Antibodies to the extracellular pore loop of TRPM8 act as antagonists of channel activation.

The mammalian transient receptor potential melastatin channel 8 (TRPM8) is highly expressed in trigeminal and dorsal root ganglia. TRPM8 is activated by cold temperature or compounds that cause a cooling sensation, such as menthol or icilin. TRPM8 may play a role in cold hypersensitivity and hyperalgesia in various pain syndromes. Therefore, TRPM8 antagonists are pursued as therapeutics. In this study we explored the feasibility of blocking TRPM8 activation with antibodies. We report the functional characterization of a rabbit polyclonal antibody, ACC-049, directed against the third extracellular loop near the pore region of the human TRPM8 channel. ACC-049 acted as a full antagonist at recombinantly expressed human and rodent TRPM8 channels in cell based agonist-induced 45Ca2+ uptake assays. Further, several poly-and monoclonal antibodies that recognize the same region also blocked icilin activation of not only recombinantly expressed TRPM8, but also endogenous TRPM8 expressed in rat dorsal root ganglion neurons revealing the feasibility of generating monoclonal antibody antagonists. We conclude that antagonist antibodies are valuable tools to investigate TRPM8 function and may ultimately pave the way for development of therapeutic antibodies

Transient receptor potential melastatin 8 (TRPM8) channels are involved in body temperature regulation

Abstract Background Transient receptor potential cation channel subfamily M member 8 (TRPM8) is activated by cold temperature in vitro and has been demonstrated to act as a ‘cold temperature sensor’ in vivo. Although it is known that agonists of this ‘cold temperature sensor’, such as menthol and icilin, cause a transient increase in body temperature (Tb), it is not known if TRPM8 plays a role in Tb regulation. Since TRPM8 has been considered as a potential target for chronic pain therapeutics, we have investigated the role of TRPM8 in Tb regulation. Results We characterized five chemically distinct compounds (AMG0635, AMG2850, AMG8788, AMG9678, and Compound 496) as potent and selective antagonists of TRPM8 and tested their effects on Tb in rats and mice implanted with radiotelemetry probes. All five antagonists used in the study caused a transient decrease in Tb (maximum decrease of 0.98°C). Since thermoregulation is a homeostatic process that maintains Tb about 37°C, we further evaluated whether repeated administration of an antagonist attenuated the decrease in Tb. Indeed, repeated daily administration of AMG9678 for four consecutive days showed a reduction in the magnitude of the Tb decrease Day 2 onwards. Conclusions The data reported here demonstrate that TRPM8 channels play a role in Tb regulation. Further, a reduction of magnitude in Tb decrease after repeated dosing of an antagonist suggests that TRPM8’s role in Tb maintenance may not pose an issue for developing TRPM8 antagonists as therapeutics.</p

Alignment of extracellular pore loop sequences.

<p>Alignment of the third extracellular loop sequences of the human, rat and mouse TRPM8 channel and the human TRPA1 and TRPV1 channels. The red line indicates the epitope sequence that ACC-049 was generated against.</p

Icilin activation of TRPM8 in CHO cells and rat DRG neurons.

<p>Antagonism of icilin induced activation of human TRPM8 recombinantly expressed by CHO cells (A) and rat DRG neurons (B) by additional poly- and monoclonal antibodies generated against the third extracellular pore loop. <b>a</b>. Alomone ACC-049. <b>b</b>. MyBiosource MBS609041. <b>c</b>. Creative Diagnostics CABT37242RH. d. Thermo Scientific OST00133W. <b>e</b>. Antibodies Online ABIN351226. <b>f</b>. Lifespan Biosciences LS-B6668. <b>g</b>. Enzo Lifesciences BML-SA664. <b>h</b>. M8-B. <b>i</b>. 1 µM icilin. <b>j</b>. 1 µM icilin + peptide (SDVD GTTYDFAHC). <b>k</b>. Buffer. A. Note the complete block of TRPM8 channel activation by ACC-049 (<b>a</b>), MyBiosource (<b>b</b>) and Enzo Lifesciences (<b>g</b>) antibodies at the single concentration tested. Small molecule positive control M8-B also completely blocked TRPM8 channel activation (<b>h</b>). B. Five out of seven antibodies tested (<b>a, b, e, f, g</b>) block icilin activation of rat DRG neurons by 70–80%, two antibodies (<b>c, d</b>) are ineffective. Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). ⁴⁵Ca²⁺ uptake of CHO-TRPM8 cells activated with 1 µM icilin and antigen peptide (<b>j</b>) was considered as 100 percent and wells with only assay buffer plus ⁴⁵Ca²⁺ were set as zero percent.</p

Icilin activation of TRPM8.

<p>Concentration dependent antagonism of icilin induced activation of the human (A), rat (B) or mouse (C) TRPM8 channels by ACC-049, control IgG and M8-B measured by ⁴⁵calcium uptake. Note the right shifted concentration response of ACC-049 on human TRPM8 (A) compared to rat (B) or mouse (C) TRPM8, while the small molecule antagonist positive control M8-B exhibited comparable responses on TRPM8 channels of all species tested (A–C). Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). Icilin induced ⁴⁵Ca²⁺ uptake was considered as 100 percent and wells with only assay buffer plus ⁴⁵Ca²⁺ were set as zero percent.</p

IC₅₀ values (nM) of cold, icilin, and menthol induced human, rat, or mouse TRPM8 channel activation by ACC-049.

<p>Values are expressed as mean ± SD of one experiment conducted in triplicate wells in a 96 well format. Concentration ranges of ACC-049 and respective agonists are described in the text. IC₅₀ of control IgG in all assays was >2500 nM.</p><p>IC₅₀ values (nM) of cold, icilin, and menthol induced human, rat, or mouse TRPM8 channel activation by ACC-049.</p

Source and characteristics of the antibodies.

<p>Except for ACC-049, the antigen sequence used for the antibodies was not disclosed by the vendors.</p><p>Source and characteristics of the antibodies.</p

Cold activation of TRPM8.

<p>Concentration dependent antagonism of cold activation (10°C) of the human (A), rat (B), or mouse (C) TRPM8 channels by ACC-049, control IgG and M8-B measured by ⁴⁵calcium uptake. Note the right shifted concentration response of ACC-049 on human TRPM8 (A) compared to rat (B) or mouse (C) TRPM8, while the small molecule antagonist positive control M8-B exhibited comparable responses on TRPM8 channels of all species tested (A–C). Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). Cold induced ⁴⁵Ca²⁺ uptake was considered as 100 percent and wells with M8-B at 1 µM plus ⁴⁵Ca²⁺ were set as zero percent.</p

ACC-049 is a selective inhibitor of TRPM8.

<p>Specificity of ACC-049 (2.5 µM) for blocking human TRPM8 activation induced by the specific natural agonist cold (A) or synthetic agonist icilin (D). No effect of ACC-049 on noxious cold induced human TRPA1 (B) or heat induced TRPV1 activation (C). Small molecule antagonists AMG9090 and AMG6541 are the positive control for TRPA1 (B) or TRPV1 (C) blockage, respectively. Note the near complete blockade of TRPM8 activation by ACC-049 at 2.5 µM, similar to that by the positive small molecule antagonist control M8-B (A). Neither control IgG, nor peptide-absorbed ACC-049, or peptide alone blocked activation of any of the channels tested (A–D). Values are means of triplicate measures in a single experiment and expressed as percent of control (POC). Agonist induced ⁴⁵Ca²⁺ uptake in the absence of antibodies (no Ab) was considered as 100 percent and wells with small molecule antagonists plus ⁴⁵Ca²⁺ were set as zero percent.</p

Pragmatic phase II clinical trial to improve depression care in a real-world diverse MS cohort from an academic MS centre in Northern California: MS CATCH study protocol.

INTRODUCTION: Depression occurs in over 50% of individuals living with multiple sclerosis (MS) and can be treated using many modalities. Yet, it remains: under-reported by patients, under-ascertained by clinicians and under-treated. To enhance these three behaviours likely to promote evidence-based depression care, we engaged multiple stakeholders to iteratively design a first-in-kind digital health tool. The tool, MS CATCH (Care technology to Ascertain, Treat, and engage the Community to Heal depression in patients with MS), closes the communication loop between patients and clinicians. Between clinical visits, the tool queries patients monthly about mood symptoms, supports patient self-management and alerts clinicians to worsening mood via their electronic health record in-basket. Clinicians can also access an MS CATCH dashboard displaying patients mood scores over the course of their disease, and providing comprehensive management tools (contributing factors, antidepressant pathway, resources in patients neighbourhood). The goal of the current trial is to evaluate the clinical effect and usability of MS CATCH in a real-world clinical setting. METHODS AND ANALYSIS: MS CATCH is a single-site, phase II randomised, delayed start, trial enrolling 125 adults with MS and mild to moderately severe depression. Arm 1 will receive MS CATCH for 12 months, and arm 2 will receive usual care for 6 months, then MS CATCH for 6 months. Clinicians will be randomised to avoid practice effects. The effectiveness analysis is superiority intent-to-treat comparing MS CATCH to usual care over 6 months (primary outcome: evidence of screening and treatment; secondary outcome: Hospital Anxiety Depression Scale-Depression scores). The usability of the intervention will also be evaluated (primary outcome: adoption; secondary outcomes: adherence, engagement, satisfaction). ETHICS AND DISSEMINATION: University of California, San Francisco Institutional Review Board (22-36620). The findings of the study are planned to be shared through conferences and publishments in a peer-reviewed journal. The deidentified dataset will be shared with qualified collaborators on request, provision of CITI and other certifications, and data sharing agreement. We will share the results, once the data are complete and analysed, with the scientific community and patient/clinician participants through abstracts, presentations and manuscripts. TRIAL REGISTRATION NUMBER: NCT05865405