The goal of this investigation was to examine clinical translation of glucose responsiveness of MK-2640, which is a novel insulin saccharide conjugate that can bind the insulin receptor or mannose receptor C type 1 (MRC1), the latter dependent upon glucose concentration. In a rising dose study in 36 healthy adults under euglycemic clamp conditions, rising exposures revealed saturation of MK-2640 clearance, likely due to saturation of clearance by MRC1. Potency of MK-2640 was ~25-fold reduced relative to regular human insulin. In a randomized, 2-period crossover trial in 16 subjects with type 1 diabetes mellitus to evaluate glucose-responsiveness of i.v. administered MK-2640, we were unable to demonstrate a glucose-dependent change in MK-2640 clearance, although a significant glucose-dependent augmentation of glucose infusion rate was observed. These pharmacokinetic (PK) and pharmacodynamic (PD) data provide crucial insights into next steps for developing an insulin saccharide conjugate as a clinically effective glucose-responsive insulin analog.
F-MK-6240 is a highly selective, subnanomolar-affinity Positron Emission Tomography (PET) tracer for imaging neurofibrillary tangles (NFTs). Plasma kinetics, brain uptake, and preliminary quantitative analysis of F-MK-6240 in healthy elderly subjects (HE), subjects with clinically probable Alzheimer disease (AD), and amnestic mild cognitive impairment (MCI) were characterized in a first-in-human study. Dynamic PET scans of up to 150 min were performed in 4 cognitively normal HE, 4 AD and 2 MCI subjects, after bolus injection of 152-169 MBq F-MK-6240 to evaluate tracer kinetics and distribution in brain. Regional standardized uptake value ratio (SUVR) and distribution volume ratio (DVR) were determined using the cerebellar cortex as a reference region. Total distribution volume () was assessed by compartmental modeling using radiometabolite corrected input function in a subgroup of 6 subjects. F-MK-6240 had rapid brain uptake with peak standardized uptake value of 3-5, followed by a uniformly quick washout from all brain regions in HE; slower clearance was observed in regions commonly associated with NFT deposition in AD. In AD, SUVR measured between 60-90 min postinjection was high (approximately 2-4) in regions associated with NFT deposition; whereas, in HE, SUVR was approximately 1 across all brain regions suggesting high tracer selectivity for binding NFTs in vivo.F-MK-6240 VT was approximately 2- to 3-fold higher in neocortical and medial temporal brain regions of AD compared with HE, and stabilized by 60 min in both groups. DVR estimated by Logan reference tissue model or compartmental modeling correlated well (R >0.9) to SUVR for AD. F-MK-6240 exhibited favorable kinetics with high-binding levels to brain regions with a plausible pattern for NFT deposition in AD. In comparison, negligible tracer binding was observed in HE. This pilot study suggests simplified ratio methods such as SUVR can be employed to quantify NFT binding. These results support further clinical development ofF-MK-6240 for potential application in longitudinal studies.
A recurring obstacle for structural genomics is the expression of insoluble, aggregated proteins. In these cases, the use of alternative salvage strategies, like in vitro refolding, is hindered by the lack of a universal refolding method. To overcome this obstacle, fractional factorial screens have been introduced as a systematic and rapid method to identify refolding conditions. However, methodical analyses of the effectiveness of refolding reagents on large sets of proteins remain limited. In this study, we address this void by designing a fractional factorial screen to rapidly explore the effect of 14 different reagents on the refolding of 33 structurally and functionally diverse proteins. The refolding data was analyzed using statistical methods to determine the effect of each refolding additive. The screen has been miniaturized for automation resulting in reduced protein requirements and increased throughput. Our results show that the choice of pH and reducing agent had the largest impact on protein refolding. Bis-mercaptoacetamide cyclohexane (BMC) and tris(2-carboxyethylphosphine) (TCEP) were superior reductants when compared to others in the screen. BMC was particularly effective in refolding disulfide-containing proteins, while TCEP was better for nondisulfide-containing proteins. From the screen, we successfully identified a positive synergistic interaction between nondetergent sulfobetaine 201 (NDSB 201) and BMC on Cdc25A refolding. The soluble protein resulting from this interaction crystallized and yielded a 2.2 Å structure. Our method, which combines a fractional factorial screen with statistical analysis of the data, provides a powerful approach for the identification of optimal refolding reagents in a general refolding screen. Keywords: protein folding; fractional factorial screen; crystal structure; inclusion bodies; highthroughput refolding; structural genomicsThe identification of 20,000-25,000 genes from the human genome project has resulted in a wealth of potential targets for structural biology investigation and pharmaceutical design (International Human Genome Sequencing Consortium 2004). Since the completion of the project, expectations have been high that the number of protein crystal structures would dramatically increase but, in reality, there has only been a moderate rise in the number of crystal structures, due largely to a lack of sufficient quantities of protein suitable for structural studies (Service 2002). Although the technology responsible for expressing recombinant proteins is highly developed , it is still difficult to produce enough soluble protein for these structural studies. The ultimate goal of determining crystal structures on a genome-wide scale requires methods designed to improve the yield of functional protein.Reprint requests to: Ted Fox, Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, MA 02139, USA; e-mail: ted_fox@vrtx.com; fax: (617) 444-6820.Abbreviations: AMP-PNP, 5 0 -adenylylimidodiphosphate; bME, b-mercaptoethanol; BMC, bis-mercaptoacetamide...
Given the recognition that disease‐modifying therapies should focus on earlier Parkinson's disease stages, trial enrollment based purely on clinical criteria poses significant challenges. The goal herein was to determine the utility of dopamine transporter neuroimaging as an enrichment biomarker in early motor Parkinson's disease clinical trials. Patient‐level longitudinal data of 672 subjects with early‐stage Parkinson's disease in the Parkinson's Progression Markers Initiative (PPMI) observational study and the Parkinson Research Examination of CEP‐1347 Trial (PRECEPT) clinical trial were utilized in a linear mixed‐effects model analysis. The rate of worsening in the motor scores between subjects with or without a scan without evidence of dopamine transporter deficit was different both statistically and clinically. The average difference in the change from baseline of motor scores at 24 months between biomarker statuses was –3.16 (90% confidence interval [CI] = –0.96 to –5.42) points. Dopamine transporter imaging could identify subjects with a steeper worsening of the motor scores, allowing trial enrichment and 24% reduction of sample size.
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