## 2 Materials and Methods
### In Vitro Assessment of the DVB Metabolic Stability  
The evaluation of Cl\({}_{\text{int}}\) and in vitro t\({}_{1/2}\) of DVB included measuring the amount of DVB that remained after performing in vitro metabolic incubation. The incubation protocol was performed by applying the HLMs matrix, which was initiated with MgCl\({}_{2}\) and NADPH as a coenzyme for the metabolic reaction. The in vitro incubation research was conducted using a four-step methodology. During the initial stage, a 1 \(\upmu\)L sample of DVB was pre-incubated with HLMs. The above-mentioned methodology was performed with a water bath maintained at 37 \({}^{\circ}\)C through thermostatic management for 10 min. At the beginning of the experiment, each sample was given a solution containing 1 mM NADPH. Subsequently, all samples were kept in a shaking water bath at 37 \({}^{\circ}\)C. In the third step of the study, 100 \(\upmu\)L of EFB (2000 ng/mL) was added prior to ACN addition, which acted as a quenching agent. The target of this step was to attain a uniform IS level and minimize any possible influence of metabolic enzymatic reactions on the IS concentration. During the fourth step, known as the cessation phase, a 2 mL sample of ACN was added at certain time points (0, 2.5, 7.5, 15, 20, 25, 30, 45, and 60 min) to quench the metabolic pathway and remove any excess proteins through precipitation. The initial step of the extraction methodology for DVB and EFB is denoted as the primary phase, as outlined in Section 2.9. An HLMS negative control was prepared to investigate the influence of omitting NADPH during the incubation of DVB with HLMs, following the previously reported approach. The target of the performed experiment was to estimate the possible effects of incubation sets and HLMs' ME on the DVB concentration in realistic in vitro metabolic incubation experiments.  
The residual DVB level was estimated by using the equation of the regression line generated from the calibration curve of DVB. The DVB metabolic stability curve was built by graphing the specified time intervals (x-axis) ranging from 0 to 60 min against the % DVB that remained compared to the DVB at time zero (100%) (y-axis). Therefore, the portion of the metabolic curve between 0 and 25 min was chosen to produce a logarithmic curve. The time span of 0-25 min, as previously mentioned, was used to plot the LN of DVB levels against the relevant time intervals. To get the rate constant for DVB metabolic stability, the slope of the previous graph was analyzed. Afterwards, the slope was used to estimate the in vitro t\({}_{1/2}\) using the formula in vitro t\({}_{1/2}\) = ln2/slope. The estimation of the Cl\({}_{\text{int}}\) in mL/min/kg was performed by consulting prior research [45]. The computation in question entailed the usage of the matrix mass of HLMs (45 mg) per gram of liver tissue and the liver tissue mass (26 g) per kilogram of body weight, as given in Equation (8) [46].  
\[\text{Cl}_{\text{int}}=0.693\times\frac{1}{\text{t}_{\text{b}_{2}}(\text{min. })}\times\frac{\text{mL incubation}}{\text{mg protein}}\times\frac{\text{mg microsomal proteins}}{\text{g of liver weight}}\times\frac{\text{g liver}}{\text{Kg b.w.}} \tag{8}\]