Objectives:To evaluate the efficacy and safety of 18 months of tafamidis treatment in patients with early-stage V30M transthyretin familial amyloid polyneuropathy (TTR-FAP). Methods:In this randomized, double-blind trial, patients received tafamidis 20 mg QD or placebo.Coprimary endpoints were the Neuropathy Impairment Score-Lower Limbs (NIS-LL) responder analysis (Ͻ2-point worsening) and treatment-group difference in the mean change from baseline in Norfolk Quality of Life-Diabetic Neuropathy total score (TQOL) in the intent-to-treat (ITT) population (n ϭ 125). These endpoints were also evaluated in the efficacy-evaluable (EE; n ϭ 87) population. Secondary endpoints, including changes in neurologic function, nutritional status, and TTR stabilization, were analyzed in the ITT population.Results: There was a higher-than-anticipated liver transplantation dropout rate. No differences were observed between the tafamidis and placebo groups for the coprimary endpoints, NIS-LL responder analysis (45.3% vs 29.5% responders; p ϭ 0.068) and change in TQOL (2.0 vs 7.2; p ϭ 0.116) in the ITT population. In the EE population, significantly more tafamidis patients than placebo patients were NIS-LL responders (60.0% vs 38.1%; p ϭ 0.041), and tafamidis patients had better-preserved TQOL (0.1 vs 8.9; p ϭ 0.045). Significant differences in most secondary endpoints favored tafamidis. TTR was stabilized in 98% of tafamidis and 0% of placebo patients (p Ͻ 0.0001). Adverse events were similar between groups. Conclusions:Although the coprimary endpoints were not met in the ITT population, tafamidis was associated with no trend toward more NIS-LL responders and a significant reduction in worsening of most neurologic variables, supporting the hypothesis that preventing TTR dissociation can delay peripheral neurologic impairment. Classification of evidence:This study provides Class II evidence that 20 mg tafamidis QD was associated with no difference in clinical progression in patients with TTR-FAP, as measured by the NIS-LL and the Norfolk QOL-DN score. Secondary outcomes demonstrated a significant delay in peripheral neurologic impairment with tafamidis, which was well tolerated over 18 months. Neurology® 2012;79:785-792 GLOSSARY AE ϭ adverse event; ANCOVA ϭ analysis of covariance; ARR ϭ absolute risk reduction; CI ϭ confidence interval; DPN ϭ diabetic polyneuropathy; EE ϭ efficacy-evaluable; ITT ϭ intent-to-treat; LS Mean ϭ least-squares mean; mBMI ϭ modified body mass index; NIS-LL ϭ Neuropathy Impairment Score-Lower Limbs; NNT ϭ number needed to treat; QOL ϭ quality of life; QOL-DN ϭ Quality of Life-Diabetic Neuropathy Questionnaire; TQOL ϭ total quality of life; TTR-FAP ϭ transthyretin familial amyloid polyneuropathy.

The transthyretin amyloidoses (ATTR) are invariably fatal diseases characterized by progressive neuropathy and/or cardiomyopathy. ATTR are caused by aggregation of transthyretin (TTR), a natively tetrameric protein involved in the transport of thyroxine and the vitamin A–retinol-binding protein complex. Mutations within TTR that cause autosomal dominant forms of disease facilitate tetramer dissociation, monomer misfolding, and aggregation, although wild-type TTR can also form amyloid fibrils in elderly patients. Because tetramer dissociation is the rate-limiting step in TTR amyloidogenesis, targeted therapies have focused on small molecules that kinetically stabilize the tetramer, inhibiting TTR amyloid fibril formation. One such compound, tafamidis meglumine (Fx-1006A), has recently completed Phase II/III trials for the treatment of Transthyretin Type Familial Amyloid Polyneuropathy (TTR-FAP) and demonstrated a slowing of disease progression in patients heterozygous for the V30M TTR mutation. Herein we describe the molecular and structural basis of TTR tetramer stabilization by tafamidis. Tafamidis binds selectively and with negative cooperativity (K d s ∼2 nM and ∼200 nM) to the two normally unoccupied thyroxine-binding sites of the tetramer, and kinetically stabilizes TTR. Patient-derived amyloidogenic variants of TTR, including kinetically and thermodynamically less stable mutants, are also stabilized by tafamidis binding. The crystal structure of tafamidis-bound TTR suggests that binding stabilizes the weaker dimer-dimer interface against dissociation, the rate-limiting step of amyloidogenesis.

A new 4-point pharmacophore method for molecular similarity and diversity that rapidly calculates all potential pharmacophores/pharmacophoric shapes for a molecule or a protein site is described. The method, an extension to the ChemDiverse/Chem-X software (Oxford Molecular, Oxford, England), has also been customized to enable a new internally referenced measure of pharmacophore diversity. The "privileged" substructure concept for the design of high-affinity ligands is presented, and an example of this new method is described for the design of combinatorial libraries for 7-transmembrane G-protein-coupled receptor targets, where "privileged" substructures are used as special features to internally reference the pharmacophoric shapes. Up to 7 features and 15 distance ranges are considered, giving up to 350 million potential 4-point 3D pharmacophores/molecule. The resultant pharmacophore "key" ("fingerprint") serves as a powerful measure for diversity or similarity, calculable for both a ligand and a protein site, and provides a consistent frame of reference for comparing molecules, sets of molecules, and protein sites. Explicit "on-the-fly" conformational sampling is performed for a molecule to enable the calculation of all geometries accessible for all combinations of four features (i.e., 4-point pharmacophores) at any desired sampling resolution. For a protein site, complementary site points to groups displayed in the site are generated and all combinations of four site points are considered. In this paper we report (i) the details of our customized implementation of the method and its modification to systematically measure 4-point pharmacophores relative to a "special" substructure of interest present in the molecules under study; (ii) comparisons of 3- and 4-point pharmacophore methods, highlighting the much increased resolution of the 4-point method; (iii) applications of the 4-point potential pharmacophore descriptors as a new measure of molecular similarity and diversity and for the design of focused/biased combinatorial libraries.

Tafamidis, a transthyretin (TTR) kinetic stabilizer, delayed neuropathic progression in patients with Val30Met TTR familial amyloid polyneuropathy (TTR-FAP) in an 18-month randomized controlled trial (study Fx-005). This 12-month, open-label extension study evaluated the long-term safety, tolerability, and efficacy of tafamidis 20 mg once daily in 86 patients who earlier received blinded treatment with tafamidis or placebo. Efficacy measures included the Neuropathy Impairment Score in the Lower Limbs (NIS-LL), Norfolk Quality of Life-Diabetic Neuropathy total quality of life (TQOL) score, and changes in neurologic function and nutritional status. We quantified the monthly rates of change in efficacy measures, and TTR stabilization, and monitored adverse events (AEs). Patients who continued on tafamidis had stable rates of change in NIS-LL (from 0.08 to 0.11/month; p = 0.60) and TQOL (from −0.03 to 0.25; p = 0.16). In patients switched from placebo, the monthly rate of change in NIS-LL declined (from 0.34 to 0.16/month; p = 0.01), as did TQOL score (from 0.61 to −0.16; p < 0.001). Patients treated with tafamidis for 30 months had 55.9 % greater preservation of neurologic function as measured by the NIS-LL than patients in whom tafamidis was initiated later. Plasma TTR was stabilized in 94.1 % of patients treated with tafamidis for 30 months. AEs were similar between groups; no patients discontinued because of an AE. Long-term tafamidis was well tolerated, with the reduced rate of neurologic deterioration sustained over 30 months. Tafamidis also slowed neurologic impairment in patients previously given placebo, but treatment benefits were greater when tafamidis was begun earlier.

Modification of the hydantoin ring in the previously described lead compound 2a has led to the discovery of compound 12a, tadalafil, a highly potent and highly selective PDE5 inhibitor. The replacement of the hydantoin in compound 2a by a piperazinedione ring led to compound cis-11a which showed similar PDE5 inhibitory potency. Introduction of a 3,4-methylenedioxy substitution on the phenyl ring in position 6 led to a potent PDE5 inhibitor cis-11c with increased cellular potency. Optimization of the chain on the piperazinedione ring led to the identification of the racemic cis-N-methyl derivative 11i. High diastereospecificity for PDE5 inhibition was observed in the piperazinedione series with the cis-(6R,12aR) enantiomer displaying the highest PDE5 inhibitory activity. The piperazinedione 12a, tadalafil (GF196960), has been identified as a highly potent PDE5 inhibitor (IC(50) = 5 nM) with high selectivity for PDE5 vs PDE1-4 and PDE6. Compound 12a displays 85-fold greater selectivity vs PDE6 than sildenafil 1. 12a showed profound and long-lasting blood pressure lowering activity (30 mmHg/>7 h) in the spontaneously hypertensive rat model after oral administration (5 mg/kg).