CYP2D plays a major role in berberine metabolism in liver of mice and humans

Abstract

Berberine is a widely used plant extract for gastrointestinal infections, and is reported to have potential benefits in treatment for diabetes and hypercholesterolemia. It has been suggested that interactions between berberine-containing products and cytochromes P450 (CYPs) exist, but little is known about which CYPs mediate the metabolism of berberine in vivo. In this study, berberine metabolites in urine and feces of mice were analyzed, and the role that CYPs play in producing these metabolites were characterized in liver microsomes from mice (MLM) and humans (HLM), as well as recombinant human CYPs. Eleven berberine metabolites were identified in mice, including 5 unconjugated metabolites, mainly in feces, and 6 glucuronide and sulfate conjugates, predominantly in urine. Three novel berberine metabolites were observed. Three unconjugated metabolites of berberine were produced by MLM, HLM, and recombinant human CYPs. CYP2D6 was the primary recombinant human CYP producing these metabolites, followed by CYP1A2, 3A4, 2E1 and CYP2C19. The metabolism of berberine in MLM and HLM was decreased the most by a CYP2D inhibitor, and moderately by inhibitors of CYP1A and 3A. CYP2D plays a major role in berberine biotransformation, therefore, CYP2D6 pharmacogenetics and potential drug-drug interactions should be considered when berberine is used.

Fig. 1. MS/MS Fragmentation Patterns of Berberine and Three Novel Metabolites

(A) MS/MS fragmentation pattern for berberine. (B) MS/MS analysis of demethylated/1,3-dioxole ring-opening metabolite VII, whichhas a [M]⁺ ion at m/z 324, 12 Daltons less than that of berberine. (C) Mono-glucuronidated metabolite IX has a [M]⁺ ion at m/z 500, 176 mass units higher than that of metabolite VII. (D) Demethylated/demethenylated metabolite XII was only observed in feces, and corresponded to a [M]⁺ion at m/z 310. Berberine is abbreviated as BBR in all the figures in the present study.

Fig. 2. Relative Amount of Berberine and Its Metabolites in Urine and Feces of Mice

Eleven metabolites were detected in urine and feces of mice, five unconjugated metabolites (metabolites II, IV, VI, VII, and XII) and six conjugated metabolites (metabolites II, V, VIII, IX, X, and XI). All the glucuronidated metabolites (metabolites III, V, VIII, IX, and X) were observed in urine, but not in feces, whereas, unconjugated metabolite XII was detected only in feces and in low abundance. Berberine (I), the parent drug administered, was the most abundant compound in urine, and was 1.88 folds higher in urine than feces. Unconjugated metabolites IV and VI were the two predominant metabolites in feces followed by metabolites II and VII, and the amount of each in feces was 1.65, 29.7, 6.83, and 5.47 folds more than that in urine, respectively. The amount of the sulfated metabolite XI in feces was 1.2 folds more than that in urine.

Fig. 3. Berberine Metabolism in MLM and HLM

Three of five unconjugated metabolites, namely demethylated metabolite IV (m/z 322), demethenylated metabolite VI (m/z 324) and demethylated/demethenylated metabolite XII (m/z 310), were produced by HLM and MLM in an NADPH-dependent manner. With MLM, metabolite VI (m/z 324) was the major product. In the 1 μM berberine incubation system, the peak area of metabolite VI (m/z 324) was 40% higher than that of metabolite IV (m/z 322) and 3.67-fold higher than that of metabolite XII (m/z 310), whereas in the 10 μM berberine incubation system metabolite VI (m/z 324) was 20% higher than that of metabolite IV (m/z 322) and 3.25-fold higher than that of metabolite XII. In HLM incubated with 1 μM berberine, the amount of metabolite VI (m/z 324) was similar to metabolite IV (m/z 322) and 8-fold more than that of metabolite XII (m/z 310), whereas in the 10 μM berberine incubation system, the amount of metabolite VI (m/z 324) was about 14% more than that of metabolite IV (m/z 322) and 12-fold more than metabolite XII (m/z 310).

Fig. 4. Berberine Metabolism in Recombinant Human CYPs

Those three unconjugated metabolites of berberine observed in liver microsomes incubated with parent compound (metabolite IV, m/z 322; metabolite VI, m/z 324; metabolite XII, m/z 310) were also produced by a panel of recombinant human CYPs. Among the nine recombinant human CYP enzymes, CYP2D6 contributed to the formation of all three unconjugated metabolites. CYP1A2 also produced these three metabolites, but less than by CYP2D6. CYP3A4, 2E1 and 2C19 catalyzed only the synthesis of metabolite VI (m/z 324).

Fig. 5. Berberine Metabolism in MLM and HLM Pre-incubated with Specific CYP Inhibitors

The effect of paroxetine (PA), furafylline (FUR), ketoconazole (KCZ), which are inhibitors for CYP2D6, CYP1A2 and CYP3A4, on berberine (I, m/z 336) biotransformation in MLM and HLM showed that the metabolism of berberine mediated by various CYPs was inhibited in mice and humans, and formation of metabolite IV (m/z 322), metabolite VI (m/z 324) and metabolite XII (m/z 310) was decreased. The number on top of each bar is test/control. Paroxetine is abbreviated as PA, furafylline is abbreviated as FUR, ketoconazole is abbreviated as KCZ.

Fig. 6. Proposed Biotransformation Pathways of Berberine

Berberine biotransformation pathways were proposed based on the previous reports (from Pan et al., 2002; Zuo et al., 2006; and Qiu et al., 2008) and the current study. New metabolites were marked by asterisks. Five unconjugated metabolites of berberine and their corresponding conjugated metabolites are noted. The metabolism of berberine (I)by human recombinant CYPs shows that CYP2D6 is the primary enzyme involved in the formation of three berberine metabolites (IV, VI and XII), whereas CYP1A2 produced the same metabolites but with lower amount. CYP3A4, 2E1 and 2C19 also participate in the formation of demethylenation metabolite VI.