Fast and efficient synthesis of Zorro-LNA type 3'-5'-5'-3' oligonucleotide conjugates via parallel in situ stepwise conjugation.

Zorro-LNA is a new class of therapeutic anti-gene oligonucleotides (ONs) capable of invading supercoiled DNA. The synthesis of single stranded Zorro-LNA is typically complex and laborious, requiring reverse phosphoramidites and a chemical linker connecting the two separate ON arms. Here, a simplified synthesis strategy based on 'click chemistry' is presented with a high potential for screening Zorro-LNA ONs directed against new anti-gene targets. Four different Zorro type 3'-5' 5'-3' constructs were synthesized via parallel in situ Cu(i) [3 + 2] catalysed cycloaddition. They were prepared from commercially obtained ONs functionalized on solid support (one ON with the azide and the other ON with the activated triple bond linker N-propynoylamino)-p-toluic acid (PATA)) and after cleavage from resin, they were conjugated in solution. Our report shows the benefit of combining different approaches when developing anti-gene ONs, (1) the ability for rapid and robust screening of potential targets and (2) refining the hits with more anti-gene optimized constructs. We present as well the first report showing double-strand invasion (DSI) efficiency of two combined Zorro-LNAs.

Evaluation of the human prediction of clearance from hepatocyte and microsome intrinsic clearance for 52 drug compounds.

We compare three different approaches to scale clearance (CL) from human hepatocyte and microsome CL(int) (intrinsic CL) for 52 drug compounds. By using the well-stirred model with protein binding included only 11% and 30% of the compounds were predicted within 2-fold and the average absolute fold errors (AAFE) for the predictions were 5.9 and 4.1 for hepatocytes and microsomes, respectively. When predictions were performed without protein binding, 59% of the compounds were predicted within 2-fold using either hepatocytes or microsomes and the AAFE was 2.2 and 2.3, respectively. For hepatocytes and microsomes there were significant correlations (P = 8.7 x 10(-13), R(2) = 0.72; P = 2.8 x 10(-9), R(2) = 0.61) between predicted CL(int in vivo) (obtained from in vitro CL(int)) and measured CL(int in vivo) (obtained using the well-stirred model). When CL was calculated from the regression, 76% and 70% of the compounds were predicted within 2-fold and the AAFE was 1.6 and 1.8 for hepatocytes and microsomes, respectively. We demonstrate that microsomes and hepatocytes are in many cases comparable when scaling of CL is performed from regression. By using the hepatocyte regression, CL for 82% of the compounds in an independent test set (n = 11) were predicted within 2-fold (AAFE 1.4). We suggest that a regression line that adjusts for systematic under-predictions should be the first-hand choice for scaling of CL.