Lemons to Lemonade: Turning a Disappointing ...

Lemons to Lemonade: Turning a Disappointing Transition from Lab to Human Skin Testing into Better Repellents. Robert H. Bedoukian ESA Annual Meeting, Denver, Colorado November 5th, 2017

Screening Potential Topical Repellents without Using Human Bait • Potential topical repellents are commonly screened using methods that employ nothing more than warmed blood and a faux skin to entice mosquitoes to land/probe or a choice type olfactometer that simply measures attraction to a source. • New ideas generally require the screening of 100’s of compounds to find a handful that may work. • Human testing is too expensive and/or IRB/HSRB approval can be too time consuming to go through these steps for new ideas that may or may not have merit. • Human screening is usually not employed until after compounds have passed a battery of tests, usually comparing to DEET (the “gold standard”, positive control) and an untreated (bait only, negative control) sample.

Standard Repellents

Compounds of Interest • Cyclic Ketones:

“nature inspired”

– Methyl Apritone – Safety • • • •

Acute Oral Toxicity – LD50 > 5000 mg/kg Acute Dermal Toxicity – LC50 >5000 mg/kg Dermal Irritation – Not an irritant for rabbits (neat) Dermal Sensitization – Not a sensitizer on guinea pigs (neat)

Compounds of Interest “nature inspired”

• Jasmonate Series:

– Methyl Dihydrojasmonate – Ethyl Dihydrojasmonate – Propyl Dihydrojasmonate – Prenyl Dihydrojasmonate – Heptyl Dihydrojasmonate

O O O R

Safety Information on Jasmonates • Methyl Dihydrojasmonate: naturally occurring; well established as Flavor/Fragrance material for over 50 years. Approx. 8,300 MT used in 2011. • Acute Oral Toxicity – LD50 > 5000 mg/kg • Acute Dermal Toxicity – LC50 >5000 mg/kg • Dermal Irritation – 20% on humans had no effects • Dermal Sensitization – 20% on humans had no effects • Propyl Dihydrojasmonate – excellent safety data, EPA registered for food use. • Data can be extrapolated to other, larger jasmonates but there is no known toxicological data specific to those compounds.

Compounds of Interest Lactone Series: ‒ ‒ ‒ ‒ ‒ ‒ ‒

“nature inspired”

Gamma Dodecalactone Gamma Methyl Dodecalactone Gamma Tridecalactone Gamma Methyl Tridecalactone Gamma Tetradecalactone Gamma Pentadecalactone Gamma Heptadecalactone

O

O

R

‒ Delta lactones also of interest (gamma shown here)

Safety Information on Lactones • Gamma Dodecalactone: naturally occurring; well established as Flavor/Fragrance material since 1940’s • Acute Oral Toxicity – LD50 > 5,000 mg/kg • Acute Dermal Toxicity – LC50 >5,000 mg/kg • Dermal Irritation – 12% on humans had no effects • Dermal Sensitization – 12% on humans had no effects • Readily biodegradable • Volumes of use: Gamma-Decalactone – 453 tons/year Gamma-Undecalactone – 2,300 tons/year Gamma-Dodecalactone – 21 tons/year

Compounds of Interest, continued • Carboxylic Acids – Lactic Acid – Salicylic Acid – Geranic Acid – Citronellic Acid

Safety Information on Carboxylic Acids • Lactic Acid is synthetically derived and has a wide variety of uses. • Acute Oral Toxicity – LD50 > 200,000 mg/kg • Acute Dermal Toxicity – LC50 >1,000 mg/kg • Sensitization– non-sensitizer • Biodegradable • Permitted in cosmetics up to 10% and with a pH ≥ 3.5 • Volumes of use: • Worldwide consumption >60,000 MT • 613,000 kg consumed in flavors in 2005

Laboratory Screening: Mosquitoes

Gupta (Modified K&D) Box

Gupta (Modified K&D) Box Testing Protocol  Five replicates of 250 adult female mosquitoes

 clear plastic cages with access to five warmed, blood-filled, membranecovered wells.  Aedes aegypti unless otherwise noted.

 Membranes were treated with repellent dilutions.  Five replicates, rotating positions within the chamber.  Each replicate used a fresh batch of mosquitoes, blood and treated membranes.  The “instant” number of mosquitoes probing each well was recorded at two minute intervals for 20 minutes.

Gupta (Modified K&D) Box Testing Protocol  The percentage repellency relative to the control was calculated  The total numbers of probes on each well were tallied at the end of the observation period and the average for each formulation was calculated.  To compare formulations from different tests, the control probes were adjusted to 100 for each test and the formula then applied to each formulation to adjust accordingly.

Screening Potential Topical Repellents - Lab

Relationship between volatility and repellency • It has often been stated that the volatility of a repellent compound is vital for its overall effectiveness and that evaporation of the active ingredient from the host’s skin surface is necessary for its efficacy. • Therefore, one is faced with the dilemma that while high volatility was likely to enhance repellency, it also shortens protection time. • DEET with a boiling point of 290 C is not very long lasting unless higher concentrations are used (30 – 75%). • We chose to repellents that are much less volatile than DEET, and thus make for very interesting options for topical repellency. (lower odor, too!) • Methyl Apritone 324 C • Propyl Dihydrojasmonate 336 C • Gamma Tridecalactone 325 C • 3-Methyl-5-Nonyl-2-Cyclohexenone 325 C

Relative Volatility – (Percent remaining)

Oven at 50C = 122F


Arm in Cage Test *1st time point was at 15 minutes post treatment. None of the BRI compounds had zero bites at the first time point.

Moving from Gupta Box to an Arm in Cage Test So what happened? – Laboratory screening tests show that the BRI compounds exhibit much better repellency than DEET. – Does their repellency actually decrease when applied to human skin? Or can their poor performance in the human testing protocol be explained in another way?

“Way?” • Looking back at the lab (non skin) results, and applying the more rigorous conditions used by EPA and WHO for human testing, it is not so surprising that the “new” repellents failed. – the lab tests run for only 20 minutes, checking only at discrete 2 minute intervals, and the mosquitoes have a choice of “less repellent” chambers.

• Arm in cage testing gives mosquitoes 3 or more full minutes to probe a larger surface with no alternative “host”. – A 99.0% repellency in the Gupta chamber would quickly fail in the human test. In fact, even a “100%” repellency under K&D conditions could fail immediately on the human test.

Moving from Gupta Box to an Arm in Cage Test So what happened (continued)? – Our repellent’s efficacy may only appear to be decreasing when applied to human skin. The poor performance in the human testing protocol may be explained by the rigor of the protocol.

– BUT what about DEET? – DEET is not very repellent in our lab tests. – How can 5% (even 10%) DEET work at all in this more rigorous test protocol when they are not 100% effective in the Gupta box?



Epiphany? • Could there be something on human skin that acts synergistically with DEET, making it actually repel better than it does in the lab? • Might our good “lab” repellents also behave synergistically with DEET?

Recapping….. • DEET, PMD, and Picaridin exhibit less repellency than many “biological” repellents in the lab. – But….. DEET, PMD, and Picaridin work well in human testing as repellents. • In one instance - in a Gupta box test when DEET was applied at a rate of 0.18 microliters/cm2 to the test well it was only 47.61% repellent over the 20 minute period, to Aedes aegypti. • However when DEET was applied at a rate of 0.10 microliters/cm2 to a human volunteer’s arm and the arm inserted into a test cage of Aedes aegypti, DEET was 100% repellent for 2.75 hours.

• Since our “repellents” are based on naturally occurring chemicals we decided to look for synergy with DEET.

Is there synergy? – Some of our new repellents work very well on their own in lab tests – These materials don’t work well at all on human skin, alone. – When combined with DEET – will the improvement is significant? – What combinations of materials will work to extend Complete Protection time?

Combinations The following data show combinations of DEET plus BRI components that are either commonly found on human skin or are produced by plants (BRI used synthetic materials).

Combination Testing: Arm in Cage testing with USDA Test results Treatment ED50 (% active) showed that % DEET % BRI - Chem % Combined combining BRI materials with DEET 0.126 conventional repellents like DEET allowed for g-D 1.8 far less conventional to be DEET + g-D 0.0129 0.1294 0.1423 used. 1 part DEET: 2.5 parts g-D

The table shows that 9.8X less DEET and 13.9X less g-D are needed in the combination than separately.

Arm in Cage Testing Complete Protection Time (CPT) • Contract Labs • BRI Lab in Melbourne, FL

Arm in Cage Testing Complete Protection Time (CPT) • Contract Labs • BRI Lab in Melbourne, FL

Arm in Cage Testing for Complete Protection Time • 3 repetitions conducted • Modified WHO protocol • Approximately 75 Aedes aegypti mosquitoes in a 12”X12”X12” (28316.85 cm3) screen cage. • Treatment area of approximately 200 cm2 marked off, between wrist and elbow, which was treated at a rate of ~1.56 mL/cm2.

Arm in Cage Testing for Complete Protection Time (CPT) • Untreated arm inserted for 30 seconds prior to test to determine activity (> 10 landings with intent required to proceed) • Treated arm inserted for 3 minutes at different time points until failure (2 landings with intent to bite). – First ‘challenge’ at 30 minutes post treatment – Second exposure at 1 hour, then every hour thereafter until ‘Failure’ or 8 hours CPT.



Arm in Cage Results in Melbourne, FL lab

Arm in Cage Results at Contract Lab

Additional Results in Melbourne, FL lab

Additional Results in Melbourne, FL lab

Theories of the Mode of Action of Repellents Five theoretical modes of action for insect repellents (DeGennaro 2015):

1. Inhibit the response of sensory neurons of host attractants, 2. Activate a receptor system that mediates a competing or inappropriate behavior, 3. Act as attractant at low concentration, but as a repellent at higher concentration, 4. Activate receptors linked to several behavioral programs to increase the noise/signal ratio in order to “jam” the relevant sensory circuit, and 5. Activating unique avoidance/aversive receptor(s).

Olfactory Mechanisms of Action • Avoidance – Theory that the “smell” of DEET causes insects to avoid the odor • Confusant – A theory that says DEET does nothing if not combined with host odors. Working with host odors, confusion is created causing insects to not be able to find the DEET covered host (DeGennaro 2015).

Electrophysiology/ Odorant Receptor • Walter Leal’s group – 2014 – “We have discovered an odorant receptor in the southern house mosquito, which is essential for repellency, thus unravelling how DEET works. Additionally, we have identified a link between the receptor for DEET and for Methyl Jasmonate, thus suggesting that DEET might work by mimicking defensive compound(s) from plants.” – This could be the rationale behind the commonality between the different “plant based” structures used in our testing!

In Conclusion • There is synergy – We were able to show that combinations of materials naturally found on human skin, as well as other biochemical, “plant based” compounds commonly used in the flavor and fragrance industry can be combined with DEET to significantly extend the complete protection time.

Patents / Partners • We were granted US Patent 9,314,029 on April 19th. • Covering a broad range of chemicals that can be used as topical mosquito repellents. • These chemicals, plus some others not included in this patent, are subject matter for a pending patent application in which these molecules can be used as synergists for DEET, para-Menthanediol, Picaridin, and other conventional repellents. (US application # 14/540,612 and PCT/US2014/065454).

• Looking for partners!

Thank you!! • And special thanks to: – Dr. Karen McKenzie whose lab (and arm) provided much of the data presented. – Dr. Lacey Jenson, who helped Karen put this presentation together. – Marie Maher, who oversaw all the testing and is the “keeper of the data” that we’ve been generating over many years.