Structural elucidation of tramadol, its derivatives, and metabolites using chemical derivatization and liquid chromatography-high-resolution tandem mass spectrometry.

RATIONALE: Tramadol (T) is a strong painkiller drug that belongs to the opioid analgesic group. Several accidental intoxication cases after oral administration of T have been reported in the past decade. Tramadol, its derivatives, and metabolites present information-limited mass spectra with one prominent peak representing the amine-containing residue; therefore, their structural determination based on both electron impact mass spectrometry (EI-MS) and ESI-MS/MS spectra could be misleading. METHODS: A novel analytical method for the structural elucidation of tramadol, its four homologs, and its two main phase I metabolites (N-desmethyltramadol and O-desmethyltramadol) was developed using chemical modification and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS) with Orbitrap technology. RESULTS: After chemical derivatization, each of the investigated T series exhibited informative mass spectra that enabled better exposition of their structures. The developed method was successfully implemented to explicitly identify the structures of tramadol and its N-desmethyltramadol metabolite in urine samples at low ng/mL levels. CONCLUSIONS: An efficient derivatization-aided strategy was developed for rapidly elucidating the structure of tramadol-like compounds. The method is intended to assist forensic chemists in better diagnosing T and its analogs and metabolites in clinical or forensic toxicology laboratories.

PHARMACOKINETICS OF TRAMADOL AND O-DESMETHYLTRAMADOL IN GIANT TORTOISES (CHELONOIDIS VANDENBURGHI, CHELONOIDIS VICINA).

The objective of this study was to determine the pharmacokinetics of two orally administered doses of tramadol (1 mg/kg and 5 mg/kg) and its metabolite, O-desmethyltramadol (M1) in giant tortoises (Chelonoidis vandenburghi, Chelonoidis vicina). Eleven giant tortoises (C. vandenburghi, C. vicina) received two randomly assigned, oral doses of tramadol (either 1 mg/kg or 5 mg/kg), with a washout period of 3 wk between each dose. The half-life (t½) of orally administered tramadol at 1 mg/kg and 5 mg/kg was 11.9 ± 4.6 h and 13.2 ± 6.1 h, respectively. After oral administration of tramadol at 1 mg/kg and 5 mg/kg, the maximum concentration (Cmax) was 125 ± 69 ng/ml and 518 ± 411 ng/ml, respectively. There were not enough data points to determine pharmacokinetic (PK) parameters for the M1 metabolite from either dose. Tramadol administered orally to giant tortoises at both doses provided measurable plasma concentrations of tramadol for approximately 48 h with occasional transient sedation. Oral tramadol at 5 mg/kg, on average, achieves concentrations of >100 ng/ml, the reported human therapeutic threshold, for 24 h. Based on the low levels of M1 seen in this study, M1 may not be a major metabolite in this taxon.

Intoxication by 3-MeO-PCP and O-desmethyltramadol: an unusual NPS mix.

Over the past few years, the new psychoactive substances' phenomenon has been continuously studied. Its dynamic context is characterized by a broad diversity of substances, including several groups, such as synthetic cathinones, synthetic opiates, and synthetic cannabinoids. However, and due both to this diversity and to the low number of detected cases, information on intoxication reports is always important, in order to understand their biological mechanisms. In this case, a male individual was found unresponsive, with some different powders and paraphernalia near him. After toxicological analysis to the powders, paraphernalia, and whole blood samples, five different compounds were identified. From these, two of them (3-MeO-PCP and o-desmethyltramadol) were identified and quantitated in the whole blood sample. The obtained results suggested that death was due to the presence and action of these two substances, in what may be considered an unusual mix of NPS. This case highlights the value of evaluating all the traces found in the scene investigation and the need of sending all the paraphernalia found for toxicological examination, together with all the possible information obtained on the scene, namely by relatives or witnesses. On the other hand, this case shows the significance of broad-spectrum analytical methods, in order to detect and identify, as specifically as possible, eventual substances present and used by victims.

Hepatotoxic effect of tramadol and O-desmethyltramadol in HepG2 cells and potential role of PI3K/AKT/mTOR.

1. The aim of this study was to compare the in vitro cytotoxic effect of tramadol and M1 metabolite in HepG2 cell line, the underlying mechanism, and PI3K/AKT/mTOR as potential target.2. Concentrations representing therapeutic level for tramadol (2 µM) and M1 metabolite (0.5 µM) were used. In addition, other increasing concentrations representing higher toxic levels were used (6, 10 µM for tramadol and 1.5, 2.5 µM for M1 metabolites). Cytotoxicity was assessed at 24, 48 and 72 h.3. Both tramadol and M1 metabolites were able to produce cytotoxicity in a dose and time dependent manner. Insignificant difference was detected between cells exposed to tramadol and M1 metabolite at therapeutic concentrations. Tramadol-induced apoptotic and autophagic cell death while M1 metabolite-induced apoptosis only. For PI3K/AKT/mTOR pathway, the therapeutic concentration of tramadol was only able to increase phosphorylation of AKT while higher toxic concentrations were able to increase phosphorylation of whole pathway; Meanwhile, M1 metabolite was able to increase the phosphorylation of the whole pathway significantly in therapeutic and toxic concentrations.4. In conclusion, both tramadol and M1 are equally cytotoxic. Apoptosis and autophagy both mediate hepatic cell death. PI3K/AKT pathway is involved in apoptosis induction while autophagy is regulated through mTOR independent pathway.

A Reversed-Phase Mode LC-MS/MS Method Using a Polysaccharide Chiral Selector for Simultaneous Quantitation of Each Enantiomer of Tramadol and its Metabolites in Human Plasma and Evaluation of CYP-Mediated Stereoselective Demethylation.

BACKGROUND: The enantiomeric pharmacokinetics and metabolism of tramadol and its metabolites have not fully been understood. This study aimed to develop a reversed-phase mode liquid chromatography coupled to a tandem mass spectrometry method for the enantiomeric quantitation of tramadol and its metabolites in human plasma and to evaluate the stereoselective demethylation. METHODS: Racemic tramadol and its metabolites in plasma specimens were separated using a chiral selector coated with cellulose tris(3,5-dimethylphenylcarbamate) on silica gel under a reversed-phase mode. The mass spectrometer ran in the positive ion multiple-reaction monitoring mode. This method was performed to quantify plasma samples from 20 cancer patients treated with oral tramadol. The stereoselective demethylation was evaluated using recombinant cytochrome P450 (CYP) enzymes. RESULTS: The calibration curves of (+)- and (-)-tramadol, (+)- and (-)-O-desmethyltramadol (ODT), and (+)- and (-)-N-desmethyltramadol (NDT) were linear over the plasma concentration ranges of 6.25-800, 1.25-160, and 3.13-400 ng/mL for the respective enantiomers. In the present method, the intra- and inter-day accuracies and imprecisions were 94.2%-108.3% and 0.5%-6.0% for all analytes. The plasma concentrations of (+)-tramadol and NDT were higher than those of (-)-enantiomers. In contrast, no differences were observed between the plasma concentrations of (+)- and (-)-ODT. In the demethylation assay, the O-demethylations of tramadol and NDT by CYP2D6 were (-)-form-selective. CONCLUSIONS: The present method can be useful in the enantiomeric evaluation of tramadol and its metabolites in human plasma. Although CYP2D6 contributed to the stereoselective demethylation of tramadol, remarkable differences between (+)- and (-)-ODT were not observed in the plasma of the cancer patients.

Intrinsic clearance rate of O-desmethyltramadol (M1) by glucuronide conjugation and phase I metabolism by feline, canine and common brush-tailed possum microsomes.

Quantitative aspects of in vitro phase II glucuronidative metabolism of O-desmethyltramadol (O-DSMT or M1), the active metabolite of the analgesic drug tramadol, by feline, canine and common brush-tailed possum hepatic microsomes are described.Whilst previous studies have focused on the phase I conversion of tramadol to M1, this is the first report in which the phase II glucuronidative metabolic pathway of M1 has been isolated by an in vitro comparative species study.Using the substrate depletion method, microsomal phase II glucuronidative in vitro intrinsic clearance (Clint) of M1 was determined.The in vitro Clint (mean ± SD) by pooled common brush-tailed possum microsomes was 9.9 ± 1.7 µL/min/mg microsomal protein whereas the in vitro Clint by pooled canine microsomes was 1.9 ± 0.07 µL/min/mg microsomal protein. The rate of M1 depletion by feline microsomes, as measured solely by high pressure liquid chromatography, was too slow to determine. Liquid chromatography-mass spectrometry identified O-DSMT glucuronide in samples generated from all three species' microsomes, although the amount detected under the feline condition was minimal.This study indicates that M1 likely undergoes in vitro phase II glucuronidation by canine and common brush-tailed possum microsomes and, to a minor extent, by feline microsomes. The rate of depletion of M1 by phase I metabolism was also undertaken.When incubated with phase I co-factors and common brush-tailed possum microsomes or canine microsomes, M1 had an in vitro Clint of 47.6 and 22.8 µL/min/mg microsomal protein, respectively. However, due to a lack of CYP2B-like activity in the feline liver, unsurprisingly, M1 did not deplete when incubated with feline microsomes. Consequently, major M1 elimination pathways, using feline microsomes, were not determined."

Inhibition by O-desmethyltramadol of glutamatergic excitatory transmission in adult rat spinal substantia gelatinosa neurons.

To reveal cellular mechanisms for antinociception produced by clinically used tramadol, we investigated the effect of its metabolite O-desmethyltramadol (M1) on glutamatergic excitatory transmission in spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons. The whole-cell patch-clamp technique was applied at a holding potential of -70 mV to SG neurons of an adult rat spinal cord slice with an attached dorsal root. Under the condition where a postsynaptic action of M1 was inhibited, M1 superfused for 2 min reduced the frequency of spontaneous excitatory postsynaptic current in a manner sensitive to a µ-opioid receptor antagonist CTAP; its amplitude and also a response of SG neurons to bath-applied AMPA were hardly affected. The presynaptic effect of M1 was different from that of noradrenaline or serotonin which was examined in the same neuron. M1 also reduced by almost the same extent the peak amplitudes of monosynaptic primary-afferent Aδ-fiber and C-fiber excitatory postsynaptic currents evoked by stimulating the dorsal root. These actions of M1 persisted for >10 min after its washout. These results indicate that M1 inhibits the quantal release of L-glutamate from nerve terminals by activating µ-opioid but not noradrenaline and serotonin receptors; this inhibition is comparable in extent between monosynaptic primary-afferent Aδ-fiber and C-fiber transmissions. Considering that the SG plays a pivotal role in regulating nociceptive transmission, the present findings could contribute to at least a part of the inhibitory action of tramadol on nociceptive transmission together with its hyperpolarizing effect as reported previously.

Oral Coadministration of Fluconazole with Tramadol Markedly Increases Plasma and Urine Concentrations of Tramadol and the O-Desmethyltramadol Metabolite in Healthy Dogs.

Tramadol is used frequently in the management of mild to moderate pain conditions in dogs. This use is controversial because multiple reports in treated dogs demonstrate very low plasma concentrations of O-desmethyltramadol (M1), the active metabolite. The objective of this study was to identify a drug that could be coadministered with tramadol to increase plasma M1 concentrations, thereby enhancing analgesic efficacy. In vitro studies were initially conducted to identify a compound that inhibited tramadol metabolism to N-desmethyltramadol (M2) and M1 metabolism to N,O-didesmethyltramadol (M5) without reducing tramadol metabolism to M1. A randomized crossover drug-drug interaction study was then conducted by administering this inhibitor or placebo with tramadol to 12 dogs. Blood and urine samples were collected to measure tramadol, tramadol metabolites, and inhibitor concentrations. After screening 86 compounds, fluconazole was the only drug found to inhibit M2 and M5 formation potently without reducing M1 formation. Four hours after tramadol administration to fluconazole-treated dogs, there were marked statistically significant (P < 0.001; Wilcoxon signed-rank test) increases in plasma tramadol (31-fold higher) and M1 (39-fold higher) concentrations when compared with placebo-treated dogs. Conversely, plasma M2 and M5 concentrations were significantly lower (11-fold and 3-fold, respectively; P < 0.01) in fluconazole-treated dogs. Metabolite concentrations in urine followed a similar pattern. This is the first study to demonstrate a potentially beneficial drug-drug interaction in dogs through enhancing plasma tramadol and M1 concentrations. Future studies are needed to determine whether adding fluconazole can enhance the analgesic efficacy of tramadol in healthy dogs and clinical patients experiencing pain.

Suppression of Human Natural Killer Cells by Different Classes of Opioids.

BACKGROUND: The use of regional and other opioid-sparing forms of anesthesia has been associated with a decrease in the recurrence of certain malignancies. Direct suppression of human natural killer cells by opioids has been postulated to explain this observation. However, the effect of different classes of opioids on suppression of natural killer cell cytotoxicity has not been systematically characterized. METHODS: After confirming that freshly isolated natural killer cells from peripheral human blood express opioid receptors, cells were incubated with increasing concentrations of clinically used or receptor-specific opioid agonists. We also evaluated the effect of pretreatment with receptor-specific antagonists or naloxone. Treated natural killer cells were then coincubated with a carboxyfluorescein succinimidyl ester-labeled target tumor cell line, K562. Annexin V staining was used to compare the percent of tumor cell apoptosis in the presence of opioid-pretreated and untreated natural killer cells. Treated samples were compared to untreated samples using Kruskal-Wallis tests with a post hoc Dunn correction. RESULTS: Morphine, methadone, buprenorphine, loperamide, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin, and U-50488 significantly decreased natural killer cell cytotoxicity. When natural killer cells were pretreated with naloxone, cyprodime, and nor-binaltorphimine before exposure to morphine, there was no difference in natural killer cytotoxicity, compared to the amount observed by untreated natural killer cells. Fentanyl, O-desmethyltramadol, and [D-Pen2,D-Pen5] enkephalin did not change natural killer cell cytotoxicity compare to untreated natural killer cells. CONCLUSIONS: Incubation of isolated natural killer cells with certain opioids causes a decrease in activity that is not observed after naloxone pretreatment. Suppression of natural killer cell cytotoxicity was observed with µ- and κ-receptor agonists but not δ-receptor agonists. These data suggest that the effect is mediated by µ- and κ-receptor agonism and that suppression is similar with many clinically used opioids.

Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6) are associated with long term tramadol treatment-induced oxidative damage and hepatotoxicity.

Our objective was to figure out whether CYP2D6 gene polymorphisms may account for long term tramadol-induced oxidative stress and hepatotoxicity in 60 patients receiving chronic tramadol treatment in Neurology and Rheumatology Outpatients Clinic, Zagazig University Hospitals, Egypt. As expected, CYP2D6*1 allele (wild type) frequency was significantly greater than CYP2D6*DUP, CYP2D6*4 and CYP2D6*10 alleles in both chronically tramadol-treated and control groups. In tramadol-treated patients, CYP2D6*DUP allele carriers followed by those carrying CYP2D6*1, displayed higher levels of urinary tramadol major active metabolite, O-desmethyltramadol (M1) and serum lipid peroxidation along with lower levels of total antioxidants than those carrying other impaired function alleles (CYP2D6*4&*10), suggesting oxidative stress. There were also significant increases in serum hepatic damage markers including alpha-glutathione transferase (α-GST) levels and liver function enzyme activities in *DUP and *1 carriers compared to carriers of other alleles. Moreover, we reported that in 42 patients with allele *1, tramadol caused mild to moderate hepatotoxicity (grades: 1-2) within 13-16 months while in 7 patients with duplicated allele (*DUP), tramadol caused moderate to severe hepatotoxicity (grades: 2-3) within 10-11 months (moderately longer period but shorter than that observed in allele *1), implying that exposure to tramadol for longer time in extensive and ultra-rapid metabolizers may contribute to hepatotoxicity development. Overall, our results suggest that CYP2D6 gene polymorphisms, particularly enhanced or normal function of CYP2D6, may increase the vulnerability to long term tramadol-induced hepatotoxicity through the enhancement of accumulation of tramadol bioactive metabolite (M1) and hence oxidative stress. Therefore, tramadol doses should be adjusted according to patient's CYP2D6 genotyping analysis to avoid hepatotoxicity.

Impact of CYP genotype and inflammatory markers on the plasma concentrations of tramadol and its demethylated metabolites and drug tolerability in cancer patients.

PURPOSE: Clinical responses to oral tramadol show a large variation in cancer patients. This study aimed to evaluate the impacts of cytochrome P450 (CYP) genotype and serum inflammatory markers on the plasma concentrations of tramadol and its demethylated metabolites and drug tolerability in cancer patients. METHODS: The predose plasma concentrations of tramadol and its demethylated metabolites were determined at day 4 or later in 70 Japanese cancer patients treated with oral tramadol. The CYP genotypes, serum interleukin-6 (IL-6) and C-reactive protein (CRP) levels, and the duration of tramadol treatment were evaluated. RESULTS: The CYP2D6 genotype did not affect the plasma tramadol concentration. The plasma concentration of O-desmethyltramadol and its ratio to tramadol were lower in the CYP2D6 intermediate and poor metabolizer (IM + PM) group than in the normal metabolizer (NM) group (P = 0.002 and P = 0.023). The plasma concentration of N-desmethyltramadol and its ratio to tramadol were higher in the CYP2D6 IM + PM group than in the NM group (P = 0.001 and P = 0.001). The CYP2B6*6 and CYP3A5*3 alleles had no effect on the plasma concentrations of tramadol and its demethylated metabolites. The serum IL-6 and CRP levels were inversely correlated with the plasma concentration ratios of N-desmethyltramadol to tramadol and of N,O-didesmethyltramadol to O-desmethyltramadol. The serum IL-6 level was associated with the treatment duration of oral tramadol. CONCLUSIONS: The CYP2D6 genotype but not the CYP2B6 and CYP3A5 genotypes affected the plasma concentrations of O- and N-desmethyltramadol through alteration of the tramadol metabolic pathway. The serum IL-6 level was associated with N-demethylation activity and tramadol tolerability.

Preliminary pharmacokinetics of tramadol hydrochloride after administration via different routes in male and female B6 mice.

OBJECTIVE: 1) To determine the pharmacokinetics of tramadol hydrochloride and its active metabolite, O-desmethyltramadol (M1), after administration through different routes in female and male C57Bl/6 mice; 2) to evaluate the stability of tramadol solutions; and 3) to identify a suitable dose regimen for prospective clinical analgesia in B6 mice. STUDY DESIGN: Prospective, randomized, blinded, parallel design. ANIMALS: A total of 18 male and 18 female C57Bl/6 mice (20-30 g). METHODS: Mice were administered 25 mg kg-1 tramadol as a bolus [intravenously (IV), intraperitoneally (IP), subcutaneously (SQ), orally per gavage (OSgavage)] over 25 hours [orally in drinking water (OSwater) or Syrspend SF (OSSyrsp)]. Venous blood was sampled at six predetermined time points over 4 to 31 hours, depending on administration route, to determine tramadol and M1 plasma concentrations (liquid chromatography and tandem mass spectrometry detection). Pharmacokinetic parameters were described using a noncompartmental model. The stability of tramadol in water (acidified and untreated) and Syrspend SF (0.20 mg mL-1) at ambient conditions for 1 week was evaluated. RESULTS: After all administration routes, Cmax was >100 ng mL-1 for tramadol and >40 ng mL-1 for M1 (reported analgesic ranges in man) followed by short half-lives (2-6 hours). The mean tramadol plasma concentration after self-administration remained >100 ng mL-1 throughout consumption time. M1 was found in the OSSyrs group only at 7 hours, whereas it was detectable in OSwater throughout administration. Tramadol had low oral bioavailability (26%). Short-lasting side effects were observed only after IV administration. Water and Syrspend SF solutions were stable for 1 week. CONCLUSIONS AND CLINICAL RELEVANCE: 1) At the dose administered, high plasma concentrations of tramadol and M1 were obtained, with half-life depending on the administration route. 2) Plasma levels were stable over self-consumption time. 3) Solutions were stable for 1 week at ambient conditions.

Influence of tramadol on acute thermal and mechanical cutaneous nociception in dogs.

OBJECTIVE: The aim of the study was to evaluate the influence of tramadol on acute nociception in dogs. STUDY DESIGN: Experimental, blinded, randomized, crossover study. ANIMALS: Six healthy laboratory Beagle dogs. METHODS: Dogs received three treatments intravenously (IV): isotonic saline placebo (P), tramadol 1 mg kg-1 (T1) and tramadol 4 mg kg-1 (T4). Thermal thresholds were determined by ramped contact heat stimulation (0.6 °C second-1) at the lateral thoracic wall. Mechanical thresholds (MT) were measured using a probe containing three blunted pins which were constantly advanced over the radial bone, using a rate of force increase of 0.8 N second-1. Stimulation end points were defined responses (e.g. skin twitch, head turn, repositioning, vocalization) or pre-set cut-out values (55 °C, 20 N). Thresholds were determined before treatment and at predetermined time points up to 24 hours after treatment. At each measurement point, blood was collected for determination of O-desmethyltramadol concentrations. The degree of sedation and behavioural side effects were recorded. Data were analysed by one-way anova and two-way anova for repeated measurements. RESULTS: Thermal nociception was not influenced by drug treatment. Mechanical nociception was significantly increased between P and T1 at 120 and 240 minutes, and between P and T4 at 30, 60, 240 and 420 minutes. T1 and T4 did not differ. O-desmethyltramadol (M1) maximum plasma concentrations (Cmax) were 4.2±0.8 ng mL-1 and 14.3±2.8 ng mL-1 for T1 and T4, respectively. Times to reach maximum plasma concentrations (Tmax) were 27.6±6.3 minutes for T1 and 32.1±7.8 minutes for T4. No sedation occurred. There were signs of nausea and mild to moderate salivation in both groups. CONCLUSION AND CLINICAL RELEVANCE: Tramadol was metabolized marginally to O-desmethyltramadol and failed to produce clinically relevant acute antinociception. Therefore, the use of tramadol for acute nociceptive pain is questionable in dogs.

SLC22A1/OCT1 Genotype Affects O-desmethyltramadol Exposure in Newborn Infants.

BACKGROUND: This study determined whether the SLC22A1 [encoding the organic cation transporter 1 (OCT1)] genotype could explain, in addition to the postmenstrual age (referring to gestational plus postnatal age) and CYP2D6 genotype, the tramadol (M) pharmacokinetic variability in early infancy. METHODS: Fifty infants, median postmenstrual age 39.5 (interquartile range: 36.8-41.3) weeks, received an i.v. M loading dose (2 mg/kg) followed by a continuous infusion (5-8 mg·kg·24 h). Blood was sampled from 4 to 24 hours after start of the M treatment, which generated 230 observations. M and O-desmethyltramadol (M1) concentrations were measured by high-performance liquid chromatography. RESULTS: Linear mixed-model analysis illustrated that the SLC22A1/OCT1 genotype was independently associated with a log-transformed M1/M ratio (P = 0.013), with carriers of <2 SLC22A1/OCT1 functional gene copies having a higher M1/M ratio (2.25; 95% CI, 2.01-2.48) than infants with 2 functional gene copies (1.86; 95% CI, 1.66-2.06). The CYP2D6/SLC22A1 combined genotype was associated with 57.8% higher M1/M ratio in carriers of ≥2 CYP2D6 functional gene copies and <2 SLC22A1/OCT1 functional gene copies compared with infants with <2 active CYP2D6 functional gene copies and SLC22A1/OCT1 normal activity (P < 0.001). CONCLUSIONS: These findings highlight the additional role of SLC22A1/OCT1 genetics in M1 exposure in neonates. They also suggest that OCT1 is already active early after birth, which may have impact on the disposition of other OCT1 substrates in this population.

Design, synthesis and biological evaluation of N-phenylalkyl-substituted tramadol derivatives as novel µ opioid receptor ligands.

AIM: Tramadol is an atypical opioid analgesic with low potential for tolerance and addiction. However, its opioid activity is much lower than classic opiates such as morphine. To develop novel analgesic and further explore the structure activity relationship (SAR) of tramadol skeleton. METHODS: Based on a three-dimensional (3D) structure superimposition and molecular docking study, we found that M1 (the active metabolite of tramadol) and morphine have common pharmacophore features and similar binding modes at the µ opioid receptor in which the substituents on the nitrogen atom of both compounds faced a common hydrophobic pocket formed by Trp2936.48 and Tyr3267.43. In this study, N-phenethylnormorphine was docked to the µ opioid receptor. It was found that the N-substituted group of N-phenethylnormorphine extended into a hydrophobic pocket formed by Trp2936.48 and Tyr3267.43. This hydrophobic interaction may contribute to the improvement of its opioid activities as compared with morphine. The binding modes of M1, morphine and N-phenethylnormorphine overlapped, indicating that the substituent on the nitrogen atoms of the three compounds may adopt common orientations. A series of N-phenylalkyl derivatives from the tramadol scaffold were designed, synthesized and assayed in order to generate a new type of analgesics. RESULTS: As a result, compound 5b was identified to be an active candidate from these compounds. Furthermore, the binding modes of 5b and morphine derivatives in the µ opioid receptor were comparatively studied. CONCLUSION: Unlike morphine-derived structures in which bulky N-substitution is associated with improved opioid-like activities, there seems to be a different story for tramadol, suggesting the potential difference of SAR between these compounds. A new type of interaction mechanism in tramadol analogue (5b) was discovered, which will help advance potent tramadol-based analgesic design.

Tramadol and its metabolite m1 selectively suppress transient receptor potential ankyrin 1 activity, but not transient receptor potential vanilloid 1 activity.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) and the transient receptor potential ankyrin 1 (TRPA1), which are expressed in sensory neurons, are polymodal nonselective cation channels that sense noxious stimuli. Recent reports showed that these channels play important roles in inflammatory, neuropathic, or cancer pain, suggesting that they may serve as attractive analgesic pharmacological targets. Tramadol is an effective analgesic that is widely used in clinical practice. Reportedly, tramadol and its metabolite (M1) bind to µ-opioid receptors and/or inhibit reuptake of monoamines in the central nervous system, resulting in the activation of the descending inhibitory system. However, the fundamental mechanisms of tramadol in pain control remain unclear. TRPV1 and TRPA1 may be targets of tramadol; however, they have not been studied extensively. METHODS: We examined whether and how tramadol and M1 act on human embryonic kidney 293 (HEK293) cells expressing human TRPV1 (hTRPV1) or hTRPA1 by using a Ca imaging assay and whole-cell patch-clamp recording. RESULTS: Tramadol and M1 (0.01-10 µM) alone did not increase in intracellular Ca concentration ([Ca]i) in HEK293 cells expressing hTRPV1 or hTRPA1 compared with capsaicin (a TRPV1 agonist) or the allyl isothiocyanate (AITC, a TRPA1 agonist), respectively. Furthermore, in HEK293 cells expressing hTRPV1, pretreatment with tramadol or M1 for 5 minutes did not change the increase in [Ca]i induced by capsaicin. Conversely, pretreatment with tramadol (0.1-10 µM) and M1 (1-10 µM) significantly suppressed the AITC-induced [Ca]i increases in HEK293 cells expressing hTRPA1. In addition, the patch-clamp study showed that pretreatment with tramadol and M1 (10 µM) decreased the inward currents induced by AITC. CONCLUSIONS: These data indicate that tramadol and M1 selectively inhibit the function of hTRPA1, but not that of hTRPV1, and that hTRPA1 may play a role in the analgesic effects of these compounds.

Regulation of cerebral CYP2D alters tramadol metabolism in the brain: interactions of tramadol with propranolol and nicotine.

1. Cytochrome P450 2D (CYP2D) protein is widely expressed across brain regions in human and rodents. We investigated the interactions between tramadol, a clinically used analgesic, and brain CYP2D regulators, by establishing concentration-time curves of tramadol and O-desmethyltramadol (M1) in rat cerebrospinal fluid (CSF) and plasma, as well as by analyzing the analgesia-time course of tramadol. 2. Propranolol (20 µg, intracerebroventricular injection), CYP2D inhibitor, prolonged the elimination t1/2 of tramadol (40 mg/kg, intraperitoneal injection) in the CSF; meanwhile, lower Cmax and AUC0-∞ values of M1 were observed. Nicotine (1 mg base/kg, subcutaneous injection, seven days), brain CYP2D inducer, induced a shorter Tmax and elevated Cmax of M1 in CSF. No differences in the peripheral metabolism of tramadol were observed following propranolol and nicotine pretreatment. Nicotine increased areas under the analgesia-time curve (AUC) for 0-45 min and 0-90 min of tramadol, which was attenuated by propranolol administration. The analgesic actions of tramadol positively correlated with cerebral M1 concentration. 3. The results suggest that the regulation of brain CYP2D by xenobiotics may cause drug-drug interactions (DDIs) of tramadol. Brain CYPs may play an important role in DDIs of centrally active substances.

Simultaneous Determination of Tramadol and its Metabolite in Human Plasma by GC/MS.

A simple and sensitive GC/MS method for the determination of tramadol and its metabolite (O-desmethyltramadol) in human plasma was developed and validated. Medazepam was used as an internal standard. The calibration curves were linear (r=0.999) over tramadol and O-desmethyltramadol concentrations ranging from 10 to 200 ng/mL and 7.5 to 300 ng/mL, respectively. The method had an accuracy of >95% and intra- and interday precision (RSD%) of ≤4.83% and ≤4.68% for tramadol and O-desmethyltramadol, respectively. The extraction recoveries were 97.6±1.21% and 96.3±1.66% for tramadol and O-desmethyltramadol, respectively. The LOQ using 0.5 mL human plasma was 10 ng/mL for tramadol and 7.5 ng/mL for O-desmethyltramadol. Stability studies showed that tramadol and O-desmethyltramadol were stable in human plasma after 8 h incubation at room temperature or after 1 week storage at -20°C with three freeze-thaw cycles. Also, this method was successfully applied to six patients who had been given an intravenous formulation of 100 mg tramadol with Cmax results of 2018.1±687.8 and 96.1±22.7 ng/mL for tramadol and O-desmethyltramadol, respectively.

Effects of tramadol and o-desmethyltramadol on canine innate immune system function.

OBJECTIVE: Tramadol is a commonly used opioid analgesic in dogs, particularly in dogs with a compromised immune system. An opioid may be selected for its immunomodulatory effects. Consequently, the objective of this study was to investigate the effects of tramadol on immune system function by evaluating the effect of tramadol and o-desmethyltramadol (M1) on the function of canine leukocytes in vitro. The hypothesis was that tramadol and M1 would not alter polymorphonuclear leukocyte (PMN) phagocytosis, PMN oxidative burst, or stimulated leukocyte cytokine production capacity of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10. STUDY DESIGN: In vitro pharmacodynamic study. ANIMALS: Six healthy dogs. METHODS: Blood from six dogs was obtained and incubated with various concentrations of tramadol and M1. Phagocytosis and oxidative burst were assessed using flow cytometry, and lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PG)-stimulated leukocyte production of TNF, IL-6, and IL-10 were measured using a canine specific multiplex assay. RESULTS: No differences were detected in phagocytosis or oxidative burst with any drug concentration. Tramadol did not alter leukocyte cytokine production, however, M1 significantly blunted IL-10 production. CONCLUSIONS: Tramadol and its metabolite M1 were sparing to PMN phagocytosis and oxidative burst in dogs in vitro. Tramadol did not alter leukocyte cytokine production, however, M1 blunted IL-10 production at clinically achievable concentrations suggesting that M1 may promote a proinflammatory shift. CLINICAL RELEVANCE: These data suggest that tramadol has minimal effect on phagocytosis and oxidative burst, and may promote a proinflammatory shift. Therefore, tramadol may be an ideal opioid analgesic in dogs at high risk of infection. Further investigation in vivo is warranted.

µ-Opioid receptor activation by tramadol and O-desmethyltramadol (M1).

Tramadol has been used as an analgesic for several decades. µ-Opioid receptors (µORs) are the major receptors that mediate the analgesic effects of opioids. Although µORs have been thought to be one of the sites of action of tramadol, there has been no report that directly proves whether tramadol is an agonist of µOR or not. In this study, we examined the effects of tramadol and its main active metabolite O-desmethyltramadol (M1), on the function of µORs using Xenopus oocytes expressing cloned human µORs. The effects of tramadol and M1 were evaluated using the Ca(2+)-activated Cl(-) current assay method for G(i/o)-protein-coupled receptors by using a µOR fused to G(qi5) (µOR-G(qi5)) in Xenopus oocytes. DAMGO [(D-Ala(2), N-MePhe(4), Gly(5)-ol)-enkephalin] evoked Cl(-) currents in oocytes expressing µOR-G(qi5) in a concentration-dependent manner. Tramadol and M1 also evoked Cl(-) currents in the oocytes expressing µOR-G(qi5); however, relatively higher concentrations (compared to DMAGO) were necessary to induce such currents. Tramadol and M1 had a direct effect on µORs expressed in Xenopus oocytes. Although the monoamine uptake system and several types of ligand-gated ion channels are thought to be one of the targets for tramadol, tramadol-induced antinociception may be mediated at least in part, by the direct activation of µORs.