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Metadichol ®. A Novel Inverse Agonist of Aryl Hydrocarbon Receptor (AHR) and NRF2 Inhibitor

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Metadichol ® a novel nano emulsion of lipid alcohols is an Inverse agonist of Vitamin D Receptor (VDR). In this communication, we show that Metadichol is an inverse agonist of the nuclear receptor AHR (Aryl Hydrocarbon receptor) and also an inhibitor of NRF2 (Nuclear factor (erythroid-derived 2)-like 2)) and is a transcription factor that is ubiquitously expressed at low levels in all human organs and regulates a primary cellular defense mechanism, tight regulation to maintain cellular homeostasis. AHR is highly expressed in a broad panel of tumors, AHR is induced by 2,3,7,8-tetrachloride-benzo-p-dioxin (TCDD) Metadichol® as a inverse agonist against AHR could be potentially useful in the treatment of such diseases. Strong in vivo evidence suggests that TCDD can stimulate cross-talk between AHR and Nrf2. The constitutive up regulation of Nrf2 signaling appears to drive the cellular proliferation and resistance to chemotherapy in various cancers. Therefore, pharmacological inhibition of Nrf2 by Metadichol ® holds promise as a therapeutic strategy in chemo resistant forms of cancer.
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OMICS International
Research Article
Raghavan, J Cancer Sci Ther 2017, 9:9
DOI: 10.4172/1948-5956.1000489
J Cancer Sci Ther
ISSN: 1948-5956 JCST, an open access journal Volume 9(9) 661-668 (2017) - 661
Journal of
Cancer Science & Therapy
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ISSN: 1948-5956
Keywords: Metadichol; Nano lipid; Long chain saturated alcohols;
VDR; Inverse agonist; Protean agonist; AHR; Aryl Hydrocarbon
Receptor; Inverse agonist; Antagonist; Chemo resistant cancers;
Proliferation; Metastasis; Constitutive activity; NRF2
Introduction
Metadichol® is a nano formulation of long chain alcohols that is
an inverse agonist of Vitamin D receptor [1]. AHR (Aryl hydrocarbon
receptor) like VDR a ligand activated transcription factor. It forms
a nuclear hetero dimer with the AHR nuclear trans locator (ARNT)
protein to activate gene expression. AHR [2] is known to mediate most
of the toxic and carcinogenic eects of a wide variety of environmental
contaminants such as dioxin TCDD (2,3,7,8-tetrachlorodibenzo-[p]-
dioxin). Many dietary compounds and products of commensal flora
activate AHR signaling at physiologically relevant doses and with
significant potency [3,4]. This allows targeting AHR to treat
inflammatory and autoimmune diseases [5]. It is conceivable therefore
that inhibition of AHR activity by antagonists could result in beneficial
anti-inflammatory actions. Evidence for such anti-inflammatory effects
has recently been identified by use of the AHR antagonist CH-223191
[6] which represses TH17. A high affinity AHR antagonist, Stemregenin
1 (SR1) has been shown to increase the proliferation of human
hematopoietic stem cells in vitro [7]. AHR provides a molecular pathway
through which environmental factors modulate the immune response
and, consequently, the development of immune-mediated disorders.
Agonists of the AHR receptor play an integral role in T-cell function,
promoting a TH2/TH1 switch resulting in a TH1 bias [8]. AHR is
usually present in epithelial cells where it is activated by different
ligands. Elevated levels of AHR are seen in breast and other cancers
where AHR is always activated by depressing anti-tumor immune
responses. AHR up regulation allows tumor cells to migrate, invade
local tissue and metastasize to other organs. AHR is highly
expressed in a wide variety of tumors. Molecules with antagonistic AHR
activity could be considered potential candidates for the treatment
of such diseases. AHR is widely expressed in Pancreatic [9], Prostate
[10], Urinary tract [11] Lung [12] Esophagus [13], Pituitary [14],
gliomas [15] For long-term survival of cancers treatments activating
the patient’s own immune system is an important approach. For a
successful treatment, other aspects of cancer progression, like cancer
stem cells, metastatic behavior and decreased tumor cell apoptosis
need to be targeted simultaneously. AHR also plays a role in producing
“cancer stem cells”, that are resistant to chemotherapies and this leads
to lethal relapse and metastasis even years aer “successful” chemo- or
radiotherapy. Inhibitors not only boost anti-tumor immunity, but they
also suppress metastasis and the formation of chemo-resistant cancer
stem cells. Inhibiting AHR activity holds the promise of a non-toxic
therapy that targets crucial tumor progressing processes at the same
time. AHR antagonists have therapeutic immunological value [16].
Nrf2
e Nuclear factor erythroid 2-related factor 2 (Nrf2) is a
transcription factor regulating a variety of genes for antioxidant and
detoxification enzymes such as glutathione S-transferase A2 (GSTA2)
and NADPH quinone oxido-reductase 1 (NQO1) in response to
oxidative and xenobiotic stress [17]. Antioxidants counteract oxidative
stress, employing a variety of techniques to diminish ROS levels in the
intracellular environment. Free radicals are elevated during cancer and
activate signaling pathways of cell proliferation and migration, and
can cause DNA damage leading to mutations. Nrf2 signaling pathway
consists of three main components: (a) Keap1, (b) Nrf2, and (c) the
Antioxidant response element (ARE). ese three elements function
respectively as the sensor, controller, and responder [18]. Constitutive
activation of Nrf2 occurs in many cancers. Aberrant activation of Nrf2
is correlated to tumor progression, chemo resistance, and radiation
resistance. NRF2 activations are regarded as benecial, but recent
work suggests otherwise [19]. Increased NRF2 activity from mutations
*Corresponding author: PR Raghavan, Founder and CEO, Nanorx Inc.,
PO Box 131, Chappaqua, New York, 10514, USA, Tel: 9146710224; E-mail:
raghavan@nanorxinc.com
Received August 16, 2017; Accepted September 25, 2017; Published September
26, 2017
Citation: Raghavan PR (2017) Metadichol ®. A Novel Inverse Agonist of Aryl
Hydrocarbon Receptor (AHR) and NRF2 Inhibitor. J Cancer Sci Ther 9: 661-668.
doi:10.4172/1948-5956.1000489
Copyright: © 2017 Raghavan PR. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Metadichol ®. A Novel Inverse Agonist of Aryl Hydrocarbon Receptor
(AHR) and NRF2 Inhibitor
Raghavan PR*
Nanorx Inc., Chappaqua, New York, USA
Abstract
Metadichol ® a novel nano emulsion of lipid alcohols is an Inverse agonist of Vitamin D Receptor (VDR). In
this communication, we show that Metadichol is an inverse agonist of the nuclear receptor AHR (Aryl
Hydrocarbon receptor) and also an inhibitor of NRF2 (Nuclear factor (erythroid-derived 2)-like 2)) and is a
transcription factor that is ubiquitously expressed at low levels in all human organs and regulates a primary
cellular defense mechanism, tight regulation to maintain cellular homeostasis. AHR is highly expressed in a
broad panel of tumors, AHR is induced by 2,3,7,8-tetrachloride-benzo-p-dioxin (TCDD) Metadichol® as a
inverse agonist against AHR could be potentially useful in the treatment of such diseases. Strong in vivo
evidence suggests that TCDD can stimulate cross-talk between AHR and Nrf2. The constitutive up regulation
of Nrf2 signaling appears to drive the cellular proliferation and resistance to chemotherapy in various cancers.
Therefore, pharmacological inhibition of Nrf2 by Metadichol ® holds promise as a therapeutic strategy in chemo
resistant forms of cancer.
Citation: Raghavan PR (2017) Metadichol ®. A Novel Inverse Agonist of Aryl Hydrocarbon Receptor (AHR) and NRF2 Inhibitor. J Cancer Sci Ther 9:
661-668. doi:10.4172/1948-5956.1000489
J Cancer Sci Ther
ISSN: 1948-5956 JCST, an open access journal Volume 9(9) 661-668 (2017) - 662
control. Performance of the human NR agonist and antagonist assays
were validated by performing a Reference Agonist (MeBIOP), and
Antagonist (GNF 351) dose response curves
Assay methods
Plasmids. is study utilized proprietary nuclear receptor reporter
cells expressing either the full-length nuclear receptor sequence (AHR,)
or a hybrid receptor comprising the N-terminal Gal4 DNA binding
domain fused to the ligand binding domain of the specic human
nuclear receptor. e reporter vectors used in these studies comprise
the rey luciferase gene functionally linked to either an upstream NR
response element (NRE) or the Gal4 activation sequence (UAS).
Compound handling
Test compound was delivered as solution dissolved in water. Stock
of test compounds was stored at room temperature as directed by the
sponsor. Setup of NR Assays. e NR Assays were performed as depicted
in Figures 1 and 2. In brief, Step 1: A suspension of Reporter cells was
prepared in cell recovery medium (CRM; containing 10% charcoal
stripped FBS). For antagonist assays, reporter cells were supplemented
with a 2x-EC80 concentration of the appropriate reference agonist. For
agonist and antagonist assays, 100 ml of the reporter cell suspension
was dispensed into the wells of white, cell culture treated, 96-well assay
plates. For agonist and antagonist assays, 2x-concentration treatment
media were prepared.
Step 2: Immediately prior to assay setup, test compounds were
diluted using compound screening medium (CSM; containing 10%
charcoal stripped FBS) to generate ‘2x-concentration’ treatment media.
100 ml of each treatment medium was dispensed into triplicate assay
wells pre-dispensed with Reporter Cells. Assay plates were incubated
at 37°C for 24 hr.
Step 3: Following the 24 hr incubation period, treatment media
were discarded and 100 μl/well of Luciferase Detection Reagent was
added. RLUs were quantied from each assay well to determine NR
activity.
in NFE2L2 or KEAP1 is contributing to chemotherapy resistance.
Excessive induction of NRF2 genes in cancer cells, and therefore
superior protection against cell stress may confer a survival advantage
[20].
NRF2 is being researched as a drug target in many diseases
like COPD and Multiple sclerosis and in enhancing cancer cell
susceptibility to chemotherapy. Sulforaphane is useful in COPD,
rescuing impaired phagocytosis in alveolar macrophages and
breaking down macrophage resistance to gluco corticosteroid
treatment via HDAC2 denitrosylation [21,22]. Dimethyl fumarate has
shown promise in clinical multiple sclerosis trials, in a mouse MS model
(experimental autoimmune encephalomyelitis, or EAE). It attenuated
some of the damaging eects of the disease in dependence on Nrf2.
Treatment enhanced Nrf2 stabilization and activity [23]. Ren et al. [24]
studied the eect of Brusatol a compound found in a plant extract that
inhibits the NRF2 pathway. On lung cancer cells Brusatol increases
ubiquitination of NRF2 leaving other signaling pathways unaected,
and renders both cancer cell lines and xenogras susceptible to the
chemotherapeutic agents like Cisplatin.
Nrf2 is essential to maintaining the cellular redox homeostasis,
providing protection against oxidative or electrophilic stresses, and
suppressing tumor promotion and progression [25]. In vi vo, Nrf2-
knockout mice show greater damage in organs such as the lung, liver,
and kidney when exposed to chemical toxins such as cigarette smoke
or hypoxia [26]. Additionally, Nrf2-knockout mice are more prone
to developing diseases related to oxidative stress and cancers [27].
Constitutive elevation of Nrf2 expression and Nrf2-ARE activity
occurs in many cancers and is involved in their chemo-resistance and
radiation resistance. Ideally, inhibitors should block the Nrf2-ARE
signaling pathway in cancer cells, while leaving healthy cells unaected.
Although expression of Nrf2 can be suppressed experimentally by gene
therapy, implementing them clinically in cancer treatment is a major
hurdle. It is important to identify small-molecular Nrf2 inhibitors that
block Nrf2 signaling pathways specifically in cancer cells. us, there is
a need for a molecule that is specic and has bioactivity and also has no
toxicity. Metadichol ® as we shall show may t that need.
NRF2 and AHR
Both AHR and NRF2 signaling regulate the expression of genes
aecting the metabolism of xenobiotics. Miao et al. [28] showed that
Nrf2 gene transcription is modulated by AHR activation. NRF2 also
directly modulates AHR signaling, highlighting the dependent nature
of interactions between these two pathways [29]. NRF2 controls AHR
signaling to expand its function of NRF2 to include the metabolism
to include xenobiotics and carcinogens via CYPs and adipogenesis.
AHR and Nrf2 double knockout mice studies oer additional evidence
for interactions between these pathways [30]. Cooperation between
AHR and Nrf2 has biological implications. AHR has to do with
carcinogenesis, whereas Nrf2 is involved with cytoprotection. e
challenge is to exploit this to a to therapeutic advantage and to improve
chemo preventive strategies.
Experimental
AHR nuclear receptor assay was outsourced and performed by
Indigibio sciences PA, USA.
Reporter cells were treated with seven concentrations of
respective test compounds, starting at 10 mg/ml and following
with six more 3.33-fold dilutions. All treatment concentrations
were performed in triplicate. Reporter cells were also treated
with vehicle only as a
Figure 2: Antagonist assays.
Figure 1: Agonist assays.
Citation: Raghavan PR (2017) Metadichol ®. A Novel Inverse Agonist of Aryl Hydrocarbon Receptor (AHR) and NRF2 Inhibitor. J Cancer Sci Ther 9:
661-668. doi:10.4172/1948-5956.1000489
J Cancer Sci Ther
ISSN: 1948-5956 JCST, an open access journal Volume 9(9) 661-668 (2017) - 663
Assay validation
Reference compounds were utilized to conrm the performance
of the specic lot of NR Reporter Cells treated with the Sponsor’s test
compounds. Reference compound and test compound assays were
performed at the same time and, hence, were exposed to the same assay
reagents and environmental conditions. Refer to individual data sets
for the identities of specic reference agonist and antagonist and their
respective treatment concentration ranges. Reference groups always
include a ‘Vehicle’ control to determine background activity in the assay
and to calculate fold-activation or percent-inhibition.
Data reduction
Microso Excel was used to manage and archive assay data, as well
as to calculate average RLU values +/- Standard Deviation (SD), Fold-
activation, Percent activation, Percent Coecients of Variation (%CV),
and Z’ values.
Graphical data methods
Dose-response curve (DRC) analyses of the reference compounds
and test compounds were performed via non-linear curve-tting of
Fold-Activation vs. Log(Compound) for agonist assays and percent
inhibition vs. log(Compound) for antagonist and inverse agonist assays,
using Graph Pad Prism soware.
Keap1-NRF2 assays
All work was outsourced and performed by DiscoverX, Fremont,
CA, using their proprietary procedure. is is a target-specic, cell-
based functional assay for NRF2 that provides a non-transcriptional
response of activation-dependent NRF2 translocation to the nucleus.
Assay measures
Nuclear translocation of NRF2 following its disassociation from
Keap1
Activation of the NRF2 response pathway
e control ligand was CDDO methyl ester.
Experimental Procedure
Compounds were tested in agonist and antagonist mode with the
Keap1‐NRF2 Biosensor Assay. For agonist assays, data was normalized
to the maximal and minimal response observed in the presence of
control ligand and vehicle. For antagonist assays, data was normalized
to the maximal and minimal response observed in the presence of EC80
ligand and vehicle. An EC80 of 12 nM CDDO methyl ester was used.
Cell handling
1. PathHunter Pathway cell lines were expanded from freezer stocks
according to standard procedures.
2. Cells were seeded in a total volume of 20 μL into white walled,
384‐well micro plates and incubated for the appropriate me prior to
testing.
Agonist format
1. For agonist determination, cells were incubated with sample to
induce response.
2. Intermediate dilution of sample stocks was performed to generate
5X sample in assay buer.
3. 5 μL of 5X sample was added to cells and incubated at 37°C or
room temperature for 1.5‐16 hours depending on the assay. Vehicle
concentration was 1%.
Antagonist format
1. For antagonist determination, cells were pre‐incubated with
antagonist followed by agonist challenge at the EC80 concentration.
2. Intermediate dilution of sample stocks was performed to generate
5X sample in assay buer.
3. 5 μL of 5X sample was added to cells and incubated at 37°C or
room temperature for 60 minutes. Vehicle concentration was 1%.
4. 5 μL of 6X EC80 agonist in assay buer was added to the cells and
incubated at 37°C or room temperature for 1.5‐16 hours depending on
the assay.
Signal detection
1. Assay signal was generated through a single addition of 12.5 or
15 μL (50% v/v) of PathHunter Detection reagent cocktail for agonist
and antagonist assays respectively, followed by a one-hour incubation
at room temperature. For some assays, activity was detected using
a high sensitivity ti reagent (PathHunter FlashKit) to improve assay
performance. In these assays, equal volume of detection reagent (25
or 30 uL) was added to the wells, followed by a one hour at room
temperature.
2. Micro plates were read following signal generation with a Perkin-
Elmer EnvisionTM instrument for chemiluminescent signal detection
Data analysis
1. Compound activity was analyzed using CBIS data analysis suite
(ChemInnovation, CA).
2. For agonist mode assays, percentage activity was calculated using
the following formula: % Activity=100% x (mean RLU of test sample-
mean RLU of vehicle control) / (mean MAX RLU control ligand- mean
RLU of vehicle control).
3. For antagonist mode assays, percentage inhibition was calculated
using the following formula:
% Inhibition=100% × (1- (mean RLU of test sample-mean RLU
of vehicle control)/(mean RLU of EC80 control- mean RLU of vehicle
control)).
Results and Discussion
e AHR raw data results for agonist and antagonist are is shown in
Tables 1 and 2, e agonist and antagonist graphs are shown in Figures
3-8. MeBio was used as a standard in the agonist assay and GNF-351
used as the standard in the antagonist assay. Keap1-NRF2 raw data are
shown in Tables 3-5. From the Nuclear receptor agonist and antagonist
assay Figures 5 and 6 it is clear that Metadichol is behaving like an
inverse agonist. ere is no known inverse agonist of AHR reported
in literature.
In the Keap-NRF2 assay shown in Figure 7 it clear that Metadichol
has no agonist activity but has an antagonist activity. But what is
interesting is that the response is not 100% even at higher concentration
and maximum response did not exceed 35%.
Nutrition has a huge role in functioning of the human organism by
aecting gene expression in specic tissues of the organism. is helps
the organism to adapt to changes in the environment. e intracellular
Nuclear receptors link diet and gene expression in response to small
Citation: Raghavan PR (2017) Metadichol ®. A Novel Inverse Agonist of Aryl Hydrocarbon Receptor (AHR) and NRF2 Inhibitor. J Cancer Sci Ther 9:
661-668. doi:10.4172/1948-5956.1000489
J Cancer Sci Ther
ISSN: 1948-5956 JCST, an open access journal Volume 9(9) 661-668 (2017) - 664
Nuclear receptors are lipids with the exception of the yroid receptor
which has Iodine present. [32].
An inverse agonist binds to the same binding site on the receptor
Compound Values
Water 0.20% 15,067 11,180 10,349 12,199 2,518 1.0 4.2 21 -
Metadichol (µg/ml)
0.0073 10,986 8,085 10,559 9,877 1,566 0.81 3.4 16 -
0.024 9,304 7,012 5,776 7,364 1,790 0.60 2.5 24 -
0.081 8,291 6,874 7,178 7,448 746 0.61 2.5 10 -
0.27 7,445 7,537 6,870 7,284 361 0.60 2.5 5.0 -
0.90 3,513 5,732 5,247 4,830 1,167 0.40 1.6 24 -
3.0 3,056 2,112 2,966 2,712 521 0.22 0.92 19 -
10 302 262 286 283 20 0.023 0.10 7.1 -
Reference Antagonist: GNF351 (nM)
0.0038 84,571 80,274 96,520 87,121 8,418 7.1 30 9.7 Z'
0.19 110628 130,696 92,272 111,199 19,218 9.1 38 17 -
0.96 184984 156,008 155,234 165,409 16,957 14 56 10 -
4.8 229832 217,538 173,853 207,074 29,420 17 70 14 0.51
24 282781 262,276 269,296 271,451 10,421 22 92 3.8 0.85
120 238531 300,398 297,017 278,649 34,784 23 95 12 0.58
600 284983 326,358 270,364 293,902 29,043 24 100 10 0.66
3,000 266265 347,989 235,103 249,786 15,658 20 85 6.3 0.77
Table 1: Agonist assay.
Compound Conc. Human AhR antagonist assays AVG SD Fold-inhibition % Inhibition % CV Value
Luc1 Luc2 Luc3
Water 0.20% 231,269 274,275 272,890 259,478 24,440 1.0 0.0 9.4 -
Metadichol (µg/ml)
0.0073 283,234 226,097 297,118 268,860 37,667 0.97 -3.6 14 -
0.024 266,507 285,157 280,584 277,416 9,720 0.94 -6.9 3.5 -
0.081 266,802 263,538 285,246 271,862 11,705 0.95 -4.8 4.3 -
0.27 231,747 225,736 243,391 233,625 8,976 1.1 10 3.8 -
0.90 140,583 147,609 127,846 138,679 10,018 1.9 47 7.2 -
3.0 47,538 39,305 26,603 37,815 10,547 6.9 85 28 -
10 1,216 766 1,264 1,082 275 240 100 25 -
Reference Antagonist:
GNF351 (nM)
0.61 267,661 254,185 214,007 245,284 27,912 1.1 5.5 11 Z'
2.4 235,944 271,376 260,974 256,098 18,212 1.0 1.3 7.1 -
9.8 269,755 247,412 204,923 240,697 32,934 1.1 7.2 14 -
39 241,635 231,420 226,931 233,329 7,536 1.1 10 3.2 -
156 175,211 154,885 188,760 172,952 17,050 1.5 33 10 -
625 67,127 70,861 94,960 77,649 15,107 3.3 70 19 -
2500 10,709 11,052 14,977 12,246 2,371 21 95 19 0.67
10000 202 128 218 183 48 1420 100 26 0.72
Table 2: Antagonist assay.
Figure 3: Microsoft Excel was used to manage and archive assay data.
lipophilic ligands derived from endocrine organs, metabolism, diet
and the environment. Metadichol is composed of C-26, C-28, C-30
long chain alcohols and is an open chain analogue of 1,25 dihydroxy
vitamin D3 the natural ligand of VDR which is a C27 alcohol. In
addition, Metadichol is ubiquitous lipid that is part of our diet [31]
and is a lipophilic ligand. Most of the ligands for the known Figure 4: Assay protocol, The NRF2 response pathway.
Citation: Raghavan PR (2017) Metadichol ®. A Novel Inverse Agonist of Aryl Hydrocarbon Receptor (AHR) and NRF2 Inhibitor. J Cancer Sci Ther 9:
661-668. doi:10.4172/1948-5956.1000489
J Cancer Sci Ther
ISSN: 1948-5956 JCST, an open access journal Volume 9(9) 661-668 (2017) - 665
as an agonist does but induces a biological response opposite to that
of an agonist. A prerequisite for an inverse agonist response is that
the receptor must have high constitutive or basal activity [33] in the
absence of an identiable ligand. An inverse agonist [34] can have eects
similar to that of an antagonist but cause a distinct set of downstream
biological responses. ey block the eects of binding agonists like a
classical antagonist but also inhibit the basal activity of the receptor.
Figure 5: Agonist assay of Metadichol.
Figure 6: Standard agonist assay of MeBio.
Figure 7: Antagonist assay of Metadichol.
CDDO Methyl Ester
Well ID Conc Raw Value Percent Efcacy
B22 5.08E-06 237800 2.0
B19 1.52E-05 232800 1.4
B20 1.52E-05 244000 2.8
B17 4.57E-05 221200 0.1
B18
4.57E-05
219000 -0.2
B15 0.00013717 226400 0.7
B16 0.00013717 241400 2.5
B13 0.00041152 253400 3.9
B14 0.00041152 262800 5.0
B11 0.0012346 367200 17.2
B12
0.0012346
352200 15.5
B9 0.0037037 685600 54.6
B10 0.0037037 624400 47.4
B7 0. 011111 927200 82.9
B8 0. 011111 942400 84.7
B5
0.033333
1E+06 94.2
B6 0.033333 1E+06 105.8
B3 0.1 1E+06 96.0
B4 0.1 1E+06 110.4
Table 3: Keap1-NRF2 raw data.
MetadichoI-Agonist assay Conc Raw value Percent efcacy
C21 9.77E-02 273400 1.3
C22 9.77E-02 275400 1.5
C19 1.95E-01 254200 -1.1
C20 1.95E-01 281200 2.3
Cu7 3.91 E-01 257000 -0.7
C18 3.91 E-01 248800 -1.7
C15 0.78125 256200 -0.8
C16 0.78125 267800 0.6
C13 1.5625 246800 -2.0
C14 1.5625 244400 -2.3
C11 3.125 255400 -0.9
C12 3.125 255200 -1.0
C9 6.25 268600 0.7
C10 6.25 279200 2.0
C7 12.5 296000 4.1
C8 12.5 277000 1.7
C5 25 269600 0.8
C6 25 266000 0.4
C3 50 238000 -3.1
C4 50 257000 -0.7
Table 4: MetadichoI-agonist assay Keap1-NRF2 raw data.
Metadichol antagonist assay Conc Raw val Percent efcacy
H21 9.77E-02 1195800 -3.5
H22 9.77E-02 1252000 -9.8
H19 1.95E-01 1239600 -8.4
H2O 1.95E-01 1124000 4.5
H17 3.91 E-01 1125000 4.4
H18 3.91 E-01 1125400 4.3
H16 0.78125 1050000 12.8
H16 0.78125 1116800 5.3
H13 1.5625 1001000 18.2
H14 1.5625 1072800 10.2
H11 3.125 977800 20.8
H12 3.125 913000 28.1
H9 6.25 984400 20.1
H10 6.25 986800 19.8
H7 12.5 871000 32.8
H8 12.5 914600 27.9
H5 25 886000 31.1
H6 25 879600 31.8
H3 50 853800 34.7
H4 50 831400 37.2
Table 5: MetadichoI-antagonist assay Keap1-NRF2 raw data.
Citation: Raghavan PR (2017) Metadichol ®. A Novel Inverse Agonist of Aryl Hydrocarbon Receptor (AHR) and NRF2 Inhibitor. J Cancer Sci Ther 9:
661-668. doi:10.4172/1948-5956.1000489
J Cancer Sci Ther
ISSN: 1948-5956 JCST, an open access journal Volume 9(9) 661-668 (2017) - 666
Antihistamines were classied previously as antagonists of histamine
H1 receptors have been reclassied as inverse agonists [35]. AHR is
constitutively active in prostate cancer [36,37]. Tumors and tumor cell
lines have elevated AHR levels. AHR is chronically activated in tumors
[38]. Masafumi et al. [39] created a transgenic mouse lines expressing
the constitutive form of the AHR in keratinocytes. In these lines of
mice, and showed that the AHR activity was constitutively enhanced
in the absence of ligands, and direct eects of PAHs (poly aromatic
hydrocarbons) and their metabolites could be ignored.
e enhanced level of expression of AHR in human tumors is the
ability of NFkB to increase AHR expression, which correlates
with inflammatory status [40]. There is a need for specific high-
affinity AHR
ligands that exhibit improved biological profiles. Metadichol, as we
have shown, is an inhibitor of NF-KB. The emerging role of the
AHR in immune tolerance, and clinical testing of AHR antagonists
like Metadichol as an adjunct to immunotherapy is an approach that
needs to be pursued. We have already shown that Metadichol is an
inverse agonist of VDR. No known inverse agonist of AHR and VDR
are known. What has been postulated is that an inverse agonist will
have effects opposite to that of an agonist. Inverse agonists not
only block constitutive responses of receptors but also to activate
and regulate seven-transmembrane receptor (GPCR) signaling [41].
Our studies in human subjects suggest that Metadichol behaves
more like a protean agonist [42,43]. For example, we have shown
that it lowers insulin levels as well as increases Insulin levels. We
believe that it also acts as a protean agonist i.e. it acts as an inverse
agonist where there is constitutive activity and as an agonist when
there is none.
NRF2 and Metadichol
In the Keap-NRF2 assay shown in Figure 9 it clear that Metadichol
has no agonist activity but has an antagonist activity. But what is
interesting is that the response is not 100% even at higher
concentration, and maximum response does not exceed 35%. The
barrier to be overcome in cancer treatment is the resistance of cancer
cells to chemotherapy. High constitutive expression of Nrf2 is found
in many types of cancers, that leads to an environment conducive to
cancer cell survival. Thus, there is a need for optimal inhibition of
NRF2 expression to overcome the resistance of cancer cells to
chemotherapy. Metadichol dose response is probably U-shaped
(Figure 10). U-shaped. Such shaped curves are seen with some chemo
preventive agents, such as vitamin D and selenium as well as for some
chemotherapeutic drugs. Metadichol does not activate NRF2 and thus
could be useful to increase the effectiveness of cancer treatment.
[44]. The nrf2 pathway is the useful target in the treatment of
diabetes [45]. We have published a case study where Metadichol
normalized blood sugar levels [46].
Conclusion
The results that we have obtained with Metadichol shows that it is
an inverse agonist of AHR. Possible signaling pathways by the binding
actions of Metadichol on AHR and NRF2 is shown in Figure 11. Thus,
Metadichol could be useful in cancers where there are increased levels
of AHR expressed. The discovery of functional cooperation between
AHR and Nrf2 has significant biological implications [47,48]. AHR
has been associated with carcinogenesis, but Nrf2 is associated with
Figure 8: Antagonist assay of standard GNF-351.
Figure 9: Keap-NRF2 assay.
Figure 10: Metadichol dose response is probably U-shaped.
Figure 11: Possible signaling pathways by the binding actions of Metadichol
on AHR and NRF2.
Citation: Raghavan PR (2017) Metadichol ®. A Novel Inverse Agonist of Aryl Hydrocarbon Receptor (AHR) and NRF2 Inhibitor. J Cancer Sci Ther 9:
661-668. doi:10.4172/1948-5956.1000489
J Cancer Sci Ther
ISSN: 1948-5956 JCST, an open access journal Volume 9(9) 661-668 (2017) - 667
cytoprotection against degenerative diseases. Cross-talk between these
two transcription factors can be exploited by Metadichol to therapeutic
advantage and improve outcomes in cancer chemotherapy where there
is a need to control AHR and NRF2 level. Metadichol by binding to
VDR could be useful given the role of Vitamin D in cancer therapy [49].
Metadichol by leveraging targets AHR, VDR, NRF2 that are synergistic
can produce greater levels of ecacy and synergy combinations and
positively impacts the complex systems biology of human disease. Being
not toxic [50-52] and is a huge advantage over all drugs in development
and allows for clinical testing in humans.
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668. doi:10.4172/1948-5956.1000489
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Metadichol® is a Nano emulsion of long-chain alcohols called as Policosanol and is present in foods such as rice, sugar cane, wheat, and peanuts. Metadichol® acts on Nuclear Vitamin D receptors (VDR) that are present in cells throughout the body to stimulate the immune system and inhibit a variety of disease processes, resulting from inflammation to infection [1]. We present a case study of a patient with Rheumatoid arthritis with the high levels of RF antibodies, CRP and ESR levels, and low bone mineral density leading to osteoporosis. The case report shows how Metadichol® by its actions on the VDR has affected key biomarkers and mitigated the disease conditions without any side effects. Also, his bone density improved dramatically. Metadichol® is safe because it consists of natural components of conventional foods and has no known adverse side effects. Its constituents are present in many foods that we consume every day. Metadichol® has the potential to serve as a novel, safe solution to help patients with RA and other autoimmune diseases that confront the world today.
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