As part of an effort to examine the utility of antibody-drug conjugates (ADCs) beyond oncology indications, a novel pyrophosphate ester linker was discovered to enable the targeted delivery of glucocorticoids. As small molecules, these highly soluble phosphate ester drug linkers were found to have ideal orthogonal properties: robust plasma stability coupled with rapid release of payload in a lysosomal environment. Building upon these findings, site-specific ADCs were made between this drug linker combination and an antibody against human CD70, a receptor specifically expressed in immune cells but also found aberrantly expressed in multiple human carcinomas. Full characterization of these ADCs enabled procession to in vitro proof of concept, wherein ADCs 1-22 and 1-37 were demonstrated to afford potent, targeted delivery of glucocorticoids to a representative cell line, as measured by changes in glucocorticoid receptor-mediated gene mRNA levels. These activities were found to be antibody-, linker-, and payload-dependent. Preliminary mechanistic studies support the notion that lysosomal trafficking and enzymatic linker cleavage are required for activity and that the utility for the pyrophosphate linker may be general for internalizing ADCs as well as other targeted delivery platforms.
In an effort to examine the utility of antibody-drug conjugates (ADCs) beyond oncology indications, a novel phosphate bridged Cathepsin B sensitive linker was developed to enable the targeted delivery of glucocorticoids. Phosphate bridging of the Cathepsin B sensitive linkers allows for payload attachment at an aliphatic alcohol. As small molecule drug-linkers, these aqueous soluble phosphate containing drug-linkers were found to have robust plasma stability coupled with rapid release of payload in a lysosomal environment. Site-specific ADCs were successfully made between these drug-linkers and an antibody against human CD70, a receptor specifically expressed in immune cells but also found aberrantly expressed in multiple human carcinomas. These ADCs demonstrated in vitro targeted delivery of glucocorticoids to a representative cell line as measured by changes in glucocorticoid receptor (GR) mediated gene mRNA levels. This novel linker expands the scope of potential ADC payloads by allowing an aliphatic alcohol to be a stable, yet cleavable attachment site. This phosphate linker may have broad utility for internalizing ADCs as well as other targeted delivery platforms.
HILIC LC-MS for the determination of 29-C-methylcytidine-triphosphate in rat liverA very accurate and selective LC-MS/MS method was developed and validated for the quantification of 29-C-modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96-well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm62.0 mm, 3 lm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 lL/min flow rate. The 29-C-modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05 -50 lM concentration range. Satisfying results, confirming the high reliability of the established LC-MS/MS method, were obtained for intraday precision (CV = 2.5 -9.1%) and accuracy (92.6 -94.8%) and interday precision (CV = 9.6 -11.5%) and accuracy (94.4 -102.4%) as well as for recovery (82.0 -112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 29-C-methyl-cytidine-triphosphate in liver tissue samples.
J. Neurochem. (2011) 116, 82–92. Abstract Elevated plasma homocysteine, a risk factor for Alzheimer’s disease, could result from increased production from methionine or by inefficient clearance by folate‐ and B‐vitamin‐dependent pathways. Understanding the relative contributions of these processes to pathogenesis is important for therapeutic strategies designed to lower homocysteine. To assess these alternatives, we elevated plasma homocysteine by feeding mutant amyloid precursor protein (APP)‐expressing mice diets with either high methionine (HM) or deficient in B‐vitamins and folate (B Def). Mutant APP mice fed HM demonstrated increased brain beta amyloid. Interestingly, this increase was not observed in mutant APP mice fed B Def diet, nor was it observed in C57Bl6 or YAC‐APP mice fed HM. Furthermore, HM, but not B Def, produced a prolonged increase in brain homocysteine only in mutant APP mice but not wild‐type mice. These changes were time‐dependent over 10 weeks. Further, by 10 weeks HM increased brain cholesterol and phosphorylated tau in mutant APP mice. Transcriptional profiling experiments revealed robust differences in RNA expression between C57Bl6 and mutant APP mice. The HM diet in C57Bl6 mice transiently induced a transcriptional profile similar to mutant APP cortex, peaking at 2 weeks, following a time course comparable to brain homocysteine changes. Together, these data suggest a link between APP and methionine metabolism.
Novel 3-cyanoisoquinoline Kv1.5 antagonists have been prepared and evaluated in in vitro and in vivo assays for inhibition of the Kv1.5 potassium channel and its associated cardiac potassium current, IKur. Structural modifications of isoquinolinone lead 1 afforded compounds with excellent potency, selectivity, and oral bioavailability.
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