Kinase experiment:

Josamycin is prepared in polymix buffer, containing 5 mM magnesium acetate, 5 mM ammonium chloride, 95 mM potassium chloride, 0.5 mM calcium chloride, 8 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate, and 1 mM dithioerythritol. Josamycin at different concentrations (2, 3, 4, and 6 μM is added to preinitiated ribosomes to start the incubation. One volume of elongation mix is added to 1 volume of reaction mix at each incubation time, and after 10 s the reaction is quenched with formic acid. The association rates are estimated from the fraction of tri-peptide-forming ribosomes[1].

Animal experiment:

Rat: Tritium-labelled Josamycin (200 mg/kg) is orally administrated to rats. The blood and tissue levels are determined at 1 h and 3 h by bioassay[2].Mouse: Tritium-labelled Josamycin (200 mg/kg) is orally administrated to mice. The blood and tissue levels are determined at 1 h and 3 h by bioassay[2].

Josamycin (EN-141) is a macrolide antibiotic exhibiting antimicrobial activity against a wide spectrum of pathogens, such as bacteria. The dissociation constant Kd from ribosome for Josamycin is 5.5 nM.

Studies show that the average lifetime on the ribosome is 3 h for Josamycin and that the dissociation constants for Josamycin binding to the ribosome is 5.5 nM. Josamycin slows down formation of the first peptide bond of a nascent peptide in an amino acid-dependent way and completely inhibits formation of the second or third peptide bond, depending on peptide sequence at a saturating drug concentration, synthesis of fulllength proteins is completely shut down by Josamycin. At a saturating drug concentration, synthesis of fulllength proteins is completely shut down by Josamycin[1].

Blood and tissue levels of Josamycin after oral administration are 200 mg/kg to rabbits. Tissue levels are generally much higher than the blood levels, and 3 h after the administration, when the blood level is very low, the tissue levels are rather higher than those 1 h after the dose. One hour after the medication, the level in the lungs is the highest of all the tissue levels[2].

[1]. Lovmar M, et al. Kinetics of macrolide action: the Josamycin and erythromycin cases. J Biol Chem. 2004 Dec 17;279(51):53506-15. [2]. Osono T, et al. Pharmacokinetics of macrolides, lincosamides and streptogramins. J Antimicrob Chemother. 1985 Jul;16 Suppl A:151-66.