If well B11 turned from yellow to BAY 73-4506 purple, Tetrazolium-Tween 80 mixture was added to all wells and incubated for another 24 h. If well B11 remained yellow, incubation was continued and the
tetrazolium-tween 80 mixture added to wells C11, D11, E11, F11, and G11 on day 7, 9, 11, 13, and 15 respectively. The MIC was defined as the lowest drug concentration that prevented a colour change of Tetrazolium dye from yellow to purple. Fractional Inhibitory Concentration (FIC) index was calculated to evaluate the drug interactions using the following formula11: FICIndex:MICofdrugincombination/MICofdrugalone The sum of the FIC Index (∑FIC) was calculated as follows11: ∑FIC:MICA(incombination)/MICA(alone)+MICB(incombination)/MICB(alone). The interaction Selleckchem Vemurafenib was expressed as synergistic if the value of ∑FIC ≤ 0.5; additive/indifferent if 0.5 < ∑FIC ≤ 4.0; and antagonistic if ∑FIC > 4.0. The augmentation of the hydrophilic isoniazid (INH) into a lipophilic compound was achieved by increasing the molecular weight (g/mol) through the addition of hydrophobic hydrocarbon chain at the amine group of INH. The increase in the molecular
mass will increase the lipophilicity/hydrophobicity of the compound. In order to further confirm this, the numerical measurement of hydrophobicity, Log Poct/wat was calculated using the software developed by Molinspiration Chemoinformatics.12 The Log Poct/wat value of 1-isonicotinoyl-2-hexadecanoyl hydrazine (INH-C16), 1-isonicotinoyl-2-heptadecanoyl hydrazine (INH-C17) and 1-isonicotinoyl-2-octadecanoyl hydrazine (INH-C18) is 6.423, 6.928 and 7.433 respectively compared to the INH value of −0.969. It should be highlighted that Log Poct/wat of INH has a negative value due to its hydrophilic characteristic. Whereas, Log Poct/wat of INH-C16, INH-C17 and INH-C18 have positive values due to the presence of hydrophobic moiety which made them more hydrophobic. The individual MICs of INH-C16, INH-C17, INH-C18, INH, streptomycin (STR), rifampicin (RIF), and ethambutol (EMB) are tabulated
in Table 1. The results showed that INH-C16, INH-C17 and INH-C18 lowered the MIC value of their the parent compound INH against M. tuberculosis H37Rv, thus surpassing the activity of INH by 2-fold. Among the clinical isolates tested, INH-C16 showed lower MIC than INH only in an isolate and INH-C17 and INH-C18 in 2 out of 7 isolates. Hence, it is very apparent that there could be other factors other than hydrophobicity properties which influence the uptake and distribution of an anti-TB drug in M. tuberculosis. Such factors could be the structural properties of the compounds and the complex microenvironment within the cell as well as cell wall permeability differences between the strains.