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Novel Treatments
for Neuromuscular Conditions
Chemical Neuro Stimulation of TRPV1 and TRPA1 Sensory
Neurons Decreases Muscle Cramps in Humans
Glenn F. Short III, Laura B. Rosen, Robin Sutherland, Jian Liu, Jennifer M. Cermak, Gary Maier and Thomas Wessel
Flex Pharma, Inc. Boston, MA 02199
Presentation Number: 537.15
Chemical Neuro Stimulation is the treatment of neurological disorders by using small molecules applied
topically to sensory neurons to alter the behavior of distinct neural circuits within the central nervous
system. We have devised one such approach whereby the co-activation of TRPV1 and TRPA1 ion
channels in the upper alimentary canal decreases muscle cramp frequency and severity. Based upon
recent evidence that α-motoneuron hyperexcitability is the underlying cause of cramps and spasticity
(1,2), we hypothesized that TRP activation could provide sufficient excitatory sensory input via the
solitary tract to modulate descending spinal pathway signaling to increase interneuronal inhibition and
dampen motor neuron hyperexcitability. We have generated data that suggests that either an oral
solution containing a mixture of naturally-derived TRP activators (TRP-Stim) or FLX-787, a synthesized
single molecule TRPV1/TRPA1 co-activator, stimulate TRP ion channels in the mouth, oropharynx and
esophagus in a local, topical fashion to inhibit muscle cramping. Efficacy studies using an electrically-
induced cramp (EIC) model demonstrated that both TRP-Stim and FLX-787 significantly reduced cramp
intensity by as much as 3-fold relative to inactive control (p<0.01). Moreover, the pharmacokinetic
profile of FLX-787 could not account for its EIC efficacy, as no systemic exposure of the parent form of
FLX-787 in human plasma was observed. In both animals and humans, FLX-787 was found to undergo
rapid phase 2 metabolism, resulting in extensive conjugation 15 minutes after ingestion, predominantly
to glucuronide and sulfate metabolites. Even at doses up to 500 mg/kg/day in rats, the conjugates of
FLX-787 accounted for >90% of circulating drug. To understand if topical exposure to mucous
membranes in the mouth, oropharynx and esophagus mediates the TRP-Stim and FLX-787 effect (given
the lack of systemic exposure to FLX-787), the TRP-Stim mixture was encapsulated in gelatin capsules.
Ingestion of the encapsulated mixture afforded no EIC efficacy relative to vehicle control. These results
suggest that the observed effect on electrically-induced muscle cramps does not depend on the
systemic bioavailability of TRP activators but rather on topical exposure of sensory neurons and
consequent neuronal signaling. Given that efficacy signals have also been observed in proof of concept
(POC) nocturnal leg cramp (NLC) studies in humans with TRP-Stim or FLX-787, Chemical Neuro
Stimulation may be a general approach to develop novel treatments for cramps, spasms and spasticity
in clinical populations. Based upon these findings, we have initiated studies with FLX-787 in MS and
ALS.
Summary
Results
NLC Exploratory POC Studies Conclusions
1. Minetto MA, Holobar A, Botter A, and Farina D. Exerc. Sport Sci. Rev. 41(1): 3-10, 2013.
2. Milanov, I. Electromyogr Clin Neurophysiol. 32 (2): 73-9, 1994.
3. Okun, M. & Lampl, I. Nature Neurosci. 11: 535–537, 2008.
4. Beilefeldt et al., Am J Physiol Gastrointest Liver Physiol 294: G130–G138, 2008.
5. Yu et al., Am J Physiol Gastrointest Liver Physiol 297: G34–G42, 2009.
6. El-Tawil S. et al.. Cochrane Database of Systematic Reviews Issue 12, Art. No.: CD005044,2010.
References
Nocturnal leg cramps (NLC)
• 50% of those over the age of 50 suffer from NLC with increasing
prevalence and frequency with age; Over 4 million in the US over age
65 suffer daily.
• Lack of clinical evidence that common “remedies” such as electrolyte
replacement, bananas and hydration afford relief.
• Quinine, prescribed in the United Kingdom for NLC, is associated with
thrombocytopenia, hypersensitivity reactions and QT prolongation
and is no longer approved in the US for NLC.
• No approved drug alternative in US to treat NLC.
Topical Chemical Neuro Stimulation
Figure 1. Overview of suspected mechanism of muscle cramps and methods of cramp inhibition by activation
of TRP ion channels. Muscle cramping is caused by the uncontrolled and repetitive firing of α-motor neurons in
the spinal cord (1), resulting in maintained contraction of the muscle. Flex Pharma’s proprietary products exploit
a general principle of neural circuits whereby strong excitatory sensory input from one source enhances overall
inhibitory tone by increased recruitment of inhibitory neurons, thereby reducing excitability in other parts of
the circuit (3). Flex Pharma’s products stimulate primary sensory neurons in the mouth, esophagus and
stomach by activating TRPV1 and TRPA1 ion channels (4,5). When activated, these sensory neurons, which
project both directly and indirectly to the spinal cord, enhance the inhibitory tone in spinal cord circuits to
reduce repetitive firing of α-motor neurons which prevents or reduces the frequency and intensity of muscle
cramps and spasms.
Muscle cramps were induced in the flexor hallucis brevis (FHB) muscle by electrical stimulation and
monitored by EMG to quantify cramp intensity and duration (Figure 2). The subject’s medial plantar
nerve was electrically stimulated by transcutaneous nerve stimulation 2Hz above the experimentally
determined cramp threshold frequency to elicit a reproducible and robust cramp. The muscle cramp
intensity and duration were measured by EMG and quantified by calculating the area under the
cramp curve (AUC) and cramp duration post cessation of electrical stimulation (Figure 3). Muscle
cramp intensity and duration were found to vary from subject-to-subject, necessitating a pre-
treatment EMG to serve as a subject-specific baseline control. After consumption of Flex Pharma
products or vehicle control, the resulting EMGs were quantified for cramp AUC and duration and
compared to baseline values. The time at which the subject received treatment or vehicle control is
referred to as time point zero.
Methods
Fig 3. Exemplary EMG
demonstrating cramp
induction and observable
AUC.
0 1 2 3
0
50
100
150
200
Time (min)
E
M
G
(u
V
)
Electrical stimulation
to cause cramp
voluntary
flex
Cramp, with
Area Under the Curve
(AUC)
Subject no longer
in cramp
Fig 2. Muscle cramps
were induced in the
flexor hallucis brevis and
monitored by EMG.
Ion channel in cell surface membrane
FLX-787, taken orally, topically stimulates primary
sensory neurons in the oropharynx and
esophagus by activating TRPV1 and TRPA1 ion
channels
Stimulated sensory neurons send impulses to
the spinal cord presumably via brainstem relays
Descending spinal pathways, similar to the
endogenous antinociceptive pathway, activate
inhibitory interneurons to increase inhibitory
tone
OUR HYPOTHESIS
Activation of inhibitory interneurons reduce
excessive firing of the a-motor neuron circuit
Reduction in a-motor neuron hyperexcitability
inhibits muscle cramp or spasm
1
5
2
4
a-motor neuron circuit
3
Figure 4. Topical exposure to oropharynx and
esophagus is required for cramp inhibition
Figure 5. FLX-787 Inhibition of electrically-Induced
cramps in healthy subjects is dose-responsive
1 1 0 1 0 0
0 . 0
0 . 5
1 . 0
0
l o g D o s e ( m g )
P
e
rc
e
n
t
R
e
d
u
c
ti
o
n
i
n
r
A
U
C
(
%
)
F L X - 7 8 7 I n h i b i t i o n o f e l e c t r i c a l l y - i n d u c e d
c r a m p s i n n o r m a l h e a l t h y s u b j e c t s
• Seven doses of FLX-787 ODT yield classic sigmoidal dose curve (n=5,
p<0.05)
• Efficacy saturates at 32 mg FLX-787-ODT
FLX-787 (29 mg), a single molecule that activates both TRPA1 and TRPV1,
afforded a greater decrease in cramp intensity compared to other treatments
tested. The effect of treatment was calculated based upon the AUC change
from a pre-treatment baseline cramp. FLX-787 treatment led to a 5-fold
reduction in AUC compared to its study specific vehicle control. Treatments
that demonstrated a significant difference from study-specific vehicle
controls are annotated with an asterisk (* p<0.05, ** p < 0.01).
Figure 6. FLX-787 is not systemically available in its parent
form in humans
• 1h after PO administration of a liquid formulation containing 19 mg FLX-787
• The parent form of FLX-787 was not detected in plasma only conjugates (LLOQ 0.100
ng/mL).
• Suggests rapid first-pass phase 2 metabolism with no measurable systemic exposure of
parent drug.
Figure 7. Primary systemic exposure of
glucuronide and sulfate conjugates of FLX-787 in all species
0 1 0 2 0 3 0
0
5 0
1 0 0
1 5 0
M e a n ( + / - S D ) P l a s m a c o n c e n t r a t i o n s ( n g / m L )
o f F L X - 7 8 7 i n d o g p l a s m a
T i m e ( h )
C
o
n
c
e
n
tr
a
ti
o
n
(
n
g
/m
L
)
P a r e n t F L X - 7 8 7 ( d o g , 6 0 m g / k g / d a y )
T o t a l F L X - 7 8 7 ( d o g , 6 0 m g / k g / d a y )
1 0 2 0 3 0
- 5
0
5
1 0
1 5
2 0
2 5
M e a n ( + / - S D ) P l a s m a c o n c e n t r a t i o n s ( n g / m L )
o f F L X - 7 8 7 i n h u m a n p l a s m a
T i m e ( h )
C
o
n
c
e
n
tr
a
ti
o
n
(
n
g
/m
L
)
P a r e n t F L X - 7 8 7 ( h u m a n , 6 0 m g O D T )
T o t a l F L X - 7 8 7 ( h u m a n , 6 0 m g O D T )
• Bioanalytical methods for FLX-787 were validated with LLOQ of 0.100 ng/mL; Total FLX-787
methods are semi-quantitative with LLOQ of <0.200 ng/mL
• The variability noted in the metabolic conversion of FLX-787 to conjugates suggests rapid
and extensive phase 2 metabolism (first pass likely at the enterocyte level)
• Study 1: Efficacy signals (cramp frequency, p=0.06) with ODT in sub-analysis (n=37) of parallel portion
– Data from 1 site (n=35) excluded
• Study 2 (dosing/formulation): Significant effect (cramp frequency, p<0.05) in first exposure parallel
analysis (repeated, sequential, multiple crossovers, n=29 from prior NLC study)
– Statistically significant on some, but not all crossover endpoints
Multi-Center Trials in NLC: Two randomized, double-blind, placebo-controlled, cross-over studies to
evaluate the effects of a FLX-787 on the frequency of nocturnal leg foot/cramps when self-administered
approximately 45 minutes before going to bed.
Figure 8. Interim data analyses of NLC Exploratory POC Studies
signal efficacy and carry-over effects
Figure 9. Effect sizes calculated for the parallel portion of the cross-over
studies are on-average larger than those reported in the quinine clinical literature
• Exploratory ANOVA analyses across studies showed a period effect within each cross-over
• To avoid the potential influence of carry-over on effect size estimates were calculated from the first
exposure in each study
• Average effect size of cramp frequency derived from quinine literature is 0.12 (95%CI[-3.5,-1.36]). (6)
• FLX-787 has demonstrated a sigmoidal dose-response curve in a
human EIC-model in the absence of systemic exposure.
• Topical Chemical Neuro Stimulation of TRPA1/TRPV1 indirectly inhibits
a-motor neuron hyperexcitability.
• FLX-787 has shown positive signals on cramp frequency in the parallel
design portion of two exploratory human POC NLC studies.
• FLX-787 is well tolerated and safe, and no SAEs have been reported.
• Consistent with FDA guidance, future FLX-787 studies in NLC will be
parallel design with emphasis on patient selection, data capture &
monitoring.
• Clinical studies in MS and ALS are underway to explore the utility of
FLX-787 in additional indications of different etiology where cramping
and/or spasticity is prevalent.
• Planned initiation of IND-opening Phase 2 parallel design study in H1
2017.
0 1 0 2 0 3 0
0
2 0 0 0
4 0 0 0
6 0 0 0
M e a n ( + / - S D ) P l a s m a c o n c e n t r a t i o n s ( n g / m L )
o f F L X - 7 8 7 i n r a t p l a s m a
T i m e ( h )
C
o
n
c
e
n
tr
a
ti
o
n
(
n
g
/m
L
)
P a r e n t F L X - 7 8 7 ( r a t 5 0 0 m g / k g / d a y )
T o t a l F L X - 7 8 7 ( r a t , 5 0 0 m g / k g / d a y )
*The effect size of Study 1 is a sub-analysis (n=37) excluding data from 1 site (n=35).