Pharmacology/Drug Development

Patients with cancer, especially breast, prostate, and lung cancer, commonly experience bone metastases that are difficult to manage and are associated with bone cancer pain (BCP). Amitriptyline is often used to treat chronic pain, such as neuropathic pain. In the present study, the effects of amitriptyline on the mechanical withdrawal threshold (MWT) and its underlying mechanisms were evaluated in rat models of BCP. Walker 256 rat mammary gland carcinoma cells were injected into the bone marrow cavity of the right tibia of rats to provoke BCP. Then, amitriptyline was intraperitoneally administered twice daily from fifth day after the operation. Rats with bone cancer showed an apparent decline in the MWT at day 11 after Walker 256 cells inoculation. The levels of the glutamate transporter GLAST in the spinal cord dorsal horn decreased remarkably, and the concentration of the excitatory amino acid (EAA) glutamate (Glu) in the cerebrospinal fluid (CSF) increased substantially. Amitriptyline injection could prevent the decline of MWT in BCP rats. In addition, GLAST was upregulated on the glial cell surface, and Glu levels were reduced in the CSF. However, amitriptyline injection could not prevent the BCP-induced reduction in GLAST in the glial cell cytosol, it further downregulated cytosolic GLAST. Amitriptyline had no significant effect on GLAST mRNA expression, and BCP-invoked PKA/PKC upregulation was prevented. Taken together, these results suggest that the intraperitoneal injection of amitriptyline can prevent the decrease of MWT in BCP rats, the underlying mechanisms may be associated with the inhibition of PKA/PKC expression, thus promoting GLAST trafficking onto the glial cell surface and reducing EAA concentrations in the CSF.

Cisplatin is a widely used platinum-derived antineoplastic agent that frequently results in peripheral neuropathy. Therapeutic strategies for neuropathic pain are limited and characterized by variable efficacy and severe adverse effects. Clinical translation of novel analgesics has proven difficult with many agents demonstrating preclinical efficacy failing in clinical trials. Preclinical studies frequently assess pain behaviors in the hind paws, however the front paws have a greater degree of the fine sensorimotor functions characteristically damaged by chemotherapy-induced neuropathy. This is the first study to assess pain responses in the front paws. Here we test the hypothesis that mouse front paws exhibit pain-related alterations in mechanical and thermal (cold) sensitivity in a murine model of cisplatin-induced neuropathy, and that pharmacological treatment with amitriptyline, gabapentin, ibuprofen and URB937 normalize pain behaviors in the front and hind paws. Cold (acetone withdrawal latencies) and mechanical (von Frey withdrawal thresholds) sensitivity were significantly decreased and increased respectively in both the front and the hind paws following initiation of weekly systemic (intraperitoneal) cisplatin injections (5 mg/kg). For the hind paws, systemic administration of amitriptyline (30 mg/kg), gabapentin (100 mg/kg), ibuprofen (0 -10 mg/kg) or URB937 (0 -10 mg/kg) resulted in a decrease in acetone withdrawal latencies and increase in von Frey withdrawal thresholds with return to normal values at the highest doses tested. For the front paws, return to baseline values for the highest doses was found for cold allodynia but not mechanical allodynia, where the highest doses failed to return to baseline values. These results indicate that mouse front paws exhibit pain-related changes in cisplatin-induced neuropathy and that drug effects can vary based on testing stimulus and location. This suggests that front paw responses across multiple modalities provide reliable and accurate information about pain-related drug effects. Future studies should be aimed at elucidating the mechanisms underlying these differential effects.

We have examined the regulation of mutually exclusive Cav2.2 exon 37a and b variants by the mouse μ-opioid receptor (mMOR) C-terminal splice variants 1, 1C and 1O in tsA-201 cells. Electrophysiological analyses revealed that both channel isoforms exhibit DAMGO-induced voltage-dependent (Gβγ-mediated) inhibition and its recovery by voltage pre-pulses, as well as a voltage-independent component. However, the two channel isoforms differ in their relative extent of voltage-dependent and independent inhibition, with Cav2.2-37b showing significantly more voltage-dependent inhibition upon activation of the three mMOR receptors studied. In addition, coexpression of either mMOR1 or mMOR1C results in an agonist-independent reduction in the peak current density of Cav2.2-37a channels, whereas the peak current density of Cav2.2-37b does not appear to be affected. Interestingly, this decrease is not due to an effect on channel expression at the plasma membrane, as demonstrated by biotinylation experiments. We further examined the mechanism underlying the agonist-independent modulation of Cav2.2-37a by mMOR1C. Incubation of cells with pertussis toxin did not affect the mMOR1C mediated inhibition of Cav2.2-37a currents, indicating a lack of involvement of Gi/o signaling. However, when a Src tyrosine kinase inhibitor was applied, the effect of mMOR1C was lost. Moreover, when we recorded currents using a Cav2.2-37a mutant in which tyrosine 1747 was replaced with phenylalanine (Y1747F), the agonist independent effects of mMOR1C were abolished. Altogether our findings show that Cav2.2-37a and Cav2.2-37b isoforms are subject to differential regulation by C-terminal splice variants of mMORs, and that constitutive mMOR1C activity and downstream tyrosine kinase activity exert a selective inhibition of the Cav2.2-37a splice variant, an N-type channel isoform that is highly enriched in nociceptors. Our study provides new insights into the roles of the MOR full-length C-terminal variants in modulating Cav2.2 channel isoform activities.