assignments were made for the other half. The lever assignments remained the same for each rat throughout the study. The order of drug and vehicle training followed a double-alternation sequence (i.e., right-left-left-right), with periods of single alternation (i.e., right-left-right-left) interposed. During the training period, the FR value was increased when the following criteria were individually met for at least four consecutive sessions: 1) a minimum of 80% of the total responses was correct and 2) the total of incorrect responses made prior to delivery of the first reinforcer was less than the FR value. The order of increases in FR value was 1, 3, 5, and 10.

C) Testing

Test sessions commenced for an individual subject under the FR10 schedule of reinforcement when the criteria were met for at least four consecutive sessions. After the first test session, subsequent test sessions were conducted when the subject satisfied the criteria for at least one drug and one vehicle training session. The order of assignments of the training drug or vehicle between tests was nonsystematic. Test sessions were identical to the training sessions except that 10 responses on either lever resulted in the delivery of food and various doses of drugs were administered. Dose-effect assessments were conducted first for each training drug in all rats, followed by tests with other compounds. Doses of test compounds were administered from doses that produced less than group averages of 20% drug-appropriate responding up to doses that produced greater than or equal to group averages of 80% drug-appropriate responding, decreased response rate to less than 20% of the vehicle control per subject, or were deemed potentially toxic or could not be increased further due to limited solubility. For the substitution tests, the following dose ranges (incremented in 0.25 log unit) and presession treatment periods were assessed: morphine (0.32–56 mg/kg, 15 minutes), mitragynine (3.2–56 mg/kg, 30 minutes), and 7HMG (0.1–5.6 mg/kg, 15 minutes). For the pretreatment tests with ketoconazole (56 mg/kg, o.g., 30 minutes; Mantsch and Goeders, 1999), the following ranges of drug doses and presession treatment periods were assessed: morphine (0.56–56 mg/kg, 15 minutes), mitragynine (0.32–56 mg/kg, 30 minutes), and 7HMG (0.01–17.8 mg/kg, 15 minutes).

In Vitro Receptor Binding

[³H]DADLE, [³H]U69,593, and [³H]DAMGO were used to label the δ- (DOR), κ- (KOR), and μ- (MOR) opioid receptors, respectively (Barrett and Vaught, 1983; Lahti et al., 1985; Onogi et al., 1995; Obeng et al., 2021). The K_D and B_max values for the radioligands at their respective receptors were first determined using a saturation assay (Supplemental Table 1). The assay was conducted using monoclonal human opioid receptors expressed in Chinese hamster ovary (CHO) cell lines for DOR (generous gift from Dr. Stephen J. Cutler, University of South Carolina) and MOR (PerkinElmer). KOR (Dr. Stephen J. Cutler, University of South Carolina) was expressed in human embryonic kidney 293 (HEK-293) cells. Each membrane protein (10 μg) was separately incubated with the corresponding radioligand in the presence of different concentrations of test compounds in TME buffer [50 mM Tris (Sigma-Aldrich), 3 mM MgCl₂ (Sigma-Aldrich), and 0.2 mM ethylene glycol-bis(β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA; Sigma-Aldrich), pH 7.7] for 60 minutes at room temperature. The bound radioligand was separated by filtration using the PerkinElmer MicroBeta unifilter-96 cell harvester (Waltham, MA) and counted for radioactivity using the MicroBeta2 microplate counter (PerkinElmer, Waltham, MA). Specific binding at the DOR, KOR, and MOR was determined as the difference in binding obtained in the absence and presence of 10 μM SNC80, U69,593, and naltrexone, respectively. The assay was conducted in triplicate and repeated at least three times.

Data Analyses

The percentage of mitragynine remaining at each time point after incubation of mitragynine with RLMs in the presence or absence of ketoconazole was calculated as the percentage ratio of the peak area ratio of mitragynine to IS at time t to the peak area ratio of mitragynine to IS at time 0 minutes. All pharmacokinetic parameters were estimated by subjecting plasma concentration-time data to a noncompartmental analysis using Phoenix WinNonlin, version 6.4 (Certara Inc., Princeton, NJ). Mitragynine formulations were also quantified for mitragynine content, and exact doses were used for the estimation of pharmacokinetic parameters.

For the radioligand binding studies, IC₅₀ values were determined using nonlinear, least-squares regression analysis (Prism 8; GraphPad Software, Inc., San Diego, CA) and then converted to inhibition constant (K_i) values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

For the in vivo functional studies, statistical analyses were conducted using GraphPad Prism version 8 for Windows (San Diego, CA) and SigmaPlot version 14.0 (Systat Software Inc., San Jose, CA). A one- or two-way repeated-measures analysis of variance (ANOVA) followed by post hoc Bonferroni t tests were used to analyze the effects of doses, sex, or training drug (P < 0.05). The hot plate latencies were normalized to the percentage of the maximum possible effect (%MPE) using the following formula: %MPE = 100 × (postinjection latency − preinjection baseline latency)/(maximum latency 60 seconds − preinjection baseline latency). For the drug discrimination study, the percentage of drug-appropriate responding was calculated by dividing the total number of responses on the drug-appropriate lever by the total number of responses on the drug- and vehicle-appropriate levers. The rate of responding was calculated by dividing the total number of responses by the session time. The rates of responding per group are expressed as the percentage of the mean control rate of responding calculated from all of the vehicle training sessions conducted between test sessions. The percentage of drug-appropriate responding was considered a potentially unreliable indication of lever selection and was not: 1) included in the analysis for a given rat when rate of responding was less than 20% of the control rate of responding or 2) plotted or analyzed when fewer than half of the rats tested responded at less than 20% of the control rate of responding. However, all response rates and MPEs were plotted and analyzed. Full substitution for the training drugs was defined as greater than or equal to 80% percent drug-appropriate responding, and partial substitution was defined as 21%–79% drug-appropriate responding. Using standard linear regression techniques (Snedecor and Cochran, 1967), ED₅₀ values with 95% confidence intervals (CIs) for the dose-effect functions were calculated when the mean of drug-appropriate responding or %MPE was increased to greater than 50% and when response rate was decreased to less than 50%. Only data from the linear portion of the dose-effect functions, as determined per group, were used. Potency ratios and corresponding 95% CIs were calculated as the ratio of ED₅₀ values calculated from the dose-effect functions (Tallarida, 2002). If the 95% CIs of the ED₅₀ values did not overlap or if the 95% CIs of the potency ratio did not include 1, the potencies were considered to be significantly different.

Results

In Vitro Metabolism of Mitragynine in RLMs

Mitragynine and 7HMG (1.0 μM) were extensively metabolized in male RLMs in the absence of ketoconazole (Fig. 1, A and C, filled symbols). Ketoconazole (3.0 μM) reduced mitragynine metabolism 4.6-fold (Fig. 1A, open symbols), and a reduction in the formation of 7HMG was observed (Fig. 1B). These results suggest that ketoconazole is an effective inhibitor of mitragynine metabolism in RLMs and that 7HMG formation from mitragynine is greatly reduced by ketoconazole (Fig. 1, A and B). Similarly, a 1.8-fold reduction in the rate of metabolism of 7HMG was observed when incubated in liver microsomes in the presence of ketoconazole (Fig. 1C). This may confirm reports that 7HMG is metabolized by CYP3A, although ketoconazole also inhibits other CYP isoforms (Eagling et al., 1998).

Fig. 1.

In vitro metabolism of mitragynine (1.0 μM) and 7HMG (1.0 μM) in RLMs with or without ketoconazole (3.0 μM). The plot represents the percentage of mitragynine remaining (mean ± S.D.) vs. time in RLM incubations supplemented with NADPH with or without ketoconazole (Fig. 1A) and the ratio of 7HMG peak area to the IS peak area (mean ± S.D.) vs. time in the same incubation (Fig. 1B). The plot represents the percentage of 7HMG remaining (mean ± S.D.) vs. time in RLM incubations supplemented with NADPH with or without ketoconazole (Fig. 1C).

Pharmacokinetics of Mitragynine in Rats

Figure 2 shows the plasma concentration of mitragynine and 7HMG over time after oral dose (13.3 mg/kg) of mitragynine in rats, with or without ketoconazole pretreatment (30 mg/kg, 30 minutes prior). The pharmacokinetic parameters of mitragynine and 7HMG were calculated using noncompartmental analysis and are shown in Table 1. With ketoconazole pretreatment, the peak plasma concentration (C_max) of mitragynine was increased by 130% from 159.9 ± 37.2 (S.E.M.) to 369.1 ± 53.1 ng/ml (Fig. 2; Table 1). The time to reach maximum plasma concentration (T_max) of mitragynine was increased from 1.0 ± 0.4 to 2.6 ± 1.2 hours in the presence of ketoconazole (Fig. 2; Table 1). Ketoconazole also increased the area under the concentration versus time curve (AUC) for plasma mitragynine by 120% (Table 1) and systematic exposure to 7HMG by 130% (Table 1).

Fig. 2.

Plasma concentration-time profile of mitragynine (upper panel) or 7HMG (lower panel) after oral dose of mitragynine (13.3 mg/kg) with or without ketoconazole (30 mg/kg, o.g.) in male rats (n = 4 per treatment). The data represents mean ± S.E.M.

TABLE 1.

Pharmacokinetic parameters of mitragynine after oral administration of mitragynine (13.3 mg/kg) with or without ketoconazole pretreatment (30 mg/kg, o.g.) in male rats

All values represent mean ± S.E.M.

Parameter	Mitragynine (13.3 mg/kg) Alone		Mitragynine (13.3 mg/kg) with Ketoconazole (30 mg/kg)
	Mitragynine	7HMG	Mitragynine	7HMG
Cmax (ng/ml)	159.9 ± 37.2	3.9 ± 1.0	369.1 ± 53.1	6.7 ± 0.6
Tmax (h)	1.0 ± 0.4	1.0 ± 0.4	2.6 ± 1.2	3.4 ± 1.5
AUClast (h*ng/ml)	974.2 ± 252.7	15.3 ± 5.7	2168.5 ± 70.5	34.6 ± 3.2
%AUC ratio (7HMG/mitragynine)	Not applicable	1.4 ± 0.2	Not applicable	1.6 ± 0.2

In Vivo Functional Assays

Baseline

During all intertest sessions, rates of responding after vehicle were stable in both training groups. For the morphine-trained group, a two-way repeated-measures ANOVA indicated that there were no significant effects of session (F_55,330 = 1.03; P = 0.417), sex (F_1,330 = 0.430; P = 0.536), or their interaction (F_55,330 = 0.907; P = 0.662). For the mitragynine-trained group, there were no significant effects of session (F_37,222 = 1.19; P = 0.227) or sex (F_1,222 = 0.00228; P = 0.865), but there was a significant interaction effect (F_37,222 = 1.60; P = 0.022). A post hoc test indicated significantly different rates of responding between females and males at 30th and 34th intertest sessions out of 38 intertest sessions. In both 30th and 34th intertest sessions, respectively, rates of responding in females [1.21 (S.E.M.: 0.0234) and 1.30 (0.126) responses per second] were higher than those in males [0.521 (0.254) and 0.708 (0.186) responses per second]. The mean rates of responding after vehicle injections were comparable across all intertest sessions in both morphine- [mean (S.E.M.): 1.02 (0.0708)] and mitragynine-trained [0.899 (0.0810)] groups. There were no significant effects of training drug (F_1,12 = 1.05; P = 0.326), sex (F_1,12 = 0.211; P = 0.654), or their interaction (F_1,12 = 0.150; P = 0.706). For the hot plate test, baseline latency across all measurements on test days did not significantly vary. The mean baseline latency was 7.69 (S.E.M.: 0.44) and 8.48 (0.42) in morphine- and mitragynine-trained groups, respectively. There were no significant effects of the training drug (F_1,12 = 0.188; P = 0.672), sex (F_1,12 = 0.948; P = 0.349), or their interaction (F_1,12 = 1.85; P = 0.199).

Effects of Ketoconazole in Rats Trained with Mitragynine

Mitragynine produced a maximum of 96.0% (S.E.M.: 1.99%) mitragynine-appropriate response at 32 mg/kg. The 56-mg/kg dose of mitragynine decreased response rate to 26.9% (10.0%) of vehicle control rates of responding and produced 7.7% (5.1%) MPE (Fig. 3, upper panels, filled symbols). The ED₅₀ values for the discriminative-stimulus, rate-decreasing, and antinociceptive effects of the test compounds and corresponding potency ratio values (ED₅₀ values for the rate-decreasing or antinociceptive effects divided by those for the discriminative-stimulus effects) are summarized in Table 2. Mitragynine was 2.2-fold more potent in producing discriminative-stimulus effects than rate-decreasing effects (Table 2). Ketoconazole (56 mg/kg, o.g.) produced 1.5% (1.3%) mitragynine-appropriate responding, 91.7% (4.2%) response rates, and −3.7% (1.7%) MPE (Fig. 3, upper panels, open squares above vehicle). Ketoconazole (56 mg/kg) produced a significant (4.2-fold) leftward shift in the dose-effect function of mitragynine to produce discriminative stimulus effects, whereas there was no significant effect of ketoconazole on the rate-decreasing and antinociceptive effects of mitragynine (Fig. 3, upper panels; Supplemental Table 2). There was no significant sex effect observed in the discriminative-stimulus, rate-decreasing, or antinociceptive effects of mitragynine, either in the presence or absence of ketoconazole (56 mg/kg) (Supplemental Table 2).

Fig. 3.

Effects of mitragynine or morphine in the presence of the CYP3A inhibitor ketoconazole in rats discriminating 3.2 mg/kg morphine or 32 mg/kg mitragynine from vehicle. Abscissae: Vehicle and compound dose in mg/kg (i.p., log scale). Ordinates: Left panels, percentage of mean responses on drug-appropriate lever; middle panels, percentage of mean rates of responding after vehicle administration during intertest sessions; right panels, percentage of MPEs. Upper panels: Rats trained with mitragynine; Lower panels: Rats trained with morphine. Each point represents the mean ± S.E.M. (n = 4 per sex per data point unless noted). The percentage of responses emitted on the drug-appropriate lever was not plotted when individual or group mean of response rates was less than 20% of the vehicle control levels or less than half of the sample size did not produce more than or equal to 20% of the control rate of responding. Mitragynine and morphine were administered i.p., respectively, at 30 and 15 minutes before sessions, whereas ketoconazole (56 mg/kg) was administered o.g. 30 minutes before sessions. Upper left: The discriminative-stimulus effects of mitragynine. Dose of mitragynine alone; vehicle, and 3.2, 5.6, 10, 17.8, and 32 mg/kg. Mitragynine doses in the presence of ketoconazole; vehicle, and 0.32, 0.56, 1.0, 1.78, 3.2, 5.6, 10, 17.8, and 32 (three rats per sex) mg/kg. Upper middle: The rate-decreasing effects of mitragynine. Dose of mitragynine alone; vehicle, and 3.2, 5.6, 10, 17.8, 32, and 56 mg/kg. Mitragynine doses in the presence of ketoconazole; vehicle, and 0.32, 0.56, 1.0, 1.78, 3.2, 5.6, 10, 17.8, 32, and 56 mg/kg. Upper right: The antinociceptive effects of mitragynine. Dose of mitragynine alone; vehicle, and 3.2, 5.6, 10, 17.8, 32 and 56 mg/kg. Mitragynine doses in the presence of ketoconazole; vehicle, and 0.32, 0.56, 1.0, 1.78, 3.2, 5.6, 10, 17.8, 32, and 56 mg/kg. Lower left: The discriminative stimulus effects of morphine. Doses of morphine alone; vehicle, and 0.56, 1.0, 1.78, 3.2, 5.6, and 10 mg/kg. Morphine doses in the presence of ketoconazole; vehicle, and 0.56, 1.0, 1.78, 3.2, 5.6, and 10 (four females and three males) mg/kg. Lower middle: The rate-decreasing effects of morphine. Doses of morphine alone and in the presence of ketoconazole; vehicle, and 0.56, 1.0, 1.78, 3.2, 5.6, 10, 17.8, 32, and 56 mg/kg. Lower right: The antinociceptive effects of morphine. Doses of morphine alone in the presence of ketoconazole; vehicle, and 0.56, 1.0, 1.78, 3.2, 5.6, 10, 17.8, 32, and 56 mg/kg. Details for statistical analyses are shown in Supplemental Table 2 and Tables 2–4.

TABLE 2.

ED₅₀ values (95% CIs in parentheses) for the discriminative stimulus, rate-decreasing, and antinociceptive effects for various compounds in rats trained to discriminate 32 mg/kg mitragynine or 3.2 mg/kg morphine from their vehicle as shown in Figs. 2 and 3

The sample sizes are described in each figure legend. Each value is for a combination of females with males unless described. In each training drug, potency ratios per compound (95% CIs in parentheses) are calculated as a division of the ED₅₀ values for the rate-decreasing or antinociceptive effects from those for the discriminative-stimulus effects. Significant differences are italicized.

Test Drug	ED50 (95% CIs)			Potency Ratio (95% CIs)
	Discriminative Stimulus	Response Rate	Maximum Possible Effect	Rate-Decreasing / Discriminative Stimulus	Antinociceptive / Discriminative Stimulus
Rats Trained to Discriminate an Injection of 32 mg/kg Mitragynine from Its Vehicle
Mitragynine alone	18.9 (14.0, 24.2)	40.7 (32.3, 55.8)	Unable to determine due to an adverse reaction (lethality) at 100 mg/kg [up to 7.69% (5.13%) @ 56 mg/kg]	2.15 (1.33, 3.99)	Not Applicable
Mitragynine in the presence of 56 mg/kg ketoconazole	4.46 (3.57, 5.54)	33.7 (27.5, 43.3)	Unable to determine due to an adverse reaction (lethality) at 100 mg/kg [up to 18.8% (5.15%) @ 56 mg/kg]	7.56 (4.96, 12.1)	Not Applicable
Rats Trained to Discriminate an Injection of 3.2 mg/kg Morphine from Its Vehicle
7HMG alone	0.275 (0.0768, 0.411)	4.73 (3.25, 6.35)	10.5 (8.89, 12.5)	17.2 (7.91, 82.7)	38.2 (21.6, 163)
7HMG in the presence of 56 mg/kg ketoconazole	0.0285 (0.0224, 0.0346)	1.84 (1.47, 2.28)	2.56 (2.17, 3.02)	64.6 (42.5, 122)	89.8 (78.0, 135)
Mitragynine alone	29.6 (18.8, 55.9)	45.7 (36.4, 64.1)	Unable to determine due to an adverse reaction at 100 mg/kg [up to 7.49% (6.46%) @ 5.6 mg/kg]	1.54 (0.651, 3.41)	Not Applicable
Mitragynine in the presence of 56 mg/kg ketoconazole	6.31 (3.93, 8.69)	33.0 (27.4, 41.2)	Unable to determine due to an adverse reaction (lethality) at 100 mg/kg [up to 18.1% (5.37%) @ 56 mg/kg]	5.23 (3.15, 10.5)	Not Applicable
Morphine alone	1.46 (0.941, 1.90)	19.9 (16.4, 23.7)	35.8 (32.7, 39.3)	13.6 (8.63, 25.2)	24.5 (17.2, 41.8)
Morphine alone (females)	1.70 (0.752, 2.77)	16.4 (12.6, 20.2)	37.9 (30.4, 46.8)	9.65 (4.55, 26.9)	22.3 (11.0, 62.2)
Morphine alone (males)	1.10 (0.762, 1.42)	16.0 (10.8, 21.5)	34.3 (30.7, 38.3)	14.5 (7.61, 28.2)	31.2 (21.6, 50.3)
Morphine in the presence of 56 mg/kg ketoconazole	2.35 (1.76, 2.93)	23.2 (18.8, 28.5)	33.5 (29.9, 38.1)	9.87 (6.42, 16.2)	14.3 (10.2, 21.6)

Effects of Ketoconazole in Rats Trained with Morphine

Morphine produced a maximum of 98.0% (1.2%) morphine-appropriate responding at 10 mg/kg, decreased response rates to 1.4% (1.2%) vehicle control at 32 mg/kg, and increased MPEs to 89.8% (4.7%) at 56 mg/kg (Fig. 3, lower panels). The ED₅₀ values (95% CIs) of morphine to produce the discriminative-stimulus, rate-decreasing, and antinociceptive effects were 1.46 (0.941, 1.90), 19.9 (16.4, 23.7), and 35.8 (32.7, 39.3) mg/kg, respectively (Table 2). The discriminative-stimulus and antinociceptive effects of morphine did not differ significantly between males and females (F values ≤ 2.03; P values ≥ 0.204, Supplemental Table 2). However, the rate-decreasing effects of morphine significantly differed between males and females (F_1,54 = 7.14; P = 0.037, Supplemental Table 2). There was no significant sex × morphine dose interaction (F_9,54 = 1.75; P = 0.100, Supplemental Table 2). Ketoconazole (56 mg/kg by mouth) produced 0.4% (0.2%) morphine-appropriate responding, 101% (4.5%) rates of responding, and −1.1% (2.5%) MPE (Fig. 3, lower panels, open squares above vehicle). Ketoconazole (56 mg/kg) did not modify the potency of morphine to produce discriminative-stimulus, rate-decreasing, or antinociceptive effects (Fig. 3, lower panels; Table 3). In the presence of ketoconazole (56 mg/kg), there was no significant effect of sex or its interaction with morphine dose on morphine-appropriate responding, rates of responding, or antinociception (Supplemental Table 2).

TABLE 3.

Comparison of potency ratios of mitragynine, morphine, or 7HMG in the presence of ketoconazole pretreated to produce the discriminative stimulus, rate-decreasing, and antinociceptive effects in mitragynine- or morphine-trained rats relative to the corresponding compounds alone

Each value (95% CIs in parentheses) is for a combination of females with males unless described and calculated as a division of the ED₅₀ values of mitragynine, morphine, or 7HMG in the presence of ketoconazole (56 mg/kg, o.g., 30 minutes prior to sessions) pretreated from those of the corresponding compounds alone, as shown in Table 2. The sample sizes are described in each figure legend (Figs. 3 and 4). Significant differences are italicized.

Rats Trained with Mitragynine
Test Compound	Discriminative Stimulus	Response Rate	Antinociception
56 mg/kg Ketoconazole + Mitragynine vs. Mitragynine Alone	4.24 (2.52, 6.78)	1.21 (0.746, 2.03)	Not Applicable
Rats Trained with Morphine
Test compound	Discriminative Stimulus	Response Rate	Antinociception
56 mg/kg Ketoconazole + Morphine vs. Morphine Alone	0.621 (0.321, 1.08)	0.858 (0.575, 1.26)	1.07 (0.858, 1.33)
56 mg/kg Ketoconazole + Mitragynine vs. Mitragynine Alone	4.69 (2.16, 14.2)	1.38 (0.883, 2.34)	Not Applicable
56 mg/kg Ketoconazole + 7HMG vs. 7HMG Alone	9.65 (2.22, 18.3)	2.57 (1.43, 4.32)	4.10 (2.94, 5.76)

In rats discriminating morphine, the 56-mg/kg dose of mitragynine produced 72.3% (S.E.M.: 23.9%) morphine-appropriate responding, decreased response rates to 30.1% (10.3%) vehicle control rates of responding, and produced 1.2% (2.7%) antinociception (Fig. 4, upper panels, filled circles). The ED₅₀ values of mitragynine to produce morphine-appropriate responding and rate-decreasing effects were 29.6 (18.8, 55.9) and 45.7 (36.4, 64.1) mg/kg, respectively (Table 2). In rats discriminating morphine, there were no significant effects of the interaction of sex on the discriminative-stimulus, rate-decreasing, or antinociceptive effects of mitragynine (Supplemental Table 2). Mitragynine did not differ in its potencies to produce the discriminative stimulus [potency ratio 0.639 (95% CIs: 0.250, 1.29)] and rate-decreasing [0.891 (0.504, 1.53)] effects across the training drugs. In the presence of ketoconazole (56 mg/kg), mitragynine produced 99.5% (S.E.M.: 0.2%) morphine-appropriate responding at 32 mg/kg; the 56-mg/kg dose of mitragynine markedly decreased the rates of responding to 2.8% (2.4%) and produced 18.1% (5.4%) antinociception (Fig. 4, upper panels, open squares). Ketoconazole (56 mg/kg) produced a significant (4.7-fold) leftward shift in the dose-effect function of mitragynine to produce morphine-appropriate responding, whereas there was no significant effect of ketoconazole on the rate-decreasing and antinociceptive effects of mitragynine (Fig. 4, upper panels; Table 3). In the presence of ketoconazole (56 mg/kg), a two-way repeated-measures ANOVA indicated no significant effect of sex or its interaction with mitragynine dose on rate-decreasing or antinociceptive effects (Supplemental Table 2). In addition, there was no significant effect of sex on morphine-lever responding for mitragynine; however, there was a significant interaction between sex and mitragynine dose for morphine-lever responding when mitragynine was combined with ketoconazole (Supplemental Table 2). Post hoc analyses indicated significant sex differences at 3.2 and 5.6 mg/kg mitragynine for morphine-lever responding (Supplemental Table 2); however, the 95% CIs for the ED₅₀ values of mitragynine, in combination with ketoconazole, to produce the morphine-like discriminative-stimulus overlapped between females and males (Table 2). Thus, there was no significant sex difference of mitragynine to produce morphine-like discriminative-stimulus effects regardless of the presence or absence of ketoconazole.

Fig. 4.

Effects of substitution of mitragynine or 7HMG for morphine in the presence of ketoconazole. Abscissae: Vehicle and compound dose in mg/kg (i.p., log scale). Ordinates: Left panels, percentage of responses on morphine-appropriate lever; middle panels, percentage of mean rates of responding after vehicle administration during intertest sessions; right panels, percentage of MPEs. Each point represents the mean ± S.E.M. (n = 4 per sex per data point unless noted). The percentage of responses emitted on the morphine-appropriate lever was not plotted, when individual or group mean of response rates was less than 20% of the vehicle control levels or less than half of the sample size did not produce more than or equal to 20% of the control rate of responding. Mitragynine and 7HMG were administered i.p. at 30 and 15 minutes before sessions, respectively, whereas ketoconazole (56 mg/kg) was administered o.g. at 30 minutes before sessions. Upper left: The morphine-like discriminative stimulus effects of mitragynine. Dose of mitragynine alone; vehicle, and 5.6, 10, 17.8, 32, and 56 mg/kg. Mitragynine doses in the presence of ketoconazole; vehicle, and 1.0, 1.78, 3.2, 5.6, 10, 17.8 (three females and four males), and 32 (three females and four males) mg/kg. Upper middle: The rate-decreasing effects of mitragynine. Dose of mitragynine alone; vehicle, and 5.6, 10, 17.8, 32, and 56 mg/kg. Mitragynine doses in the presence of ketoconazole; vehicle, and 1.0, 1.78, 3.2, 5.6, 10, 17.8, 32, and 56 mg/kg. Upper right: The antinociceptive effects of mitragynine. Dose of mitragynine alone; vehicle, and 5.6, 10, 17.8, 32 and 56 mg/kg. Mitragynine doses in the presence of ketoconazole; vehicle, and 1.0, 1.78, 3.2, 5.6, 10, 17.8, 32, and 56 mg/kg. Lower left: The morphine-like discriminative stimulus effects of 7HMG. Doses of 7HMG alone; vehicle, and 0.1, 0.178, 0.32, 0.56 (three females and four males), 1.0 (three females and four males), 1.78 (three females and four males), and 3.2 (two females and three males) mg/kg. 7HMG doses in the presence of ketoconazole; vehicle, and 0.01, 0.0178, 0.032, 0.056, 0.1, 0.178, 0.32 (three females and four males), 0.56 (three females and four males), 1.0 (three females and four males), and 1.78 (one female and three males) mg/kg. Lower middle: The rate-decreasing effects of 7HMG. Doses of 7HMG alone; vehicle, and 0.1, 0.178, 0.32, 0.56, 1.0, 1.78, 3.2, 5.6, 10, and 17.8 mg/kg. 7HMG doses in the presence of ketoconazole; vehicle, and 0.01, 0.0178, 0.032, 0.056, 0.1, 0.178, 0.32, 0.56, 1.0, 1.78, 3.2, and 5.6 mg/kg. Lower right: The antinociceptive effects of 7HMG. Doses of 7HMG alone; vehicle, and 0.1, 0.178, 0.32, 0.56, 1.0, 1.78, 3.2, 5.6, 10, and 17.8 mg/kg. 7HMG doses in the presence of ketoconazole; vehicle, and 0.01, 0.0178, 0.032, 0.056, 0.1, 0.178, 0.32, 0.56, 1.0, 1.78, 3.2, and 5.6 mg/kg. Details for statistical analyses are shown in Supplemental Table 2 and Tables 2–4.

7HMG at 1.0 mg/kg produced a maximum of 99.7% (0.1%) morphine-appropriate responding and at 17.8 mg/kg eliminated responding and produced 78.6% (8.4%) antinociception (Fig. 4, lower panels, filled downward triangles). The ED₅₀ values of 7HMG to produce morphine-appropriate responding, rate-decreasing, and antinociceptive effects were 0.28 (0.08, 0.41), 4.73 (3.25, 6.35), and 10.5 (8.89, 12.5) mg/kg, respectively (Table 2). There was no significant interaction of sex on morphine-appropriate responding, rate-decreasing effects, and antinociception (Supplemental Table 2). In the presence of ketoconazole (56 mg/kg), 7HMG produced 94.8% (S.E.M.: 3.4%) morphine-appropriate responding at 1.78 mg/kg; the 5.6-mg/kg dose of 7HMG markedly decreased response rates to 0.01% (S.E.M.: 0.01%) and produced 100% antinociception (Fig. 4, lower panels, open squares). Ketoconazole (56 mg/kg) produced significant 9.7- and 4.1-fold leftward shifts in the dose-effect functions of 7HMG to produce morphine-like discriminative-stimulus effects and antinociception; however, there was no significant effect of 56 mg/kg ketoconazole on the rate-decreasing effect of 7HMG (Fig. 4, lower panels, open squares; Table 3). In the presence of ketoconazole (56 mg/kg), there was no significant effect of sex or its interaction with 7HMG dose on its discriminative-stimulus, rate-decreasing, or antinociceptive effects (Supplemental Table 2).

In Vitro Receptor Binding

The receptor binding affinity of ketoconazole and kratom alkaloids at the human opioid receptor subtypes was compared with those of the reference opioid receptor-ligand morphine (Fig. 5; Table 4). Ketoconazole was found to have negligible affinities for the KOR (>10,000 nM) and MOR (>10,000 nM) and higher yet still relatively low affinity at the DOR [K_i = 4390 (2120–9650) nM]. The lack of MOR binding of ketoconazole compared with mitragynine and 7HMG (Table 4) suggests no MOR pharmacodynamic interactions of ketoconazole with mitragynine and 7HMG.

Fig. 5.

Displacement of radioligands for opioid receptor subtypes by various compounds studied. Ordinates: Percentage of specific radiotracer bound to membrane preparations as described in the Materials and Methods section and Supplemental Table 1. Abscissae: Concentrations of each competing compound (log scale). The left panel shows the displacement of [3H]DAMGO for MORs. The middle panel shows the displacement of [3H]U69,593 for KORs. The right panel shows the displacement of [3H]DADLE for DORs. Each data point represents the mean of the results of three repeated experiments with vertical bars representing S.E.M.s (n = 3) from at least three independent triplicate replications per sample. The results were selected from at least three independent replications per sample as mean percent specific binding values resulting from a global modeling of the entire concentration-effect functions. The data for 7HMG, mitragynine, and morphine were from the literature (Obeng et al., 2021).

TABLE 4.

Inhibition of binding of the radioligands labeling DOR, KOR, and MOR

Values are K_i values (nM) for displacement of the listed radioligands. The displacement curves are shown in Fig. 5.

Compound	DOR Ki Value ± S.E.M.	ΚΟR Ki Value ± S.E.M.	MOR Ki Value ± S.E.M.	KOR/MOR	DOR/MOR	DOR/KOR
7HMGa	243 ± 70.4	220 ± 20.0	77.9 ± 16.6	2.82	3.12	1.15
Ketoconazole	4390 ± 638	>10,000	>10,000	Not Applicable	Not Applicable	Not Applicable
Mitragyninea	6800 ± 721	1530 ± 143	709 ± 91.2	2.40	9.60	4.00
Morphinea	250 ± 18.0	40.4 ± 13.9	4.04 ± 0.560	9.64	59.6	6.19

^a(Obeng et al., 2021).

Discussion

The goal of this study was to assess the contribution of the CYP3A isoform to the metabolism of the low-efficacy MOR agonist mitragynine, particularly to its 7-hydroxy metabolite 7HMG, which has higher potency and efficacy than mitragynine. We hypothesized that the CYP3A inhibitor ketoconazole would increase the plasma concentration of mitragynine by preventing its metabolism and concomitantly decrease plasma concentrations of 7HMG. If conversion to 7HMG drove the behavioral effects of mitragynine (i.e., mitragynine is a prodrug), one outcome might have been a decrease in the potency of mitragynine to produce MOR-related behavioral effects in the presence of ketoconazole. On the other hand, ketoconazole-induced increases in potency would indicate that the activity of mitragynine at MOR is important for behavioral effects.

The in vitro metabolism study in RLMs demonstrated that ketoconazole decreased the metabolism of mitragynine and subsequent formation of 7HMG. These results are consistent with previous findings in mouse and human liver microsomes (Kamble et al., 2019; Kruegel et al., 2019), and demonstrate that CYP3A plays a predominant role in the metabolism of mitragynine in rats. Based on this in vitro observation, ketoconazole was expected to reduce the clearance of mitragynine. In turn, this was expected to reduce formation of 7HMG, which would help determine the contribution of this higher-efficacy MOR agonist metabolite to the behavioral effects of mitragynine. In drug-naïve male rats, ketoconazole pretreatment (30 mg/kg, o.g.) in combination with a behaviorally active dose of mitragynine (13.3 mg/kg, o.g.) resulted in a 2.3-fold increase in the C_max and a 2.6-fold increase in the T_max of mitragynine. The systemic exposure of mitragynine was increased 2.2-fold by ketoconazole, the likely result of reduced metabolic clearance. However, the decreased metabolic clearance of mitragynine did not result in a reduction of the exposure of the CYP3A-mediated metabolite 7HMG. Alternatively, there was a 2.2-fold increase in AUC_last of 7HMG after ketoconazole pretreatment. Overall, there was no net change in the %AUC ratio of 7HMG to mitragynine upon ketoconazole pretreatment (Table 1). We therefore evaluated the involvement of CYP3A in the metabolism of 7HMG. The incubation of 7HMG with RLMs supplemented with NADPH in the presence of ketoconazole showed a 1.8-fold reduction in the rate of metabolism of 7HMG. Collectively, the current results suggest that CYP3A isoforms are involved in the metabolism of both mitragynine and 7HMG in rats (Fig. 1). Thus, ketoconazole pretreatment in rats appears to have reduced the systemic clearance of both mitragynine and 7HMG. This could explain the lack of discernable net change in the %AUC ratio of 7HMG to mitragynine in vivo after ketoconazole pretreatment. Overall, these results suggest that unlike the in vitro observations in mice and humans (Kamble et al., 2019; Kruegel et al., 2019), in vivo 7HMG formation is either not solely dependent on CYP3A in rats and/or the subsequent metabolism of 7HMG is also dependent on CYP3A enzyme.

Ketoconazole was expected to modify the potency of mitragynine through pharmacokinetic mechanisms. To examine potential pharmacodynamic mechanisms, we assessed the in vitro receptor binding affinity of ketoconazole at human opioid receptor subtypes. The results demonstrated that ketoconazole has negligible binding affinity for the human opioid receptor subtypes (Fig. 5) and suggested that ketoconazole and opioid receptor agonists would not interact at opioid receptors to modify each other’s effects. In agreement with the pharmacokinetic results, ketoconazole significantly shifted the dose-effect function for discriminative stimulus effects of mitragynine in both the mitragynine and morphine discrimination assays 4.2-fold and 4.7-fold leftward, respectively. The pharmacological selectivity of ketoconazole for CYP3A inhibition was strongly suggested by its failure to change the potency of morphine to produce discriminative stimulus or antinociceptive effects. Morphine is metabolized predominantly through the UDP-glucuronosyltransferase (UGT) UGT2B1 isoform in rats (Kimura et al., 2017). The insensitivity of the in vivo MOR activity of morphine to ketoconazole suggests that any allosteric modulatory effects of ketoconazole on MOR activity are at least orthosteric agonist specific. Collectively, these results strongly suggest that CYP3A is a key isoform for metabolism of mitragynine and that inhibition of this isoform has potential to create drug-drug interactions and an increase in the potency of mitragynine.

When designing this study, we did not anticipate that CYP3A also plays an important role in the metabolism of 7HMG. Based on the results obtained from RLMs, it appeared that ketoconazole might also inhibit metabolism of 7HMG in rats. The measurement of plasma 7HMG formed after administration of mitragynine and quantitative comparisons of AUC in the presence versus the absence of ketoconazole were consistent with the 7HMG metabolism being inhibited by ketoconazole. To test this hypothesis more directly, ketoconazole was combined with administration of 7HMG. In morphine-trained rats, ketoconazole increased the potency of 7HMG 9.7-fold to produce discriminative stimulus effects and 4.1-fold to produce antinociceptive effects. Because pharmacological inhibition of CYP3A inhibited the metabolism of both mitragynine and 7HMG, it was not possible to use this approach to assess the extent to which mitragynine is a prodrug; that is, to determine whether conversion to and subsequent pharmacological actions of 7HMG are essential to the effects of mitragynine administration. Because 7HMG was formed even when ketoconazole was combined with mitragynine, it appears that isoforms besides CYP3A are involved in the formation of 7HMG. No matter the mechanism by which mitragynine is converted to 7HMG, the formation of 7HMG would not be affected by ketoconazole-induced inhibition of CYP3A.

The effects of ketoconazole in combination with mitragynine and 7HMG on rates of lever-pressing were smaller than its effects on the discriminative stimulus effects of the two compounds. If the increases in potency were simply due to increased compound exposure, then ketoconazole might have been expected to influence the two effects similarly. The pharmacological mechanisms underlying discriminative-stimulus effects are generally more pharmacologically specific than those underlying disruptions in response rates. The discriminative-stimulus effects of morphine and mitragynine are mediated by MOR, whereas additional receptor types mediate the rate-decreasing effects of these two drugs (Obeng et al., 2020, 2021). Ketoconazole itself may be impacting response rates through mechanisms unrelated to its inhibitory actions on CYP3A; these direct effects of ketoconazole may be producing unexpected interactions with the rate-decreasing of mitragynine and 7HMG that do not generalize to discriminative-stimulus effects.

Mitragynine lacked antinociceptive effects in rats even in the presence of ketoconazole, consistent with the notion that the MOR agonist efficacy of mitragynine is too low to exert antinociceptive effects against acute noxious heat, which requires relatively high MOR efficacy for effects. In fact, mitragynine and another low-efficacy MOR agonist, nalbuphine, antagonized the antinociceptive effects of higher efficacy MOR agonist morphine using the same hot plate assay, whereas both mitragynine and nalbuphine potentiated the discriminative-stimulus effects of morphine (Obeng et al., 2021). Thus, the lack of antinociceptive effects of mitragynine even in the presence of ketoconazole further supports that mitragynine has low, if any, efficacy at MORs (Obeng et al., 2020, 2021). However, mitragynine might increase hot plate latency if a lower plate temperature is used.

Limitations in the design of the present in vivo pharmacokinetic and functional studies are as follows. Naïve rats were used for the pharmacokinetic study, whereas the behavioral tests included rats that had an extensive history of exposure to morphine or mitragynine. The drug discrimination assay was employed because it is a highly selective bioassay for MOR activity (e.g., Obeng et al., 2021). In addition, different routes of administration of mitragynine (o.g. and i.p. for the present pharmacokinetic and functional studies, respectively) were used. The i.p. route of administration is preferred for long-term repeated drug administration often required for drug discrimination (Katagiri et al., 1988; Harun et al., 2015). Kratom is commonly consumed orally; therefore, pharmacokinetic studies of mitragynine were assessed after o.g. dose. These methodological parameters notwithstanding, the present results identify CYP3A as an important isoform for the metabolism of mitragynine as well as 7HMG. In summary, the present pharmacokinetic and pharmacodynamic results convergently suggest that CYP3A is responsible for the in vivo metabolism of both mitragynine and 7HMG in rats. These data suggest that CYP3A inhibitors could increase the exposure and MOR-mediated activity of both mitragynine and 7HMG. Further studies in other species are warranted to translate these observations into human kratom users.

Abbreviations

AUC

area under the plasma concentration-time curve

CI

confidence interval

CYP3A

cytochrome P450 3A

DADLE

[D-Ala², D-Leu⁵]-enkephalin

DAMGO

[D-Ala², N-MePhe⁴, Gly-ol]-enkephalin

DOR

δ-opioid receptor

FR

fixed ratio

IS

internal standard

K_i

inhibition constant

KOR

k-opioid receptor

LED

light-emitting diode

MOR

μ-opioid receptor

MPE

maximum possible effect

o.g.

oral gavage

RLM

rat liver microsome

T_max

time to reach C_max

UPLC-MS/MS

ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry

Authorship Contributions

Participated in research design: Kamble, Obeng, McMahon, Sharma, Hiranita.

Conducted experiments: Kamble, Obeng, Restrepo, King, Berthold, Kanumuri, Gamez-Jimenez, Pallares, Patel, Ho.

Contributed new reagents or analytic tools: León, McCurdy.

Performed data analysis: Kamble, Obeng, Restrepo, Sharma, Hiranita.

Wrote or contributed to the writing of the manuscript: Kamble, Obeng, León, Restrepo, King, Berthold, Kanumuri, Gamez-Jimenez, Pallares, Patel, Ho, Hampson, McCurdy, McMahon, Wilkerson, Sharma, Hiranita.