Human metabolism of the experimental cancer therapeutic agentd-limonene

Abstract

d-Limonene has efficacy in preclinical models of breast cancer, causing > 80% of carcinomas to regress with little host toxicity. We performed a pilot study on healthy human volunteers to identify plasma metabolites of limonene and to assess the toxicity of supradietary quantities of d-limonene. Seven subjects ingested 100 mg/kg limonene in a custard. Blood was drawn at 0 and 24 h for chemistry-panel analysis and at 0, 4, and 24 h for limonene-metabolite analysis. On-line capillary gas chromatography/mass spectrometry (GC/MS) analysis indicated that at least five compounds were present at 4 h that were not present at time zero. Two major peaks were identified as the rat limonene metabolites dihydroperillic acid and perillic acid, and two minor peaks were found to be the respective methyl esters of these acids. A third major peak was identified as limonene-1,2-diol. Limonene was a minor component. At a dose of 100 mg/kg, limonene caused no gradable toxicity. Limonene is metabolized by humans and rats in a similar manner. These observations and the high therapeutic ratio of limonene in the chemotherapy of rodent cancers suggest that limonene may be an efficacious chemotherapeutic agent for human malignancies.

R-Limonene metabolism in humans and metabolite kinetics after oral administration

“…This finding is in accordance with Crowell et al (1994), who found LMN-1,2-OH, PA, and dihydroperillic acid (DHPA) but not the unmetabolised precursor in blood, after a single oral LMN dose of 100 mg kg −1 bw. POH was not detectable in the blood samples, which may be either because it is quickly oxidised to PA or it is excreted rapidly.…”

“…After ingestion, LMN is completely absorbed and undergoes extensive metabolism in humans (Kodama et al 1976;Vigushin et al 1998). methyl-3-cyclohexene-1-yl)-1,2-propanediol, , limonene-1,2-diol [1-methyl-4-(1-methylethenyl) cyclohexane-1,2-diol, LMN-1,2-OH] (Kodama et al 1976;Poon et al 1996), and perillic acid [(1-methylethenyl)-1-cyclohexene-1-carboxylic acid, PA] (Crowell et al 1994) which may originate from the intermediate product perillyl alcohol [(4-(1-methylethenyl)-1-cyclohexene-1-methanol; POH] (Zhang et al 1999) were found to be prominent LMN metabolites. Another previously identified metabolite is dihydroperillic acid [4-(1-methylethenyl)-cyclohexanecarboxylic acid, DHPA] (Crowell et al 1994).…”

“…methyl-3-cyclohexene-1-yl)-1,2-propanediol, , limonene-1,2-diol [1-methyl-4-(1-methylethenyl) cyclohexane-1,2-diol, LMN-1,2-OH] (Kodama et al 1976;Poon et al 1996), and perillic acid [(1-methylethenyl)-1-cyclohexene-1-carboxylic acid, PA] (Crowell et al 1994) which may originate from the intermediate product perillyl alcohol [(4-(1-methylethenyl)-1-cyclohexene-1-methanol; POH] (Zhang et al 1999) were found to be prominent LMN metabolites. Another previously identified metabolite is dihydroperillic acid [4-(1-methylethenyl)-cyclohexanecarboxylic acid, DHPA] (Crowell et al 1994). In contrast, the monohydroxylated metabolites POH as well as the diastereomers cis-and trans-carveol [(1SR,5S)-2-methyl-5-(1-methylethenyl)-2-cyclohexene-1-ol, cCAR, and tCAR] have only been shown in vitro Shimada et al 2002).…”

“…Moreover, DHPA was already shown to be a human metabolite of LMN byCrowell et al (1994). Summing up the main methyl side-chain oxidation products PA and DHPA, this metabolic pathway outranks the endo-cyclic trans-dihydroxylation branch (LMN-1,2-OH).…”

C-erbB-2/ HER-2 upregulates fascin, an actin-bundling protein associated with cell motility, in human breast cancer cell lines

“…Exogenous agents included: (1) Ras pathway antagonist HFP (a-hydroxyfarnesyl-phosphoric acid) (Gibbs et al, 1993), cells cultured at 1 mM for 0, 6, 12, 24, 36 and 48 h prior to cell lysis and Western blotting; (2) P13 kinase antagonist Ly-294002 (Vlahos et al, 1994), cells cultured at 50 mM for 0, 6, 12, 24, 36 and 48 h prior to cell lysis and Western blotting; (3) Small G-protein and p21 ras antagonist perillic acid (Crowell et al, 1991), cells cultured at 1 mM for 0, 6, 12, 24, 36 and 48 h prior to cell lysis and Western blotting; (4) Src family PTK antagonist Pp1/2 (Calbiochem, La Jolla, CA, USA), cells cultured at 500 nM for 2, 6, 12 and 24 h prior to cell lysis and Western blotting; (5) NF-kB agonists IL-1 and TNF-a (Roche, Indianapolis, IN, USA), cells cultured at 10 ng/ml for 0, 2, 6, 8, 16, 24 and 36 h prior to cell lysis and Western blotting; and (6) glucocorticoid agonist dexamethasone (Wong et al, 1999), cells cultured at 1 mM for 48 h prior to cell lysis and Western blotting. Some of these antagonists/agonists were further tested with respect to their ability to inhibit/stimulate transcription of our exogenously introduced (transiently transfected) luciferase based fascin reporter constructs (data not shown).…”