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1 Both the 5-HT 1D and 5-HT 1B receptors are implicated in migraine pathophysiology. Recently isochromans have been discovered to bind primate 5-HT 1D receptors with much higher a nity than 5-HT 1B receptors. In the guinea-pig, a primary animal model for anti-migraine drug testing, however, isochromans bound the 5-HT 1D receptor with lower a nity than the gorilla receptor. 2 This species-speci®c pharmacology was investigated, using site-directed mutagenesis on cloned guinea-pig receptors heterologously expressed in human embryonic kidney 293 cells. Mutations of threonine 100 and arginine 102 at the extracellular side of transmembrane II of the guinea-pig 5-HT 1D receptor to the corresponding primate residues, isoleucine and histidine, respectively, enhanced its a nity for isochromans to that of the gorilla receptor, with little e ects on its a nities for serotonin, sumatriptan and metergoline. Free energy change from the R102H mutation was about twice as much as that from the T100I mutation. 3 For G protein-coupling, serotonin marginally enhanced GTPg 35 S binding in membranes expressing the guinea-pig 5-HT 1D receptor and its mutants, but robustly in membranes expressing the gorilla receptor. Sumatriptan enhanced GTPg 35 S binding in the latter nearly as much as serotonin, and several isochromans by 30 ± 60% of serotonin. 4 We discovered key di erences in the function and binding properties of guinea-pig and gorilla 5-HT 1D receptors, and identi®ed contributions of I100 and H102 of primate 5-HT 1D receptors to isochroman binding. Among common experimental animals, only the rabbit shares I100 and H102 with primates, and could be useful for studying isochroman actions in vivo.

The present study describes the preclinical pharmacology of a highly selective 5-HT1D receptor agonist PNU-142633. PNU-142633 binds with a Ki of 6 nm at the human 5-HT1D receptor and a Ki of> 18 000 nm at the human 5-HT1B receptor. The intrinsic activity of PNU-142633 at the human 5-HT1D receptor was determined to be 70% that of 5-HT in a cytosensor cell-based assay compared with 84% for that of sumatriptan. PNU-142633 was equally effective as sumatriptan and a half-log more potent than sumatriptan in preventing plasma protein extravasation induced by electrical stimulation of the trigeminal ganglion. Like sumatriptan, PNU-142633 reduced the increase in cat nucleus trigeminal caudalis blood flow elicited by electrical stimulation of the trigeminal ganglion compared with the vehicle control. The direct vasoconstrictor potential of PNU-142633 was evaluated in vascular beds. Sumatriptan increased vascular resistance in carotid, meningeal and coronary arteries while PNU-142633 failed to alter resistance in these vascular beds. These data are discussed in relation to the clinical findings of PNU-142633 in a phase II acute migraine study.

ObjectivesSurvival Motor Neuron (SMN) protein levels may become key pharmacodynamic (PD) markers in spinal muscular atrophy (SMA) clinical trials. SMN protein in peripheral blood mononuclear cells (PBMCs) can be quantified for trials using an enzyme-linked immunosorbent assay (ELISA). We developed protocols to collect, process, store and analyze these samples in a standardized manner for SMA clinical studies, and to understand the impact of age and intraindividual variability over time on PBMC SMN signal.MethodsSeveral variables affecting SMN protein signal were evaluated using an ELISA. Samples were from healthy adults, adult with respiratory infections, SMA patients, and adult SMA carriers.ResultsDelaying PBMCs processing by 45 min, 2 hr or 24 hr after collection or isolation allows sensitive detection of SMN levels and high cell viability (>90%). SMN levels from PBMCs isolated by EDTA tubes/Lymphoprep gradient are stable with processing delays and have greater signal compared to CPT-collected samples. SMN signal in healthy individuals varies up to 8x when collected at intervals up to 1 month. SMN signals from individuals with respiratory infections show 3–5x changes, driven largely by the CD14 fraction. SMN signal in PBMC frozen lysates are relatively stable for up to 6 months. Cross-sectional analysis of PBMCs from SMA patients and carriers suggest SMN protein levels decline with age.ConclusionsThe sources of SMN signal variability in PBMCs need to be considered in the design and of SMA clinical trials, and interpreted in light of recent medical history. Improved normalization to DNA or PBMC subcellular fractions may mitigate signal variability and should be explored in SMA patients.

Condensation of racemic keto-ester 2 and 1.1 equiv of (R)-(−)-phenylglycinol in toluene with azeotropic removal of water and methanol gave rise to a tetracyclic lactam in greater than 90% yield. Examination of the crude reaction product by 1H and 13C NMR, capillary GC, and HPLC revealed this product to be a single isomer, the absolute configuration of which was determined to be that illustrated by structure 3. The tetracyclic lactam 3 was stereospecifically reduced to either the cis-fused bicyclic pyrrolidine 4 or the cis-fused bicyclic pyrrolidinone 9. The chiral auxiliaries in both 4 and 9 were removed using different, novel methodologies. This highly stereoselective reaction establishes two contiguous chiral centers via the deracemization of an achiral keto-ester. This methodology has been applied to the synthesis of (1R,5R)-2-azabicyclo[3.3.0]octane 15. This important amine is now readily available from commercial materials in only three steps.