Similar to previous observations in lysosomal lipid storage disease cells, prolonged exposure to lipidosis-inducing CADs caused a pronounced hyperaccumulation of amine-containing compounds that are substrates for lysosomal ion trapping.15, 16, 37
Imipramine was found to specifically increase the punctate vesicular staining and cellular accumulation of amine-containing compounds that are substrates for ion trapping-based accumulation in lysosomes but had no impact on the cellular accumulation of molecules that are not substrates (). These results are suggestive of an increase in the ability of cells to accumulate amine-containing drugs in lysosomal structures following imipramine exposure and argue against a non-specific increase in cellular amine accumulation by increasing membrane permeability or by increasing the non-specific cellular binding sites for any amine-containing compounds.
Initial studies were conducted using a human fibroblast cell line. Their outstretched cellular morphology is conducive to microscopic evaluation, thus allowing greater visualization of intracellular fluorescent probe distribution. In addition, it allowed for comparisons between the CAD-induced phenotype and that observed in fibroblasts obtained from NPC disease patients. The similarity between the distribution and accumulation of LTR in NPC disease and CAD-treated fibroblasts was notable as can be seen by the TOC Figure and Supplemental Figure 4
. A 48 h CAD-treatment was initially chosen based on previous studies showing a significant time-dependent induction of the lipidosis in human fibroblasts that is most pronounced at this time point.43
Subsequent studies sought to illustrate that our findings are not specific to one cell type, but instead are representative of cells regardless of their source. The squamous cell carcinoma cell line MDA-1986 was chosen for this purpose. The high proliferation rate of these cells allowed for rapid growth in culture which made them preferable for high throughput analysis. Therefore, multiple evaluations were undertaken using this cell line including the time-dependency of the CAD-induced increase in LTR accumulation. It was found that maximal response was achieved within 24 hours of drug exposure (). Therefore, subsequent experiments conducted in this cell line were carried out using the optimized 24 h drug treatment, which was also found to be adequate in the fibroblast cell line (Supplemental Figure 4
). A 24 h imipramine treatment was used in the determination of lysosomal ion trapping-based accumulation of LTR () and changes in lysosomal pH (Supplemental Figure 3
) that were used to calculate changes in apparent lysosomal volume.
In these studies, a variety of lysosomotropic amine compounds were used to probe for this drug interaction pathway, including: LTR, LTG, daunorubicin, propranolol and methylamine. The diversity of compounds used was to ultimately illustrate the broad implications of this observed phenomenon. Not only did this work illustrate the capacity of CAD treatment to affect the cellular accumulation of model lysosomotropic probes, like LTR and LTG, but it also showed that it had implications for therapeutic compounds, such as daunorubicin and propranolol.
Because imipramine served as a model CAD, we wanted to be sure that its effect was reflective of its cationic amphiphilic properties and not some other innate pharmacological activity. Therefore, we tested eight other known CADs from a variety of pharmacological classes for their ability to influence cellular accumulation of LTR and found that they all significantly enhance LTR accumulation (). The ability of CADs to increase the cellular accumulation of lysosomotropic amine drugs illustrates a novel pathway through which drug co-administration can lead to a distribution-based pharmacokinetic drug interaction. Although a comprehensive structure activity relationship was not evaluated, the series of tested CADs that were capable of causing a significant expansion of the lysosomal volume were characterized as cationic, with predicted pKa values between 7.8 and 9.7, as well as, lipophilic with a predicted logP between 2.5 and 7.8 ().
The observed increase in amine-containing drug accumulation following CAD treatment would at first glance appear to directly contradict earlier work that has illustrated the capacity of lysosomotropic amine drugs to inhibit the cellular uptake of each other by competing for uptake into lysosomes.23, 24, 44–49
This competition was presumed to occur through the ability of lysosomotropic amine drugs to buffer the lysosomal pH and inhibit the lysosomal uptake of secondarily applied lysosomotropic amine drugs. We believe these apparent discrepancies originate from differences in drug dosing and duration of exposure leading to differential physiological effects on cells and the lysosomal system. These earlier studies relied on short-term, high-dose drug treatments which are known to cause acute lysosomal alkalinization and therefore reduced ion trapping of amines in lysosomes.49
These experiments were also conducted in purified lysosomes where it is not clear if the V-ATPase was fully active under the experimental conditions. Our studies relied on longer durations of drug exposure in intact cells with relatively lower treatment doses which is more closely associated with the induction of a cellular lipidosis.50–52
In fact, we carried out additional experiments with treatments known to cause lysosomal alkalinization6, 53
by exposing cells to either 30 µM chloroquine, 10 mM ammonium chloride or 200 nM of the vacuolar-type H+
-ATPase inhibitor concanamycin A, concurrent with the 2 h LTR accumulation assay (Supplemental Figure 6
). Ammonium chloride is a weak base that requires millimolar drug concentrations to cause a significant alkalinization of lysosomes, whereas chloroquine is a dibasic amine that is capable of causing a significant buffering of the lysosome at concentrations in the micromolar range.4, 6, 54
All lysosomal alkalinizing treatments were found to drastically decrease the cellular accumulation of LTR under these conditions. Thus, our results don’t argue against previous observations of potential drug interactions occurring through drug-induced lysosomal alkalinization, but offers an additional pathway through which amine-containing drugs that accumulate in lysosomes can influence the pharmacokinetic properties of each other depending on their physiological effect on the lysosomal apparatus. Since the therapeutic concentrations of most CADs are well below the concentrations necessary to cause lysosomal alkalinization and since most of the typical CADs are used as chronic therapies we would argue that CAD-induced lipidosis is a more likely clinical scenario than lysosomal alkalinization.
By enhancing the cellular retention of this secondarily applied drug, one would expect changes in the drug’s pharmacokinetic profile, such as an increase in the drug’s volume of distribution and half-life. Following the initial characterization of this novel drug interaction pathway we set out to understand the mechanism through which CADs cause the increased cellular accumulation of compounds that are substrates for ion trapping in lysosomes. We found that imipramine’s ability to increase LTR accumulation was strongly dependent on imipramine exposure time (). Long exposure times were found to be needed for the maximum observed effect (>15 hrs). In addition, we found at low temperature (4°C) imipramine was unable to induce the cellular changes needed for the increased LTR accumulation observed at the physiological temperature of 37°C (). Our results are unable to rule out the possibility that a reduction in the cellular accumulation of imipramine at 4°C is responsible for the observed temperature-dependency, but we believe that together these results argue against the possibility that imipramine is in some way inhibiting an acute cellular efflux pathway for LTR and is consistent with imipramine inducing an energy-dependent cellular remodeling process that results in enhanced LTR accumulation.
The predominant mechanism for uptake of lysosomotropic amines, such as LTR, is thought to be through ion trapping,1
but realizing that work in our lab and others have shown that amines often accumulate in cells to a degree greater than that predicted by ion trapping alone suggests the potential for other mechanisms of intracellular and intralysosomal retention.31, 49, 55, 56
The observation that elimination of the lysosome-to-cytosol pH gradient reversed the ability of imipramine to influence LTR accumulation suggested that this remodeling process resulted in an increase in the lysosomal ion trapping capacity of cells (). A comparison of the amount of LTR accumulated in cells by ion trapping in lysosomes in control cells and cells treated with imipramine showed a significant increase in lysosomal ion trapping-based accumulation of LTR in imipramine treated cells ().
The assumption that lysosomal pH dependent accumulation of LTR is solely mediated by the ion trapping mechanism led us to speculate that imipramine can cause one of two effects, 1) increased lysosomal volume or 2) decreased lysosomal pH, either of which would increase the amount of LTR accumulating in the cell by ion trapping in lysosomes. To distinguish the two possibilities, measurements of lysosomal pH were undertaken and showed a slight but insignificant increase in lysosomal pH following imipramine treatment (Supplemental Figure 3
). Therefore we concluded that imipramine causes the increased cellular accumulation of LTR by causing an expansion of the lysosomal volume, but because definitive lysosomal volume measurements were not undertaken we have denoted this change as an increase in the apparent lysosomal volume. These observations were consistent with our microscopy studies that suggested an increase in the vesicular volume that stained with the various lysosomotropic dyes ().
A rather simple relationship is predicted to exist between the amount of drugs in lysosomes sequestered by ion trapping, lysosomal volume and lysosomal pH. Therefore, measurement of the effect of imipramine on the ion trapping-based accumulation of an LTR () and lysosomal pH (Supplemental Figure 3
) allows for an approximation of the imipramine-induced expansion of the lysosomal volume. The equations for these simple relationships are detailed in the Experimental
section. Using the data collected in MDA-1986 cells following a 24 h exposure to 10 µM imipramine we found an approximately 3-fold increase in the steady state accumulation of LTR by ion trapping in lysosomes and a 0.1 unit increase in the lysosomal pH. Together, these results would predict an approximately 4-fold increase in lysosomal volume in the imipramine treated cells.
In order to assess if imipramine’s mechanism of increasing amine-drug accumulation was through a decrease in lysosomotropic amine efflux kinetic measurements of both lysosomotropic amine uptake and release were undertaken. Using two model lysosomotropic amine compounds, LTG (Supplemental Figure 2
) and propranolol (), we found that imipramine treatment doesn’t inhibit lysosomotropic amine efflux but rather increases uptake. These results suggest that it is the enhanced steady-state volume of lysosomes associated with imipramine exposure that contributes to increased accumulation.
Based on our hypothesis that imipramine’s capacity to induce a cellular lipidosis is related to its ability to cause an expansion of the apparent lysosomal volume and since the time-dependency of this effect follows quite well to previous reports on the time dependency of CAD induced lipid trafficking defects50, 51, 57
we tested the ability of lipidosis-correcting treatments to prevent lysosomal volume expansion. We found that both, growth in lipoprotein-depleted media and treatment with HPCD, as lipidosis reversing therapies, prevented imipramine from increasing the cellular accumulation of our model lysosomotropic amine probe, LTR (). These results suggest that the lysosomal volume expansion is secondary to the capacity of the perpetrator to inhibit the normal trafficking of LDL-derived cholesterol out of lysosomes.
Our results are in agreement with recent work that has shown a progressive, non-steady state accumulation profile for the phospholipidosis-inducing drug chloroquine.17
Similar to our results these authors found that chloroquine at concentrations between 25 and 200 µM induced the formation of an expanded acidic vesicular compartment that resulted in enhanced cellular accumulation of the lipidosis-inducing drug. Their data shows a 10- to 30-fold increase in vesicular volume with up to a 1 unit upward shift in the vesicular pH. Although our CAD treatments showed a less pronounced increase in vesicular volume and no significant increase in vesicular pH, we believe that such effects would be observed with increasing drug concentrations.
Drug-drug interactions involving lysosomes examined in this work are projected to have clinical significance. On a cellular level, we predict that drug-induced changes in the extent of lysosomal sequestration can directly influence activity by altering the availability of the drug to bind with intended extralysosomal targets. We also predict that this drug-interaction could cause significant changes in the pharmacokinetic behavior of drugs. In an intact animal the extensive sequestration of a drug in lysosomes is the cause for an extremely large apparent volume of distribution and a prolonged elimination half-life. Accordingly, we anticipate that pre-administration of a perpetrator in an animal could cause a significant increase in the apparent volume of distribution and half-life of a subsequently administered victim drug. This could have a significant impact on therapeutic outcomes, particularly for those drugs that have a narrow therapeutic index.
It is interesting to entertain the likely possibility that many of the drugs that we have shown to act as perpetrators in the interaction would also fall victim to it. In a clinical setting this could result in volume of distribution and half-life parameters that appear to increase with the time of administration. In support of this notion, kinetic studies of amiodarone in humans indicated that half-life was 10–17 hours after a single i.v. dose and 8–21 days after chronic administration.58
Collectively, this work illustrates an intracellular drug interaction pathway resulting from the ability of CADs to cause an expansion of the apparent lysosomal volume of cells. The perpetrator drug in such an interaction is the CAD, which causes an increase in the volume of the lysosomal compartment. The victim drug would include any drug that accumulates in lysosomes by the ion trapping-based mechanism, where lysosomal volume is an important factor in determining total cellular drug accumulation. Therefore, victim drugs can include both CAD and non-CAD lysosomotropic drugs. This lysosomal volume expansion was found to be directly related to the CAD-induced cellular lipidosis and to result in the increased cellular uptake of drugs that are substrates for ion trapping-based accumulation in lysosomes. Our evaluations in this manuscript were performed using two different cell lines from separate lineages. The fact that our results were consistently observed in these different cell lines suggests that this drug-interaction could be universal and apply to any cells chronically exposed to CADs at relatively low concentrations. Further in vivo studies will help reveal if this is the case.