An efficient CRISPR interference-based prediction method for synergistic/additive effects of novel combinations of anti-tuberculosis drugs.

Tuberculosis (TB) is treated by chemotherapy with multiple anti-TB drugs for a long period, spanning 6 months even in a standard course. In perspective, to prevent the emergence of antimicrobial resistance, novel drugs that act synergistically or additively in combination with major anti-TB drugs and, if possible, shorten the duration of TB therapy are needed. However, their combinatorial effect cannot be predicted until the lead identification phase of the drug development. Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a powerful genetic tool that enables high-throughput screening of novel drug targets. The development of anti-TB drugs promises to be accelerated by CRISPRi. This study determined whether CRISPRi could be applicable for predictive screening of the combinatorial effect between major anti-TB drugs and an inhibitor of a novel target. In the checkerboard assay, isoniazid killed Mycobacterium smegmatis synergistically or additively in combinations with rifampicin or ethambutol, respectively. The susceptibility to rifampicin and ethambutol was increased by knockdown of inhA, which encodes a target molecule of isoniazid. Additionally, knockdown of rpoB, which encodes a target molecule of rifampicin, increased the susceptibility to isoniazid and ethambutol, which act synergistically with rifampicin in the checkerboard assay. Moreover, CRISPRi could successfully predict the synergistic action of cyclomarin A, a novel TB drug candidate, with isoniazid or rifampicin. These results demonstrate that CRISPRi is a useful tool not only for drug target exploration but also for screening the combinatorial effects of novel combinations of anti-TB drugs. This study provides a rationale for anti-TB drug development using CRISPRi.

Pharmacokinetic-pharmacodynamic modelling of the hypoglycaemic effect of pulsatile administration of human insulin in rats.

The relationship between the plasma insulin (INS) concentration-time course and plasma glucose concentration-time course during and after pulsatile INS administration to rats was characterized using a pharmacokinetic-pharmacodynamic (PK-PD) model. A total INS dose of 0.5 IU/kg was intravenously injected in 2 to 20 pulses over a 2-h period. Compared with the single bolus administration, the area under the effect-time curve (AUE) increased depending on the number of pulses, and the AUEs for more than four pulses plateaued at a significantly larger value, which was similar to that after the infusion of a total of 0.5 IU/kg of INS over 2 h. No increase in plasma INS concentration occurred after pulsatile administration. Two indirect response models primarily reflecting the receptor-binding process (IR model) or glucose transporter 4 (GLUT4) translocation (GT model) were applied to describe the PK-PD relationship after single intravenous bolus administration of INS. These models could not explain the observed data after pulsatile administration. However, the IR-GT model, which was a combination of the IR and GT models, successfully explained the effects of pulsatile administration and intravenous infusion. These results indicate that the receptor-binding process and GLUT4 translocation are responsible for the change in AUE after pulsatile administration.