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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: 

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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. 

[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- 

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. 

[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 ). 

[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- 

