1. Monitoring Nano-entities of Compounds for Human
Consumption using the NMR Aggregation Assay
Introduction
In designing compounds destined for human
consumption the pharmaceutical and food
industries take every precatution into consideration
to prevent undesirable secondary effects.
Some could be attributed to off-target effects
caused by the physical phenomenon of aggregation
by these compounds at different concentrations.
Sunset Yellow FCF (2783-94-0)
Allura Red AC (25956-17-6)
Materials And Methods
• Allura Red AC (25956-17-6)
• Sunset Yellow FCF (2783-94-0)
• Erythrosin B (16423-68-0)
• Tartrazine (1934-21-0)
• Acid Violet (1694-09-3)
• Drug 1
• D2
O Buffer
• A Tween 20 10% v/v solution was prepared in D2
O; it was then
added to the maximum concentration of each compound following
centrifugation at 2000 RPM for 10 minutes.
• The samples were run on a 500 MHz NMR with a scan number of
1500 each.
• Observations were focused primarly on the aromatic region of the
NMR between 9 and 6 ppm.
Aim
This experirment deals with the monitoring
of self-aggregation using NMR with increasing
concentrations of organic compounds in aqueous
solvent.
How do we monitor for aggregation?(1)
Some of the unusual features seen in aggregates are
Broad resonances due to micelle-like assemblies
causing it to behave as a slow thumbling aggregate.
Under different concentrations it is possible to observe
shift resonance and more resonances (unexpected
peaks) than corresponding hydrogens on the
compound.
Should aggregates form, the break up of assemblies can
also be monitored by the“detergent effect”of Tween 20.
Studied adverse effects
• Significantly reduced reproductive success, parental and offspring weight, brain weight, survival and female vaginal
patency development in rats at doses of up to 10% of the diet.(2)
• At high levels (86 – 1430 times greater than human ADI) it produced adverse effects in reproductive and neurobehavioral
parameters in mice.(3)
• Genotoxic potential in yeast cells at 37°C. Risk of affecting the fermentation process of baking.(4)
Studied adverse effects
• “CSPI reports that those dyes (Red 40, Yellow 5 and Yellow 6) contain known carcinogens and contaminants […]
unnecessarily increase the risks of cancer, hyperactivity in children and allergic reactions.(5)
• Alters relative expression of CD3e/CD4/CD8 receptors in T cells and CD19 in B-Cells at concentrations above 250 µg/mL.
SY exposure suppresses functional properties of T-cells and B-Cells. Decline in cytokines production.(6)
• Patchy degeneration of testis in rats when fed 0.5% and 3% SY mixed with pelled diet for 90 days.(7)
Observed changes
• Steady reasonance shift as concentration
increases.
• There are no particular changes on the shape of
the resonance; no broad peak shape.
• Increasing concentration allows to better
identify the peaks corresponding to this
compound.
• Detergent made an aggregate peak disappear
in favour of the peaks corresponding to the actual
structure
Observed changes
• Minor resonance shift on the early stages but
increasing rapidly as concentration reaches 110
µM.
• There are no particular changes on the shape of
the resonance; no broad peaks.
• Increase in concentration allows to better
identify the peaks corresponding to this
compound.
• Detergent didn’t cause any additional effects
other than making the peaks sharper
References
1. LaPlante S. R.; Carson, R.; Gillard, J.; Aubry, N.; Coulombe R.; Bordeleau, S.; Bonneau, P.; Little, M.; O’ Meara, J.; Beaulieu, P. L. Compound Aggregation in Drug Discovery: Implementing a
Practical NMR Assay for Medicinal Chemists. J. Med. Chem. 2013, 56(12), pp 5142-5150.
For a full list of references please refer to the following website:
http://goo.gl/sR4QLs
Ernesto Cuadra-Foy, Steve Laplante, Pat Forgione*.
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec.
400 µM
110 µM
220 µM
440µM
350 µM
55 µM
200 µM
36 µM
175 µM
22 µM
133 µM
116 µM
400 µM+
TW20
440 µM+
TW20
E
D
A
G
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F
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D
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C
B
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A
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B B’
D’
A
C
E
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BD
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CE
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