Intracellular drug distribution-based targeting: Exploiting lysosomes to enhance the selectivity of drugs towards cancer cells

Under the most ideal circumstances, anticancer agents should be minimally toxic to normal cells and maximally noxious to cancer cells. Unfortunately, an optimal degree of selectivity is not typically achieved and chemotherapy is often prematurely stopped due to potentially life threatening side effects. For this reason, various approaches have been explored in an attempt to enhance the selectivity of anticancer drugs. For the most part, these techniques are based on Paul Ehrlich's concept of a "magic bullet" which is the attempt to target drugs to a disease site while avoiding healthy tissues. These approaches therefore share a common requirement that the active drug achieves greater concentration in or around tumor cells relative to normal cells. However, many of these approaches have achieved limited success due to the difficulty of achieving site-specific accumulation of conventional anticancer agents. A rarely considered option in enhancing drug selectivity lies in optimizing the intracellular distribution of drugs to achieve favorable distribution in cancer cells (i.e. in compartments that allow drug-target interactions), and unfavorable distribution in normal cells (i.e. in compartments that diminish drug-target interactions), essentially an intracellular drug distribution-based (IDB) targeting approach. The IDB targeting approach presents a paradigm shift from the classical approaches to enhance selectivity, since the active drug is not expected to achieve higher concentrations in cancer cells relative to normal cells. Instead the drug accumulates to the same extent in both normal and cancer cells, but the aforementioned differences in intracellular drug distribution result in selectivity. In the work presented here, we investigated whether the defective lysosomal acidification associated with some cancer cells can be exploited to enhance selectivity of weakly basic anticancer agents. Normal cells typically have very acidic lysosomes, which provide a driving force for the accumulation of weakly basic drugs (with appropriate physicochemical properties) into lysosomes. Some cancer cells have been shown to have defective acidification of lysosomes, leading to a reduction in the extent of lysosomal trapping of such weakly basic drugs. Our hypothesis is that the reduced sequestration of weakly basic drugs in lysosomes of cancer cells would increase cytosolic drug concentration, thus enhancing drug-target interactions, compared to the case in normal cells, where extensive sequestration would diminish drug-target interactions. We proposed that these differences in drug localization patterns between normal and cancer cells, and the resultant difference in drug activity, would enhance selectivity of lysosomotropic anticancer drugs to cancer cells. In order to establish the potential for broad therapeutic application of this approach, we assessed the prevalence of defective lysosomal acidification in cancer cells, and whether lysosomal targeting of anticancer drugs could reduce their systemic toxicity. We also evaluated whether IDB selectivity can be optimized according to relevant physicochemical parameters of drug candidates, specifically the ionization constant (pKa). These evaluations provide a rationale for the design or modification of anticancer drugs with physicochemical properties that maximize lysosomal trapping in order to enhance selectivity. Collectively, our results demonstrate that drugs with optimal lysosomotropic properties are more selective to cells with defective lysosomal acidification. Therefore, intracellular drug-distribution based (IDB) targeting provides a viable approach to enhance anticancer drug selectivity. As mentioned previously, the major limitation to enhancing selectivity through site-directed targeting of conventional anticancer drugs to tumors is the difficulty of achieving site-specific localization. Enhancing selectivity through IDB targeting represents a rational approach that will not be subject to the limitations faced by site-directed targeting approaches since there is no requirement that drugs achieve tumor-specific localization

Lysosomotropic Properties of Weakly Basic Anticancer Agents Promote Cancer Cell Selectivity In Vitro

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author’s publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Drug distribution in cells is a fundamentally important, yet often overlooked, variable in drug efficacy. Many weakly basic anticancer agents accumulate extensively in the acidic lysosomes of normal cells through ion trapping. Lysosomal trapping reduces the activity of anticancer drugs, since anticancer drug targets are often localized in the cell cytosol or nucleus. Some cancer cells have defective acidification of lysosomes, which causes a redistribution of trapped drugs from the lysosomes to the cytosol. We have previously established that such differences in drug localization between normal and cancer cells can contribute to the apparent selectivity of weakly basic drugs to cancer cells in vitro. In this work, we tested whether this intracellular distribution-based drug selectivity could be optimized based on the acid dissociation constant (pKa) of the drug, which is one of the determinants of lysosomal sequestration capacity. We synthesized seven weakly basic structural analogs of the Hsp90 inhibitor geldanamycin (GDA) with pKa values ranging from 5 to 12. The selectivity of each analog was expressed by taking ratios of anti-proliferative IC50 values of the inhibitors in normal fibroblasts to the IC50 values in human leukemic HL-60 cells. Similar selectivity assessments were performed in a pair of cancer cell lines that differed in lysosomal pH as a result of siRNA-mediated alteration of vacuolar proton ATPase subunit expression. Optimal selectivity was observed for analogs with pKa values near 8. Similar trends were observed with commercial anticancer agents with varying weakly basic pKa values. These evaluations advance our understanding of how weakly basic properties can be optimized to achieve maximum anticancer drug selectivity towards cancer cells with defective lysosomal acidification in vitro. Additional in vivo studies are needed to examine the utility of this approach for enhancing selectivity

Intracellular Distribution-based Anticancer Drug Targeting: Exploiting a Lysosomal Acidification Defect Associated with Cancer Cells

This is the published version, also available here: http://mcpharmacol.com/index.php/Journals/article/view/103.The therapeutic usefulness of anticancer agents relies on their ability to exert maximal toxicity to cancer cells and minimal toxicity to normal cells. The difference between these two parameters defines the therapeutic index of the agent. Towards this end, much research has focused on the design of anticancer agents that have optimized potency against a variety of cancer cell types; however, much less effort is spent on the design of drugs that are minimally toxic to normal cells. We have previously described a concept for a novel drug delivery platform that relies on the propensity of drugs with optimal physicochemical properties to distribute differently in normal versus cancer cells due to differences in intracellular pH gradients. Specifically, we demonstrated in vitro that certain weakly basic anticancer agents had the propensity to distribute to intracellular locations in normal cells that prevent interaction with the drug target, and to intracellular locations in cancer cells that promote drug-target interactions. We refer to this concept broadly as intracellular distribution-based drug targeting. Here we will discuss current in vivo work from our laboratory that examined the role of lysosome pH on the intracellular distribution and toxicity of inhibitors of the Hsp90 molecular chaperone in mice

The Role of Lysosomes in Limiting Drug Toxicity in MiceS⃞

The distribution behavior of a drug within a cell is an important, yet often overlooked, variable in both activity and differential selectivity. In normal cells, drugs with weakly basic properties are known to be extensively compartmentalized in acidic organelles such as lysosomes via ion trapping. Several cancer cell lines have been shown to have defective acidification of endocytic organelles and therefore have a diminished capacity to sequester such lysosomotropic agents. In this study, we tested the hypothesis that the low lysosomal pH of normal cells plays an important role in protecting normal tissues from the toxic effects of lysosomotropic anticancer drugs. The influence of lysosomal pH status on the toxicity of inhibitors of the molecular chaperone Hsp90 that did or did not possess lysosomotropic properties was evaluated in mice. Toxicity of Hsp90 inhibitors was evaluated in normal mice and in mice treated with chloroquine to elevate lysosomal pH by assessing morbidity and utilizing biochemical assays to diagnose hepatic and renal toxicity. Toxicity of the lysosomotropic inhibitor 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) was significantly enhanced in mice with elevated lysosomal pH relative to mice with normal lysosomal pH. In contrast, elevation of lysosomal pH had no significant impact on toxicity of the nonlysosomotropic inhibitor geldanamycin. These results support the notion that the low lysosomal pH of normal cells plays an important role in protecting these cells from the toxic effects of anticancer agents with lysosomotropic properties and has implications for the design/selection of anticancer drugs with improved safety and differential selectivity

Overall selectivity comparisons for geldanamycin and its derivatives with varying pKa.

<p>Overall selectivity represents the IC₅₀ for a derivative in normal human fibroblasts divided by the value obtained in HL60 human leukemic cells. Bars represent mean±s.d. (n = 3); *, <i>p</i><0.05 compared to GDA.</p

Theoretical plot of lysosomal sequestration as a function of weak base pKa and alpha (α).

<p>The equation used for these simulations can be found in the text. The pH of lysosomes and extracellular space were set at 4.4 and 7.4, respectively. The alpha parameter was varied as indicated.</p

Structures and properties of geldanamycin derivatives.

<p>The seven derivatives with different weak base modifications at the 17-position of GDA are shown. The pKa values for derivatives 1, 2, 3, 5 and 6 was measured experimentally using proton NMR (n = 1). The pKa for compound 4 was measured experimentally in an earlier manuscript (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049366#s2" target="_blank">Results</a>). The pKa for compound 7 was estimated using software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049366#s4" target="_blank">Materials and Methods</a>). The binding affinity for each of the inhibitors with rHsp90 is shown (n = 2), and was based on a previously established fluorescence polarization assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049366#s4" target="_blank">Materials and Methods</a>).</p

Intracellular distribution-based drug selectivity comparisons for geldanamycin and its derivatives with varying pKa.

<p>IDB selectivity is defined as the IC₅₀ in MD-MB-231 cells treated with scrambled shRNA, which have low lysosomal pH, divided by the value obtained from the same cell line treated with shRNA against the V1E1 subunit of the vacuolar proton ATPase, which have elevated lysosomal pH. Bars represent mean±s.d. (n = 3); *, <i>p</i><0.05 compared to GDA.</p

Characterization of MDA-MB-231 cells with reduced V-H⁺-ATPase subunit V1E1 expression and increased lysosomal pH.

<p>A. Western blot analysis of the V1E1 subunit expression is shown along with the actin loading control. Experimentally determined lysosomal pH values in untreated, scrambled shRNA- and V1E1 shRNA-treated cells is shown (values represent the mean±s.d., n = 3) B. V-ATPase subunit knockdown does not alter the growth rate of MDA-MB 231 cells. The filled circles represent cells treated with the scrambled shRNA vector and the open circles represent cells treated with the V-ATPase V1E1 shRNA. Cells were plated at a density of 3×10⁵ cells/well on a 6-well plate and were trypsinized, counted and replated every 24 hours for 3 days (data points represent the mean±s.d, n = 3).</p

Only lysosomotropic anticancer agents possess significant intracellular distribution-based drug selectivity.

<p>Mitoxantrone and daunorubicin are weakly basic with pKa values near 8 and have significant IDB selectivity. Non-lysosomotropic anticancer agents 5-fluorouracil and chlorambucil have IDB selectivity values near 1, which demonstrates that their activity is not influenced by lysosomal pH. Bars represent mean±s.d. (n = 3).</p