Safety and efficacy of bosutinib (SKI-606) in chronic phase Philadelphia chromosome-positive chronic myeloid leukemia patients with resistance or intolerance to imatinib

Bosutinib, a dual Src/Abl kinase inhibitor, has shown potent activity against chronic myeloid leukemia (CML). In this phase 1/2 study we evaluated bosutinib in patients with chronic phase imatinib-resistant or imatinib-intolerant CML. Part 1 was a dose-escalation study to determine the recommended starting dose for part 2; part 2 evaluated the efficacy and safety of bosutinib 500 mg once-daily dosing. The study enrolled 288 patients with imatinib-resistant (n = 200) or imatinib-intolerant (n = 88) CML and no other previous kinase inhibitor exposure. At 24 weeks, 31% of patients achieved major cytogenetic response (primary end point). After a median follow-up of 24.2 months, 86% of patients achieved complete hematologic remission, 53% had a major cytogenetic response (41% had a complete cytogenetic response), and 64% of those achieving complete cytogenetic response had a major molecular response. At 2 years, progression-free survival was 79%; overall survival at 2 years was 92%. Responses were seen across Bcr-Abl mutants, except T315I. Bosutinib exhibited an acceptable safety profile; the most common treatment-emergent adverse event was mild/moderate, typically self-limiting diarrhea. Grade 3/4 nonhematologic adverse events (> 2% of patients) included diarrhea (9%), rash (9%), and vomiting (3%). These data suggest bosutinib is effective and tolerable in patients with chronic phase imatinib-resistant or imatinib-intolerant CML. This trial was registered at http://www.clinicaltrials.gov as NCT00261846.     

High prevalence of aquagenic wrinkling of the palms in patients with cystic fibrosis and association with measurable increases in transepidermal water loss

Aquagenic wrinkling of the palms (AWP) is a cutaneous phenomenon marked by the transient formation of edematous, translucent papules and plaques on the palms and fingertips within minutes of water exposure. AWP is anecdotally reported in patients with cystic fibrosis (CF) and several studies have recently confirmed this association. The primary aim of this study was to determine the prevalence of aquagenic wrinkling of the palms in subjects with cystic fibrosis (CF) compared to controls, and secondarily to evaluate for genotype-phenotype correlations among CF subjects found to have AWP. Fifty-one children with CF and 25 control children who were being treated for asthma underwent a 5-minute hand immersion in lukewarm water. The test for AWP was positive if subjects demonstrated >30% wrinkling over the palm. Secondary analyses explored associations with genotype, pancreatic and pulmonary function, body mass index (BMI), and sweat chloride levels. Palmar transepidermal water loss (TEWL) was also measured for all subjects with and without AWP. Forty-three of the subjects (84%) with CF demonstrated aquagenic wrinkling, in contrast to none (0%) of the controls. These results remained statistically significant when stratified for by age and race. TEWL was significantly higher in CF subjects with AWP compared to CF subjects without AWP and controls. No genotype-phenotype correlations were detected in patients with AWP, nor were there associations of AWP with other phenotypic features of CF, although these analyses were likely underpowered. Aquagenic wrinkling of the palms is prevalent in children with CF and is associated with increased TEWL.     

Interaction of daptomycin with two recombinant thromboplastin reagents leads to falsely prolonged patient prothrombin time/International Normalized Ratio results.

A cluster of patients experiencing elevations of International Normalized Ratio without clinical bleeding in temporal association with daptomycin therapy was identified during postmarketing safety surveillance. A common element was the thromboplastin reagent used for the laboratory assay. The present study evaluated the effect of daptomycin on measured prothrombin time using commercially available thromboplastin reagent kits commonly used in the United States. Thirty reagent kits were obtained. Daptomycin was added to pooled normal human plasma samples to achieve final concentrations of 0-200 microg/ml. Quality control ranges were established for each reagent kit using normal and abnormal control plasmas. Triplicate assays of the prothrombin time were performed on the daptomycin-spiked plasma samples using each of the 30 kits. The activated partial thromboplastin time and thrombin time were also assessed. Statistical comparisons of interest were performed using analysis of variance with the Bonferroni t-test for multiple comparisons; alpha = 0.05 was used. Addition of daptomycin to human plasma samples dose-dependently prolonged measured prothrombin times when two recombinant thromboplastin reagents were utilized. The findings were statistically and clinically significant. No clinically meaningful effect was observed with the other reagents. The activated partial thromboplastin time and thrombin time were not affected. Prolonged International Normalized Ratio patient values were an artifact caused by the interaction of daptomycin with the in-vitro prothrombin time test reagent; an in-vivo anticoagulant effect was not observed. Healthcare providers should consider a possible drug-laboratory test interaction if prolonged prothrombin time or elevated International Normalized Ratio values are observed in patients receiving daptomycin.     

Binding of small molecules to an adaptive protein-protein interface

Understanding binding properties at protein-protein interfaces has been limited to structural and mutational analyses of natural binding partners or small peptides identified by phage display. Here, we present a high-resolution analysis of a nonpeptidyl small molecule, previously discovered by medicinal chemistry [Tilley, J. W., et al. (1997) J. Am. Chem. Soc. 119, 7589-7590], which binds to the cytokine IL-2. The small molecule binds to the same site that binds the IL-2 alpha receptor and buries into a groove not seen in the free structure of IL-2. Comparison of the bound and several free structures shows this site to be composed of two subsites: one is rigid, and the other is highly adaptive. Thermodynamic data suggest the energy barriers between these conformations are low. The subsites were dissected by using a site-directed screening method called tethering, in which small fragments were captured by disulfide interchange with cysteines introduced into IL-2 around these subsites. X-ray structures with the tethered fragments show that the subsite-binding interactions are similar to those observed with the original small molecule. Moreover, the adaptive subsite tethered many more compounds than did the rigid one. Thus, the adaptive nature of a protein-protein interface provides sites for small molecules to bind and underscores the challenge of applying structure-based design strategies that cannot accurately predict a dynamic protein surface.     

Phase 1, single‐dose escalating study of marzeptacog alfa (activated), a recombinant factor VIIa variant,in patients with severe hemophilia

Essentials

• Marzeptacog alfa (activated) [MarzAA] is a novel variant of activated human factor VII.
• A phase 1 dose escalation trial of MarzAA was conducted in subjects with severe hemophilia.
• MarzAA was safe and tolerated at intravenous doses up to 30 μg kg^?1
• Data observed support further trials for hemophilia patients with inhibitors to factors VIII/IX.

Summary

Background

Marzeptacog alfa (activated) (MarzAA), a new recombinant activated human factor VII (rFVIIa) variant with four amino acid substitutions, was developed to provide increased procoagulant activity and a longer duration of action in people with hemophilia.

Objectives

To investigate the safety, tolerability, immunogenicity, pharmacokinetics (PK) and pharmacodynamics (PD) of single ascending intravenous bolus doses of MarzAA in non‐bleeding patients with congenital hemophilia A or B with or without inhibitors.

Methods

This international, phase 1, open‐label study (NCT01439971) enrolled males aged 18–64 years with severe hemophilia A or B, with or without FVIII or FIX inhibitors. Subjects were assigned to single‐dose MarzAA cohorts (0.5, 4.5, 9, 18 or 30 μg kg^?1). Blood sampling was performed predose and postdose, and subjects were monitored for 60 days postdose. Safety endpoints included adverse events, vital sign changes, electrocardiograms, laboratory abnormalities, and immunogenicity; secondary endpoints included evaluation of PK and PD.

Results

Overall, in 25 patients, MarzAA was well tolerated at all dose levels tested, and was not associated with dose‐limiting toxicity. No treatment‐emergent severe or serious adverse events occurred. MarzAA showed linear dose–response PK across the 4.5–30 μg kg^?1 dose range, with a terminal half‐life of ? 3.5 h. Dose‐dependent shortening of the activated partial thromboplastin time and prothrombin time, and evidence of an increase in peak thrombin as determined with a thrombin generation assay, were observed at all doses.

Conclusions

MarzAA was tolerated at doses up to 30 μg kg^?1. The safety profile and pharmacological effects observed support further clinical trials for the treatment of hemophilic patients with inhibitors.

     

Altered cofactor regulation with disease-associated p97/VCP mutations

Dominant mutations in p97/VCP (valosin-containing protein) cause a rare multisystem degenerative disease with varied phenotypes that include inclusion body myopathy, Paget’s disease of bone, frontotemporal dementia, and amyotrophic lateral sclerosis. p97 disease mutants have altered N-domain conformations, elevated ATPase activity, and altered cofactor association. We have now discovered a previously unidentified disease-relevant functional property of p97 by identifying how the cofactors p37 and p47 regulate p97 ATPase activity. We define p37 as, to our knowledge, the first known p97-activating cofactor, which enhances the catalytic efficiency (k_cat/K_m) of p97 by 11-fold. Whereas both p37 and p47 decrease the K_m of ATP in p97, p37 increases the k_cat of p97. In contrast, regulation by p47 is biphasic, with decreased k_cat at low levels but increased k_cat at higher levels. By deleting a region of p47 that lacks homology to p37 (amino acids 69–92), we changed p47 from an inhibitory cofactor to an activating cofactor, similar to p37. Our data suggest that cofactors regulate p97 ATPase activity by binding to the N domain. Induced conformation changes affect ADP/ATP binding at the D1 domain, which in turn controls ATPase cycling. Most importantly, we found that the D2 domain of disease mutants failed to be activated by p37 or p47. Our results show that cofactors play a critical role in controlling p97 ATPase activity, and suggest that lack of cofactor-regulated communication may contribute to p97-associated disease pathogenesis.The p97 AAA [ATPase associated with diverse cellular activities; also called VCP (valosin-containing protein)] participates in key steps in ubiquitin-dependent protein quality control (1), autophagy (2, 3), membrane remodeling (4, 5), and numerous other fundamentally important cellular functions. p97 directs proteins to two major degradation systems: the proteasome and autophagy pathways. This key role of p97 underscores its importance in protein homeostasis and strongly implicates p97 in neurodegenerative diseases (6). Dominantly inherited missense mutations in p97 were initially associated with a rare degenerative disorder termed inclusion body myopathy with Paget’s disease of bone and frontotemporal dementia (IBMPFD) (7, 8). Subsequently, it has been found that mutations in p97 cause a wider spectrum of degenerative diseases that includes amyotrophic lateral sclerosis (9–12) and parkinsonism (13). Thus, the acronym IBMPFD is insufficient. Moreover, mutations in the heterogeneous nuclear ribonucleoproteins hnRNPA2B1 and hnRNPA1 also cause a syndrome similar to conditions associated with VCP mutations (14). To account for the varied phenotypes and genetic etiologies of IBMPFD, it has been suggested that the term multisystem proteinopathy type 1 (MSP1) (14) be used to describe the phenotypic syndrome associated with mutations in p97. Disease mutants of p97 contribute to pathology by disrupting autophagosome (2, 3) and endosome maturation, which leads to vacuolation, weakness, and muscle atrophy (15, 16). However, the molecular disease mechanisms associated with mutant p97 are still being investigated, with the eventual hope of developing therapeutic agents for these serious illnesses.p97 assembles into a homohexamer, as revealed by X-ray crystallography (17, 18). A single monomer is composed of an N domain, which interacts with p97 cofactor proteins, and two ATPase domains, D1 and D2. The D1 and D2 domains form stacked hexameric rings, whereas the N domains extend outward, coplanar with the D1 ring. Most of the disease mutations occur in the N domain, whereas some occur in the D1 or the N–D1 linker region (see SI Appendix, Fig. S1A for examples). All p97 disease mutants tested thus far can form stable hexamers (19, 20) and exhibit increased D2 ATPase activity (19–23). Disease mutations lead to increased proteolytic susceptibility of the D2 ring (19). Structural and biochemical studies suggest that disease mutations alter N-domain and D1 conformations (20, 23, 24) and cause defects in interdomain communication between neighboring subunits.A major role of the N domain is to recruit cofactors (25, 26), such as the Npl4 (nuclear protein localization homolog 4) and Ufd1 (ubiquitin fusion degradation 1) heterodimers (27), and an array of 13 UBX (ubiquitin regulatory X) domain cofactors (28). By recruiting certain cofactor proteins, the N domain may link the mechanochemical activity of ATP hydrolysis to the unfolding or disassembly of substrate proteins. p47, the first p97 UBX cofactor discovered, is required for p97-mediated membrane fusion (29). Binding of p47 (also called NSFL1 cofactor p47 or UBX domain-containing protein 2C) to the N domain of p97 significantly reduces the diameter of the p97 ring (29) and inhibits wild-type p97 ATPase activity (30). Although actively studied, the physiological functions of p97–cofactor complexes and their mechanisms are largely unknown.X-ray crystallography of p97 has revealed that the N domain of p97 is conformationally flexible (17, 18), adopting two primary conformations. In the up conformation, the N domain extends above the D1 ring, whereas in the down conformation, the N domain lies coplanar with the D1 ring. The conformation is nucleotide-dependent, that is, determined by the binding state of the D1 domain (17). It has been proposed that the flexibility of the N domain is crucial to ATP hydrolysis, because modifying the N domain reduces ATPase activity. Specifically, reducing N-domain mobility inhibits wild-type p97 ATPase activity (20). Moreover, removing the N domain (1∼209) altogether was shown to block the enhanced ATPase activity of a disease mutant (20).In wild-type p97, the N domains exist in a tightly regulated, heterogeneous arrangement of up and down conformations. In contrast, disease mutants exhibit dysregulated N-domain conformations (12, 17, 24). Crystal structures of two disease mutants showed all six N domains of the complex in the up conformation, a behavior that has been observed in only disease mutants (23). A recent study found that this uniform arrangement is a secondary effect of reduced ADP binding by the D1 domain, whose state controls N-domain conformation (23).Altered conformation of the N domain in p97 disease mutants is further supported by atypical p97 cofactor binding in cells (31–33). Decreased binding to a UBX cofactor, UBXD1, is observed in 293T cells expressing p97 disease mutants and leads to a blockade of caveolin 1 trafficking (33). Intriguingly, disease mutants can coimmunoprecipitate more p47 and Npl4/Ufd1 heterodimers than WT p97, suggesting elevated binding affinities for p47 and Npl4/Ufd1 in mutant cells (31, 33). However, the consequences of altered binding to cofactors in cells that express mutant p97 have not been investigated biochemically. To provide a mechanistic understanding of cofactor-regulated ATPase activity, we analyzed the effect of p37 and p47 on the ATPase activity of WT and disease mutants of p97 in this study.     

Identification of genes involved in the toxic response of Saccharomyces cerevisiae against iron and copper overload by parallel analysis of deletion mutants.

Iron and copper are essential nutrients for life as they are required for the function of many proteins but can be toxic if present in excess. Accumulation of these metals in the human body as a consequence of overload disorders and/or high environmental exposures has detrimental effects on health. The budding yeast Saccharomyces cerevisiae is an accepted cellular model for iron and copper metabolism in humans primarily because of the high degree of conservation between pathways and proteins involved. Here we report a systematic screen using yeast deletion mutants to identify genes involved in the toxic response to growth-inhibitory concentrations of iron and copper sulfate. We aimed to understand the cellular responses to toxic concentrations of these two metals by analyzing the different subnetworks and biological processes significantly enriched with these genes. Our results indicate the presence of two different detoxification pathways for iron and copper that converge toward the vacuole. The product of several of the identified genes in these pathways form molecular complexes that are conserved in mammals and include the retromer, endosomal sorting complex required for transport (ESCRT) and AP-3 complexes, suggesting that the mechanisms involved can be extrapolated to humans. Our data also suggest a disruption in ion homeostasis and, in particular, of iron after copper exposure. Moreover, the identification of treatment-specific genes associated with biological processes such as DNA double-strand break repair for iron and tryptophan biosynthesis for copper suggests differences in the mechanisms by which these two metals are toxic at high concentrations.