Mechanisms of Herb-Drug Interactions Involving Cinnamon and CYP2A6: Focus on Time-Dependent Inhibition by Cinnamaldehyde and 2-Methoxycinnamaldehyde.

Information is scarce regarding pharmacokinetic-based herb-drug interactions (HDI) with trans-cinnamaldehyde (CA) and 2-methoxycinnamaldehyde (MCA), components of cinnamon. Given the presence of cinnamon in food and herbal treatments for various diseases, HDIs involving the CYP2A6 substrates nicotine and letrozole with MCA (KS = 1.58 µM; Hill slope = 1.16) and CA were investigated. The time-dependent inhibition (TDI) by MCA and CA of CYP2A6-mediated nicotine metabolism is a complex process involving multiple mechanisms. Molecular dynamic simulations showed that CYP2A6's active site accommodates two dynamic ligands. The preferred binding orientations for MCA and CA were consistent with the observed metabolism: epoxidation, O-demethylation, and aromatic hydroxylation of MCA and cinnamic acid formation from CA. The percent remaining activity plots for TDI by MCA and CA were curved, and they were analyzed with a numerical method using models of varying complexity. The best-fit models support multiple inactivator binding, inhibitor depletion, and partial inactivation. Deconvoluted mass spectra indicated that MCA and CA modified CYP2A6 apoprotein with mass additions of 156.79 (142.54-171.04) and 132.67 (123.37-141.98), respectively, and it was unaffected by glutathione. Heme degradation was observed in the presence of MCA (48.5% ± 13.4% loss; detected by liquid chromatography-tandem mass spectrometry). In the absence of clinical data, HDI predictions were made for nicotine and letrozole using inhibition parameters from the best-fit TDI models and parameters scaled from rats. Predicted area under the concentration-time curve fold changes were 4.29 (CA-nicotine), 4.92 (CA-letrozole), 4.35 (MCA-nicotine), and 5.00 (MCA-letrozole). These findings suggest that extensive exposure to cinnamon (corresponding to ≈ 275 mg CA) would lead to noteworthy interactions. SIGNIFICANCE STATEMENT: Human exposure to cinnamon is common because of its presence in food and cinnamon-based herbal treatments. Little is known about the risk for cinnamaldehyde and methoxycinnamaldehyde, two components of cinnamon, to interact with drugs that are eliminated by CYP2A6-mediated metabolism. The interactions with CYP2A6 are complex, involving multiple-ligand binding, time-dependent inhibition of nicotine metabolism, heme degradation, and apoprotein modification. An herb-drug interaction prediction suggests that extensive exposure to cinnamon would lead to noteworthy interactions with nicotine.

Critical functions of the polyamine putrescine for proliferation and viability of Leishmania donovani parasites.

Polyamines are metabolites that play important roles in rapidly proliferating cells, and recent studies have highlighted their critical nature in Leishmania parasites. However, little is known about the function of polyamines in parasites. To address this question, we assessed the effect of polyamine depletion in Leishmania donovani mutants lacking ornithine decarboxylase (Δodc) or spermidine synthase (Δspdsyn). Intracellular putrescine levels depleted rapidly in Δodc mutants and accumulated in Δspdsyn mutants, while spermidine levels were maintained at low but stable levels in both cell lines. Putrescine depletion in the Δodc mutants led to cell rounding, immediate cessation of proliferation, and loss of viability, while putrescine-rich Δspdsyn mutants displayed an intermediate proliferation phenotype and were able to arrest in a quiescent-like state for 6 weeks. Supplementation of Δodc mutants with spermidine had little effect on cell proliferation and morphology but enabled parasites to persist for 14 weeks. Thus, putrescine is not only essential as precursor for spermidine formation but also critical for parasite proliferation, morphology, and viability.

Forecasting academic success through implementation of an online prerequisite review tutorials program for first year pharmacy students.

OBJECTIVE: Online prerequisite review (OPR) tutorials were designed and implemented to reinforce foundational scientific material in order to protect in-class time, foster self-directed learning, and ensure all students have similar baseline knowledge. METHODS: Twenty-one tutorials covering undergraduate prerequisite material were developed by faculty and organized into six core modules, comprising basic biology, chemistry, and physiology topics. A quiz on this material was given on the first day of each course. This score was correlated with the final exam score at course completion. Additional student and faculty feedback was collected through surveys. RESULTS: 2372 quiz-exam pairings were collected over three consecutive fall semesters. A one point increase in the quiz score was associated with a 3.6 point (95% confidence interval 3.1-4.0) higher exam score, as well as a greater probability of passing the exam (P<0.0001). Furthermore, simple linear regression revealed a positive correlation between quiz and exam scores (P<0.0001). Three full years of student survey data revealed an overwhelmingly positive perception of the OPR tutorials, and surveyed faculty reported better use of class time and improved student competency and participation. CONCLUSIONS: Implementation of OPR tutorials may give faculty more efficient use of class time, and their associated quizzes serve as an early indicator for students at-risk of not passing who are candidates for early interventions. Furthermore, the OPR tutorial design gives it great transferability to biomedical post-graduate programs.

Expanding the view of breast cancer metabolism: Promising molecular targets and therapeutic opportunities.

The changes in breast cancer cells that contribute to tumor evolution, heterogeneity, metastasis and ultimately drug resistance are shaped by numerous genetic changes including alterations in cellular metabolism. These include intermediary metabolic pathways such as glycolysis, the citric acid cycle oxidative phosphorylation, amino acid synthesis and lipid metabolism. However, cancer cells also exhibit key alterations in other metabolic pathways involved in drug metabolism such as cytochrome P450 enzymes, sulfotransferase and steroid sulfatases that are involved in the synthesis of estrogens and themselves serve as drug targets. In this review we bring together these two sides of metabolism, discuss the evidence underpinning their role in breast cancer development and bring to light promising therapeutic targets and up and coming pharmacologic agents.

Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole.

Human exposure to trans-cinnamic aldehyde [t-CA; cinnamaldehyde; cinnamal; (E)-3-phenylprop-2-enal] is common through diet and through the use of cinnamon powder for diabetes and to provide flavor and scent in commercial products. We evaluated the likelihood of t-CA to influence metabolism by inhibition of P450 enzymes. IC50 values from recombinant enzymes indicated that an interaction is most probable for CYP2A6 (IC50 = 6.1 µM). t-CA was 10.5-fold more selective for human CYP2A6 than for CYP2E1; IC50 values for P450s 1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were 15.8-fold higher or more. t-CA is a type I ligand for CYP2A6 (KS = 14.9 µM). Inhibition of CYP2A6 by t-CA was metabolism-dependent; inhibition required NADPH and increased with time. Glutathione lessened the extent of inhibition modestly and statistically significantly. The carbon monoxide binding spectrum was dramatically diminished after exposure to NADPH and t-CA, suggesting degradation of the heme or CYP2A6 apoprotein. Using a static model and mechanism-based inhibition parameters (K(I) = 18.0 µM; k(inact) = 0.056 minute(-1)), changes in the area under the concentration-time curve (AUC) for nicotine and letrozole were predicted in the presence of t-CA (0.1 and 1 µM). The AUC fold-change ranged from 1.1 to 3.6. In summary, t-CA is a potential source of pharmacokinetic variability for CYP2A6 substrates due to metabolism-dependent inhibition, especially in scenarios when exposure to t-CA is elevated due to high dietary exposure, or when cinnamon is used as a treatment of specific disease states (e.g., diabetes).

Allosteric modulation of substrate motion in cytochrome P450 3A4-mediated xylene oxidation.

Many cytochrome P450 enzymes (CYPs) exhibit allosteric behavior reflecting a complex ligand-binding process involving numerous factors: conformational selection, protein-protein interactions, substrate/effector/protein structure, and multiple-ligand binding. The interplay of CYP plasticity and rigidity contributes to substrate/product selectivity and to allosterism. Detailed evidence describing how protein motion modulates product selectivity is incomplete as are descriptions of effector-induced modulation of substrate dynamics. Our intent was to discover details of allosteric behavior and CYP3A4 flexibility and rigidity by investigating substrate motion using low-molecular weight ligands. Steady state kinetics and product ratios were measured for oxidation of m-xylene-(2)H3 and p-xylene; intramolecular isotope effects were measured for m-xylene-(2)H3 oxidation as a function of m-xylene-(2)H3 and p-xylene concentration. Biphasic kinetic plots indicated homotropic cooperative behavior with xylene isomers. Selectivity for aromatic hydroxylation over benzylic hydroxylation of m-xylene-(2)H3 supports a model in which the region near the CYP3A4 active oxidizing species limits substrate dynamics. p-Xylene impedes the motion of m-xylene-(2)H3 substrates that have access to the active oxidizing species: (kH/kD)obs values for m-xylene-(2)H3 decreased with p-xylene concentration. m-Xylene-(2)H3 and p-xylene do not have simultaneous access to the active oxidizing species: deuterium-labeled and unlabeled p-xylene exhibited similar effects on the (kH/kD)obs values for m-xylene-(2)H3 oxidation. p-Xylene and m-xylene-(2)H3 bind at different sites: m-xylene-(2)H3 oxidation rates and product selectivity were consistent across the p-xylene concentration range. Overall, this study indicates that the intramolecular isotope effect experimental design provides a unique opportunity to investigate allosteric mechanisms as it provides information about substrate motion when the enzyme is primed to oxidize substrates.

Covalent modification and time-dependent inhibition of human CYP2E1 by the meta-isomer of acetaminophen.

The hypothesis that N-acetyl-m-aminophenol (AMAP), the meta isomer of acetaminophen, will covalently bind to and inhibit human CYP2E1 in a time- and NADPH-dependent manner was investigated. Liquid chromatography/electrospray ionization-mass spectrometry analysis indicated that AMAP metabolites (i.e., AMAP*) selectively and covalently modified CYP2E1 apoprotein in a ratio of 1.4:1 (AMAP*/CYP2E1) in a reconstituted system. The deconvoluted spectra of CYP2E1 apoprotein from incubations containing NADPH and AMAP displayed mass shifts of 167.2 ± 7.1 and 334.4 ± 6.5 Da, suggesting the addition of one and two hydroxylated AMAP metabolites to CYP2E1, respectively. Mass shifts in cytochrome P450 reductase, cytochrome b(5), and heme from these samples were not observed. CYP2E1 inhibition by AMAP increased with time in the presence of NADPH; a reversible inhibition component was also observed. The results support a bioactivation process that involves formation of a hydroquinone metabolite that undergoes further oxidation to a quinone, which reacts with CYP2E1 nucleophilic residues. The data are consistent with evidence from previous studies that identified hydroxylated AMAP glutathione conjugates collected from mice and indicate that cysteine residues are the most likely sites for adduct formation. This study reports the first direct evidence of AMAP-derived hydroquinone metabolites bound to human CYP2E1.

Predictions of cytochrome P450-mediated drug-drug interactions using cryopreserved human hepatocytes: comparison of plasma and protein-free media incubation conditions.

Cryopreserved human hepatocytes suspended in human plasma (HHSHP) have previously provided accurate CYP3A drug-drug interaction (DDI) predictions from a single IC(50) that captures both reversible and time-dependent inhibition. The goal of this study was to compare the accuracy of DDI predictions by a protein-free human hepatocyte system combined with the fraction unbound in plasma for inhibitor(s) with those obtained with protein-containing incubations. Seventeen CYP3A, CYP2C9, or CYP2D6 inhibitors were incubated with hepatocytes in human plasma or hepatocyte maintenance medium (HMM) for 20 min over a range of concentrations after which midazolam 1'-hydroxylation, diclofenac 4'-hydroxylation or (R)-bufuralol 1'-hydroxylation were used to quantify the corresponding cytochrome P450 (P450) catalytic activities. Two methods were used to predict the human exposure ratio of the victim drug in the presence and absence of inhibitor. The HMM K(i, app) values were combined with the free average systemic plasma concentration ("free [I] with HMM K(i, app)") and the plasma K(i, app) values were combined with the total average systemic plasma concentration ("total [I] with plasma K(i, app)"). Of 63 clinical DDI studies, the total [I] with plasma K(i, app) method predicted 89% of cases within 2-fold of the reported interaction whereas the free [I] with HMM K(i, app) method predicted only 59%. There was a general underprediction by the free [I] with HMM K(i, app) method, which is consistent with an underestimation of in vitro inhibition potency in this system. In conclusion, the HHSHP system proved to be a simple, accurate predictor of DDIs for three major P450s and superior to the protein-free approach.

Prediction of CYP3A-mediated drug-drug interactions using human hepatocytes suspended in human plasma.

Cryopreserved human hepatocytes suspended in human plasma (HHSHP) represent an integrated metabolic environment for predicting drug-drug interactions (DDIs). In this study, 13 CYP3A reversible and/or time-dependent inhibitors (TDIs) were incubated with HHSHP for 20 min over a range of concentrations after which midazolam 1'-hydroxylation was used to measure CYP3A activity. This single incubation time method yielded IC(50) values for the 13 inhibitors. For each CYP3A inhibitor-victim drug pair, the IC(50) value was combined with total average plasma concentration of the inhibitor in humans, fraction of the victim drug cleared by CYP3A, and intestinal availability of the victim drug to predict the ratio of plasma area under the curve of the victim drug in the presence and absence of inhibitor. Of 52 clinical DDI studies using these 13 inhibitors identified in the literature, 85% were predicted by this method within 2-fold of the observed change, and all were predicted within 3-fold. Subsequent studies to determine mechanism (reversible and time-dependent inhibitors) were performed by using a range of incubation periods and inhibitor concentrations. This system differentiated among reversible inhibitors, TDIs, and the combination of both. When the reversible and inactivation parameters were incorporated into predictive models, 65% of 52 clinical DDIs were predicted within 2-fold of the observed changes and 88% were within 3-fold. Thus, HHSHP produced accurate DDI predictions with a simple IC(50) determined at a single incubation time regardless of the inhibition mechanism; further if needed, the mechanism(s) of inhibition can be identified.

Multiple-ligand binding in CYP2A6: probing mechanisms of cytochrome P450 cooperativity by assessing substrate dynamics.

The contribution of ligand dynamics to CYP allosterism has not been considered in detail. On the basis of a previous study, we hypothesized that CYP2A6 and CYP2E1 accommodate multiple xylene ligands. As a result, the intramolecular ( k H/ k D) obs values observed for some xylene isomers are expected to be dependent on ligand concentration with contributions from [CYP.xylene] and [CYP.xylene.xylene], etc. To explore this possibility and the utility of kinetic isotope effects in characterizing allosteric CYP behavior, steady state kinetics, product ratios, and ( k H/ k D) obs values for CYP2E1 and CYP2A6 oxidation of m-xylene-alpha- (2)H 3 and p-xylene-alpha- (2)H 3 were determined. Evidence is presented that CYP2A6 accommodates multiple ligands and that intramolecular isotope effect experiments can provide insight into the mechanisms of multiple-ligand binding. CYP2A6 exhibited cooperative kinetics for m-xylene-alpha- (2)H 3 oxidation and a concentration-dependent decrease in the m-methylbenzylalcohol:2,4-dimethylphenol product ratio (9.8 +/- 0.1 and 4.8 +/- 0.3 at 2.5 microM and 1 mM, respectively). Heterotropic effects were observed as well, as incubations containing both 15 microM m-xylene-alpha- (2)H 3 and 200 microM p-xylene resulted in further reduction of the product ratio (2.4 +/- 0.2). When p-xylene (60 microM) was replaced with deuterium-labeled d 6- p-xylene (60 microM), an intermolecular competitive inverse isotope effect on 2,4-dimethylphenol formation [( k H/ k D) obs = 0.49] was observed, indicating that p-xylene exerts heterotropic effects by residing in the active site simultaneously with m-xylene. The data indicate that there is a concentration-dependent decrease in the reorientation rate of m-xylene, as no increase in ( k H/ k D) obs was observed in the presence of an increased level of metabolic switching. That is, the accommodation of a second xylene molecule in the active site leads to a decrease in substrate dynamics.

A comparison of substrate dynamics in human CYP2E1 and CYP2A6.

Considering the dynamic nature of CYPs, methods that reveal information about substrate and enzyme dynamics are necessary to generate predictive models. To compare substrate dynamics in CYP2E1 and CYP2A6, intramolecular isotope effect experiments were conducted, using deuterium labeled substrates: o-xylene, m-xylene, p-xylene, 2,6-dimethylnaphthalene, and 4,4'-dimethylbiphenyl. Competitive intermolecular experiments were also conducted using d(0)- and d(6)-labeled p-xylene. Both CYP2E1 and CYP2A6 displayed full isotope effect expression for o-xylene oxidation and almost complete suppression for dimethylbiphenyl. Interestingly, (k(H)/k(D))(obs) for d(3)-p-xylene oxidation ((k(H)/k(D))(obs)=6.04 and (k(H)/k(D))(obs)=5.53 for CYP2E1 and CYP2A6, respectively) was only slightly higher than (k(H)/k(D))(obs) for d(3)-dimethylnaphthalene ((k(H)/k(D))(obs)=5.50 and (k(H)/k(D))(obs)=4.96, respectively). One explanation is that in some instances (k(H)/k(D))(obs) values are generated by the presence of two substrates-bound simultaneously to the CYP. Speculatively, if this explanation is valid, then intramolecular isotope effect experiments should be useful in the mechanistic investigation of P450 cooperativity.