Indole-3-CarbinolProperties, Targets, Required Animal Data

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Dr. Bhaswat S. Chakraborty

• Indole-3-carbinol (I3C) belongs to dietary polyphenols which include flavonoids, stillbenes, lignas, and phenolic acids

• I3C is produced by the breakdown of the glucosinolate viz. glucobrassicin– found at relatively high levels in cruciferous vegetables, e.g., broccoli, cabbage,

cauliflower, brussels sprouts, collard greens and kale– cruciferous vegetables differ from other classes of vegetables in that they are rich

sources of sulfur-containing compounds (glucosinolates)– cruciferous vegetables are associated with lower risk of several types of cancer

(epidemiologic evidence)

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• I3C:– Off-white powder; mixes with water– Average Molecular Weight 147.1739– Melting Point 90 °C– Water Solubility 3.75 g/L– pKa ? (can act as both an electron donor & acceptor)– pH (1% solution) ?– storage temp. 2-8°C– Oral formulations can be made

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• Hydrolysis of glucobrassicin by myrosinase at neutral pH results in an unstable indole isothiocyanate that degrades to form I3C and a thiocyanate ion

• At acidic pH (stomach), I3C condenses to dimers (3,3'-diindolylmethane, DIM) & trimers (Cyclononal tri-indole, CT)

• When cruciferous vegetables are cooked (inactivating myrosinase), glucobrassicin hydrolysis by intestinal bacteria still results in some I3C formation, whereas DIM & CT do not form in alkaline pH of intestine

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QSAR & CoMFA • At least one 3D-QSAR Comparative Molecular Field Analysis (CoMFA)

study of 14 DIM derivatives is reported• The CoMFA model derived from DIM analogues proved a good predictive

ability • Some newly designed compounds exhibited 3-fold more potent radical

scavenging activity than reference substance Vitamin E in DPPH model expressed by IC50 values

• The primary antitumor screening essay showed that some DIM derivatives designed exhibited the inhibitory activities to some tumor cell growth at relatively high concentration, and DIM was the most effective among them

• Relaiable 3D-QSAR model can be developed for radical scavenging activities and anticancer assays in vitro.

5Benabadji SH et al., Acta Pharmacol Sin 2004; 25: 666-671

PK of I3C• Very little data• Gastric acid converts I3C to active metabolites DIM and indolylcarbazole,

which are further metabolized in the liver– most metabolites are excreted through the feces– Conversion from I3C into DIM not only requires a precise acidity, it requires

time. This conversion may proceed slowly, requiring more time than most foods typically spend in the intestinal tract

• Following an oral dose of I3C in humans, only DIM and no I3C was found circulating in the bloodstream of human subjects

• Over 90% of an oral dose of I3C is converted into non-DIM condensation products of uncertain structure, uptake and activity

• I3C induces CYP 1A2 and can reduce serum concentration of drugs metabolized by this enzyme

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Arneson, DW et al., Proceedings of the American Association for Cancer Research, 1999 Mar; (40): #2833Cover CM, et al. Cancer Res 1999; 59:1244-51Yoshida M et al., Carcinogenesis. 2004 Nov;25(11):2257-64

Anti-Inflammatory Properties and Mechanisms

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• Several dietary polyphenol compounds (e.g. resveratrol, genistein, catechin, and indole-3-carbinol) have been studied for inflammation and cancer.

• The potential molecular mechanisms of their anti-inflammatory activities have also been suggested to include inhibition of enzymes related to inflammation:– cyclooxygenase and lipoxygenase– PPAR– NOS– NF-κB– NAG-1

• Mainly– AA-Dependent pathway– AA-Independent pathway

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Baek SJ et al. Carcinogenesis, 2004;25:2425-32.Baek SJ et al. Carcinogenesis, 2002;23:425-34.Lee SH, et al. Biochem Biophys Res Commun, 2005;328:63-9.Wilson LC et al. Int J Cancer 2003;105:747-53.

AA- Dependent Mechanisms• Aspirin and other NSAIDs block COX-1 and COX-2• The role of COX inhibition by I3C is not very clear but all NF-κB-regulated gene

products including COX-2 are down-regulated by I3C– however, some dietary polyphenols, such as galangin and luteolin, and curcumin inhibit AA

peroxidation

• Flavonols, including kaempferol, quercetin, morin and myricetin, were found to be 5-LOX inhibitors. Hamamelitannin and the galloylated proanthocyanidins were found to be the most potent inhibitors of 5-LOX.

• In contrast, there are few reports regarding 12-LOX inhibition. Using bovine PMNs and 12-LOX from bovine platelets, kuwanson C and quercetin potently inhibit 12-LOX activity

• Again, LOX inhibition by I3C is not very clear but all NF-κB-regulated gene products including LOX are down-regulated by I3C and DIM

• Both COX and LOX are blocked by curcumin• Quercetin was found to be an effective inhibitor of PLA2 in human38 and rabbit39

leukocytes. Quercetagetin, kaempferol-3-O-galactoside, and scutellarein inhibited human recombinant synovial PLA2.

9Yoon J-H & Baek SJ, Yonsei Medical Journal 2005, 46, 585 - 596

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AA- Independent Mechanisms• PPARs (nuclear hormone receptors) regulate many inflammatory cytokines at the transcriptional

level, either enhancing or inhibiting inflammation process• Genistein increased the expression of lipid catabolism genes (this effect is not estrogen receptor-

dependent, but PPARα-dependent). EGCG also binds to PPARα. Amentoflavone up-regulates PPARγ expression in A549 human lung epithelial cells.

• Few studies regarded polyphenols as PPAR ligands, but it is probable that polyphenols may also affect PPAR protein expression, which results in the activation of the PPAR pathway

• Effect of I3C on PPARs not reported• Nitric oxide synthase (NOS) endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS

(iNOS): many polyphenols, including 6-gingerol,64 EGCG,65 resveratrol, indole-3-carbinol, and oroxylin A, inhibit NOS expression in LPS-induced RAW264.7 cells (these effects are probably mediated by NF-κB)

• Nuclear transcription factor κB (NF-κB): Quercetin was reported to suppress TNF-α induced expression of IL-8 and monocyte chemoattractant protein (MCP-1) due to its ability to inhibit the activation of NF-κB. Quercetin also inhibited the NF-κB pathway without any modification of c-Jun N- terminal kinase activity both in vivo & in vitro

• Recently, it has been reported that I3C inhibits NF-κB and NF-κB-regulated gene expression.• NAG-1 (NSAID activated gene-1): I3C and other polyphenols induce NAG-1 expression, except (-) -

epicatechin (EC) and (-)-epigallocatechin (EGC), indicating the structure-specific expression of NAG-1 induction

11Yoon J-H & Baek SJ, Yonsei Medical Journal 2005, 46, 585 - 596

Anticancer Properties & Mechanisms

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Anticancer Properties• I3C & DIM are good candidates for cancer prevention. Several studies

demonstrate that they – can cause cell cycle arrest and apoptosis in cancer cell lines– have antiangiogenic activity– inhibit tobacco smoke-induced lung adenocarcinoma in mice– I3C enhances efficacy of gemcitabine and acts synergistically with bortezomib in vitro.

• Clinical trial data show that I3C is effective in treatment of precancerous cervical dysplasia and vulvar intraepithelial neoplasia

– In premenopausal women, a supplement containing I3C and 7-hydroxymatairesinol significantly increased the urinary 2:16-hydroxyestrone ratio, a known biomarker for the reduction of breast cancer risk

• I3C also stimulates detoxification enzymes in the gut and liver• I3C is generally well tolerated when taken orally

– it may, however, promote tumor growth in animals that have been exposed to carcinogens

– because it induces cytochrome P450 enzymes, I3C may interact with several medications

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Nuclear factors modulated by I3C• Four nuclear transcription factors interact with I3C and DIM

– estrogen receptor (ER)– Sp1– nuclear factor кB (NFкB)– aryl hydrocarbonreceptor (AhR)

• These may account for changes in downstream events in normal and neoplastic cells.

14Y.S. Kim, J.A. Milner. J Nutri Biochem 16 (2005) 65–73

15DIM Resource Centre, University of California at Berkeley

16DIM Resource Centre, University of California at Berkeley

17DIM Resource Centre, University of California at Berkeley

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Chemical structures of I3C and DIM and summary of their key modulatory effects on hormone responsiveness and receptor signaling in human cancer cells. EGF, Epidermal growth factor.

I3C Targets Multiple Pathways• I3C and DIM have potent growth inhibitory and apoptosis inducing effects

on human and animal cancer cells by targeting multiple cellular signaling pathways in vitro

• Their inhibitory effects on cancer cell growth are through the modulation of genes that are related to the control of cell proliferation, cell cycle, apoptosis, signal transduction, oncogenesis, and transcription regulation

• Their pleiotropic effects on cancer cells through targeting multiple cellular signaling pathways including NF-κB, Akt, MAPK, Wnt, Notch, and androgen receptor (AR)

• Could be useful either alone or in combination with conventional therapeutics for the prevention of tumor progression and/or treatment of human malignancies

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Sarkar & Li, Cancer Treat Rev. 2009 November ; 35(7): 597–607Chinni SR et al., Oncogene 2001;20:2927–36Li Y et al., J Nutr 2003;133:1011–9Mukhopadhyay A et al., Oncogene 2001;20:7597–609.Shao ZM et al., Int J Cancer 2002;98:234–40.

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NF-κB, Akt, and Notch signaling pathways altered by isoflavone, I3C, DIM, and curcumin

Targeting Multiple Signals • Inactivation of NF-κB is mechanistically linked with growth inhibitory and

apoptosis promoting activity– Inhibition of NF-κB through modulation of IKK and IκB

• Inhibition of Akt signaling (Akt responsible for cancer development and progression)– Decreased phosphorylated Akt protein at Ser473 and the Akt kinase activity

• inhibition of Wnt signaling– By up-regulating GSK-3β, enhanced GSK-3β binding to β-catenin, and

increased the phosphorylation of β-catenin

• Also inhibitory effects on Notch signaling, resulting in the inhibition of cancer cell growth

Sarkar & Li, Cancer Treat Rev. 2009 November ; 35(7): 597–607Akiyama T et al., J Biol Chem 1987;262:5592–5Li Y et al., J Biol Chem 2008;283:27707–16Wang Z et al., Mol Cancer Ther 2006;5:483–93

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Wnt and AR signaling pathways altered by natural products such as isoflavone, I3C, DIM, etc.

Reported Phase I Study• Phase I trial of indole-3-carbinol (I3C) in 17 women (1 postmenopausal and 16

premenopausal) from a high-risk breast cancer cohort• After a 4-week placebo run-in period, subjects ingested 400 mg I3C daily for 4 weeks

followed by a 4-week period of 800 mg I3C daily– these chronic doses were tolerated well by all subjects– hormonal variables were measured near the end of the placebo and dosing periods,

including determination of the urinary 2-hydroxyestrone/16alpha-hydroxyestrone ratio– serum estradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, and sex

hormone binding globulin showed no significant changes in response to I3C– comparing the results of placebo and the 800 mg daily dose period, CYP1A2 was elevated

by I3C in 94% of the subjects, NAT-2 activities decreased, Xanthine oxidase was not affected. Lymphocyte glutathione S-transferase activity increased by 69% ; also inrease in the urinary 2-hydroxyestrone/16alpha-hydroxyestrone ratio in response to I3C

– maximal increase was observed with the 400 mg daily dose of I3C, with no further increase found at 800 mg daily

– with hydroxylated estrone metabolites is a biomarker for chemoprevention, 400 mg I3C daily will elicit a maximal protective effect.

23Reed GA at al., Cancer Epidemiol Biomarkers Prev. 2005 Aug;14,1953-60.

Example of Reported Phase II Studies• Therapeutic benefits of indole-3-carbinol (I3C) in the management of vulvar intraepithelial

neoplasia (VIN)– Women (n=12) with histologically confirmed high-grade VIN were randomized to receive 200 and

400 mg/day of I3C– Symptomatology by visual analog scale and vulvoscopic appearance were assessed at recruitment, 6

weeks, 3 months, and 6 months– Tissue biopsy to determine histologic response was obtained at completion of the study period– Urine samples were obtained at each visit to determine 2-hydroxyestrone to 16-alpha-

hydroxyestrone ratios. – There was a significant improvement in symptomatology with the introduction of I3C (itch, P= 0.018;

pain, P= 0.028). Lesion size and severity were also significantly reduced (size, P= 0.005; appearance, P= 0.046)

– also a significant increase in 2-hydroxyestrone to 16-alpha-hydroxyestrone ratio following commencement of I3C, P= 0.05

– tissue biopsy from the worst-affected vulval areas revealed no improvement in grade of VIN during the 6-month period, P= 0.317

– no significant differences in results between those women taking 200 mg/day of I3C and those on 400 mg/day

– study has shown significant clinical improvement in symptomatology and vulvoscopic appearance of VIN with I3C therapy. Further clinical and scientific investigations are required to support these preliminary findings.

24Naik R et al.,Int J Gynecol Cancer, 2006 Mar-Apr;16(2):786-90

Example of Reported Phase II Studies..• Randomized, double-blind, placebo-controlled, parallel group study enrolled pre-

(n=47) and post-menopausal (n=49) women who were not on contraceptives or hormone replacement therapy

– A supplement containing 200 mg of I3C & 10 mg of HMR lignan along with other minor herbal constituents, was given to the treatment group

– placebo group received capsules containing microcrystalline cellulose– at baseline and study end (day 28), blood samples were analyzed for serum

enterolactone concentrations, and morning urine samples were evaluated for estrogen metabolites

– pre-menopausal women receiving the HMR lignan/I3C upplement had a significant increase (p=0.003) in urinary 2-hydroxyestrone (2-OHE) concentrations and in 2:16 OHE ratio (p=0.016)

– post-menopausal women receiving the supplement had a significant increase in urinary 2-OHE concentrations (p=0.035)

– pre- and post-menopausal groups combined, a significant increase in urinary 2-OHE concentrations (p=0.001) and a trend (p=0.074) toward increased 2:16-OHE ratio were observed

– authors concluded that HMR lignan/I3C supplementation significantly increased estrogen C-2 hydroxylation, which may reduce the risk for breast and other estrogen-related cancers.

25Laidlaw M et al., Breast Cancer. 2010 Dec 16;4:85-95.

Indole-3-Carbinol in Treating Patients With PSA Recurrence After Surgery for Prostate Cancer

• OBJECTIVES: 1) Determine the feasibility of Brassica vegetable intake and indole-3-carbinol supplement use in patients with prostate cancer with prostate-specific antigen (PSA) recurrence after prostatectomy. 2) Identify adverse events in these patients. 3) Quantify the effects of each intervention on PSA in these patients.

• Patients (n not given) are stratified by pretreatment prostat-specific antigen (PSA) growth rate (low [0.00-0.15] vs medium [0.16-0.30] vs high [> 0.30]). They are randomized to 1 of 3 treatment arms, and randomization status to arms II and III is double-blinded.

• Arm I (Brassica vegetables): Patients consume Brassica vegetables at least 2 servings (½ cup/serving) daily for 6 months

• Arm II (Placebo): Patients receive oral placebo once daily for 6 months.• Arm III (Indole-3-carbinol supplement): Patients receive oral indole-3-carbinol supplement

(capsules) once daily for 6 months.• Blood and urine samples (for urinary isothiocyanate levels) are collected at baseline and at 2,

4, and 6 months. Medical records are reviewed for prostate cancer-related information, surgical dates, dose and type of radiation, and PSA history.

• RESULTS NOT PUBLISHED YET

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Peculiarities• Protocol dependent effects

– I3C is a very powerful inhibitor of tumor formation in rats, mice, and other species, including trout

– It has very interesting protocol-dependent effects• e.g., if given to trout before and during exposure to aflatoxin (potent liver

carcinogen), it inhibits liver cancer• if given the aflatoxin first, and then stop treatment and give indole-3-carbinol, the

indole-3-carbinol actually promotes liver cancer

• Low affinity for nuclear factors– despite clear modulating effects on the trancrition factors– dynamics among I3C/DIM & and promoter regions of ER, AhR and Sp1 may arise from

interactive regulation of transcription factors– other potential sites where I3C/DIM may modulate the expression of a variety of cancer-

related genes?

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Disease Targets• Cancer

– Prostate– Breast– Colon– HPV related

• Cervical Intraepithelial Neoplasia• Vulvar Intraepithelial Neoplasia• Recurrent Respiratory Papillomatosis

• Autoimmune and Inflammatory– Systemic lupus erythematosus (SLE)– Obesity– Rheumatoid & Osteo- Arthritis– IBS– Biochemical failure

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ClinicalTrials.Gov

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Non-clinical & Animal Studies Required• For an IND (up to Phase I only)• Demonstrate through screening tests

– Ames (mutagenicity) is negative– Clastogenicity (chromosome damage) is negative– hERG (cardiac safety) is negative

• Animal Studies– Pharmacology (efficacy; animal proof of principle) studies– A rodent general toxicology study of at least 14 days or more– A non-rodent general toxicology study of at least 14 days or more– Genotoxicity studies (GLP preferred)– Safety pharmacology studies– ADME– Analytical assays

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Animal Studies contd..• Dose levels

– Usually 3 dose groups and a control• Want to see toxicity at highest dose• Would like to see no toxicity at low does that are near the clinical dose• Typical safety factor between a “clean” dose level (NOAEL = no observed

adverse effects level) and the clinical starting dose is 10X based on mg/m2

• Rat or Mouse? Dog or Monkey?– Relevant species

• Metabolism similar to humans• Pharmacology

– Species should have the pharmacological target– Some species very sensitive to some drugs

» eg rats and NSAIDS, dogs and glucocorticoid steroids

• Required amount of drug should be available

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Ideas on Preclinical Efficacy Studies • First, a comparative list of desirable anti-inflammatory and undesirable

pro-inflammatory properties be prepared• Cytokines to be measured

– anti-inflammatory: IL-2, IL-6 & IL-10– pro-inflammatory: TNFα, IL-1, IFNγ

• Undesirable properties of glucocorticoids– weight gain, glucose tolerance, LFT elevation, decreased bone density,

decreased lymphocyte count, …

• Study in rats – validated model• End-points: measurement of anti-inflammatory parameters for validation

of efficacy, body weight, Cytokines, LFT, and lymphocytes• Study duration: > 30 days

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Thanks

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