Antileishmanial Activity of the Antiulcer Agent Omeprazole

Parasites. Leishmania donovani Sudan 1S promastigotes were maintained in medium 199 with 15% fetal bovine serum at pH 7.2 as described previously (12). Parasites were acid adapted by growing them in medium 199 supplemented with 15% fetal bovine serum and 20 mM MES (2-[N-morpholino]ethanesulfonic acid) at pH 5.1. They have been maintained in the acidic medium at 37°C with 5% CO₂ for several years. These cells are ovoid, mostly nonflagellated, and amastigote-like.

Macrophages.Peritoneal macrophages were obtained from CBA/caj mice without thioglycolate stimulation and cultivated in eight-well chamber slides at a density of 2.5 × 10⁵/ml in RPMI 1640 medium with 15% fetal bovine serum. Nonadherent cells were washed off after a 24-h incubation at 37°C and 5% CO₂. Promastigotes were loaded in each well at a ratio of 10 promastigotes to 1 macrophage. After overnight infection, the macrophages were washed to remove all external promastigotes. Omeprazole was added at different dosages. The slides were washed after 48 h, dried, and stained with Wright-Giemsa stain (Diff-QuiK; Baxter Scientific Products).

Intracellular pH determination.Intracellular pH (pHi) was determined by measuring the fluorescence of the pHi indicator BCECF (2′,7′-bis [carboxyethyl-5 (and -6)-carboxy] fluorescein) as previously described (12, 16). In brief, promastigotes were harvested at stationary phase and loaded with BCECF in fresh medium 199 at pH 7.2. They were treated with nigericin (10 μg/ml) for acid loading, and treatment was stopped by the addition of bovine serum albumin (5 mg/ml). The cells were washed twice with choline buffer containing 115 mM choline chloride, 10 mM glucose, 5 mM MgCl₂, 10 mM MES, 10 mM MOPS (3-[N-morpholino]propanesulfonic acid) (pH 7.1. adjusted with Trizma base) to remove excess dye, nigericin, and BSA. Cells treated with omeprazole were incubated for 10 min before they were scanned in the choline buffer (pH 5.7) with a fluorescence spectrophotometer (Perkin-Elmer pmf-66) at excitation and emission wavelengths of 503 and 535 nm, respectively. Once fluorescence intensity was stabilized, 5 mM KCl was added and the change in fluorescence was recorded. Procedures for parasites cultured at pH 5.1 were the same except that after BCECF loading at pH 7.2, they were washed with choline buffer at pH 5.7.

ATPase assay.Activity of purified ATPase was determined by measuring the release of inorganic phosphate (P_i) from [γ-³²P]ATP as described previously (3). Purified enzyme (0.5 μg) was incubated for 20 min at room temperature in 100 μl of buffer containing 1 mM MES, 1 mM MOPS, and 50 mM sucrose at pH 7.4. The reaction was started by adding 100 μl of reaction buffer containing 40 mM MOPS, 40 mM sucrose, 4 mM MgCl₂, 2 mM [γ-³²P]ATP, and 20 mM KCl at pH 7.4. In experiments to determine the effect of omeprazole, the enzyme was incubated with various amounts of omeprazole for 20 min at room temperature at pH 5.5 before adding the reaction mixture with [γ-³²P]ATP. After a 10-min incubation at 37°C, the reaction was stopped by the addition of 5% (final) trichloroacetic acid, and 50 μl of the mixture was removed for analysis of P_i (3).

Effect of omeprazole on parasites in vitro.Parasites cultured at neutral pH as well as those that were acid adapted long term were used to investigate the effect of omeprazole on growth in vitro. Growth was followed at various pHs and at different concentrations of omeprazole. Omeprazole was not active at pH 7.2, and although it may permeate the outer membrane, its effect on the parasite was negligible even at the high dose of 150 μM (Fig. 1A). In contrast, at pH 5.5 (Fig. 1B) it was in its pharmacologically active form. At 50 μM, growth is reduced by 50% while at 150 μM growth is inhibited 90 to 95% in 48 h. Acid-adapted cells also suffered similarly high levels of growth inhibition, with omeprazole concentrations of 50, 100, and 150 μM when tested at pH 5 (Fig. 1C). During a 96-h incubation, parasites grown at neutral pH showed no growth inhibition in the presence of 100 to 150 μM omeprazole at pH 7.2 (Fig. 2A) but the same cells suffered severe growth inhibition at pH 5.5 (Fig. 2B). Parasites cultured long term in pH-5.1 medium and then treated with omeprazole in pH-5.0 medium were also severely inhibited (Fig. 2C). It is clear that omeprazole in its active protonated form limits parasite growth severely in vitro.

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FIG. 1.

Effect of pH on inhibition of growth by omeprazole. Parasites from pH-7.2 medium were washed and inoculated at a starting density of 5.7 × 10⁶ cells/ml in fresh medium at pH 7.2 (A) and at pH 5.5 (B) with various doses of omeprazole (50, 100, and 150 μM) in 12-well tissue culture plates. Acid-adapted parasites (C) were incubated at a starting density of 7.5 × 10⁶ cells/ml in the presence of 50, 100, and 150 μM omeprazole. All were incubated at 26°C with 5% CO₂. Cells were counted in triplicate from each well, and the experiments were repeated four times. This figure represents results after 48 h of incubation.

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FIG. 2.

Effect of omeprazole on the growth of L. donovani at various pHs. Growth was followed by inoculating the cells into media with or without omeprazole in wells at starting densities of 2.5 × 10⁶/ml at pH 7.0 (A), 7.5 × 10⁶/ml at pH 5.5 (B), and 2.5 × 10⁶/ml at pH 5.0 (C). Cells from each well were counted at regular intervals. (A) Promastigotes grown at pH 7.0 and incubated in pH 7.0 medium with or without drug. (B) Promastigotes grown at pH 7.0 and incubated in pH 5.5 medium with or without drug. (C) Acid-adapted parasites incubated in pH 5.0 medium with or without drug. Each point represents the average for three experiments.

Effect of omeprazole on parasites within cultured macrophages.To test the antileishmanial property of omeprazole further, macrophages infected with L. donovani were treated with various concentrations of omeprazole, ranging from 0 to 150 μM. As shown in Fig. 3, intracellular parasites (amastigotes) suffer significant inhibition in a dose-dependent manner. Figure 4 shows the results of a typical experiment: untreated control cultures (Fig. 4A) show 1 to 30 parasites per macrophage, but infected cultures treated with 150 μM omeprazole (Fig. 4B) show very few or no intracellular parasites. In another set of experiments, peritoneal macrophages were infected in vivo by injecting 2 × 10⁹ parasites into the peritoneal cavity of CBA/caj mice. Two days postinfection, macrophages were harvested by flushing the peritoneal cavity with physiological saline followed by centrifugation at 2,000 × g. They were washed twice and incubated in eight-well chamber slides in RPMI 1640 medium with 15% fetal bovine serum. Unattached cells were washed off, and the slides were incubated in the same medium containing 150 μM omeprazole. The results after 48 h of incubation were essentially similar to those shown in Fig. 4B. Omeprazole thus shows a marked effect on amastigote survival within the macrophages.

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FIG. 3.

Dose-related response of intracellular amastigotes to omeprazole.

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FIG. 4.

Effect of omeprazole on L. donovani in infected macrophages in vitro. (A) Leishmania-infected macrophages (control). The arrow points to amastigotes within the acidic phagolysosomes of the macrophage. (B) Leishmania-infected macrophages treated with 150 μM omeprazole for 48 h.

Effect of omeprazole on the K⁺,H⁺-ATPase.We have previously demonstrated the presence of a K⁺,H⁺-ATPase on the surface membrane of L. donovani (12). This pump plays a key role in the maintenance of the pH_i in L. donovani. Intracellular pH changes can be monitored with various fluorescent dyes (13), among which BCECF is the most widely used. It was thought that inhibition of the K⁺,H⁺-ATPase by omeprazole could block H⁺ extrusion, resulting in proton accumulation and intracellular acidification, which would terminate or limit the growth of the parasite. To explore this possibility, the effect of omeprazole on intracellular pH changes was examined.

Parasites cultivated both at pH 7.2 and pH 5.1 were used in this experiment. They were initially loaded with protons as described in Materials and Methods so that the pH_i declined to approximately pH 6.4. Protons are not released unless K⁺ is added to the suspension (12). Upon addition of 5 mM KCl, the acid-loaded parasites pumped out H⁺ and fully recovered within 5 min (Fig. 5). However, cells incubated with omeprazole for 10 min at pH 5.7 following acid loading suffered inhibition of proton extrusion in a concentration-dependent manner (Fig. 5). It is worth noting that once the cells are acid loaded, omeprazole inhibits proton extrusion even when the cells are suspended at neutral pH. The drug is protonated to its active form as the intracellular pH drops below neutral.

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FIG. 5.

Effect of omeprazole on H⁺ extrusion and pH_i in L. donovani. Tracings show fluorescence in promastigotes treated with various amounts of omeprazole (30, 50, 80, and 100 μM).

Since omeprazole is a specific inhibitor of the gastric K⁺,H⁺-ATPase both in vitro and in vivo (11, 13), its effect on the purified plasma membrane K⁺,H⁺-ATPase in L. donovani was tested. The enzyme is indeed sensitive to omeprazole at acid pH. At 80 μM omeprazole, activity is inhibited about 50%. Activity (nmol of P_i/min/mg of protein) in the absence and presence of the drug was 3,710 ± 62 and 1,900 ± 47, respectively (n = 4). Blockage of the enzyme by omeprazole confirms not only that the ATPase is indeed a K⁺,H⁺-ATPase but also that this enzyme is responsible for the K⁺-induced proton extrusion in L. donovani. When omeprazole blocks the K⁺,H⁺-ATPase on the plasma membrane, excess protons cannot be extruded, leading to intracellular acidification. Growth of the parasite is seriously impeded or terminated as the intracellular pH drops.