US 2019/0262453 Al  27  Aug. 29, 2019  TABLE 6-continued  Model Parameters  Baseline Input Range of Input  Symbol  Description  Value  Value  Units  CLC_H  CLC7 H+  Fixed  stoichiometry  R  Gas constant  8.314  Fixed  J • K- 1  • mo1- 1  T  Absolute  0  Fixed  Kelvin  temperature  F  Faraday’s  96485  Fixed  J/volt  constant  Nav  Avogadro’s  6.02  X  10 23  Fixed  molecules/mo!  number  [0266]  More specifically, membrane proton permeability  was varied from physiological baseline input value of  6xl0- 5  emfs to 6 emfs in geometric intervals of 1.78, the  number ofV-ATPase molecules per lysosome was manually  varied from O to physiological value of 300 in arbitrarily  chosen intervals, the number of CLC7 molecules per lyso› some was varied using parametric feature of BM from O to  physiological value of 5000 in geometric intervals of 2.09,  and the cytoplasmic chloride concentration was manually  varied from Oto physiological value of 10 mM in arbitrarily  chosen intervals. For cytoplasmic chloride concentration,  and V-ATPase and CLC7 numbers the individual ranges of  input values were chosen to represent the corresponding  [0261]  Next, to mimic disc-shaped lysosomes as have  been reported in different cell types under other biologically  relevant conditions (Agarwal R, et al., Proc Natl Acad Sci U  SA. 2013;110( 43):17247-52; Doshi N, et al., Proc NatlAcad  Sci US A. 2009;106(51):21495-9; Mitragotri S, et al., Nat  Mater. 2009;8(1):15-23), lysosomal radius in the range 422  nm to 10 um and height in the range 0.5 nm to 294.9 nm  were modeled using a rage of lysosomal surface area 1.90x  10- 3  cm 2  to 6.28xl0- 6  cm 2 . The dimensional relationship  between the disc-shaped lysosomal radius and height, at  constant lysosomal volume of 1.65xl0-$^{16}$L (equivalent to  the volume of a spherical lysosome of 0.34 um in radius, as  previously mentioned in the study of spherical lysosomes),  [0265]  Parametric simulations were run to study the  effects of the following four drug-induced stresses on lyso› somal physiology: V-ATPase inhibition, CLC7 inhibition,  lysosomal membrane permeabilization, and decreased mem› brane potential dissipation based on lower cytoplasmic  chloride concentration (FIG.  22).  [0264]  Simulating Drug Induced Lysosomal Stress  [0260]  For comparison, tubular lysosomes (represented in  FIG.  22B)  possessing different radii in the range 40 nm to  270 nm and heights in the range 585 nm to 5.73 um were  modeled using a range of volumes 2.88xl0-$^{17 }$L to 1.34x  10-$^{16 }$L. The dimensional relationship between the tubular  radius and height, at constant lysosomal surface area of  1.45xl o-s cm 2  (  equivalent to the surface area of a spherical  lysosome of 0.34 um in radius), was calculated using  cylindrical equation (V=itrh, where r is radius and h is  height). These morphologies are consistent with measure› ments by other publications (Swanson  J, et al., J Cell Biol.  1987;104(5):1217-22; Knapp PE, et al.,  J  Cell Sci. 1990;95  (Pt 3):433-9).  [0259]  To study how drug-induced changes in lysosomal  morphology affect ion homeostasis, simulations were per› formed in lysosomes of different surface areas and volumes.  First, lysosomes were modeled as perfect spheres. Assuming  there are approximately around 100 lysosomes in a cell,  which occupy 1  % of cellular volume, the volume of a single  lysosome was set to 1.65xl0-$^{16 }$L. For a spherical vesicle,  this volume corresponds to a lysosomal radius of 0.34 um  and a surface area of 1.45x10+cm 2 .  [0258]  Simulating Drug Induced Changes in Lysosomal  Morphology  [0263]  To understand how lysosomes possessing different  structural and functional characteristics may respond differ› ently to drugs, the number of V-ATPase and membrane  proton permeability were individually varied. These two  particular lysosomal parameters are referred to "stress tol› erance inducers". Thus, the number ofV-ATPase molecules  per lysosome was increased from physiological baseline  input value of 300 to 1.3xl 0 $^{5 }$in arbitrarily chosen intervals,  while proton permeability was decreased from physiological  baseline input value of 6x10- 5  emfs to 1.38xl0- 7  emfs in  arbitrarily chosen intervals. These ranges of input values for  both V-ATPase number and proton permeability allowed us  to quantitatively compare and contrast the relationship of  lysosomal surface area to the number of V-ATPase mol› ecules and membrane proton permeability per lysosome. For  example, different fold increments in lysosomal surface area  (say, 20- or 400-fold) were chosen and in one simulation at  a time set the corresponding lysosomal surface area as an  input. So, in the case of a lysosome with a 20-fold lysosomal  surface area expansion, its surface area was set to 2.9x10- 7  cm 2 .  Then, to study the effect of the number of V-ATPase  molecules on this expanded lysosome, parametric simula› tion was performed for the V-ATPase number ranging O to  6000, which corresponds to O to 20-fold increment m  V-ATPase number per lysosome.  [0262]  Simulating  Stress  Tolerance  Following Drug  Induced Changes in Lysosomal Morphology  was calculated using cylindrical equation (S=2itr2+2mh,  where r is radius and h is height).  [0257]  * estimated intrinsic surface potentials for inner  (Win) and outer (Wout) leaflets of the lysosomal membrane  accounted for when modeling membrane transporter medi› ated dynamic lysosomal and cytoplasmic ion concentrations  at the surface (Grabe et al., 2001, supra).  [0256]  Baseline input values are literature values (Van  Dyke R  W.  Am  J  Physiol. 1993;265(4 Pt 1):C901-17;  Gambale et al, 1994, supra; Heuser et al, 1993, supra;  Sonawane et al, 2003, supra; Alberts et al., 2008, supra)  representing physiological lysosomes and are in agreement  with previously published model (Ishida et al., 2013, supra;  Grabe et al., 2001, supra). 