Identification of a Tricyclic PIII Chiral Auxiliary for Solid-Supported Synthesis of Stereopure Phosphorothioate-Containing Oligonucleotides.

Since the recognition of oligonucleotides as a therapeutic modality, significant work has been devoted to improving therapeutic properties, including nuclease stability. Phosphorothioate (PS) modifications of phosphodiesters are one of the most explored chemical modification and integral to currently approved oligonucleotide therapeutics, including antisense oligonucleotides (ASOs) and short interfering RNAs (siRNAs). Insertion of sulfur into the phosphate bridge in an n-mer leads to 2n isomeric mixtures of PSs, with different nuclease stability and protein-binding properties. Efforts to create stereopure PS-containing oligonucleotides has spurred interest in identifying new synthetic methods. Herein, work on a novel and practical tricyclic PIII chiral auxiliary and its application in solid-supported synthesis of stereopure PS-containing oligonucleotides is reported.

Investigation of unknown impurities of paromomycin in a 15% topical cream by liquid chromatography combined with mass spectrometry.

RATIONALE: Characterization of unknown impurities in degraded paromomycin 15% topical cream samples by ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) gas-phase fragmentation is described. METHODS: A volatile reversed-phase high-performance liquid chromatography/charged aerosol detection (RP-HPLC/CAD) method was developed and validated for fast and consistent analysis of the paromomycin potency and impurity values in drug substance and drug product samples. A Veo charged aerosol detector was chosen for routine analysis. The method shows separation of two paromomycin isomers as well as twelve unknown impurity peaks. The identity of the unknown impurities was investigated by LC/MS/MS using a quadrupole time-of-flight (QTOF) mass spectrometer. RESULTS: Six of the HPLC/CAD impurity peaks met the ICH Q3B(R2) threshold for identification and were investigated by coupling the developed LC method with a QTOF instrument. Structures for the impurities were proposed based on gas-phase fragmentation patterns of the paromomycin reference standard. Impurity fragmentation pathways were proposed based on literature of other structurally related aminoglycoside compounds. CONCLUSIONS: Nine unknown paromomycin impurities were identified and the observed modifications included acetylation, carbamylation, gain of a hexose, and loss of glucosamine. The most abundant paromomycin modification was carbamylation by urea (a cream excipient) characterized by a 43 Da mass increase.

Obesity, metabolic syndrome and cardiovascular prognosis: from the Partners coronary computed tomography angiography registry.

OBJECTIVE: To investigate the relationship among body mass index (BMI), cardiometabolic risk and coronary artery disease (CAD) among patients undergoing coronary computed tomography angiography (CTA). METHODS: Retrospective cohort study of 1118 patients, who underwent coronary CTA at two centers from September 2004 to October 2011. Coronary CTA were categorized as normal, nonobstructive CAD (<50%), or obstructive CAD (≥50%) in addition to segment involvement (SIS) and stenosis scores. Extensive CAD was defined as SIS > 4. Association of BMI with cardiovascular prognosis was evaluated using multivariable fractional polynomial models. RESULTS: Mean age of the cohort was 57 ± 13 years with median follow-up of 3.2 years. Increasing BMI was associated with MetS (OR 1.28 per 1 kg/m2, p < 0.001) and burden of CAD on a univariable basis, but not after multivariable adjustment. Prognosis demonstrated a J-shaped relationship with BMI. For BMI from 20-39.9 kg/m2, after adjustment for age, gender, and smoking, MetS (HR 2.23, p = 0.009) was more strongly associated with adverse events. CONCLUSIONS: Compared to normal BMI, there was an increased burden of CAD for BMI > 25 kg/m2. Within each BMI category, metabolically unhealthy patients had greater extent of CAD, as measured by CCTA, compared to metabolically healthy patients.

Development of a potential high-throughput workflow to characterize sites of bioconjugation by immuno-affinity capture coupled to MALDI-TOF mass spectrometry.

The characterization of conjugation sites in bioconjugates is critical in the early discovery phase because site-specific conjugation improves in vivo stability and drug efficacy. We previously developed an engineered monoclonal antibody (mAb) scaffold which enables site-specific conjugation toward a reactive lysine (Lys) residue on each heavy chain (HC) by using an azetidinone (AZD) linker. In order to explore conjugations in other location which avoids potential interference with target binding, other chemical linkers have been studied and the investigation of N-hydroxysuccinimade (NHS) linker is reported here. The complexity of identifying the sites lies in part to the large number of Lys residues available for conjugation on the mAb scaffold. This has posed technical challenges to standard peptide mapping approaches. Therefore, an alternative strategy intended for a rapid analysis has been investigated by coupling immuno-affinity capture to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). In this study, we have employed a novel application of two different capture formats: Surface enhanced laser dissociation/ionization (SELDI) and mass spectrometry immunoassay (MSIA) tips to reduce the analysis time. An antibody against the pharmacophore portion was immobilized to capture the conjugated peptides, and subsequently provide characterization of the conjugation sites on the scaffold. Multiple sites for the AZD and NHS linkers have been easily identified and confirmed by MS2 sequencing. Lysine99 is the predominant site for the AZD linker, and Lysine55 is the primary site for the NHS linker with Lysine193 and Tyrosine37 being minor sites as shown in the abstract figure. We have also demonstrated the use of conjugation mapping to compare the distribution pattern between the AZD and NHS linkers as well as to study the stability of conjugation sites in a rapid way.

Development of a long acting human growth hormone analog suitable for once a week dosing.

Human growth hormone was conjugated to a carrier aldolase antibody, using a novel linker by connecting a disulphide bond in growth hormone to a lysine-94 amine located on the Fab arm of the antibody. The resulting CovX body showed reduced affinity towards human growth hormone receptor, reduced cell-based activity, but improved pharmacodynamic properties. We have demonstrated that this CovX-body, given once a week, showed comparable activity as growth hormone given daily in an in vivo hypophysectomized rat model.

Complications and outcomes of diaphyseal forearm fracture intramedullary nailing: a comparison of pediatric and adolescent age groups.

BACKGROUND: Flexible intramedullary nailing (IMN) has become a popular technique for the management of unstable or open forearm fractures. Recent publications have suggested an increased incidence of delayed union and poor outcomes in older children and adolescents. The objective of this study was to review forearm fractures treated with IMN, comparing the rate of complications and outcomes between the 2 age groups. Our hypothesis was that IMN is an effective technique with a similar rate of complications in both age groups. METHODS: An Institutional Review Board-approved retrospective review was conducted of pediatric forearm fractures treated from 1998 to 2008 at a single institution. Over the study time period, 4161 pediatric forearm fractures were managed nonoperatively (92%) and 353 were treated operatively with plate, cross-pin, or intramedullary fixation (8%). Patients with inadequate follow-up, cross-pin, or plate fixation were excluded. Medical records were reviewed for indications and complications. Complications were graded with a modification of the Clavien-Dindo classification. Outcomes were judged by a new grading system. RESULTS: A total of 205 forearm fractures treated with IMN in 203 patients were identified. The mean age was 9.7 years (range, 1.7 to 16.2 y) and mean follow-up was 42 weeks. Operative indications were failure of closed treatment in 165 (80%) and open fracture in 40 (20%). Mean time from injury to IMN was 5.9 days (range, 0 to 25 d). Single bone IMN was performed in 40 of 185 both bone fractures (26%); there were 20 single-bone forearm fractures treated with IMN. Open reduction was required in 61/165 (37%) of closed fractures. Asymptomatic delayed union (grade 1 complication) was observed in 9 fractures (4%). More severe complications were noted in 17% (grade 2 to 4 complications). Postoperative compartment syndrome occurred in 3 isolated forearm fractures with a significant younger mean age (6.0 vs. 10 y, P=0.031). Overall, complications were significantly more frequent in children older than 10 years of age (25/101) as compared with younger children (13/104, P=0.031). In particular, delayed union was more common in children over the age of 10 years (9/101 vs. 1/104, OR=9.99, P=0.009). Outcomes were good or excellent in 91% of fractures. There was no statistical association of patient age with a fair or poor outcome. CONCLUSIONS: IMN is an effective technique for pediatric forearm fractures with good to excellent outcomes in 91%. Complications are not infrequent with this technique, with complications of grade 2 to 4 severity in 17%. There was a 2-fold increase in the rate of complications in children over the age of 10 years. Compartment syndrome was more common in younger children. Patients and families should be counseled about the risks preoperatively.

A Plasma-Based Protein Marker Panel for Colorectal Cancer Detection Identified by Multiplex Targeted Mass Spectrometry.

INTRODUCTION: Colorectal cancer (CRC) testing programs reduce mortality; however, approximately 40% of the recommended population who should undergo CRC testing does not. Early colon cancer detection in patient populations ineligible for testing, such as the elderly or those with significant comorbidities, could have clinical benefit. Despite many attempts to identify individual protein markers of this disease, little progress has been made. Targeted mass spectrometry, using multiple reaction monitoring (MRM) technology, enables the simultaneous assessment of groups of candidates for improved detection performance. MATERIALS AND METHODS: A multiplex assay was developed for 187 candidate marker proteins, using 337 peptides monitored through 674 simultaneously measured MRM transitions in a 30-minute liquid chromatography-mass spectrometry analysis of immunodepleted blood plasma. To evaluate the combined candidate marker performance, the present study used 274 individual patient blood plasma samples, 137 with biopsy-confirmed colorectal cancer and 137 age- and gender-matched controls. Using 2 well-matched platforms running 5 days each week, all 274 samples were analyzed in 52 days. RESULTS: Using one half of the data as a discovery set (69 disease cases and 69 control cases), the elastic net feature selection and random forest classifier assembly were used in cross-validation to identify a 15-transition classifier. The mean training receiver operating characteristic area under the curve was 0.82. After final classifier assembly using the entire discovery set, the 136-sample (68 disease cases and 68 control cases) validation set was evaluated. The validation area under the curve was 0.91. At the point of maximum accuracy (84%), the sensitivity was 87% and the specificity was 81%. CONCLUSION: These results have demonstrated the ability of simultaneous assessment of candidate marker proteins using high-multiplex, targeted-mass spectrometry to identify a subset group of CRC markers with significant and meaningful performance.

Combined use of immunoassay and two-dimensional liquid chromatography mass spectrometry for the detection and identification of metabolites from biotherapeutic pharmacokinetic samples.

Peptides and monoclonal antibodies have both emerged as important therapeutic modalities, but each has challenges which limit their use. Non-recombinant chemical conjugation of peptides onto antibodies has the potential to minimize or eliminate altogether many of these limitations. Once such approach, pioneered by CovX has created the possibility for rapid stoichiometric fusion of pharmacophores to a single antibody platform. These molecules, called CovX-Bodies, maintain both the pharmacologic properties of a given peptide and the pharmacokinetic properties of a monoclonal antibody. The result is a new class of molecules wherein each component contributes desirable traits. In this paper, we demonstrate the use of immunoassay and two-dimensional liquid chromatography mass spectrometry (2DLC/MS) in combination to investigate the antibody conjugates of Glucagon-like peptide-1 (GLP-1) and analogs for intact protein metabolite identification directly from mouse serum. The information gained from combining these approaches has helped guide and expedite the optimization of our drug product development efforts.