Proposed In Silico/In Vitro Approach for Botanical Mixtures - Presenter name: Dr. Catherine Mahony Affiliation: Procter & Gamble Contact ...
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Proposed In Silico/In Vitro Approach for Botanical Mixtures Presenter name: Dr. Catherine Mahony Affiliation: Procter & Gamble Contact information: mahony.c@pg.com
Conflict of Interest Statement Presenter is employed by Procter & Gamble. Procter & Gamble provided financial support to the research presented.
Outline of Presentation • Goals and overall strategic approach • Constituent characterisation and in silico approach • High content and high throughput in vitro approaches • Conclusions and outlook
Goals of Botanical Safety Research
⇒ Tools and Strategies for Botanical Safety Assessment = Better predictions of
toxicity and risk
Support the increasing business interest in botanicals
Focused on challenges that botanicals represent
Identity/adulteration and complex chemistry
Data gaps/limitations
Herb-Drug Interactions (HDIs)
Botanical Safety Strategic Approach
Raw Material Identity and Quality are cornerstones
Tier 1: Based on low level of exposure & Threshold of Toxicological Concern (TTC)
approach (for botanical mixture)
Tier 2: Based on significant human use history (consider delayed effects and
concomitant meds)
Tier 3: Based on the in-depth characterization of
botanical constituents & evaluation of toxicityFocus on Tier 3
Safety
Assessment of
Botanical
Preparation
Pre-Work
Safety Dossier to
Support Human Use
in Market Botanical Cleared
Pre-Work
Botanical
Preparation
Where data is insufficient from Tier 1 and Tier 2
Safety
Assessment
(Published Data)
assessment, i.e., higher exposures and
Unresolved
Endpoint Gaps or
Different
Preparation/Dose*
Sample Acquisition &
Characterization
Sample
Acquisition
&
Characterization
toxicological data are lacking; differing
(Analytical Data)
Advanced Analytical
Instrumentation
UHPLC w/UV, CAD,
and HRMS detection
Obtain Comprehensive
compositional, structural and
quantification data*
extractions etc.
(B) (A) NO
(F) YES YES
Commonly Constituent
Dose at dietary
consumed in Structure
exposure
diet? known?
levels?
NO
sse Hazard Analysis:
(C) Complete Safety ocr Identify
Established Endpoint Assessment P Constituent of the
tn
tox profile and Known Data e Botanical
suitable MoS/ (Gentox, DART m
ss Driving Safety
MoE? Systemic, etc) es
Generate data via s Assessment
A (In Silico Data)
High Throughput coi
NO Expert Systems li
S
(i.e. DEREK) nI
(YES) (D) Complete:
Constituent(s) SAR Analogue Identification
possible & & SAR Assessment for Verify Identity
Clearedǂ
suitable MoS/ Constituents of and Exposure
MoE? Concern Levels for Any
Unfavorable
Alerts
NO
Use Expert Tools: Assess Constituents
(E)
Cramer Classification with Structural Alerts
Exposure level
DEREK Alerts or Unfavorable Data
below relevant
DART Decision Tree
TTC?
Expert Opinion
NO
Resolution,
(STOP) Redesign or
Reduce dose to supportable level (based on % Testing
constituent(s) driving safety assessment) or obtain
additional safety dataPre-Work
Botanical
Preparation
Safety
Assessment
(Published Data)
Sample
Acquisition
&
Characterization
(Analytical Data)
Hazard Analysis:
Identify
Constituent of the
Botanical
Driving Safety
Assessment
4 Key Steps
(In Silico Data)
Resolution,
Redesign or
TestingFocus on Tier 3
Safety Safety Dossier to
Pre-Work
Assessment of Support Human Use
Pre-Work Botanical Cleared Botanical
Botanical in Market
Preparation
Preparation Safety
Complete Pre-Work Assessment
(Published Data)
1. Build hazard assessment dossier from reliable sources
(Evidence based support not absence of evidence)
Unresolved
Endpoint Gaps or
2. Evaluate botanical for endpoints (Toxicology Review)
Different Sample Acquisition & 3. Determine: Adequate or Gap for each end point
Sample
Preparation/Dose* Characterization
Acquisition
&
Characterization
(Analytical Data)
Advanced Analytical Obtain Comprehensive
Instrumentation compositional, structural and
UHPLC w/UV, CAD, quantification data*
and HRMS detection
(B) (A) NO
(F) YES YES
Commonly Constituent
Dose at dietary
consumed in Structure
exposure
diet? known?
levels?Focus on Tier 3
Safety Safety Dossier to
Pre-Work
Assessment of Support Human Use
Pre-Work Botanical Cleared Botanical
Botanical in Market
Preparation
Preparation Safety
Assessment
(Published Data)
Unresolved
Endpoint Gaps or
Different Sample Acquisition &
Sample
Preparation/Dose* Characterization
Acquisition
&
Unresolved Gaps
Characterization
(Analytical Data)
Advanced Analytical
Instrumentation
Obtain Comprehensive
compositional, structural and Examples:
UHPLC w/UV, CAD, quantification data*
• Different Solvent Systems
and HRMS detection
• Natural Variation
• Target Audience
• Change dose/exposure > dietary/traditional
• Efficacy vs. Safety
(B) (A) NO
(F) YES YES
Commonly Constituent
Dose at dietary
consumed in Structure
exposure
diet? known?
levels?Focus on Tier 3
Constituent Characterization and
Safety Safety Dossier to
Identification
(CCID)
Pre-Work
Assessment of Support Human Use
Pre-Work Botanical Cleared Botanical
Botanical in Market
Preparation
Preparation • Need sample intended for human exposureSafety
• Reference samples helpful to confirm ID of
Assessment
(Published Data)
botanical and any constituents
• Need both qualitative data and quantitative data
Unresolved • Results of genetox data and level of indented dose
will drive lower limit of detection required—”no
Endpoint Gaps or
Different Sample Acquisition &
Sample
need to chase ZERO”.
Preparation/Dose* Characterization
Acquisition
&
Characterization
(Analytical Data)
Advanced Analytical Obtain Comprehensive
Instrumentation compositional, structural and
UHPLC w/UV, CAD, quantification data*
and HRMS detection
(B) (A) NO
(F) YES YES
Commonly Constituent
Dose at dietary
consumed in Structure
exposure
diet? known?
levels?Instrumentation
Focus on Tier 3
Safety Safety Dossier to
Pre-Work
Assessment of Support Human Use
Pre-Work Botanical Cleared Botanical
Botanical in Market
Preparation
Preparation Safety
Assessment
(Published Data)
Unresolved
Endpoint Gaps or
Different Sample Acquisition &
Sample
Preparation/Dose* Characterization
Acquisition
&
Characterization
(Analytical Data)
Advanced Analytical Obtain Comprehensive
Instrumentation compositional, structural and
UHPLC w/UV, CAD, quantification data*
and HRMS detection
(B) (A) NO
(F) YES YES
Commonly Constituent
Dose at dietary
consumed in Structure
exposure
diet? known?
levels?Focus on Tier 3
(B) (A) NO
(F) YES YES
Commonly Constituent
Dose at dietary
consumed in Structure
exposure
diet? known?
levels?
For each structure defined
NO by Analytical you must know:
1. If constituent is > TTC, structure and dose ss
e Hazard Analysis:
c
2. If constituent is < TTC follow
(C)
TTC Rules Endpoint
Established
Complete Safety
Assessment
o
r
P
Identify
Constituent of the
tn
tox profile and Known Data e Botanical
suitable MoS/ (Gentox, DART m
ss Driving Safety
MoE? Systemic, etc) e
ss
Generate data via Assessment
A (In Silico Data)
High Throughput o
NO ic
Expert Systems ilS
(i.e. DEREK) n
I
(YES) (D) Complete:
Constituent(s) SAR Analogue Identification
possible & & SAR Assessment for Verify Identity
Clearedǂ
suitable MoS/ Constituents of and Exposure
MoE? Concern Levels for Any
Unfavorable
Alerts
NO
Use Expert Tools: Assess Constituents
(E)
Cramer Classification with Structural Alerts
Exposure level
DEREK Alerts or Unfavorable Data
below relevant
DART Decision Tree
TTC?
Expert Opinion
NO
Resolution,
(STOP) Redesign or
Reduce dose to supportable level (based on % Testing
constituent(s) driving safety assessment) or obtain
additional safety dataFocus on Tier 3
and HRMS detection
(B) (A) NO
(F) YES YES
Commonly Constituent
Dose at dietary
consumed in Structure
exposure
diet? known?
levels?
NO
ss
e
c Hazard Analysis:
(C) Complete Safety o Identify
r
Established Endpoint Assessment P Constituent of the
tn
tox profile and Known Data e Botanical
suitable MoS/ (Gentox, DART sm
se Driving Safety
MoE? Systemic, etc)
Generate data via ss Assessment
A (In Silico Data)
High Throughput o
NO For eachExpert
structure
Systemsdefined byicliS Analytical you must know:
1. If constituent is > TTC, then need
(i.e. DEREK) n
I structure and dose
(YES) (D) Complete: 2. If constituent is < TTC, follow TTC Rules
Constituent(s) SAR Analogue Identification
possible & & SAR Assessment for Verify Identity
Clearedǂ
suitable MoS/ Constituents of and Exposure
MoE? Concern Levels for Any
Unfavorable
Alerts
NO
Use Expert Tools: Assess Constituents
(E)
Cramer Classification with Structural Alerts
Exposure level
DEREK Alerts or Unfavorable Data
below relevant
DART Decision Tree
TTC?
Expert Opinion
NO
Resolution,Focus on Tier 3
and HRMS detection
(B) (A) NO
(F) YES YES
Commonly Constituent
Dose at dietary
consumed in Structure
exposure
diet? known?
levels?
NO
ss
e
c Hazard Analysis:
(C) Complete Safety o Identify
r
Established Endpoint Assessment P Constituent of the
tn
tox profile and Known Data e Botanical
suitable MoS/ (Gentox, DART sm
se Driving Safety
MoE? Systemic, etc)
Generate data via ss Assessment
A (In Silico Data)
High Throughput o
ic
Determine if exposures is already
NO Expert Systems li
S
(i.e. DEREK) n
I
(YES)
Constituent(s)
(D)
SAR
Complete:
established in diet:
Analogue Identification
possible & & SAR Assessment for Verify Identity
Clearedǂ
suitable MoS/ Constituents of and Exposure
MoE? • What Concern
qualifies as dietary intake? Levels for Any
• “Dietary exposures are supported by aAlerts
genetically
Unfavorable
diverse
NO
and sufficiently large population”
Use Expert Tools: Assess Constituents
(E)
Cramer Classification with Structural Alerts
Exposure level
DEREK Alerts or Unfavorable Data
below relevant
DART Decision Tree
TTC?
Expert Opinion
NO
Resolution,If exposure cannot be supported by dietary intake,
(B) (A) NO
(F)
With adequate toxicology
YES
data for each
Commonly YES Constituent
Focus on Tier 3
Dose at dietary
consumed in Structure
endexposure
point
levels?
diet? known?
then safe human use must be estimated based on
toxicology data.
NO
Two Challenges:
1. Adequate data w/o over reliance on history
sse of use
Hazard Analysis:
Complete Safety2. Generating more data to fill gaps w/oc
r defaulting to Identify
traditional tox studies
(C) o
Established Endpoint Assessment P Constituent of the
tn
tox profile and Known Data e Botanical
suitable MoS/ (Gentox, DART m
ss Driving Safety
MoE? Systemic, etc) se
s Assessment
Generate data via A
High Throughput o (In Silico Data)
NO ic
Expert Systems ilS
(i.e. DEREK) In
(YES) (D) Complete:
Constituent(s) SAR Analogue Identification
possible & & SAR Assessment for Verify Identity
Clearedǂ
suitable MoS/ Constituents of and Exposure
MoE? Concern Levels for Any
Unfavorable
Alerts
NO
Use Expert Tools: Assess Constituents
(E)
Cramer Classification with Structural Alerts
Exposure level
DEREK Alerts or Unfavorable Data
below relevant
DART Decision Tree
TTC?
Expert Opinion
NO
Resolution,
(STOP) Redesign or
Reduce dose to supportable level (based on % Testing
constituent(s) driving safety assessment) or obtain
additional safety data(B)
When data is not adequate to
(A) NO
(F) YES YES
Commonly Constituent
Focus on Tier 3
Dose at dietary
consumed in Structure
support safe human exposure than
exposure
diet? known?
levels?
additional assessment approaches
NO may
s
be needed.
1. In silico Approaches
se Hazard Analysis:
(C) Complete Safety c
o
r Identify
Established Endpoint Assessment E.g., DEREK
P
tn and customized
Constituent alerts
of the
tox profile and Known Data
suitable MoS/ (Gentox, DART E.g., SARemss
Botanical
Driving Safety
MoE? Systemic, etc) se
s Assessment
Generate data via A
High Throughput o (In Silico Data)
NO ic
Expert Systems ilS
(i.e. DEREK) In
(YES) (D) Complete:
Constituent(s) SAR Analogue Identification
possible & & SAR Assessment for Verify Identity
Clearedǂ
suitable MoS/ Constituents of and Exposure
MoE? Concern Levels for Any
Unfavorable
Alerts
NO
Use Expert Tools: Assess Constituents
(E)
Cramer Classification with Structural Alerts
Exposure level
DEREK Alerts or Unfavorable Data
below relevant
DART Decision Tree
TTC?
Expert Opinion
NO
Resolution,
(STOP) Redesign or
Reduce dose to supportable level (based on % Testing
constituent(s) driving safety assessment) or obtain
additional safety data(YES) (D) Complete:
Constituent(s) SAR Analogue Identification
possible & & SAR Assessment for Verify Identity
Clearedǂ
Focus on Tier 3
suitable MoS/ Constituents of and Exposure
MoE? Concern Levels for Any
Options and next steps:
Unfavorable
Alerts
NO
Use Expert Tools: Assess Constituents
• Reduce dose until safety data can
(E)
Cramer Classification with Structural Alerts
Exposure level
DEREK Alerts or Unfavorable Data
support exposure
below relevant
DART Decision Tree
TTC?
Expert Opinion
NO
• Data generated to this point is useful to
design further studies if needed Redesign
Resolution,
or
• Genetox data is often needed
(STOP)
Reduce dose to supportable level (based on % Testing
constituent(s) driving safety assessment) or obtain
additional safety dataExamples Where In Silico Decision Tree is Used
1. Benchmarking constituent safety between the diet and botanical
supplements
2. Applying constituent analysis to justify bridging safety data between
different methods of botanical preparation
3. Establishing exposure thresholds for individual constituents with
limited human use data using in silico toxicology assessment tools
4. Informing design of follow up safety studies where neededSummary of CCID-In Silico Approach
• Thoroughly vet the scientific literature─define the question(s)!
• Advanced multi-detector analytical characterization technique to
establish botanical constituent composition (simultaneous ID and
quantitation)
• Each identified constituents is processed through a decision-tree to
resolve questions
─ Close safety gaps, inform supportable exposure levels, or need for safety
studies
• ⇒ a focused approach for detecting possible bad actors in botanical
extracts, the variables involvedCase Study─Artichoke Leaf
O
CH2
O
H2C
CH2
O
HO OH
O
CH2
Cynaropicrin
(Sesquiterpene lactone)
Food-Like Tannins
Insoluble UnknownIn Vitro Testing Approaches: Informing DART Potential at a MOA Level • High-throughput approaches • HTS batteries: Customised Cerep Panel • Global gene expression analysis: CMAP in 4 cell types • Decision-making • By lack of response on developmentally relevant targets • By functional comparisons
How Many Developmentally Relevant MOAs?
• Cataloging via DART
ontology project
• 19 major categories,
multiple subcategories
• Focus on
─Mechanisms not
involving reactivity
─Mechanisms that are
unique to development
or where development
is most sensitive or
response most severeCerep Assay Selections
Receptor Binding Assays
• Steroid and retinoic acid receptors (8)
• Neurotransmitter receptors and transporters (32)
• Ca, K, Na ion channels (3) Enzyme and Uptake Assays
• DART-relevant enzymes
• Cox 1 and 2
• ACE
• Cholesterol synthesis
• Tubulin
• MAO-A
• Phosphodiesterases
• Run botanical extracts with and without human
liver S9 (with NADPH)Botanicals Tested in Cerep • Selected based on ─Presumed lack of DART because of dietary use ─Anecdotal information of DART ─Pharmacology that might preclude use in pregnancy • Concentration based on a multiple of measured Cmax of an active marker compound, or conservative assumptions to estimate a Cmax
Cerep Big Picture
Top two. The more red, the
higher the inhibition
Bottom. Difference between parent
and metabolite where blue means
less activity from the metabolites,
and red means more activity with
the metaboliteCerep Results Case study interpretation─artichoke leaf–receptor activity consistent with traditional use (relief of digestive disorders) BUT lack of response on developmentally relevant targets
Cerep Results
IC50 Follow Up─PR Activity
IC50 (ug/mL)
Levonorgestrel Norgestimate
(Plan B) Chaste Tree T0
Progesterone Cat’s
Claw Ginger T90
3-Keto Desogestrel
17-Deacetylated Norgestimate T0 Ashwagandha T0
Gestodene Ashwagandha T90
Dihydrotestosterone Ginger T0
Mifepristone Chaste Tree T90
Cat’s Claw T90
(Abortifacient)
0.00001 0.0001 0.001 0.01 0.1 1 10 100 1000
Can inform potency differences; early pregnancy abortifacient →
contraceptives → endogenous hormones → botanicals (food vs. non-food)Connectivity Mapping (CMAP) Approach
MCF7 cells, rich in nuclear hormone receptors
HepG2 cells, hepatic characteristics, including limited
xenobiotic metabolism
A549 cells, respiratory epithelium
CDI (iPS-derived) cardiomyocytes, numerous ion channels
and neurotransmitter receptorsSt. John Wort (SJW)
• Cerep activity and CMAP – where is the commonality?
Botanical Test Parent hits Metabolite hits St. John's Wort
article FDRComparing Active Concentrations
• CMAP effect concentration
─ 25-250 ug/ml, or 1.8-18 uM hyperforin, a marker compound for SJW
• Ki for key receptor for functional analog
─ 2uM Chlorpromazine
• Relative potency is 0.9 - 9
• Developmental NOAEL for functional analog
─ E.g., chlorpromazine: 20 mg/kg/day (rat)
o Cmax at this dose is approx. 0.5-1uM
• Adjust for potency: 0.45 - 9 uM hyperforin
• Therapeutic dose of SJW gives a Cmax for hyperforin of 280 nM
─ Margin is ~1.6
─ Animal studies have shown equivocal results. The potential risk for humans is unknown.
In the absence of sufficient clinical data, the use during pregnancy and lactation is not
recommended.Summary of In Vitro MoA Approach
• Biological activity of botanicals can be characterized through in vitro
approaches
• Best approach will include both methods
─CMAP to identify functional analogs (but more complex analysis)
─Cerep to provide better focus (esp. to rule out false positives)
• Further work ongoing to utilize data for risk assessment purposes
─Comparing concentrations to assess relative potency
o CMAP effect/no-effect concentration; Ki/IC50 for key receptors
─Extending both panels for greater coverage of systemic toxicity MoAConclusions and Outlook • Constituent level characterization and in silico approach can resolve botanical safety gaps, refine supportable exposure levels, or inform need for safety studies • High content and high throughput in vitro approaches can inform botanical mode of action or lack of response on toxicologically relevant targets • Botanically-derived ingredients are extremely challenging from a Safety POV, but good science can save time and money!
References • Vandermolen, K. et, al. Safety Assessment of Mushrooms in Dietary Supplements by Combining Analytical Data with In Silico Toxicology Evaluation. Food and Chemical Toxicology 103 (2017) 133-147 • Little, J. et, al. In Silico Approach to Safety of Botanical Dietary Supplement Ingredients Utilizing Constituent-Level Characterization. Food and Chemical Toxicology 107 (2017) 418-429
Acknowledgements
• CCID/In silico approach • Botanical MoA/DART Project
─ Jason Little ─ Karen VanderMolen
─ Tim Baker ─ Eurofins
─ Brian Regg o Annie Otto-Bruc (AnnieOtto-
─ Jason Price Bruc@eurofins.com)
─ Kady Krivos o Kevin Kennedy
─ Vincent Sica (KevinKennedy@eurofins.co
m)
─ Bob Strife ─ Jorge Naciff
─ Karen VanderMolen ─ George Daston
─ Bin Sun
─ Dan MarsmanThank you for your attention!
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