Our results demonstrate that single doses of BR-DIM of up to 200 mg are well-tolerated by healthy subjects, and that even at a dose of 300 mg adverse effects were infrequent and of minimal severity. Although adverse effects were observed, several features suggest that these effects may not be caused by DIM. First, the time of onset and duration of some reported adverse effects are difficult to associate with the time of BR-DIM administration and its pharmacokinetics. Moreover, the incidence of adverse effects does not correlate with BR-DIM dose (). As shown in , the incidence of adverse effects also does not correlate with actual DIM exposure, based either on Cmax or on AUC. Finally, the most extensive range of adverse effects was from a subject who had received placebo, rather than active BR-DIM.
This tolerability of BR-DIM is consistent with the results of a pilot study in which nineteen post-menopausal women took BR-DIM (108 mg DIM daily) for thirty days (17
). Of those subjects, one reported a rash that resolved with discontinuing the supplement and taking antihistamines, two reported increased hot flashes but continued to take the supplement, and one subject complained of nausea when BR-DIM was taken without food. BR-DIM also has been well-tolerated in our ongoing multiple-dose study. To date, fourteen healthy subjects have received twice-daily doses of either 100 mg or 200 mg BR-DIM for a four week period. This is a double-blind study, so no assignment by dose can be made prior to completion of all analyses, but adverse effects have not been reported by our subjects. The results of the pilot study in post-menopausal women (17
), of our single ascending dose study reported here, and the preliminary findings of our current multiple-dose study with BR-DIM all support the tolerability of BR-DIM in this dose range.
Although most studies of the effects on I3C and related compounds on carcinogenesis in animal models have demonstrated chemopreventive effects (1
), there have been reports of apparent tumor promotion by I3C in certain systems and at relatively high doses (21
). A single report of tumor promotion by DIM, in an aflatoxin B1-initiated liver tumor model in trout, also has been published (24
). A unifying feature of the studies showing tumor promotion by either I3C or DIM is the use of high doses of these supplements, and these doses appear to be associated with toxicity (25
). These reports of possible tumor promoting activity for these indole derivatives is noteworthy, however the high doses employed in those studies and the associated toxicity both differ from the dosing regimens we have used and from our observations on tolerability and adverse effects reported here and in our current multiple dose study.
Examination and calculation of pharmacokinetic parameters for DIM from BR-DIM produce a tmax
similar to those for DIM following ingestion of I3C (13
). A comparison of Cmax and AUC values, however, shows that, when normalized to dose administered, BR-DIM produces 2-3 times higher values than does I3C. This could represent both the fractional conversion of I3C to DIM under acid conditions (26
) and the lower bioavailability of DIM without the absorption-enhancing BR-DIM formulation.
Our inclusion and exclusion criteria were intended to minimize differences in general health, diet, social habits, and drug exposure as variables affecting DIM pharmacokinetics. Despite these restrictions, marked inter-individual variability in Cmax and AUC for DIM were noted (). When these values were compared among subjects at a given dose level no correlation or trend could be found with sex, age, or BMI. Normalizing Cmax and AUC to dose, expressed as mg/kg body weight, had minimal effects on the coefficient of variation for each dose group. Inter-individual variability in DIM metabolism might also contribute to the observed variability in pharmacokinetic parameters, however DIM metabolites have not been reported from in vivo
studies. Staub et al. recently reported the formation of hydroxylated DIM sulfates by human breast cancer cells in culture (27
), but the relevance of this finding to the intact organism is not known. It is noteworthy that Anderton et al. did not observe any DIM metabolites in either tissues or plasma of mice dosed with I3C (28
) or DIM (19
). The relative contributions to inter-individual variability in DIM pharmacokinetics of genetic variability and of additional dietary or environmental factors not addressed by our criteria cannot be assessed from this study.
Our pharmacokinetics data demonstrate a more linear dose-exposure relationship for BR-DIM, over the range from 50 mg to 300 mg, than was observed using the DIM precursor I3C (13
). The mean Cmax for DIM is a linear function of BR-DIM up to the 200 mg dose (r2
= 0.9552), and the mean AUC is a linear function of BR-DIM dose up to the 300 mg dose (r2
= 0.9682). In contrast, DIM Cmax and AUC following ingestion of I3C deviated dramatically from linearity (). Such markedly dose-dependent pharmacokinetics presents a major challenge to standardization of dose and predictability of responses, thus the linearity of pharmacokinetics support BR-DIM as the more favored supplement for development as a chemopreventive agent.
The linearity of BR-DIM pharmacokinetics demonstrated here prompts a reexamination of our consideration of DIM pharmacokinetics when I3C, the precursor, is administered (13
). We suggested that the increasing dose-normalized Cmax and AUC at increasing doses of I3C could represent a saturation of MDR1 and other efflux transporters in the enterocytes, thus increasing the net uptake of DIM. Given the increased linear range of Cmax and AUC with BR-DIM administration suggests that saturation of efflux is less likely. Rather, the superlinear increases observed with I3C may reflect increased DIM formation in a bimolecular reaction of I3C-derived reactants to produce DIM.
In our Phase 1 study of I3C we noted that DIM was the only detectable I3C-derived compound in plasma, and that no adverse effects were reported or observed at doses of 200 and 400 mg administered twice daily for 4 weeks (13
). The latter dose generated a Cmax of 69 ± 42 ng/mL and an AUC of 372 ± 180 h*ng/mL (13
). These Cmax and AUC determined for DIM after four weeks of twice-daily 400 mg doses of I3C are only 13% higher than the corresponding values for a single dose of 400 mg, indicating no alteration in kinetics from the single dose case. This four week I3C treatment at 400 mg twice daily resulted in marked induction of CYP1A2, and in a doubling of the urinary 2-hydroxyestrone: 16α-hydroxyestrone ratio (14
). Moreover, the change in the estrone hydroxylation ratio was obtained after four weeks at 200 mg I3C twice daily. Both of these changes elicited by I3C treatment fit with proposed mechanisms of chemoprevention by this agent, and if DIM is the active species eliciting these changes then we also have a target plasma concentration and AUC for chemoprevention. Our current findings with BR-DIM show that this target Cmax would be obtained at single dose of less than 150 mg, and that the target AUC would be achieved from a single dose between 150 and 200 mg. Based on this analysis, we are currently carrying out a multiple dose study with BR-DIM at doses of 100 mg and 200 mg administered twice daily. This study will assess the influence of BR-DIM on the activity of multiple hepatic enzymes including CYP1A2, CYP3A4, CYP2C9, and CYP2D6.