Background & Aims

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a family of non-selective voltage-gated cation channels (the associated current is called Ih current), that are important in modulating the excitability of neurons. Specifically, the HCN isoforms 1, 2 and 3 are expressed in the peripheral sensory neurons. HCN4 channels are predominantly expressed in the heart. Clinically, non-selective HCN inhibitors such as ivabradine have limited analgesic potential and are used to treat angina pain while preclinically, HCN2-inhibition has been demonstrated to produce analgesia in mice (Emery et al., 2011). We aimed to investigate and contrast in vivo target modulation and heart rate (HR) effects produced by a novel nonselective HCN inhibitor (MSD-1) and a novel HCN2 selective inhibitor (MSD-2) using the rat threshold-tracking (rTT), spinal nerve ligation model and ectopic firing assays.

Methods

Whole-cell currents were recorded from recombinant cell lines stably expressing human HCN1, HCN2, HCN3 or HCN 4 channels using the Qube® technology. Cells were held at -40 mV. HCN1, HCN2 and HCN3 currents were elicited by 2s test pulses to -120 mV. HCN4 currents were elicited by 4s test pulses to -140 mV.
Threshold Tracking: In anesthetized rats, 2 stimulating electrodes were placed in the muscle of the leg, while 2 recording electrodes were placed on the tail. Recordings were made using QTRAC. HR changes were monitored.
Spinal nerve ligation: Rats were assessed for baseline mechanosensitivity with von Frey filaments. They underwent left lumbar 5 spinal nerve (L5) ligation under deep anesthesia and were used for behavior testing following a 1-week recovery period.
Ectopic firing recordings: In anesthetized rats with CCI injury, the L4/L5 dorsal root was exposed, and teased to make electrical recordings. Data was digitized with CED Power 1401 and analyzed using Spike 2 software

Results

MSD-1 has an IC50 of 2.7 µM, 1.5 µM, 2.5 µM and 20.6 µM on HCN1, HCN2, HCN3 and HCN4, respectively. Whereas MSD-2 is highly potent in inhibiting HCN2 with an IC50= 23 nM and is 490x, 122x and 80x selective over HCN1 (IC50= 11.2 µM), HCN3 (IC50 = 2.8 µM) and HCN4 (IC50= 1.8 µM) ion channels. Administration of MSD-1 and MSD-2 resulted in a leftward shift of the compound muscle action potential (CMAP)-derived IV curve as measured via rat threshold tracking demonstrating in-vivo target modulation. For both compounds, maximal efficacy was associated with significant bradycardia. In contrast, Only MSD-1 showed efficacy in the rat spinal nerve ligation assay. MSD-1 also inhibited spontaneous/ectopic firing in the L5 dorsal root of rats with CCI injury.

Conclusions

Our results demonstrate a lack of improved analgesic efficacy or therapeutic index by selectively inhibiting HCN2 channels in rats. Non-selective HCN inhibition (using MSD-1) resulted in attenuation of ectopia in peripheral nerves and induces analgesia in rodents with nerve injury.

References

HCN2 ion channels play a central role in inflammatory and neuropathic pain.
Emery EC, Young GT, Berrocoso EM, Chen L, McNaughton PA.
Science. 2011 Sep 9;333(6048):1462-6. PMID: 21903816

Presenting Author

Parul S. Pall

Poster Authors

Parul Pall

PhD

Merck and Co., Inc.

Lead Author

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Xiaoping Zhou

MS

Lead Author

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Dillon McDevitt

Merck & Co.

Lead Author

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Yuxing Li MS

Lead Author

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Richard L. Kraus PhD

Lead Author

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Jason Cox PhD

Lead Author

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Ashok Arasappan PhD

Lead Author

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Darrell Henze

Merck Research Laboratories

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Topics

  • Mechanisms: Biological-Systems (Physiology/Anatomy)