US 2019/0262453 Al  18  Aug. 29, 2019  from that of CFZ-HCl contammg lysosome in order to  quantify the physiological difference between CFZ-HCl  containing and CFZ-HCl free lysosomes. The lysosomal pH  difference of the CFZ-HCl containing lysosome and CFZ› HCl free lysosome under baseline physiological V-ATPase  (300 V-ATPase molecules per lysosome) and CLC7 (5000  CLC7 molecules per lysosome ), symbolized by ApHb, was  used as the baseline pH difference associated with maximum  drug uptake. Moreover, as the lysosomal pH difference  (ApH) between CFZ-HCl containing and CFZ-HCl free  lysosomes increases, (llpH>llpHb ), it indicates deviation  from maximum drug uptake. Thus, using the inverse rela› tionship of llpH to% drug uptake,% drug uptake ofCFZ in  the presence of the defined V-ATPase and CLC7 inhibition  ranges associated with viable lysosome can be calculated as  follows:  867 2015). To confirm if CFZ-HCl is defined by this  reported bathochromicity in fluorescence of the biocrystals,  confocal fluorescence microspectroscopy was performed on  synthesized CFZ-HCl crystals. The CFZ-HCl crystals also  had peak fluorescence activity in a similar spectral range as  measured from biocrystals (peak excitation: 560-600 nm,  peak emission: 650-690 nm) (FIG.  4a).  When visualized  using conventional epifluorescence microscopy using the  appropriate filters, CFZ-HCl had high far-red fluorescence  activity and negligible green fluorescence (FIG.  4a).  [0173]  Lysosomal accumulation of CFZ in RAW264.7  cells. To demonstrate that CFZ bioaccumulation and even› tual biocrystallization is associated with lysosomes, CFZ  was incubated with RA W264.7 cells and the type and spatial  distribution of fluorescence in the cells was monitored. From  the results described above, it was determined that CFZ  fluoresces in the green and red fluorescent range while  CFZ-HCl would fluoresce in the red and far-red fluorescence  range. Green fluorescent, red fluorescent and far-red fluo› rescent punctuate spots, indicative of vesicular accumulation  of CFZ were visible in the cells relative to the control cells  (FIG.  4b).  Notably, there was minimal colocalization of the  far-red fluorescent spots with the green fluorescent spots  (Pearson’s Colocalization Coeflicient=0.30–0.29) while the  red fluorescent spots colocalized to a higher degree (p<0.  005) with both the green (Pearson’s Colocalization Coefli› cient=0.60–0.22) and far-red (Pearson’s Colocalization  Coeflicient=0.61–0.20) fluorescent spots. This is indicative  of the fact that vesicles accumulating CFZ and CFZ-HCl  were distinct from each other.  Results  [0171]  Estimating and measuring the weakly basic chemi› cal characteristics of CFZ (Ksp, pKa). CFZ is identified as  a weakly basic, lipophilic drug and has four amine groups.  While its biochemistry has been described previously,  experimental evidence of its chemical nature is sparse.  Using ChemAxonfi, it was estimated that two amines can be  protonated within the pH range of 0-9 with the pKa, 1 =2.3  and pKa,2=9.29 (FIG.  3A).  This indicated that within a cell,  the majority of the drug would be present and bioaccumulate  as monoprotonated species within the cell (FIG.  3B).  To  confirm this value using an experimental approach, a pH› dependent solubility study was performed using Gordon› Flynn (Kramer and Flynn, 1972, supra) approach, which  allowed one to accurately measure the total solubility of  CFZ-HCl as a function of pH. From the experimental  measurements, the solubility properties of CFZ including its  apparent pKa,2 (pKa=6.08–2.43x10-3; 95% Confidence  Interval=6.07, 6.09) and intrinsic free base solubility (S0=0.  48–4.05x10-6 µM; 95% CI=0.48, 0.48) were calculated  (FIG.  3C).  Moreover, the pH-dependent solubility experi› mental measurement revealed that CFZ precipitation as a  hydrochloride salt was highly sensitive to small, but physi› ologically-relevant variations in pH and chloride concentra› tions (pHmax=4.5–7.llxl0- 15 ,  95% CI=4.5, 4.5; and  Ksp=332.3–3.71 µM2, 95% CI=323.1, 341.5) (FIG.  3C).  Thus, at the pharmacologically-relevant CFZ concentrations  that have been measured in serum (1 to 20 µM), local  variations in pH (-7.2 extracellular; -7.4 cytosolic and -4.5  lysosomal) and chloride concentrations (-80 mM extracel› lular; -10-20 mM in cytosol; and -50-200 mM in lyso› somes) can lead to significant differences in CFZ precipita› tion and solid-state protonated chemistry in extracellular vs.  cytosolic vs. lysosomal microenvironments.  [017 4]  In silica analysis----Computationally defining a  lysosome as a localized region of chloride and proton  activity. In order to simulate a lysosomal compartment, a  well-established and published systems-based mathematical  model of lysosomal ion regulation, which assumes a spheri› cal lysosomal vesicle, with radius of 0.34 um, containing  300 V-ATPase and 5000 CLC7 molecules, along with mem› brane proton permeability of 6x10-5 emfs and cytoplasmic  chloride concentration of 10 mM, was used. These and other  lysosomal ion concentration values which are detailed in  Table 1 are in agreement with physiological lysosomal  conditions (Heuser et al.,  J Cell Biol  121:1311-1327 1993;  Van Dyke,  Am J Physiol  265:C901-917 1993; Gambale et  al.,  Eur Biophys J Biophy  22:399-403 1994; Sonawane et al.,  Journal of Biological Chemistry  277:5506-5513 2002;  Alberts,  Moelcular Biology of the Cell. Fifth edition.  Gar› land Science, New York 2008) consistent with lysosomal ion  homeostasis.  [0175]  In silica analysis: The role of lysosomal proton  transport dominates that of chloride transport on the bioac› cumulation of CFZ within lysosomes. To computationally  study the roles of these lysosomal membrane proteins on the  lysosomal bioaccumulation of CFZ, the number of active  V-ATPase and CLC7 molecules per lysosome (resulting in 0  to 100% inhibition of both parameters from baseline physi› ological input values of 300 and 5000, respectively) were  simultaneously varied in the presence and absence of CFZ› HCl accumulating in a dose-dependent manner (0 to 0.01  picomoles/cell/day ).  [0172]  Bathochromic shift in fluorescence ofCFZ. CFZ’s  inherent fluorescence is dependent upon whether it is in  free-base form in solution and as a solid crystal (green and  red fluorescent; peak excitation: 540-560 nm, peak emis› sion: 560-600 nm) or when present as biocrystals (red and  far red fluorescence; peak excitation: 560-600 nm, peak  emission: 650-690 nm) (Keswani et al.,  Cytometry A  87:855- [0176]  To accommodate lysosomal CFZ-HCl accumula› tion at the rate of0.01 picomoles/cell/day, there needed to be  at least 9 V-ATPase molecules per lysosome (which corre› sponds to 97% V-ATPase inhibition), even while maintain› ing all of the other lysosomal parameters at baseline physi- Ll.pHb  % Drug Uptake= --*100%  Ll.pH  32 