5β-Reduced neuroactive steroids are novel voltage-dependent blockers of T-type Ca²⁺ channels in rat sensory neurons in vitro and potent peripheral analgesics in vivo

T-type Ca²⁺ channels are believed to play an important role in pain perception, and anesthetic steroids such as alphaxalone and allopregnanolone, which have a 5α-configuration at the steroid A, B ring fusion, are known to inhibit T-type Ca²⁺ channels and cause analgesia in a thermal nociceptive model (Soc Neurosci Abstr 29:657.9, 2003). To define further the structure-activity relationships for steroid analgesia, we synthesized and examined a series of 5β-reduced steroids for their ability to induce thermal antinociception in rats when injected locally into the peripheral receptive fields of the nociceptors and studied their effects on T-type Ca²⁺ channel function in vitro. We found that most of the steroids completely blocked T-type Ca²⁺ currents in vitro with IC₅₀ values at a holding potential of -90 mV ranging from 2.8 to 40 μM. T current blockade exhibited mild voltage-dependence, suggesting that 5β-reduced neuroactive steroids stabilize inactive states of the channel. For the most potent steroids, we found that other voltage-gated currents were not significantly affected at concentrations that produce nearly maximal blockade of T currents. All tested compounds induced dose-dependent analgesia in thermal nociceptive testing; the most potent effect (ED₅₀, 30 ng/100 μl) obtained with a compound [(3β,5β,17β)-3- hydroxyandrostane-17-carbonitrile] that was also the most effective blocker of T currents. Compared with previously studied 5α-reduced steroids, these 5β-reduced steroids are more efficacious blockers of neuronal T-type Ca²⁺ channels and are potentially useful as new experimental reagents for understanding the role of neuronal T-type Ca²⁺ channels in peripheral pain pathways.