Jennifer L. Donovan

Cocoa and health: a decade of research

It has been over 10 years since the first mention in a medical journal about cocoa and chocolate as potential sources of antioxidants for health. During this time, cocoa has been found to improve antioxidant status, reduce inflammation and correlate with reduced heart disease risk; with these results, and its popularity, it has received wide coverage in the press. However, after 10 years of research, what is known about the potential health benefits of cocoa and what are the important next steps in understanding this decadent source of antioxidants?

Two CES1 Gene Mutations Lead to Dysfunctional Carboxylesterase 1 Activity in Man: Clinical Significance and Molecular Basis

The human carboxylesterase 1 (CES1) gene encodes for the enzyme carboxylesterase 1, a serine esterase governing both metabolic deactivation and activation of numerous therapeutic agents. During the course of a study of the pharmacokinetics of the methyl ester racemic psychostimulant methylphenidate, profoundly elevated methylphenidate plasma concentrations, unprecedented distortions in isomer disposition, and increases in hemodynamic measures were observed in a subject of European descent. These observations led to a focused study of the subjects CES1 gene. DNA sequencing detected two coding region single-nucleotide mutations located in exons 4 and 6. The mutation in exon 4 is located in codon 143 and leads to a nonconservative substitution, p.Gly143Glu. A deletion in exon 6 at codon 260 results in a frameshift mutation, p.Asp260fs, altering residues 260-299 before truncating at a premature stop codon. The minor allele frequency of p.Gly143Glu was determined to be 3.7%, 4.3%, 2.0%, and 0% in white, black, Hispanic, and Asian populations, respectively. Of 925 individual DNA samples examined, none carried the p.Asp260fs, indicating it is an extremely rare mutation. In vitro functional studies demonstrated the catalytic functions of both p.Gly143Glu and p.Asp260fs are substantially impaired, resulting in a complete loss of hydrolytic activity toward methylphenidate. When a more sensitive esterase substrate, p-nitrophenyl acetate was utilized, only 21.4% and 0.6% catalytic efficiency (V(max)/K(m)) were determined in p.Gly143Glu and p.Asp260fs, respectively, compared to the wild-type enzyme. These findings indicate that specific CES1 gene variants can lead to clinically significant alterations in pharmacokinetics and drug response of carboxylesterase 1 substrates.

The brain entry of risperidone and 9-hydroxyrisperidone is greatly limited by P-glycoprotein.

P-glycoprotein (P-gp) in the brain capillary endothelial cell limits the entry of many drugs into the brain. Our previous in-vitro study using ATPase as a marker of P-gp activity suggested that risperidone might be effectively transported by P-gp. In the present study, we compared the concentrations of risperidone and its major pharmacologically active metabolite 9-hydroxyrisperidone (9-OH-risperidone), in plasma, brain and various other tissues between abcb1ab-/- knockout mice which are functionally devoid of P-gp in their blood-brain barrier vs. FVB wild-type mice. One hour after intraperitoneal injection of 4 microg/g risperidone, the brain concentrations and ratios of brain:plasma concentrations of risperidone (13.1-fold and 12-fold respectively, p<0.05) and 9-OH-risperidone (29.4-fold and 29-fold respectively, p<0.01) were significantly higher in the abcb1ab-/- mice than those in the FVB mice. These results indicate that P-gp in the blood-brain barrier significantly influences the brain concentrations of risperidone and 9-OH-risperidone by limiting their CNS access.

Milk decreases urinary excretion but not plasma pharmacokinetics of cocoa flavan-3-ol metabolites in humans

BACKGROUND Cocoa drinks containing flavan-3-ols are associated with many health benefits, and conflicting evidence exists as to whether milk adversely affects the bioavailability of flavan-3-ols. OBJECTIVE The objective was to determine the effect of milk on the bioavailability of cocoa flavan-3-ol metabolites. DESIGN Nine human volunteers followed a low-flavonoid diet for 2 d before drinking 250 mL of a cocoa beverage, made with water or milk, that contained 45 micromol (-)-epicatechin and (-)-catechin. Plasma and urine samples were collected for 24 h, and flavan-3-ol metabolites were analyzed by HPLC with photodiode array and mass spectrometric detection. RESULTS Milk affected neither gastric emptying nor the transit time through the small intestine. Two flavan-3-ol metabolites were detected in plasma and 4 in urine. Milk had only minor effects on the plasma pharmacokinetics of an (epi)catechin-O-sulfate and had no effect on an O-methyl-(epi)catechin-O-sulfate. However, milk significantly lowered the excretion of 4 urinary flavan-3-ol metabolites from 18.3% to 10.5% of the ingested dose (P = 0.016). Studies that showed protective effects of cocoa and those that showed no effect of milk on bioavailability used products that have a much higher flavan-3-ol content than does the commercial cocoa used in the present study. CONCLUSIONS Most studies of the protective effects of cocoa have used drinks with a very high flavan-3-ol content. Whether similar protective effects are associated with the consumption of many commercial chocolate and cocoa products containing substantially lower amounts of flavan-3-ols, especially when absorption at lower doses is obstructed by milk, remains to be determined.

Effects of garlic (Allium sativum L.) supplementation on cytochrome P450 2D6 and 3A4 activity in healthy volunteers.

Garlic (Allium sativum L.) is a commonly used food and herbal supplement. The objective of this study was to assess in healthy volunteers (N = 14) the influence of a garlic extract on the activity of cytochrome P450 (CYP) 2D6 and 3A4. Probe substrates dextromethorphan (CYP2D6) and alprazolam (CYP3A4) were administered orally at baseline and again after treatment with garlic extract (3 × 600 mg twice daily) for 14 days. Urinary dextromethorphan/dextrorphan ratios and alprazolam plasma concentrations were determined by HPLC at baseline and after garlic extract treatment. The ratio of dextromethorphan to its metabolite was 0.044 ± 0.48 at baseline and 0.052 ± 0.095 after garlic supplementation. There were no significant differences between the baseline and garlic phases (P ≥ .05). For alprazolam, there were no significant differences in pharmacokinetic parameters at baseline and after garlic extract treatment (all P values ≥ .05; maximum concentration in plasma, 27.3 ± 2.6 ng/mL versus 27.3 ± 4.8 ng/mL; time to reach maximum concentration in plasma, 1.9 ± 1.4 h versus 2.4 ± 1.8 h; area under the time‐versus‐concentration curve, 537 ± 94 h · ng · mL−1 versus 548 ± 159 h · ng · mL−1; half‐life of elimination, 13.7 ± 4.4 h versus 14.5 ± 4.3 h). Our results indicate that garlic extracts are unlikely to alter the disposition of coadministered medications primarily dependent on the CYP2D6 or CYP3A4 pathway of metabolism.

(+)-Catechin is more bioavailable than (−)-catechin: Relevance to the bioavailability of catechin from cocoa

Catechin is a flavonoid present in fruits, wine and cocoa products. Most foods contain the (+)-enantiomer of catechin but chocolate mainly contains ( − )-catechin, in addition to its major flavanol, ( − )-epicatechin. Previous studies have shown poor bioavailability of catechin when consumed in chocolate. We compared the absorption of ( − ) and (+)-catechin after in situ perfusion of 10, 30 or 50 μmol/l of each catechin enantiomer in the jejunum and ileum in the rat. We also assayed 23 samples of chocolate for (+) and ( − )-catechin. Samples were analyzed using HPLC with a Cyclobond I-2000 RSP chiral column. At all concentrations studied, the intestinal absorption of ( − )-catechin was lower than the intestinal absorption of (+)-catechin (p < 0.01). Plasma concentrations of ( − )-catechin were significantly reduced compared to (+)-catechin (p < 0.05). The mean concentration of ( − )-catechin in chocolate was 218 ± 126 mg/kg compared to 25 ± 15 mg/kg (+)-catechin. Our findings provide an explanation for the poor bioavailability of catechin when consumed in chocolate or other cocoa containing products.

Multiple-dose administration of Ginkgo biloba did not affect cytochrome P-450 2D6 or 3A4 activity in normal volunteers.

Standardized extracts from the Ginkgo biloba tree are purported to exert positive neurocognitive effects and may also be useful in the treatment of a variety of vascular and other disorders. This dietary supplement is among the most commonly used herbal preparations in the world. The objective of this study was to assess in normal volunteers (n = 12) the influence of standardized Ginkgo biloba (GB) on the activity of cytochrome P-450 (CYP) 2D6 and 3A4 normal volunteers phenotyped as CYP2D6 extensive metabolizers. Probe substrates dextromethorphan (CYP2D6 activity) and alprazolam (CYP 3A4 activity) were co-administered orally at baseline, and following treatment with GB (120 mg twice daily) for 14 days. Urinary concentrations of dextromethorphan and dextrorphan were quantified and dextromethorphan metabolic ratios (DMRs) were determined at baseline and after GB treatment. Likewise, plasma samples were collected (0–60 hrs) for alprazolam pharmacokinetics at baseline and after GB treatment to assess effects on CYP 3A4 activity. Validated HPLC methods were used to quantify all compounds and relevant metabolites. No statistically significant differences were found between baseline and post-GB treatment DMRs indicating a lack of effect on CYP2D6. For alprazolam there was a 17% decrease in the area under the plasma concentration versus time curve (AUC); (P <0.05). However, the half-life of elimination was not significantly different after GB administration indicating a lack of hepatic CYP3A4 induction. We conclude that standardized extracts of GB at recommended doses are unlikely to significantly alter the disposition of co-administered medications primarily dependent on the CYP2D6 or CYP3A4 pathways for elimination.

Short-term administration of (-)-epigallocatechin gallate reduces hepatic steatosis and protects against warm hepatic ischemia/reperfusion injury in steatotic mice.

Hepatic steatosis increases the extent of cellular injury incurred during ischemia/reperfusion (I/R) injury. (‐)‐Epigallocatechin gallate (EGCG), the major flavonoid component of green tea (camellia sinensis) is a potent antioxidant that inhibits fatty acid synthase (FAS) in vitro. We investigated the effects of EGCG on hepatic steatosis and markers of cellular damage at baseline and after I/R injury in ob/ob mice. Animals were pretreated with 85 mg/kg EGCG via intraperitoneal (ip) injection for 2 days or oral consumption in the drinking water for 5 days before 15 minutes of warm ischemia and 24 hours of reperfusion. After EGCG administration, total baseline hepatic fat content decreased from baseline. Palmitic acid and linoleic acid levels also were reduced substantially in all ECGC‐treated animals before I/R. Alanine aminotransferase (ALT) levels decreased in all EGCG‐treated animals compared with control animals after I/R. Histologic analysis demonstrated an average decrease of 65% necrosis after EGCG administration. EGCG administration also increased resting hepatic energy stores as determined by an increase in cellular adenosine triphosphate (ATP) with a concomitant decrease in uncoupling protein 2 (UCP2) before I/R. Finally, there was an increased level of glutathione (GSH) in the EGCG‐treated mice compared with the vehicle‐treated mice both at baseline and after I/R. In conclusion, taken together, this study demonstrates that treatment with ECGC by either oral or ip administration, significantly protects the liver after I/R, possibly by reducing hepatic fat content, increasing hepatic energy status, and functioning as an antioxidant. (Liver Transpl 2005;11:298–308.)

Modafinil and Cocaine Interactions

This Phase I trial evaluated the interaction between modafinil steady-state and cocaine. Twelve non-treatment seeking, cocaine dependent volunteers received four sets of randomized blinded infusions of saline, 20 mg IV cocaine, and 40 mg IV cocaine. Modafinil was given open label at 0 mg, 400 mg, or 800 mg. Modafinil combined with IV cocaine did not result in any significant hemodynamic interactions. Modafinil significantly dampened scores on Visual Analog Scale measures as compared to baseline cocaine conditions. No significant alterations in labs occurred. Further outpatient trials of modafinil appear to be warranted.

Risperidone and paliperidone inhibit p-glycoprotein activity in vitro.

Risperidone (RSP) and its major active metabolite, 9-hydroxy-risperidone (paliperidone, PALI), are substrates of the drug transporter P-glycoprotein (P-gp). The goal of this study was to examine the in vitro effects of RSP and PALI on P-gp-mediated transport. The intracellular accumulation of rhodamine123 (Rh123) and doxorubicin (DOX) were examined in LLC-PK1/MDR1 cells to evaluate P-gp inhibition by RSP and PALI. Both compounds significantly increased the intracellular accumulation of Rh123 and DOX in a concentration-dependent manner. The IC50 values of RSP for inhibiting P-gp-mediated transport of Rh123 and DOX were 63.26 and 15.78 μM, respectively, whereas the IC50 values of PALI were >100 μM, indicating that PALI is a less potent P-gp inhibitor. Caco-2 and primary cultured rat brain microvessel endothelial cells (RBMECs) were utilized to investigate the possible influence of RSP on intestinal absorption and blood–brain barrier (BBB) transport of coadministered drugs that are P-gp substrates. RSP, 1–50 μM, significantly enhanced the intracellular accumulation of Rh123 in Caco-2 cells by inhibiting P-gp activity with an IC50 value of 5.87 μM. Following exposure to 10 μM RSP, the apparent permeability coefficient of Rh123 across Caco-2 and RBMECs monolayers was increased to 2.02 and 2.63-fold in the apical to basolateral direction, but decreased to 0.37 and 0.21-fold in the basolateral to apical direction, respectively. These data suggest that RSP and PALI, to a lesser extent, have a potential to influence the pharmacokinetics and hence the pharmacodynamics of coadministered drugs via inhibition of P-gp-mediated transport. However, no human data exist that address this issue. In particular, RSP may interact with its own active metabolite PALI by promoting its brain concentration through inhibiting P-gp-mediated efflux of PALI across endothelial cells of the BBB.