ABSTRACT
Progress toward the improvement of meglumine antimoniate (MA), commercially known as Glucantime, a highly effective but also toxic antileishmanial drug, has been hindered by the lack of knowledge and control of its chemical composition. Here, MA was manipulated chemically with the aim of achieving an orally effective drug. MA compounds were synthesized from either antimony pentachloride (MA-SbCl5) or potassium hexahydroxyantimonate [MA-KSb(OH)6] and prepared under a low polymerization state. These compounds were compared to Glucantime regarding chemical composition, permeation properties across a cellulose membrane and Caco-2 cell monolayer, and uptake by peritoneal macrophages. MA-SbCl5 and MA-KSb(OH)6 were characterized as less polymerized and more permeative 2:2 Sb-meglumine complexes than Glucantime, which consisted of a mixture of 2:3 and 3:3 Sb-meglumine complexes. The antileishmanial activities and hepatic uptake of all compounds were evaluated after oral administration in BALB/c mice infected with Leishmania infantum chagasi, as a model of visceral leishmaniasis (VL). The synthetic MA compounds given at 300 mg Sb/kg of body weight/12 h for 30 days significantly reduced spleen and liver parasite burdens, in contrast to those for Glucantime at the same dose. The greater activity of synthetic compounds could be attributed to their higher intestinal absorption and accumulation efficiency in the liver. MA-SbCl5 given orally was as efficacious as Glucantime by the parenteral route (80 mg Sb/kg/24 h intraperitoneally). These data taken together suggest that treatment with a less-polymerized form of MA by the oral route may be effective for the treatment of VL.
INTRODUCTION
Leishmaniasis, one the most significant vector-borne neglected tropical diseases (NTDs), encompasses a wide spectrum of clinical manifestations, ranging from self-healing skin lesions, associated with infection by Leishmania (Leishmania) major and L. (L.) amazonensis, to fatal visceral infections by L. (L.) infantum and L. (L.) donovani, which occur mostly in tropical and subtropical areas around the globe (1–3). Leishmania, a protozoan parasite and the causative agent for leishmaniasis, is transmitted to the mammalian host by the bite of infected female sandflies. Leishmaniasis is prevalent in 98 countries, with an estimated 900,000 to 1.3 million new cases and a mortality rate of 20,000 to 30,000 a year, leaving 350 million people at risk. Visceral leishmaniasis (VL), the most severe form of the disease, leads to death in 95% of the cases if left untreated and accounts for an estimated 200,000 to 400,000 new cases per year. In 2014, more than 90% of new VL cases were reported to occur in Brazil, Ethiopia, India, Somalia, South Sudan, and Sudan (2, 4, 5).
Even though pentavalent antimonials are still the first-line drugs in several developing countries against all forms of leishmaniasis, their use in the clinical setting has several limitations (6–8). Pentavalent antimonials are often considered prodrugs in which the metal is reduced to the more active and toxic trivalent form (8). These compounds have to be given parenterally, daily, for at least 3 weeks (typically, 20 mg of Sb/kg of body weight/day for 20 to 30 days). Antimony therapy is often accompanied by local pain during intramuscular injection and by severe side effects that include cardiotoxicity, pancreatitis, hepatotoxicity, nephrotoxicity, arthralgias, and myalgias (6). Consequently, careful medical supervision is required, and compliance problems are common. Antimony resistance has also emerged, with the most severe situation in the Indian subcontinent (9).
As alternatives, liposomal amphotericin B and oral miltefosine, despite their high cure rates, also present drawbacks, including toxicities and inaccessible cost (10). Therefore, the development of alternative antileishmanial therapeutic strategies has become critically significant and is strongly recommended by the World Health Organization (WHO). In this context, improvement of the efficacy and minimization of the toxicity and cost of the existing drugs should be investigated, in addition to the design of new chemical entities. Moreover, the development and modification of the existing drugs to orally administrable drugs may add to ease of use and improvement of drug efficacy (8).
Previous attempts to improve the oral bioavailability of antimonial drugs comprise the complexation of meglumine antimoniate (MA) with β-cyclodextrin (βCD) and the synthesis of amphiphilic antimony(V) complexes (11, 12). Although these novel chemical forms were active by the oral route, the MA-βCD complex showed limited efficacy in a murine model of VL (13) and the use of amphiphilic antimony(V) complexes may be limited by their toxicity (14).
The ability of βCD to improve the oral bioavailability of MA has been attributed to the formation of a ternary meglumine (N-methyl-d-glucamine [NMG])-Sb-cyclodextrin complex, which maintains the antimonial compound in a depolymerized state and subsequently releases the low-molecular-weight 1:1 Sb-NMG complex (15). In that study (15), it was also observed that when a solution of MA was heated at 55°C, the compound suffered dissociation, from high-molecular-weight Sb complexes into species of lower molecular weight. Strikingly, freeze-drying of this solution was found to preserve a less-polymerized state of MA, resulting in a higher serum level of Sb than that in MA freshly prepared at room temperature, after administration in mice by the oral route (15). Our hypothesis here is that freeze-dried MA under a low polymerization state may be orally active against leishmaniasis.
In the present work, MA compounds were synthesized from either antimony pentachloride (SbCl5) or potassium hexahydroxyantimonate [KSb(OH)6] and prepared under a low polymerization state. These were compared to the commercial MA, Glucantime, regarding chemical composition and permeation properties and then evaluated by the oral route for serum pharmacokinetics and antileishmanial activity in a mouse model of VL.
RESULTS
Polymerization state of MA compounds.Table 1 displays the chemical composition of MA compounds synthesized from SbCl5 and KSb(OH)6 and freeze-dried Glucantime. Compared to Glucantime, the synthetic compounds exhibited a much higher level of K+ and a lower level of Na+. This is due to the use of KSb(OH)6 and KOH during the synthetic processes (16). From these data, the NMG/Sb molar ratio could be estimated and was found to be close to 1 for synthetic MA and to 1.3 for Glucantime (Table 1).
Analyses of Na, Cl, K, Sb, and carbon in synthetic MA compounds and lyophilized Glucantime
To characterize the state of polymerization of the antimonial compounds (synthetic MAs and Glucantime) in a concentrated aqueous solution (0.7 M Sb), the osmolarity was measured just after dilution in water from 0.7 to 0.1 M Sb, taking advantage of the low rate of dissociation of Sb(V) complexes (17). As shown in Fig. 1, the solutions of synthetic MAs had greater osmolarity than Glucantime. From this result, the concentrations of the complex in the concentrated solutions were estimated as 0.38 M for synthetic MAs and 0.28 M for Glucantime. Thus, the average numbers of NMG molecule per complex are about 2 in synthetic MAs and 3 in Glucantime. Furthermore, the average numbers of Sb atom per complex are about 2 in synthetic MAs and between 2 and 3 (2.4) in Glucantime.
Osmolarity of solutions of synthetic MAs and Glucantime just after dilution in water at 25°C from 0.7 to 0.1 M Sb. Data are presented as means ± standard errors (SE) (n = 3 or 4).
These data taken together strongly suggest that synthetic MAs consist essentially of 2:2 Sb-NMG complexes and that Glucantime is a mixture of 2:3 and 3:3 Sb-NMG complexes, as illustrated in Fig. 2. Thus, the solutions of synthetic MAs contained less-polymerized antimony(V) complexes than Glucantime.
Representation of the predominant species in synthetic MA compounds [MA-SbCl5 and MA-KSb(OH)6] (top) and in Glucantime (bottom).
Efficacy by the oral route in a murine VL model.Figure 3 shows the parasite burdens in the liver (Fig. 3A) and spleen (Fig. 3B) of BALB/c mice infected with L. (L.) infantum chagasi following oral administration of the different antimonial compounds at 300 mg Sb/kg/12 h for 30 days. In the spleen, both of the synthetic MA compounds significantly reduced the parasite load (P < 0.05), in comparison to the saline-treated control. In the liver, MA obtained from SbCl5 showed significant antileishmanial activity (P < 0.05). In contrast, Glucantime given orally was not effective in reducing the parasite load in either the spleen or the liver. As expected, Glucantime given intraperitoneally (i.p.) at 80 mg Sb/kg/day for 30 days significantly reduced the parasite loads in the spleen (P < 0.05) and the liver (P < 0.01). The oral treatment with MA-SbCl5 (300 mg Sb/kg/12 h) showed efficacy equivalent to that of parenteral treatment with Glucantime (80 mg Sb/kg/24 h).
Antileishmanial activity of synthetic MAs and Glucantime given orally in a mouse model of VL, based on parasite suppression in the liver (A) and spleen (B). BALB/c mice were infected intravenously with L. (L.) infantum chagasi. After 7 days, the animals were subjected to the following treatments for 30 days: synthetic MA [obtained from SbCl5 or KSb(OH)6] and Glucantime (Glu), given by gavage at 300 mg Sb/kg/12 h; Glucantime given by the i.p. route at 80 mg Sb/kg/day (Glu IP); and saline treatment (control). Three days after the end of treatment, the animals were euthanized and the liver and spleen were processed to determine the parasitic load by the limiting dilution assay. Data are medians with 95% confidence intervals (CI) (n = 5 or 6). *, P < 0.05; **, P < 0.01 (for comparison with results for the saline-treated control by the Kruskal-Wallis test with Dunn's posttest).
Serum pharmacokinetics and hepatic level of Sb.To evaluate whether the greater antileishmanial activity of synthetic MA compounds may be related to higher oral drug bioavailability and/or tissue uptake of Sb, we compared the different antimonial compounds regarding their serum pharmacokinetics in mice after a single oral dose and their hepatic accumulation at the end of the multiple-dose treatment as described above.
Figure 4A and Table 2 display the resulting serum pharmacokinetics and pharmacokinetic parameters, respectively. The synthetic MA compounds showed a shorter time to reach the peak Sb concentration (Tmax) values than Glucantime, suggesting a higher rate of intestinal absorption. This was also supported by the significantly higher concentration of Sb at 30 min for MA-SbCl5 than for Glucantime. On the other hand, Glucantime showed a slightly greater area under curve (AUC) than the synthetic MAs, suggesting no marked difference in oral bioavailability between these compounds.
Serum pharmacokinetics of Sb after a single oral dose of synthetic MA compounds or Glucantime (A) and accumulation of Sb in the liver of mice following a multiple-dose treatment by the oral route with the antimonial compounds (B). (A) Swiss mice received by gavage a single dose of synthetic MA [MA-SbCl5 or MA-KSb(OH)6] or Glucantime at 300 mg Sb/kg and were euthanized after different time intervals for serum recovery and Sb determinations. Data are means ± SE (n = 5/point). **, P < 0.01 (for comparison with results for Glucantime, by the Kruskal-Wallis test with Dunn's posttest). (B) BALB/c mice infected with L. (L.) infantum chagasi were subjected to treatment for 30 days with synthetic MA compounds or Glucantime as follows: MA synthesized from SbCl5 or KSb(OH)6 at 300 mg Sb/kg/12 h by the oral route; Glucantime at 300 mg Sb/kg/12 h by the oral route (Glu oral) or 80 mg Sb/kg/24 h by the i.p. route (Glu IP). Animals were euthanized 3 days after treatment, and the livers were recovered for determination of Sb by graphite furnace atomic absorption spectroscopy (GFAAS). Data are means ± SE (n = 5 or 6). *, P < 0.05 (for comparison by one-way ANOVA, followed by Tukey's posttest).
Serum pharmacokinetic parameters of synthetic MA compoundsa and Glucantimeb
As shown in Fig. 4B, MA synthesized from SbCl5 promoted a significantly higher level of Sb in the liver than did Glucantime. It is also noteworthy that the hepatic levels of Sb after oral treatment with the antimonial compounds (300 mg Sb/kg/12 h) were equivalent to that after parenteral treatment with Glucantime (80 mg Sb/kg/24 h), in accordance with the efficacy results.
Drug permeation of a Caco-2 cell monolayer or synthetic membrane and uptake by peritoneal macrophages.To get insight into the mechanisms responsible for the higher rate of intestinal absorption and hepatic accumulation of the synthetic MAs, these compounds were compared to Glucantime for their abilities to permeate a human colon carcinoma Caco-2 cell monolayer or a cellulose membrane (molecular weight cutoff [MWCO], 3 kDa), as well as for their uptake by peritoneal macrophages.
Figure 5A shows that after 10 and 20 min, the synthetic MA compounds exhibited significantly higher values of apparent permeability (Papp) across a Caco-2 cell monolayer than Glucantime. The fact that the synthetic compounds also permeated the cellulose membrane more effectively (Fig. 5B) suggests an influence of the MA polymerization state, the most polymerized compound being the least able to permeate.
Comparison of synthetic MA compounds and Glucantime with respect to permeation efficiency across a Caco-2 cell monolayer (A) or a synthetic membrane (B) and uptake by peritoneal macrophages (C). (A) Solutions of MA-SbCl5, MA-Sb(OH)6, or Glucantime diluted in Hanks' balanced salt solution at 50 mM Sb were applied at the apical side of a Caco-2 cell monolayer at 37°C. After different time intervals, samples were collected at the basolateral side for determination of the amount of Sb that had permeated the monolayer, through GFAAS. The apparent permeability coefficient (Papp) values were calculated (n = 4 to 6). (B) Solutions of MA-SbCl5, MA-Sb(OH)6, or Glucantime at 0.7 M Sb were applied to an ultrafiltration cellulose membrane with an MWCO of 3 kDa. The concentration of Sb was determined in the ultrafiltrate by GFAAS after 15 min of centrifugation (n = 5). (C) Peritoneal macrophages were exposed to the antimonial compounds for 4 h, and intracellular Sb was determined by GFAAS after cell washing (n = 3). Data are means ± SE. *, P < 0.05; **, P < 0.01, ***, P < 0.001 (for comparison with results for Glucantime, using one-way ANOVA, followed by the Bonferroni posttest).
A remarkable contribution of this study is the demonstration that the synthetic MA compounds are significantly more efficiently captured by macrophages than Glucantime (Fig. 5C). This property may also explain the higher accumulation of the synthetic compounds by macrophage-rich organs, such as the liver.
DISCUSSION
This work represents a continuation of our prior efforts to find antimonial drugs that may be used for the oral treatment of leishmaniasis. Here, the chemical composition of synthetic and commercial MA, in the form of concentrated solutions, has been characterized for the first time. When prepared at the same Sb concentration of 0.7 M, the synthetic MA compounds obtained under a low polymerization state were characterized as 2:2 Sb-meglumine complexes, whereas Glucantime consisted of a mixture of 2:3 and 3:3 Sb-meglumine complexes. This difference in polymerization state can be attributed at least in part to the lower meglumine/Sb ratio used to obtain the synthetic compounds (molar ratio, ∼1), compared to that of Glucantime (molar ratio, 1.28).
This study also reports for the first time the antileishmanial activity of synthetic MA under a low polymerization state when given by the oral route in a murine model of VL. This is in contrast to Glucantime given orally at the same dose, which did not produce significant parasite suppression. From the pharmacokinetic data, one can propose that the greater activity of synthetic compounds may be due to their greater intestinal absorption and accumulation efficiency in the liver. From the mechanistic point of view, our data suggest that the lower molecular weight of synthetic MAs contributed to their higher rate of permeation and intestinal absorption. Furthermore, the higher accumulation efficiency of synthetic MAs in the liver may be related to their higher uptake by macrophages, presumably because of the greater availability of Sb for binding to the cell surface and subsequent internalization by endocytosis. This interpretation is consistent with our observation that Sb(V) is reduced at a higher rate by cysteine in synthetic MAs than by cysteine in Glucantime (data not shown).
It is noteworthy that MA-SbCl5 given orally (300 mg Sb/kg/12 h) is as effective as Glucantime by the parenteral route (80 mg Sb/kg/24 h) against VL. Considering a ratio of body surface area of 12 between mouse and human (18), the dose of 300 mg Sb/kg/12 h in mouse would be equivalent to 25 mg Sb/kg/12 h in human. This dose in human is 2.5 times as high as the dose administered parenterally in conventional antimonial therapy (20 mg Sb/kg/24 h). Even though the oral treatment with antimonials may require the administration of a higher total drug dose than that of conventional therapy, the cost of treatment would not necessarily be increased if it was carried out at home without hospitalization.
The Leishmania infantum-infected BALB/c mouse used here is a well-established and commonly used model, showing parasitological and clinical features of human VL, such as high parasite burdens in the liver and spleen and hepatosplenomegaly. Therefore, it is appropriate for the purpose of comparison of the efficacies of the synthetic antimonials and Glucantime. However, this experimental model does not reproduce the human disease outcome, because of organ-specific immune responses (19). Thus, to guide future drug development, it is important to further validate the therapeutic efficacy of the synthetic compounds using an experimental model that more closely mimics the human disease, such as Leishmania infection in Syrian golden hamsters or dogs (20).
Another important aspect to be addressed in future studies is the safety of the oral therapy, compared to the parenteral treatment. Preliminary evaluations of the histopathological changes in the livers of mice just after treatment suggest that the synthetic MA compounds given by the oral route are less toxic for hepatocytes than parenteral Glucantime (data not shown), even though similar levels of Sb were found in the liver and treatment with MA-SbCl5 showed the same efficacy. However, these data should be complemented with more complete toxicological evaluations to confirm the reduction in global drug toxicity.
In conclusion, as our main contribution, this study suggests that treatment with a less-polymerized form of MA by the oral route is effective for the treatment of VL.
MATERIALS AND METHODS
Materials.N-methyl-d-glucamine (NMG; ≥98%) and antimony pentachloride (SbCl5; ≥99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA, and Milwaukee, WI, USA), and potassium hexahydroxoantimonate(V) [KSb(OH)6; ≥99%] was from Fluka Chemie GmbH (Switzerland). All other reagents were of at least reagent grade. Double-distilled deionized water, obtained using a Milli-Q system (Millipore Corp., Bedford, MA, USA), was used to prepare all the solutions throughout the experiments. A commercial sample of meglumine antimoniate (Glucantime), 5-ml vials at 300 mg/ml (batch 6050; Sanofi-Aventis, São Paulo, Brazil), was obtained from Instituto René Rachou, Fundação Oswaldo Cruz (IRR/Fiocruz Minas, Belo Horizonte, Brazil).
Synthesis and preparation of meglumine antimoniate compounds.Synthetic MA was obtained, as previously described (16), from equimolar amounts of NMG and pentavalent antimony hydroxide, using either antimony pentachloride (SbCl5) (21) or potassium hexahydroxyantimonate [KSb(OH)6] (C. P. Demicheli and F. J. G. Frézard, BR patent application PI 0106505-7) as a source of Sb(V). The compounds obtained from acetone precipitation exhibit a high degree of polymerization (15). Reduction of the polymerization state of MA was achieved as described previously (15; F. J. G. Frézard, A. L. De Melo, P. S. Martins, and C. Demicheli, BR patent application PI 0602371-1), with the following modifications. Briefly, the resulting MA compounds were dissolved in deionized water at a concentration of 0.7 M Sb. The solutions were incubated at 60°C for 3 h under stirring and then immediately freeze-dried. All subsequent measurements or experiments were performed just after preparation of fresh solutions through dissolution of freeze-dried compounds in water at a final Sb concentration of 0.7 M.
Characterization of meglumine antimoniate compounds.The quantification of Sb, Na, and K in the commercial and synthetic MA compounds was performed using inductively coupled plasma optical emission spectrometry (ICP-OES). The quantification of carbon element and Cl was carried out by inductively coupled plasma atomic absorption spectrometry (ICP-AES).
Determination of the state of polymerization.To determine the state of polymerization of synthetic and commercial MA compounds, osmolarity measurements were performed at 25°C using a vapor pressure osmometer (Vapro5520; Wescor, Logan, UT, USA), as described previously (17), just after dilution with water (25°C) of all tested solutions from 0.7 to 0.1 M Sb. The results were obtained from three or four independent experiments. The concentration of the complex could be estimated by subtracting the osmolarity from the molar concentration of free ions (Na+, K+, and Cl−).
In vivo treatment, efficacy, and toxicity studies.The animals were handled according to the protocols approved by the Ethical Committee for Animal Experimentation of the Universidade Federal de Minas Gerais (UFMG) (protocol no. 215/09 and 199/2011) and the IRR/Fiocruz Minas (protocol P-0321/06; license no. L-0024/8).
Parasites and animals.L. (L.) infantum chagasi (MHOM/BR/70/BH46) parasites, originally obtained from the IRR/Fiocruz Minas, were isolated from golden (Syrian) hamsters (Mesocricetus auratus) and routinely maintained and grown as promastigotes. Promastigotes were maintained in vitro at 22 ± 1°C in Schneider's medium supplemented with 10% fetal bovine serum (FBS) by serial subcultures after every 48 to 72 h.
The BALB/c mice (female, 6 to 8 weeks old, 18 to 20 g) used in the drug activity assay were obtained from the Centro de Bioterismo of IRR/Fiocruz Minas. The Swiss mice (female, 5 to 8 weeks old, 25 ± 5 g) used in the pharmacokinetic study were obtained from the Animal Facility Center of the Institute of Biological Sciences of the Federal University of Minas Gerais (UFMG). Free access to a standard diet was allowed, and tap water was supplied ad libitum.
Infection and treatment of animals.BALB/c mice were inoculated intravenously (via tail vein) with 2 × 107 late-log-phase L. (L.) infantum chagasi promastigotes. After 7 days of infection, mice (6 per group) were treated for 30 consecutive days with the pentavalent antimonial compounds, as follows: (i) commercial MA (Glucantime), given by the oral route at 300 mg of Sb/kg/12 h; (ii) reconstituted freeze-dried MA synthesized from SbCl5, given by the oral route at 300 mg of Sb/kg/12 h; (iii) reconstituted freeze-dried MA synthesized from KSb(OH)6, given by the oral route at 300 mg of Sb/kg/12 h; (iv) commercial MA given by the intraperitoneal route at 80 mg of Sb/kg/day (positive control); and (v) saline given twice a day by the oral route (negative control). Mice were sacrificed on day 40 postinfection in a CO2 chamber, and the liver and spleen were collected from each animal.
Determination of parasite burden after oral treatment.Three days after the interruption of treatment, the numbers of viable parasites in the liver and spleen were determined using the quantitative limiting dilution assay (22), with modifications. Briefly, organs were weighed and aseptically fragmented, and a tissue homogenate was obtained with 2 ml of Schneider's medium supplemented with 10% FBS, 50 U/ml penicillin, and 50 mg/ml streptomycin at pH 7.0. Each tissue homogenate was serially diluted (10-fold) into 96-well flat-bottom microtiter plates in triplicate and incubated at 26°C for 10 days. The wells containing motile promastigotes were identified with an inverted microscope (Axiovert 25; Zeiss), and the parasite burden was determined from the highest dilution at which promastigotes had grown after 10 days of incubation; i.e., the parasite burden equals 10highest dilution. Data are presented as the parasite burden per milligram of organ.
Hepatic concentration of Sb after oral treatment.The concentration of Sb was determined in the liver as previously described (12) by graphite furnace atomic absorption spectrometry (GFAAS) using a Perkin-Elmer AA600 spectrometer (Shelton, CT, USA). An aliquot of the liver homogenate was digested with nitric acid in a dry block MA 4004 (Marconi, Sao Paulo, Brazil). The analytical method for the determination of Sb in the liver was validated and showed suitable levels of precision (coefficient of variation, <5%), accuracy (80 to 120% analyte recovery), and linearity (range, 10 to 180 μg of Sb/liter). The quantification limit of the analytical method was 0.93 μg of Sb/g of wet organ.
Serum pharmacokinetics after oral administration.Swiss mice received through an oral inoculum, with the aid of a needle gauge, 0.2 ml of an aqueous solution of synthetic MA [prepared from either SbCl5 or KSb(OH)6] or commercial MA at 300 mg of Sb/kg of body weight. Mice from each group were euthanized (n = 5/time) at different time intervals: 15 and 30 min and 1, 2, 3, 4, 6, and 8 h after administration. Animal sacrifice was done by cervical dislocation after ketamine-xylazine anesthesia. Blood samples were then collected from the brachial plexus, and the serum was recovered and frozen at −20°C. The amount of Sb was determined in the serum diluted 1:40 with 0.2% nitric acid by GFAAS using a Perkin-Elmer AA600 spectrometer, as described previously (12). The analytical method for determination of Sb in the serum was validated and showed suitable levels of precision (coefficient of variation, <5%), accuracy (80 to 120% analyte recovery), and linearity (range, 10 to 180 μg of Sb/liter). The quantification limit of the analytical method was 240 μg of Sb/liter. Pharmacokinetic parameters were determined using the R-STRIP 4.03 computer program. Experimental Sb concentration-time data were subjected to iterative weighted nonlinear least-squares regression. A bi-exponential model with oral bolus input was chosen. Fitted parameters included the maximum concentration of Sb (Cmax), the time to reach the peak Sb concentration (Tmax), the mean residence time of Sb in the 0–∞ interval (MRT), and the area under the concentration-time curve in the 0–∞ interval (AUC).
Drug permeation of a cellulose membrane.Freshly prepared aqueous solutions of synthetic MA [from either SbCl5 or KSb(OH)6] or commercial MA, at 0.7 M Sb, were subjected to an ultrafiltration process, using an Amicon Ultra regenerated ultrafiltration cellulose membrane (MWCO, 3 kDa), by centrifugation at 14,000 × g for 15 min at 25°C. The amount of Sb in each sample under study (before and after filtration) was quantified by GFAAS using a Perkin-Elmer AA600 spectrometer, and the percentage of Sb that crossed the membrane was calculated. Data were obtained from five independent experiments.
Drug permeation of a Caco-2 cell monolayer.The drug permeation assay, using a Caco-2 cell monolayer as a model of intestinal epithelium, was performed as previously described (23–25). Briefly, human colon carcinoma Caco-2 cells were cultured in Dulbecco's modified Eagle medium supplemented with 20% FBS, 1% nonessential amino acids, and 0.2% penicillin-streptomycin, at 37°C in an atmosphere of 5% CO2 and 95% humidity. Caco-2 cells with 40 to 45 passages were used. The medium was changed every 2 to 3 days.
To prepare the Caco-2 monolayer, cells were seeded in 12-well polyester Corning Transwell plates (multiwell insert system; Sigma-Aldrich, St, Louis, MO, USA) at an initial density of 1 × 105 cells/well and grown for 21 days until the formation of a monolayer in each well. For the permeation study, the medium was removed and the cells were washed twice with prewarmed (37°C) Hanks' balanced salt solution (HBSS; pH 7.4) (Sigma-Aldrich) buffer. Solutions of synthetic and commercial MA, prepared at 50 mM Sb through dilution of the stock solutions with HBSS without phenol, were applied to the apical side of the Caco-2 monolayer (2.0 ml), followed by incubation at 37°C for 120 min. The integrity of the monolayer was checked just before incubation and at the end of the experiment by measuring the transepithelial electrical resistance (TEER) with the aid of a voltmeter coupled to microelectrodes placed in both compartments (apical and basolateral) of the inserts (Millicell ERS-2 epithelial volt-Ω meter). TEER measurements were greater than approximately 280 Ω/cm2. At time intervals of 10, 20, 40, 60, 90, and 120 min, 200-μl samples were retrieved from the donor compartment and replaced with the same volume of HBSS buffer. The amount of permeated Sb was determined by GFAAS using a Perkin-Elmer AA600 spectrometer, and the apparent permeability coefficient (Papp) was determined as described previously, according to the following equation (23): Papp = ΔQ/(ΔT × A × C0), where ΔQ is the amount of permeated drug in the receptor compartment during the time interval, ΔT is the time interval, A is the surface area of the membrane (1.13 cm2), and C0 is the initial drug concentration in the donor compartment (50 mM Sb).
Intracellular incorporation of Sb by peritoneal macrophages.Peritoneal macrophages were obtained from Swiss mice after intraperitoneal injection of 2 ml of 3% sodium thioglycolate medium. Mice were euthanized after 72 h by cervical dislocation following ketamine-xylazine anesthesia, just before removal of peritoneal macrophages by washing with cold RPMI 1640 medium. The cells were washed and resuspended at 1 × 106 cells/ml in RPMI 1640 medium supplemented with 10% FBS, 50 U/ml penicillin, and 50 mg/ml streptomycin. The cells were then incubated in centrifuge tubes at 37°C in a humidified 5% CO2 atmosphere for 4 h in the presence of the antimonial compounds at 0.8 mM Sb. After incubation, the cells were submitted to centrifugation (2,000 × g) for 10 min at 4°C and washed three times with cold phosphate-buffered saline (PBS). Concentrated nitric acid (65%) was then added to the cell pellet, and after overnight incubation at room temperature, the digested sample was diluted 100-fold with 0.2% nitric acid. The concentration of Sb was then determined by GFAAS. The uptake of Sb from each antimonial compound was evaluated in triplicate.
Statistical analysis.All experiments were performed in triplicate independently. Statistical analyses of the data were performed using GraphPad Prism software, version 6.0 (La Jolla, CA, USA). To compare the results obtained for different experimental groups, the Kruskal-Wallis test (followed by Dunn's multiple-comparison test) was used in cases of data with a nonnormal distribution. One-way analysis of variance (ANOVA) with the Bonferroni posttest was used to perform comparisons between normally distributed data. Differences with P values of <0.05 were considered statistically significant.
ACKNOWLEDGMENTS
We thank Flaviana R. Fernandes, Lídia M. de Andrade, Vicente Almeida Freitas, and Adriel A. F. Ferreira for technical support.
This work was financially supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant no. 472468/2013-8), Coordenação de Aperfeicoamento de Pessoal de Nível Superior (CAPES; grant no. PNPD20131163), and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG; grant no. RED-00007-14, APQ-01542-13, and BPD-00222-14). F.F. and A.R. were recipients of fellowships from CNPq (grant no. 303227/2013-3 and 303798/2013-0).
F.F., K.C.K., J.D.C.-J., W.V.D.C., A.R., M.F.L., and C.D. participated in the experimental design. K.C.K. and E.D.M.T. performed the experiments. K.C.K., F.F., A.R., J.D.C.-J., M.F.L., and A.I. analyzed the data. K.C.K. and F.F. interpreted the results. F.F., K.C.K., and A.I. prepared the manuscript draft. All authors reviewed, edited, and approved the final manuscript.
The authors declare no conflict of interest.
FOOTNOTES
- Received 18 March 2018.
- Returned for modification 3 April 2018.
- Accepted 30 May 2018.
- Accepted manuscript posted online 4 June 2018.
- Copyright © 2018 American Society for Microbiology.
