Research Background

3,3-diindolylmethane (DIM) is a major derivative of anticancer agent indole-3-carbinol (I3C), which presents in cruciferous
vegetables. Due to its anticancer properties, DIM is proposed as possible chemopreventive supplement. However, DIM is poorly
absorbed from the gastrointestinal tract. So to address this problem, a formulation of DIM with higher bioavailability, BR-DIM
was made. This study was aimed to examine the safety, tolerability and pharmacokinetics of single ascending doses of BR-DIM.

Experimental Method
In this study, men and women ages 22 to 58 y old who have negative result for tobacco and drug use were enrolled. Subjects
must also free of acute, unstable, chronic medical condition with Body mass index between 18-30.
Strict vegetarians and individuals who eat more than 3 medium servings of cruciferous vegetables per week were excluded from
this study. The characteristics of the enrolled subjects can be seen in Table 1.

Subjects were divided into 6 groups of different administration dose level which were 50, 100, 150, 200 and 300 mg, with the
300-mg dose repeated in an additional group. Each group consist of four subjects, 3 were administered with functional BR-DIM
and 1 subject administered with placebo.

At least 48 hours prior to DIM administration, caffeine and grapefruit containing foods and beverages were avoided by subjects.
After overnight fasting, subjects were administered their oral dose of BR-DIM or matching placebo. Blood samples were collected
before administration and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 hours after BR-DIM administration.
From this sample, the concentration of DIM in plasma were measured and pharmacokinetic analysis will be done to obtain:
1. Area under the concentration-time curve up to the last measurable time point
2. Maximum plasma concentration (Cmax)
3. Time to reach Cmax
4. Elimination half-time for DIM

Results
Adverse effects of DIM dosing
After administration of BR-DIM, all the adverse events that occurred in subjects are observed and listed in Table 2. Based on
subject examination and considering the time of onset and resolution of adverse effects relative to DIM administration, only the
vomiting after the 300-mg dose administration was classified as probably related to DIM.
This result shows that single doses of BR-DIM of up to 200 mg are well tolerated by healthy subjects and that even at 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, Time of onset and the duration of some reported adverse effects are difficult
to associate with the time of BR-DIM administration and its pharmacokinetics. Second, Incidence of adverse effects does not
correlate with BR-DIM dose (Table 2) and actual DIM exposure (Figure 3).

DIM Pharmacokinetics
The mean plasma DIM concentrations over sampling time are shown for each dose group in Figure 1. No subjects had detectable
DIM in their pre-dose plasma, and all subjects DIM plasma concentrations had dropped to below limit of detection after 12
hours for BR-DIM doses of 150 mg or less, and after 24 hours for all doses.

From Figure 1, the pharmacokinetic variables Cmax and time to reach Cmax can be determined and AUC as well as elimination
half-life values can be calculated for each subject as seen in Table 3. Mean values are reported for all doses except for 50-mg
dose, where only one subject showed detectable DIM concentration in plasma. Although inclusion and exclusion criteria were set
up to minimize the differences in subjects that could affect the DIM pharmacokinetics, significant inter-individual variability in C
max and AUC for DIM can still be noted.

From these pharmacokinetic variables, the dose-exposure relationship can be observed as seen in Figure 2. Figure 2 A and C
showed the Cmax and AUC on different doses of BR-DIM respectively. Figure 2 B and D showed the Cmax and AUC on different
doses of I3C, which is the precursor of DIM. The data showed more linear dose-exposure relationship for BR-DIM compared to
I3C. The mean Cmax for DIM is a linear function up to 200 mg dose, and the mean AUC is a linear function up to 300-mg dose. In
contrast, DIM Cmax and AUC following ingestion of I3C deviated dramatically from linearity, which present major challenge to
standardization of dose and predictability of response. Therefore, the linearity of pharmacokinetics supports BR-DIM as more
favored supplement for development as chemopreventive agent.