## 4 Prediction of Human Pharmacokine-Pharmacodynamic Relationship
Because of their transparency and easy genetic modification, fluorescence microscopy of pathogen and immune cells can be leveraged to follow infection and treatment [104]. With the small size and high fecundity of the zebrafish, high-throughput assays are available to test large numbers of compounds in short amounts of time with enough statistical power [105]. Methods to quantify internal drug exposure have also been established [106; 107; 108]. Recently, an exposure-response relationship has been developed for isoniazid in the zebrafish, which translated well to humans [109]. In general, non-invasive imaging of lesion pathology by computed tomography (CT) and positron emission tomography (PET) has the potential to improve the comparison between preclinical and clinical measurements of disease progression and treatment [102]. [PERSON] et al have demonstrated this by using dynamic [11C]rifampicin PET-CT imaging in patients newly diagnosed with pulmonary TB and rabbits infected with cavitary TB to noninvasively measure intralesional drug concentration-time profiles and, consequently, time to bacterial extinction [110]. They also employed integrated modeling of the PET-captured concentration-time profiles in hollow-fiber bacterial kill curve experiments to predict the rifampicin dose required to achieve a cure in 4 months, which has a huge potential in antimicrobial drug development to shorten TB treatments [110]. It is clear that no single animal model represents a heterogeneous disease such as TB. A mechanistic understanding of TB in humans will identify which elements are characterized best by which animal model [103]. Independently of which preclinical experimental method is utilized, the sampling design of both PK (e.g., drug and/or metabolite concentration) and PD (e.g., infection, bacterial burden) biomarkers is of the utmost importance. The careful selection of datapoints over the duration of the experiment and at different drug concentrations is essential for a reliable quantification of the exposure-response relationship. Regulatory agencies suggest determining PK/PD indices based on preclinical data for antibiotics, e.g., the area under the concentration curve over MIC (AUC/MIC), the maximum concentration (Cmax) over MIC (Cmax/MIC), and the percent of a 24-hour time period that the drug concentration is above MIC (T > MIC), for the establishment of the PK-PD profile of antimicrobials and for deciding the most optimal dosing regimens. PK/PD indices are based on preclinical studies that describe the PK-PD relationships of antimicrobials [111]. However, PK/PD indices suffer from several clear limitations, some of which are inherent to their use of MIC, the limitations of which are discussed above. Using PK/PD indices ignores information about the time-course of individual PK and PD processes [112]. As summary endpoints, they lack the ability to track the changes in the bacterial load over time [113]. Furthermore, when using AUC/MIC as a PK/PD index, the rate of drug administration is ignored, while, when using Cmax/MIC, bacterial killing is assumed to depend solely on the maximum drug concentration, ignoring drug half-life and infusion duration [99]. Using T > MIC assumes that the maximal drug effect has been reached when MIC is reached, regardless of whether higher concentrations were given [99]. Additionally, the colony-forming units (CFU) versus PK/PD indices profile shows great variability in the CFU observations for the same PK/PD indices value [99]. These PK/PD indices are selected and predicted as PD targets using HFS-TB to quantify a more realistic _in vitro_ exposure-responserelationship that is translatable to _in vivo_[98; 99; 114]. However, despite EMA's qualification of the preclinical HFS-TB to be used to complement existing methodologies, it still suffers from a number of limitations. The EMA advises caution when interpreting HFS-TB results, as many instances of over- and under-estimates of the drug's anti-TB activity have been reported [115].