Single-Shot Solid-Phase Synthesis of Full-Length H2 Relaxin Disulfide Surrogates.

Chemical synthesis of insulin superfamily proteins (ISPs) has recently been widely studied to develop next-generation drugs. Separate synthesis of multiple peptide fragments and tedious chain-to-chain folding are usually encountered in these studies, limiting accessibility to ISP derivatives. Here we report the finding that insulin superfamily proteins (e.g. H2 relaxin, insulin itself, and H3 relaxin) incorporating a pre-made diaminodiacid bridge at A-B chain terminal disulfide can be easily and rapidly synthesized by a single-shot automated solid-phase synthesis and expedient one-step folding. Our new H2 relaxin analogues exhibit almost identical structures and activities when compared to their natural counterparts. This new synthetic strategy will expediate production of new ISP analogues for pharmaceutical studies.

Chemical synthesis of disulfide surrogate peptides by using beta-carbon dimethyl modified diaminodiacids.

The replacement of disulfide bridges with metabolically stable isosteres is a promising strategy to improve the stability of disulfide-rich polypeptides towards reducing agents and isomerases. A diaminodiacid-based strategy is one of the most effective methods to construct disulfide bond mimics, but modified diaminodiacids have not been developed till now. Inspired by the fact that alkylation of disulfide bonds can regulate the activity of polypeptides, herein, we report the first example of thioether bridged diaminodiacids incorporating Cys Cß dimethyl modification, obtained by penicillamine (Pen)-based thiol alkylation. The utility of these new diaminodiacids was demonstrated by the synthesis of disulfide surrogates of oxytocin containing a short-span disulfide bond and of KIIIA with large-span disulfide bonds. This new type of synthetic bridge further extends the diaminodiacid toolbox to facilitate the study of the structure-activity relationship of disulfide-rich peptides.

Synthesis of A11Cys-B11Cys Disulfide Surrogates of H2 Relaxin through an Intermolecular Native Chemical Ligation-Assisted Diaminodiacid Strategy.

We report an intermolecular native chemical ligation-assisted diaminodiacid strategy for the flexible construction of A11Cys-B11Cys disulfide surrogates of H2 relaxin. The practicality of this strategy was evidenced by the synthesis of four new H2 relaxin analogs, among which H2-2a-B28Ile is found to exhibit improved potency, selectivity, and stability compared with native H2 relaxin.

New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process.

Predicting clinically significant drug interactions during drug development is a challenge for the pharmaceutical industry and regulatory agencies. Since the publication of the US Food and Drug Administration's (FDA's) first in vitro and in vivo drug interaction guidance documents in 1997 and 1999, researchers and clinicians have gained a better understanding of drug interactions. This knowledge has enabled the FDA and the industry to progress and begin to overcome these challenges. The FDA has continued its efforts to evaluate methodologies to study drug interactions and communicate recommendations regarding the conduct of drug interaction studies, particularly for CYP-based and transporter-based drug interactions, to the pharmaceutical industry. A drug interaction Web site was established to document the FDA's current understanding of drug interactions (http://www.fda.gov/cder/drug/drugInteractions/default.htm). This report provides an overview of the evolution of the drug interaction guidances, includes a synopsis of the steps taken by the FDA to revise the original drug interaction guidance documents, and summarizes and highlights updated sections in the current guidance document, Drug Interaction Studies-Study Design, Data Analysis, and Implications for Dosing and Labeling.

Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions.

Pharmaceutical industry investigators routinely evaluate the potential for a new drug to modify cytochrome p450 (p450) activities by determining the effect of the drug on in vitro probe reactions that represent activity of specific p450 enzymes. The in vitro findings obtained with one probe substrate are usually extrapolated to the compound's potential to affect all substrates of the same enzyme. Due to this practice, it is important to use the right probe substrate and to conduct the experiment under optimal conditions. Surveys conducted by reviewers in CDER indicated that the most common in vitro probe reactions used by industry investigators include the following: phenacetin O-deethylation for CYP1A2, coumarin 7-hydroxylation for CYP2A6, 7-ethoxy-4-trifluoromethyl coumarin O-dealkylation for CYP2B6, tolbutamide 4'-hydroxylation for CYP2C9, S-mephenytoin 4-hydroxylation for CYP2C19, bufuralol 1'-hydroxylation for CYP2D6, chlorzoxazone 6-hydroxylation for CYP2E1, and testosterone 6 beta-hydroxylation for CYP3A4. We reviewed the validation information in the literature on these reactions and other frequently used reactions, including caffeine N3-demethylation for CYP1A2, S-mephenytoin N-demethylation for CYP2B6, S-warfarin 7'-hydroxylation for CYP2C9, dextromethorphan O-demethylation for CYP2D6, and midazolam 1'-hydroxylation for CYP3A4. The available information indicates that we need to continue the search for better probe substrates for some enzymes. For CYP3A4-based drug interactions it may be necessary to evaluate two or more probe substrates. In many cases, the probe reaction represents a particular enzyme activity only under specific experimental conditions. Investigators must consider appropriateness of probe substrates and experimental conditions when conducting in vitro drug interaction studies and when extrapolating the results to in vivo situations.