Presentation Number: 606.20
TRPV1 and TRPA1 Activators Reduce Human Muscle Cramping
Novel Treatments for Neuromuscular Condi4ons Glenn F. Short III, Brooke W. Hegarty, Christoph H. Westphal and Jennifer M. Cermak
Flex Pharma, Inc. Boston, MA 02199
FLX$788'
Methods 60
40
20
0
2
4
6
TRPA1&
(EC50&patch1&
&&clamp)&
1
2
Fig 3. Exemplary EMG demonstra2ng cramp induc2on and observable AUC. FLX1788&
0.1
0.25 1
2
4
6
8
Time (h)
Figure 4.
0
-500
TRPV1&(EC50)&
FLIPR&
FLX6787&
20.8&µM&
1.2&µM&
FLX6788&
29.1&µM&
7.5&µM&*&
Error bars represent
standard deviation
-1500
Vehicle
Extract Mixture
Figure 5.
1.0
0.5
'
'
'
'
e'
e'
98 797 2788 787
ur
icl
7
t
2
2
2
h
x
X
X FLX
X
Ve 'Mi
FL
FL
FL
t
c
tra
x
E
Delayed$onset$
Delayed onset
150
40
28%$
150
EMG (uV)
60
100
2%$
100
1
2
3
4
0
0
1
2
3
4
High intensity and sustained
High$intensity$and$sustained$
20%$
100
0
3
4
Time (min)
16%$
150
50
2
Mul67phasic$
200
EMG (uV)
EMG (uV)
150
1
Multi-phasic
Time (min)
Time (min)
0
100
50
0
0
1
2
Time (min)
3
4
0
1
2
3
4
Time (min)
Figure 6. Review of baseline EMGs from the study above showed that cramps were reproducibly induced with liTle intra-­‐subject varia2on of cramp threshold and EMG cramp profile. However, EMG cramp profiles varied considerably between subjects and could be categorized into several sub-­‐
types: 1) low intensity but sustained for several minutes, 2) high intensity with a rapid return to baseline, 3) delayed onset, 4) high intensity and sustained, and 5) mul2-­‐phasic sustained. Most subjects fell into subtype 1 (34%, remaining percentages displayed on EMG’s). There was no correla2on between the threshold seqngs used to induce a cramp and the resul2ng cramp profile. Cramps may be a consequence of disequilibrium of the α-­‐motor neuron circuit favoring disinhibi2on and hyperexcitability, while cramp inhibi2on may be a consequence of enhanced inhibitory interneuron firing. ΔAUC from Baseline (uV•sec)
0
0
Figure 9. FLX-­‐787, FLX-­‐788 and combina2ons containing two compounds capable of ac2va2ng both TRPA1 and TRPV1 (FLX-­‐797 and FLX-­‐798) were observed to decrease cramp intensity. FLX-­‐compounds were synthe2cally-­‐manufactured under GMP condi2ons. In normal healthy volunteers (n=9), cramps were electrically elicited in the FHB 1h prior to treatment followed by 1h and 2h measurements aper treatment. The ra2o of the cramp AUC’s (treatment/baseline) were calculated and the mean ra2o AUC derived across 2me points for each treatment. FLX-­‐787, FLX-­‐788, FLX-­‐797 and FLX-­‐798 treatment arms were found to significantly reduce cramp intensity rela2ve to vehicle control (p<0.01, generalized linear mixed model (SAS), Tukey-­‐Kramer post-­‐hoc). Addi2onally, FLX-­‐787 and FLX-­‐788 significantly reduced cramp intensity rela2ve to the TRP-­‐
s2m extract mixture (p<0.02 and p<0.05, respec2vely)(generalized linear mixed model (SAS), Tukey-­‐Kramer post-­‐hoc) sugges2ng that the single compound embodiments may likely be responsible for efficacy in the mixture. Error bars represent standard error of the mean. FLX-­‐787 demonstrates dose-­‐response human efficacy 50
50
20
0
P<0.05&
0.0
0
A"
-1000
-2000
-3000
FLX-787
-4000
0.000
0.002
0.004
Concentration (M)
0.006
v  A mixture of natural extracts significantly inhibited the intensity and dura4on of electrically-­‐induced cramps by 3-­‐fold (p<0.0001) within minutes of inges4on, las4ng up to 6-­‐8 hours. v  Disequilibrium of the α-­‐motor neuron circuit favoring disinhibi4on and hyperexcitability may underlie cramping. v  GMP-­‐synthesized single agent molecules, FLX-­‐787 and FLX-­‐788, as well as doublet combina4ons of GMP-­‐synthesized TRPA1 and TRPV1 agonists, FLX-­‐797 and FLX-­‐798, significantly decreased cramp intensity rela4ve to vehicle (p<0.01). v  FLX-­‐787 and FLX-­‐788 demonstrated improved efficacy at decreasing cramp intensity compared to the parental extract formula4on by 2-­‐fold. v  FLX-­‐787 displayed significant dose-­‐response human efficacy (p<0.01). v  S4mula4on of TRPA1 and TRPV1 channels in the mucous membranes by one or two ac4vators is an effec4ve strategy to inhibit cramping. v  Chemical neuro s4mula4on may be a generally applicable method to treat disorders stemming from α-­‐motor neuron hyperexcitability such as cramping or spas4city due to ALS and MS. Future Clinical Direc4ons • 
• 
P<0.01&
1.5
Conclusions v  Mul4ple Sclerosis (MS) associated spas4city 2.0
High$intensity$and$rapid$return$
High intensity and rapid return
EMG (uV)
EMG (uV)
34%$
10
TRPA1&(EC50)&
Ephys&
200
80
1.0
-1000
250
100
0.1
1000
FLX-­‐787, FLX-­‐788, FLX-­‐797 and FLX-­‐798 Ratio
significantly reduce muscle of cramp AUC after treatment
relative to pre-treatment
baseline
cramping in humans Figures 4 and 5. In three independent studies, the TRP-­‐s2m extract mixture showed a sta2s2cally significant treatment effect rela2ve to vehicle control in each study. The aggregated data from (n=37 normal healthy volunteers) these three studies also demonstrated a significant overall treatment efficacy (one-­‐way ANOVA, p<0.0001). Aggregated study data in Figure 4 depicts a reduc2on in muscle cramp intensity at various 2me points following administra2on of the extract mixture compared to vehicle control. Figure 5 depicts an overall 3-­‐fold reduc2on in the average muscle cramp intensity when data from all three studies is combined. Low$intensity$and$sustained$
10
ΔAUC Effect Size relative to vehicle
-2000
Error bars represent
standard
error of the mean
min
Figure 8. FLX-­‐787 and FLX-­‐788 were assayed for the ability to ac2vate either TRPA1 or TRPV1. Both molecules were confirmed as agonists of the channels displaying similar potencies. TRPA1 EC50’s were determined by automated Patch clamp (Patchliner) in HEK cells stably transfected with human TRPA1. TRPV1 EC50’s were measured by monitoring Ca2+ flux in CHO cells stably transfected with human TRPV1 pre-­‐loaded with a calcium-­‐
sensi2ve fluorescent dye (FLIPR). *Denotes an EC50 es2mate due to not reaching Effmax. FLX-787 (one cmpd)
-1500
1.0
FLX-788 (one cmpd)
-1000
10
FLX-789 (two cpmd mixture)
-500
0.1
1000
FLX-798 (two cmpd mixture)
0
10
Extract Mixture
ΔEMG-AUC from Baseline (uV•sec)
TRP-­‐s4m extract mixture decreases electrically-­‐induced cramp intensity Extract Mixture
Vehicle
16
TRPV1&
(EC501FLIPR)&
Concentration (µM)
Results 500
14
RFU&
0.1
3
Time (min)
Fig 2. Muscle cramps were induced in the flexor hallucis brevis and monitored by EMG. 12
RFU&
0
Vehicle
0
Norm[Avg(outward)]&[%]&
EMG (uV)
FLX1787&
Subject no longer
in cramp
50
10
FLX-­‐787 and FLX-­‐788 display similar TRP potencies Cramp, with
Area Under the Curve
(AUC)
voluntary
flex
8
Figure 7. The TRP-­‐s2m extract mixture was analyzed by HPLC and peak reten2on 2mes compared to known molecules present in the natural extracts used to prepare the mixture. FLX-­‐787 and FLX-­‐788 shared HPLC elu2on profiles with two known molecules. In comparison to FLX-­‐788, FLX-­‐787 was a minor component of the TRP-­‐s2m extract mixture. Reten%on'Time'(min)'
200
100
- 10-Gingerol
A9.039
re
a:
25
6.
46
1
0
Electrical stimulation
to cause cramp
150
- 10-Shogaol
A11.328
re
a:
60
.2
70
4
- 6-Gingerdiol
A3.315
re
a:
32
.8
86
4
80
EMG-­‐derived baseline cramp profiles are heterogeneous and may suggest disequilibrium of the α-­‐motor neuron circuit Figure 1. Overview of suspected mechanism of muscle cramps and methods of cramp inhibi4on by ac4va4on of TRP ion channels. Muscle cramping is caused by the uncontrolled and repe22ve firing of α-­‐motor neurons in the spinal cord (1), resul2ng in maintained contrac2on 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 s2mulate primary sensory neurons in the mouth, esophagus and stomach by ac2va2ng TRPV1 and TRPA1 ion channels (4,5). When ac2vated, these sensory neurons, which project both directly and indirectly to the spinal cord, enhance the inhibitory tone in spinal cord circuits to reduce repe22ve firing of α-­‐motor neurons which prevents or reduces the frequency and intensity of muscle cramps and spasms. - 8-Gingerol
A6.872
re
a:
14- 6-Shogaol
A7.304
re
6.
a:
34
1
13
2.
07
2
100
- 8-Shogaol
A9.495
re
a:
28
.4
83
6
120
Ratio AUC (Treatment/Baseline)
Background 140
Muscle cramps were induced in the flexor hallucis brevis (FHB) muscle by electrical s2mula2on and monitored by EMG to quan2fy cramp intensity and dura2on (Figure 2). The subject’s medial plantar nerve was electrically s2mulated by transcutaneous nerve s2mula2on 2Hz above the experimentally determined cramp threshold frequency to elicit a reproducible and robust cramp. The muscle cramp intensity and dura2on were measured by EMG and quan2fied by calcula2ng the area under the cramp curve (AUC) and cramp dura2on post cessa2on of electrical s2mula2on (Figure 3). Muscle cramp intensity and dura2on were found to vary from subject-­‐to-­‐subject, necessita2ng a pre-­‐
treatment EMG to serve as a subject-­‐specific baseline control. Aper consump2on of Flex Pharma products or vehicle control, the resul2ng EMGs were quan2fied for cramp AUC and dura2on and compared to baseline values. The 2me at which the subject received treatment or vehicle control is referred to as 2me point zero. ΔEMG-AUC from Baseline (uV•sec)
Muscle cramps can be painful and debilita2ng for healthy athletes as well as those suffering from nocturnal leg cramps, mul2ple sclerosis, amyotrophic lateral sclerosis, and other neurological diseases. Studies have demonstrated that hyperexcitability of α-­‐motor neurons in the spinal cord is likely the underlying cause of cramps and spas2city (1,2). We hypothesized that transient receptor poten2al channel (TRP) ac2va2on in the mouth, esophagus and stomach could yield sufficient excitatory input to dampen motor neuron hyperexcitability. To test this hypothesis, we conducted human studies to assess the effec2veness of TRP ac2va2on to inhibit electrically-­‐induced cramps of the foot. An oral solu2on containing a mixture of TRPA1 and TRPV1 ac2vators derived from natural extracts (TRP-­‐s2m extract mixture), was shown to decrease cramp intensity by 3-­‐fold (p<0.0001) across three independent, randomized, blinded clinical studies. Review of EMG baseline recordings demonstrated very reproducible intra-­‐subject cramp profiles compared to inter-­‐subject profiles which varied considerably. The inter-­‐subject profiles were categorized into five dis2nct sub-­‐types each displaying par2cular paTerns of increased and decreased periods of EMG ac2vity. While the TRP-­‐s2m extract mixture was effec2ve at reducing cramp intensity regardless of cramp sub-­‐type, the paTerns of EMG ac2vity are likely a consequence of disequilibrium between disinhibi2on and inhibi2on of the α-­‐motor neuron circuit. To iden2fy the likely ac2ve components, the extract mixture was analyzed by HPLC in rela2on to a series of molecules known to be present in the natural extracts. Compounds confirmed to be present in the extract mixture were assayed for TRP agonist ac2vity. The data showed that two compounds in par2cular, FLX-­‐787 and FLX-­‐788, were able to ac2vate both TRPA1 and TRPV1 with similar potencies. When tested in an electrically-­‐induced cramp model in normal healthy subjects, GMP-­‐
synthesized FLX-­‐787 and FLX-­‐788, as well as FLX-­‐797 and FLX-­‐798, each consis2ng of two GMP-­‐
synthesized TRPA1 and TRPV1 agonists, significantly reduced cramp intensity rela2ve to vehicle control (p<0.01). Moreover, FLX-­‐787 and FLX-­‐788 provided significant improvement in efficacy over the TRP-­‐s2m extract mixture (FLX-­‐787: p<0.01, FLX-­‐788: p<0.05). An exploratory analysis of two concentra2ons of FLX-­‐787 demonstrated an increase in the cramp inhibi2on effect size at higher concentra2on consistent with significant dose-­‐response efficacy (p< 0.01). These results suggest that both the single agent molecules and the doublet combina2ons are efficacious at decreasing cramp intensity in a poten2ally dose-­‐responsive manner. Flex Pharma is moving toward further clinical studies to evaluate the effec2veness of these compounds to treat nocturnal leg cramps and spas2city associated with mul2ple sclerosis. Taken together, these data demonstrate the u2lity of Flex Pharma’s proprietary products to treat cramps and the general strategy of chemical neuro s2mula2on to limit α-­‐motor neuron hyperexcitability in other neurological diseases. FLX$787'
3.961 -A 6-Gingerol
re
a:
83
9.
03
9
DAD1 B, Sig=282,4 Ref =500,25 (D2315\D2315_37.D)
mAU
Norm[Avg(outward)]&[%]&
Norm[Avg(outward)]&
Summary FLX-­‐787 and FLX-­‐788 confirmed components of parental extract mixture 0.0
Between 250,000 and 350,000 people in the US suffer from MS, approximately 84% of whom experience spas2city. Neuronal dysfunc2on associated with inflammatory and degenera2ve processes in the brain and spinal cord lead to hyperac2ve muscle stretch reflexes, resul2ng in involuntary contrac2ons and spas2city. Mul4-­‐Center MS Trial (EX-­‐USA): a randomized, blinded, double cross-­‐over study to inves2gate the effects of Flex clinical candidate in subjects with symptoms of cramps, spasms and spas2city !
Recruitment)and)
Screening)
(SpasEcity!of!at!least!
3!months,!that!is!
not!completely!
relieved!by!current!
therapy)!!
!
★!
• 
• 
• 
Cross1Over)Period)2)
FLX2candidate!
!(14!days)!
!
Cross1Over)Period)2)
Placebo!!
(14!days)!
!
7)
d)
)
W
a)
S)
h)
)
o)
u)
t)
★!
!
!
★!
Cross1Over)Period)3)
Placebo!!
(14!days)!
!
!!
!
Cross1Over)Period)3)
FLX2candidate!!
(14!days)!
!
!
N=50!to!be!enrolled!
• 
Mul4-­‐Center Trial in NLC: A randomized, double-­‐blind, placebo-­‐controlled, double cross-­‐over study to evaluate the effects of a Flex product on the frequency of nocturnal foot and/or leg cramps when self-­‐administered approximately 45 !
★!
minutes before going to bed. ★!
!
-0.2
-0.3
!
-0.4
(cramps!for!≥!4!
nights!per!week!
for!4!consecu5ve!
weeks!prior!to!
Screening!!
! Study Visit
★
0.006
Figure 10. Increasing concentra2ons of FLX-­‐787 improve efficacy and demonstrate dose-­‐response. Panel A depicts the aggregate data from two single-­‐blind, cross-­‐over electrically-­‐induced cramp studies in which 2.75 mM and 5.5 mM single agent FLX-­‐787 were tested in normal healthy volunteers (n=9). At the highest dose tested, 5.5 mM FLX-­‐787 significantly improved efficacy decreasing cramp intensity across all subjects tested rela2ve to the 2.75 mM concentra2on (Wilcoxon rank sum test, p < 0.01). In an exploratory analysis (Panel B), comparison of the effect size response between 2.75 mM and 5.5 mM of FLX-­‐787 demonstrated a marked increase in response at the higher concentra2on (Effect size es2mates are calculated by the Cohen’s d effect size method). This observa2on of improved efficacy at higher concentra2ons of FLX-­‐787 supports the no2on of dose-­‐response efficacy. Error bars represent the standard error of the mean. ★!
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 hydra2on afford relief. Quinine, prescribed in the United Kingdom for NLC, is associated with thrombocytopenia, hypersensi2vity reac2ons and QT prolonga2on and is no longer approved in the US for NLC. No approved drug alterna2ve in US to treat NLC. Recruitment'
and'Screening'
Concentration (M)
7)
d)
)
W
a)
S)
h)
)
o)
u)
t)
★!
Exclude!Non2
responders!
v  Nocturnal leg cramps (NLC) B"
0.004
!
Enrollment)
Period)1:)
All!Subjects!
receive!FLX2!
candidate!!
(7!days)!
!
Study Visit
!★!
0.002
★!
★!
-0.1
-0.5
0.000
★!
★!
!
!
★!
PERIOD'1/Run,in''
Placebo''
(2!weeks)!and!
Randomiza5on!
!
!
Period'2'
FLX9product!
!(2!weeks)!
Period'3'
FLX9product!
!(2!weeks)!
!
Period'2'
Placebo!
(2!weeks)!
Period'3'
Placebo!
(2!weeks)!
!
!
!
!
!
Exclude!Placebo!
Responders!
At!least!40!
enrolled!
References 1. 
2. 
3. 
4. 
5. 
Minetto MA, Holobar A, Botter A, and Farina D. Exerc. Sport Sci. Rev. 41(1): 3-10, 2013.
Milanov, I. Electromyogr Clin Neurophysiol. 32 (2): 73-9, 1994.
Okun, M. & Lampl, I. Nature Neurosci. 11: 535–537, 2008.
Beilefeldt et al., Am J Physiol Gastrointest Liver Physiol 294: G130–G138, 2008.
Yu et al., Am J Physiol Gastrointest Liver Physiol 297: G34–G42, 2009.