US 2019/0262453 Al  17  Aug. 29, 2019  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  [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  [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:  [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.  [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.  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).  [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.  cal pH gradient of up to 4.6 pH units, associated with the  hydrolysis of 3 ATP molecules, was maintained.  (  Lysosomal pH- J  Baseline 1 ysosomal pH  % Cell Viability= 100% - B  r  l  l H  *  100%  ase 1ne ysosoma p  31 