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cal pH gradient of up to 4.6 pH units, associated with the hydrolysis of 3 ATP molecules, was maintained. 

[0165] Generating 3D Plots. All of the readout values in the absence and presence of CFZ-HCI were obtained as a function of a lysosomal parameter that was varied using the parametric plot feature in BM while manually adjusting one simulation at a time an additional lysosomal parameter to a fixed value from a chosen range of values. Thus, multiple two-dimensional datasets, corresponding to the total number of values chosen for the additional lysosomal parameter, were saved as Notepad files and exported to Microsoft Excel. The 2D datasets were further categorized in an Excel spreadsheet depending on the additional lysosomal param› eter being varied. For example, when simultaneously inhib› iting V-ATPase and CLC7 in the presence or absence of lysosomal CFZ-HCI accumulation, 2D lysosomal readout values were obtained as a function of the total number of CLC7 molecules per lysosome for each adjusted input value of the total number of V-ATPase molecules per lysosome ranging from 0 to 300. Thus, each set of 2D lysosomal readout values as a function of the total number of CLC7 molecules per lysosome is categorized in the Excel spread› sheet under a row clearly identified with the associated value of the total number of V-ATPase molecules per lysosome. 

[0166] Furthermore, a matrix was generated for each indi› vidual lysosomal readout value. For example, in the case of simultaneous V-ATPase and CLC7 inhibition in the presence of CFZ-HCI accumulation at lx, the total number of vari› ables for the total number ofV-ATPase and CLC7 molecules per lysosome were 7 and 16, respectively. Thus, the matrix was 16 by 7, where the lists of the total number of CLC7 and V-ATPase molecules per lysosome were chosen as the row and colunm of the matrix, respectively, and the lysosomal readout values corresponding to the different combinations of the total number of CLC7 and V-ATPase molecules per lysosome were contained within the matrix itself. The matrix containing the calculated results was then exported to Sig› maP!otfi to generate a three-dimensional plot. In the 3D plot, the Z axis corresponds to the calculated result, while the X and Y axes correspond to the varied lysosomal parameters. 

[0167] Cell Viability. Viability of RAW264.7 cells were performed using Cell Proliferation Kit II (XTT) (Roche Applied Science, Mannheim, Germany, Cat. No. 11465015001) using the manufacturer’s instructions. RAW264.7 cells at a very high seeding density of 50,000 cells/well, grown in a 96 well tissue culture plate in 280 µI/well of Dulbecco’s Modified Eagles Medium (DMEM) +10% Fetal Bovine Serum (FBS) +1% Penicillin/Strepto› mycin (P/S) (growth media), were pre-incubated with vary› ing concentrations of BafAl (Sigma-Aldrich, St. Louis, Mo., Cat. No. Bl 793) (0-l0nM) and NPPB (Sigma-Aldrich, St. Louis, Mo., Cat. No. N4779) (0-200 µM) for 4 hours before adding CFZ dissolved in DMSO such that final concentrations of CFZ were in the range of 0-10 µM. Post 24 hours exposure to CFZ, the cells were incubated with the yellow XTT solution (final concentration 0.3 mg/ml) for 4 h. After this incubation period, orange formazan solution was formed, which was spectrophotometrically quantified using a Synergy 2 plate reader (BioTek, Winooski, Vt.) at 500 nm and 700 nm (for background). An increase in number of living cells directly correlates to the amount of orange formazan formed, as monitored by the solution absorbance at 500 nm (Abs500). The following formula was used to 

determine viability of drug and inhibitor treated cells using untreated cells (blank) as a control----Cell Viability= (Abs500, treated-Abs700,treated)/(Abs500, blank› Abs700,blank). 

[0168] Drug Uptake. Measurement of CFZ uptake in RAW264.7 cells was performed using a modified absor› bance spectroscopy method using 9 M H2SO4 (pH<<0.1) to digest the entire cell population and extract CFZ from the cells. Cells at a very high seeding density of 50,000 cells/ well, grown in a 96 well tissue culture plate in 280 µI/well of DMEM +10% FBS +1% P/S (growth media), were pre-incubated with varying concentrations of BafAl (0-4 nM) and NPPB (0-200 µM) for 4 hours before adding CFZ dissolved in DMSO such that final concentrations of CFZ were in the range of 0-8 µM. Post 4 and 24 hours exposure to CFZ, growth media was aspirated out and the cells were washed with PBS twice before adding 100 µI/well of 9 M H2SO4 and incubated at room temperature for 30 minutes before spectrophotometric quantification using a Synergy 2 plate reader (BioTek, Winooski, Vt.) at 540 nm (Abs540) and 750 nm (Abs750) (for background). Standards were prepared on the same plate by adding 100 µI/well pre› determined standards of CFZ. Total uptake was reported as total picomoles of intracellular drug as measured and cal› culated using the standard curve. 

[0169] Cell Viability Measurement: Comparing Wet Lab Experimental Study with Simulation Study. In order to compare wet lab experimental cell viability determination with that of model simulation, the rate at which drug accumulation occurred in the experimental study was cal› culated. This was done assuming the doubling time of 50,000 cells/well to be approximately 11 hours (Sakagami et al., Anticancer Res 29:343-347 2009), and calculating the amount of drug in individual cells in units of picomole per cell for each extracellular CFZ concentration in the absence of any inhibitor mentioned earlier. Then, the extracellular CFZ concentration that’s closely associated with the rate of drug accumulation at either 0.01 picomole/cell/day or 0.1 picomole/cell/day, which are the rates used in the modeling and simulation study as mentioned earlier, was chosen as the comparable experimental dose to the modeling and simula› tion dose. Then, cell viability was calculated using the following equation: 

<img src='content_image/926985.jpg'>

Where the baseline lysosomal pH, is as mentioned before, one of the lysosomal readout values, specifically used in this case as a direct indicator of cell viability, and is associated with baseline physiological lysosomal parameters including 300 V-ATPase and 5000 CLC7 molecules per lysosome. Whereas, the lysosomal pH corresponds to all of the readout lysosomal pH values in the presence as well as absence of V-ATPase and CLC7 inhibitors. 

[0170] Drug Uptake Measurement: Comparing Wet Lab Experimental Study with Simulation Study. First, simulated V-ATPase and CLC7 inhibition ranges associated with viable lysosomes which can accommodate CFZ-HCI accu› mulation at lx or !Ox were determined. For these ranges, the lysosomal pH of CFZ-HCI free lysosome was subtracted 

