Modulation of spinal GABAergic analgesia by inhibition of chloride extrusion capacity in mice.

UNLABELLED: Spinal gamma-aminobutyric acid receptor type A (GABA(A)) receptor modulation with agonists and allosteric modulators evokes analgesia and antinociception. Changes in K(+)-Cl(-) cotransporter isoform 2 (KCC2) expression or function that occur after peripheral nerve injury can result in an impairment in the Cl(-) extrusion capacity of spinal dorsal horn neurons. This, in turn, alters Cl(-)-mediated hyperpolarization via GABA(A) receptor activation, contributing to allodynia or hypersensitivity associated with nerve injury or inflammation. A gap in knowledge exists concerning how this loss of spinal KCC2 activity differentially impacts the analgesic efficacy or potency of GABA(A) agonists and allosteric modulators. We utilized intrathecal drug administration in the tail flick assay to measure the analgesic effects of general GABA(A) agonists muscimol and Z-3-[(aminoiminomethyl)thio]prop-2-enoic acid (ZAPA), the ∂-subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP), and allosteric modulators of the benzodiazepine (midazolam) and neurosteroid (ganaxolone) class, alone or in the presence of K(+)-Cl(-) cotransporter isoform (KCC) blockade. Intrathecal muscimol, ZAPA, THIP midazolam, and ganaxolone all evoked significant analgesia in the tail flick test. Coadministration of either agonists or allosteric modulators with [(dihydroindenyl)oxy] alkanoic acid (DIOA) (a drug that blocks KCC2) had no effect on agonist or allosteric modulator potency. On the other hand, the analgesic efficacy of muscimol and ZAPA and the allosteric modulator ganaxolone were markedly reduced whereas THIP and midazolam were unaffected. Finally, in the spared nerve injury model, midazolam significantly reversed tactile hypersensitivity while ganaxolone had no effect. These results indicate that the KCC2-dependent Cl(-) extrusion capacity differentially regulates the analgesic efficacy of agonists and allosteric modulators at the GABA(A) receptor complex. PERSPECTIVE: Our work suggests that drug discovery efforts for the treatment of chronic pain disorders should target benzodiazepine or ∂-subunit-containing sites at the GABA(A) complex.

Cannabilactones: a novel class of CB2 selective agonists with peripheral analgesic activity.

The identification of the CB2 cannabinoid receptor has provided a novel target for the development of therapeutically useful cannabinergic molecules. We have synthesized benzo[ c]chromen-6-one analogs possessing high affinity and selectivity for this receptor. These novel compounds are structurally related to cannabinol (6,6,9-trimethyl-3-pentyl-6 H-benzo[ c]chromen-1-ol), a natural constituent of cannabis with modest CB2 selectivity. Key pharmacophoric features of the new selective agonists include a 3-(1',1'-dimethylheptyl) side chain and a 6-oxo group on the cannabinoid tricyclic structure that characterizes this class of compounds as "cannabilactones." Our results suggest that the six-membered lactone pharmacophore is critical for CB2 receptor selectivity. Optimal receptor subtype selectivity of 490-fold and subnanomolar affinity for the CB2 receptor is exhibited by a 9-hydroxyl analog 5 (AM1714), while the 9-methoxy analog 4b (AM1710) had a 54-fold CB2 selectivity. X-ray crystallography and molecular modeling show the cannabilactones to have a planar ring conformation. In vitro testing revealed that the novel compounds are CB2 agonists, while in vivo testing of cannabilactones 4b and 5 found them to possess potent peripheral analgesic activity.

Tryptophan depletion selectively reduces CSF 5-HT metabolites in healthy young men: results from single lumbar puncture sampling technique.

Plasma tryptophan (Trp) depletion is a commonly used tool for determining the role of brain serotonin (5-HT) function in a variety of psychiatric disorders. This study measured the cerebrospinal fluid (CSF) monoamine metabolite response to Trp depletion and control testing in five healthy subjects utilizing a single lumbar puncture. Testing was done in a placebo-controlled, double-blind, randomized, cross-over design. Plasma-free and total Trp levels and behavioural ratings were obtained prior to and 5 h after ingestion of each amino-acid drink. CSF was obtained by performing a standard lumbar puncture 7 h after ingestion of the drink. Compared to control testing, Trp depletion caused a significant decrease of CSF 5-hydroxyindoleacetic acid (5-HIAA) (p = 0.03), but not of homovanillic acid or 3-methoxy-4-hydroxy-phenylglycol. Behavioural ratings were minimally affected in all subjects. This confirms that plasma Trp depletion reduces central nervous system measures of 5-HT function and suggests that the single lumbar puncture technique may be sufficient to detect the extent of CSF 5-HIAA changes during Trp depletion studies.