Discovery of a selective TRPM8 antagonist with clinical efficacy in cold-related pain

Article abstract of DOI:10.1021/ml500479v

The transient receptor potential (TRP) family of ion channels comprises nonselective cation channels that respond to a wide range of chemical and thermal stimuli. TRPM8, a member of the melastatin subfamily, is activated by cold temperatures (<28 °C), and antagonists of this channel have the potential to treat cold induced allodynia and hyperalgesia. However, TRPM8 has also been implicated in mammalian thermoregulation and antagonists have the potential to induce hypothermia in patients. We report herein the identification and optimization of a series of TRPM8 antagonists that ultimately led to the discovery of PF-05105679. The clinical finding with this compound will be discussed, including both efficacy and its ability to affect thermoregulation processes in humans.

Full text of DOI:10.1021/ml500479v

Letter
pubs.acs.org/acsmedchemlett
Discovery of a Selective TRPM8 Antagonist with Clinical Efficacy in
Cold-Related Pain
Mark D. Andrews,§ Kerry af Forselles,∥ Kevin Beaumont,‡ Sebastien R. G. Galan,§ Paul A. Glossop,§
́
Mathilde Grenie,§ Alan Jessiman,§ Amy S. Kenyon,§ Graham Lunn,§ Graham Maw,§ Robert M. Owen,*,†
David C. Pryde,† Dannielle Roberts,§ and Thien Duc Tran§
†Worldwide Medicinal Chemistry, Pfizer Neusentis, Granta Park, Cambridge CB21 6GS, United Kingdom
‡Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, 610 Main Street, Cambridge,
Massachusetts 02139, United States
∥
§Worldwide Medicinal Chemistry and Biology, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road,
Sandwich, Kent CT13 9NJ, United Kingdom
S
* Supporting Information
ABSTRACT: The transient receptor potential (TRP) family of ion channels
comprises nonselective cation channels that respond to a wide range of chemical
and thermal stimuli. TRPM8, a member of the melastatin subfamily, is activated by
cold temperatures (<28 °C), and antagonists of this channel have the potential to treat
cold induced allodynia and hyperalgesia. However, TRPM8 has also been implicated in
mammalian thermoregulation and antagonists have the potential to induce
hypothermia in patients. We report herein the identification and optimization of a
series of TRPM8 antagonists that ultimately led to the discovery of PF-05105679. The
clinical finding with this compound will be discussed, including both efficacy and its
ability to affect thermoregulation processes in humans.
KEYWORDS: TRPM8, clinical tool, hypothermia, PF-05105679, thermoregulation, TRP channel, ion channel, pain
he transient receptor potential (TRP) family of ion
the translation of these thermoregulation studies from preclinical
Tchannels are nonselective cation channels that respond to species to humans.
a wide range of chemical and thermal stimuli.1,2 In recent years,
these ion channels have attracted attention as potential targets
for the treatment of a variety of diseases, in part due to their
ability to transmit noxious (or painful) stimuli, e.g., TRPA1 and
TRPV1.3−6 TRPM8, a member of the melastatin subfamily, is
activated by cold temperatures (8−28 °C) and cooling agents
such as menthol and icilin.7,8 Preclinical studies have linked
TRPM8 to pain in both oxaliplatin-induced9 and chronic nerve
injury neuropathic pain models.10,11 Consequently, antagonists
of this channel have the potential to treat cold induced allodynia
(pain caused by normally innocuous stimuli) and hyperalgesia (a
prolonged or more intense pain response) in human patients.
Beyond its role in transmitting cold induced pain signals,
TRPM8 is also implicated in mammalian thermoregulation.12,13
Studies in preclinical species have demonstrated that the TRPM8
agonist menthol can induce hyperthermia in vivo.14 By analogy to
TRPV1 agonists and antagonists, which cause hypothermia and
hyperthermia respectively in humans,15,16 TRPM8 antagonists
have the potential to induce hypothermia; an effect that has been
demonstrated in preclinical species.17 A variety of small molecule
TRPM8 antagonists covering a broad range of chemical and
physical property space have been published in the litera-
ture.18−32 However, until very recently,33 no clinical data for
these compounds had been disclosed, which might help to
understand the role of TRPM8 in human physiology including
In order to address this key thermoregulation concern, we
adopted a project strategy that focused on rapidly identifying a
compound that could be used as a clinical tool to understand the
role of TRPM8 in humans from both a pain and thermoreg-
ulation standpoint. Herein we describe our efforts, which led to
the identification of PF-05105679, an antagonist of TRPM8, and
a key clinical tool for exploring both the role of TRPM8 in
regulating core body temperature and the potential of TRPM8
antagonists for the treatment of pain in humans.
As a starting point, the project team identified compound 1
from a high throughput screen (HTS) of the Pfizer compound
collection (Figure 1). Although a relatively weak inhibitor
(TRPM8 FLIPR IC50 = 1173 nM), this hit was singled out as it
was in good lipophilic efficiency (LipE or LLE = 4.9 as
determined from logD)34−37 space and provided access to novel
physicochemical space and biopharmaceutical properties, as
acidic TRPM8 antagonists were unknown in the literature when
we started this project.38 In addition, the chemistry available to
construct this scaffold provided a convenient method for
systematically varying each of the three vectors around the
central nitrogen core. On the basis of these factors, the project
Received: November 22, 2014
Accepted: January 30, 2015
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
the bicyclic system proved unsuccessful (5 and 9) and did not
result in any strong enhancements in potency or LipE.
Once compound 8 had been identified, the project team next
evaluated the structural requirements of the acidic portion of the
scaffold on TRPM8 potency (Table 2). Modification of the key
Table 2. TRPM8 Potency SAR for Modification of the Acid
Vector
Figure 1. Initial HTS hit (1).
team initiated medicinal chemistry efforts to optimize compound
1 with the goal of identifying a clinical tool.
Separation of 1 into its two enantiomers indicated that
potency at TRPM8 resided in the R-enantiomer (R-1) (TRPM8
FLIPR IC50 = 702 nM), while the S-enantiomer (S-1) was
generally inactive (TRPM8 FLIPR IC50 > 10,000 nM). With this
in mind, we designed a series of compounds to systematically
explore the SAR of this scaffold. Optimization of the amide
portion of the scaffold (Table 1) quickly demonstrated that
Table 1. TRPM8 Potency SAR for Modification of the Amide
Vector
structural features of compound 1 generally led to decreases in
potency. For example, the acid isostere 19 or homologation to a
CH2 linked carboxylate (18) provided weaker inhibitors. More
subtle modification around the benzoic acid moiety suggested
that substituents ortho to the carboxylate, e.g., fluoro (15) or
methyl (16), were not well tolerated. Para substituents, such as
the fluorine in compound 17, were tolerated, although they did
not give any potency or efficiency advantages over compound 8.
Introducing polarity into the ring in the form of a pyridyl
carboxylic acid (14) led to a drop in potency but maintained the
compound’s overall efficiency.
In parallel to evaluating the compounds shown in Table 2, we
also explored the changes to the chiral benzyl substituent (Table
3). Subtle changes to substituents around the phenyl ring were
generally well tolerated (22 and 26) but did not lead to any
improvements in either potency or overall efficiency. An electron
rich furan motif (27) was well tolerated, but introduction of a
para-methoxy phenyl group (25) led to a large drop in potency.
As previously mentioned, the chirality of the benzyl substituent
was important for TRPM8 activity with all the activity residing in
the R-enantiomer. Introduction of a gem-dimethyl group (23)
significantly decreased potency, as did removal of the methyl
group (20). Larger aliphatic substituents were tolerated (24)
although they led to a drop in LipE, but similarly sized, more
polar groups were not (21). These observations coupled with the
large improvement in potency and LipE observed moving from
20 to 8 suggested the chiral methyl group may help to enforce a
conformation that approaches the bound conformation of the
antagonist in the TRPM8 binding site.
fused, bicyclic heteroaromatic systems provided the best potency
and efficiency against TRPM8, with other types of amides (2,
rac-7, 12, and 11) only leading to less active compounds.
Compound 3 suggested that larger groups such as biaryls could
be tolerated; however, because of the poor LipE of this
compound, further examples were not explored. Two of the
best improvements in potency and LipE in the 6,6-biaryl amides
came from replacing the 6-quinolone nitrogen (1) with the 3-
quinoline (8) or the 3-isoquinoline (13). Modifying the
quinoline to a 5,6-ring system (4, 6, and 10) was also tolerated.
Unfortunately, attempts to introduce additional polarity within
Following on from these first three rounds of design, we
looked to combine some of the fragments contained in the
compounds with better LipE values in order to see if we could
improve on the overall efficiency of our compounds (Table 4).
Gratifyingly, each of these recombinations provided potent
TRPM8 antagonists in good LipE space. For example, combining
B
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ACS Medicinal Chemistry Letters
Letter
Table 3. TRPM8 Potency SAR for Modification of the Chiral
Benzyl Substituent
Figure 2. LipE overview for project compounds. Key project
compounds are marked with a red “X”; see Table 5.
optimized to a good cluster of efficient compounds of around
LipE = 6. This analysis also suggested that the project had
reached a plateau in terms of its ability to improve the efficiency
of the series above a LipE ≈ 6.1. As a result, we focused on
generating additional data on the best compounds identified
from our design and screening efforts in the hope of identifying a
suitable compound to advance to the clinic.
The results from a standard set of in vitro ADME assays39−41
are summarized in Table 5. All compounds tested did not show
any potential risks with respect to either hERG inhibition (as
measured in a dofetilide binding assay) or drug−drug
interactions, e.g., <20% inhibition at 3 μM for CYP3A4,
CYP2D6, CYP1A2, CYP2C8, and CYP2C9. As the project was
looking to quickly progress a compound to the clinic, our ideal
tool compound required good antagonist potency at TRPM8
along with excellent membrane permeation and in vitro
metabolic stability in both human and rat in order to facilitate
pre-clinical toxicity studies. Although a number of compounds
were superior to compound 8 in specific aspects of their in vitro
profiles, overall 8 provided the best combination of potency and
in vitro metabolic stability measurements in both human and rat
and was therefore selected for further evaluation.
Table 4. Recombination of the Best LipE Fragments
Single cell patch clamp electrophysiology (Ephys) studies,
confirmed the potency of 8 against TRPM8 (Table 6).33 In
addition, 8 demonstrated >100-fold selectivity across a range of
different receptors, ion channels, and enzymes including the
closely related TRPV1 and TRPA1 channels. As a P-gp substrate,
compound 8 also exhibited some CNS restriction (CSF to free
plasma ratio of 0.3 in rat and guinea pigs) but was still progressed
as the project believed that clinical efficacy would be obtained by
targeting peripheral TRPM8.
In order to obtain an estimate of the efficacious concentration
(Ceff) for clinical studies, compound 8 was tested in a cold-
induced bladder contractility model in guinea pigs.33,42 While not
a direct measure of the compounds ability to act as an analgesic,
the assay did provide a direct measure of TRMP8-mediated
pharmacology. Results from these studies indicated that a
maximum unbound concentration (Cmax) of drug between 254−
450 nM (∼3 × IC50) would be sufficient to test the mechanism in
clinical studies. Preclinical PK studies demonstrated that
compound 8 should exhibit moderate blood clearance with
respect to hepatic blood flow and essentially complete oral
absorption in both rat and dog. Volume of distribution values
(Vdss) were much higher than would be expected for an acidic
compound and may be driven by enterophepatic recirculation of
either compound 8 or its primary metabolite, the acyl
the 3-isoquinoline motif from compound 13 with the pryidyl
carboxylic acid moiety in 14 resulted in 31, which suggested the
pyridyl acid motif could be a viable replacement for the benzoic
acid moiety based on the similar LipE values between
compounds 28 and 31. In addition, compounds 28 and 29
both exhibited potency and efficiency values that were on par
with the best compounds identified in our first rounds of design.
However, as a whole, these modifications did not provide any
large improvements in efficiency.
Examining the full set of compounds made by this stage of the
project (Figure 2) demonstrated that, in a few rounds of design,
the project had successfully taken the initial HTS hit (1) and
C
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Letter
a
,
Table 5. Key TRPM8 Compounds Evaluated in an in Vitro ADME Panel39−41
cmpd no.
IC50
logD7.4
HLM
HHEP
RLM
Dof Ki (nM)
CYP % inhib @ 3 μM
4
94
0.9
1.0
ND
0.6
1.1
1.3
1.5
1.1
1.2
0.9
0.4
42
<8
<8
ND
16
<8
10
11
28
23
11
23
325
17
>9182
>15857
ND
ND
8
181
340
307
90
<15
ND
ND
21
0 > 20%
ND
9
76
10
13
24
26
28
29
30
31
113
166
<14
102
31
>9182
>9182
>15219
ND
ND
ND
202
62
28
ND
45
0 > 20%
0 > 20%
0 > 20%
ND
60
<2
ND
123
234
291
48
41
ND
31
85
ND
<2.2
31
ND
0 > 20%
a
HLM, HHEP, and RLM values are reported in units of intrinsic clearance (μL/min/mg for microsomes and μL/min/million cells for hepatocytes).
ND = not determined.
temperature at efficacious concentrations, the lack of a
therapeutic index between an adverse event (feeling hot) and
efficacy and its short half-life in humans limit the further
progression of 8 in the clinic. Further studies are required to
understand if TRPM8 remains a viable target for the treatment of
cold-related pain in patients.
Table 6. Further in Vitro and in Vivo PK Evaluation of
Compound 8 (PF-05105679)
a
TRPM8 Ephys
IC50 (nM)
CEREP
hERG
protein
polypharmacology (μM) permeability binding (fu)
103
>100-fold
selectivity
>30
RRCK:
6.8 × 10−6
human:
0.012
rat: 0.033
ASSOCIATED CONTENT
* Supporting Information
Synthetic details for all compounds described. This material is
available free of charge via the Internet at http://pubs.acs.org.
MDCK ER: guinea pig:
■
4.8
0.07
S
dog: 0.067
rat
dog
Cl (mL/min/kg)
(Clu)
19.8 (618)
31 (462)
AUTHOR INFORMATION
Corresponding Author
*E-mail: robert.owen@pfizer.com.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
■
V
dss (L/kg)
1/2 (h)
6.2
3.6
7.4
3.9
T
%F (absorption)
29% @ 2 mg/kg (46%) 92% @ 20 mg/kg
(∼100%)
62% @ 10 mg/kg
(100%)
a
Blood Clu values were determined by correcting the clearance by
fraction unbound. Absorption estimated from the clearance relative to
hepatic blood flow.
Notes
The authors declare no competing financial interest.
glucuronide, which was observed in both in vitro and bile-duct
cannulated rat studies. On the basis of the worse case assumption
that enterohepatic recirculation may not be a prevalent process in
humans, human dose predictions using a more typical Vdss for
acids of 0.2 L/kg43 indicated that a dose of 1 g would provide
sufficient exposure of 8 to reach the predicted Ceff in the clinic. In
addition, preclinical toxicity studies allowed for progression of 8
to the clinic with sufficient toxicology margins relative to the
predicted Ceff to safely test the effect of TRPM8 antagonism in
humans.
ACKNOWLEDGMENTS
■
The authors wish to acknowledge the contributions of A. Cronin,
W. Klute, D. Lovering, W. McCarte, A. Nortcliffe, S. Wheeler, L.
Watson, and A. Warren towards the synthesis of compounds
described in this manuscript. The authors also wish to thank W.
Winchester, D. Blakemore, J. Warmus, C. Rose, and J. Bian for
assistance in assembling this manuscript and M. J. Palmer for his
assistance in working up the project HTS.
Evaluation of compound 8 (PF-05105679) in humans
demonstrated that a single 900 mg dose provided efficacy in a
cold pressor test that was comparable to oxycodone. At this dose,
compound 8 reached circulating concentrations of about 3-fold
the TRPM8 IC50 at Cmax, supporting the predicted Ceff from
preclinical species and the dose prediction strategy described
above.33 Importantly, doses up to 900 mg did not result in
significant changes to core body temperature. However, this
efficacious dose did result in unexpected adverse events,
including a nontolerated, hot sensation localized to the mouth,
face, upper body, arms, and hands.
ABBREVIATIONS
■
HTS, high throughput screen; Ceff, efficacious concentration;
CYP, cytochrome P450; HLM, human liver microsomes; RLM,
rat liver microsomes; HHEP, human hepatocytes; Dof,
dofetilide; hERG, human ether-a-go-go related gene; LipE,
lipophilic efficiency; MDCK ER, MDCK efflux ratio
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D
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