Mixed-Lipid Storage Disorder Induced in Macrophages and Fibroblasts by Oritavancin (LY333328), a New Glycopeptide Antibiotic with Exceptional Cellular Accumulation

Oritavancin, a semisynthetic derivative of vancomycin endowedwith a cationic amphiphilic character, accumulates to largeextent in the lysosomes of eukaryotic cells (F. Van Bambeke,S. Carryn, C. Seral, H. Chanteux, D. Tyteca, M. P. Mingeot-Leclercq,and P. M. Tulkens, Antimicrob. Agents Chemother. 48:2853-2860,2004). In the present study, we examined whether this accumulationcould cause cell alterations in phagocytic (J774 mouse macrophages)and nonphagocytic (rat embryo fibroblasts) cells exposed toclinically meaningful (0- to 40-mg/liter) concentrations oforitavancin. Optical and electronic microscopy evidenced conspicuousalterations of the vacuolar apparatus in both cell types, characterizedby the deposition of concentric lamellar structures, finelygranular material, or other less-defined osmiophilic material,often deposed in giant vesicles. Biochemical studies showedan accumulation of phospholipids (1.5x control values) and freeand esterified cholesterol (3 to 4x control values for totalcholesterol). Accumulation of these lipids was in close relationto that of oritavancin (excess phospholipid/oritavancin andexcess cholesterol/oritavancin molar ratios of 2 to 3 and 3to 5, respectively). Cholesterol accumulation was rapid andreversible, and that of phospholipids was slower and poorlyreversible. Vancomycin and teicoplanin, used as controls (50and 100 mg/liter, respectively), did not cause any significantchange in the lipid content of fibroblasts. The data thereforesuggest that oritavancin has the potential to cause a mixed-lipidstorage disorder in eukaryotic cells.

INTRODUCTION

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Introduction

There is now a growing need for agents directed against multiresistantgram-positive organisms such as enterococci and Staphylococcusaureus (27). Oritavancin, originally discovered in the Eli LillyResearch Laboratories (4), is a new glycopeptide antibioticthat shows highly concentration-dependent bactericidal activitytowards both extracellular and intracellular forms of enterococciand S. aureus (1, 2, 21). In a previous study, we showed thatoritavancin (see its structure in reference 26) accumulatesin very large amounts in macrophages and other cultured cells,reaching apparent cellular concentrations as high as 350-foldthe extracellular concentrations after 24 h of incubation (26).We also showed that oritavancin is not homogeneously distributedin cells but is predominantly, if not exclusively, localizedin lysosomes (26). This specific tropism is probably the reasonfor its high activity against S. aureus, which is believed totake refuge and to thrive within these organelles. However,such a huge and specific accumulation also raises issues regardingthe potential for cytotoxicity of oritavancin. We thereforedecided to examine the morphology of cells exposed to clinicallymeaningful concentrations of oritavancin, using both phagocytic(J774 macrophages) and nonphagocytic (fibroblasts) cell lines.We found, and report here, that oritavancin causes in both celltypes conspicuous morphological alterations of the lysosomesand related vacuoles that are associated with a marked accumulationof cholesterol and phospholipids.

MATERIALS AND METHODS

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Materials and Methods

Cells, cell culture conditions, uptake and efflux studies, and assessment of cell viability. J774 mouse macrophages and rat embryo fibroblasts were obtainedand cultured exactly as previously described (26). Uptake andefflux studies, cell collection, and assessment of cell viabilitywere also performed according to published methods (24, 25).

Optic and electron microscopy. For observations in the optic microscope ("plastic sections"technique), sections that were approximately 0.5 µm thickwere obtained with a glass knife, stained with toluidine blue,mounted in Spur, and examined with a Zeiss microscope underoil immersion at a lens objective of 63x. For electron microscopy,we followed exactly the methods described earlier (25).

Biochemical and antibiotic assays. Collected cells were disrupted by sonication, and the lysateswere then used to determine the oritavancin, phospholipid, cholesterol,and protein contents. Oritavancin was measured by scintillationcounting (using ¹⁴C radiolabeled drug; this assay has been validatedpreviously in comparison with a bioassay [26]). The apparentcellular-to-extracellular concentration ratio of oritavancinwas calculated assuming a cell volume of 5 µl/mg of protein(26). Phospholipids were extracted and total lipid phosphorusmeasured as in our previous publication (25). Total cholesterolwas assayed with a ready-to-use commercial kit (Sigma-Aldrich,St. Louis, MO). Free cholesterol was determined using the sameprocedure except that cholesterol esterase was omitted whenpreparing the reagent (all other components were present atthe same concentration as in the ready-to-use kit). The cholesterylester content was then calculated by subtracting the free cholesterolcontent from the total cholesterol content. All experimentalvalues were expressed as nanomoles of lipid phosphorus or cholesterolper mg of cell protein, by comparison with standards of phosphorusor cholesterol. Proteins were assayed by the Folin-Ciocalteu/biuretmethod (11).

Materials. Oritavancin (LY333328, supplied as diphosphate salt fully hydrated;potency, 80.6%) and [¹⁴C]oritavancin (3.5 µCi/mg) wereobtained from Eli Lilly & Co., Indianapolis, IN. The labeleddrug was mixed with unlabeled oritavancin to obtain a specificactivity of 0.6 µCi/mg. Vancomycin and teicoplanin wereprocured as Vancocin and Targocid (the commercial products registeredfor clinical use in Belgium and supplied by Glaxo-SmithKlineBelgium [on behalf of Eli Lilly Benelux] and Aventis Belgium,respectively). Cell culture media and fetal calf serum werepurchased from Gibco Biocult (Paisley, Scotland). Unless statedotherwise, all other reagents were of analytical grade and werepurchased from E. Merck AG (Darmstadt, Germany) or from Sigma-Aldrich(St. Louis, MO).

RESULTS

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Results

Determination of conditions causing gross cytotoxicity. In a first series of studies, we systematically examined whichconditions of incubation would cause gross cell toxicity, asassessed by the release of lactate dehydrogenase in the culturemedium. Using a release threshold corresponding to twice theamount found in control cells at the beginning of the experiments(16), we observed that macrophages could not be maintained formore than 24 h with 25 mg/liter of oritavancin and that fibroblastscould not be maintained for more than 72 h with 40 mg/literof oritavancin. All experiments described hereunder used, therefore,incubation times and drug concentrations that did not exceedthese values.

Morphological studies. Macrophages and fibroblasts incubated with 20 mg/liter of oritavancinand examined under a microscope revealed conspicuous modifications,which are illustrated in Fig. 1 and 2. These consisted of theappearance not only of small, heavily stained granules containingmembraneous materials, but also of large electron-lucent (macrophages)or heterogeneously stained structures suggestive of the formationof some sort of giant digestive vacuole. Other organelles (inparticular, endoplasmic reticulum and mitochondria) seemed unaffected.

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FIG. 1. Appearance of macrophages and fibroblasts under optic microscopy (plastic sections technique); all pictures were taken at an objective magnification of 63x. (A) Control macrophages; (B and C) macrophages exposed for 24 h to 20 mg/liter of oritavancin and showing evidence of accumulation of heavily stained material in granules (B) or the appearance of large electron lucent vacuoles (C); (D) control fibroblasts; (E through H) fibroblasts exposed for 72 h to 20 mg/liter of oritavancin and showing evidence of accumulation of heterogeneous, heavily stained material in small granules (E and F), the appearance of large, heterogeneously stained structures (F and H), or structures filled with a finely granular, moderately homogenously stained material (G).

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FIG. 2. Appearance under electron microscopy of cultured cells exposed to oritavancin at an extracellular concentration of 20 mg/liter. Panels illustrate the main alterations seen. (A) Mouse macrophages (24 h of incubation); (B through E) rat embryo fibroblasts (72 h of incubation). Both cell types show an abundance of electron-dense structures of various sizes and shapes (A and B) which, upon high magnification, display a clearly lamellar aspect (illustrated for macrophages in the inset of panel A). Treated fibroblasts also showed large structures, sometimes with a highly heterogeneous appearance (C) but also with a moderately osmiophilic granular content (D) or a mixture of electron-lucent and amorphous material (E). Bars, 1 µm (100 nm for the inset of panel A).

Biochemical analyses and determination of cell accumulation of oritavancin. Since part of the material accumulated by cells was structurallyevocative of what is commonly observed in the tissues of patientssuffering from disorders of the lysosomal catabolism of polarlipids, we examined the influence of oritavancin on the cellcontent of total phospholipids and cholesterol. For this purpose,cells were incubated with increasing concentrations of oritavancinfor 1 day (macrophages) or 3 days (fibroblasts). Figure 3 shows(i) that phospholipids accumulated in a concentration-dependentfashion in both types of cells, reaching a content of about150% that of controls at the highest concentrations tested (25mg/liter and 40 mg/liter for macrophages and fibroblasts, respectively),and (ii) that accumulation of cholesterol was even more extensive,reaching values about 3.5 times those of controls under thesame conditions. In control cells, cholesterol was found mostlyin its free form (about 85% of the total content) (Fig. 3, middlepanels). In macrophages exposed to oritavancin, both free andesterified cholesterol increased roughly in parallel. In fibroblasts,similar simultaneous increases were seen up to an extracellularconcentration of 20 mg/liter, after which an increase was seenalmost exclusively in the esterified form. In parallel, we measuredthe cell accumulation of oritavancin (lower panels of Fig. 3).As previously described (26), the accumulation of oritavancinoccurred in a cooperative fashion with respect to its extracellularconcentration. Based on the data obtained at the largest extracellularconcentration tested, we calculated that the apparent cellularconcentrations of oritavancin reached values about 400-fold(macrophages) and 600-fold (fibroblasts) higher than in theextracellular medium.

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FIG. 3. Accumulation of total cholesterol and phospholipids (top panel), free and esterified cholesterol (middle panels), and oritavancin (lower panels) in mouse J774 macrophages (left panels) after 24 h of incubation or in rat embryo fibroblasts (right panels) after 72 h of incubation with increasing extracellular concentrations of oritavancin. All data are means ± the standard deviations of three independent experiments (error bars that are not visible are included in the symbols). Data for phospholipids and total cholesterol are expressed as percentages of the original content of control cells collected at the same time (160.0 ± 5.3 nmol/mg of protein for phospholipids and 40.2 ± 2.6 nmol/mg of protein for cholesterol in macrophages; 259.9 ± 21.7 nmol/mg of protein for phospholipids and 86.1 ± 8.8 nmol/mg of protein for cholesterol in fibroblasts).

The kinetics of the accumulation of phospholipids and cholesterolwere then examined in fibroblasts maintained for up to 5 daysin the presence of 20 mg/liter of oritavancin. At the same time,we examined the reversibility of this accumulation by transferringcells incubated for 3 days in the presence of 20 mg/liter oforitavancin to drug-free medium for 3 additional days. (Thisdesign was adopted after preliminary experiments had disclosedthat confluent fibroblasts exposed to 20 mg/liter of oritavancincould not be maintained for more than a total of 6 days in culture.These experiments also showed that confluent macrophages couldnot be maintained viable in culture for more than 24 to 36 h.)As shown in Fig. 4 (upper panel), the cell phospholipid contentincreased continuously as a function of the time of incubationand was only partially reversible upon transfer of the cellsto drug-free medium. In contrast with phospholipids, the accumulationof cholesterol reached its maximum after only 1 day of incubationand slightly decreased thereafter (middle panel). The accumulationof cholesterol was largely reversible upon transfer of the cellsto drug-free medium. The lower panel of Fig. 4 shows that theaccumulation of oritavancin proceeded continuously during theperiod of exposure, with, however, a trend towards a plateauat the 5th day (at an apparent cellular-to-extracellular concentrationratio of about 700). Efflux of oritavancin from fibroblastsproceeded at a rate apparently higher than its uptake for thefirst 24 h but remained thereafter largely incomplete, sinceabout half of the cell-associated drug was still present after3 days of efflux.

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FIG. 4. Kinetics of accumulation and loss of phospholipids (upper panel), total cholesterol (middle panel), and oritavancin (lower panel) in fibroblasts exposed to 20 mg/liter of oritavancin. All cells were incubated in the presence of 20 mg/liter of oritavancin for a first period of 3 days. At this time point (indicated by a vertical arrow), cultures were either maintained in the continuing presence of oritavancin (solid lines) or washed and reincubated in drug-free medium (broken lines). All data are means ± the standard deviations of three independent experiments (error bars that are not visible are included in the symbols). Data for phospholipids and cholesterol are expressed as percentages of the content of control cells at the same day (values were 221.3 ± 28.2 nmol/mg of protein for phospholipids and 58.5 ± 6.7 nmol/mg of protein for cholesterol at day 0, and values increased during the culture to reach 252.9 ± 11.3 nmol/mg of protein [prot] for phospholipids and 94.9 ± 4.1 nmol/mg of protein for cholesterol at day 5).

The data generated by these studies were then used to examinewhether constant molecular relationships could be deduced fromour observations. For this purpose, all phospholipids, cholesterol,and oritavancin accumulation data were converted into nmol permg protein, and for phospholipids and cholesterol, data wereexpressed as amounts in excess of control values. Figure 5 showsa highly significant correlation between the excess of totalcholesterol and the cell accumulation of oritavancin. The correlationwas less significant for phospholipids, partly due to theirlack of disappearance upon drug efflux. The correlation betweenthe molar excess of cholesterol and that of phospholipids wasalso only partial, but the slope of the correlation suggesteda constant molar ratio close to 1, disregarding the type ofcells studied.

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FIG. 5. Correlation between the cell accumulation of oritavancin and the excess of total cholesterol (left panel) and phospholipids (middle panel) and between the excess of cholesterol and the excess of phospholipids (right panel) in macrophages (open symbols) and fibroblasts (closed symbols) exposed to oritavancin. The data used to construct this figure are those of Fig. 3 and 4, but all data are expressed in molar amounts (for cholesterol and phospholipids, the values found in the matched control cells have been subtracted). Regression parameters: cholesterol versus oritavancin, R² = 0.987 and slope = 4.73 ± 0.17 in macrophages and R² = 0.930 and slope = 3.07 ± 0.13 in fibroblasts; phospholipids versus oritavancin, R² = 0.488 and slope = 2.82 ± 0.91 in macrophages and R² = 0.663 and slope = 1.90 ± 0.21 in fibroblasts; cholesterol versus phospholipids, R² = 0.507 and slope = 0.85 ± 0.11. prot, protein.

Specificity versus other glycopeptides. The specificity of the effects of oritavancin on lipid accumulationin fibroblasts was tested by repeating these experiments withvancomycin and teicoplanin (at 50 and 100 mg/liter, respectively).Table 1 shows that neither vancomycin nor teicoplanin significantlyaffected the cholesterol and phospholipid content of cells.

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TABLE 1. Accumulation of phospholipids and cholesterol in rat fibroblasts incubated for 3 days with glycopeptides

DISCUSSION

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Discussion

While accumulation of antibiotics in cells and tissue may givecells and tissue a potential for activity against intracellularbacteria, it may also lead to the development of cellular alterations.This is well exemplified here by the behavior of oritavancinif the present data are compared with data dealing with intracellularactivity (21, 26). We know that oritavancin accumulates in lysosomesof cultured cells (26), which strongly suggests that the alterationsseen here are related to some sort of drug-induced storage disorderat the level of these organelles. One of the key functions oflysosomes is to degrade, through the concerted action of a largearray of acid hydrolases, both endogenous and exogenous materialsthat reach them through the processes of autophagy and heterophagy(5). The highly heterogeneous aspect of the material seen incells incubated with oritavancin suggests that several lysosomalcatabolic pathways are affected. Our images are indeed reminiscentof two situations in which the activities of several lysosomalenzymes are simultaneously decreased, namely, (i) mucolipidosistype II (I-cell disease [6, 9]), a situation in which newlysynthesized lysosomal enzymes are diverted to the extracellularmilieu (18) so as to result in a multiple lysosomal enzyme defect(10), and (ii) the impairment of the activities of several lysosomalhydrolases by means of antibodies (23). We may, however, alsoenvisage that oritavancin, as a cationic amphiphile (4), bindsto membranes and, indirectly, causes the alterations seen. Cationicamphiphilic drugs indeed form complexes with membranes (12)but usually tend to induce a specific accumulation of phospholipids(7). Accumulation of cholesterol has, however, been reportedfor some agents (14, 19) and could be related to a stimulationof its synthesis and/or an inhibition of its transport. Futurestudies will need to further examine these non-mutually exclusivepossibilities.

The present work raises critical questions regarding the potentialtoxicity of oritavancin to patients. Our observations were madewith concentrations of oritavancin close to the expected maximumconcentration of drug in the sera of humans but were maintainedcontinuously for several days, creating an area under the concentration-timecurve considerably larger that what could be seen during conventional,discontinuous administration. Oritavancin accumulated by cells,however, leaks out only very slowly and partially, so that discontinuousadministration will also eventually result in a sustained accumulationin the long term. Oritavancin is also reported to be highlyprotein bound in human serum (about 90%) (P. A. Rowe and T.J. Brown, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother.,abstr. A-2193, 2001), so that cell uptake in vivo could be muchlower if related to free concentration only. We, unfortunately,lack data concerning the tissular accumulation of oritavancinin animals or humans. As discussed earlier (26), however, J774macrophages incubated with 20 mg/liter oritavancin in the presenceof 10% calf serum (as in the present conditions) accumulateroughly the same amount of drug as those maintained with 3 to5 mg/liter oritavancin in the absence of serum. Extrapolationto human cells remains, however, largely uncertain. Yet, cellularaccumulation seems to be a key event, since no significant cellalteration has been reported for vancomycin and teicoplanin,which accumulate to much lower amounts in cultured cells (13,28). More importantly, perhaps, we do not know from the presentstudy whether the lysosomal alterations induced by oritavancinwill lead to organ dysfunction and toxicity. Drug-induced phospholipidaccumulation and lysosomal alterations have been associatedwith renal, lung, and liver dysfunction for aminoglycosides(15), amiodarone (20), or diethylaminoethoxyhexestrol (29),respectively. Excesses of both free and esterified cellularcholesterol are toxic, as illustrated by their involvement inthe pathogeny of the Niemann-Pick disease type C (17, 19) oratherosclerosis (8, 22). In this respect, the screening of thelipidogenic capacity of drugs on cell culture models is nowadaysconsidered to be an important aspect in the evaluation of theirsafety profiles (3). The present observations may thereforetrigger more-comprehensive studies aimed at better delineatingthe true toxic potential of oritavancin.

ACKNOWLEDGMENTS

M. C. Cambier and F. Renoird provided dedicated technical assistance.

F.V.B. is Chercheur Qualifié of the Belgian Fonds nationalde la Recherche Scientifique. We thank Eli Lilly (Indianapolis,IN) for the kind gift of oritavancin. This work was supportedby the Fonds Spéciaux de Recherches (FSR) of the Universitécatholique de Louvain.

FOOTNOTES

* Corresponding author. Mailing address: UCL 7370 avenue Mounier 73, 1200 Brussels, Belgium. Phone: 32-2-764.73.78. Fax: 32-2-764.73.73. E-mail: vanbambeke{at}facm.ucl.ac.be.

REFERENCES

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