Article Figures & Data
Figures
- Fig. 1.
Tramadol metabolic pathways evaluated in this study. Tramadol and the M1 and M2 metabolites have two chiral centers in the cyclohexane ring. All pharmaceutical preparations of tramadol are a racemic mixture of (+)-(1R,2R)-tramadol and (−)-(1S,2S)-tramadol, also known as (+)-tramadol and (−)-tramadol, respectively. In humans, racemic tramadol is O-demethylated by CYP2D6 to O-desmethyltramadol (M1) and N-demethylated by CYP2B6 and CYP3A4 to N-desmethyltramadol (M2).
- Fig. 2.
Species differences in formation rates of M1 (A) and M2 (B) and in the ratios of M1/M2 (C) from racemic (±)-tramadol by pooled dog (n = 27), human (n = 48), and cat (n = 16) liver microsomes. Bars represent the mean ± S.D. of triplicate independent determinations. *P < 0.001 versus DLMs (ANOVA with the Tukey test).
- Fig. 3.
Formation rates of M1 (A) and M2 (B) from racemic (±)-tramadol, (+)-tramadol, and (−)-tramadol by pooled (n = 27) DLMs. Bars represent the mean ± S.D. of triplicate independent determinations. *P < 0.001 versus DLMs (ANOVA with the Tukey test).
- Fig. 4.
Michaelis–Menten enzyme kinetic plots of M1 (A) and M2 (C) formation from (+)-tramadol and from (−)-tramadol by pooled (n = 27) DLMs. Also shown are Eadie-Hofstee plots of these same data (B and D, respectively). Each data point represents the mean of two independent determinations performed in duplicate, and the curves represent the model of best fit to the data. V/S, reaction velocity divided by substrate concentration.
- Fig. 5.
Formation rates of M1 (A and C) and M2 (B and D) from (+)-tramadol (A and B) and from (−)-tramadol (C and D) by recombinant canine P450s measured at the 5- and 100-µM substrate concentrations. Bars represent the mean ± S.D. of triplicate determinations. CYP, cytochrome P450.
- Fig. 6.
Michaelis–Menten enzyme kinetic plots of M1 formation by CYP2D15 (A) and M2 formation by CYP2B11 (C) from (+)-tramadol and from (−)-tramadol. Also shown are Eadie-Hofstee plots of these same data (B and D, respectively). Each data point represents the mean of three independent determinations performed in duplicate, and the curves represent the model of best fit to the data. V/S, XXX
- Fig. 7.
Estimated relative contributions of canine P450s to M1 and M2 formation from tramadol in the liver from (+)-tramadol and from (−)-tramadol. Intrinsic clearance estimates from Table 2 were normalized to hepatic P450 content using average published estimates for CYP2B11, CYP2C21, CYP2D15, and CYP3A12 in Beagle liver microsomes. Note that this evaluation does not include a possible contribution from CYP2C41 since a hepatic abundance estimate was not available for this P450.
- Fig. 8.
Selective inhibition of M1 formation from (+)-tramadol (A) and (−)-tramadol (C) by quinidine in pooled (n = 27) DLMs and CYP2D15, and selective inhibition of M2 formation from (+)-tramadol (B) and (−)-tramadol (D) by chloramphenicol in pooled (n = 27) DLMs and CYP2B11. Shown are the rates of metabolite formation (mean ± S.D. of triplicate determinations) in the presence of inhibitor (0.01–1000 µM) expressed as a percentage of the formation rate without inhibitor (control activity).
- Fig. 9.
Selective inhibition of M2 (but not M1) formation from (+)-tramadol and (−)-tramadol by anti-CYP2B11 immune serum in pooled (n = 27) DLMs. Shown are the rates of metabolite formation (mean ± S.D. of triplicate determinations) in the presence of anti-CYP2B11 immune serum (5:1 to 20:1 antiserum to microsome protein ratio) expressed as a percentage of the formation rate without antiserum (control activity).
- Fig. 10.
Effect of P450 inducers on the rate of M1 (A) and M2 (B) formation from (+)-tramadol and (−)-tramadol in pooled liver microsomes from dogs treated with rifampin, β-naphthoflavone, phenobarbital, and clofibric acid. Shown are the rates of metabolite formation (mean ± S.D. of triplicate determinations) in microsomes prepared from inducer-treated male Beagle dogs (pooled from two dogs per treatment) expressed as a ratio of the formation rate in microsomes from vehicle-treated dogs (control activity).
Tables
- TABLE 1
Enzyme kinetic parameters determined by nonlinear regression for formation of M1 and M2 from (+)-tramadol and (−)-tramadol by pooled DLMs (n = 27)
Activity High-Affinity Activity Low-Affinity Activity Km Vmax Vmax/Km Km Vmax Vmax/Km ΣVmax/Km μM pmol/min per mg protein ml/min per g protein μM pmol/min per mg protein ml/min per g protein (+)-M1 7.0 248 35 554 329 0.6 36 (−)-M1 9.8 92 9.3 117 13 0.11 9.4 (+)-M2 69 440 6.4 5442 843 0.15 6.6 (−)-M2 49 235 4.6 2494 318 0.12 4.7 The data points used for fitting were the average of two independent experiments performed in duplicate (data points shown in Fig. 4 with the curves of best fit). Fitted parameters included Km and Vmax, while intrinsic clearance (Vmax/Km) values were calculated. Data for (+)-M1 and (−)-M1 formation were best fit by a two-enzyme model. Kinetic parameters for high- and low-affinity activities, as well as the sum of the high and low intrinsic clearance values (ΣVmax/Km), are given.
- TABLE 2
Enzyme kinetic parameters determined by nonlinear regression for formation of M1 and M2 from (+)-tramadol and (−)-tramadol by dog recombinant P450s
Activity P450 High-Affinity Activity Low-Affinity Activity Km Vmax Vmax/Km Km Vmax Vmax/Km ΣVmax/Km μM pmol/min per pmol P450 μl/min per nmol P450 μM pmol/min per pmol P450 μl/min per nmol P450 (+)-M1 CYP2D15 5.5 1.0 182 66 2.1 32 214 CYP2B11 173 0.3 1.7 (−)-M1 CYP2D15 3.0 0.12 40 59 1.3 22 62 CYP2B11 445 0.18 0.4 (+)-M2 CYP2B11 10.4 0.5 48 430 1.5 3.5 51.5 CYP2D15 474 0.6 1.3 CYP2C21 30 0.4 13 CYP2C41 8.5 1.1 130 CYP3A12 23 1.5 65 (−)-M2 CYP2B11 7.2 0.2 28 765 1.9 2.5 30.5 CYP2D15 351 0.6 1.7 CYP2C21 68.5 0.3 4.4 CYP2C41 61 0.8 13 CYP3A12 55 0.4 7.3 The data points used for fitting were the average of three independent experiments performed in duplicate (data points shown in Fig. 5 with the curves of best fit). Fitted parameters included Km and Vmax, while intrinsic clearance (Vmax/Km) values were calculated. Data for (+)-M1 and (−)-M1 formation by CYP2D15 were best fit by a two-enzyme model. Kinetic parameters for high- and low-affinity activities, as well as the sum of the high and low intrinsic clearance values (ΣVmax/Km), are given.
In this issue
Tramadol Metabolism in Dogs
